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current medical treatment of hyperthyroidism consists of the antithyroid medication such as, methimazole (mz) or propylthiouracil (ptu). in most cases, these medications restore euthyroidism in 1 to 2 months. when conventional managements have failed, surgery or radioactive iodine is usually considered for alternative treatment modality. literature reviews have reported a few cases that showed poor response to conventional therapy and required additional management. cholestyramine (cs), a bile salt sequestrant, has been used to lower cholesterol since the 1960s. cs forms an insoluble complex with the bile acids, preventing the reabsorption from the intestines. it is also an ion exchange resin, which binds to iodothyronines and would lower serum thyroid hormone level. several studies demonstrated that using cs in combination with conventional antithyroid medication, mz, or ptu, produces a more rapid decline of serum thyroid hormone. we recently reported one thyrotoxic female patient who showed refractoriness to nearly all conventional antithyroid medications and finally underwent total thyroidectomy after short - term administration of cs and following achievement of near euthyroid state. herein we retrospectively evaluated the efficacy and safety of high - dose cs added to maximal dose of antithyroid drugs used in a short period to control thyrotoxicosis and not to increase the dose of antithyroid drugs. we retrospectively reviewed patients who were prescribed cs to control dyslipidemia, hyperbilirubinemia, and thyrotoxicosis from january 2014 to december 2014. among these patients we finally enrolled five patients (three women and two men, aged 18 to 64) because they took both high dose mz (at least 30 mg) and cs (4 g three times a day, totally 12 g per day) during the initial short term to control severe thyrotoxicosis and checked thyroid function tests in a timely manner. we made up of control group in which patients with the age, gender, and initial free thyroxine (t4) levels matched to combination therapy group, received nearly the same dose of mz within the same period, to evaluate the efficacy and safety of adding cs to high dose antithyroid drug. totally, five patients in combination therapy group and 12 in control group were included in this study and their clinical characteristics were described in table 1. among them, one patient who presented severe nausea and vomiting after taking high - dose mz led to administration of cs to lower total dose of antithyroid drugs. compared to age and gender matched control group with mz - only use during the same period of treatment, combination therapy group showed more rapid reduction of free t4 and triiodothyronine (t3) levels in nearly all subjects (fig. 1), even though statistically insignificant differences due to the small number of study subjects. judging from the changes in thyroid hormone levels associated with mz plus cs, reduction of t3 levels was higher than that of free t4 levels (table 1). combination therapy group reported only one minor pruritus which might be due to thyrotoxicosis by itself or adverse events of mz so no cs - related adverse events were observed. on the other hand, control group showed one hepatitis and three pruritus, which were resolved after short - term medication. cs 's ionic exchange properties could decrease excessive thyroid hormone level by enhancing thyroid hormone clearance by enterohepatic circulation. by this mechanism of action, cs control thyrotoxic symptom effectively by reducing circulating hormone levels. unfortunately, it is uncertain whether cs influences the clinical prognosis, such as remission rates and relapse rate. this study showed more rapid improvement of thyroid hormone level by adding high - dose cs to conventional mz therapy without more adverse events, compared to mz therapy only. sebastian - ochoa. reported that totally 12 g per day of cs with mz for 4 weeks resulted in more rapid decline in all thyroid hormone levels. in our study, we adopted the same protocol, which is 12 g a day of cs combination with mz for 4 weeks. rapid decline in all thyroid hormones, especially total t3 decline was observed after 4 weeks of treatment as a consequence of the combination treatment. who reported that total t3 reduction as well as free t4 was observed in cs treated group. however, in the study of mercado. t3 levels declined for the second 2 weeks of the study after 1 week of washout period, not for the first 2 weeks. unfortunately, we were unable to observe the declining pattern of t3 level during the study. our study subjects took nearly 30 mg of mz, which might be conventional maximal dose, without any dose escalation above that. usually, adverse events of antithyroid drug are dose - dependent so those may have more adverse events who should take more antithyroid drug above maximal conventional dose as in 30 mg of mz. due to the combination of cs to patients who already took high dose of mz, we could control thyrotoxicosis without adding another doses of mz or experiencing adverse effects. combination therapy by antithyroid drugs with different side effect profiles will be a safer choice to reduce high levels of thyroid hormone. fortunately, none of the patients complained of typical symptoms during the treatment with cs. considering one of the clinical manifestations of thyrotoxicosis is diarrhea, constipation as an adverse effect of cs seems to be even favorable. however, we need more sample size to validate the effect of cs and to estimate the risk and benefit of use of cs. limitations of our study include that the sample size is too small to demonstrate statistically significance of the efficacy and safety of cs. our result only implicates the tendency to rapid decrease in free t4 and t3 level with cs use without major side effects. another limitation is that we measured the change of tsh - receptor - antibody (r - ab) level for only two patients in case group and three patients in control group. tsh - r - ab measurement is another critical cues to evaluate the improvement of thyrotoxicosis. unfortunately, tsh - r - ab levels were not measured at follow - up in most cases so we could not obtain any clinical meaning from these facts. in conclusion, cs could be safely applicable adjunctive therapy for 4 weeks when first - line antithyroid medications are not enough to adequately control severe thyrotoxicosis or side effects of antithyroid drug would be of great concern at the initial stage of treatment.	cholestyramine (cs) is an ion exchange resin, which binds to iodothyronines and would lower serum thyroid hormone level. the use of cs added to conventional antithyroid drugs to control thyrotoxicosis has been applied since 1980 's, and several studies indicate that using cs in combination with methimazole (mz) produces a more rapid decline in serum thyroid hormones than with only mz treatment. our recent retrospective review of five patients taking high dose mz and cs, compared to age-, gender-, initial free thyroxine (t4) level-, and mz dose - matched 12 patients with mz use only, showed more rapid decline of both free t4 and triiodothyronine levels without more adverse events. cs could be safely applicable short - term adjunctive therapy when first - line antithyroid medications are not enough to adequately control severe thyrotoxicosis or side effects of antithyroid drug would be of great concern.
physical attractiveness is an important social issue in our culture, and the face is one of its key features.1 facial attractiveness and smile attractiveness appear to be strongly connected to each other, since attention is mainly directed toward the mouth and eyes of the speaker 's face in social interactions. the mouth is the center of facial communication, and the smile plays important roles in facial expression and appearance.2 the demand for improved dental appearance is one of the major motives of orthodontic treatment. many adults and adolescents perceive their smile as unpleasant and seek orthodontic treatment in order to improve it. an esthetically pleasing smile is not only dependent on components such as tooth position, size, shape, and color, but also on the amount of gingiva displayed and the framing of the lips.3 all these components form a harmonic and symmetric entity. kokich and colleagues4 were the first to use computer - based image alterations to quantify the acceptability of smile characteristics. because of the consistency of variable manipulation and controlled presentation, computerized alteration appears to be an effective method to explore esthetics. through this method, it was possible to assess the perceptive abilities of dentists and laypersons under various conditions such as dental midline shift, asymmetry of crown width / height, canting of the occlusal plane, buccal corridor minimization, and excessive gingival display, and showed that these factors affected the attractiveness of the smile.4,5,6 among these factors, laypersons showed different perceptive abilities in maxillary to mandibular midline discrepancy, asymmetrical central incisor crown length, lateral incisor width, and canting of the occlusal plane compared to dentists, demonstrating a larger acceptable range. esthetic perception varies from person to person and is influenced by each person 's experiences and social environment. miller7 stated that the trained and observant eye readily detects that which is out of balance and not in harmony with its environment. there are several studies reporting that not only level of education, but also gender, age, and culture can influence the esthetic perception of dental features.8,9,10,11,12,13 however, there are few studies regarding the effects of orthodontic treatment experience on esthetic self - perception. tuominen.14 reported that after orthodontic treatment, adult patients showed higher perception in treatment demand than others who did not receive orthodontic treatment. kerosuo.15 and bernab.16 reported that orthodontic treatment had a positive effect on the self - perception of dental treatment needs in young adults. the studies used the aesthetic component of the index of orthodontic treatment need and visual analog scale (vas), respectively, and reported that treated young adults showed a higher self - perception of dental appearance. feu.17 found that in adolescents, orthodontic treatment with a fixed appliance significantly improved esthetic self - perception. however, these studies focused largely on evaluating gross esthetic discrepancies related to debilitating malocclusions by using various intraoral photographs of anterior dental crowding, crossbite, and so on. moreover, a previous study18 examined the perception of esthetic smile components such as gingival exposure and maxillary dental midline deviation by laypersons with and without orthodontic treatment by using only 3 modified images for the evaluation of these variables. the purpose of this study was to examine whether the experience of orthodontic treatment affects the individual 's perception of smile esthetics and to evaluate differences among orthodontically treated patients, non - treated laypersons, and dentists by using computerized image alterations. the non - treated layperson group (group 1 : n = 50, 12 men and 38 women ; mean age, 22.0 2.9 years) consisted of wonkwang university (iksan, korea) college students who had never received orthodontic treatment. the treated layperson group (group 2 : n = 50, 6 men and 44 women ; mean age, 23.8 3.6 years) consisted of patients who had completed orthodontic treatment at wonkwang university dental hospital (iksan, korea). the dentist group (group 3 : n = 30, 17 men and 13 women ; 27.4 5.1 years) consisted of residents at wonkwang university dental hospital. a photograph of a posed smile of young korean woman was digitally altered by using adobe photoshop (version cs3 ; adobe systems, inc. variables such as maxillary right central incisor gingival margin height, maxillary left lateral incisor crown width and length, incisal plane canting, and dental midline shift were altered gradually from the original photograph. after manipulation, the nose and chin were erased to reduce the number of variables in the images. however, for the dental midline shift, part of the nose was left to help evaluate the facial midline. the clinical crown length of the maxillary right central incisor was shortened in 0.5-mm increments by changing the free gingival margin while maintaining the incisal edge at the same level (figure 1). the maxillary left lateral incisor crown width and length were altered in 1.0-mm increments. as the crown width decreased, the gingival margin moved incisally while the crown width / height proportion was maintained (figure 2). canting of the incisal plane was manipulated by altering the plane from the right canine to the left canine (figure 3). since it was impossible to see the interpupillary line in the photographs, the lower lip line and incisal plane were positioned parallel to the lower border of the original photograph as a reference. by rotating around the central point of the incisal embrasure between the central incisor crowns, gradual canting was performed in 1.0-mm increments for the entire anterior dental segment while positioning the left side inferior and the right side superior. the maxillary dental midline was shifted to the patient 's right side in 0.5-mm increments, and the mandibular dental midline was shifted to the patient 's left side in 0.5-mm increments, for a total of 1.0 mm of gradual change while keeping the adjacent soft tissue intact (figure 4). the 20 photographs, consisting of 16 digitally altered photographs and 4 original images, were compiled into a catalog of 5 pages. each rater received the catalog and an evaluation sheet with a 100-mm vas. on the vas, the leftmost position indicated " very unattractive " and the rightmost position " very attractive. " the vas score ranged from 0 to 100, with 0 being the minimum and 100 the maximum esthetic value. the scores were measured from the leftmost point to the mark made by the rater. the data were statistically analyzed with pasw statistics software (version 18.0 ; ibm co., armonk, ny, usa). differences in the mean esthetic scores among the levels of asymmetries were analyzed by using a one - way repeated analysis of variance (anova). the hypothesis that there was no difference between the various altered smiles among each group of raters was tested. measurement error was calculated by the formula where d is the difference between 2 measurements made by the same observer within a 2-week interval, and n is the size of the sample. means, standard deviations, and the result of the newman - keuls test for each group are shown in figure 5 and tables 1,2,3,4. in the most altered photograph for each variable, 0.0001) were significantly lower when the gingival margin height of the central incisor was changed 1.0 mm or more. the vas scores of group 1 (f = 18.60 ; p < 0.0001) and group 2 (f = 16.65 ; p < 0.0001) were significantly lower when the gingival margin height of the central incisor was changed 1.5 mm or more (table 1). regarding the lateral incisor crown width and length, the vas scores of group 3 (f = 9.78 ; p < 0.0001) were significantly lower for changes of 3.0 mm or greater. similarly, group 1 (f = 4.04 ; p = 0.0003) and group 2 (f = 10.46 ; p < 0.0001) could distinguish a 3.0-mm change in crown width, although each group showed overlapping of the newman - keuls groups for 2 photographs (the 1.0-mm and 2.0-mm altered photographs in group 1 and the 2.0-mm and 3.0-mm altered photographs in group 2) (table 2). 0.0001) were significantly lower when the canting of the incisal plane was 1.0 mm, while they were lower in group 2 (f = 16.90 ; p < 0.0001) when the canting was 2.0 mm. group 1 (f = 6.83 ; p < 0.0001) perceived a change when the amount of canting was 3.0 mm or greater, although the newman - keuls groups overlapped for 2 photographs (the 1.0-mm and 2.0-mm altered photographs) (table 3). group 1 (f = 1.41 ; p = 0.23) showed no significant difference among all photographs with dental midline shifts, unlike group 2 (f = 4.62 ; p = 0.001) and group 3 (f = 3.72 ; p = 0.006), which showed significantly different vas scores and demonstrated overlap of the newman - keuls groups for 2 photographs (the 2.0-mm and 3.0-mm altered photographs in both groups). both groups perceived a change when the amount of shift was 3.0 mm or greater (table 4). when the perceptive ability among the three groups was compared, statistical differences in most situations were observed. however, no difference was shown among the three groups in perceiving the original image versus an image in which the central incisor gingival margin height had been altered by 0.5 mm. groups 1 and 2 showed differences in perceiving the image in which the incisal plane was canted 4.0 mm and the dental midline was shifted 3.0 mm (tables 1,2,3,4). regarding gender, there was no statistically significant difference in the reported vas scores (p < 0.05). in order to investigate the effects of orthodontic treatment experience on one 's perception of smile esthetics, we used four variables in this study : maxillary central incisor gingival margin height, maxillary lateral incisor crown width and length, canting of the incisal plane, and dental midline shift. although other various factors affect the smile, these four variables are easy to perceive and showed differences between laypersons and dentists in other studies.4,5 in most situations, dentists were more critical of dental esthetics than laypersons (tables 1,2,3,4). this finding corroborates the results of previous studies regarding esthetic perceptions showing that dentists and laypersons have different perceptions of smile esthetics and that dentists are less tolerant of some dental conditions than the general public.4,5,19,20,21,22 recent studies reported that laypersons could not detect an asymmetric crown length unless 1 crown was 1.5 - 2.0 mm shorter than the other.5,22 similarly, in our study, the non - treated group perceived a smile as unattractive when the gingival margin height was 1.5 mm or greater, while the dentist group perceived it as such when the margin height was 1.0 mm or greater (table 1). this suggests that correcting gingival margin asymmetries of the maxillary central incisors in the range between 0.5 mm and 1.5 mm might be not necessary, since it is not a concern of patients.23,24 however, correcting such an asymmetry might be justifiable after it is fully discussed with the patient. in clinical situations, we frequently encounter patients with a lateral incisor that is shorter or narrower than the contralateral incisor. in this study, the dentist group perceived a smile as unattractive when the crown width difference between the maxillary lateral incisors was 3.0 mm (table 2). this result in the dentist group was the same as in the study of kokich.,5 but the results from the layperson (non - treated) group showed some differences. in their study, the layperson group perceived a 4.0-mm discrepancy ; but in our study, the non - treated group could not perceive such a minor change. they also reported that it was difficult for laypersons to perceive change when the crown length was decreased along with the crown width (4.0 mm) while maintaining the same ratio, and more difficult than when only the crown width was decreased (2.0 mm). in this study, unlike the non - treated group, the treated group was able to distinguish between a 1.0 - 2.0 mm change and a change of 4.0 mm, meaning that the perceptive ability of the treated group to distinguish changes was greater than in the non - treated group (table 2). in this study, the dentist group perceived a smile as unattractive when the canting of the incisal plane was 1.0 mm. the treated and non - treated groups perceived it as unattractive when the cantings were 2.0 mm and 3.0 mm, respectively (table 3). these results are similar to those of kokich.,4 who reported that the threshold was 1.0 mm among orthodontists and general dentists, while it was 3.0 mm among laypersons. ker.25 also found that laypersons accepted as much as 4 degrees of canting (equivalent to 3.0 mm). however, there was no report regarding the increased perceptive ability of a treated group. the threshold difference among dentists, treated laypersons, and non - treated laypersons in our study could mean that the experience of orthodontic treatment affects perceptive ability regarding incisal plane canting. in contrast, padwa.26 reported that the perception of occlusal plane canting depends on the degree of inclination, and not necessarily on the levels of experience of the observers. the dentist group perceived a difference when the midline was shifted 3.0 mm in this study (table 4). similarly, kokich.4 reported that orthodontists classified smiles as least attractive only when midline shifts reached 4.0 mm. on the contrary, pinho.22 reported that orthodontists and prosthodontists were less tolerant of dental midline shifts, and rated 1.0 mm and 3.0 mm shifts as less attractive, respectively. the non - treated layperson group could not perceive dental midline shifts in this study, similar to the results of other studies.4,22 however, some studies reported that midline shifts greater than 2.0 mm are perceived by most people,20 and 56% of laypersons noticed 2.0-mm midline shifts.21 moreover, ker.25 reported that maxillary and mandibular dental midline deviations were deemed acceptable by laypersons until they exceeded 2.9 mm and 2.1 mm, respectively. these differing results can be explained by the different digital alterations, specialist participants, statistical analyses, and questionnaires. in these studies, only the maxillary or mandibular dental midline was shifted in the facial photographs, including the eyes and nose. moreover, laypersons evaluated the photographs only as " acceptable " or " not acceptable " and did not score them. our study is notable because the perceptions of dental midline shift were similar between the treated group and the dentist group. this finding is different from another study18 concluding that orthodontic treatment experience did not influence a layperson 's perception of maxillary dental midline shifts. these discrepant results might be explained by differences in the methods used, and by the degrees and numbers of image alterations. based on this result, we speculate that a patient 's esthetic perception is improved through orthodontic treatment because of the increased attention paid during frequent appointments with an orthodontist. previous studies regarding the influence of age on esthetic perceptions showed discrepant findings.1,10,12,27 some studies reported that young adults paid more attention to the appearance of anterior teeth,12,27 while other studies reported that age did not influence esthetic perceptions.1,10 therefore, we restricted the age range of the laypersons to exclude the influence of age from this study. the male - to - female ratio in our study was 12:38 for non - treated laypersons, 6:44 for treated laypersons, and 17:13 for dentists, respectively. there was no statistically significant difference in the mean scores given by men and women raters within each group. this agreed with the findings of kokich.5 and rosenstiel and rashid,6 but is in contrast to the findings of geron and atalia,8 where women were found to be more tolerant of upper gingival exposure. this difference in results between the two studies is likely attributable to variations in esthetic perceptions among different populations. in order to simulate smile esthetics, we used a computer program to produce various dental conditions in this study. it was possible to assess various dental features independently using the same photograph. in our study, laypersons with orthodontic treatment experience showed more keen perceptive abilities than those without treatment experience. however, we are not suggesting that the results of our research should be interpreted as a general fact that can be applied to every patient in practice. this study was a cross - sectional study with different participants included in the non - treated and treated groups. therefore, a longitudinal study using the same participants in a non - treated group before treatment and in a treated group after treatment should be conducted in order to confirm whether orthodontic treatment experience affects perceptions. to examine whether the experience of orthodontic treatment affects the individual 's perception of smile esthetics, computerized image alterations and questionnaires with a vas were used to compare perceptions among laypersons with and without orthodontic treatment experience and dentists. laypersons with and without orthodontic treatment experience and dentists had different perceptions of smile esthetics. while laypersons perceived a 1.5 mm decrease in the gingival margin height of the maxillary central incisor, dentists perceived a 1.0 mm decrease. laypersons and dentists classified smiles as least attractive when alterations of the crown width and length of the maxillary lateral incisor reached 3.0 mm. non - treated laypersons perceived changes in the incisal plane when the canting was 3.0 mm, while treated laypersons and dentists perceived canting at 2.0 mm and 1.0 mm, respectively. the results of the present study suggest that orthodontic treatment experience improved esthetic perceptions among laypersons.	objectivethe purpose of this study was to examine whether orthodontic treatment experience affects the individual 's perception of smile esthetics and to evaluate differences among orthodontically treated laypersons, non - treated laypersons, and dentists by using computerized image alterations.methodsa photograph of a woman 's smile was digitally altered using a software image editing program. the alterations involved gingival margin height, crown width and length, incisal plane canting, and dental midline of the maxillary anterior teeth. three groups of raters (orthodontically treated laypersons, non - treated laypersons, and dentists) evaluated the original and altered images using a visual analog scale.resultsthe threshold for detecting changes in maxillary central incisor gingival margin height among laypersons was 1.5 mm ; the threshold of dentists, who were more perceptive, was 1.0 mm. for maxillary lateral incisor crown width and height, the threshold of all three groups was 3.0 mm. canting of the incisal plane was perceived when the canting was 3.0 mm among non - treated laypersons, 2.0 mm among treated laypersons, and 1.0 mm among dentists. non - treated laypersons could not perceive dental midline shifts ; however, treated laypersons and dentists perceived them when the shift was 3.0 mm.conclusionslaypersons with and without orthodontic treatment experience and dentists have different perceptions of smile esthetics. orthodontically treated laypersons were more critical than non - treated laypersons regarding incisal plane canting and dental midline shifts. based on these findings, it is suggested that orthodontic treatment experience improved the esthetic perceptions of laypersons.
receptors of melanocortins are encoded by a gene family consisting of five members (mc1r mc5r). the encoded receptors bind four ligands : -, - and -melanocyte - stimulating hormone (-, -, -msh) and the adrenocorticotropic hormone (acth). among them, mc1r binds preferentially -msh, while mc2r binds acth. expression of the mcr genes is tissue - specific : mc1r is mainly expressed in melanocytes, mc2r in the adrenal cortex, mc3r and mc4r in the nervous system, and mc5r in sebaceous glands and other tissues, e.g. the brain, muscles, lung and kidney (yang 2011). the physiological role of the melanocortin receptors has been previously reviewed several times (cone 2006 ; eves and haycock 2010 ; yang 2011). also, the effect of their mutations and polymorphisms in humans was reviewed, especially with regard to cutaneous pigmentation, mc1r (dessinioti. 2011), and obesity, mc3r (tao 2010b) and mc4r (santini. 2009 ; tao 2010a ; loos 2011). the mcr genes, mainly mc1r and mc4r, were also extensively studied in domestic mammals. polymorphism of these genes was analysed in terms of coat colour variability or the association with production traits related to fat tissue deposition and feed conversion ratio. thus, this article is focused on studies of the mcr gene family polymorphisms in domestic mammals. the mcr genes contain only a single exon and the encoded number of amino acids varies from 296 (mc2r) to 332 (mc4r). some of them (mc2r, mc4r and mc5r) are located on a single chromosome : 18 in humans and the mouse, 24 in cattle and 1 in the dog. in the case of the pig karyotype, the location is slightly different, since the mc1r, mc2r and mc5r genes reside on chromosome 6, but not mc4r, which is not syntenic with mc2r and mc5r. this difference reflects chromosome rearrangements which took place during pig karyotype evolution (goureau. 1996).table 1chromosomal location of the mcr genes in mammalian speciesgenehumanmousepigcattledog mc1r 1686185 mc2r 18186241 mc3r 202171324 mc4r 18181241 mc5r 18186241 chromosomal location of the mcr genes in mammalian species a comparison of the coding sequences of the two most frequently studied mcr genes (mc1r and mc4r) revealed a higher evolutionary conservatism of the mc4r protein (tables 2 and 3). in the case of the mc1r protein, the amino acid similarity varied between 74.0 % (mouse vs. dog) and 83.6 % (cattle vs. dog), while for mc4r, it varied between 90.7 % (mouse vs. cattle) and 96.1 % (pig vs. dog). a comparison of nucleotide sequences revealed practically the same level of similarity. for mc1r, it ranged between 75.1 % (mouse vs. cattle) and 85.3 % (pig vs. cattle), while for mc4r, it was between 84.0 % (mouse vs. cattle) and 91.3 % (human vs. pig).table 2identity (%) of the nucleotide (above the diagonal) and amino acid (below the diagonal) sequences of the mc1r gene in five mammalian specieshuman (953 nt)mouse (947 nt)pig (963 nt)cattle (953 nt)dog (952 nt)human (317 aa)76.984.682.781.9mouse (315 aa)75.974.675.175.6pig (320 aa)78.572.485.382.9cattle (317 aa)81.474.682.681.3dog (317 aa)80.474.082.083.6 nucleotide, amino acidtable 3identity (%) of the coding sequence (999 nucleotides, above the diagonal) of the mc4r gene and the encoded polypeptide (323 amino acids, below the diagonal) in five mammalian specieshumanmousepigcattledoghuman87.291.387.288.4mouse93.487.884.086.8pig95.894.088.589.5cattle92.890.794.087.2dog95.294.396.193.1 identity (%) of the nucleotide (above the diagonal) and amino acid (below the diagonal) sequences of the mc1r gene in five mammalian species nucleotide, amino acid identity (%) of the coding sequence (999 nucleotides, above the diagonal) of the mc4r gene and the encoded polypeptide (323 amino acids, below the diagonal) in five mammalian species knowledge on the genetic variants of melanocortin receptor genes and their phenotypic effects is most advanced for the mc1r and mc4r genes, and to a lesser extent for mc3r. two other genes (mc2r and mc5r) thus, its polymorphism was studied in terms of its effect on hair and skin colour in humans and coat colour in animals. it is estimated that human cutaneous pigmentation (skin, hair and eye) is controlled by approximately 120 genes, but a crucial role in this process is played by mc1r, for which over 100 missense polymorphisms were identified (dessinioti. altogether, 16 causative polymorphisms in seven species (pig 5, dog 3, cattle 2, sheep 2, arctic fox 2, horse 1 and red fox 1) were identified (table 4). the polymorphic sites were located in extracellular (6), transmembrane (5) or intracellular (5) domains.table 4polymorphic variants of the mc1r gene influencing coat colour in domestic animal speciesspeciespolymorphismfunction phenotypereferencesnucleotideamino aciddogc916targ306stop e (wild type) g.916cc and g.916ct p.306arg and p.306arg / p.306stop brown / black coat e 916 tt p.360stop red / yellow coatbreeds : golden retriever, yellow labrador, irish setternewton. (2000)g233tgly78val e g.233tt and g.233ct p.78val or p.78gly / p.78valand aa in asip gene phenotype grizzle or dominobreeds : saluki, afghan hounddreger and schmutz (2010)a790gmet264val e g.790gg or g.790ag p.264val or p.264val / p.264met black melanistic mask e g.790aa p.264met dogs without black melanistic maskbreeds : akita, bullmastiff, great dane, german shepherd, bouvier, whippet, english setter, dachshund, doberman pinscher, cocker spaniel, miniature poodle, irish setterschmutz. (2003)cattlet296cp.leu99pro e g.296cc p.99pro dominant black coat colour e g.296tt p.99leu wild type combination of red or reddish brown and reddish black coat colour ee p.155 (premature stop codon) red coat colourorder of dominance : e > e > e klungland. (1996)sheept218ag361ap.met73lysp.asp121asn e g.218aa and g.361aa p.73lys and p.121asn dominant black phenotypebreeds : norwegian dalavge. (1999)horsec901tp.ser83phe ee g.901cc p.83ser non - chestnut ee g.901ct p.83ser / p.83phe non - chestnut ee g.901tt p.83phe chestnutmarklund. p.99leu allows full expression of both pheomelanin and eumelanin e g.296c p.99pro dominant blackbreeds : asiankijas. (1998)g361ap.asp121asn e (wild type) g.361gg p.121asp e g.361 aa p.121asn dominant blackbreeds : europeankijas. (1998)c491tg727ap.ala164valala243thr e (wild type) g.491c g.727 g p.164ala p.243ala e g.491 t g.727a p.164 val p.243 thr red coat colourkijas. p.23 ala e black spotting on red or white backgroundkijas. (2001)red foxt373cgly5cys e g.373c p.125arg alaskan silver coatingvge. (2005) polymorphic variants of the mc1r gene influencing coat colour in domestic animal species in cattle, the black / red coat colour depends on two polymorphic sites, which give a series of three alleles : e, e and e. the e (p.99pro) and e wild type (p.99leu) substitutions are located in the first extracellular loop of the mc1r protein and are responsible for black coat colour and a combination of red or reddish brown / black coat colours, respectively (klungland. 1995). interestingly, the same mutation was observed in asian pigs and variant e (p.99pro) also caused black coat colour, while the wild allele (e) facilitates the full expression of both pheomelanin and eumelanin (kijas. the third allele (e) of the bovine mc1r gene was created by a deletion of guanine nucleotide at position 310/311, resulting in a premature stop codon, instead of the presence of tyrosine at the 155 position in the polypeptide (the second intracellular loop). interestingly, the genetic background of black colour is different in asian and european breeds. in asian breeds, black coat colour depends on the occurrence of the above - mentioned e allele (p.99pro), while in european breeds, the black coating is controlled by the e allele (p.121asn) (kijas. two substitutions are present (p.164val and p.243thr), which are located in the fourth and sixth transmembrane domains, respectively. it is not clear if one or both substitutions are responsible for this coat colour (kijas. the e allele responsible for black spotting on the red or white background was identified (kijas. 2001). this phenotype is a consequence of two c nucleotide insertions, at the position of the 67 nucleotide, leading to the frameshift and premature stop codon. studies on the molecular background of coat colour variation in canids revealed 22 polymorphic sites : ten in the dog, eight in the red fox, three in the arctic fox and one in the chinese raccoon dog (nowacka - woszuk. 2013). among them, six are responsible for coat colour : three in dogs, two in arctic foxes and one in red foxes (table 4). in dogs, four main alleles were identified : e (wild type), e, e and e. the recessive e allele was independently identified by two teams (newton. it is a c > t transition at the 916 nucleotide position, causing a premature stop codon, instead of arginine at the 306 position of the encoded polypeptide. the missense variant (p.306stop, allele e) leads to a reduction of the cytoplasmic tail of the receptor and, as a consequence, results in red / yellow coat colour in dogs. this allele was found in three breeds : golden retriever, yellow labrador and irish setter. the e (p.78val) allele produces the so - called grizzle phenotype in saluki and the domino phenotype in afghan hound breeds (dreger and schmutz 2010). the occurrence of a black melanistic mask in 12 dog breeds is controlled by the e allele, p.264val (schmutz. 2003). polymorphisms in the canine mc1r gene are located in the intracellular c - terminal extension (allele e), the second transmembrane domain (e) and the third extracellular loop (e). analysis of data collected from commercial and research canadian laboratories, performing dna tests to detect coat colour alleles in dogs, revealed the occurrence of the e and e alleles also in other breeds : the german wirehaired pointer, german shorthaired pointer and the great dane, while the e allele occurred in the basset hound, boxer and the chinese shar - pei (schmutz and melekhovets 2012). interestingly, the authors described white dogs with the e / e genotype in the chow, german shepherd dog, miniature schnauzer and puli breeds instead of the expected red or yellow coat colour. it was suggested that an interaction of an unknown gene with the e / e genotype causes elimination of the red pigment. in red foxes, the e allele (p.125arg), responsible for alaskan silver coating, was reported by vge. studies on the mc1r polymorphism in the arctic fox revealed two non - synonymous substitutions (p.gly5cys and p.phe280cys) in a highly conserved region of the protein, which is associated with a constitutive activation of the receptor (vge. n - terminus) and p.280cys (third extracellular loop) variants were observed in blue coat variants, which are rare in wild populations (35 %) and very frequent in farm populations. until now, only one silent polymorphism in the coding sequence of the mc1r gene (g.759c > t) was identified in the chinese raccoon dog (nowacka - woszuk. mc2r, also known as the adrenocorticotropic hormone receptor gene (acthr), encodes a receptor for the hormone, which plays a crucial role in the regulation of glucocorticoid secretion. the adrenocorticotropic hormone (acth) selectively activates the mc2r and induces glucocorticoid production and its secretion in the adrenal cortex, especially in zona fasciculata (mountjoy. recent studies revealed that small single - pass transmembrane proteins, called melanocortin receptor accessory proteins (mrap and mrap2), are essential for the expression of the melanocortin receptor type 2 and its transport to the plasma membrane (for reviews, see webb and clark 2010 ; novoselova. 2013). a crucial insight into the role of mc2r comes from knockout mice (chida. it was also concluded that mc2r knockout mice is a useful model for a rare, autosomal human hereditary disease, familial glucocorticoid deficiency (fgd). a majority of these mutations result in an unsuccessful protein traffic to the cell surface (webb. furthermore, some of the human mc2r polymorphisms (e.g. g.-184a, rs2186944) have a protective effect against heroin addiction in the spanish population (proudnikov. 2008). moreover, four snps (rs1893219, rs1893220, rs2186944 and g.-2t > c) showed an association with responsiveness to acth therapy in some types of epileptic encephalopathy, infantile spasms. the tcct haplotype results in an increased expression of mc2r and a stronger response to acth (liu. 2008 ; ding. 2010). the g.-2t > c mutation is also associated with higher levels of dehydroepiandrosterone, androstenedione and plasma acth in children with premature adrenarche (lappalainen. it is rather unlikely that functional polymorphisms of the mc2r gene may significantly contribute to the phenotypic variability of production traits in livestock. thus, it is not surprising that studies on the mc2r polymorphism in domestic animals are very scarce. according to the single nucleotide polymorphism database (dbsnps ; ncbi platform), only 11 snps in dogs, four in pigs and none in cattle, sheep and horse were identified. in the pig, the mc2r locus was mapped within a qtl region for intramuscular fat content and back fat thickness (jacobs. the authors showed that the distribution of a silent t > g substitution is different (p t and 549c > t) further studies of one of these snps (549c > t), carried out on a small sample (n = 101) of czech large white sows, revealed its association with the estimated breeding value for average daily weight gain (weisz. extensive studies on the mc3r gene in four species of the family canidae (dog, red fox, arctic fox and chinese raccoon dog) showed a variable level of its polymorphism (skorczyk. 2011). in total, 16 polymorphisms were described and a majority of them were found in the 5-flanking (8) and 3-flanking (2) regions. the mc3r gene of the red fox (eight polymorphic sites) and the chinese raccoon dog (six polymorphisms) appeared to be the most polymorphic. in the dog, only two polymorphisms association studies carried out in red foxes (n = 376) for two polymorphisms (silent substitution c.957a > c and c.185c > t in the 3-flanking region), revealed a significant relationship with body weight. the melanocortin receptor type 4 is a well known, major controller of food intake and energy expenditure (for reviews, see adan. 2006 ; tao 2010a). altogether, more than 150 variants were identified in this gene (tao 2009 ; loos 2011). the variants are classified into five groups according to the phenotypic effects they evoke (tao 2009). class i contains mutations causing defective protein synthesis or its accelerated degradation, resulting in dramatically decreased expression. class ii mutations cause receptor retention inside a cell, probably due to a misfolding of the receptor. class iii represents variants which are present on the cell surface, but their binding capacity or ligand affinity are impaired. variants causing defective signalling properties (decreased efficacy and/or potency) are categorised as class iv. variants causing unknown effects form class v. among the known variants, there are two (val103ile and ile251leu) which are considered as having a protective role against obesity (loos 2011). however, sometimes, this effect is not pronounced, especially if small populations are analysed (nowacka - woszuk. on the other hand, extensive genome - wide association studies (gwas) revealed that an snp located close to the mc4r gene is associated with a predisposition to polygenic obesity. this variant (rs17782313), mapped 188 kb downstream of the gene (loos. 2008), shows a strong association with an elevated bmi (for a review, see xi. association of the mc4r gene variants with human obesity have initiated studies on the relationship with fat tissue accumulation in livestock species. the most extensive studies were carried out in the pig, resulting in the identification of eight polymorphic sites (table 5). among them, the missense substitution c.1426 g > a (asp298asn) was the most extensively studied in terms of its association with production traits, mainly fatness, feed intake and feed conversion ratio. (2000a) and was originally named c.892 g > a.table 5genetic variants identified in the porcine mc4r gene. positions numbered according to nm_214173 (positions in brackets numbered according to ab021664)locationpositioneffectreferencesproximal promoterc.-780c > gpossible disruption of transcription factor binding sitefan. (2009)exon 1c.175c > t(c.706c > t)leu59leuovilo. (2006)exon 1c.707 g > positions numbered according to nm_214173 (positions in brackets numbered according to ab021664) the asp298asn substitution is located in a highly conserved motif within the seventh transmembrane domain. functional studies revealed that both polymorphic forms of mc4r bind its agonist with similar affinity. (2004) reported the asp298 variant s inability to generate signals, in contrast to fan. (2008), who proved a similar signalling force for both variants. in a majority of studies, it was claimed that allele asp298 is strongly associated with lower back fat thickness, higher lean meat percentage, slower growth rate and lower feed intake, while the asn298 allele had the opposite effects, i.e. higher fat deposition and faster growth, which seems to be a result of greater feed intake (table 6). association studies on meat quality traits and fatty acid composition were also performed (table 6). for example, ovilo. (2006) concluded that animals homozygous for the asn298 allele exhibit lower meat redness and a higher content of saturated fatty acids compared to the gg homozygote. further studies revealed that allele frequencies differ greatly among pig breeds and lines, probably due to long - term artificial selection. for example, pigs representing lines of the same breed and raised for fresh meat production showed an increased asp298 allele frequency when compared to those bred for cured ham and loin production (burgos. 2006). despite those promising reports, some of the subsequent studies failed to confirm an association of asp298asn snp in the mc4r gene with performance and quality traits in pigs (schwab. 2009 ; munoz. 2011) or reported breed - related differences in the observed effects (stachowiak. it is possible that this mutation might not be the causative one, only closely related to the real quantitative trait nucleotide (qtn), or there might be an epistatic interaction (bruun. 2006).table 6effects of the missense substitution c.892 g > a (presently described as c.1426 g > a) causing amino acid substitution (asp298asn) on pig production traitstraiteffect of allele g (asp) compared to allele a (asn)breedreferencesaverage daily gaindurockim. (2012)back fat thicknesslandrace ; large white ; large white duroc ; large white meishankim. (2006)landrace ; large white ; large white duroc ; large white meishankim. (2010)growth ratelandrace ; large white ; large white x duroc ; large white meishankim. (2006) effects of the missense substitution c.892 g > a (presently described as c.1426 g > a) causing amino acid substitution (asp298asn) on pig production traits another missense substitution (707a > g, arg236his) was detected in pietrain, vietnamese pigs and berkshire yorkshire crossbreds (kim. 2004 ; meidtner. animals carrying a minor allele a are fatter and grow more slowly than those carrying allele g. according to fan. (2009), this polymorphism co - segregates with four other snps (780c > g in promoter, 135c > t in 5utr, 175c > t synonymous substitution in exon 1 and 430a > t in putative 3utr), forming three haplotypes, which exhibit a significant association with average back fat thickness and average daily weight gain. as predicted by the in silico study, the occurrence of 780c > g and 135c > t snps may disrupt several transcription factor binding sites. the influence of these polymorphisms may be an explanation for the above - mentioned inconsistent data on the asp298asn association with production traits (fan. 17 polymorphic sites were discovered and, among them, 13 were silent mutations and four were missense substitutions. they occurred in the following positions : 293c > g, 193a > t, 192 t > g, 129a > g (zhang. 2009), 84 t > c (liu. 2010), 19c > a, 20a > t, 83 t > c, 128 g > a (huang. 2010), 709 g > a (val166met) (seong. 2012), 747 g > a, 927c > t (valle. 2004), 1069c > g (leu286val) (thue. 2001), 1343c > a, 1786c > t (seong. 2012), 145val > ala and 172ala > thr (haegeman. a majority of them were reported only once, of which six were reported to be associated with production traits (table 7). the most extensively studied substitution was 1069c > g, for which strong associations with back fat thickness, marbling, carcass and live weight were reported (huang. 2012).table 7genetic variants of the bovine mc4r gene and their association with production traitspositioneffect onbreedreferences293c > g129a > g(linked snps)body weightaverage daily gain293c/129ananyang, qinchuan, jiaxian red, jinnanzhang. (2012)989 g > aser330asnback fatgrade fata angus, holsteinmclean and schmutz (2011)length of longissimus dorsi (2012)simmental, angus, hereford, charolais, limousine, qinchuan, luxi, jinnanhuang. (2012)marblingt genetic variants of the bovine mc4r gene and their association with production traits studies of the canine mc4r gene revealed the presence of four snps, 637 g > t, 777 t > c, 33c > g (skorczyk. 2007) and 868c > t (van den berg. 2010). among them, only one snp resulted in amino acid substitution, namely, 637 g > t, changing valine to phenylalanine at position 213 (skorczyk. analysis of this polymorphism disclosed no association with morphological measures (van den berg. 2010), probably because ligand binding and signalling abilities are not disturbed when compared to the wild variant of the mc4r gene (yan and tao 2011). further studies focused on 5utr revealed the presence of two novel indels and three novel snps (nowacka - woszuk. there was an 11-bp indel within a putative upstream open reading frame (uorf). association studies (n = 381) did not show any relationship of the haplotypes with body weight. the mc5r gene is expressed in the central nervous system and in a variety of peripheral tissues, especially in the skin. the encoded protein is involved in different physiological processes, including lipid metabolism, exocrine function (yang. together with other members of the melanocortin receptor family, the mc5r expression down - regulates leptin secretion in the in vitro cultured adipocytes (hoggard. 2003), as well as mediates in the interleukin 6 (il6) production (jun. because a high level of the il6 circulating in blood correlates with insulin resistance (kristiansen and mandrup - poulsen 2005), and leptin takes part in regulating food intake and energy expenditure, melanocortin receptor 5 is a functional candidate gene for obesity in humans or fatness in domestic animals. also an. (2007) demonstrated the involvement of mcr subtype 5 in inducing fatty acid oxidation in skeletal muscles. despite a broad range of functions, only several polymorphisms of the mc5r gene were described in both humans and the domestic animals (table 8). in the pig genome, the mc5r gene was mapped closely to marker s0059, which is within a qtl for fatness and meat quality. several reports confirmed an association between porcine back fat thickness or feed intake and polymorphic variants of the mc5r gene (kovik. also, in humans the mc5r polymorphisms were reported to be associated with obesity (chagnon. 1997 ; valli - jaakola. 2008). due to a variety of physiological processes involving mc5r, it was also studied in relation to skin condition, metabolic and mental disorders. as a result of these studies, associations with type 2 diabetes, schizophrenia and bipolar disorder were documented (valli - jaakola. 2009).table 8polymorphisms of the mc5r gene and their phenotypic effects in humans and pigsspeciespositionstudied traitseffect onvariant present in breed / populationreferenceshumans849c > gphe209leuskin condition, acne vulgarisassociation not foundnegro, south indian, japanese, polynesian, caucasianhatta. (2001)ala81alanegro, south indian, japanese, polynesian, caucasianasp108aspnegro, inuit, japanese, caucasianser125sercaucasianthr248thrnegro, south indian, japanese, polynesian, caucasianpsti, pvuiiobesitybmi, fat mass, resting metabolic ratecaucasian (canada)chagnon. (2008)849c > g phe209leutype 2 diabetestype 2 diabetescaucasian (finland)valli - jaakola. (2008)mental disordersg allele predisposes to schizophrenia and bipolar disordercaucasian (usa), african americanmiller. (2009)pigs303a > g ala109thrfatness traitsaverage daily gain, feed intake, feed conversation g large white landracekovik. (2000b)fat deposition, carcass quality traitstenth rib back fat thicknessberkshire, duroc, hampshire, landraceemnett. (2000b) polymorphisms of the mc5r gene and their phenotypic effects in humans and pigs thus, its polymorphism was studied in terms of its effect on hair and skin colour in humans and coat colour in animals. it is estimated that human cutaneous pigmentation (skin, hair and eye) is controlled by approximately 120 genes, but a crucial role in this process is played by mc1r, for which over 100 missense polymorphisms were identified (dessinioti. altogether, 16 causative polymorphisms in seven species (pig 5, dog 3, cattle 2, sheep 2, arctic fox 2, horse 1 and red fox 1) were identified (table 4). the polymorphic sites were located in extracellular (6), transmembrane (5) or intracellular (5) domains.table 4polymorphic variants of the mc1r gene influencing coat colour in domestic animal speciesspeciespolymorphismfunction phenotypereferencesnucleotideamino aciddogc916targ306stop e (wild type) g.916cc and g.916ct p.306arg and p.306arg / p.306stop brown / black coat e 916 tt p.360stop red / yellow coatbreeds : golden retriever, yellow labrador, irish setternewton. (2000)g233tgly78val e g.233tt and g.233ct p.78val or p.78gly / p.78valand aa in asip gene phenotype grizzle or dominobreeds : saluki, afghan hounddreger and schmutz (2010)a790gmet264val e g.790gg or g.790ag p.264val or p.264val / p.264met black melanistic mask e g.790aa p.264met dogs without black melanistic maskbreeds : akita, bullmastiff, great dane, german shepherd, bouvier, whippet, english setter, dachshund, doberman pinscher, cocker spaniel, miniature poodle, irish setterschmutz. (2003)cattlet296cp.leu99pro e g.296cc p.99pro dominant black coat colour e g.296tt p.99leu wild type combination of red or reddish brown and reddish black coat colour ee p.155 (premature stop codon) red coat colourorder of dominance : e > e > e klungland. (1996)sheept218ag361ap.met73lysp.asp121asn e g.218aa and g.361aa p.73lys and p.121asn dominant black phenotypebreeds : norwegian dalavge. (1999)horsec901tp.ser83phe ee g.901cc p.83ser non - chestnut ee g.901ct p.83ser / p.83phe non - chestnut ee g.901tt p.83phe chestnutmarklund. (1996)pigt296cp.leu99pro e (wild type) g.296tt p.99leu allows full expression of both pheomelanin and eumelanin e g.296c p.99pro dominant blackbreeds : asiankijas. (1998)g361ap.asp121asn e (wild type) g.361gg p.121asp e g.361 aa p.121asn dominant blackbreeds : europeankijas. (1998)c491tg727ap.ala164valala243thr e (wild type) g.491c g.727 g p.164ala p.243ala e g.491 t g.727a p.164 val p.243 thr red coat colourkijas. (1998)nt67inscccodon 23 e (wild type) g.67_68cc p.23 ala e black spotting on red or white backgroundkijas. (2001)red foxt373cgly5cys e g.373c p.125arg alaskan silver coatingvge. (2005) polymorphic variants of the mc1r gene influencing coat colour in domestic animal species in cattle, the black / red coat colour depends on two polymorphic sites, which give a series of three alleles : e, e and e. the e (p.99pro) and e wild type (p.99leu) substitutions are located in the first extracellular loop of the mc1r protein and are responsible for black coat colour and a combination of red or reddish brown / black coat colours, respectively (klungland. interestingly, the same mutation was observed in asian pigs and variant e (p.99pro) also caused black coat colour, while the wild allele (e) facilitates the full expression of both pheomelanin and eumelanin (kijas. the third allele (e) of the bovine mc1r gene was created by a deletion of guanine nucleotide at position 310/311, resulting in a premature stop codon, instead of the presence of tyrosine at the 155 position in the polypeptide (the second intracellular loop). interestingly, the genetic background of black colour is different in asian and european breeds. in asian breeds, black coat colour depends on the occurrence of the above - mentioned e allele (p.99pro), while in european breeds, the black coating is controlled by the e allele (p.121asn) (kijas. two substitutions are present (p.164val and p.243thr), which are located in the fourth and sixth transmembrane domains, respectively. it is not clear if one or both substitutions are responsible for this coat colour (kijas., the e allele responsible for black spotting on the red or white background was identified (kijas. 2001). this phenotype is a consequence of two c nucleotide insertions, at the position of the 67 nucleotide, leading to the frameshift and premature stop codon. studies on the molecular background of coat colour variation in canids revealed 22 polymorphic sites : ten in the dog, eight in the red fox, three in the arctic fox and one in the chinese raccoon dog (nowacka - woszuk., six are responsible for coat colour : three in dogs, two in arctic foxes and one in red foxes (table 4). in dogs, four main alleles were identified : e (wild type), e, e and e. the recessive e allele was independently identified by two teams (newton. it is a c > t transition at the 916 nucleotide position, causing a premature stop codon, instead of arginine at the 306 position of the encoded polypeptide. the missense variant (p.306stop, allele e) leads to a reduction of the cytoplasmic tail of the receptor and, as a consequence, results in red / yellow coat colour in dogs. this allele was found in three breeds : golden retriever, yellow labrador and irish setter. the e (p.78val) allele produces the so - called grizzle phenotype in saluki and the domino phenotype in afghan hound breeds (dreger and schmutz 2010). the occurrence of a black melanistic mask in 12 dog breeds is controlled by the e allele, p.264val (schmutz. polymorphisms in the canine mc1r gene are located in the intracellular c - terminal extension (allele e), the second transmembrane domain (e) and the third extracellular loop (e). analysis of data collected from commercial and research canadian laboratories, performing dna tests to detect coat colour alleles in dogs, revealed the occurrence of the e and e alleles also in other breeds : the german wirehaired pointer, german shorthaired pointer and the great dane, while the e allele occurred in the basset hound, boxer and the chinese shar - pei (schmutz and melekhovets 2012). interestingly, the authors described white dogs with the e / e genotype in the chow, german shepherd dog, miniature schnauzer and puli breeds instead of the expected red or yellow coat colour. it was suggested that an interaction of an unknown gene with the e / e genotype causes elimination of the red pigment. in red foxes, the e allele (p.125arg), responsible for alaskan silver coating, was reported by vge. studies on the mc1r polymorphism in the arctic fox revealed two non - synonymous substitutions (p.gly5cys and p.phe280cys) in a highly conserved region of the protein, which is associated with a constitutive activation of the receptor (vge. 2005). the p.5cys (extracellular n - terminus) and p.280cys (third extracellular loop) variants were observed in blue coat variants, which are rare in wild populations (35 %) and very frequent in farm populations. until now, only one silent polymorphism in the coding sequence of the mc1r gene (g.759c > t) was identified in the chinese raccoon dog (nowacka - woszuk. mc2r, also known as the adrenocorticotropic hormone receptor gene (acthr), encodes a receptor for the hormone, which plays a crucial role in the regulation of glucocorticoid secretion. the adrenocorticotropic hormone (acth) selectively activates the mc2r and induces glucocorticoid production and its secretion in the adrenal cortex, especially in zona fasciculata (mountjoy. recent studies revealed that small single - pass transmembrane proteins, called melanocortin receptor accessory proteins (mrap and mrap2), are essential for the expression of the melanocortin receptor type 2 and its transport to the plasma membrane (for reviews, see webb and clark 2010 ; novoselova. 2013). a crucial insight into the role of mc2r comes from knockout mice (chida. it was also concluded that mc2r knockout mice is a useful model for a rare, autosomal human hereditary disease, familial glucocorticoid deficiency (fgd). a majority of these mutations result in an unsuccessful protein traffic to the cell surface (webb. furthermore, some of the human mc2r polymorphisms (e.g. g.-184a, rs2186944) have a protective effect against heroin addiction in the spanish population (proudnikov. 2008). moreover, four snps (rs1893219, rs1893220, rs2186944 and g.-2t > c) showed an association with responsiveness to acth therapy in some types of epileptic encephalopathy, infantile spasms. the tcct haplotype results in an increased expression of mc2r and a stronger response to acth (liu. 2008 ; ding. 2010). the g.-2t > c mutation is also associated with higher levels of dehydroepiandrosterone, androstenedione and plasma acth in children with premature adrenarche (lappalainen. it is rather unlikely that functional polymorphisms of the mc2r gene may significantly contribute to the phenotypic variability of production traits in livestock. thus, it is not surprising that studies on the mc2r polymorphism in domestic animals are very scarce. according to the single nucleotide polymorphism database (dbsnps ; ncbi platform), only 11 snps in dogs, four in pigs and the mc2r locus was mapped within a qtl region for intramuscular fat content and back fat thickness (jacobs. the authors showed that the distribution of a silent t > g substitution is different (p t and 549c > t) further studies of one of these snps (549c > t), carried out on a small sample (n = 101) of czech large white sows, revealed its association with the estimated breeding value for average daily weight gain (weisz. 2011). extensive studies on the mc3r gene in four species of the family canidae (dog, red fox, arctic fox and chinese raccoon dog) showed a variable level of its polymorphism (skorczyk. 2011). in total, 16 polymorphisms were described and a majority of them were found in the 5-flanking (8) and 3-flanking (2) regions. the mc3r gene of the red fox (eight polymorphic sites) and the chinese raccoon dog (six polymorphisms) appeared to be the most polymorphic. in the dog, only two polymorphisms association studies carried out in red foxes (n = 376) for two polymorphisms (silent substitution c.957a > c and c.185c > t in the 3-flanking region), revealed a significant relationship with body weight. the melanocortin receptor type 4 is a well known, major controller of food intake and energy expenditure (for reviews, see adan. 2006 ; tao 2010a). altogether, more than 150 variants were identified in this gene (tao 2009 ; loos 2011). the variants are classified into five groups according to the phenotypic effects they evoke (tao 2009). class i contains mutations causing defective protein synthesis or its accelerated degradation, resulting in dramatically decreased expression. class ii mutations cause receptor retention inside a cell, probably due to a misfolding of the receptor. class iii represents variants which are present on the cell surface, but their binding capacity or ligand affinity are impaired. variants causing defective signalling properties (decreased efficacy and/or potency) are categorised as class iv. variants causing unknown effects form class v. among the known variants, there are two (val103ile and ile251leu) which are considered as having a protective role against obesity (loos 2011). however, sometimes, this effect is not pronounced, especially if small populations are analysed (nowacka - woszuk. on the other hand, extensive genome - wide association studies (gwas) revealed that an snp located close to the mc4r gene is associated with a predisposition to polygenic obesity. this variant (rs17782313), mapped 188 kb downstream of the gene (loos. 2008), shows a strong association with an elevated bmi (for a review, see xi. association of the mc4r gene variants with human obesity have initiated studies on the relationship with fat tissue accumulation in livestock species. the most extensive studies were carried out in the pig, resulting in the identification of eight polymorphic sites (table 5). among them, the missense substitution c.1426 g > a (asp298asn) was the most extensively studied in terms of its association with production traits, mainly fatness, feed intake and feed conversion ratio. (2000a) and was originally named c.892 g > a.table 5genetic variants identified in the porcine mc4r gene. positions numbered according to nm_214173 (positions in brackets numbered according to ab021664)locationpositioneffectreferencesproximal promoterc.-780c > gpossible disruption of transcription factor binding sitefan. positions numbered according to nm_214173 (positions in brackets numbered according to ab021664) the asp298asn substitution is located in a highly conserved motif within the seventh transmembrane domain. functional studies revealed that both polymorphic forms of mc4r bind its agonist with similar affinity. (2004) reported the asp298 variant s inability to generate signals, in contrast to fan. (2008), who proved a similar signalling force for both variants. in a majority of studies, it was claimed that allele asp298 is strongly associated with lower back fat thickness, higher lean meat percentage, slower growth rate and lower feed intake, while the asn298 allele had the opposite effects, i.e. higher fat deposition and faster growth, which seems to be a result of greater feed intake (table 6). association studies on meat quality traits and fatty acid composition were also performed (table 6). for example, ovilo. (2006) concluded that animals homozygous for the asn298 allele exhibit lower meat redness and a higher content of saturated fatty acids compared to the gg homozygote. further studies revealed that allele frequencies differ greatly among pig breeds and lines, probably due to long - term artificial selection. for example, pigs representing lines of the same breed and raised for fresh meat production showed an increased asp298 allele frequency when compared to those bred for cured ham and loin production (burgos. 2006). despite those promising reports, some of the subsequent studies failed to confirm an association of asp298asn snp in the mc4r gene with performance and quality traits in pigs (schwab. 2009 ; munoz. 2011) or reported breed - related differences in the observed effects (stachowiak. it is possible that this mutation might not be the causative one, only closely related to the real quantitative trait nucleotide (qtn), or there might be an epistatic interaction (bruun. 2006).table 6effects of the missense substitution c.892 g > a (presently described as c.1426 g > a) causing amino acid substitution (asp298asn) on pig production traitstraiteffect of allele g (asp) compared to allele a (asn)breedreferencesaverage daily gaindurockim. (2012)back fat thicknesslandrace ; large white ; large white duroc ; large white meishankim. (2006)landrace ; large white ; large white duroc ; large white meishankim. (2010)growth ratelandrace ; large white ; large white x duroc ; large white meishankim. (2006) effects of the missense substitution c.892 g > a (presently described as c.1426 g > a) causing amino acid substitution (asp298asn) on pig production traits another missense substitution (707a > g, arg236his) was detected in pietrain, vietnamese pigs and berkshire yorkshire crossbreds (kim. 2004 ; meidtner. animals carrying a minor allele a are fatter and grow more slowly than those carrying allele g. according to fan. (2009), this polymorphism co - segregates with four other snps (780c > g in promoter, 135c > t in 5utr, 175c > t synonymous substitution in exon 1 and 430a > t in putative 3utr), forming three haplotypes, which exhibit a significant association with average back fat thickness and average daily weight gain. as predicted by the in silico study, the occurrence of 780c > g and 135c > t snps may disrupt several transcription factor binding sites. the influence of these polymorphisms may be an explanation for the above - mentioned inconsistent data on the asp298asn association with production traits (fan. 17 polymorphic sites were discovered and, among them, 13 were silent mutations and four were missense substitutions. they occurred in the following positions : 293c > g, 193a > t, 192 t > g, 129a > g (zhang. 2009), 84 t > c (liu. 2010), 19c > a, 20a > t, 83 t > c, 128 g > a (huang. 2010), 709 g > a (val166met) (seong. 2012), 747 g > a, 927c > t (valle. 2004), 1069c > g (leu286val) (thue. 2001), 1343c > a, 1786c > t (seong. 2012), 145val > ala and 172ala > thr (haegeman. 2001). a majority of them were reported only once, of which six were reported to be associated with production traits (table 7). the most extensively studied substitution was 1069c > g, for which strong associations with back fat thickness, marbling, carcass and live weight were reported (huang. 2012).table 7genetic variants of the bovine mc4r gene and their association with production traitspositioneffect onbreedreferences293c > g129a > g(linked snps)body weightaverage daily gain293c/129ananyang, qinchuan, jiaxian red, jinnanzhang. (2012)989 g > aser330asnback fatgrade fata angus, holsteinmclean and schmutz (2011)length of longissimus dorsi arealean meatg angus, holsteinmclean and schmutz (2011)1069c > gleu 286valback fat thicknessc (2012)simmental, angus, hereford, charolais, limousine, qinchuan, luxi, jinnanhuang. (2012)marblingt genetic variants of the bovine mc4r gene and their association with production traits studies of the canine mc4r gene revealed the presence of four snps, 637 g > t, 777 t > c, 33c > g (skorczyk. 2007) and 868c > t (van den berg. 2010). among them, only one snp resulted in amino acid substitution, namely, 637 g > t, changing valine to phenylalanine at position 213 (skorczyk. analysis of this polymorphism disclosed no association with morphological measures (van den berg. 2010), probably because ligand binding and signalling abilities are not disturbed when compared to the wild variant of the mc4r gene (yan and tao 2011). further studies focused on 5utr revealed the presence of two novel indels and three novel snps (nowacka - woszuk., there was an 11-bp indel within a putative upstream open reading frame (uorf). association studies (n = 381) did not show any relationship of the haplotypes with body weight. the mc5r gene is expressed in the central nervous system and in a variety of peripheral tissues, especially in the skin. the encoded protein is involved in different physiological processes, including lipid metabolism, exocrine function (yang. together with other members of the melanocortin receptor family, the mc5r expression down - regulates leptin secretion in the in vitro cultured adipocytes (hoggard. 2003), as well as mediates in the interleukin 6 (il6) production (jun. 2010). because a high level of the il6 circulating in blood correlates with insulin resistance (kristiansen and mandrup - poulsen 2005), and leptin takes part in regulating food intake and energy expenditure, melanocortin receptor 5 is a functional candidate gene for obesity in humans or fatness in domestic animals. also an. (2007) demonstrated the involvement of mcr subtype 5 in inducing fatty acid oxidation in skeletal muscles. despite a broad range of functions, only several polymorphisms of the mc5r gene were described in both humans and the domestic animals (table 8). in the pig genome, the mc5r gene was mapped closely to marker s0059, which is within a qtl for fatness and meat quality. several reports confirmed an association between porcine back fat thickness or feed intake and polymorphic variants of the mc5r gene (kovik. also, in humans the mc5r polymorphisms were reported to be associated with obesity (chagnon. 1997 ; valli - jaakola. 2008). due to a variety of physiological processes involving mc5r, it was also studied in relation to skin condition, metabolic and mental disorders. as a result of these studies, associations with type 2 diabetes, schizophrenia and bipolar disorder were documented (valli - jaakola. 2009).table 8polymorphisms of the mc5r gene and their phenotypic effects in humans and pigsspeciespositionstudied traitseffect onvariant present in breed / populationreferenceshumans849c > gphe209leuskin condition, acne vulgarisassociation not foundnegro, south indian, japanese, polynesian, caucasianhatta. (2001)ala81alanegro, south indian, japanese, polynesian, caucasianasp108aspnegro, inuit, japanese, caucasianser125sercaucasianthr248thrnegro, south indian, japanese, polynesian, caucasianpsti, pvuiiobesitybmi, fat mass, resting metabolic ratecaucasian (canada)chagnon. (2008)849c > g phe209leutype 2 diabetestype 2 diabetescaucasian (finland)valli - jaakola. (2008)mental disordersg allele predisposes to schizophrenia and bipolar disordercaucasian (usa), african americanmiller. (2009)pigs303a > g ala109thrfatness traitsaverage daily gain, feed intake, feed conversation g large white landracekovik. (2000b)fat deposition, carcass quality traitstenth rib back fat thicknessberkshire, duroc, hampshire, landraceemnett. (2000b) polymorphisms of the mc5r gene and their phenotypic effects in humans and pigs studies on the melanocortin receptor gene family revealed numerous functional variants, especially in the mc1r and mc4r genes. it is not surprising that extensive polymorphism of the mc1r gene exists in humans and domestic mammals, since skin or coat colour is a variable trait in human ethnic groups, as well as in domestic animal breeds. thus, further studies on mc1r gene polymorphism in domestic animals demonstrating a unique coat colour should be continued. on the other hand, the mc4r gene is highly polymorphic in humans (more than 150 variants) and much less polymorphic in domestic mammals. a low level of mc4r polymorphism in pigs and cattle may reflect a selection pressure on the decrease of fat tissue content in a carcass. comparative studies on the polymorphism of this gene, which will include breeds predisposed to adiposity (e.g. pigs of mangalica and ossabaw breeds), could verify this hypothesis. since the role of mc3r polymorphism in the development of human obesity is not clear, it seems reasonable to extend studies of this gene in domestic animals, mainly in pigs and dogs, which are considered as valuable model organisms for human hereditary diseases. finally, we showed that knowledge on the polymorphism of the remaining genes of the mcr family (mc2r and mc5r) is scarce, even in humans. it seems that mc5r is worthy of further study due to its potential role in lipid metabolism and may bring new insight to knowledge on the association with adipose tissue accumulation in mammals. finally, the application of functional genomic approaches, including epigenetic modification of mc3r, mc4r and mc5r genes in domestic animals, may elucidate their potential role in the phenotypic variability of production traits related to fatness, daily gain of body mass and feed conversion ratio.	the melanocortin receptor gene family consists of five single - exon members, which are located on autosomes. three genes (mc2r, mc4r and mc5r) are syntenic in the human, mouse, cattle and dog genomes, while in the pig, the syntenic group comprises mc1r, mc2r and mc5r. two genes (mc1r and mc4r) have been extensively studied due to their function in melanogenesis (mc1r) and energy control (mc4r). conservative organisation of these genes in five mammalian species (human, mouse, cattle, pig and dog), in terms of the encoded amino acid sequence, is higher in the case of mc4r compared to mc1r. polymorphisms of these two genes are responsible or associated with variation of pigmentation (mc1r) and adipose tissue deposition (mc4r). polymorphic variants in mc1r, causing coat colour variation, were described in humans and domestic mammals (cattle, horse, pig, sheep, dog), as well as farm red and arctic foxes. the mc4r gene is very polymorphic in humans and it is well known that some variants cause monogenic obesity or significantly contribute to the development of polygenic obesity. such relationships are not so evident in domestic mammals ; however, at least one missense substitution (298asp > asn) in the porcine mc4r significantly contributes, at least in some breeds, to fat tissue accumulation, feed conversion ratio and daily weight gain. knowledge on the phenotypic effects of polymorphisms of mc2r, mc3r and mc5r in domestic mammals is scarce, probably due to the small number of reports addressing these genes. thus, further studies focused on these genes should be undertaken.
the online version of this article (doi:10.1007/s40264 - 016 - 0501 - 2) contains supplementary material, which is available to authorized users. regular risk evaluation and mitigation strategies (rems) patient survey data indicate high levels of understanding / retention of rems safe - use messages.high compliance rates with rems program contraception requirements were also reflected. the exposure of thousands of unborn fetuses to thalidomide in the 1950s and 1960s resulted in major changes to the us pharmacovigilance and risk management legislation and regulations [1, 2 ]. thalidomide was first marketed in europe as a non - barbiturate sedative in the late 1950s, and preclinical and clinical studies had led to the conclusion that it was non - toxic in humans. thalidomide was found to be antiemetic and was used extensively to treat and prevent morning sickness associated with pregnancy. in 1961, two physicians independently associated thalidomide use in pregnancy with congenital malformations including phocomelia, amelia, syndactyly, and underdeveloped long bones [4, 5 ]. fetal thalidomide exposure has also been associated with a range of internal defects, resulting in an early mortality rate of around 40%. once the link between thalidomide and birth defects was established, the drug was withdrawn worldwide and us pharmaceutical regulatory processes were strengthened. interest in thalidomide revived when it was shown to be effective in treating erythema nodosum leprosum (enl), an inflammatory reaction that complicates lepromatous leprosy. subsequently, thalidomide was found to inhibit the expression of tumor necrosis factor (tnf), also known as the hiv / aids community began to use thalidomide to treat hiv - associated wasting, creating a need to allow access to thalidomide for those who might benefit, while preventing access by those who could be at risk from the drug s teratogenic effects. in 1998, thalidomide was approved by the us fda for acute treatment of the cutaneous manifestations of moderate to severe enl. prior to its approval, thalidomide was found to be anti - angiogenic, leading to research into its use to treat angiomas and malignancies. subsequent clinical studies determined thalidomide s activity in multiple myeloma (mm) [11, 12 ]. in 2006, thalidomide (thalomid ; celgene corporation, summit, nj, usa) in combination with dexamethasone was approved by the fda to treat newly diagnosed mm. while celgene was developing thalidomide, it created chemical congeners called imid products that shared many of the immune modulatory and other properties of thalidomide. celgene discovered that each imid product had slightly different properties from each other and from thalidomide. the first imid product to be commercialized was lenalidomide (revlimid, celgene corporation), which was approved by the fda in 2005 for the treatment of transfusion - dependent anemia due to low- or intermediate-1-risk myelodysplastic syndromes associated with a deletion 5q abnormality and in 2006 in combination with dexamethasone for mm in patients who have received at least one prior therapy. the approvals of lenalidomide and thalidomide were achieved in a different regulatory environment from that of the 1950s and 1960s and, in tandem with approval of these drugs, systems were implemented to prevent fetal exposure [13, 14 ]. since the 2007 fda amendments act, the fda has required sponsors of new drug applications (ndas), biologics license applications (blas), generic and biosimilar applications to submit a risk evaluation and mitigation strategies (rems) program if there is concern that the risks of the product can not be addressed by routine product labelling. a rems program is intended to ensure that, through the implementation of additional risk - mitigation measures, the benefits of the drug outweigh the risks. all rems programs include a timetable for submission of assessments, and the fda may require that a product - specific rems program includes a communication plan, medication guide, patient package insert, elements to assure safe use (etasu), and an implementation system. lenalidomide and thalidomide have rems programs with etasus that have two goals : (1) to prevent the risk of embryo - fetal exposure and (2) to inform prescribers, patients, and pharmacists of the serious risks and safe - use conditions of the product. to achieve these goals, some key safe - use messages in the rems programs are repeated at each physician visit. the repeated messages vary according to patient risk categories but include warnings on birth defects, sexual intercourse, birth control, drug sharing, and donations to blood and sperm banks. one of the etasu measures introduced to achieve these goals is to ensure the product is dispensed to patients enrolled in the rems only after they have received, and confirm they understand, comprehensive safe - use education messages and agree to participate in frequent mandatory knowledge, attitude, and behavior (kab) surveys. additional voluntary patient surveys were conducted up to 2013 to examine patient understanding and compliance with safe - use measures. while the fda has been able to mandate rems programs since 2007, published data available on the effectiveness of these programs are limited. in an evaluation of rems medication guides, 30.3% of medication guide assessments led to acceptable knowledge (80% correct responses), with higher correct response rates reported for rems programs including etasus. previous analysis of voluntary survey data from the lenalidomide revassist risk management program (from 2006) reported over 95% comprehension of safe - use messaging for male patients and females of childbearing potential. this study was conducted to evaluate the effectiveness of patient education activities in the lenalidomide and thalidomide rems programs by analyzing the results of the mandatory kab surveys and additional voluntary patient surveys to assess comprehension of safe - use messages and patient - reported compliance with birth control measures. the surveys described here were conducted in support of a regulatory commitment of the rems programs to serve as a practical indicator of safe - use conditions rather than for research purposes. mandatory surveys from june 2012 to june 2013 were interrogated alongside data from the voluntary surveys from june 2012 to february 2013 to establish receipt of patient brochures, patient understanding of safe - use messages, and reported compliance with birth control requirements. the main elements of the celgene rems programs are presented in fig. 1. every prescriber intending to prescribe lenalidomide or thalidomide must first become certified into the respective rems program. a certified prescriber may then enroll patients into the rems program in a similar manner. the prescriber provides the patient with educational material highlighting the serious risks and safe - use conditions of the product and describes the frequent interactions with the rems program during therapy. all patient materials were written to be comprehensible by a patient at 8th grade literacy levels. the prescriber and patient choose a unique identifier that is used in future rems interactions, and information about the patient risk category, age, and indication is recorded in the celgene rems database. throughout therapy, both the prescriber and the patient are required to provide the rems program with information that confirms continued patient understanding of the safe - use measures before each subsequent prescription can be filled.fig. 1flowchart highlighting the main components of the celgene risk evaluation and mitigation strategies (rems) programs. physician agreement form flowchart highlighting the main components of the celgene risk evaluation and mitigation strategies (rems) programs. physician agreement form the safe - use messages provided to patients prior to enrollment into the rems program are as follows : (1) these products can cause birth defects, (2) contraceptive measures must be taken while receiving therapy, (3) these drugs can not be shared, (4) patients must not donate blood to a blood bank, (5) any unused product must be returned to a healthcare professional. physician agreement form (ppaf) that is signed by patients and returned to celgene. the ppaf also includes patient demographics and international classification of diseases (icd) diagnosis codes, which are provided by the prescriber. the serious risk messages are also conveyed in the prescriber brochure to ensure prescribers are sufficiently informed to educate their patients about the risks before initiating therapy. safe - use messages appropriate to the patients risk categories are repeated at each physician visit. other patient resources include a patient resource kit, which contains a rems patient guide and an emergency contraception brochure. these materials are provided to the patient at the time of enrollment by the prescriber in support of the ppaf discussion. they are also available publicly on the rems websites (http://www.revlimidrems.com for lenalidomide ; http://www.thalomidrems.com for thalidomide). additionally, each patient is counseled by a rems - certified counselor with a rems education and counseling checklist for pharmacies before each dispense. importantly, lenalidomide and thalidomide are dispensed to patients enrolled in the rems only after they have received, and confirmed they understand, comprehensive safe - use messages. all patients must agree to participate in frequent kab surveys initially, as well as during treatment, with the frequency depending on patient risk category. these surveys may be taken online, via an interactive voice - response system, or by calling the celgene customer care center. mandatory surveys occur monthly for boys, men, girls who have not undergone menarche, and females of reproductive potential, and every 6 months for women not of reproductive potential. mandatory surveys for adult males and females of reproductive potential include questions on the use of birth control methods and drug sharing. the survey for females of reproductive potential also includes questions that confirm their risk category, obtains confirmation of acceptable contraception behaviors as outlined by rems, and affirms the patient understands the potential for the product to cause birth defects. responses were identified as discrepant if (1) they diverged from the desired answers or (2) there was discrepancy between patient and prescriber responses. if a flag occurs to halt the rems authorization process, an outbound call from celgene is placed to the prescriber to provide the appropriate patient information to remedy the flag and verify safe - use conditions. patients in the rems program indicated on the ppaf whether they would be willing to participate in additional voluntary surveys (an initial survey for patients 14 days from initial dispense and a follow - up for patients > 90 days from enrollment) that further examined their understanding and compliance with safe - use measures in relation to the rems program. the patients who volunteered were categorized into one of four risk categories (children, females of childbearing potential, females not of childbearing potential, and adult males). of these patients, one child (if available) and at least 77 other patients were randomly selected each week for the voluntary patient population. all eligible females of childbearing potential were included in the initial survey, whereas one - third of eligible females of childbearing potential were included in the follow - up survey (with no minimum or maximum number for patients in this category in either survey). the remaining patients were randomly selected from the females not of childbearing potential (40%, minimum of four patients) and adult males (60%, minimum of six patients). the survey queried patients whether they were provided with the rems program education materials and engaged in sufficient conversations with their healthcare providers to understand the contents. patients were then classified into pregnancy risk groups similar to those in the mandatory kab survey. short- and long - term understanding and retention of educational materials was evaluated by sampling patients within 14 days of the first lenalidomide or thalidomide prescription (short - term retention) and after they had received therapy for at least 3 months (long - term retention). acceptable forms of birth control for patients receiving lenalidomide and thalidomide include true abstinence or one highly effective and one additional effective method of contraception. highly effective contraception methods include an intrauterine device, hormonal birth control pills or devices, tubal ligation, or partner s vasectomy. the thalomid (thalidomide) voluntary patient survey for female patients of childbearing potential is provided in the electronic supplementary material. information provided during enrollment into the mandatory rems program and the optional voluntary patient survey, together with the results from the mandatory surveys, was analyzed to determine the effectiveness of patient education activities. the main elements of the celgene rems programs are presented in fig. 1. every prescriber intending to prescribe lenalidomide or thalidomide must first become certified into the respective rems program. a certified prescriber may then enroll patients into the rems program in a similar manner. the prescriber provides the patient with educational material highlighting the serious risks and safe - use conditions of the product and describes the frequent interactions with the rems program during therapy. all patient materials were written to be comprehensible by a patient at 8th grade literacy levels. the prescriber and patient choose a unique identifier that is used in future rems interactions, and information about the patient risk category, age, and indication is recorded in the celgene rems database. throughout therapy, both the prescriber and the patient are required to provide the rems program with information that confirms continued patient understanding of the safe - use measures before each subsequent prescription can be filled.fig. 1flowchart highlighting the main components of the celgene risk evaluation and mitigation strategies (rems) programs. physician agreement form flowchart highlighting the main components of the celgene risk evaluation and mitigation strategies (rems) programs. physician agreement form the safe - use messages provided to patients prior to enrollment into the rems program are as follows : (1) these products can cause birth defects, (2) contraceptive measures must be taken while receiving therapy, (3) these drugs can not be shared, (4) patients must not donate blood to a blood bank, (5) any unused product must be returned to a healthcare professional. physician agreement form (ppaf) that is signed by patients and returned to celgene. the ppaf also includes patient demographics and international classification of diseases (icd) diagnosis codes, which are provided by the prescriber. the serious risk messages are also conveyed in the prescriber brochure to ensure prescribers are sufficiently informed to educate their patients about the risks before initiating therapy. safe - use messages appropriate to the patients risk categories are repeated at each physician visit. other patient resources include a patient resource kit, which contains a rems patient guide and an emergency contraception brochure. these materials are provided to the patient at the time of enrollment by the prescriber in support of the ppaf discussion. they are also available publicly on the rems websites (http://www.revlimidrems.com for lenalidomide ; http://www.thalomidrems.com for thalidomide). additionally, each patient is counseled by a rems - certified counselor with a rems education and counseling checklist for pharmacies before each dispense. importantly, lenalidomide and thalidomide are dispensed to patients enrolled in the rems only after they have received, and confirmed they understand, comprehensive safe - use messages. all patients must agree to participate in frequent kab surveys initially, as well as during treatment, with the frequency depending on patient risk category. these surveys may be taken online, via an interactive voice - response system, or by calling the celgene customer care center. mandatory surveys occur monthly for boys, men, girls who have not undergone menarche, and females of reproductive potential, and every 6 months for women not of reproductive potential. mandatory surveys for adult males and females of reproductive potential include questions on the use of birth control methods and drug sharing. the survey for females of reproductive potential also includes questions that confirm their risk category, obtains confirmation of acceptable contraception behaviors as outlined by rems, and affirms the patient understands the potential for the product to cause birth defects. responses were identified as discrepant if (1) they diverged from the desired answers or (2) there was discrepancy between patient and prescriber responses. if a flag occurs to halt the rems authorization process, an outbound call from celgene is placed to the prescriber to provide the appropriate patient information to remedy the flag and verify safe - use conditions. patients in the rems program indicated on the ppaf whether they would be willing to participate in additional voluntary surveys (an initial survey for patients 14 days from initial dispense and a follow - up for patients > 90 days from enrollment) that further examined their understanding and compliance with safe - use measures in relation to the rems program. the patients who volunteered were categorized into one of four risk categories (children, females of childbearing potential, females not of childbearing potential, and adult males). of these patients, one child (if available) and at least 77 other patients were randomly selected each week for the voluntary patient population. all eligible females of childbearing potential were included in the initial survey, whereas one - third of eligible females of childbearing potential were included in the follow - up survey (with no minimum or maximum number for patients in this category in either survey). the remaining patients were randomly selected from the females not of childbearing potential (40%, minimum of four patients) and adult males (60%, minimum of six patients). the survey queried patients whether they were provided with the rems program education materials and engaged in sufficient conversations with their healthcare providers to understand the contents. patients were then classified into pregnancy risk groups similar to those in the mandatory kab survey. short- and long - term understanding and retention of educational materials was evaluated by sampling patients within 14 days of the first lenalidomide or thalidomide prescription (short - term retention) and after they had received therapy for at least 3 months (long - term retention). acceptable forms of birth control for patients receiving lenalidomide and thalidomide include true abstinence or one highly effective and one additional effective method of contraception. highly effective contraception methods include an intrauterine device, hormonal birth control pills or devices, tubal ligation, or partner s vasectomy. the thalomid (thalidomide) voluntary patient survey for female patients of childbearing potential is provided in the electronic supplementary material. information provided during enrollment into the mandatory rems program and the optional voluntary patient survey, together with the results from the mandatory surveys, was analyzed to determine the effectiveness of patient education activities. between june 2012 and june 2013, a total of 73,645 patients enrolled into the rems programs, including 56,709 patients receiving lenalidomide and 16,936 patients receiving thalidomide. the vast majority of patients (82.9%) enrolled in the rems were treated for mm, with lower numbers of patients treated for myelodysplastic syndrome (6.4%), enl (0.1%), and other conditions (10.5%). consistent with published epidemiological data for mm, there were more males (40,774 [55.4% ]) than females (32,871 [44.6% ]). of the female patients, 2216 (6.7%) were of reproductive potential, and the majority (93.1%) were adult females not of reproductive potential, indicating the older population expected for patients with the diseases associated with the use of these drugs. 2characteristics of the patient populations. a distribution of patients enrolled in the lenalidomide and thalidomide risk evaluation and mitigation strategies (rems) programs between june 2012 and june 2013 by risk category (n = 73,645). b distribution of patients in the voluntary survey population by risk category (n = 2790). c proportion of patients in the voluntary survey population who received and read the patient education brochure by risk category characteristics of the patient populations. a distribution of patients enrolled in the lenalidomide and thalidomide risk evaluation and mitigation strategies (rems) programs between june 2012 and june 2013 by risk category (n = 73,645). b distribution of patients in the voluntary survey population by risk category (n = 2790). c proportion of patients in the voluntary survey population who received and read the patient education brochure by risk category of the 73,645 patients enrolled in the rems programs, 10,391 (14.1%) agreed to participate in the additional voluntary survey. of these, 3981 patients were randomly selected for inclusion in the voluntary survey sample, of whom 2790 (70.1%) responded. the proportion of patients in each risk category was generally consistent with those recorded for the whole population enrolled in the rems programs (fig. 2a), with more females of reproductive potential, as expected from the sampling strategy used (see methods). the voluntary survey confirmed that 89.4% of patients received the initial education materials from their healthcare professional and 85.9% read them before starting treatment with lenalidomide or thalidomide (fig. 264,417 mandatory patient surveys were performed and 14,848 (5.6%) were flagged as a potential cause for concern (table 1). females of reproductive potential had the highest number of flagged surveys (6062 [45.0% ]), all of which were resolved before the patient received her prescription. follow - up determined that 696 (11.5%) were due to the patient not understanding or not complying with risk / safe - use messages. these reports were investigated and resolved prior to drug dispense, as described in the methods section. a further 4025 (66.4%) of the surveys for females of reproductive potential were flagged because, since their initial enrollment into the rems program, the patient had either had their uterus removed, or their menstrual periods had stopped for more than 24 months. these patients were re - classified as females not of reproductive potential.table 1distribution of risk evaluation and mitigation strategies (rems) patient survey flags by pregnancy risk category (june 2012june 2013)risk categorysurveys completed (n)number (%) of surveys flaggedfemales of reproductive potential (adult and child)13,4816062 (45.0)females not of reproductive potential (adult and child)33,3645289 (15.9)males (adult and child)217,5723497 (1.6)total264,41714,848 (5.6) distribution of risk evaluation and mitigation strategies (rems) patient survey flags by pregnancy risk category (june 2012june 2013) the voluntary surveys for the same period indicated that 97.9% of the women of reproductive potential and adult females not of reproductive potential (n = 1126) surveyed understood that women should not get pregnant while taking the product, and 94.0% complied with birth control requirements at initial survey (91.2% at follow - up), demonstrating the effectiveness of these interventions in ensuring patient understanding of, and compliance with, safe - use messages. patient - reported compliance with pregnancy - prevention measures was determined in the voluntary survey. compliance with the required two forms of birth control was assessed when starting therapy and 3 months later (table 2). reported birth control compliance was high : 96.3% of patients used the required birth control when starting therapy, and 96.4% reconfirmed this later.table 2patient birth control compliance by risk category (voluntary survey), june 2012february 2013risk categoryinitial surveysinitial birth control compliance (%) follow - up surveysfollow - up birth control compliance (%) females of reproductive potential233219 (94.0)147134 (91.2)males (adult and child)15321480 (96.6)14431399 (97.0)total17651699 (96.3)15901533 (96.4) patient birth control compliance by risk category (voluntary survey), june 2012february 2013 the impact of repeated educational messages on patient understanding and compliance with safe - use messages was assessed by reviewing surveys conducted in the voluntary survey population several months after starting therapy (table 3).table 3patient understanding of messages by risk category (voluntary survey), june 2012february 2013messagefemales of reproductive potentialadult females not of reproductive potentialmales aged 18 yearsmales aged 1217 yearschildren aged < 12 yearstotal (%) total surveyed1479791441272576product can cause birth defects1469581417272530 (98.2)sharing product with others can be dangerous1469691417272541 (98.6)women should not get pregnant while taking product1449581102 (97.9)men should use condoms while on product if they are sexually active with a woman who is able to become pregnant141821420 (98.4)unused product needs to be returned1298661239272243 (87.1) patient understanding of messages by risk category (voluntary survey), june 2012february 2013 overall understanding of safe - use messages was very high : around 98% of patients demonstrated an understanding of most of these messages. importantly, 98.2% of patients knew that lenalidomide and thalidomide can cause birth defects, which is part of the repeated educational messaging, whereas 87.1% recalled that unused product should be returned to their healthcare professional, which is not included in repeated messaging (table 3). between june 2012 and june 2013, a total of 73,645 patients enrolled into the rems programs, including 56,709 patients receiving lenalidomide and 16,936 patients receiving thalidomide. the vast majority of patients (82.9%) enrolled in the rems were treated for mm, with lower numbers of patients treated for myelodysplastic syndrome (6.4%), enl (0.1%), and other conditions (10.5%). consistent with published epidemiological data for mm, there were more males (40,774 [55.4% ]) than females (32,871 [44.6% ]). of the female patients, 2216 (6.7%) were of reproductive potential, and the majority (93.1%) were adult females not of reproductive potential, indicating the older population expected for patients with the diseases associated with the use of these drugs. 2characteristics of the patient populations. a distribution of patients enrolled in the lenalidomide and thalidomide risk evaluation and mitigation strategies (rems) programs between june 2012 and june 2013 by risk category (n = 73,645). b distribution of patients in the voluntary survey population by risk category (n = 2790). c proportion of patients in the voluntary survey population who received and read the patient education brochure by risk category characteristics of the patient populations. a distribution of patients enrolled in the lenalidomide and thalidomide risk evaluation and mitigation strategies (rems) programs between june 2012 and june 2013 by risk category (n = 73,645). b distribution of patients in the voluntary survey population by risk category (n = 2790). c proportion of patients in the voluntary survey population who received and read the patient education brochure by risk category of the 73,645 patients enrolled in the rems programs, 10,391 (14.1%) agreed to participate in the additional voluntary survey. of these, 3981 patients were randomly selected for inclusion in the voluntary survey sample, of whom 2790 (70.1%) responded. the proportion of patients in each risk category was generally consistent with those recorded for the whole population enrolled in the rems programs (fig. 2a), with more females of reproductive potential, as expected from the sampling strategy used (see methods). the voluntary survey confirmed that 89.4% of patients received the initial education materials from their healthcare professional and 85.9% read them before starting treatment with lenalidomide or thalidomide (fig. 264,417 mandatory patient surveys were performed and 14,848 (5.6%) were flagged as a potential cause for concern (table 1). females of reproductive potential had the highest number of flagged surveys (6062 [45.0% ]), all of which were resolved before the patient received her prescription. follow - up determined that 696 (11.5%) were due to the patient not understanding or not complying with risk / safe - use messages. these reports were investigated and resolved prior to drug dispense, as described in the methods section. a further 4025 (66.4%) of the surveys for females of reproductive potential were flagged because, since their initial enrollment into the rems program, the patient had either had their uterus removed, or their menstrual periods had stopped for more than 24 months. these patients were re - classified as females not of reproductive potential.table 1distribution of risk evaluation and mitigation strategies (rems) patient survey flags by pregnancy risk category (june 2012june 2013)risk categorysurveys completed (n)number (%) of surveys flaggedfemales of reproductive potential (adult and child)13,4816062 (45.0)females not of reproductive potential (adult and child)33,3645289 (15.9)males (adult and child)217,5723497 (1.6)total264,41714,848 (5.6) distribution of risk evaluation and mitigation strategies (rems) patient survey flags by pregnancy risk category (june 2012june 2013) the voluntary surveys for the same period indicated that 97.9% of the women of reproductive potential and adult females not of reproductive potential (n = 1126) surveyed understood that women should not get pregnant while taking the product, and 94.0% complied with birth control requirements at initial survey (91.2% at follow - up), demonstrating the effectiveness of these interventions in ensuring patient understanding of, and compliance with, safe - use messages. patient - reported compliance with pregnancy - prevention measures was determined in the voluntary survey. compliance with the required two forms of birth control was assessed when starting therapy and 3 months later (table 2). reported birth control compliance was high : 96.3% of patients used the required birth control when starting therapy, and 96.4% reconfirmed this later.table 2patient birth control compliance by risk category (voluntary survey), june 2012february 2013risk categoryinitial surveysinitial birth control compliance (%) follow - up surveysfollow - up birth control compliance (%) females of reproductive potential233219 (94.0)147134 (91.2)males (adult and child)15321480 (96.6)14431399 (97.0)total17651699 (96.3)15901533 (96.4) patient birth control compliance by risk category (voluntary survey), june 2012february 2013 the impact of repeated educational messages on patient understanding and compliance with safe - use messages was assessed by reviewing surveys conducted in the voluntary survey population several months after starting therapy (table 3).table 3patient understanding of messages by risk category (voluntary survey), june 2012february 2013messagefemales of reproductive potentialadult females not of reproductive potentialmales aged 18 yearsmales aged 1217 yearschildren aged < 12 yearstotal (%) total surveyed1479791441272576product can cause birth defects1469581417272530 (98.2)sharing product with others can be dangerous1469691417272541 (98.6)women should not get pregnant while taking product1449581102 (97.9)men should use condoms while on product if they are sexually active with a woman who is able to become pregnant141821420 (98.4)unused product needs to be returned1298661239272243 (87.1) patient understanding of messages by risk category (voluntary survey), june 2012february 2013 overall understanding of safe - use messages was very high : around 98% of patients demonstrated an understanding of most of these messages. importantly, 98.2% of patients knew that lenalidomide and thalidomide can cause birth defects, which is part of the repeated educational messaging, whereas 87.1% recalled that unused product should be returned to their healthcare professional, which is not included in repeated messaging (table 3). effectiveness of the rems programs for lenalidomide and thalidomide in driving patient understanding and retention of safe - use messages was assessed using data from mandatory kab assessments and voluntary patient surveys. patients surveyed had high levels of compliance with birth control requirements, both initially and at follow - up, as well as very high levels of understanding of safe - use messages. this study determined that mandated rems programs are effective tools for educating patients in the safe use of lenalidomide and thalidomide and re - enforcing compliance with safe - use measures by repeated messaging. previous studies have determined that levels of fetal exposure to both lenalidomide and thalidomide are very low in patients enrolled in us risk management programs for these drugs [13, 14 ]. a third celgene imid product, pomalidomide (pomalyst, approved in the usa for advanced mm in 2013) is distributed through an rems identical to that for lenalidomide. patient understanding of safe - use messages in the celgene rems programs reported in this study (approximately 98% for key messages related to pregnancy, teratogenicity, and sharing the product) is consistent with the high levels of understanding reported in a previous analysis of voluntary surveys conducted as part of the original lenalidomide risk management program (revassist). however, published data on the effectiveness of messaging in other rems programs are limited. a recent study investigating patient understanding of key risks based on medication guide assessments for 66 drugs between september 2008 and june 2012 found that primary drug risk knowledge questions were answered correctly by 63.8% of respondents on average. higher rates were recorded for rems that also included etasu or a communication plan and etasu (89.8 and 82.7%, respectively). the continued high understanding of safe - use messages for patients in the celgene rems programs is consistent with the higher rate of patient understanding in rems programs with etasus and indicates the relative effectiveness of the celgene programs. the high reported adherence to birth control measures for the celgene rems programs is particularly important given the teratogenic potential of the drugs involved. thalidomide is a proven human teratogen, and lenalidomide caused thalidomide - like malformations in newborn monkeys in pre - clinical studies. a limitation of the current study is that the surveys do not report on patient behavior, which may not necessarily reflect survey responses. in a study of oral contraception use in over 6 million us women of childbearing age, only 59.8% of those receiving both category x medications and oral contraceptives were considered to be adherent to oral contraception. consequently, an estimated 5.8% of pregnancies in the usa occur in women receiving medications of known teratogenic risk (categories d or x). in this context, the high reported adherence to birth control requirements in this study (consistently over 90%) indicates the effectiveness of repeated messaging enabling understanding of birth control requirements in the rems programs for lenalidomide and thalidomide. crucially, celgene risk management measures have been executed over 18 years, with no reported births with teratogenicity due to celgene products. in this study, understanding was particularly high for safe - use messages that were part of repeated messaging. for each of these safety messages, around 98% of patients demonstrated understanding, considerably higher than for a message not included in repeated messaging (table 3). this demonstrates the crucial role of repeated messaging in the rems programs, particularly for messages related to teratogenicity and the need for adequate birth control measures. the high rate of understanding of these messages is reflected in a high rate of reported adherence to birth control measures. repeated messaging has previously been shown to be effective in influencing prescriber behavior in a study of the provision of calcium and vitamin d supplements to uk patients receiving anti - epileptic drugs. in the celgene rems programs, the use of mandatory monthly (for patients of reproductive potential) or 6-monthly (for other patients) kab surveys allows patients continued understanding of safe - use messages to be regularly and continually assessed prior to dispensing. approximately 5% of the 264,417 surveys completed during this study were flagged, providing the opportunity to investigate and resolve concerns before lenalidomide or thalidomide was dispensed. it should be noted that the surveys described here were conducted in support of a regulatory commitment of the rems programs to serve as a practical indicator of safe - use conditions rather than for research purposes. consequently, the goals and objectives of these surveys differ from those of surveys conducted as purely research exercises. one limitation of this study is the limited duration of follow - up. fatigue with repeated messaging, which could potentially diminish patients attention to serious risk and safe - use messaging. there was no evidence in this study that the repeated messaging in the lenalidomide and thalidomide rems programs led to a reduction in patients attention to safety messages ; however, there was no follow - up beyond 3 months. additional studies are required to assess whether repeated messaging leads to diminution of patient attention. in addition, although analysis of the patient surveys allowed us to assess patient understanding of rems messaging, it did not provide any data on the impact of the rems programs on prescribers. we believe that the results presented in this report, the record of birth control compliance with other category x drugs, and the overarching rems programs rationale should be considered when evaluating the burden incurred by healthcare providers in relation to rems programs. in a survey of 364 healthcare providers in southern california, usa, most respondents (63%) considered that rems improved patient safety, whereas 62% considered that rems was very or somewhat meaningful in improving patient safety. among oncology prescribers (n = 28), 68% considered that rems improved patient safety, whereas 75% responded that rems was meaningful. despite this, the additional administrative burden incurred by rems programs is a recognized concern among oncology practitioners [21, 23 ]. to mitigate this, online enrollment systems have been developed to minimize the administrative burden on healthcare providers enrolling patients in the rems programs for lenalidomide and thalidomide. further studies are required to assess the burden of rems programs and to evaluate potential improvements to the rems process. the necessary extra administrative burden incurred by these rems programs must be considered in the context of the risk of fetal exposure and the effectiveness of the programs in preventing this exposure. data presented here and elsewhere [13, 14 ] clearly demonstrate that these programs drive the high levels of patient understanding and compliance necessary to prevent fetal exposure. lenalidomide and thalidomide are both important treatments for an increasing variety of malignant conditions. for these and other teratogenic compounds to be used safely, it is crucial that both the pharmaceutical industry and regulatory authorities work to minimize the risk of fetal exposure. the data from this study support the conclusion that the lenalidomide and thalidomide rems programs in the usa enhance patient understanding of safe - use messages, leading to high levels of compliance with the birth control precautions essential to prevent fetal exposure to these known and potential human teratogens. the 3-month follow - up with the voluntary survey means there are no data on longer - term patient understanding of the rems. however, the mandatory surveys show that overall compliance was maintained after 3 months of follow - up and throughout therapy. below is the link to the electronic supplementary material. supplementary material 1 (pdf 1043 kb) supplementary material 1 (pdf 1043 kb) robert bwire, john freeman, florence houn, and paul sheehan are employees of celgene corporation. all patients included in the study gave consent for their data to be used on the ppaf.	introductionthe effectiveness of patient education activities conducted within the lenalidomide and thalidomide risk evaluation and mitigation strategies (rems) programs was evaluated by measuring understanding of serious risk and safe - use messages.methodsresults from mandatory knowledge, attitude, and behavior surveys and voluntary patient surveys completed between june 2012 and june 2013 were analyzed, and responses to questions relating to compliance with birth control measures and understanding of safe - use messages are presented by patient risk category.resultsin total, 73,645 patients were enrolled into the rems programs for lenalidomide and thalidomide and completed mandatory surveys prior to medication dispense. of these, 2790 (3.8%) completed an additional voluntary survey. among voluntary survey participants, for all patient pregnancy risk categories, reported compliance with birth control requirements was above 90% when starting therapy and at follow - up. at the beginning of therapy, complete compliance was 96.3% ; 3 months later it was 96.4%. patient understanding of safe - use messages was very high in all pregnancy risk groups, notably for messages repeated at each physician visit. overall, 98.2% of patients knew that lenalidomide and thalidomide could cause birth defects, which is part of the repeated educational messaging. in contrast, 87.1% recalled that unused product should be returned to their healthcare professional, which is not included in repeated messaging.conclusionthe lenalidomide and thalidomide rems programs enhance patient understanding of safe - use messages, resulting in high levels of compliance with the birth control precautions essential to prevent fetal exposure to these known and potential human teratogens. overall compliance was maintained after 3 months of follow - up and throughout therapy.electronic supplementary materialthe online version of this article (doi:10.1007/s40264 - 016 - 0501 - 2) contains supplementary material, which is available to authorized users.
microscopic evaluation of myocardial tissue is commonly conducted at pathological and forensic departments, usually by means of bright - field microscopy of hematoxylin and eosin - stained (h and e) microsections. a part of the evaluation is a subjective estimate of the basic condition of the heart as perceived, e.g., amount of fibrosis, cell size, nuclear size, and presence of lipofuscin. in clinical settings, subjective estimates of histological parameters may work, but in scientific settings the need for reproducible parametric representation of data is not satisfied. furthermore, with the current introduction of whole slide scanners in clinical settings, a demand for automated analysis of digitized microsections is increasingly manifested. this study explores the digital analysis of autofluorescence from unstained microsections as a means to quantitate basic histological parameters of myocardial tissue. the versatility of autofluorescence is reflected in the broadness of previous application studies ranging from noninvasive methods for diagnosing cancer and metabolic diseases to histological diagnostics of functional disorders. the common feature is quantitation of tissue components and/or metabolites by means of fluorescent properties. methods for automated quantitation of histological tissue components in myocardial microsections using digital image analysis have been widely published. three principal approaches for quantitating fibrous tissue have been applied : (i) discrimination by staining color, (ii) discrimination by polarization properties, and (iii) discrimination by fluorescence properties. regarding (i) specific staining is necessary to provide robust detectable contrast in color, e.g., by sirius red or masson 's trichrome staining. regarding (ii) it is well established that birefringence of sirius - stained fibrous tissue is suitable for digital quantitation. regarding (iii) fibrous tissue has been quantitated with a combination of two - photon excitation of autofluorescence and second harmonic generation imaging of collagen, which requires a confocal fluorescence microscope mounted with a femtosecond laser. this setup is limited in availability and has so far been tested only on experimentally acquired myocardial tissue. in addition to fibrous tissue, cardiac lipofuscin has been the subject of autofluorescence - based quantitation, specifically by discriminating lipofuscin with autofluorescence intensity. in this study, we aimed to test autofluorescence as the base of an objective high - throughput method for quantitating the histological presentation of tissue structures in myocardial microsections. this was performed by quantitative analysis of histological autofluorescence intensities obtained at different wavelengths with a commercially available slide scanner. the method quantitates both fibrous tissue, myocytes, lipofuscin, and the extracellular phase (ecp) in unstained microsections. in addition, the method was tested at various fixation times and microsection thicknesses as autofluorescence is known to increase with fixation time and microsection thickness is expected to affect the autofluorescence. we identified twenty autopsies performed at the pathology department, rigshospitalet, university of copenhagen, denmark, in the period 20092013 with archived samples of myocardial tissue. the regional ethics committee classified the study as a methodology study requiring no ethical approval. in all cases, sampling was from the left ventricle and with the intention of verifying an infarction. all samples included areas with infarction as well as areas with noninfarcted myocardial tissue with various amounts of fibrosis. the age range of the included was 3577 years (median = 65 years), the male / female gender ratio 14/6, and the postmortem interval at autopsy 24136 h (median = 68 h). for testing the impact of fixation time and microsection thickness, an additional sample was taken from the left ventricle of a 59-year - old male autopsied 48 h postmortem in 2015. all samples were formalin fixated (neutral buffered 4% solution) and paraffin embedded following routine procedures. the additional sample was parted in two and fixated for 1 and 4 days, respectively. each part was cut in microsections of 2 and 8 m thickness following paraffin embedding. all microsections for autofluorescence were kept in the dark with minimal exposure to light when handled, which included 45 min of drying at 45c, paraffin dissolution by 2 15 min of xylene immersion and mounting of coverslips using xylene - based glue (pertex, histolab products ab, gteborg, sweden). upon drying, fluorescence images were acquired, and the microsections were immersed in xylene until loosening of the cover slips and finally van gieson (vg) stained using a standard protocol. areas of normal myocardium without infarction were identified on the h and e - stained microsections. the autofluorescence of these areas in the neighboring unstained microsections were imaged at two wavelengths within the visible spectrum : the blue area (using a dapi - filter, excitation = 365 nm, emission = 445) and the red area (using a rhodamine filter, excitation = 555 nm, emission = 605 nm) using a zeiss axio scan z1 slide scanner (zeiss, oberkochen, germany) with an light emitting diode (led) illumination module (zeiss colibri ii) and an orca flash 4.0 camera. ring aperture contrast (rac) focus mode was used for both coarse (20 objective, 6 points per area) and fine autofocus (20 objective, onion skin mode with the parameter set to 0.1). scanning was done with the 20 objective and exposure times were 100 ms in the blue area and 200 ms in the red area. we selected these exposure times through testing as they produced ample and similar autofluorescence intensities from the myocytes. during the scanning, both wavelengths were photographed immediately in each tile position by photographing first the blue area and then the red area. stitching, which is a built - in function of the zeiss microscope software that smooths the transition between imaged tiles, was disabled to avoid manipulation at the tile borders. after vg - staining of the digitally quantitated microsections, random parts of the quantitated areas were imaged at 100 magnification using a standard light microscope (leica dm2000 led, leica microsystems, wetzlar, germany) mounted with a charge - coupled devices camera, resulting in 1.2 1.2 mm images. a point grid was applied to these images with random offset using an imagej grid plugin (wayne rasband, nih) with a spacing of approximately 50 m, giving a total of approximately 450 counting points in each image. the spacing corresponds to a higher sampling intensity than the 100 m spacing suggested in a previous publication. by eye observation on screen, each counting point was determined as representing either fibrous tissue (stained red), ecp (unstained), or cells (stained yellow). all samples were formalin fixated (neutral buffered 4% solution) and paraffin embedded following routine procedures. the additional sample was parted in two and fixated for 1 and 4 days, respectively. each part was cut in microsections of 2 and 8 m thickness following paraffin embedding. all microsections for autofluorescence were kept in the dark with minimal exposure to light when handled, which included 45 min of drying at 45c, paraffin dissolution by 2 15 min of xylene immersion and mounting of coverslips using xylene - based glue (pertex, histolab products ab, gteborg, sweden). upon drying, fluorescence images were acquired, and the microsections were immersed in xylene until loosening of the cover slips and finally van gieson (vg) stained using a standard protocol. areas of normal myocardium without infarction were identified on the h and e - stained microsections. the autofluorescence of these areas in the neighboring unstained microsections were imaged at two wavelengths within the visible spectrum : the blue area (using a dapi - filter, excitation = 365 nm, emission = 445) and the red area (using a rhodamine filter, excitation = 555 nm, emission = 605 nm) using a zeiss axio scan z1 slide scanner (zeiss, oberkochen, germany) with an light emitting diode (led) illumination module (zeiss colibri ii) and an orca flash 4.0 camera. ring aperture contrast (rac) focus mode was used for both coarse (20 objective, 6 points per area) and fine autofocus (20 objective, onion skin mode with the parameter set to 0.1). scanning was done with the 20 objective and exposure times were 100 ms in the blue area and 200 ms in the red area. we selected these exposure times through testing as they produced ample and similar autofluorescence intensities from the myocytes. during the scanning, both wavelengths were photographed immediately in each tile position by photographing first the blue area and then the red area. stitching, which is a built - in function of the zeiss microscope software that smooths the transition between imaged tiles, was disabled to avoid manipulation at the tile borders. after vg - staining of the digitally quantitated microsections, random parts of the quantitated areas were imaged at 100 magnification using a standard light microscope (leica dm2000 led, leica microsystems, wetzlar, germany) mounted with a charge - coupled devices camera, resulting in 1.2 1.2 mm images. a point grid was applied to these images with random offset using an imagej grid plugin (wayne rasband, nih) with a spacing of approximately 50 m, giving a total of approximately 450 counting points in each image. the spacing corresponds to a higher sampling intensity than the 100 m spacing suggested in a previous publication. by eye observation on screen, each counting point was determined as representing either fibrous tissue (stained red), ecp (unstained), or cells (stained yellow). [figure 1a ], the intensity of fibrous tissue was high and the intensity of lipofuscin was low. the inserted intensity histograms include fitted standard deviations corresponding to the myocytes. in both wavelengths, (a and b) the same area in the blue and the red filter, respectively. (c and d) the corresponding ratio images with (c) being the ratio of blue to red and (d) the ratio of red to blue. intensity histograms with fitted standard deviations corresponding to the myocyte peaks are shown in the small insets. van gieson - staining and quantitations of the central parts of this microsection are displayed in figure 2 in keeping with the objective high throughput commitment, it was considered mandatory to develop a reproducible quantitation algorithm suitable for fully automated processing. the quantitation algorithm was based on parametric data analysis of the autofluorescence intensities. in particular, the fibrous tissue was determined by analysis of pixel intensity ratios of blue - to - red autofluorescence, lipofuscin by analysis of pixel intensity ratios of red - to - blue autofluorescence, and ecp by analysis of pixel intensities of blue autofluorescence. figure 1c shows an image where pixel values are ratios of blue - to - red autofluorescence intensities from the corresponding pixels in figure 1a, b, and d show the reciprocal image consisting of red - to - blue ratios. in the blue - to - red ratio image [figure 1c ], the myocytes are seen as a featureless background with uniform intensity, whereas the fibrous tissue is amplified and leveled. in the red - to - blue ratio image [figure 1d ], lipofuscin is amplified and leveled. all image analysis was done using imagej, origin (origin lab corporation, northampton, ma, usa) and excel (microsoft, redmond, wa, usa). the following steps 15 present the autofluorescence - based quantitation, which has been tested on various area sizes. if the area is below 1 1 mm, the included fitting procedures become unstable due to irregular, spikey intensity histograms. if the area is too large, variations in the microsection thickness may theoretically provide instability. this problem, however, was not encountered analyzing bigger areas up to 3 3 mm. it is consequently recommended to perform the analysis on square sections of at least 1 1 mm and be cautious with squares more than 3 3 mm. step 1 : creation of a mask of the ecp is performed using a standard distribution fitted to the myocyte peak in the histogram of the blue autofluorescence image. this was done by exporting the intensity histogram from imagej to origin and fit a single peak in the fit module with gauss function checked. pixels with intensities below the fitted peak value-2 standard deviations are thresholded from the remaining image and used as a mask representing ecp [figure 2 ] step 2 : the ecp mask is subtracted from the blue and the red images using the image calculator function in imagej resulting in ecp being represented with the value 0step 3 : the images are leveled with respect to the peak intensity of the myocytes : the blue / red ratio of the myocyte peak intensities is identified from histograms (please see small insets in panels a - b of [figure 1 ]) and used to align the red with the blue image using the math multiply function. for instance, the red image is multiplied by 2 if the myocyte peak intensity value of the blue image is twice that of the red imagestep 4 : creation of a mask of the fibrous tissue by generating and analyzing the blue - to - red ratio image. in summary, the standard deviation (sd) of the myocyte peak in the ratio image is used to approximate the upper - intensity level that includes pixels covering myocytes, and a transition analysis is performed to reproducibly identify that particular level. in the case of the blue to red ratio image, fibrous tissue is considered all pixels with intensity above the upper intensity level of the myocytes, and in the case of the red - to - blue ratio image, lipofuscin is considered all pixels with intensity above the level of the myocytes. the detailed procedure for identifying fibrous tissue is described in the following : the blue image is divided with the red image using the image calculator function ; 32-bit float must be checked. in the resulting ratio image, the intensity variation of all tissue elements is leveled, and the fibrous tissue intensity is amplified, and lipofuscin intensity is diminished. typically, fibrous tissue is too small a representation to be represented by a peak in the intensity histogram of the generated ratio image [figure 1c ]. hence, fibrous tissue must be defined based on the myocyte peak as follows : a standard distribution is fitted to the myocyte peak of the ratio image, which in theory is located at 1.0. by eye observation, it was established that the transition from myocytes to fibrous tissue is approximately at 5 sd above the myocyte peak intensity, where intensity values above represent fibrous tissue and intensities below represent myocytes. thus, peak intensity + 5 sd is used as the starting point of the myocyte - to - fibrous tissue transition analysis. the transition analysis considers the drop in pixels covering myocytes as the threshold approaches the signal emanating from fibrous tissue. all single - pixel representations above threshold are quantitated for each 0.02 threshold interval in the range : starting point (myocyte peak intensity + 5 sd) 0.40, which covers approximately 10% of the intensity range of the generated 32-bit ratio image. the ratio of change in single pixel presentations to the number of single pixel presentation at the initial point of the analysis (n0.02 /nstartingpoint - 0.40) is plotted against the intensities of the analyzed range. it is noted that the change in single pixel presentations approach zero with increasing threshold [figure 3 ]. in the plots, an exponential equation approaching zero asymptotically the adjusted r of the 20 fits performed in this study was typically around 0.95 and 0.88 at lowest. the fit provides the basis for selecting the threshold reproducibly, which was defined in this study as the intensity of the fit corresponding to the value n / nstartingpoint - 0.40 = 0.015step 5 : creation of a mask for lipofuscin is done by repeating step 4 only replacing the blue - to - red ratio with the red - to - blue ratio. step 1 : creation of a mask of the ecp is performed using a standard distribution fitted to the myocyte peak in the histogram of the blue autofluorescence image. this was done by exporting the intensity histogram from imagej to origin and fit a single peak in the fit module with gauss function checked. pixels with intensities below the fitted peak value-2 standard deviations are thresholded from the remaining image and used as a mask representing ecp [figure 2 ] step 2 : the ecp mask is subtracted from the blue and the red images using the image calculator function in imagej resulting in ecp being represented with the value 0 step 3 : the images are leveled with respect to the peak intensity of the myocytes : the blue / red ratio of the myocyte peak intensities is identified from histograms (please see small insets in panels a - b of [figure 1 ]) and used to align the red with the blue image using the math multiply function. for instance, the red image is multiplied by 2 if the myocyte peak intensity value of the blue image is twice that of the red image step 4 : creation of a mask of the fibrous tissue by generating and analyzing the blue - to - red ratio image. in summary, the standard deviation (sd) of the myocyte peak in the ratio image is used to approximate the upper - intensity level that includes pixels covering myocytes, and a transition analysis is performed to reproducibly identify that particular level. in the case of the blue to red ratio image, fibrous tissue is considered all pixels with intensity above the upper intensity level of the myocytes, and in the case of the red - to - blue ratio image, lipofuscin is considered all pixels with intensity above the level of the myocytes. the detailed procedure for identifying fibrous tissue is described in the following : the blue image is divided with the red image using the image calculator function ; 32-bit float must be checked. in the resulting ratio image, the intensity variation of all tissue elements is leveled, and the fibrous tissue intensity is amplified, and lipofuscin intensity is diminished. typically, fibrous tissue is too small a representation to be represented by a peak in the intensity histogram of the generated ratio image [figure 1c ]. hence, fibrous tissue must be defined based on the myocyte peak as follows : a standard distribution is fitted to the myocyte peak of the ratio image, which in theory is located at 1.0. by eye observation, it was established that the transition from myocytes to fibrous tissue is approximately at 5 sd above the myocyte peak intensity, where intensity values above represent fibrous tissue and intensities below represent myocytes. thus, peak intensity + 5 sd is used as the starting point of the myocyte - to - fibrous tissue transition analysis. the transition analysis considers the drop in pixels covering myocytes as the threshold approaches the signal emanating from fibrous tissue. all single - pixel representations above threshold are quantitated for each 0.02 threshold interval in the range : starting point (myocyte peak intensity + 5 sd) 0.40, which covers approximately 10% of the intensity range of the generated 32-bit ratio image. the ratio of change in single pixel presentations to the number of single pixel presentation at the initial point of the analysis (n0.02 /nstartingpoint - 0.40) is plotted against the intensities of the analyzed range. it is noted that the change in single pixel presentations approach zero with increasing threshold [figure 3 ]. in the plots, an exponential equation approaching zero asymptotically the adjusted r of the 20 fits performed in this study was typically around 0.95 and 0.88 at lowest. the fit provides the basis for selecting the threshold reproducibly, which was defined in this study as the intensity of the fit corresponding to the value n / nstartingpoint - 0.40 = 0.015 step 5 : creation of a mask for lipofuscin is done by repeating step 4 only replacing the blue - to - red ratio with the red - to - blue ratio. high magnification image of a van gieson - stained area (a) and the corresponding black / white masks from the digital quantitations including fibrous tissue (b), extracellular phase (c), and lipofuscin (d). this particular section was selected as both solid fibrous tissue with strands down to a few micrometers (lower half) as well as hardly discernible network - like strands below 1 m (e.g. the capillarized area central left) are represented transition analysis plot of drop in the ratio of change in single pixel presentations to the number of single pixel presentation at the initial point of the analysis (n0,02/nstarting point - 0,40) plotted against the intensities of the analyzed range. the red line is an exponential fit asymptotically approaching zero the output of the digital quantitation algorithm is best presented as black / white masks of the quantitated tissue components (black pixels) [figure 2 ]. regarding fibrous tissue [figure 2b ] and ecp [figure 2c ], differences between the vg - stained section and the autofluorescence - based digital quantitations are inconspicuous. lipofuscin [figure 2d ] can not be identified in the vg - stained section. the solid fibrous tissue is readily recaptured, whereas recapturing of the thin strands is less certain. the thin strands are probably in the proximity of the lower limit of what can be quantitated with the presented technique. the vg - stainings were used both for point grid counting for correlation with the digital quantitations and an analytical comparison between color depth and autofluorescence intensities. figure 4a shows an example of a point grid quantitated area along with corresponding digital quantitation of ecp [figure 4b ], fibrous tissue [figure 4c ], and lipofuscin [figure 4d ]. it was not possible to quantify lipofuscin using the vg - stain because the lipofuscin could not be differentiated from surrounding myocyte cytosol. scatter plots displaying the correlations between point grid quantitations and digital quantitations are presented in figure 5. the digital quantitation of cells is the entire area of the section with fibrous tissue and ecp subtracted. the function y = x is entirely included in confidence intervals of the cell and ecp quantitations. the confidence interval of the fibrous tissue quantitation is slightly below x = y in the central part of the fit corresponding to a small systematic underestimation of fibrous tissue in the digital quantitation. panel (a) displays a van gieson - stained section with an superimposed point grid, whereas panels (b - d) displays outlines of the corresponding digital quantitations of extracellular phase, fibrous tissue, and lipofuscin, respectively. this particular section includes a higher amount of dispersive fibrous tissue than normal myocardium scatterplots of point grid counting versus digital quantitations of cells (a), extracellular phase (b), and fibrous tissue (c). x = y is added as a red dashed line, black full line is least squares fitted line, and the green lines display the 95% confidence intervals thereof as a further means to validate the digitally quantitated fibrous tissue and ecp, an analytical proof of concept is presented in figures 6 and 7. in figure 6, part of a vg - stained section is shown that includes strands of fibrous tissue. the red color corresponding to fibrous tissue is demarcated by including increasing intensities of the red spectrum using the rgb color threshold feature of imagej, where figure 6d was set to fit the fibrous tissue as perceived by the author. figure 6e - h show the same section of the blue - to - red ratio image, where pixels in panels f - h are increasingly demarcated going from highest to lowest intensity with panel h representing the digitally determined threshold. it is noted that deeper red in the vg staining corresponds to higher gray scale intensity of the ratio image. the area was chosen, as it includes solely fibrous tissue because myocytes in the vg - staining could not be digitally distinguished from fibrous tissue. images of the same section demonstrating the analytical verification of the fibrous tissue quantitation. in (a - d), which are van gieson - stained, increasing areas of red are demarcated with red going from deep red to lighter nuances of red. in (e - h), which is the blue to red ratio, image pixels with the highest intensity are demarcated in (f) and decreasing intensities are included toward (h) with (h) representing the digital quantitation images of a section demonstrating the analytical verification of the extracellular phase quantitation. in panels a - d, which is van gieson - stained, areas of decreasing intensity are demarcated with red to cover the manually perceived extracellular phase completely (d). in the blue fluorescence image (e - h), areas of increasing intensity are demarcated with red with (h) representing the digital quantitation a similar approach was used for ecp. figure 7 displays a vg - stained area [figure 7a - d ] along with the corresponding area in the blue autofluorescence image [figure 7e - h ]. in the vg - images, decreasing pixel intensities are demarcated resulting in the lightest areas of the section, which is regarded as ecp, being increasingly demarcated. in figure 7d, the demarcation has been set to fit ecp as perceived by the author. in panels e - h, increasing intensities it is noted that the highest intensities of the vg - stained section correspond to the darkest pixels in the blue autofluorescence image. this analytical approach verifies the point grid quantitation by proving that the autofluorescence intensity, through quantitative analysis, correlates to the as - perceived fibrous tissue, and ecp of a vg - stained section. as for verification of the lipofuscin quantitation the outline mask of the corresponding digitally quantitated lipofuscin is shown in panel b. panel c is panel a with a slightly translucent panel b on top. three images of the same section, where (a) is the autofluorescence image in the red area, (b) is the quantitated lipofuscin, and (c) is a combination of (a and b) with (b) made slightly translucent and layered on top of (a) for overview reasons microsections cut at 2 and 8 m from samples fixated for 1 and 4 days, respectively, were digitally analyzed and quantified with point grid quantification as described above. long fixation time as well as thicker microsections resulted in an overall higher autofluorescence intensity in both the blue and the red area. at both fixation times and both microsection thicknesses the digital quantitation algorithm was applied successfully with satisfying correlations to the point grid quantitations. the outer boundaries of this additional experiment were the thin microsections fixated for 1 day and the thick microsections fixated for 4 days, which had the lowest and highest impacts, respectively, of thickness and fixation time on autofluorescence. close - up examples of these boundary samples with vg - staining and corresponding fibrous tissue quantitation are shown in figure 9. panels 9a - b is a thin microsection fixated for 1 day and 9c - d is a thick microsection fixated for 4 days. as expected, the borders between stained and unstained components are unclear in the thick microsection (9c) compared to the thin microsection (9a). the fibrous tissue outline of the thin microsection (9b) is by inspection in clear correspondence with the vg - staining, whereas the outline of the thick section (9d) is in less obvious correspondence. this ambiguity was also present in thick microsections fixated for 1 day, but not in thin microsections fixated for 4 days. it should be noted that larger tissue component presentations were clearly outlined in the thick microsections. panel a is a thin (2 m) microsection cut from the sample fixated for 1 day and panel c is a thick microsection (8 m) from the sample fixated for 4 days. panel b and d are the corresponding outline masks of the digital quantitations of fibrous tissue. panels a and b are representative of thin microsections fixated for 4 days and panels c and d are representative of thick microsections fixated for 1 day in keeping with the objective high throughput commitment, it was considered mandatory to develop a reproducible quantitation algorithm suitable for fully automated processing. the quantitation algorithm was based on parametric data analysis of the autofluorescence intensities. in particular, the fibrous tissue was determined by analysis of pixel intensity ratios of blue - to - red autofluorescence, lipofuscin by analysis of pixel intensity ratios of red - to - blue autofluorescence, and ecp by analysis of pixel intensities of blue autofluorescence. figure 1c shows an image where pixel values are ratios of blue - to - red autofluorescence intensities from the corresponding pixels in figure 1a, b, and d show the reciprocal image consisting of red - to - blue ratios. in the blue - to - red ratio image [figure 1c ], the myocytes are seen as a featureless background with uniform intensity, whereas the fibrous tissue is amplified and leveled. in the red - to - blue ratio image [figure 1d ], lipofuscin is amplified and leveled. all image analysis was done using imagej, origin (origin lab corporation, northampton, ma, usa) and excel (microsoft, redmond, wa, usa). the following steps 15 present the autofluorescence - based quantitation, which has been tested on various area sizes. if the area is below 1 1 mm, the included fitting procedures become unstable due to irregular, spikey intensity histograms. if the area is too large, variations in the microsection thickness may theoretically provide instability. this problem, however, was not encountered analyzing bigger areas up to 3 3 mm. it is consequently recommended to perform the analysis on square sections of at least 1 1 mm and be cautious with squares more than 3 3 mm. step 1 : creation of a mask of the ecp is performed using a standard distribution fitted to the myocyte peak in the histogram of the blue autofluorescence image. this was done by exporting the intensity histogram from imagej to origin and fit a single peak in the fit module with gauss function checked. pixels with intensities below the fitted peak value-2 standard deviations are thresholded from the remaining image and used as a mask representing ecp [figure 2 ] step 2 : the ecp mask is subtracted from the blue and the red images using the image calculator function in imagej resulting in ecp being represented with the value 0step 3 : the images are leveled with respect to the peak intensity of the myocytes : the blue / red ratio of the myocyte peak intensities is identified from histograms (please see small insets in panels a - b of [figure 1 ]) and used to align the red with the blue image using the math multiply function. for instance, the red image is multiplied by 2 if the myocyte peak intensity value of the blue image is twice that of the red imagestep 4 : creation of a mask of the fibrous tissue by generating and analyzing the blue - to - red ratio image. in summary, the standard deviation (sd) of the myocyte peak in the ratio image is used to approximate the upper - intensity level that includes pixels covering myocytes, and a transition analysis is performed to reproducibly identify that particular level. in the case of the blue to red ratio image, fibrous tissue is considered all pixels with intensity above the upper intensity level of the myocytes, and in the case of the red - to - blue ratio image, lipofuscin is considered all pixels with intensity above the level of the myocytes. the detailed procedure for identifying fibrous tissue is described in the following : the blue image is divided with the red image using the image calculator function ; 32-bit float must be checked. in the resulting ratio image, the intensity variation of all tissue elements is leveled, and the fibrous tissue intensity is amplified, and lipofuscin intensity is diminished. typically, fibrous tissue is too small a representation to be represented by a peak in the intensity histogram of the generated ratio image [figure 1c ]. hence, fibrous tissue must be defined based on the myocyte peak as follows : a standard distribution is fitted to the myocyte peak of the ratio image, which in theory is located at 1.0. by eye observation, it was established that the transition from myocytes to fibrous tissue is approximately at 5 sd above the myocyte peak intensity, where intensity values above represent fibrous tissue and intensities below represent myocytes. thus, peak intensity + 5 sd is used as the starting point of the myocyte - to - fibrous tissue transition analysis. the transition analysis considers the drop in pixels covering myocytes as the threshold approaches the signal emanating from fibrous tissue. in particular, all single - pixel representations above threshold are quantitated for each 0.02 threshold interval in the range : starting point (myocyte peak intensity + 5 sd) 0.40, which covers approximately 10% of the intensity range of the generated 32-bit ratio image. the ratio of change in single pixel presentations to the number of single pixel presentation at the initial point of the analysis (n0.02 /nstartingpoint - 0.40) is plotted against the intensities of the analyzed range. it is noted that the change in single pixel presentations approach zero with increasing threshold [figure 3 ]. in the plots, an exponential equation approaching zero asymptotically the adjusted r of the 20 fits performed in this study was typically around 0.95 and 0.88 at lowest. the fit provides the basis for selecting the threshold reproducibly, which was defined in this study as the intensity of the fit corresponding to the value n / nstartingpoint - 0.40 = 0.015step 5 : creation of a mask for lipofuscin is done by repeating step 4 only replacing the blue - to - red ratio with the red - to - blue ratio. step 1 : creation of a mask of the ecp is performed using a standard distribution fitted to the myocyte peak in the histogram of the blue autofluorescence image. this was done by exporting the intensity histogram from imagej to origin and fit a single peak in the fit module with gauss function checked. pixels with intensities below the fitted peak value-2 standard deviations are thresholded from the remaining image and used as a mask representing ecp [figure 2 ] step 2 : the ecp mask is subtracted from the blue and the red images using the image calculator function in imagej resulting in ecp being represented with the value 0 step 3 : the images are leveled with respect to the peak intensity of the myocytes : the blue / red ratio of the myocyte peak intensities is identified from histograms (please see small insets in panels a - b of [figure 1 ]) and used to align the red with the blue image using the math multiply function. for instance, the red image is multiplied by 2 if the myocyte peak intensity value of the blue image is twice that of the red image step 4 : creation of a mask of the fibrous tissue by generating and analyzing the blue - to - red ratio image. in summary, the standard deviation (sd) of the myocyte peak in the ratio image is used to approximate the upper - intensity level that includes pixels covering myocytes, and a transition analysis is performed to reproducibly identify that particular level. in the case of the blue to red ratio image, fibrous tissue is considered all pixels with intensity above the upper intensity level of the myocytes, and in the case of the red - to - blue ratio image, lipofuscin is considered all pixels with intensity above the level of the myocytes. the detailed procedure for identifying fibrous tissue is described in the following : the blue image is divided with the red image using the image calculator function ; 32-bit float must be checked. in the resulting ratio image, the intensity variation of all tissue elements is leveled, and the fibrous tissue intensity is amplified, and lipofuscin intensity is diminished. typically, fibrous tissue is too small a representation to be represented by a peak in the intensity histogram of the generated ratio image [figure 1c ]. hence, fibrous tissue must be defined based on the myocyte peak as follows : a standard distribution is fitted to the myocyte peak of the ratio image, which in theory is located at 1.0. by eye observation, it was established that the transition from myocytes to fibrous tissue is approximately at 5 sd above the myocyte peak intensity, where intensity values above represent fibrous tissue and intensities below represent myocytes. thus, peak intensity + 5 sd is used as the starting point of the myocyte - to - fibrous tissue transition analysis. the transition analysis considers the drop in pixels covering myocytes as the threshold approaches the signal emanating from fibrous tissue. all single - pixel representations above threshold are quantitated for each 0.02 threshold interval in the range : starting point (myocyte peak intensity + 5 sd) 0.40, which covers approximately 10% of the intensity range of the generated 32-bit ratio image. the ratio of change in single pixel presentations to the number of single pixel presentation at the initial point of the analysis (n0.02 /nstartingpoint - 0.40) is plotted against the intensities of the analyzed range. it is noted that the change in single pixel presentations approach zero with increasing threshold [figure 3 ]. in the plots, an exponential equation approaching zero asymptotically the adjusted r of the 20 fits performed in this study was typically around 0.95 and 0.88 at lowest. the fit provides the basis for selecting the threshold reproducibly, which was defined in this study as the intensity of the fit corresponding to the value n / nstartingpoint - 0.40 = 0.015 step 5 : creation of a mask for lipofuscin is done by repeating step 4 only replacing the blue - to - red ratio with the red - to - blue ratio. high magnification image of a van gieson - stained area (a) and the corresponding black / white masks from the digital quantitations including fibrous tissue (b), extracellular phase (c), and lipofuscin (d). this particular section was selected as both solid fibrous tissue with strands down to a few micrometers (lower half) as well as hardly discernible network - like strands below 1 m (e.g. the capillarized area central left) are represented transition analysis plot of drop in the ratio of change in single pixel presentations to the number of single pixel presentation at the initial point of the analysis (n0,02/nstarting point - 0,40) plotted against the intensities of the analyzed range. the red line is an exponential fit asymptotically approaching zero the output of the digital quantitation algorithm is best presented as black / white masks of the quantitated tissue components (black pixels) [figure 2 ]. regarding fibrous tissue [figure 2b ] and ecp [figure 2c ], differences between the vg - stained section and the autofluorescence - based digital quantitations are inconspicuous. lipofuscin [figure 2d ] can not be identified in the vg - stained section. the solid fibrous tissue is readily recaptured, whereas recapturing of the thin strands is less certain. the thin strands are probably in the proximity of the lower limit of what can be quantitated with the presented technique. the vg - stainings were used both for point grid counting for correlation with the digital quantitations and an analytical comparison between color depth and autofluorescence intensities. figure 4a shows an example of a point grid quantitated area along with corresponding digital quantitation of ecp [figure 4b ], fibrous tissue [figure 4c ], and lipofuscin [figure 4d ]. it was not possible to quantify lipofuscin using the vg - stain because the lipofuscin could not be differentiated from surrounding myocyte cytosol. scatter plots displaying the correlations between point grid quantitations and digital quantitations are presented in figure 5. the digital quantitation of cells is the entire area of the section with fibrous tissue and ecp subtracted. the function y = x is entirely included in confidence intervals of the cell and ecp quantitations. the confidence interval of the fibrous tissue quantitation is slightly below x = y in the central part of the fit corresponding to a small systematic underestimation of fibrous tissue in the digital quantitation. panel (a) displays a van gieson - stained section with an superimposed point grid, whereas panels (b - d) displays outlines of the corresponding digital quantitations of extracellular phase, fibrous tissue, and lipofuscin, respectively. this particular section includes a higher amount of dispersive fibrous tissue than normal myocardium scatterplots of point grid counting versus digital quantitations of cells (a), extracellular phase (b), and fibrous tissue (c). x = y is added as a red dashed line, black full line is least squares fitted line, and the green lines display the 95% confidence intervals thereof as a further means to validate the digitally quantitated fibrous tissue and ecp, an analytical proof of concept is presented in figures 6 and 7. in figure 6, part of a vg - stained section is shown that includes strands of fibrous tissue. the red color corresponding to fibrous tissue is demarcated by including increasing intensities of the red spectrum using the rgb color threshold feature of imagej, where figure 6d was set to fit the fibrous tissue as perceived by the author. figure 6e - h show the same section of the blue - to - red ratio image, where pixels in panels f - h are increasingly demarcated going from highest to lowest intensity with panel h representing the digitally determined threshold. it is noted that deeper red in the vg staining corresponds to higher gray scale intensity of the ratio image. the area was chosen, as it includes solely fibrous tissue because myocytes in the vg - staining could not be digitally distinguished from fibrous tissue. images of the same section demonstrating the analytical verification of the fibrous tissue quantitation. in (a - d), which are van gieson - stained, increasing areas of red are demarcated with red going from deep red to lighter nuances of red. in (e - h), which is the blue to red ratio, image pixels with the highest intensity are demarcated in (f) and decreasing intensities are included toward (h) with (h) representing the digital quantitation images of a section demonstrating the analytical verification of the extracellular phase quantitation. in panels a - d, which is van gieson - stained, areas of decreasing intensity are demarcated with red to cover the manually perceived extracellular phase completely (d). in the blue fluorescence image (e - h), areas of increasing intensity are demarcated with red with (h) representing the digital quantitation a similar approach was used for ecp. figure 7 displays a vg - stained area [figure 7a - d ] along with the corresponding area in the blue autofluorescence image [figure 7e - h ]. in the vg - images, decreasing pixel intensities are demarcated resulting in the lightest areas of the section, which is regarded as ecp, being increasingly demarcated. in figure 7d, the demarcation has been set to fit ecp as perceived by the author. in panels e - h, it is noted that the highest intensities of the vg - stained section correspond to the darkest pixels in the blue autofluorescence image. this analytical approach verifies the point grid quantitation by proving that the autofluorescence intensity, through quantitative analysis, correlates to the as - perceived fibrous tissue, and ecp of a vg - stained section. as for verification of the lipofuscin quantitation the outline mask of the corresponding digitally quantitated lipofuscin is shown in panel b. panel c is panel a with a slightly translucent panel b on top. three images of the same section, where (a) is the autofluorescence image in the red area, (b) is the quantitated lipofuscin, and (c) is a combination of (a and b) with (b) made slightly translucent and layered on top of (a) for overview reasons microsections cut at 2 and 8 m from samples fixated for 1 and 4 days, respectively, were digitally analyzed and quantified with point grid quantification as described above. long fixation time as well as thicker microsections resulted in an overall higher autofluorescence intensity in both the blue and the red area. at both fixation times and both microsection thicknesses the digital quantitation algorithm was applied successfully with satisfying correlations to the point grid quantitations. the outer boundaries of this additional experiment were the thin microsections fixated for 1 day and the thick microsections fixated for 4 days, which had the lowest and highest impacts, respectively, of thickness and fixation time on autofluorescence. close - up examples of these boundary samples with vg - staining and corresponding fibrous tissue quantitation are shown in figure 9. panels 9a - b is a thin microsection fixated for 1 day and 9c - d is a thick microsection fixated for 4 days. as expected, the borders between stained and unstained components are unclear in the thick microsection (9c) compared to the thin microsection (9a). the fibrous tissue outline of the thin microsection (9b) is by inspection in clear correspondence with the vg - staining, whereas the outline of the thick section (9d) is in less obvious correspondence. this ambiguity was also present in thick microsections fixated for 1 day, but not in thin microsections fixated for 4 days. it should be noted that larger tissue component presentations were clearly outlined in the thick microsections. panel a is a thin (2 m) microsection cut from the sample fixated for 1 day and panel c is a thick microsection (8 m) from the sample fixated for 4 days. panel b and d are the corresponding outline masks of the digital quantitations of fibrous tissue. panels a and b are representative of thin microsections fixated for 4 days and panels c and d are representative of thick microsections fixated for 1 day we present an autofluorescence - based technique intended for quantitation of tissue components in histological sections of the myocardium. the study acknowledges autofluorescence intensity as a tissue property suitable for quantitation of tissue components in histological sections. the digital quantitations are verified by comparison to point grid counting performed on the same microsections vg - stained. the method separates itself from previous studies on digital quantitation of myocardial tissue components on two major points : (i) several tissue components are quantitated from a single slide and (ii) no staining is required. the method may serve as a stand - alone part of any routine microscopic evaluation providing accurate quantitations and digital outlines of myocardial tissue components. this would provide immediate access to any desired distribution analysis of the microsection in question. the quantitation algorithm is based on parametric analysis using no image manipulation such as contrast enhancing filtering. although the approach provided satisfying quantitations in all cases, it is recognized that alternative analysis approaches may be applicable. in particular, filtering may replace the transition analysis, which would alleviate the computational strain. using autofluorescence ratios for identifying tissue components, however, is considered indispensable for similar autofluorescence - based techniques. the digital quantitations of fibrous tissue, ecp and myocytes were satisfyingly correlated to point grid quantitations performed on the vg - staining. the digitally quantitated fibrous tissue, however, was marginally smaller than the point grid quantitation. a similar difference was noted in a previous study comparing digital quantitation and point grid counting of cardiac fibrous tissue. the difference is possibly a result of thinly occupied areas of fibrous tissue tending to be regarded as fibrous tissue in the point grid quantitation, whereas in the digital quantitation they may be mostly ecp. it is recognized that digital quantitation of the vg - stained microsections instead of point grid counting may have served the purpose of verifying the autofluorescence - based quantitation more appropriately. indeed, we attempted to develop a color deconvolution algorithm for quantifying the vg - stained tissue components. however, vigorous image manipulation to the point of hardly recognizable tissue with considerable loss of detail was necessary. the attempt was eventually deemed a failure and point grid counting was selected for verification of the autofluorescence based quantitation. along with the analytical verification, this approach was considered sufficient. analytical verification of the quantitation of fibrous tissue and ecp was included because autofluorescence as presented is relatively unexplored in the context of digital quantitation. thus, a rational approach in addition to the point grid counting was considered necessary to exclude a coincidental correlation between the point grid counting and the digital quantitation. fixation time is a known parameter of relevance to autofluorescence intensity in that autofluorescence increases with fixation time. in the current study, the quantitation was based on relative differences in the autofluorescence intensities of different tissue components. it was reported that increase in autofluorescence with fixation time was accompanied by only small changes in the autofluorescence spectrum, which indicates an even autofluorescence increase regardless of the tissue component. in the additional study evaluating fixation time, the method was applied successfully and validated with point grid counting on microsections from samples fixated for 1 and 4 days. within the limited scope of the study, the quantitation algorithms are based on intrasection parametric data analysis. assuming a linear relationship between section thickness and autofluorescence intensity, this can be regarded as a shelter against bias. this linear relationship, however, has not been established. in the additional study evaluating microsection thickness, the method was applied successfully and validated with point grid counting on microsections cut at 2 and 8 m. the study, however, was too small to establish the detailed relationship between microsection thickness and autofluorescence intensity. it was noted that thick microsections resulted in unclear borders between tissue components in the vg - staining and less obvious correspondence between the digital quantitations and vg - staining. bleaching of the slides during handling, i.e., destruction of fluorescence resulting from light exposure, is a possible source of bias. slides were handled identically and with minimal exposure to light, thereby minimizing a potential bleaching effect. as a part of the scanning process, the slide scanner automatically sets the focus guided by a prescan identification of the focus plane in selected tiles. when using the rac - mode for autofocus, no bleaching could be detected digitally, whereas when using fluorescence - based autofocus, clear bleaching was seen in the autofocus tiles. rac - mode was used to avoid bleached tiles, which would otherwise have to be omitted from the analysis. the slide scanner used was mounted with an led illumination source, which produces an excitation beam of more uniform wavelength as compared to traditional illumination sources. this is considered an advantage in that a narrow - banded excitation beam is more likely to produce autofluorescence contrast resulting in images suitable for analysis. the presented method could be valuable in diagnosing arrhythmogenic right ventricle cardiomyopathy, which requires quantitation of fibrous tissue. cardiomyopathy research may benefit from the method considering the increasing evidence that fibrous tissue presence is a marker of severity and distribution a marker of the phenotype. the method may be applicable in other organs of interest, for example, when quantitating fibrosis in kidney or liver. in addition, the accurate digital outlines of tissue components may provide a framework for the digital analysis of stainings performed after the autofluorescence analysis, for instance of immunohistological stains. we have shown that autofluorescence intensity of unstained microsections may provide ample contrast to quantitate and outline multiple components in cardiac tissue. quantitative analysis of autofluorescence may prove useful in the diagnosis of cardiomyopathies and other areas of histology, for example, in analyzing fibrosis in hepatic or renal tissue. the perspectives, however, of a fully automated histological multicomponent quantitation and outlining tool based on unstained microsections are best appreciated in a digital light - an optimistic view sees quantitative autofluorescence analysis as the prestain starting point of any digital microscopy evaluation, providing a digital framework for image analysis of the staining to come.	background : the opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. to go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar to that of hematoxylin and eosin staining in conventional pathology. this study presents an automated fluorescence - based microscopy approach providing highly detailed morphological data from unstained microsections. this data may provide a basic histological starting point from which further digital analysis including staining may benefit.methods:this study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. data acquisition using a commercially available whole slide scanner and an image - based quantitation algorithm are presented.results:it is shown that the autofluorescence intensity of unstained microsections at two different wavelengths is a suitable starting point for automated digital analysis of myocytes, fibrous tissue, lipofuscin, and the extracellular compartment. the output of the method is absolute quantitation along with accurate outlines of above - mentioned components. the digital quantitations are verified by comparison to point grid quantitations performed on the microsections after van gieson staining.conclusion:the presented method is amply described as a prestain multicomponent quantitation and outlining tool for histological sections of cardiac tissue. the main perspective is the opportunity for combination with digital analysis of stained microsections, for which the method may provide an accurate digital framework.
intertrochanteric and femoral neck fractures constitute the majority of hip fractures in the elderly. early surgical intervention and ambulation are crucial to minimize complications123). femoral nail antirotation is commonly used to treat intertrochanteric fractures due to shorter operation time, less intraoperative blood loss, less limb shortening and shorter hospitalization in comparison with dynamic hip screw4567). advantages of intramedullary nailing include biomechanical superiority and structural stability enabling immediate postoperative rehabilitation regardless of the fracture type456). previous studies have reported favorable clinical and radiographic outcomes for proximal femoral nail antirotation (pfna ; synthes, oberdorf, switzerland) with a helical blade and a single - screw - system (gamma 3 locking nail system ; stryker, schoenkirchen, germany)8910). despite a number of intramedullary nailing advantages, 0 - 11.8% lag screw back - out this is the most common reason for treatment failure, and may occur due to varus deformity of the femoral neck - shaft angle and superior cut - out of the lag screw. baumgaertner.13) defined tip - apex distance (tad) as the summation of the distance between the tip of the lag screw and the apex of the femoral head on the anteroposterior (ap) and lateral images. they showed that an increased tad is a strong predictor of the lag screw cut - out14). in the past along with the rapid advancement in computer technology and digital imaging, picture archiving and communication systems (pacs) is widely used for transmitting radiographic images in the digital format15). introduction of pacs has allowed easy access for adjusting screen brightness, measuring distances and angles and observing magnified regions of interest. these technological advances have shifted time - consuming manual measurements to simple and convenient measurements within a few clicks. it has enhanced the work efficiency in orthopedic surgery at several points including preoperative templating of the hip arthroplasty and measurement of postoperative tad16). it is difficult to take postoperative radiographic images in neutral position immediately after emergence due to pain. in addition, x - ray source to image distance and the location of the center of an image may vary in different clinical settings. moreover, focus - film distance (ffd) is not consistently maintained due to different body contours. therefore, we conducted this study to analyze the difference between measured and actual tad values based on the degree of rotation and abduction using pacs. we used a femur replica (synbone ag, malans, switzerland) with a femoral head diameter of 50 mm, a neck shaft angle of 135 and a femoral neck anteversion of 10. similar to intramedullary nail insertion into the tip of the greater trochanter, pfna nail and a helical blade were inserted into the replica (fig. the femur neck - shaft angle was 130. the tip of the blade was placed at the inferior - center of the femoral head, the cleveland zone 5 (fig. the tad concept was first introduced for lag screw placement. in our study, the blade was located at a shallow - deep position to prevent cut - out of the nail18). ap radiographs were taken while the femur was held in neutral position and lateral radiographs were taken at 15 external rotation. ffd was consistently maintained at 1 m. the replica - film distance was also maintained at 1 m. the replica - film distance was first fixed at 10 cm and radiographs were taken at neutral position and after applying 10, 20, 30 and 40 internal / external rotation to the mechanical axis. subsequently, radiographs were also taken at replica - film distance of 20 cm under the same conditions. in the next step, radiographs were taken at replica - film distance of 10 cm and 20 cm after applying 10 abduction, and 10 and 40 adduction (fig. these conditions were based on different ffds that clinically happen for the patient 's radiographic positioning after reduction and internal fixation of an intertrochanteric fracture. we used pacs (marosis ; maro tech, seoul, korea) for analysis. pacs measures distances through magnification correction in radiographs. to acquire x - ray images with precise magnification, ffd was maintained at 100 cm and 100% magnification was applied to display the actual size. tad values in neutral position were considered as standard values. an orthopedic surgeon and a radiologist, each measuring the tad twice using pacs, measured the tad in each image (fig. the average error was 2 mm (4.5%) and the standard error was 3.04. percent relative error was calculated using the following formula : percent relative error=(experimental value - accepted value)/accepted value100%. tad values showed a percentage error of 3.9% after applying 10 external rotation at replica - film distance of 10 cm compared with the standard value. at 20, 30 and 40 external rotation, percentage errors were 9.3%, 11.6% and 13.8%, respectively. tad values showed a percentage error of 7.3%, 14.4%, 16.4% and 21.1%, respectively, after applying 10, 20, 30 and 40 external rotation at replica - film distance of 20 cm. the tad showed an increasing tendency compared to the standard value when external rotation increased at both 10 cm and 20 cm replica - film distances. in external rotation of the femur, a greater error was found at replica - film distance of 20 cm than 10 cm. in internal rotation of the femur, the tad had a relative error of -1.3% compared to the standard value at replica - film distance of 10 cm. errors were -2.3%, -1.1% and 1.2% after applying 20, 30 and 40 internal rotation, respectively. errors were -0.6%, -1.5%, -0.2% and 0.8% after applying 10, 20, 30 and 40 internal rotation, respectively, at replica - film distance of 20 cm (table 1). in internal rotation of the femur, although the tad decreased with an increase of internal rotation up to 20, tad values were close to standard value when the internal rotation angle was above 30 (fig. tad values had a percentage error of 6.7% at replicafilm distance of 10 cm and 6.6% at 20 cm after applying 10 external rotation. in internal rotation of the femur, the tad had a relative error of -1.4% and -9.6% compared to the standard value after applying 10 and 40 adduction, respectively, at replica - film distance of 10 cm, and a relative error of -1.5% and -9.8%, respectively, at 20 cm (table 2). the tad increased as the abduction angle increased and had a higher error at replica - film distance of 20 cm than 10 cm. moreover, the tad showed a decreasing tendency when the adduction angle increased, and a greater error was found at replica - film distance of 20 cm than 10 cm (fig. tad values increased consistently when rotation was applied from an internal rotation of 20 to an external rotation of 40, while decreased with adduction compared to abduction. the tad increased compared to the standard value when external rotation and abduction of the replica increased, while the tad decreased when internal rotation (less than 30) and adduction of the replica increased. baumgaertner.13) defined tad as the summation of the distance from the tip of the lag screw to the apex of the femoral head on the ap and lateral images after magnification correction. since then, a large number of studies have been performed to insert the lag screw into an appropriate location of the femoral head, and a tad of less than 25 mm has been considered to reduce the risk of lag screw cut - out19). after baumgaertner and solberg14) proposed that an increased tad is a strong predictor of lag screw cut - out, they verified that the use of the tad concept improves the surgical outcome. pervez.20) suggested that a tad of less than 20 mm is ideal to lower the risk of lag screw cut - out. we found that a significant error can occur in tad measurement after internal rotation, external rotation, adduction and abduction of femur, and changes in replica - film distance. we found that tad values were measured smaller than the actual size when the femur was adducted and internally rotated., the tad increased when replica - film distance increased during internal rotation and abduction. this is attributable to the tip of a helical blade being closer to the x - ray source in external rotation and abduction. contrary observations with 30-40 internal rotation could be the result of a change in the actual incidence angle of the helical blade tip. when the helical blade is not placed at the center of the femoral head, changes and errors in tad measurement may occur. deviation from the actual tad with a consistent trend were seen on ap and lateral images based on the radiographic conditions. in addition, differences in tad were observed with images taken under the same condition, which reflect the patient 's size and replica - film distance increase. in addition, it is difficult to take postoperative radiographs in the neutral position immediately after emergence due to pain. an error of above 10% was observed at an external rotation of above 30 in our study. for accurate tad measurement, orthopedic surgeons should consider the external rotation of the femur when radiographs are taken in patients who tend to externally rotate their hips to reduce the pain. the essence of our findings shows that surgeons need to be careful with many variables that may lead to errors in tad measurement in the clinical settings. the actual tad values are higher when the femur is adducted and internally rotated, and lower when the femur is abducted and externally rotated. since the femur is internally rotated and adducted for reduction of most intertrochanteric fractures, the lag screw should be inserted closer to the apex of the femur because the actual tad value is greater than what is measured on the c - arm image. reflecting the patient 's size therefore, the actual tad is typically smaller in patients with obesity. in obese patients, maintaining a consistently close distance between the c - arm source and the femur is proposed to be helpful in reducing errors in tad measurement. first, femoral neck - shaft angle (135), femoral neck anteversion (10) and location of the tip of a helical blade were inconsistent because the authors used a single femur replica. hence, we were unable to identify changes and errors in tad values according to changes in the femoral neck - shaft angle and placement of the tip of the helical blade. second, the tad showed a greater tendency to produce errors compared to our standard value (tad=43.61 mm), since the blade was located at a shallow - deep position in the replica, while a favorable tad is considered to be less than 25 mm in the clinical setting. third, the average femoral neck - shaft angle is 125 and the average angle of femoral neck anteversion is 15 in koreans21). the replica that was used in our study does not represent the femur in koreans. the actual tad values need to be considered more than the measured tad when the femur is adducted and internally rotated, and less when the femur is abducted and externally rotated. in addition, the actual tad values are assumed to be less in obese patients. for better evaluation of the tad on intraoperative radiographs, orthopedic surgeons need to consider the insertion of the lag screw closer to the apex of the femur, because the femur is internally rotated and adducted for reduction. to accurately assess the tad values after fixation of intertrochanteric fractures, ap and lateral radiographs should be taken in neutral position, and measurement error should be considered based on the patient 's size.	purposethe tip - apex distance (tad) is used to predict the clinical outcome of intertrochanteric fracture fixation. we aimed to measure the changes in tad by position and film distance using picture archiving and communication system (pacs).materials and methodswe used a femur replica with a 10 femoral neck anteversion and a 130 neck shaft angle. proximal femoral nail antirotation nail and a helical blade were inserted into the replica. radiographs were taken at the neutral position and after applying 10, 20, 30, 40 internal / external rotation, 10 abduction, and 10 and 40 adduction to the mechanical axis. radiographs were taken at the replica - film distance of 10 cm and 20 cm under the same conditions, mimicking the differences in focus - film distance (ffd), which reflect the patient 's contour in clinical settings. a radiologist and an orthopedic surgeon measured the tad twice using pacs. the average error was 2 mm (4.5%) and the standard error was 3.04. tads in the neutral position constituted the standard values to measure the relative errors.resultstads increased with an increase in the external rotation and abduction of the replica. tads decreased with an increase in the internal rotation and adduction of the replica. for comparable measurements, relative errors were higher at ffds of 20 cm compared to ffds of 10 cm.conclusionsince the femur is internally rotated and adducted for reduction, orthopedic surgeons would assess the lag screw to be closer to the apex of femur on intraoperative radiographs. to have a correct measurement of the tad after fixation of intertrochanteric fractures, radiographs should be taken in neutral position and measurement errors should be considered based on the patient 's size.
it is usually associated with two phenomena, ie, hyperalgesia, or increased pain sensitivity, and allodynia, or a painful response to a normally innocuous stimulus. typically these two phenomena fade as the initiating noxious stimulus is removed and/or the resultant tissue injury resolves. in some instances this pain persists in the absence of stimuli or tissue injury. at this point, the pain is no longer adaptive and becomes a disease itself ; this is generally termed chronic pain. chronic pain can result from a myriad of causes, including arthritis and rheumatism, back or neck problems, frequent headaches, painful neuropathies, trigeminal neuralgia or mandibular joint disorder, pelvic and abdominal disorders, and fibromyalgia or complex regional pain syndrome. the current estimate of the number of adult americans suffering from chronic pain is approximately 100 million.1,2 one recent internet - based survey showed that 30.7% of respondents reported some form of chronic pain lasting 6 months or longer.3 of these subjects, 32% reported their chronic pain as severe (7 on a scale from 0 to 10). the financial cost of chronic pain in the us is over $ 600 billion, of which half are direct medical expenditures and half lost productivity.1 clearly, chronic pain represents a major cause of morbidity, significantly impairing individual quality of life and imposing a substantial burden on society. nociceptive pain results from activation of neuronal pain pathways from damage to non - neuronal tissues, such as muscles, joints, viscera, and skin. this type of damage excites local primary afferent nociceptors (pans) that are activated by specific receptors or ion channels sensitive to heat, mechanical stimuli, protons, or cold.4 the central projections of pans, which have their cell bodies in the dorsal root ganglia, synapse with nociceptive - specific and wide dynamic range projection neurons in the dorsal horn of the spinal cord, which, in turn, project centrally to transmit the affective and discriminatory components of pain. these components of pain processing are further influenced by excitatory and inhibitory interneurons within the dorsal horn.5 neuropathic pain results from a lesion, disease, or dysfunction of the nociceptive pathway itself, either in the presence or absence of other tissue damage, although not all lesions of this pathway result in pain.6,7 injury to tissues leading to nociceptive pain cause the release of several inflammatory molecules capable of stimulating pans, including hydrogen and potassium ions, serotonin, substance p, glutamate, histamine, bradykinins / tachykinins, prostaglandins, and leukotrienes.8,9 in a process known as peripheral sensitization, these compounds alter the response characteristics of the primary nociceptors, made up of unmyelinated c - fibers and thinly myelinated a fibers, by activating second messenger systems such as protein kinase a and protein kinase c (pkc) in primary nociceptors, causing increased sensitivity of endogenous receptors and altered sodium channel distribution, resulting in a lowered activation threshold and increased sensitivity to pain.4,10 this may also have the added effect of activating previously silent nociceptors. the end result of all these changes is an increase in nociceptive input into the spinal cord. prolonged nociceptive input results in similar changes in the spinal cord, resulting in central sensitization. activation of intracellular kinases within projection neurons causes phosphorylation of ion channels and receptors, resulting in immediate central sensitization.4 in addition, the same second messenger systems activate transcription factors, causing altered gene expression and long - lasting changes in the physiology of projection neurons, resulting in delayed central sensitization. central sensitization is characterized by an increase in receptive field size, a reduction in threshold, and an increase in responsiveness in individual projection neurons which can cause nociceptive - specific neurons to behave like wide dynamic range neurons and respond to normally nonpainful stimuli, resulting in allodynia.4,1113 the phenomenon of windup in wide dynamic range neurons is also involved in central sensitization, and is known to involve activation of the n - methyl - d - aspartate subtype glutamate receptor.14 peripheral nerve injury can lead to neuropathic pain through mechanisms that are not completely understood.7 injured afferent neurons develop spontaneous neural activity and ectopic discharges15 while the uninjured afferent neurons can undergo peripheral sensitization as described above.16 changes in trophic factor support affect both injured and uninjured afferents, and can alter gene expression in dorsal root ganglia neurons, resulting in increased pain sensitivity.17,18 central sensitization also occurs in response to nerve injury, but unlike that involved with inflammation, which is dependent on ongoing nociceptive input, the central sensitization associated with neuropathic pain can involve different sets of afferents.7 for example, low - threshold a fibers responsive to touch shift to a nociceptor - like phenotype or synapse on sensitized nociceptive - specific and wide dynamic range neurons, resulting in a painful sensation to a light touch stimulus (allodynia),19 and there is a change in the subtypes of voltage - gated sodium channels that are expressed in injured pans, resulting in altered signal propagation.20 peripheral nerve damage is also associated with apoptosis of dorsal horn interneurons.21 these largely inhibitory neurons synapse with both the pans and projection neurons and express gamma aminobutyric acid (gaba), glycine, and endogenous opioid peptides such as enkephalin and endomorphin in order to modulate pain signaling from the periphery. a downregulation in the receptors for these inhibitory neurotransmitters also occurs in response to nerve injury.21 in addition to the above mechanisms, substance p and glutamate released by the pans and prostaglandins and nitrous oxide released by the nociceptive - specific and wide dynamic range neurons activate glia within the dorsal horn, which in turn release proinflammatory cytokines and growth factors that have hyperalgesic effects, resulting in a positive feedback mechanism of increased and prolonged pain.22 chronic pain is a debilitating disease because, for many patients, currently available analgesics either do not offer adequate pain relief or produce intolerable side effects. in regards to nociceptive pain, the best treatment is repair of the injured tissue ; however, for many chronic conditions, such as arthritis or cancer, this may not be possible. while nonsteroidal anti - inflammatory drugs are used to treat mild inflammatory pain, opioids or narcotic analgesics are an essential component of chronic nociceptive pain management. these drugs, eg, morphine, act at opioid mu, delta, and kappa receptors in the dorsal horn to inhibit nociceptive transmission.23 unfortunately, these receptors are found throughout the neuraxis and body tissues, so the long - term use of opioid drugs is limited by adverse systemic effects, including nausea, constipation, dizziness, drowsiness, and respiratory depression, the development of tolerance, and ultimately dependence on such drugs. the diverse mechanisms underlying neuropathic pain make it difficult to establish a unified clinical management approach because patients who present with the same symptoms may respond differently to the same medication. generally, patients with neuropathic pain respond poorly to nonsteroidal anti - inflammatory drugs or opioids. the classes of medications that have proven most beneficial to date are anticonvulsants and antidepressants, no doubt reflecting the role that abnormal neuronal activity plays in the pathophysiology of neuropathic pain.24 while some patients respond well to these types of drugs, it is estimated that half of all patients suffering from neuropathic pain receive little, if any, pain relief from currently available drugs.25 the medical and economic burden of chronic pain coupled with insufficient treatment options make the development of novel approaches necessary, and gene therapy is one such approach. gene therapy (or gene transfer) refers to the introduction and expression of dna or rna sequences in tissues and/or cells. it can be used to express proteins, or other gene products, that directly target the neurologic components underlying nociception, such as receptors, neurotransmitters, ion channels, second messenger systems, and biochemical mediators of inflammation.26,27 gene therapy has several benefits compared with traditional pharmaceuticals in the treatment of chronic pain. therapeutic genes can be delivered to specific regions and/or cells in the neuraxis, resulting in a localized concentration of the gene product. small molecule drugs typically require large systemic doses to ensure adequate local concentrations of drug products, often resulting in side effects. through targeted delivery and/or the use of specialized gene products (eg, transcription factors, dominant / negative genes, small interfering (si)rna), a far greater number of potential therapeutic targets become available to act upon and inhibit pain signaling. loss of efficacy due to tolerance is very unlikely to occur with gene therapy due to the relatively small amount of therapeutic protein expressed and the tendency to utilize endogenous protein products, which are not normally subject to tolerance. gene therapy offers the possibility of long - term expression of therapeutic proteins in specific cells, an especially attractive benefit for treating chronic conditions. lastly, there is almost no possibility for abuse or addiction because the effect remains local and does not interact with higher brain centers. any location along the pain pathway can be targeted by gene therapy, but the pans and their synapse with second order neurons in the dorsal horn are ideal because they are integral in the pathophysiology of chronic pain. three gene therapy strategies have been employed to deliver analgesic genes to the nervous system : cell - based therapies, usually transplantation of transformed cells into the subarachnoid space;2830 use of plasmids or oligonucleotides, sometimes encapsulated in liposomes to facilitate entry into cells;3133 and use of viral - based vectors. viral vector gene therapy takes advantage of the natural ability of viruses to infect and have their genes expressed by host cells and has been the most utilized system for gene delivery into the nervous system.34,35 among the several viral - based vector systems capable of gene delivery, herpes simplex virus-1 (hsv)-based vectors are especially useful for delivery to the nervous system and pans. hsv is a large human virus containing 152 kb of linear double - stranded dna encoding at least 75 gene products. the virus particle consists of a nucleocapsid surrounded by an envelope containing glycoproteins essential for virus attachment and penetration into cells. the virus genome consists of two segments, the unique long (ul) and unique short (us), each flanked by inverted repeats. once these nonessential genes have been deleted, 4050 kb of foreign dna can be accommodated within the virus. in its natural life cycle it infects and replicates in skin or mucous membrane and released viral particles are taken up by sensory nerve terminals.36 these particles travel via retrograde transport along axons to the neuronal perikaryon in the dorsal root ganglia, where wild - type hsv may either re - enter the lytic cycle or alternatively establish a latent state that is characterized by the persistence of viral genomes as nonreplicating intranuclear episomal elements. in the lytic life cycle, viral immediate early genes transactivate expression of viral early genes, the products of which principally code for proteins required for viral dna replication.37 replication - defective hsv vectors can be engineered by removing one or more genes that code for immediate early gene products. these vectors can only replicate in specialized cell lines that provide the missing gene product but are still capable of infecting sensory neurons and expressing an inserted transgene product.3840 transgene expression is mediated by the choice of promoter. short - term (weeks) expression is achieved using a strong promoter such as the immediate early gene promoter derived from human cytomegalovirus. hsv possesses a natural promoter system that is uniquely active during latency when all the other viral promoters are repressed.41 this latency active promoter has been used for the long - term (months) expression of transgenes in sensory neurons of the peripheral and central nervous systems.42,43 due to the natural biology of the parent virus, hsv - based vectors injected into the dermal layer of the skin will infect the sensory nerves innervating that area. thus, by selecting the correct dermatome for direct vector inoculation, one can specifically target the sensory neurons in which one wants to express the therapeutic product. this restricts the therapy to the most appropriate region of the body, both minimizing the possibility of side effects while maximizing therapeutic efficacy. because hsv - based vectors can easily be delivered to sensory neurons, several potential therapeutic targets are available that could interfere with the processing of noxious stimuli from the periphery to the central nervous system (figure 1). one such target is the synapse between the pan and projection neuron within the dorsal horn. projections from the brainstem can alter pain perception through interaction with interneurons located in the dorsal horn.44 these interneurons use inhibitory neurotransmitters to modulate the activity of both primary nociceptors and projection neurons, and as discussed above, pathologic changes in these neurons have been observed with chronic pain. our group and others have used replication - defective hsv - based vectors to overexpress and/or increase the activity of these inhibitory neurotransmitters in pans, and thus increase the overall inhibitory tone at this synapse. the first of these transmitters to be expressed was the endogenous opioid peptide enkephalin (enk), which is the natural ligand for the delta opioid receptor.45 hsv vector - mediated transfer of the enk gene to lumbar dorsal root ganglia of rodents via footpad inoculation has been demonstrated to reduce c - fiber - evoked pain behaviors in response to acute inflammatory stimuli, such as injection of capsaicin46 or formalin,47 and decrease hyperalgesia associated with chronic inflammation48,49 and bone cancer.50 similarly, footpad inoculation has been shown to reduce nociceptive behavior in rodent models of chronic neuropathic pain, including spinal nerve ligation51 and painful diabetic neuropathy,52 while application to the rat vibrissal pad has reduced neuropathic pain associated with trigeminal neuralgia.53 others have since shown that enk - expressing hsv vectors directly applied to the bladder or pancreas produce analgesia in visceral pain models of cystitis and pancreatitis5456 and extended findings to primates.57 all of these studies led to the first human gene therapy trial for the treatment of pain. in a ten - person, open - label, dose - escalation trial, np2, an enk - expressing hsv vector, demonstrated a positive safety profile with no serious adverse events in patients with severe focal pain due to cancer. analysis of daily numeric pain scores over the course of the study (28 days) suggested a dose - related analgesic response to the drug.58 two other inhibitory neurotransmitters that play a role in nociceptive transmission at the dorsal horn synapse have been utilized as hsv vector transgenes, ie, gaba and endomorphin. gaba is the dominant inhibitory neurotransmitter in the spinal cord and exerts tonic inhibitory control over nociceptive transmission primarily via gabaa and gabab receptors found presynaptically and postsynaptically on dorsal horn neurons. the evidence suggests that prolonged nociceptor excitation reduces the gaba - mediated tonic inhibition within the dorsal horn by reducing presynaptic gaba production and/or release.59 gaba synthesis in pans can be increased using hsv vectors expressing the rate - limiting enzyme in the biosynthesis of gaba from glutamate, ie, glutamic acid decarboxylase. hsv vectors expressing glutamic acid decarboxylase are more effective in reducing neuropathic pain in rodent models of spinal nerve ligation, spinal cord injury, or painful diabetic neuropathy6063 compared with enk - expressing vectors, but less effective in reducing pain - related behaviors in animal models of inflammatory pain (formalin test, bone cancer ; jrg, personal observation). endomorphin is a natural ligand of the mu opioid receptor, which is the major site of action for narcotic drugs such as morphine. while no natural endomorphin gene has been identified, our group engineered an hsv vector containing a synthetic endomorphin gene construct based on the known amino acid sequence. this vector demonstrated antinociceptive properties in the spinal nerve ligation model of neuropathic pain64 and the complete freund s adjuvant model of inflammatory pain.65 interestingly, this vector demonstrated an analgesic effect in rats with opiate tolerance induced by daily treatments with morphine, suggesting that there is no cross - tolerance between morphine and vector - mediated endomorphin. aside from increasing inhibitory tone at the dorsal horn synapse, hsv vectors can be used to release anti - inflammatory molecules at this site to decrease the effects of glia - mediated neuroimmune activation that occur in response to chronic pain. anti - inflammatory cytokines such as interleukin-4 (il-4), il-10, and il-13 have been shown to have analgesic properties in several different animal models of acute and chronic pain.6670 along the lines of those experiments, hsv - mediated expression of il-4 or tumor necrosis factor alpha soluble receptor in lumbar dorsal root ganglia neurons following footpad inoculation significantly decreases pain - related behavior in the spinal nerve ligation model of neuropathic pain71,72 and an hsv vector expressing il-10 decreases spontaneous paw flinches in the formalin model of acute inflammatory pain.73 other studies have used direct intrathecal administration to the lumbar spinal cord of adenovirus or adenoassociated virus - based vectors expressing il-10 to reduce neuropathic pain in animals.74,75 another set of potential therapeutic targets accessible to hsv vectors are the receptors that transduce noxious stimuli to electrical potentials within the pans. these targets are only now being examined, but could provide a more fine - tuned approach to pain control, ie, decrease pain from a specific stimuli such as heat. for example, the transient receptor potential vanilloid type 1 (trpv1) receptor is an ion channel activated by high temperature and involved in painful heat sensation. transduction of rodent dorsal root ganglia by footpad inoculation with an hsv vector expressing a mutant trpv1 gene that does not form the channel results in a significantly attenuated thermal response,76 but does not affect an animal s response to cold temperature (jrg, personal observation). a similar effect has been achieved by using an hsv vector that expresses a dominant negative protein kinase c epsilon. trpv1 activity is regulated by phosphorylation via protein kinase c epsilon and introduction of a dominant negative form of the enzyme alters the response of rats to capsaicin.77 another approach involves the insertion of novel receptors into sensory neurons. glycine is an inhibitory neurotransmitter used throughout the nervous system but primary sensory neurons do not express glycine receptors. inoculation into the footpad in rats with a glycine receptor vector resulted in a reduction in inflammatory pain - related behavior following application of glycine to the paw.78 this method could be refined by using vectors that express channels or receptors that respond to other ligands or drugs. other attractive therapeutic targets within the pans are the ion channels responsible for propagating the transduced stimuli at the periphery to the terminals located in the dorsal horn. voltage - gated sodium channels are especially interesting candidates because these proteins lie at points of convergence and are necessary for normal nociceptive processing. it is known that the voltage - gated sodium channels nav1.7, nav1.8, and nav1.9 play roles in nociception, and modifying their function has become a natural focus in recent studies.79 the sodium channel subunit nav1.7 plays a significant role in action spike initiation, and natural gain - of - function and loss - of - function mutations are associated with clinical pain syndromes of paroxysmal extreme pain disorder and congenital insensitivity to pain, respectively.80,81 further, the reductions in diabetic neuropathy pain - related behavior associated with the hsv - glutamic acid decarboxylase and hsv - enk gene therapy studies discussed above appear to be partly attributable to decreased expression of nav1.7 in dorsal root ganglia neurons.52,63 not surprisingly, knockdown of nav1.7 via hsv - mediated expression of a microrna or an antisense oligodeoxynucleotide has been shown to reduce hyperalgesia in models of painful diabetic neuropathy and inflammation.82,83 nav1.8 and nav1.9 are tetrodotoxin - resistant voltage - gated sodium channels expressed in nociceptors. nav1.8-null mice have decreased behavioral responses to thermal and mechanical stimuli, indicating a role for nociception for this particular channel,84 but a clearer role for nav1.9 has been more difficult to discern.79 in pilot data collected by our group, we demonstrated that hsv vectors expressing zinc finger protein transcription repressors targeted against nav1.8 can reduce transcription of this protein in vitro and reduce nociceptive behaviors in a rat model of neuropathic pain.85 as chronic pain continues to be a major health issue that is underserved with currently available drugs, new treatment approaches will be developed and tested. gene therapy with hsv - based vectors is one such approach and offers the ability to directly act upon the specific neurons and area of the nervous system that are involved in the processing of nociception. however, there remain dosing, safety, and cost issues that will need to be addressed before gene therapy becomes a fully accepted treatment option for the management of pain. treatment for chronic pain will depend on long - term expression of the therapeutic gene product, which could be achieved by using a long - term promoter but will more likely involve repeat dosing of the vector product. our group has demonstrated that readministration of hsv - based vectors is both safe and effective in re - establishing analgesia in rodent models of nociceptive and neuropathic pain.47,51,60,71 long - term expression of a transgene carries its own risks. once applied, many viral vector - based gene transfer vectors, hsv included, could persist in cells for the life of the patient. this could be quite problematic if the patient had an adverse reaction to the expressed transgene. for this reason, it may be preferential to use short - duration promoters in vector constructs. alternatively, a regulatable system capable of turning gene expression on and off could be used.86 while such a system has been used in hsv vectors to control the expression of erythropoietin in the treatment of diabetic neuropathy, it has not yet been utilized to treat pain.87 a practical concern is one of cost. the process and subsequent costs associated with growing, isolating, and purifying gene transfer vectors are substantially different from those associated with traditional small molecule therapeutics. the ultimate cost per treatment can only be known once indications and target populations have been identified. we believe that the potential increased efficacy of this approach or the ability to treat otherwise intractable patient populations will drive the acceptance of gene therapy to treat pain, and enhanced manufacturing processes and market forces will stabilize the costs to a point acceptable to both patients and payers. drug products based on hsv vectors that utilize a short - duration promoter such as the human cytomegalovirus promoter would be expected to be readministered every 23 months. the choice of transgene would depend on the indication and preclinical animal model studies ; as stated above, some transgenes are more efficacious than others in specific pain models. several targets, especially within the pan, have been examined to date, resulting in a substantial amount of preclinical data supporting an hsv - based gene therapy approach in pain management. hsv vectors enable retrograde transduction of pans via intradermal application but direct injection to nervous system structures is also possible. zou demonstrated that direct injection of an enk - expressing hsv amplicon vector into the rat ventral periaqueductal gray region resulted in an increased pain threshold in chronic constriction injury - induced neuropathic pain.88 intradermal inoculation of hsv vectors may be sufficient for well localized focal pain, but application to higher nervous system structures such as the periaqueductal gray may be needed to treat more global pain disorders, such as complex regional pain syndrome. to date, preclinical animal studies have focused on targets aiming to treat pain symptoms. increasing the inhibitory tone at the primary synapse using enk, endomorphin, or periaqueductal gray - expressing vectors are intended to interfere with pain transmission, but do not alter the underlying pathology. the use of anti - inflammatory vectors may affect central sensitization, but do not necessarily treat the causative problem. as our understanding of the pathophysiology of chronic pain increases, new targets will reveal themselves and vectors designed to treat the cause and not just the symptoms will be developed, tested, and moved into the clinic.	chronic pain represents a major medical burden not only in terms of suffering but also in terms of economic costs. traditional medical approaches have so far proven insufficient in treating chronic pain and new approaches are necessary. gene therapy with herpes simplex virus (hsv)-based vectors offers the ability to directly target specific regions of the neuraxis involved in pain transmission including the primary afferent nociceptor. this opens up new targets to interact with that are either not available to traditional systemic drugs or can not be adequately acted upon without substantial adverse off - target effects. having access to the entire neuron, which hsv - based vector gene therapy enables, expands treatment options beyond merely treating symptoms and allows for altering the basic biology of the nerve. in this paper, we discuss several hsv - based gene therapy vectors that our group and others have used to target specific neuronal functions involved in the processing of nociception in order to develop new therapies for the treatment of chronic pain.
transitional cell carcinoma (tcc) of the upper urinary tract (uut) represents approximately 5% of urothelial malignancies and less than 10% of all renal tumors. although open radical nephroureterectomy with excision of an ipsilateral periureteral bladder cuff has been considered the standard management for upper tract tcc, the operation is associated with a lengthy hospitalization and convalescence. the morbidity associated with the open approach has led to the investigation of minimally invasive techniques that may serve as alternatives. antegrade and retrograde endoscopic management of upper - tract tcc have been reported for treatment of this disease. however, the indications for these approaches are highly select and should be reserved for patients with a solitary kidney, renal insufficiency, bilateral disease, or low - volume tumors. the introduction of the laparoscopic nephroureterectomy, first performed in 1991 at washington university, has provided another minimally invasive alternative to open surgery. compared with open nephroureterectomy, early results with the laparoscopic approach were encouraging with decreased postoperative analgesia requirements, shorter hospitalization, better cosmesis, and improved convalescence. despite these patient advantages, drawbacks of the laparoscopic approach include the lengthy operative time, the steep learning curve, and the need for a skilled laparoscopic surgeon. the hand - assisted transperitoneal laparoscopic approach has been part of the evolution in minimally invasive alternatives for patients with upper - tract tcc. keeley and colleagues reported the initial results with this approach and noted a significantly shorter operative duration than their series of laparoscopic nephroureterectomy. several other small series have subsequently demonstrated the safety and efficacy of this procedure with comparable short - term oncologic control. to date, 38 patients with upper - tract transitional cell carcinoma have been treated with a hand - assisted transperitoneal laparoscopic nephroureterectomy (haln) at our institution. we report our single institution experience and compare our outcomes data with that of a contemporary series of 52 consecutive patients who underwent an open radical nephroureterectomy (on). we retrospectively reviewed the records of 90 consecutive patients who underwent an open (on) or hand - assisted laparoscopic transperitoneal (haln) radical nephroureterectomy between august 1996 and may 2003 at the new york - presbyterian hospital. all laparoscopic procedures were performed by 1 of 2 surgeons (jdp and res), while various surgeons were involved in the open procedures. diagnosis of upper - tract transitional cell carcinoma was established by computed tomography, excretory urogram, retrograde ureteropyelogram, and/or ureteroscopy with tissue biopsies. postoperative follow - up comprised regular interval history and physical examination, urinary cytologies, chest x - rays, and abdominopelvic ct scan. surveillance cystoscopy was performed every 3 months for 2 years and every 6 months for the next 2 years. demographic, operative, and follow - up data were recorded and compared between the 2 groups. haln = hand - assisted laparoscopic nephroureterectomy ; on= open nephroureterectomy ; tcc = transitional cell carcinoma. patients with a history of bladder cancer with surveillance studies revealing an upper urinary tract lesion. patients in each group underwent a bowel preparation with a clear liquid diet and a bottle of magnesium citrate. perioperative antibiotics were infused in the operating room, and general anesthesia with endotracheal intubation was used in all cases. an oral gastric tube was used to decompress the stomach, and venous insufflation boots were used to prevent lower extremity stasis. the patient was placed in the flank position with adequate padding for the brachial plexus and the dependent hip, knees, and ankles. the lower leg was flexed while the upper leg was gently extended and a pillow placed between them. the operating table was flexed and the kidney rest was raised. for right - sided haln, 4 ports were utilized : a 12-mm trocar at the midclavicular line 2 cm below the umbilicus (working instrument), a 5-mm periumbilical trocar (working instrument), a 5-mm trocar at the midline 5-cm above the umbilicus (camera), and a second 5-mm trocar just under the costal margin for the liver retractor. left - sided haln utilized a 12-mm trocar at the midclavicular line 2-cm inferior to the umbilicus (working instrument), a 5-mm trocar adjacent to the umbilicus (working instrument), a 5-mm trocar at the midline 2 finger breaths below the xyphoid process (camera), and a 7-cm vertical lower midline incision for the hand device (figure 1). (b) left hand - assisted laparoscopic nephroureterectomy for the right nephroureterectomy, the right colon and hepatic flexure were medially mobilized, and the right lobe of the liver was released from the body wall. the coronary ligament was incised to expose the upper pole of the kidney and the inferior vena cava. the duodenum was mobilized medially with the kocher maneuver for exposure of the renal hilum. the renal hilum was then adequately dissected for identification of the renal artery and vein. the artery and the remaining attachments of the kidney were then dissected from surrounding tissues with the harmonic scalpel. gerota 's fascia was kept intact and the adrenal gland was left in place. for left - sided nephroureterectomy, the colon was medialized from the level of the iliac vessels to the splenic flexure. the colon, spleen, and pancreas were mobilized en bloc resulting in exposure of the anterior aspect of gerota 's fascia. in a similar fashion to the right, the hilum was exposed, the artery and then the vein were isolated and divided, and the kidney with gerota 's fascia intact was separated from the adrenal gland. the ureter was dissected to the level of the bladder by a combination of blunt dissection, clips, and electrocautery. the open extravesical technique involves applying clips across the distal ureter to prevent tumor spillage during manipulation of the specimen. the hand - assist device is replaced by a self - retaining retractor, and traction is used to facilitate dissection of the transmural ureter. the detrusor muscle is then opened circumferentially from outside the bladder, and a 2-cm cuff of bladder with mucosa is removed en bloc with the distal ureter. the opening in the bladder is closed in 2 layers with 2 0 and 3 0 absorbable sutures. surgical exposure of the bladder was obtained either through the hand - assist device incision, pfannensteil, or gibson incision. an anterior cystotomy was made and the ipsilateral ureteral orifice was circumferentially excised with traction placed on the extravesical ureter. two centimeters of bladder cuff was removed, and these defects, as well as the anterior cystotomy, were closed in 2 layers with absorbable sutures. in the cystoscopic technique, the ureter was completely mobilized to the level of the bladder laparoscopically. an endoscopic stapling device was used to transect the distal ureter across the detrusor muscle. the ipsilateral ureteral orifice was unroofed with a collins knife attached to a resectoscope on cutting current. the incision was carried through the detrusor wall until extravesical fat, and the staple line were cauterized. a foley catheter was left in place for a minimum of 7 days with the defect healing primarily. the open surgical approach used an extraperitoneal flank approach to remove the kidney and the upper ureter. the distal ureter was excised either through a pfannenstiel or a gibson incision with similar management of the intramural ureter as described above. end - points of comparison included operative duration, estimated blood loss (ebl), intraoperative and postoperative complications, length of hospital stay, pathologic grade and stage of tumor, and tumor recurrence. statistical analysis was performed using the unpaired, 2-tailed student t test and the chi - square () log - rank test with the yates correction factor. the excel 2000 (microsoft, redmond, washington) and sas for windows, version 9.1 (sas institute, cary, north carolina) software programs were used for statistical calculations. we retrospectively reviewed the records of 90 consecutive patients who underwent an open (on) or hand - assisted laparoscopic transperitoneal (haln) radical nephroureterectomy between august 1996 and may 2003 at the new york - presbyterian hospital. all laparoscopic procedures were performed by 1 of 2 surgeons (jdp and res), while various surgeons were involved in the open procedures. diagnosis of upper - tract transitional cell carcinoma was established by computed tomography, excretory urogram, retrograde ureteropyelogram, and/or ureteroscopy with tissue biopsies. postoperative follow - up comprised regular interval history and physical examination, urinary cytologies, chest x - rays, and abdominopelvic ct scan. surveillance cystoscopy was performed every 3 months for 2 years and every 6 months for the next 2 years. demographic, operative, and follow - up data were recorded and compared between the 2 groups. haln = hand - assisted laparoscopic nephroureterectomy ; on= open nephroureterectomy ; tcc = transitional cell carcinoma. patients with a history of bladder cancer with surveillance studies revealing an upper urinary tract lesion. patients in each group underwent a bowel preparation with a clear liquid diet and a bottle of magnesium citrate. perioperative antibiotics were infused in the operating room, and general anesthesia with endotracheal intubation was used in all cases. an oral gastric tube was used to decompress the stomach, and venous insufflation boots were used to prevent lower extremity stasis. the patient was placed in the flank position with adequate padding for the brachial plexus and the dependent hip, knees, and ankles. the lower leg was flexed while the upper leg was gently extended and a pillow placed between them. the operating table was flexed and the kidney rest was raised. for right - sided haln, 4 ports were utilized : a 12-mm trocar at the midclavicular line 2 cm below the umbilicus (working instrument), a 5-mm periumbilical trocar (working instrument), a 5-mm trocar at the midline 5-cm above the umbilicus (camera), and a second 5-mm trocar just under the costal margin for the liver retractor. left - sided haln utilized a 12-mm trocar at the midclavicular line 2-cm inferior to the umbilicus (working instrument), a 5-mm trocar adjacent to the umbilicus (working instrument), a 5-mm trocar at the midline 2 finger breaths below the xyphoid process (camera), and a 7-cm vertical lower midline incision for the hand device (figure 1). (b) left hand - assisted laparoscopic nephroureterectomy for the right nephroureterectomy, the right colon and hepatic flexure were medially mobilized, and the right lobe of the liver was released from the body wall. the coronary ligament was incised to expose the upper pole of the kidney and the inferior vena cava. the duodenum was mobilized medially with the kocher maneuver for exposure of the renal hilum. the renal hilum was then adequately dissected for identification of the renal artery and vein. the artery and the remaining attachments of the kidney were then dissected from surrounding tissues with the harmonic scalpel. gerota 's fascia was kept intact and the adrenal gland was left in place. for left - sided nephroureterectomy, the colon was medialized from the level of the iliac vessels to the splenic flexure. the colon, spleen, and pancreas were mobilized en bloc resulting in exposure of the anterior aspect of gerota 's fascia. in a similar fashion to the right, the hilum was exposed, the artery and then the vein were isolated and divided, and the kidney with gerota 's fascia intact was separated from the adrenal gland. the ureter was dissected to the level of the bladder by a combination of blunt dissection, clips, and electrocautery. the open extravesical technique involves applying clips across the distal ureter to prevent tumor spillage during manipulation of the specimen. the hand - assist device is replaced by a self - retaining retractor, and traction is used to facilitate dissection of the transmural ureter. the detrusor muscle is then opened circumferentially from outside the bladder, and a 2-cm cuff of bladder with mucosa is removed en bloc with the distal ureter. the opening in the bladder is closed in 2 layers with 2 0 and 3 0 absorbable sutures. surgical exposure of the bladder was obtained either through the hand - assist device incision, pfannensteil, or gibson incision. an anterior cystotomy was made and the ipsilateral ureteral orifice was circumferentially excised with traction placed on the extravesical ureter. two centimeters of bladder cuff was removed, and these defects, as well as the anterior cystotomy, were closed in 2 layers with absorbable sutures. in the cystoscopic technique, an endoscopic stapling device was used to transect the distal ureter across the detrusor muscle. the ipsilateral ureteral orifice was unroofed with a collins knife attached to a resectoscope on cutting current. the incision was carried through the detrusor wall until extravesical fat, and the staple line were cauterized. a foley catheter was left in place for a minimum of 7 days with the defect healing primarily. the open surgical approach used an extraperitoneal flank approach to remove the kidney and the upper ureter. the distal ureter was excised either through a pfannenstiel or a gibson incision with similar management of the intramural ureter as described above. end - points of comparison included operative duration, estimated blood loss (ebl), intraoperative and postoperative complications, length of hospital stay, pathologic grade and stage of tumor, and tumor recurrence. statistical analysis was performed using the unpaired, 2-tailed student t test and the chi - square () log - rank test with the yates correction factor. the excel 2000 (microsoft, redmond, washington) and sas for windows, version 9.1 (sas institute, cary, north carolina) software programs were used for statistical calculations. no significant difference existed between the hand - assisted laparoscopic and open surgical groups with respect to patient age, gender distribution, preoperative american society of anesthesiology (asa) score, presenting symptoms, or tumor location. there was also no difference between the number of left - sided and right - sided surgical procedures performed between the groups. the hand - assisted laparoscopic group benefited from less blood loss (191 ml vs 478 ml) and a shorter hospital duration (4.6 vs 7.1 days) with an almost identical mean operative duration (244 vs 243 minutes) (table 2). no conversions were needed from the hand - assisted approach to the open surgical approach. of the hand - assisted group, no intraoperative complications occurred ; however, 4/38 (11%) patients had a postoperative complication. two patients had postoperative bleeding (one requiring re - exploration on postoperative day 1 without identification of a distinct source), one patient developed an enterocutaneous fistula that was managed conservatively by parenteral nutrition, and another patient had a myocardial infarction that required cardiac catheterization and angioplasty with coronary stenting. the open surgical group also had no intraoperative complications, but 2 patients had complications postoperatively. one patient developed an occipital cerebrovascular infarction requiring postoperative anticoagulation with no residual deficits at this time, and the other patient had a postoperative arrhythmia requiring pacemaker placement. there was no significant difference in the complication rate between the hand - assisted and open surgical groups (11% vs 4%, p=0.65). comparison of hand - assisted laparoscopic nephroureterectomy and open nephroureterectomy operative and postoperative data haln = hand - assisted laparoscopic nephroureterectomy ; on= open nephroureterectomy ; tur = transurethral resection. there was no difference in the pathologic grade and stage distribution of tumors between the 2 surgical groups (table 3). the mean follow - up for the haln group was significantly shorter (31.7 vs 51.0 months) than the on group. this was expected as the haln was first performed at our institution in 1999, and our series reflects the evolution of the laparoscopic experience since that time. at a mean follow - up of 31.7 months and 51.0 months, respectively, 58% (22/38) of the haln and 44% (23/52) of the on group had no evidence of disease recurrence. pathologic and follow - up data haln = hand - assisted laparoscopic nephroureterectomy ; on= open nephroureterectomy ; tcc = transitional cell carcinoma ; ned = no evidence of disease ; awd = alive with disease ; dod = dead of disease ; dwod = dead without disease. in the haln group, the overall recurrence rate of tcc in our series was 40% (15 of 38 patients) at a mean interval of 7.7 months after surgery. eight of these 11 patients had a history of transitional cell carcinoma of the bladder that was present before the diagnosis of upper - tract disease. in considering the management of the distal ureter, no difference was noted in bladder recurrences based on whether the technique was extravesical, intravesical, or cystoscopic (p=0.65). also no recurrences were noted at the port sites, incision sites, or in the location of the resected distal ureter. the on group had a 50% overall recurrence rate (26 of 52 patients) at a mean interval of 9.1 months after surgery. eighteen of these recurrences were in the bladder, and 10 of these patients had a prior history of bladder tcc. once again, the management of the distal ureter had no bearing on the bladder tumor recurrence rate. the hand - assisted laparoscopic group benefited from less blood loss (191 ml vs 478 ml) and a shorter hospital duration (4.6 vs 7.1 days) with an almost identical mean operative duration (244 vs 243 minutes) (table 2). no conversions were needed from the hand - assisted approach to the open surgical approach. of the hand - assisted group, no intraoperative complications occurred ; however, 4/38 (11%) patients had a postoperative complication. two patients had postoperative bleeding (one requiring re - exploration on postoperative day 1 without identification of a distinct source), one patient developed an enterocutaneous fistula that was managed conservatively by parenteral nutrition, and another patient had a myocardial infarction that required cardiac catheterization and angioplasty with coronary stenting. the open surgical group also had no intraoperative complications, but 2 patients had complications postoperatively. one patient developed an occipital cerebrovascular infarction requiring postoperative anticoagulation with no residual deficits at this time, and the other patient had a postoperative arrhythmia requiring pacemaker placement. there was no significant difference in the complication rate between the hand - assisted and open surgical groups (11% vs 4%, p=0.65). comparison of hand - assisted laparoscopic nephroureterectomy and open nephroureterectomy operative and postoperative data haln = hand - assisted laparoscopic nephroureterectomy ; on= open nephroureterectomy ; tur = transurethral resection. there was no difference in the pathologic grade and stage distribution of tumors between the 2 surgical groups (table 3). the mean follow - up for the haln group was significantly shorter (31.7 vs 51.0 months) than the on group. this was expected as the haln was first performed at our institution in 1999, and our series reflects the evolution of the laparoscopic experience since that time. at a mean follow - up of 31.7 months and 51.0 months, respectively, 58% (22/38) of the haln and 44% (23/52) of the on group had no evidence of disease recurrence. pathologic and follow - up data haln = hand - assisted laparoscopic nephroureterectomy ; on= open nephroureterectomy ; tcc = transitional cell carcinoma ; ned = no evidence of disease ; awd = alive with disease ; dod = dead of disease ; dwod = dead without disease. in the haln group, the overall recurrence rate of tcc in our series was 40% (15 of 38 patients) at a mean interval of 7.7 months after surgery. eight of these 11 patients had a history of transitional cell carcinoma of the bladder that was present before the diagnosis of upper - tract disease. in considering the management of the distal ureter, no difference was noted in bladder recurrences based on whether the technique was extravesical, intravesical, or cystoscopic (p=0.65). also no recurrences were noted at the port sites, incision sites, or in the location of the resected distal ureter. the on group had a 50% overall recurrence rate (26 of 52 patients) at a mean interval of 9.1 months after surgery. eighteen of these recurrences were in the bladder, and 10 of these patients had a prior history of bladder tcc. once again, the management of the distal ureter had no bearing on the bladder tumor recurrence rate. historically, the standard management of upper urinary tract transitional cell carcinoma has been open nephroureterectomy with excision of an ipsilateral bladder cuff. usually, a single large incision or 2 incisions in the flank and abdomen are utilized. the morbidity, prolonged patient convalescence, and duration of hospitalization have been documented and are significant. the introduction of laparoscopic techniques was developed to reduce the morbidity of the procedure while maintaining the basic principles of surgical oncology. multiple series have documented that laparoscopic radical nephroureterectomy has helped improve patient convalescence and decrease hospitalization when compared with the open surgical approach. however, the procedure is technically difficult and is associated with a steep learning curve. we feel that the hand - assisted approach offers several advantages over the pure laparoscopic approach. firstly, it allows the less experienced urologist a more feasible alternative to nephroureterectomy than open surgery. having a hand in the operative field allows for tactile sensation, spatial orientation, and blunt dissection, which are currently limited using the standard laparoscopic technique. it may be the most reasonable alternative to open surgery for urologists not facile with pure laparoscopic surgery. the other advantage is the ability to remove the specimen intact without requiring tissue morcellation. in a comparison of hand - assisted and standard laparoscopic nephroureterectomy, landman and colleagues found that haln decreased operative times without altering short - term measures of convalescence compared with standard laparoscopy. to date, several series have noted the effectiveness of transperitoneal hand - assisted laparoscopic surgery for managing upper - tract tcc. in a prospective, nonrandomized study, seifman and colleagues noted a longer operative duration, but shorter hospitalization, with similar short - term oncologic control and hospital costs when comparing hand - assisted laparoscopic to open nephroureterectomy. our series is the largest to date with the longest follow - up that has compared the transperitoneal hand - assisted laparoscopic approach to the open approach for nephroureterectomy. we found that while there was a comparable operative duration between the 2 procedures, the hand - assisted group benefiting from less blood loss (191 ml vs 478 ml) and shorter hospital duration (4.6 vs 7.1 days). no differences existed in the intraoperative or postoperative complication rates between the groups, and no patients had positive surgical margins. from an oncologic perspective, almost 60% of patients treated by haln had no evidence of disease at a mean follow - up of 2.5 years. the majority of tcc recurrences occurred in the bladder, thus reflecting the field change effect in the urothelium. given the high intravesical recurrence rate in our series, it is imperative that these patients have close surveillance following management of the primary upper - tract tcc. when comparing the haln and on groups, no significant difference was found in either the tcc recurrence rate (40% vs 50%) or the interval to recurrence (7.7 vs 9.1 months). the higher recurrence rate and mortality rate in the on group is most likely a function of the longer follow - up in this patient cohort. in fact, actuarial disease specific survivals show a similar 5-year disease specific survival (81% haln vs. 77% on, p=0.81) between the 2 surgical groups (data not shown). the commonly reported techniques for treating the distal ureter and bladder cuff include extravesical excision with bladder cuff, intravesical excision with bladder cuff, and cystoscopic unroofing of the intramural tunnel with extravesical excision of the distal ureter. the hand - assisted approach affords a significant advantage in that the surgeon can facilitate dissection and resection by providing gentle countertraction on the distal ureter. our series noted no differences in tcc recurrence rates based on the management of the distal ureter. of note, it is difficult to specifically compare each technique for management of the distal ureter given the propensity for development of bladder tumors in 30% to 50% of patients with negative margins following nephroureterectomy. furthermore, the small number of patients in each subgroup of distal ureteral management makes it difficult to draw significant conclusions between efficacy of one technique versus another. in our experience, the extravesical ureteral excision through the original hand - assist port was the most time efficient approach as it avoided repositioning the patient or making a new incision. for distal ureteral tumors, however, we still prefer the intravesical approach to optimally visualize that a negative bladder cuff margin is obtained. arguments against the transperitoneal approach include the potential for intraperitoneal seeding of cancer cells, manipulation of bowel yielding a prolonged ileus, as well as the potential for an intraperitoneal urinoma or seroma. in our series of patients, no recurrences occurred in the peritoneal cavity, and there was no delay in the return of bowel function compared with that in a series performed with a retroperitoneoscopic approach. we acknowledge several limitations in this manuscript including the heterogeneity of surgeons performing the on procedure, the limited cohort size in the haln group, and the difference in follow - up interval between the 2 surgical groups. clearly, as the haln approach becomes increasingly performed, larger cohorts and longer - term follow - up will be available for comparison. hand - assisted laparoscopic transperitoneal radical nephroureterectomy is an effective surgical option for the management of upper urinary tract transitional cell carcinoma. compared with open nephroureterectomy, patients benefited from less intraoperative blood loss and a shorter hospitalization with similar operative duration and intermediate - term cancer control.	objective : we report our experience with hand - assisted laparoscopic nephroureterectomy (haln) for upper urinary tract transitional cell carcinoma and compare our results with a contemporary series of open nephroureterectomy (on) performed at our institution.methods:between august 1996 and may 2003, 90 patients underwent nephroureterectomy for upper - tract transitional cell carcinoma (tcc). thirty - eight patients underwent haln, while 52 had an on. end - points of comparison included operative time, estimated blood loss (ebl), intraoperative and postoperative complications, length of hospital stay, pathologic grade and stage of tumor, and tumor recurrence.results:the mean patient age was 72.3 and 70.6 years in the on and haln groups, respectively. mean operative duration was 243 minutes (on) and 244 minutes (haln), with an ebl of 478ml in the open group versus 191ml in the hand - assisted group (p<0.001). no intraoperative complications occurred, but postoperative complications occurred in 4% and 11% of the on and haln groups, respectively (p=0.21). the mean hospital duration was 7.1 days (on) versus 4.6 days (haln) (p<0.01). no difference existed in the pathologic grade or stage distribution of urothelial tumors between the 2 groups. the mean follow - up was 51.0 months in the on group and 31.7 months in the haln group. recurrence of urothelial carcinoma occurred in 50% of patients who underwent on and 40% treated by haln (p=0.38) at a median interval of 9.1 and 7.7 months, respectively, after surgery.conclusion:hand-assisted laparoscopic nephroureterectomy is an effective modality for the treatment of upper urinary tract urothelial carcinoma. patients benefited from less intraoperative blood loss and a shorter hospitalization with an equivalent intermediate - term oncologic outcome compared with that of the open approach.
nitric oxide (no) plays multiple roles in the lung in both injury and repair ; it is an airway and vascular smooth muscle cell signaling molecule, an inhibitory nonadrenergic noncholinergic (inanc) signaling molecule, a modulator of apoptosis, and a component of the bactericidal arsenal of lung inflammatory cells. the primary molecular sources of no are the nos enzymes, including the inducible nos2 isoform, which is upregulated in the ovalbumin- (ova-) induced allergic airway inflammation model [13 ] and the constitutively expressed nos1 (neuronal nos) and nos3 (endothelial nos) isoforms, which contribute to the generation of no in the murine airway epithelium [4, 5 ]. no can be further metabolized to produce the more stable products, nitrate and nitrite. both of these products are considered bioactive, capable of enzyme - dependent and enzyme - independent reconversion into no [6, 7 ]. in asthma, increased no concentration in exhaled breath is considered a disease biomarker and is supported by current guidelines for use in clinical settings. the conventional treatment of asthmatics using inhaled steroids decreases exhaled no, but whether alleviation of the asthmatic symptoms is a result of decreasing exhaled no or if the decreased exhaled no is a byproduct of decreasing overall lung inflammation is still debated. in the cell, maintenance of a stable no concentration, or no homeostasis, is essential, as fluctuations in the concentration of no can alter intra and intercellular signaling and affect survivability., for example, detected an increase in the concentration of no in exhaled breath correlating to an increase in nos2 expression in nos3 mice, thus indicating a compensatory mechanism for the regulation of baseline no production. also, exposure of astrocytes to no - scavenging hemoglobin increased nos2 expression and was dependent on nf-b activation. in addition to maintaining a baseline of no production, it is necessary to upregulate no production under specific conditions such as during oxidative stress, release of heme - containing compounds [13, 14 ], or the launching of an inflammatory response. thus, the ability to further regulate nos2 expression in constitutive nos isoform knockout strains may be necessary for the organism to modulate its reaction to an insult and also resolve the response. a study by connelly., using lps stimulation, determined that nf-b translocation was necessary for nos2 expression under inflammatory conditions and the study by gobeil jr. we previously identified nos2 mice as being more susceptible to severe allergic inflammation and subepithelial fibrosis than their c57bl/6 wild - type counterparts indicating that nos2 expression and activity are necessary for regulating the intensity of the inflammatory response. thus, we chose to examine no homeostatic dysregulation in the development of allergic airway disease. in this paper, we hypothesized that under normal conditions, deleting the nos1 or nos3 gene would upregulate nos2 protein expression. thus, the lung function and total cellular population of filtered air - exposed nos1 and nos3 mice should be comparable to filtered air - exposed c57bl/6 wt mice, while the nos2 mice should have a heightened inflammatory cell profile as observed in our previous study. furthermore, ten broeke and colleagues showed that nos2 expression was nos3 dependent, and we therefore hypothesized that exposure of mice to ovalbumin (ova) would inhibit this increase in nos2 protein expression in the nos3 mice resulting in lower nos2 protein concentrations compared to nos3 filtered air controls. we also hypothesized that this would result in increased lung inflammation in the nos3 mice compared to both ova - exposed c57bl/6 wt and nos1 mice. to this end, we performed lung function analysis with a methacholine challenge protocol and collected bronchoalveolar lavage for the calculation of lung inflammatory cell influx and inflammatory cell profile. we also examined tissue - specific nos2 expression patterns in the lungs and changes in nitric oxide production of both filtered air - exposed and ova - exposed mice from nos1, nos2, nos3, and c57bl/6 wt genotype. all procedures were performed as per our iacuc - approved protocol, following their standards and regulations. all animals were maintained in an hepa - filtered laminar flow cage rack with a 12-hour light / dark cycle and allowed free access to food and water. animals were housed and cared for by the veterinary staff of animal resource services at ucd in aalac- accredited facilities, in plastic cages over autoclaved bedding in hepa - filtered cage racks. animals were routinely screened for health status by serology and histology by our veterinary animal resources facility. nos1, nos3, and the wild type strain c57bl/6 mice were purchased from jackson laboratories. the nos2 mice were initially purchased from taconic laboratories, germantown, ny and were rederived by embryo transfer to establish a breeding colony in the targeted genomics laboratory of the mouse biology barrier facility at uc davis. mice were sensitized by intraperitoneal (ip) injection of chicken egg albumin (ovalbumin, grade v, 98% pure, sigma, st. louis, mo, 2 10 g/0.1 ml, 2 weeks apart) with alum as an adjuvant. exposure to ova aerosols was performed using chambers and generators we have described elsewhere. exposures to ova aerosol, 10 ml of a 10 mg / ml (1%) solution, were begun on day 28. mice were exposed for 30 minutes, three times per week for the duration of a given experiment. age - matched control animals were injected ip with ova (sensitized) but were then exposed to filtered air. histological evaluations of c57bl/6 wt mice ova - exposed and filtered air - exposed control mice demonstrated that we were able to induce airway inflammation, epithelial cell sloughing, and goblet cell hyperplasia in the exposed mice. dynamic compliance and resistance of the respiratory system were measured using a plethysmograph for restrained animals. mice were deeply anesthetized and sedated with medetomidine, 0.5 mg / kg (domitor, orion pharma, finland), and tiletamine / zolpidem, 50 mg / kg (telazol, fort dodge laboratories, fort dodge, ia) and ventilated at 7 - 8 cc / kg with a mouse ventilator (minivent, harvard apparatus, cambridge, ma) for the duration of the procedure. compliance and resistance measurements were made at baseline and immediately following serial 3-minute nebulizations of saline and methacholine (0, 0.5, 1.0, and 2.0 mg / ml). after the physiological measurements, animals were euthanized with an overdose of phenobarbital and dilantin administered via intraperitoneal injection. animals were placed on a restraining board and their lungs lavaged with two 1 ml aliquots of phosphate - buffered saline (pbs), ph 7.4, and each aliquot was passaged twice through the lungs. cells were pelleted at 2500 rpm for 10 minutes, and the acellular supernatant was removed and stored at 20c for nitrate and nitrite (nox) analysis. the remaining cell pellet was treated with a lysis buffer (0.15 m nh4cl, 1 mm khco3, 0.1 mm edta, and ph 7.3), repelleted, and resuspended in 0.5 ml pbs. total lavage cell number was determined using a hemocytometer and 100 l aliquots of the remaining cell suspension processed onto slides using a cytocentrifuge at 1650 rpm for 15 minutes. slides were air dried and stained with a hema3 stain set as described in the manufacturer 's instructions (biochemical sciences, swedesboro, nj) and sealed using cytoseal (stephens scientific, kalamazoo, mi). cells were classified as alveolar macrophage, neutrophil, eosinophil, lymphocyte, or other based upon morphological characteristics and staining profile. five - minute samples of exhaled gases were collected into a specially constructed mylar bag from the cannulated mice via the ventilator exhalation port immediately after insertion of a mouse into the plethysmograph. this 5-minute sample was adequate for the measurement of no concentration in the expired air, using a sievers nitric oxide analyzer (sievers inst., boulder, co). nitrate and nitrite (nox) was measured in lavage fluid with this analyzer as previously described. the isolated airways were prepared by microdissecting the left lung lobe to obtain a preparation of the larger airways distal to the carina from the left bronchus through the third generation of conducting airway, separate from adhering parenchyma. samples containing 20 g total protein were electroporated under reducing conditions and transferred to a polyvinylidene difluoride (pvdf) membrane. membranes were incubated in 0.6 g / ml rabbit antimouse nos1, 0.8 g / ml nos2, 0.6 g / ml nos3, or 0.4 g / ml -actinin igg (used as a gell loading control) in 5% dry milk in pbs overnight at 4c and incubated in 40 ng / ml horseradish peroxidase- (hrp-) conjugated goat antirabbit igg (pierce biotechnology, rockford, il) in 5% milk in pbs. bands were visualized using immobilon western chemiluminescent hrp substrate kit (millipore, billerica, massachusetts) and band intensity - assessed using the kodak 1d version 3.5.4 scientific imaging system (eastman kodak co, ct). half of the animals had their lungs fixed for histological evaluation at 30 cm pressure using 1% paraformaldehyde in pbs (ph 7.5). after 24 hours of fixation, the left lung was placed in 70% ethanol and embedded in paraffin. lung sections of 5-um thickness were made with special attention to cutting through the larger lobar bronchi in parallel then dried at 37c overnight. lung sections were deparaffinized and processed for hematoxylin and eosin (h&e) staining for evaluation of total inflammation, or nos2-specific immunohistochemistry for semiquantitative assessment of tissue - specific nos2 expression or alcian blue - periodic acid - schiff (pas) staining for quantitation of mucus - containing goblet cells. left lung sections were prepared as described. slides were incubated in 1 mm etda, ph 7.5 at 100c for 20 minutes to decloak antigen. sections were processed using the r&d systems cell and tissue staining kit hrp - dab system (minneapolis, mn) for rabbit antibodies. sections were incubated overnight at 4c in 0.2 g / ml rabbit anti - mouse nos2 igg diluted in 5% goat serum in 1% bsa, 0.2 g / ml rabbit igg (isotype control), or serum + bsa only (negative control). incubation times were as follows : 1 hour in biotinylated goat antirabbit secondary antibody, 30 minutes in hss - conjugated hrp, and 15 minutes in diaminobenzidine. a grading system of 010 was established prior to the grading based upon a series of prestained slide standards. the lung tissue compartments were divided into airway epithelium, smooth muscle, and macrophages and were scored under 200x power. the linear intensity grading scale used was 0no nos2 stain as compared to a primary antibody negative control to 10dramatically increased nos2 staining comparable to a lung section from an lps - treated mouse. left lung sections, prepared as described above, were immersed in a 1% alcian blue, 3% glacial acetic acid solution (ph2.5) for 30 minutes, rinsed in tap water, and then immersed in a 1% periodic acid solution for 7 minutes. slides were then rinsed again in distilled water and immersed in schiff reagent solution consisting in 0.45% basic fuchsin, 10% hcl, and 0.45% sodium bisulfite for 15 minutes. slides were cleared in running tap water, counterstained using harris ' hematoxylin, and then dehydrated and mounted in cytoseal. each animal was represented by a single section of lung selected for maximal visualization of the main airway. each section had 5 randomly selected regions evaluated (two segments of the 1 conducting airway, two segments from separate 2 conducting airways, and one segment from a 3 conducting airway). a minimum of 100 sequential airway epithelial cells were counted from each region and the total number of pas positive cells per total epithelial cells was determined for each region. means were compared by t - test or by anova, with tukey 's correction for multiple comparisons applied where appropriate. analysis of the compliance and resistance changes was done by both two - way anova with bonferroni correction and by linear regression analysis, using the prism software package (graphpad prism 5.0, san diego, ca). we believe that the combination of these methods allowed for better understanding of the interaction between the effect of ova exposure and methacholine aerosol challenge. r, a common open source statistical computing package (url : http://www.r-project.org), was used to perform this analysis. nos2 mice exposed only to filtered air contained 8.65 6.90 10 total cells per lavage sample (figure 1(a)), of which 92 2% were pulmonary alveolar macrophages, significantly more total cells than were found in the nos1, nos3, and c57bl/6 mouse strains exposed only to filtered air (p <.01). after two weeks of ova exposure, the total inflammatory cell number recovered by lavage increased significantly in all strains of mice evaluated. nos2 mice exposed to ova for two weeks had significantly more lung lavage cells than the nos1, nos3, or c57bl/6 mice exposed to ova (figure 1(b), p <.05). the normal lung lavage from a healthy mouse contains more than 90% alveolar macrophages, and our observations in this study were consistent with this finding. there were < 1% eosinophils (88 230 eosinophils) in all of the strains of mice tested after exposure to filtered air (figure 1(c)). nos2 mice exposed to ova had 11.90 1.76 10 eosinophils in their lavage fluid, which was significantly more than were observed in nos1, nos3, or c57bl/6 mice exposed to ova (p <.01). there was a small (< 17,000 cells) but significant increase in the number of lymphocytes in all groups of ova - exposed mice compared to filtered air - exposed mice (data not shown), but no significant differences between the four different strains exposed to ova. in addition, the percentage of neutrophils was < 1% in all mouse strains exposed to ova. these results extend our previous finding that mice lacking the inducible nos2 gene are more susceptible to allergic airway inflammation than wild - type or constitutive nos knockout mice strains. to determine which nos isoform is responsible for the production of no in the expired breath, we compared the concentration of exhaled no between c57bl/6 mice and the three nos mouse strains exposed to filtered air. all nos strains exposed to filtered air had similar exhaled no concentrations (figure 2(a)) ; nos1 (5.1 1.6 ppb), nos2 (5.0 0.5 ppb), and nos3 mice (7.5 1.8 ppb), respectively. c57bl/6 mice exhaled a significantly greater no concentration than nos2 animals (9.7 0.5 versus 5.0 0.5 ppb, p <.001). after exposure for two weeks to ova aerosol, there was no significant change in exhaled no concentrations in any of the knockout mice strains compared to their respective filtered air - exposed groups (figure 2(a)). however, the nos2 animals exposed to ova had significantly lower no concentrations in exhaled breath than the nos3 mice exposed to ova (4.9 0.5 versus 7.7 1.1 ppb, resp., p <.05). in contrast, there was a decrease in exhaled no in the c57bl/6 mice exposed to ova compared to their controls exposed to filtered air (9.7 0.5 versus 7.2 0.6 ppb, p <.05). lung lavage nox concentrations of filtered air - exposed mice (figure 2(b)) from mice of all four strains were not significantly different from each other. after exposure to ova, both nos3 and c57bl/6 mice had significant increases in nox levels compared to their respective filtered air groups. c57bl/6 mice exposed to ova also had significantly higher nox levels compared to nos1 and nos2 mice exposed to ova. we examined whether nos isoform protein expression, no levels in exhaled breath, or nox concentrations in bronchoalveolar lavage were correlated by linear regression analysis. we found that exhaled no levels were lower in c57bl/6 mice after exposure to ovalbumin compared to their matched controls exposed only to filtered air. total nitrate / nitrite, the more stable products of no metabolism, was increased in the c57bl/6 mice exposed to ovalbumin. it is unclear to us why this might be a strain - related result. there is no obvious reason to believe that no consumption is greater in lungs of inflamed wild type mice compared to others. overall, we have some evidence that total lung no content was increased in the mice after ovalbumin exposure. there was no correlation between nos2 protein expression in the isolated airways and exhaled no in any of the strains examined (data not shown). however, there was a significant correlation between nos2 protein expression in the isolated airways and bronchoalveolar lavage nitrate / nitrite concentration in the filtered air and ova - treated groups of the nos3 strain (band intensity versus nox concentration m = 18.02 6.17, r = 0.36, and p <.05, figure 3). there were no significant correlations between nox concentration and nos protein expression in the other mouse strains (data not shown). to measure the development of airway hyperreactivity (ahr) in our model, we compared the total lung resistance and dynamic compliance at baseline and after inhalation of nebulized methacholine using serial doses of methacholine (0.5, 1.0, and 2.0 mg / ml). lung compliance decreased significantly in nos2 mice exposed to ova compared to filtered air (figure 4(c)). the difference in lung compliance between the air and ova - exposed nos2 mice was significant at each dose of methacholine (p <.001 for 02.0 mg / ml methacholine). the slope of the mch response by linear regression analysis for nos2 mice exposed to ova (2.6 0.27 10, f = 96.35) was significantly different from that of the nos2 mice exposed to air (1.8 0.12 10, f = 226.9 ; p <.05). measured lung compliance after exposure to methacholine also significantly differed for nos3 mice exposed to ova versus air (figure 4(d)). the slopes of the best fit lines by linear regression analysis for the methacholine dose - response curve for ova - exposed nos3 mice, 0.5 0.2 10, f = 4.627, and for filtered air - exposed nos3 mice was 3.0 0.3 10, f = 95.07 (p =.004). neither the c57bl/6 nor the nos1 mice demonstrated significant decreases in lung compliance with methacholine challenge after exposure to ova (figures 4(a) and 4(b)). lung resistance measurements from the four different strains of mice examined did not show significant differences when analyzed by two - way anova (figures 5(a)5(d)) but differed significantly compared to their corresponding filtered air exposures when analyzed by linear regression analysis. upon challenge with increasing doses of mch, nos1 mice exposed to ova (slope = 0.266 0.027, f = 94.09) had a significantly greater increase in lung resistance compared to the same strain of mice exposed to filtered air (slope = 0.079 0.008, f = 93.15 air- and ova - exposed nos2 mice had similar increases in lung resistance upon mch challenge testing (slopes = 0.12 0.01 and 0.15 0.02, resp.). these results suggest that these strains of mice behaved differently than the nos1 or nos3 mice after their respective exposures, which could reflect differences in their inflammatory responses, differences in airway remodeling, or both factors. the relative amount of the different nos isoforms in airways isolated from each of the mouse strains was assessed using western blot band intensity analysis. protein blots of airways from both the nos1 (figure 6(a)) and nos3 (figure 6(b)) mice exposed to filtered air showed a higher nos2 protein expression than the c57bl/6 controls (nos1 60.2 10.7 versus wt c57 bl/6 6.5 3.6, p <.01) and (nos395.6 28.1 versus wt c57 bl/6 26.6 5.1, p <.05). upon ova exposure, the nos2 expression in nos1 animals was further upregulated (96.5 30.1 (ova) versus 60.2 10.7 (fa), p =.019), while nos3 animals (figure 6(b)) showed no change in nos2 with ova treatment compared to their filtered air controls. in contrast, the nos2 mice (figures 7(a) and 7(b)) showed no significant change in the protein levels of either nos1 or nos3 in the airway samples from mice that were exposed to filtered air or ova. we analyzed smooth muscle tissue, airway epithelium, and macrophage populations to evaluate differences in nos2 protein content between these three lung tissue compartments. consistent with the western blot data derived from the isolated airways, nos1 and nos3 mice exposed to filtered air had a greater nos2 protein content than c57bl/6 mice exposed to filtered air (figures 8 and 9). quantitative assessment by immunohistochemistry of these air - exposed mice showed that increases in nos2 protein content were limited to the airway epithelium (nos1 versus c57bl/6 : p <.05 and nos3 versus c57bl/6 : p <.05) and the smooth muscle of the airways and vasculature (nos1 versus c57bl/6 : p <.01 and nos3 versus c57bl/6 : p <.01), but no change in nos2 content was detected in the tissue macrophage population (figure 9). ova exposure significantly reduced total nos2 protein in the airway epithelium and smooth muscle tissue compared to the filtered air - exposed animals in the nos3 mice (figures 9(a) and 9(b), p <.01). in contrast to the nos3 mice exposed to ova, the nos1 mice exposed to ova maintained the nos2 protein content in the airway epithelium and also showed significant increases in nos2 protein content in the macrophage population (figures 9(a) and 9(c), p <.05 compared to ova - exposed nos3 and filtered air - exposed nos1 mice). to determine if the decrease in nos2 protein content in the airway epithelium was due to the replacement of ciliated epithelium with mucus - producing goblet cells in the nos3 animals exposed to ova, we used the periodic acid - schiff (pas) to stain for mucus - containing cells in the airway epithelium and determined the overall ratio of mucus - containing cells to total cell population by cell counting. all of the filtered air - exposed groups contained essentially no mucus - producing cells in the airways (see, e.g., c57bl/6 animals ; figure 10(a)). in contrast, exposure to ova significantly increased the number of mucus - containing cells in all three strains of mice tested (figures 10(b)10(d)). we observed that 937% of the total cell population of the upper airway epithelium was mucus - producing cells, and that the observed value depended on the genotype (figure 11) with nos1 mice exposed to ova having significantly fewer pas positive - stained cells compared to the ova - exposed c57bl/6 (p <.01) or nos3 (p <.05) mice. no modulates pulmonary vascular tone, non - adrenergic non - cholinergic mediated bronchodilation, the lung inflammatory response, and apoptosis [2629 ]. in previous work using an nos2 strain, we observed increased airway inflammation as compared to wild - type mice exposed only to air, and especially upon exposure to ova. we concluded that the ability to upregulate active nos2 enzyme in response to ova exposure is necessary for the anti - inflammatory effect imparted by no. however, these earlier findings could not define the mechanistic basis for these observations, for example, whether nos2 was itself the required source for no in allergen - induced airway inflammation or whether the effects were caused by increased l - arginine turnover by the other nos isoforms or the reconversion of oxidization products, nitrate and nitrite, into no. to attempt to answer these questions, we examined the effects of deleting individual nos isoforms on the overall dynamic of total lung no balance in allergic airway disease. measurements of exhaled no in the various mouse strains tested focused our attention on nos2 as the most likely source of no in exhaled air from mice exposed to ovalbumin (figure 3). examination of the correlation between nos2 protein expression in isolated airways and total concentration of nox in the lung lavage fluid showed that the nos3 strain was the only strain studied, where nos2 protein expression was tightly coupled to nox production, and such coupling was observed in the nos3 mice exposed to either air or ovalbumin. in contrast, the nos1 and nos3 mice actually exhaled more no than their wild - type counterparts. the upregulation of nos2 we observed in the two constitutive nos knockout strains may explain this finding. in previous work using the nos2 strain, we observed increased numbers of cells in the lavage fluid (predominantly macrophages), which raised the question of whether deletion of either of the other individual nos isoforms (nos1 and nos3) would have similar effects on the lavagable alveolar macrophage population. because we hypothesized that there would be an upregulation of nos2 in the constitutive knockouts, we expected to find no significant increase in total lung cells in lavage fluid from the nos1 and nos3 animals exposed to filtered air. our results were consistent with this hypothesis, as we found similar bal total cell counts in the constitutive knockout strains and the c57bl/6 mice. we conclude that the deletion of individual nos isoforms affects no output by specific isoforms, but that the inability to normalize the total lung no concentration by upregulating nos2 results in an increase in the recruitment of bone marrow - derived cells as illustrated by an increase in the population of lavagable macrophages only in the nos2 mice. in this study, nos1 animals exposed to filtered air demonstrated a significant change in lung compliance and resistance upon challenge with methacholine. with increased nos2 protein expression induced by ova exposure we also saw a significant decrease in the respiratory system compliance of nos2 mice exposed to ova compared to their counterparts exposed to filtered air. we interpret these findings to suggest that changes in lung compliance in this model directly reflect lung inflammation. only the nos1 animals exposed to ova demonstrated a significant decrease in lung compliance associated with a concomitant increase in lung resistance that reflects a bronchoconstrictive response. these data suggest that no from a specific enzyme source in this case from nos1 in the subepithelial smooth muscle and/or the epithelium is responsible for the bronchodilatory properties of no, but that the induction of nos2 may be sufficient to mitigate some aspects of ahr. attribute decreased nos1 activity in pulmonary and tracheal smooth muscle after ova challenge in guinea pigs to airway hyperresponsiveness, and our data are consistent with these results. de sanctis. also examined changes in inflammatory response in the nos2 strain compared to the c57bl/6 strain but observed no significant increase in total inflammatory cell number. although our current and previous observations appear to conflict with the findings of de sanctis, substantial variations in the exposure protocol may have caused the differing outcomes. nos2 is capable of producing one thousand times more no than either of the constitutive isoforms and has multiple levels of regulation that are dependent upon no concentration [10, 3541 ]. we hypothesized that under normal noninflammatory conditions, deletion of the nos1 or nos3 gene would upregulate nos2 protein expression. knocking out individual constitutive nos isoforms would lower overall cellular no concentration, resulting in increased nos2 expression in order to maintain a minimal total lung no concentration. to test this hypothesis, we measured lung no and nox concentrations, total airway nos protein content, and localized the expression of nos2 protein in the four strains of mice (c57bl/6 wild - type strain and nos1, nos2, and nos3) exposed to filtered air only. the constitutive nos knockout strains, nos1 and nos3, exposed to filtered air showed significant upregulation of nos2 protein expression compared to the c57bl/6 wild - type control. as predicted, the nos1 mice showed no change in nos3 expression and nos3 mice showed no change in nos1 expression. these results support our hypothesis that no homeostasis is established by maintaining a concentration of available total no independent of nos isoform origin. the nos2 strain showed no significant increase in protein expression of either of the two constitutively expressed nos isoforms (nos1 or nos3). these results indicate different upstream regulatory mechanisms inducing gene expression and potential translational control of the two constitutive isoforms and indicate that the control of constitutive nos expression is not no dependent. thus, we conclude that at baseline, nos2 enzyme production can be induced to maintain total lung no homeostasis via a mechanism that is capable of detecting decreased concentrations of cellular no and activating nos2 expression. this conclusion is in accordance with observations by cook., who noted increased nos2 expression and exhaled no concentrations in nos3 mice. immunohistochemical analysis of whole lung sections identified the cellular origins of nos2 expression in the filtered air - exposed genotypes. consistent with the western blot results, the c57bl/6 mice exposed to filtered air showed low expression of nos2 protein, with only light staining present in the smooth muscle and airway epithelium. in contrast, both the nos1 and nos3 animals displayed a significant increase in nos2 protein expression compared to the c57bl/6 mice by western blot analysis, and these increases were limited to the airway epithelium and smooth muscle tissue of the airway and vasculature (see figures 8 - 9). as the airway epithelium constitutively expresses both nos1 and nos3 and is capable of upregulating nos2 under inflammatory conditions, this result implies a tight regulatory control of no homeostasis in these particular cell types. the smooth muscle tissue of the lung expresses low levels of nos2 protein in the c57bl/6 mice and may benefit from vectorial production of no derived from adjacent epithelial and endothelial cells. as localized no concentrations decrease, nos2 expression, which suggest that induction of nos2 gene expression by nf-b is dependent upon nos3 activity (figure 8), we hypothesized that exposure to ovalbumin would inhibit the increase in nos2 protein expression in the nos3 mice. we examined the upregulation of nos2 in nos1 and nos3 mice exposed to ova to determine if the no - dependent mechanism to induce nos2 expression is isoform specific, that is, derived solely from the nos3 isoform. if the ova - induced increase in nos2 expression is driven by the nos3, we would expect no significant increase in nos2 protein expression in the nos3 animals while we would expect an increase in nos2 protein expression in the nos1 animals. in our study, the patterns of nos2 expression in the ova - exposed nos1 and nos3 animals were consistent with this mechanism being nos3 specific. in fact, we observed a surprising lack of nos2 expression in the lungs of nos3 animals overall by immunohistochemistry. analysis of nos2 expression in the nos1 and nos3 lung compartments of mice exposed to ova indicated a significant reduction of nos2 in the airway epithelium and smooth muscle compartments of the nos3 mice only, with nos1 mice maintaining nos2 protein expression in both compartments, including upregulation of macrophage nos2 expression. while examining nos2 protein content in the airway epithelium, we observed a difference in the cellular population of the airway epithelium between the different mouse genotypes with exposure to ova that may have contributed to changes in nos2 protein content. one limitation of our study is that we used nos gene knockout strains only and did not use overexpressing mice to confirm our findings. however, we are encouraged by the complimentary data of ten broeke and colleagues. they found that transgenic mice overexpressing nos3 increased no production in the lungs, decreased lung lavage inflammatory cell counts, and improved airway hyperresponsiveness compared to littermate controls after exposure to ovalbumin. one possible explanation for the seeming overlap between ten broeke 's results and ours is the interrelationship among the noss in the lung. this suggests that important feedback mechanisms to nos3 regulate much of the measurable no that is produced in the lung in response to allergen. the nos 1 mice had a significantly reduced number of goblet cells present compared to the other three strains. nos1 mice exposed to ova also had reduced airway reactivity and airway inflammation compared to the other strains, which may indicate roles for nos1 in inflammatory cell recruitment signaling and, potentially, airway epithelial cell apoptosis. in addition to its effects on airway hyperreactivity, no has pro- and antiapoptotic effects that are concentration and cellular compartment dependent. the nos1 isoform is also localized to the mitochondria (mtnos) and despite the ubiquitous nature of no in the lung during inflammation, deletion of the nos1/mtnos isoform may contribute to the reversal of airway goblet cell metaplasia seen in the other isoform knockout and c57bl/6 strains exposed to ovalbumin. although we were able to identify increased goblet cell metaplasia in the airways of nos3 mice exposed to ovalbumin as compared to nos1 mice, this change in epithelial cell content did not account for the lack of nos2 protein in the remaining intact epithelial cells. the cells that were pas - negative still comprised 50% of the airway cell population. in addition, the lack of nos2 protein in the intact smooth muscle of the airways and vasculature of the nos3 mice was also noteworthy. though our hypothesis indicates that knockout of nos3 results in an inability to signal for increased nos2 expression upon allergen exposure, an alternative explanation exists for these results that we could not address within the scope of our studies in intact animals. nf-b - dependent transcription can also be affected by nitrosation of the rel and p50 components of the active nf-b complex. in high no environments such as cell culture medium with high concentrations of no donor compounds added, cysteine residues of both p65 and p50 have been shown to be nitrosated, resulting in reduced dna - binding capacity [35, 3941 ] (figure 9). increased no production by nos2 under inflammatory conditions may be sufficient to nitrosate these residues resulting in the activation of this negative feedback loop. despite the lack of nos2 in the lungs of nos3 animals exposed to ova, increases in bal nox concentrations in the nos3 mice may indicate an accumulation of no metabolites in the lung from another source. there is increasing evidence suggesting that nitrite serves as a bioavailable pool of no to act as a vasodilator, as deoxyhemoglobin may have nitrite reductase activity, promoting this conversion. while we did not measure the arterial oxygen saturation of our mice during this experiment, it is possible that the ventilation - perfusion mismatching as a result of dense inflammatory infiltrate and/or methacholine challenge could lead to regional hypoxemia and have an effect on nox to no conversion. however, we did not see changes in either no or nox concentrations in nos2 animals after exposure to ova that would be consistent with this pathway. in addition, it has been theorized that alterations in the ph of the airway lining fluid as a result of inflammatory cell influx and oxidative damage may result in nitrite conversion to no. both the nos3 and nos2 mice had more inflammatory cells in their bal fluid but their exhaled no concentrations were relatively unaffected. examination of individual nos isoform knockout and c56bl/6 strains exposed to filtered air supports the hypothesis that there is an nos - dependent mechanism in the cell that maintains a baseline no production in both airway epithelium and airway and vascular smooth muscle and is unable to distinguish between nos isoforms of origin. under inflammatory conditions, the expression of the nos2 isoform is essential for reducing lung inflammation and may also contribute to the normalization of airway reactivity. nos3 isoform activity is essential for the upregulation of nos2 in response to ovalbumin exposure. in contrast, nos2 can be expressed independently of nos1 activity in the inflamed lung, but nos1 activity contributes to goblet cell metaplasia in the airways of ovalbumin - exposed animals.	objectives and design. the function of the airway nitric oxide synthase (nos) isoforms and the lung cell types responsible for its production are not fully understood. we hypothesized that no homeostasis in the airway is important to control inflammation, which requires upregulation, of nos2 protein expression by an nos3-dependent mechanism. materials or subjects. mice from a c57bl/6 wild - type, nos1/, nos2/, and nos3/ genotypes were used. all mice strains were systemically sensitized and exposed to filtered air or ovalbumin (ova) aerosol for two weeks to create a subchronic model of allergen - induced airway inflammation. methods. we measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled no, nitrate and nitrite concentration, and airway nos1, nos2, and nos3 protein content. results. deletion of nos1 or nos3 increases nos2 protein present in the airway epithelium and smooth muscle of air - exposed animals. exposure to allergen significantly reduced the expression of nos2 protein in the airway epithelium and smooth muscle of the nos3/ strain only. this reduction in nos2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express nos2. nos1/ animals had significantly reduced goblet cell metaplasia compared to c57bl/6 wt, nos2/, and nos3/ allergen - exposed mice. conclusion. the airway epithelial and smooth muscle cells maintain a stable airway no concentration under noninflammatory conditions. this homeostatic mechanism is unable to distinguish between nos derived from the different constitutive nos isoforms. nos3 is essential for the expression of nos2 under inflammatory conditions, while nos1 expression contributes to allergen - induced goblet cell metaplasia.
radial artery puncture is an invasive procedure that is frequently used for arterial blood gas analysis or invasive blood pressure monitoring. although known to be safe, radial artery cannulation can cause thrombosis, leading to severe ischemia of the hand and even subsequent gangrene and tissue loss. the purposes of this case report are to share the treatment course of finger necrosis after accidental radial artery puncture and review the literature. a previously healthy 63-year - old woman presented to the emergency department (ed) complaining of aggravating pain on her left second and third fingers along with a color change. she had visited a local clinic for a diagnostic esophagoduodenoscopy the day before as part of her annual checkup that was accompanied by procedural sedation. after having trouble establishing intravenous (iv) access for medication, the providers managed to insert an iv catheter in the left wrist on the lateral palmar side, a less common site for iv access. during the procedure, she experienced a sharp and extreme pain that persisted even after removal of iv access. however, the treating physician did not verify the distal circulation of her hand and discharged her after prescribing a painkiller. after returning home, her fingers turned pale and eventually started to show a bluish discoloration (fig. the pain became intolerable ; therefore, she visited our ed, 12 hours after the procedure. her initial vital signs were as follows : blood pressure, 127/77 mmhg ; heart rate, 101/min ; respiratory rate, 16/min ; and body temperature, 36.7c. although seemed stable, she complained of excessive pain (8/10 on the numeric rating scale) in her left second and third fingers. emergency doppler ultrasonography performed in the ed showed normal blood flow from her radial artery to the common palmar digital artery. decreased flow at the second and third proper palmar digital arteries was also found. computed tomography angiography showed a pseudoaneurysm in her left radial artery and no sign of blood flow from her palmar digital artery (fig. subcutaneous enoxaparin (40 mg) and oral nifedipine (30 mg) were administered. the occluded branch was distal, which limited the usefulness of surgery ; therefore, angiography was performed instead. angiography showed patent radial and ulnar arteries, with a patent common palmar digital artery and sufficient collateral circulation. however, the second and third proper palmar digital arteries were not visualized at all. vasodilation was induced with nitroglycerin (300 g) but failed. a small pseudoaneurysm observed in the radial artery near the puncture site suggested that the second and third finger occlusion was caused by a microembolism from the pseudoaneurysm. intra - arterial thrombolysis was performed with the infusion of urokinase (960,000 u) over 4 hours. however aspirin (100 mg per day [qd ]), clopidogrel (75 mg qd), and alprostadil (10 g qd) were prescribed. after close observation for 1 week, the circulation on the third finger almost completely recovered, but the second finger turned gangrenous, which forced us to amputate it (fig. the patient was discharged 12 days after admission in a tolerable state and was scheduled for outpatient surgery. the middle and distal phalanges of her second finger were amputated 25 days after the event, and she was discharged without additional significant complications. radial artery puncture is frequently performed in many eds. arterial blood gas analysis and invasive blood pressure monitoring require radial artery puncture. the radial artery is favored because of the existence of the collateral and ulnar arteries as well as the ease of access. the radial artery is also a popular access site for coronary angiography and cerebral angiography. doppler ultrasonography or a modified allen s test can be used to evaluate the patency of collateral flow. common complications after radial artery cannulation include temporary radial artery occlusion (19.7%), hematoma (14.4%), infection (0.72%), hemorrhage (0.53%), and bacteremia (0.13%). permanent ischemic damage and pseudoaneurysm are very rare adverse effects, with rates of 0.09% each. risk factors for complications involve the cannula size, vasospasm, patient age, female sex, high number of puncture attempts, and duration of catheter placement [5 - 8 ]. in this case, the patient s providers attempted iv access for procedural sedation. probably because of poor vein access, they chose an unfamiliar site for the iv, which resulted in critical pseudoaneurysm and thromboembolism. the optimal treatment of patients who develop ischemia after radial artery cannulation is controversial ; therefore, it should be individualized. systemic anticoagulation, intra - arterial verapamil, and balloon angioplasty with localized abciximab or low molecular weight dextran are the first - line therapies for patients without bleeding risks. invasive angiography can visualize the injured vessels directly and provide a vehicle for the direct injection of vasodilatory agents to the site. the limitation is that it can not reach very distal portions as in this case, which attenuates its efficacy. when a substantially large portion of tissue is ischemic, surgical treatments as thromboembolectomy, arterial repair, patch angioplasty, or vein grafts can be considered. in conclusion, radial artery puncture must be conducted in proper sites and accompanied by collateral flow check - up, carefully conducted procedures, follow - up, and continuous monitoring of distal blood flow. when a complication is suspected, rapid assessment and optimal treatments are required to prevent fatal complications.	radial artery puncture, an invasive procedure, is frequently used for critical patients. although considered safe, severe complications such as finger necrosis can occur. herein, we review the clinical course of finger necrosis after accidental radial artery puncture. a 63-year - old woman visited the emergency department (ed) with left second and third finger pain after undergoing intravenous (iv) access in her wrist for procedural sedation. during the iv access, she experienced wrist pain, which increased during the 12 hours prior to her ed presentation. emergency angiography revealed a pseudoaneurysm in her left radial artery and absence of blood flow to the proper palmar digital artery. subsequent angiointervention and urokinase thrombolysis failed. the second finger was eventually amputated owing to gangrene. radial artery puncture can occur accidentally during iv wrist access, resulting in severe morbidity. providers should carefully examine the puncture site and collateral flow, followed by multiple examinations to ensure distal circulation.
myotonic dystrophy in a complex multisystemic autosomal dominant disorder characterized by two different mutations resulting in an unstable ctg repeat expansion in the 3(untranslated region of the dystrophia myotonica protein kinase (dmpk) gene on chromosome 19 (myotonic dystrophy type 1) and a cctg repeat expansion in intron 1 on zinc finger protein 9 on chromosome 3 (myotonic dystrophy type 2). it is the most common form of muscular dystrophy with an incidence of 1:8,000 worldwide. symptoms are multisystemic and include muscle hyperexcitability, progressive muscle weakness, cataract development, testicular atrophy and cardiac arrhythmias. insulin resistance in myotonic dystrophy correlates with a failure to express the insulin receptor splice variant that normally predominates in adult skeletal muscle, leading to the expression of an insulin receptor variant with a lower signaling capability. muscles of myotonic dystrophy 1 patients contain lower than normal levels of insulin receptor rna and protein. this results in reduced responsiveness of muscle to insulin compared to normal individuals, confirming previous studies which suggested that metabolic abnormalities which may lead to glucose intolerance were related to insulin response defects. hepatic dysfunction in patients with myotonic dystrophy is common and includes chronic liver disease and gallbladder dismotility. one study showed that an approximate 66% of patients with myotonic dystrophy 1 had at least one abnormality of liver enzymes. in this article we describe the case of a patient with myotonic dystrophy and non - alcoholic steatohepatitis (nash) and discuss the possible links between the myotonic dystrophy genetic defect and development of steatohepatitis. the patient was a 39-year - old male with a history of myotonic dystrophy who was being evaluated for abnormal liver function tests. abnormal laboratory values included total bilirubin 2.5 mg / dl (normal 0.2 - 1.2), indirect bilirubin 2.1 mg / dl, ast 71 u / l (normal 10 - 40), alt 96 u / l (normal 9 - 60), gamma - gt 126 u / l (normal 3 - 90). viral hepatitis serologies and autoimmune panel were negative. a liver biopsy was performed which showed a moderate degree of macrovescicular steatosis with foci of hepatocellular inflammation suggestive of steatohepatitis. we suggest that patients with myotonic dystrophy are at increased risk of developing hepatocytic inflammation / nash through the same pathways that are seen in patients with metabolic syndrome, i.e. abnormalities in insulin receptor and glucose intolerance leading to elevated free fatty acid oxidation and reactive oxygen species. these abnormal metabolic pathways promote hepatocyte inflammation which in turn can increase the risk of development of hepatic cirrhosis and carcinoma. nash, a common cause of cryptogenic cirrhosis and hepatocellular carcinoma, is characterized by fatty infiltration of the liver, inflammation, hepatocellular damage and fibrosis. it is seen in a subset of patients with non - alcoholic fatty liver disease (nafld) which is a well - known hepatic manifestation seen in patients with the metabolic syndrome (which includes insulin resistance, hypertriglyceridemia, hypertension and obesity). the etiology of elevated liver tests in patients with myotonic dystrophy has been suggested to arise from two possible mechanisms : (1) elevated ast from skeletal muscle breakdown as correlated with creatine kinase elevations and (2) biliary contraction / bile excretion abnormalities. these laboratory abnormalities in patients with myotonic dystrophy type 1 include changes in serum creatine kinase, ast / alt and fasting blood glucose. increase in alt, alkaline phosphatase and gamma - gt activity has been reported to be specific of liver involvement in patients with myotonic dystrophy. biochemical abnormalities seen in myotonic dystrophy patients have been described to be mainly secondary to muscular damage and falsely attributed to the liver, leading to costly and unnecessary liver investigations. some have suggested alterations in the contractility of bile canaliculi and bile ductules that may lead to incomplete biliary obstruction and impaired bile excretion as an etiology. a recent study showed decreased insulin receptor rna and protein not only in skeletal muscle but also in the liver (up to an 80% decrease). we suggest that the resulting insulin resistance in hepatocytes and other tissues leads to hepatocyte damage similar to that seen in nash. the mechanism of damage in nash / nafld has been related to insulin resistance even in the absence of obesity or diabetes. insulin resistance leads to an increase in circulating free fatty acids because of the decreased antilipolytic effects of insulin in adipose tissue. also, mitochondrial dysfunction secondary to insulin resistance leads to increased formation of reactive oxygen species in hepatocytes leading to inflammation. in vitro studies have identified a cug - binding protein that binds to the anomalous triplet expansions in myotonic dystrophy 1. cug - binding protein activity is increased as a result of this interaction in myotonic dystrophy 1 which leads to aberrant splicing defects in chloride channel 1 and insulin receptor rna resulting in myotonia and insulin receptor defects [5, 18, 19 ].	myotonic dystrophy is a multisystemic disorder characterized by repeat expansion mutations of the dystrophia myotonica protein kinase (dmpk) gene resulting in a defective muscular insulin receptor and insulin resistance. we describe a patient with myotonic dystrophy who developed biopsy - proven non - alcoholic steatohepatitis. we suggest that patients with myotonic dystrophy are at risk of developing steatohepatitis. the relationship between defective insulin receptor and development of steatohepatitis should be further investigated.
	in the bacterial isc system for iron sulfur cluster assembly, iscu acts as a primary scaffold protein, and the molecular co - chaperones hsca and hscb specifically interact with iscu to facilitate atp - driven cluster transfer. in this work, cluster transfer from azotobacter vinelandii [fe2s2]2 + cluster - bound iscu to apo - grx5, a general purpose monothiol glutaredoxin in a. vinelandii, was monitored by circular dichroism spectroscopy, in the absence and in the presence of hsca / hscb / mg - atp. the results indicate a 700-fold enhancement in the rate of [fe2s2]2 + cluster transfer in the presence of the co - chaperones and mg - atp, yielding a second - order rate constant of 20 000 m1 min1 at 23 c. thus, hsca and hscb are required for efficient atp - dependent [fe2s2]2 + cluster transfer from iscu to grx5. the results support a role for monothiol grx s in storing and transporting [fe2s2]2 + clusters assembled on iscu and illustrate the limitations of interpreting in vitro cluster transfer studies involving [fe2s2]-iscu in the absence of the dedicated hsca / hscb co - chaperone system.
this work was supported by the japan securities scholarship foundation, the technology and new energy and industrial technology development organization (nedo), and special coordination funds for promoting science.	we fabricated a ph - sensitive device on a glass substrate based on properties of carbon nanotubes. nanotubes were immobilized specifically on chemically modified areas on a substrate followed by deposition of metallic source and drain electrodes on the area. some nanotubes connected the source and drain electrodes. a top gate electrode was fabricated on an insulating layer of silane coupling agent on the nanotube. the device showed properties of ann - type field effect transistor when a potential was applied to the nanotube from the top gate electrode. before fabrication of the insulating layer, the device showed that thep - type field effect transistor and the current through the source and drain electrodes depend on the buffer ph. the current increases with decreasing ph of the cnt solution. this device, which can detect ph, is applicable for use as a biosensor through modification of the cnt surface.
reports of violence and aggression related with epilepsy receive considerable attention in the medical literature. although aggression associated with epilepsy is well documented, reports of criminal behavior associated with seizure activity are rare. even though seizure control may be possible with the use of anti - epileptic medications, behavioral symptoms usually persist.1 aggressive behavior in epilepsy patients can be interictal, ictal or postictal.2 ictal aggressive behavior is usually resistive, not targeted, stereotyped and more commonly originates from the frontal or temporal regions. more frequently, aggressive behavior occurs during the postictal period.3,4 postictal aggression usually occurs when the patient is in a confusional state and manifests as resistive violence when attempts are made to restrain the patient.3 here we report the rare case of a patient with intractable epilepsy and escalating aggression resulting in murder who had complete resolution of her seizures and explosive behavior following a right temporal lobectomy. the patient was charged with second - degree murder and was put on trial ; however, the patient underwent successful epilepsy surgery with complete resolution of her symptoms, so her charge was downgraded to manslaughter and she was transferred to a psychiatric facility. in this article a previously healthy 40-year - old right - handed female presented with new onset complex partial seizures and occasional secondary generalization. over the next 5 years, she rapidly became refractory to medical treatment with seizure frequency increasing to almost daily despite attempts to control seizures with multiple anti - epileptic drugs. the patient failed initial monotherapy with valproic acid and progressed to require polytherapy with carbamazepine 600 mg twice a day (bid), lamotrigine 150 mg bid, clobazam 10 mg bid and levetiracetam 1000 mg bid (which was discontinued when the behavioral issues came to light) and yet did not achieve adequate seizure control. over this same period, the patient developed increasing emotional lability, outbursts of anger and escalating violent behavior coinciding with seizure onset and which was especially worse postictally, lasting 5 to 10 minutes. these explosive episodes led to numerous altercations with police, resulting in multiple arrests for violent behavior. the patient was convicted a total of 32 times prior to the murder for various minor offences and violent incidents, the majority of these occurring in the last 4 years when the patient developed intractable epilepsy. the patient s past medical history was otherwise only remarkable for chronic alcoholism and depression. although it was offered, the patient repeatedly refused psychiatric evaluation during her disease evolution and was intermittently treated by her family physician for depression with venlafaxine and citalopram. the most common were complex partial seizures characterized by lack of aura, staring, bimanual and oral automatisms and postictal confusion. the frequency of these complex partial seizures varied but was on average six per month, with one cluster per month. the patient also had complex partial seizures with secondary generalization (one per month). video - eeg telemetry was performed and 6 seizures were recorded ; all of them had clear onset over the right temporal region at f8-t4 with involvement of the ipsilateral scalp sphenoidal electrode. six months after the video - eeg telemetry investigation was performed, the patient had a disagreement with her basement tenant. during this dispute unfortunately, the patient is amnestic of the event and there were no other witnesses, so both the duration of the aggressive episode and whether it was preceded by seizure are unknown. while in jail, the patient and her family wished to revisit the possibility of neurosurgery. at age 46, the patient underwent a standard right temporal lobectomy, twelve months after the video - eeg telemetry investigation. the patient remained in hospital for two weeks post - operatively and was then transferred back to prison. after six months in a maximum security facility, she was transferred to a psychiatric center due to the noted positive behavior change. at two years post - surgical follow up, the patient reported no seizure activity and was on monotherapy with carbamazepine 400 mg bid. the patient s behavior improved dramatically after the temporal resection with no evidence of emotional lability or aggressive outbursts since. this patient was administered a standard battery of neuropsychological tests, assessing the domains of attention, working memory, language, learning and memory and psychological / emotional functioning, on two separate occasions. her first neuropsychological assessment occurred about 22 months prior to neurosurgery and indicated significantly compromised cognitive functioning, with difficulties most evident on tasks assessing verbal comprehension, working memory and learning of novel material, particularly if the material was visual in nature. she still demonstrated good reasoning and problem solving skills, and adequate learning if provided with an opportunity to first deal with hands - on material. she was also experiencing significant levels of psychological distress due to the limitations imposed by her seizure disorder. the second neuropsychological assessment was undertaken about 15 months post neurosurgery and indicated that this patient was no longer psychologically distressed as she was no longer experiencing seizures. however, her neuropsychological profile indicated that she had experienced further global decline in cognitive functioning with marked impairment and limited function. this decline was attributed to the longstanding history of epilepsy, but could also be partially due to the resection. this patient was administered a standard battery of neuropsychological tests, assessing the domains of attention, working memory, language, learning and memory and psychological / emotional functioning, on two separate occasions. her first neuropsychological assessment occurred about 22 months prior to neurosurgery and indicated significantly compromised cognitive functioning, with difficulties most evident on tasks assessing verbal comprehension, working memory and learning of novel material, particularly if the material was visual in nature. she still demonstrated good reasoning and problem solving skills, and adequate learning if provided with an opportunity to first deal with hands - on material. she was also experiencing significant levels of psychological distress due to the limitations imposed by her seizure disorder. the second neuropsychological assessment was undertaken about 15 months post neurosurgery and indicated that this patient was no longer psychologically distressed as she was no longer experiencing seizures. however, her neuropsychological profile indicated that she had experienced further global decline in cognitive functioning with marked impairment and limited function. this decline was attributed to the longstanding history of epilepsy, but could also be partially due to the resection. we describe the case of a young woman with medically refractory epilepsy that developed increasing emotional lability, outbursts of anger and escalating aggressive behavior culminating in a violent murder. our patient underwent a temporal lobectomy with complete resolution of the seizures, anger and violence. we have reviewed all the available cases reported from 1880 to 2013 using medline, index medicus, cochrane database and bibliographies of pertinent reviews and original articles using the following search terms : epilepsy, aggression, violence, criminal, crime, criminality, law, legal, murder, homicide, matricide, epileptic automatism, epileptic furor, epileptic equivalent, larvate epilepsy and rage. of 178 citations identified, 46 potentially eligible articles were reviewed in full text. from these, we found 30 articles reporting 176 original cases of various aggressive behaviors in patients with epilepsy. we selected only those case reports and case series of epileptic patients that committed murder and were placed on trial. the largest available series in the literature was that of treiman,7 who reported 75 crimes of violence in which epilepsy was used as a defense. from this review we selected 31 patients with a clear history of epilepsy before the crime. in our review, the mean age of patients was 31.5 years (range 1952). in 58% of cases, in the group of patients with focal epilepsy, 80% had temporal epilepsy and 20% frontal epilepsy. our case is unusual because the patient is a female, in contrast to the predominance of males in the majority of reports in the literature, although is similar in the reported age group and in the type of epilepsy (focal - temporal). in our review our patient is only the second case reported in the literature about a temporal lobectomy being curative for aggression resulting in murder. only walker1 reported a patient who underwent a left temporal lobectomy after committing murder in a state of postictal aggression. similar to our case, this patient improved after surgery with fewer seizures and less aggressive behavior.1 regarding the timing of the violent event with respect to the seizure, our review shows that 39 patients (78%) did not have a clear temporal relation between the violent behavior and seizure. in only 11 patients (22%) did the violent episodes have a clear temporal relationship to the seizures. in 9 of 11 patients (82%) the violent episodes occurred in the postictal period, one in the ictal period (9%) and the last one in the interictal period. in our patient, the violent act was not clearly related with a seizure (interictal), but we can not rule out a seizure immediately preceding the event. in a larger series, gerard describe six patients with targeted aggression similar to that of our patient. in these patients, postictal violence was intentionally directed towards an individual or an object.8 ito, marsh and akuffo have described a different, more common pattern of postictal behavior : that of resistive violence.3,4,10 in these patients, the violence is passive and undirected, occurring in the form of resistance at attempts at restraint. this pattern was also seen in our patient during previous attempts at arrest by police. in the majority of reported cases, the victim is a family member (50%), usually the wife (60%).1,7,1113,15 of the 7 women we found who committed an aggressive act,3,7,16 60% of these were committed against family members, either against their baby3 or husband.7 our patient attacked a non - family member, which occurs less frequently, but likely represents the fact that family members are often physically closest to the patient during the ictal / postictal period. of note is the ferocious and excessive nature of the violence in these cases. we found 5 similar cases of vicious murders in which the aggressor inflicted more injuries than required to kill the victim.1,7,13 in one report, a woman was stabbed 42 times with butcher knife, hatchet, and sharpened screwdriver.7 another case outlines a women who was stabbed 30 times in the chest and back.1 these violent episodes can be precipitated by alcohol ingestion or stressful situations.10,17 in two separate series, 50% and 65% of patients were intoxicated before the crime.2,18 our patient s history is significant for alcohol abuse, and she had been drinking prior to the event, although it is unknown whether she was intoxicated, and the altercation occurred after an arguably stressful situation. after reviewing the literature, a pattern emerges regarding the characteristics of people with epilepsy who commit violent crimes : they are usually young adults, males, with low average intelligence and a history of behavioral difficulties starting commonly in childhood.8,19 the aggressive episodes are stereotyped and repetitive, more common in the postictal period, occur more frequently after a cluster of seizures and in patients with focal epilepsy (table 2).8 with regards to the legal situations in these cases, our review shows that 72% of patients were initially charged with homicide, and 22% with manslaughter. after trial, 62% of patients were convicted of homicide and 38% were found not criminally responsible for their actions (insanity plea) and were discharged to a psychiatric facility for hospitalization. the eminent british psychiatrist henry maudsley suggested that, when a murder has been committed without apparent motive and the reason of it seems inexplicable, it may chance that the perpetrator is found on inquiry to be afflicted with epilepsy.21 put into the context of our case, our patient s charge was downgraded from second - degree murder to manslaughter as epilepsy may have been the inciting event. using her disease as a defense allowed our patient to be moved from a high security prison to a psychiatric center for appropriate treatment.15,22 the higher risk of violent crimes in patients with epilepsy has not been demonstrated in many studies. in a swedish epidemiological study, patients with epilepsy did not have an increased risk of violent crimes compared with the general population.23 accordingly, epilepsy and criminality is rarely reported. reuber reported a series of 13 cases identified between 1975 and 2001 in the uk that were found not guilty by reason of insanity because of epilepsy.18 charges included murder (1), attempted murder (1), assault (7), arson (2), abduction / kidnapping (3) and burglary (1).18 93% were males, 84.6% had psychiatric comorbidity and psychotic symptoms may have been present when the offenses were committed in 52.8% of cases. over two - thirds of offenses probably occurred during the postictal period.18 few studies have proposed specific criteria to determine whether a violent crime was the result of an epileptic seizure. delgado - escueta assessed 33 events of aggressive behavior during seizures in 19 patients recorded on video - eeg.17 after analysis of the spells, the international team of epileptologists noted some common characteristics : all aggressive acts occurred suddenly, without evidence of planning, and lasted an average of 29 seconds. all patients were easily restrained, and automatic acts were short - lived, fragmentary and unsustained.17 this international panel developed a set of five criteria to determine whether an aggressive act was the result of an epileptic seizure (table 3). although we never documented aggressiveness during the video - eeg investigation in our patient, the rest of the criteria were fulfilled. aggressiveness related with epilepsy has been well described in the literature for more than a century. these patients share some common characteristics : usually are young men with a long history of drug - resistant epilepsy and lower than average intelligence. the violent act is postictal, usually occurs after a cluster of seizures, is of sudden onset and is related with stressful situations and alcohol abuse. our case is unique because the patient had interictal aggressiveness, probably related with temporal epilepsy, that markedly improved after epilepsy surgery. to our knowledge, there is only one other published case in the literature in which an epileptic patient s aggressive behavior improved after surgical resection of the epileptogenic lesion. it is unclear if our patient had a seizure before the incident that could have potentially triggered the abnormal behavior. our case is also unique because the defense lawyer was able to use the patient s medical condition and the positive outcome post - surgery as a defense to downgrade the charges against the patient.	purposewe report the rare case of a patient with intractable epilepsy and escalating aggression, resulting in murder, who had complete resolution of her seizures and explosive behavior following a right temporal lobectomy.patients and methodswe searched the available literature from 1880 to 2013 for cases of epilepsy being used as a court defense for murder and collected information regarding the final sentencing outcomes. we selected 15 papers with a total of 50 homicides.resultswe describe the case of a 47-year - old woman with drug - resistant right temporal epilepsy who developed increasing emotional lability, outbursts of anger and escalating violent behavior culminating in a violent murder. the patient was imprisoned while awaiting trial. in the interim, she underwent a successful temporal lobectomy with full resolution of seizures, interictal rage and aggressive behaviors. after the surgery, her charges were downgraded and she was transferred to a psychiatric facility.conclusionthe aggressive behavior associated with epilepsy has been described in the literature for over a century. a link between epilepsy and aggression has been disproportionally emphasized. these patients share some common characteristics : they are usually young men with a long history of epilepsy and lower than average intelligence. the violent act is postictal, sudden - onset, more likely to occur after a cluster of seizures and is usually related with alcohol abuse.
evolutionary conservation is a powerful feature to identify functionally important regions of a genome. among the most highly conserved our starting point for the design of the svc tool was a collaboration in which we have searched for novel candidate sites subject to rna editing (1). as shown recently, exceptionally conserved exonic sequence guide us to editing sites in the transcript (2). for visual exploration and detailed analyses of alignments of evolutionarily related sequences versatile software tools are indispensable (3,4). the vista tools, for example, provide the user with the ability to visualize and explore the degree of conservation and the alignments along genomic regions. one main difficulty in detailed genome analyses of single loci is the variability of the scales. in particular, many mammalian genes consist of many exons of a few hundred bases or even fewer that are interrupted by large introns of up to several hundred thousand bases. so, if drawn to scale, the usual visualization of such a gene in a genome browser results in a line representing the genomic sequence with the exons appearing as thin vertical marks (5). the detailed investigation of sequence conservation in the genomic context, at splice sites for example, usually requires a lot of zooming in and out or moving along the genomic sequence. we introduce another approach that is tailored to investigating the conservation of the gene structure and regulatory sequences. we exploit the fact that distant homology can more easily be identified between protein - coding sequences. mapping the aligned sequences back onto their genomes pairwise nucleotide sequence alignments of the exons and introns are performed and fitted into the scaffold. our aim is to display evolutionary conservation in a more structured way. especially for researchers who are interested mainly in alternative gene products and their regulation, our tool helps to focus on relevant regions and to explore the genome more efficiently. as primary data source we use the following ensembl databases (6) : homo_sapiens_core_30_35c, mus_musculus _ core_30_33f, rattus_norwegicus_core_29_3f, fugu_rubripes_core_30_2e, danio_rerio_core_30_4c. the lists of orthologous genes for each pair of organisms are obtained from ensembl via ensmart (7) and are based on the dataset ensembl_compara_27_1. for the identification of conserved sequences between human and puffer fish exonic sequence we used the following sequence file : fugu_rubripes.fugu2.jul.dna_rm.scaffold.fa. we employ the blast family of alignment programs obtained from the ncbi website (8,9). as input the user enters a gene name or identifier, a text string for an advanced keyword search, or uploads a list of gene identifiers from file. the advanced search scans the available gene descriptions in the ensembl database and returns all genes that match the query. the first step in the analysis pipeline consists of the collection of all the necessary gene, transcript and exon identifiers. then the orthologs in each organism of interest are identified and the sequences and the annotation are downloaded from ensembl. for each pair of orthologous genes we compute pairwise local alignments between the exons to obtain the best reciprocal hits. in this procedure the exonic as well as the intronic sequences are aligned at the nucleotide level using blastn in order to determine evolutionarily highly conserved regions. then we perform a sequence search of the exons against the introns of the orthologous genes in the other species using tblastx again. the purpose of this step is the identification of alternative and conserved exons that were observed in one organism but not in the other. after some trials we decided to choose an e - value cutoff of 10 in all the blast analyses. we have also adapted our analysis pipeline to search for candidate rna editing sites. as an additional step, the length of the window and the required percentage identity can be specified by the user. for a human mouse comparison we recommend 80 bp window length and a cutoff of 95% identity. as supporting evidence we align the flanking introns to identify highly similar sequence regions, and we compare the exonic windows to the puffer fish sequence to illustrate the degree of conservation. to get an overview of complex gene structures we developed a new schematic visualization. it is important to emphasize that exons and introns are not drawn to scale, but all at fixed lengths. sequence similarities between introns and between introns and exons are displayed by blue and green boxes between the genes, respectively. a detailed description of the options, the visualization and the output has been included in the online help page. the main focus of our work is the investigation of alternative gene products and their differential regulation. to support this, the detection of the orthologs is not part of this work, and for this we rely on the compara database (10). we chose not to align the whole genomic sequence but to concentrate on the protein - coding part of orthologous genes first. if the structure of the two genes is found to be conserved we obtain a scaffold to map the orthologous exons and introns onto the underlying genomes. we know from single - gene studies that many genomic loci are not yet entirely resolved (11,12). rare splice variants or alternative transcripts are often not represented in the available databases. if a splice variant is not displayed as such in svc it still might be that there is already evidence for this transcript from large - scale efforts to investigate alternative splicing (1315). we believe that, if we do not display the exons and introns at their relative extensions, the visualization becomes more focused and conserved features stand out more clearly. this could stem from exons of alternative, but not yet observed, transcripts or regulatory sequences. consider, for instance, an intronic region that shows sequence similarity to an exon in another organism. if one wished to test this in the lab, one could download the sequence and input it into a primer design program. another interesting observation is that alternatively spliced exons often come with conserved regulatory regions in the flanking introns (16). if this is the case for an exon of interest, it should be apparent in the svc visualization. rna editing is a phenomenon whereby single nucleotides in the mrna are transformed adenosine to inosine, for example (1). high evolutionary sequence conservation can indicate potential rna editing sites (2). applying our analysis pipeline, exonic sites are considered candidates if the surrounding 80 bp window is extraordinarily similar between the human and the mouse lineage and if we find conservation in the flanking introns. a number of identified candidates are currently being tested in the lab. as a case study we present the results for human glutamate receptor 5 (glur5) (17,18). the encoding gene is edited at a specific site in exon 14 of the ensembl transcript enst00000309434, which corresponds to exon number 15 in figure 1. from the connecting red line and the blue bars in the vicinity it is obvious that the corresponding exons are conserved between human and mouse as well as many regions in the flanking introns. another interesting observation is that human glur5 exon 11 has not been observed in any mouse transcript in the ensembl database. however, the sequence similarity to an intronic region at the right genomic location strongly suggests that this variant also exists in mice. the effect of drawing the introns not to scale but to standard length is also seen in figure 1. the first intron is more then 200 kb long, which is more than 10 times the length of intron 4, for example. the conservation pattern of the first intron looks much denser, but this in fact might stem from its shear length. in conclusion, we believe that svc is a helpful tool to investigate sequence conservation in the context of structure in complex vertebrate genes. we have introduced a new visualization that displays the structural elements of a gene with standard size in order to emphasize evolutionarily conserved features. svc graphical output for comparison of human and mouse glutamate receptor 5 (fragment). green dashed lines indicate similarity between exonic and intronic sequences. in mouse exon 2,	we have developed a web application for the detailed analysis and visualization of evolutionary sequence conservation in complex vertebrate genes. given a pair of orthologous genes, the protein - coding sequences are aligned. when these sequences are mapped back onto their encoding exons in the genomes, a scaffold of the conserved gene structure naturally emerges. sequence similarity between exons and introns is analysed and embedded into the gene structure scaffold. the visualization on the svc server provides detailed information about evolutionarily conserved features of these genes. it further allows concise representation of complex splice patterns in the context of evolutionary conservation. a particular application of our tool arises from the fact that around mrna editing sites both exonic and intronic sequences are highly conserved. this aids in delineation of these sites. svc is available at.
a 54-year - old male presented with painful, swollen bilateral knees following a minor motorcycle accident where he fell from a motorcycle traveling at a relatively low speed. the accident occurred when a driver backed into the street in front of him riding a motorcycle in an alleyway. radiographs revealed schatzker type iv tibial plateau fractures involving medial condyles in both knees (fig. two years and four months before the accident, the patient had undergone an arthroscopic acl reconstruction using double - bundle technique with tibialis posterior allograft and medial collateral ligament (mcl) repair on his left knee at a hospital. in spite of our efforts to obtain more information, such as the type of the screw used and tunnel size created in the previous operation, additional information from that hospital was not available. he had the same surgery on his right knee about eight months ago at another hospital. an arthroscopic acl reconstruction was performed using single - bundle technique with a 25 mm femur endobutton (smith & nephew endoscopy, andover, ma, usa), a 9 mm in length and 25 mm in diameter bio - absorbable interference screw (conmed linvatec, largo, fl, usa), and tibialis posterior allograft. mcl femoral tensioning using an 11 mm bone staple was performed during the operation with removal of a spiked washer and a screw on the left tibia that had been implanted in the previous acl reconstruction. due to severe pain, knee joint stability and range of motion could not be assessed. computed tomography was performed to evaluate the fracture pattern in relation to the acl tibial tunnel, which demonstrated intra - articular involvement of split fractures on the bilateral medial tibial plateaus. 1). considering the unique nature of the fractures, we decided to attempt treatment and confirm the state of the previously reconstructed acl later by second - look arthroscopy. fracture reduction on the left side was achieved intraoperatively under fluoroscopic control, which was followed by fixation using a six - hole medial locking compression plate (lcp medial proximal tibial plate ; depuy synthes, oberdorf, switzerland) with a 6.5 cancellous screw and a washer (depuy synthes) for anatomical reduction of the intra - articular surface. subsequently, removal of the spiked washer and screw on the right tibia was done. in the same way, utilizing a four - hole medial locking compression plate (lcp medial proximal tibial plate) with a 6.5 cancellous screw and a washer (depuy synthes), fracture reduction and fixation on the right side was performed. the planned second - look arthroscopic examination for confirming the previously reconstructed acl state was achieved three weeks afterwards because the patient had suffered from fever and urinary tract infection since immediately after surgery. examination under general anesthesia revealed negative anterior lachmann test and pivot shift test with feeling of firm as the endpoint. however, we could not identify the degree of screw resorption or tunnel widening. physical therapy with continuous passive motion was started immediately postoperatively, and protected weight bearing was maintained for two postoperative months. at five months follow - up, follow - up radiographs of the patient showed callus formations with healed fractures in both tibias (fig. tibial plateau fractures presenting as a serious complication after acl reconstruction are rare, and only isolated cases have been reported. interestingly, in most of these cases, the tibial plateau fractures occurred through the tibial tunnel. authors of these reports suggested that the presence of the tibial tunnel could be the main factor in predisposing the patient to the fracture acting as a stress riser3,4,5,6,7). more recently, a case described by a. gobbi.6) had some interesting features. tibial plateau fractures had occurred after primary anatomic double - bundle acl reconstruction following minimal trauma of running backwards during a soccer game in their case. they suggested that two tunnels made in the proximal tibia might have caused a greater increase in local stresses around the tunnels with less bone available to withstand the applied load compared with a single tunnel. we could not find any studies that specifically measured bone strength of the tibia following tibial tunnel drilling or compared the mechanical effect of two tunnel creation to one tunnel creation. however, it has been documented that bone defects, such as screw holes, can concentrate stress and significantly decrease bone resistance to bending and torsional forces8,9). for instance, diaphyseal drill holes with a diameter greater than 20% of the bone have been shown to reduce bone strength by 55%-90%8,9,10). in our case, the fracture pattern had some unique and distinct features. although the patient fell from a motorcycle while riding it at a relative low speed, the fractures involved bilateral tibias and occurred through tibial tunnels that had been formed during previous acl reconstruction. furthermore, we could not identify other injuries to the articular structures in our patient. he had undergone an arthroscopic acl reconstruction using double - bundle technique on his left knee and the same surgery using single - bundle technique on his right knee. fortunately, the acls on both knees were found to be intact when examined by second - look arthroscopy. although we were not able to find differences between the effect of single and double - bundle reconstructions on this unique fracture in our patient, it was obvious that the fracture lines on the patient 's tibias extended into the tibial tunnel. our study suggests that a transosseous tibial tunnel alone can somehow act as a stress - riser that decreases strength at the level of proximal tibial metaphysis. we would like to emphasize that the surgeon must be very careful in tunnel placement and should not perform extensive bone removal during acl reconstruction. also, it is crucial to be aware of the possible occurrence of this, albeit rare, postoperative complication considering most cases required surgical fixation of the fractures.	tibial plateau fractures after arthroscopic anterior cruciate ligament (acl) reconstruction are rare, and only isolated cases have been reported. the authors describe a case of bilateral medial tibial plateau fracture following a minor motorcycle accident in a patient who underwent arthroscopic acl reconstruction in the past. two years and four months before the accident, the patient underwent an arthroscopically assisted acl reconstruction using double - bundle technique on his left knee at a hospital. he had the same surgery using single - bundle technique on his right knee about eight months ago at another hospital. the fractures in his both involved knees occurred through the tibial tunnel and required open reduction with internal fixation. at three weeks after fixation, a second - look arthroscopy revealed intact acls in both knees. at five months follow - up, he was able to walk without instability on physical examination. follow - up radiographs of the patient showed callus formations with healed fractures.
physical, sensory or cognitive limitations, health complaints such as fatigue or pain, psychological distress or medical requirements may hinder the performance of work tasks or even lead to work disability (lerner. chronically ill employees themselves state that, apart from work accommodations, they need acceptance of having a disease, coping strategies and support from their supervisor in order to stay at work (detaille. 2003). this suggests that vocational rehabilitation aimed at changing personal attitudes and improving personal skills, including communication skills, is needed. we developed a theory - driven group training programme for employees with chronic disease who experience work - related problems. the programme provided participants with knowledge, skills and insight regarding their values and needs, and we called it an empowerment programme (feste and anderson 1995). it focused on solving work - related problems and aimed at job retention and maintenance and an increase in job satisfaction. in this article, we present a process evaluation of eight training courses with a total of 64 participants. a systematic process evaluation can tell us whether the intervention was feasible and describe potential barriers to its implementation. furthermore, it may clarify how the intervention works and gives insight into factors that influence its effectiveness (swanborn 2004 ; baranowski and stables 2000 ; saunders. 2005 ; jonkers. the research questions for the process evaluation are : did the recruitment go as planned?was the target group reached?did participants follow the programme as it was intended?was the programme administered as intended?was the programme tailored to the group of participants?were participants satisfied with the program?was the programme perceived as effective?the medical ethics committee of the academic medical center in amsterdam approved of the study idea, but deemed ethical review unnecessary because they did not perceive the study to be medical research. the effectiveness of this intervention is studied with a randomized controlled trial (rct) design. did participants follow the programme as it was intended ? was the programme administered as intended ? was the programme tailored to the group of participants ? were participants satisfied with the program ? was the programme perceived as effective ? the training programme consisted of six three - hour sessions every 2 weeks and a seventh session 2 months after the sixth session., there was an actor present for practicing role - playing. to discuss personal problems and progress at more length, three individual consultations also took place, one at the beginning, one halfway through the training and one after the sixth session. the trainers were experienced in working with groups, had psychotherapeutic knowledge of the principles of rational emotive therapy (ret) and occupational psychology, and a basic understanding of chronic disease and its consequences. the pilot version was adapted based on the trainers experiences, the researcher s observations, a pre- and post - test questionnaire and interviews with the participants by telephone. the programme had a stepwise approach : first, exploring and clarifying work - related problems ; second, a focus on communication at work ; and third, developing and realizing solutions. work - related problems were clarified with the help of the quality of work model, which emphasizes the energizing or distressing influences of work tasks, social relationships at work, working conditions and work - home interference. a seventy - page course book accompanied the training, and the sessions consisted of four to seven components, including discussion of the homework and preparations for the next session. each session focused on one theme : exploration and clarification of practical and psychosocial work - related problems with the help of the model quality of work;insight into feelings and thoughts about having a chronic disease and how these may influence communication;communication in daily work situations : theory and role play with an actor;practical matters : the occupational physician, the employment expert, legislation and facilities for disabled employees;communication and assertiveness : theory and role play with an actor;a smart plan to solve problems ; andfollow - up : what works and what does not.participants were eligible for the intervention if they had a chronic physical disease, had a paid job, experienced problems at work and feared losing their job or job satisfaction. workers with predominant psychiatric conditions were excluded ; people with a chronic physical disease in combination with depression were not excluded. workers on long - term full sick leave that was expected to extend into the following months were excluded. the candidates themselves had to apply to the programme by telephone, also when they were referred by medical professionals. exploration and clarification of practical and psychosocial work - related problems with the help of the model quality of work; insight into feelings and thoughts about having a chronic disease and how these may influence communication ; communication in daily work situations : theory and role play with an actor ; practical matters : the occupational physician, the employment expert, legislation and facilities for disabled employees ; communication and assertiveness : theory and role play with an actor ; a smart plan to solve problems ; and follow - up : what works and what does not. a comprehensive description of the set - up and contents of the training programme, its development and theoretical framework is published elsewhere (varekamp. 2008), as well as a systematic review of interventions of the same kind (varekamp. the various elements of the process evaluation, their operationalisation and measurement are presented in table 1 (baranowski and stables 2000 ; jonkers. specific barriers or other observations could be noted on the form, in addition to the structured items. participants filled in a questionnaire at baseline, and again after 4, 8, 12 and 24 months. opinions about the importance of several themes, satisfaction with various methods used in the training and overall satisfaction were measured on a 110 scale. participant opinions of the trainers capacities were assessed with an adapted version of the satisfaction with occupation rehabilitation (2004), consisting of six subscales : expertise (2 items), advice given (4 items), friendly treatment (3), personal attention (3), usefulness of programme (2) and information about programme (2). 24 mn.recruitment of participantskind of agencies approached and ways and frequency of approachxreach of target populationcharacteristics of participantsxparticipation in programmefrequency and reasons for dropoutxxfrequency and reasons for not attending meetingsxextent to which the programme was implementedextent to which all components were administered as plannedxdose received or fit with the participants needs and capabilitiestrainers opinion on participants ability to cognitively and emotionally grasp the components of the programxtrainers opinion on commitment and goal attainment of components of the programxsatisfaction of participants with the training programme and the traineroverall opinion on programme (110)xxxopinion on various themes (110)xopinion on various methods (110)xxopinion on various skills of the trainerxeffectiveness as perceived by participantsopinion on effectiveness with regard to three phasesxopinion on effectiveness of consultation with supervisor with regard to problem solvingxpersonal goal and opinion on goal attainmentxxopinion on effectiveness with regard to attitudes, skills, work accommodations and situation at homexopinion on permanence of effectxnotes res. notes of the researcher about recruitment and on drop outs after contact with trainers and participantsproc. questionnaire filled in by participants in advance, after 4, 8, 12 and 24 months elements of process evaluation notes res. notes of the researcher about recruitment and on drop outs after contact with trainers and participants proc. questionnaire filled in by participants in advance, after 4, 8, 12 and 24 months the medical ethics committee of academic medical center in amsterdam approved the study design and deemed ethical review unnecessary due to the non - medical nature of the research. participants were recruited for the training programme and study from late spring 2006 to january 2008. participants were recruited via outpatient clinics, occupational health services, patient organizations, companies and so on. presentations were given to patient organizations, doctors, nurses and social workers in outpatient clinics, professionals at occupational health centres and to a national conference on chronic diseases. in addition, mailings were sent to several large companies and one patient organization sent a recruitment mailing to their members. advertisements were published in patient organization magazines, electronic newsletters and/or websites, in staff magazines at large companies and in magazines from an occupational health centre. about 3,500 paper leaflets were distributed via outpatient clinics, an occupational health centre and a patient information centre. it is difficult to assess the relative success of the various recruitment strategies, as we had no reports of the actions of medical professionals after hearing our presentations or reading about the project. presentations at outpatient clinics were seldom successful ; when they were, it was due to interested nurses who advised patients to contact us. contacts with companies were successful if they paid attention to the project in the staff magazine. table 2 presents figures on the sources of information about the project that the participants encountered (control group included). recruitment took considerably more time than expected ; we estimate roughly that it took 810 months of full - time effort for one person to complete. one of the reasons for recruitment problems, according to some professionals of outpatient clinics and occupational health services, was that these professionals felt restrained from referring persons to the project because of the possibility of randomization to the control group (personal communications to iv). another possible reason was that occupational physicians were afraid to lose patients when they referred them to the training programme (personal communication to fvd).table 2source of information about the training programme for the participants of the study (training participants and controls)sources of information%patient organization : magazine, presentation, website, mailing34companies : house organ or supervisor21occupational health service20outpatient clinic13conference on chronic diseases : magazine or presentation7other10more than one answer was possible (n = 122) source of information about the training programme for the participants of the study (training participants and controls) more than one answer was possible (n = 122) the personal, work and medical characteristics of the participants of the programme are presented in table 3. mean age was 46 years, most participants were women, and highly educated people were over - represented. mean disease duration was 10 years and almost half had more than one chronic disease. fourteen per cent had categories of diseases, such as renal failure, poor eyesight, hiv and chronic fatigue syndrome. the great majority of the participants worked in the commercial or non - commercial service sector, for 30 h weekly, on average.table 3personal, medical and work characteristics of the training programme participants (n = 64)mean (sd) or % age46.1 (8.8)women 83living alone (not with partner, children or parents) 33education lower3 middle36 higher61chronic disease icd classification 1. diseases of the musculoskeletal system and connective tissue28 2. diseases of the nervous system20 3. diseases not otherwise specified14disease duration in years10.2 (9.6)an additional chronic disease % (co morbidity)48branch of industry agriculture and fishing0 industry and building industry0 commercial services27 non - commercial services73appointment hours per week30 (8.6) personal, medical and work characteristics of the training programme participants (n = 64) from november 2006 to march 2008, eight training courses took place, including three trainers and 64 participants in total. three participants withdrew halfway, one due to medical treatment that interfered and two because they were not satisfied with the programme. overall, there were 55 missed sessions, but in the majority of cases, participants called to say they were unable to attend. forty - eight per cent participated in the training programme during working hours, 31% used days off and 20% combined these. generally, all the planned components of the sessions were discussed, although some only briefly because of lack of time. quality of work, which emphasizes energizing and fatiguing or distressing factors, took too much time. another trainer observed that the participants preferred to have time to exchange experiences with each other rather than listen to theoretical explanations, which they felt they could read in the course book. when discussing homework, it was often not possible to discuss each participant s work. when discussing work - related problems in the group using the quality of work model, only one instead of the planned two participants was often discussed. it was often impossible to have all participants practice role - playing in one session. one of the reasons was that discussing role - playing afterwards took a lot of time. according to the trainers, participants had rarely cognitive difficulties with understanding the various components of the training. one thing that some people found difficult to grasp was reflection on their work in terms of subjective perceptions instead of objective facts. slight or more severe emotional difficulties were met when discussing the consequences of having a chronic disease, feelings and thoughts on having a chronic disease and practical matters. one homework exercise presented difficulties for several participants ; they were asked twice in the course of the programme to arrange a consultation with their supervisor. the first session was intended to be a discussion of how the supervisor judged their work performance, the second to discuss work - related problems and solutions. pointless, because of their supervisor s attitude, or they wanted to practice such a consultation beforehand in order to be prepared (see also last paragraph of the results section). the participants were asked to score how important the sessions themes were for them on a 110 scale (table 4). the themes insight into feelings and thoughts about having a chronic disease (session 2) and communication and assertiveness (sessions 3 and 5), were valued highest, with a mean score of 8.0. the theme exploration of practical and psychosocial work - related problems, which included the explanation of the model quality of work (session 1), scored 7.6. practical matters ; the occupational physician, the employment expert, legislation and facilities for disabled employees was evaluated as lowest, with a mean score of 7.0, and a high standard deviation. the training programme as a whole was evaluated with a mean score of 8.1 immediately after completion ; this dropped 0.2 points 8 months later and 0.3 points 24 months later.table 4opinion of the training programme participants on the overall training programme, significance of themes, course book and methods (n = 64)rating (110) mean (sd)overall training programme opinion after 4 months8.1 (1.1) opinion after 12 months7.9 (1.1) opinion after 24 months7.8 (1.3)themes exploration and clarification of practical and psychosocial problems ; quality of work model (session 1)7.6 (1.7) insight into feelings and thoughts about having a chronic disease (session 2)8.0 (1.4) communication in daily work situations and standing up for oneself (sessions 3 and 5)8.0 (1.4) practical matters ; the occupational physician, the employment expert, legislation and facilities for disabled employees (session 4)7.0 (2.0) a smart plan to solve problems (session 6)7.5 (1.7) the course book7.9 (1.2)methods theory explanation7.2 (1.6) exchanging experiences8.3 (1.4) filling in and discussing quality of work model7.5 (1.2) discussing others quality of work model7.7 (1.5) role play with actor8.1 (1.6) questioning occupational physician and employment expert7.1 (1.7) having a consultation with the supervisor (homework)7.2 (1.9) having a consultation with an occupational physician (homework)6.7 (2.2) individual consultation with trainer halfway7.9 (1.4) individual consultation with trainer at the end7.9 (1.2)including opinion of three persons that dropped out halfwaylow response, n = 57low response, n = 49 opinion of the training programme participants on the overall training programme, significance of themes, course book and methods (n = 64) including opinion of three persons that dropped out halfway eighty - six per cent of the participants always read the short introductions in the course book to prepare for the group sessions, whereas 95% had read the entire course book at the end of the training course. most valued were the chapters on communication and assertiveness, and on feelings and thoughts about having a chronic disease. lowest valued, with the highest standard deviation, was the chapter on legislation and work accommodations. a variety of methods was used in the training programme : theoretical explanation, exchange of experiences, role - playing, and homework, such as completing the model quality of work, or arranging a consultation with a supervisor and occupational physician. role - playing and seeing and discussing others role - playing was also highly appreciated, as were the individual consultations with the trainers. non - response on these two questionnaire items was high, 7 and 15, respectively, which indicates that these arrangements not always took place. the expertise of the trainers was overall judged very positively (mean score 68 on a 1680 scale), and the advice given by the trainers was felt to be helpful. the participants felt well - treated and felt that they received personal attention during the programme. they considered introductory information to be sufficient, although this could have been better for a minority. satisfaction with the trainers was not lower in the three groups in which the trainers acted for the first time, when compared to the five groups for which trainers were more experienced. the training programme used a stepwise approach : first exploring and clarifying work - related problems, then focusing on communication at work, and finally working on developing and realizing solutions. eight months after the start, 84% of the participants found that the first phase worked well, while 69% found that the second phase and 65% found that the third phase worked well (table 5).table 5success of three steps of the training programme, as perceived by the training programme participants after 8 months (n = 64)not successful at all % a little successful % amply successful % completely successful % 1clarifying bottlenecks (model quality of work)016.455.727.92discussing bottlenecks at work3.327.945.923.03developing and realizing solutions6.728.345.020.0 success of three steps of the training programme, as perceived by the training programme participants after 8 months (n = 64) the majority of the participants, 53 persons, had, as part of the training, discussed matters with their supervisor in order to find a solution for work - related problems. fifty - three per cent of them felt this contributed a great deal to solving problems, 40% said that it contributed somewhat, whereas 6% said that it did not contribute and 2% felt these discussions had worked negatively. table 6 presents the effects of the programme on various aspects of working with a chronic disease, as perceived at 12 months follow - up. the participants noticed positive effects most often with regard to how they experienced and dealt with disease and work. this was followed by how matters at work were discussed and how they dealt with the supervisor and colleagues. after 24 months, 79% perceived a lasting effect of the training programme, 10% perceived an effect that had faded away, 3% were not sure whether it had lasted, and 8% perceived none or only a limited effect.table 6effect of training programme on work as perceived by the training programme participants after 12 months (n = 64)effect training on large negative effectsmall negative effectno effectsmall positive effectlarge positive effecthow i experience my disease and my work03.311.748.336.7how i deal with my disease and my work03.38.345.043.3how i discuss matters at work01.726.741.730.0how i deal with my supervisor0023.351.725.0how i deal with my colleagues0028.356.715.0how my supervisor deals with me0038.343.318.3how my colleagues deal with me0041.738.320.0the situation at home0043.330.026.7accommodations of my workplace or work tasks1.71.753.326.716.7 effect of training programme on work as perceived by the training programme participants after 12 months (n = 64) in the course of the programme, the participants formulated a plan of action with one or more personal goals. these goals related to work - home interference (78%), feelings and thoughts about having a chronic disease (59%), communication at the workplace (44%), leisure time (33%), work accommodations (29%) or other topics (18%). one year after the start of the programme, 6 per cent felt that they had not reached the goal that they set in the course of the programme, 38% reached it participants were recruited for the training programme and study from late spring 2006 to january 2008. participants were recruited via outpatient clinics, occupational health services, patient organizations, companies and so on. presentations were given to patient organizations, doctors, nurses and social workers in outpatient clinics, professionals at occupational health centres and to a national conference on chronic diseases. in addition, mailings were sent to several large companies and one patient organization sent a recruitment mailing to their members. advertisements were published in patient organization magazines, electronic newsletters and/or websites, in staff magazines at large companies and in magazines from an occupational health centre. about 3,500 paper leaflets were distributed via outpatient clinics, an occupational health centre and a patient information centre. it is difficult to assess the relative success of the various recruitment strategies, as we had no reports of the actions of medical professionals after hearing our presentations or reading about the project. presentations at outpatient clinics were seldom successful ; when they were, it was due to interested nurses who advised patients to contact us. contacts with companies were successful if they paid attention to the project in the staff magazine. table 2 presents figures on the sources of information about the project that the participants encountered (control group included). recruitment took considerably more time than expected ; we estimate roughly that it took 810 months of full - time effort for one person to complete. one of the reasons for recruitment problems, according to some professionals of outpatient clinics and occupational health services, was that these professionals felt restrained from referring persons to the project because of the possibility of randomization to the control group (personal communications to iv). another possible reason was that occupational physicians were afraid to lose patients when they referred them to the training programme (personal communication to fvd).table 2source of information about the training programme for the participants of the study (training participants and controls)sources of information%patient organization : magazine, presentation, website, mailing34companies : house organ or supervisor21occupational health service20outpatient clinic13conference on chronic diseases : magazine or presentation7other10more than one answer was possible (n = 122) source of information about the training programme for the participants of the study (training participants and controls) more than one answer was possible (n = 122) the personal, work and medical characteristics of the participants of the programme are presented in table 3. mean age was 46 years, most participants were women, and highly educated people were over - represented. mean disease duration was 10 years and almost half had more than one chronic disease. fourteen per cent had categories of diseases, such as renal failure, poor eyesight, hiv and chronic fatigue syndrome. the great majority of the participants worked in the commercial or non - commercial service sector, for 30 h weekly, on average.table 3personal, medical and work characteristics of the training programme participants (n = 64)mean (sd) or % age46.1 (8.8)women 83living alone (not with partner, children or parents) 33education lower3 middle36 higher61chronic disease icd classification 1. diseases not otherwise specified14disease duration in years10.2 (9.6)an additional chronic disease % (co morbidity)48branch of industry agriculture and fishing0 industry and building industry0 commercial services27 non - commercial services73appointment hours per week30 (8.6) personal, medical and work characteristics of the training programme participants (n = 64) from november 2006 to march 2008, eight training courses took place, including three trainers and 64 participants in total. three participants withdrew halfway, one due to medical treatment that interfered and two because they were not satisfied with the programme. overall, there were 55 missed sessions, but in the majority of cases, participants called to say they were unable to attend. forty - eight per cent participated in the training programme during working hours, 31% used days off and 20% combined these. generally, all the planned components of the sessions were discussed, although some only briefly because of lack of time. one trainer mentioned that explaining the model quality of work, which emphasizes energizing and fatiguing or distressing factors, took too much time. another trainer observed that the participants preferred to have time to exchange experiences with each other rather than listen to theoretical explanations, which they felt they could read in the course book. when discussing homework, it was often not possible to discuss each participant s work. when discussing work - related problems in the group using the quality of work model, only one instead of the planned two participants was often discussed it was often impossible to have all participants practice role - playing in one session. one of the reasons was that discussing role - playing afterwards took a lot of time. according to the trainers, participants had rarely cognitive difficulties with understanding the various components of the training. one thing that some people found difficult to grasp was reflection on their work in terms of subjective perceptions instead of objective facts. slight or more severe emotional difficulties were met when discussing the consequences of having a chronic disease, feelings and thoughts on having a chronic disease and practical matters. one homework exercise presented difficulties for several participants ; they were asked twice in the course of the programme to arrange a consultation with their supervisor. the first session was intended to be a discussion of how the supervisor judged their work performance, the second to discuss work - related problems and solutions. pointless, because of their supervisor s attitude, or they wanted to practice such a consultation beforehand in order to be prepared (see also last paragraph of the results section). the participants were asked to score how important the sessions themes were for them on a 110 scale (table 4). the themes insight into feelings and thoughts about having a chronic disease (session 2) and communication and assertiveness (sessions 3 and 5), were valued highest, with a mean score of 8.0. the theme exploration of practical and psychosocial work - related problems, which included the explanation of the model quality of work (session 1), scored 7.6. smart personal plan, scored 7.5. practical matters ; the occupational physician, the employment expert, legislation and facilities for disabled employees was evaluated as lowest, with a mean score of 7.0, and a high standard deviation. the training programme as a whole was evaluated with a mean score of 8.1 immediately after completion ; this dropped 0.2 points 8 months later and 0.3 points 24 months later.table 4opinion of the training programme participants on the overall training programme, significance of themes, course book and methods (n = 64)rating (110) mean (sd)overall training programme opinion after 4 months8.1 (1.1) opinion after 12 months7.9 (1.1) opinion after 24 months7.8 (1.3)themes exploration and clarification of practical and psychosocial problems ; quality of work model (session 1)7.6 (1.7) insight into feelings and thoughts about having a chronic disease (session 2)8.0 (1.4) communication in daily work situations and standing up for oneself (sessions 3 and 5)8.0 (1.4) practical matters ; the occupational physician, the employment expert, legislation and facilities for disabled employees (session 4)7.0 (2.0) a smart plan to solve problems (session 6)7.5 (1.7) the course book7.9 (1.2)methods theory explanation7.2 (1.6) exchanging experiences8.3 (1.4) filling in and discussing quality of work model7.5 (1.2) discussing others quality of work model7.7 (1.5) role play with actor8.1 (1.6) questioning occupational physician and employment expert7.1 (1.7) having a consultation with the supervisor (homework)7.2 (1.9) having a consultation with an occupational physician (homework)6.7 (2.2) individual consultation with trainer halfway7.9 (1.4) individual consultation with trainer at the end7.9 (1.2)including opinion of three persons that dropped out halfwaylow response, n = 57low response, n = 49 opinion of the training programme participants on the overall training programme, significance of themes, course book and methods (n = 64) including opinion of three persons that dropped out halfway eighty - six per cent of the participants always read the short introductions in the course book to prepare for the group sessions, whereas 95% had read the entire course book at the end of the training course. most valued were the chapters on communication and assertiveness, and on feelings and thoughts about having a chronic disease. lowest valued, with the highest standard deviation, was the chapter on legislation and work accommodations. a variety of methods was used in the training programme : theoretical explanation, exchange of experiences, role - playing, and homework, such as completing the model quality of work, or arranging a consultation with a supervisor and occupational physician. role - playing and seeing and discussing others role - playing was also highly appreciated, as were the individual consultations with the trainers. non - response on these two questionnaire items was high, 7 and 15, respectively, which indicates that these arrangements not always took place. the expertise of the trainers was overall judged very positively (mean score 68 on a 1680 scale), and the advice given by the trainers was felt to be helpful. the participants felt well - treated and felt that they received personal attention during the programme. they considered introductory information to be sufficient, although this could have been better for a minority. satisfaction with the trainers was not lower in the three groups in which the trainers acted for the first time, when compared to the five groups for which trainers were more experienced. the training programme used a stepwise approach : first exploring and clarifying work - related problems, then focusing on communication at work, and finally working on developing and realizing solutions. eight months after the start, 84% of the participants found that the first phase worked well, while 69% found that the second phase and 65% found that the third phase worked well (table 5).table 5success of three steps of the training programme, as perceived by the training programme participants after 8 months (n = 64)not successful at all % a little successful % amply successful % completely successful % 1clarifying bottlenecks (model quality of work)016.455.727.92discussing bottlenecks at work3.327.945.923.03developing and realizing solutions6.728.345.020.0 success of three steps of the training programme, as perceived by the training programme participants after 8 months (n = 64) the majority of the participants, 53 persons, had, as part of the training, discussed matters with their supervisor in order to find a solution for work - related problems. fifty - three per cent of them felt this contributed a great deal to solving problems, 40% said that it contributed somewhat, whereas 6% said that it did not contribute and 2% felt these discussions had worked negatively. table 6 presents the effects of the programme on various aspects of working with a chronic disease, as perceived at 12 months follow - up. the participants noticed positive effects most often with regard to how they experienced and dealt with disease and work. this was followed by how matters at work were discussed and how they dealt with the supervisor and colleagues. after 24 months, 79% perceived a lasting effect of the training programme, 10% perceived an effect that had faded away, 3% were not sure whether it had lasted, and 8% perceived none or only a limited effect.table 6effect of training programme on work as perceived by the training programme participants after 12 months (n = 64)effect training on large negative effectsmall negative effectno effectsmall positive effectlarge positive effecthow i experience my disease and my work03.311.748.336.7how i deal with my disease and my work03.38.345.043.3how i discuss matters at work01.726.741.730.0how i deal with my supervisor0023.351.725.0how i deal with my colleagues0028.356.715.0how my supervisor deals with me0038.343.318.3how my colleagues deal with me0041.738.320.0the situation at home0043.330.026.7accommodations of my workplace or work tasks1.71.753.326.716.7 effect of training programme on work as perceived by the training programme participants after 12 months (n = 64) in the course of the programme, the participants formulated a plan of action with one or more personal goals. these goals related to work - home interference (78%), feelings and thoughts about having a chronic disease (59%), communication at the workplace (44%), leisure time (33%), work accommodations (29%) or other topics (18%). one year after the start of the programme, 6 per cent felt that they had not reached the goal that they set in the course of the programme, 38% reached it a little, 36% reached it amply and 20% completely. the recruitment for this intervention yielded enough participants but was time - consuming. we enrolled a sample in which higher - educated women working in the service sector are over - represented. the majority of the participants were satisfied with the programme, and only a few dropouts were noted. for the most part, the programme was administered as planned, although some components took too much time. quality of work models and/or homework were not always discussed and not everybody had the opportunity to do role - playing as planned. the participants had no or only minor difficulties with understanding the materials discussed, but were more often emotionally upset, particularly when consequences of disease or feelings and thoughts were discussed, or during role - playing. generally, the participants completed their homework, but when asked to organize a consultation with their supervisor, many hesitated to do so ; a minority did not complete this assignment. among those who completed these consultations, the perceived effectiveness of the training programme was highest in how it shaped participants personal attitudes and lowest in matters that are more practical. the forms were completed by the trainers themselves and were likely correct as far as objective facts are concerned. the validity of some answers may be questionable, however, as trainers gave subjective judgments on whether the programme s components were tailored to the participants. furthermore, they give an overall response for the whole group, rather than individuals. however, the forms are of special value when the three trainers showed consensus on less positive aspects or when they noted barriers. for instance, there was consensus on the lack of time for some components, all three observed that some components are likely to raise emotional difficulties and all noted that consultations with the supervisor are often met with resistance. another weakness of this study is that we do not know what proportion of the target group was reached., information about the project was disseminated through various channels and potential participants had to contact researchers to participate. the consequence is that we do not know how many employees who experience serious work - related problems were not interested in our programme or did not enrol for other reasons. we do know that the group we reached was a selected group in terms of socio - demographic characteristics. we know that our programme is implementable, although we have to keep in mind that the majority in this study was highly educated. at some sessions the time to discuss personal experiences is not an option. because participants have three individual consultations with a trainer, and because lack of personal attention appeared not to be a problem, it is presumed to be better to accept this programme design but to indicate at the beginning of the sessions that not everyone may receive equal attention in all components of the programme. we found in the pilot phase that participants with a variety of chronic physical diseases could be put together in the same group. people experience the general aspects of chronic diseases as more important than the disease specifics. the trainers observed that many of the components raised emotional feelings, and it is interesting to note that these components were often highly valued. apparently, many participants realized that going through a phase of mourning and learning to accept having a chronic disease is difficult, but it assists in learning to cope. this brings us to our assumption that participants needed to pass through three phases : clarifying, communicating and solving problems. we understood the earlier phases as necessary to accomplish the last essential phase and understood this final phase implicitly as organizing work accommodations. however, it appears that organizing work accommodations may be the primary problem for some persons ; for others, the main problem is in the earlier phases of accepting the chronic disease and learning to communicate about it and/or in maintaining an enjoyable life outside work. another remarkable phenomenon was that many participants showed resistance to a consultation with their supervisor, but in the end, the majority felt that it helped in solving problems. 2003 ; post. 2005), that a good relationship with the supervisor is very important. for future use, it is important to discuss with the participants that this consultation often is successful in the end, the more so when it is practiced beforehand with role - playing. one reason is the randomization procedure, but the fact that the majority of the participants needed to use days of might have played a part as well. recruitment through professionals in outpatient clinics was problematic compared to recruitment with the help of patient organizations. disseminating this kind of programme through normal health care channels appears not to work ; lack of interest in work - related problems among many health care professionals is a primary reason (van weel. physicians and nurses should be encouraged in the course of their education and by post - graduate courses to pay attention to the working life of their patients ; there is little chance for referral of patients to vocational rehabilitation programmes without conversations about these matters. it is positive that practice guidelines for physicians increasingly pay attention to work - related problems of patients. maybe incentives like co - authorship of a scientific article may help to raise interest in this kind of research and development projects. in addition, focus on specialized nurses as collaborating partners may prove beneficial, as these professionals concentrate more on the social consequences of chronic disease. working together more intensively with outpatient clinics in the future would have the added advantage of contact with a more diverse group of potential participants. heavy manual work and low education are prognostic factors for work disability among employees with chronic disease (detaille. we do not know why we had only a few participants working in industry, and fewer men and less - educated people than expected. research into whether similar communication - focused programmes are attuned to the culture and working conditions outside of the service sector is necessary. we need to know why less - educated people seldom applied for the study, as well as whether and how more men can be convinced to participate in empowerment programmes, which focus on sometimes emotionally disturbing topics. several vocational rehabilitation approaches aimed at job retention for people with chronic or longstanding disease have recently been developed, varying widely in approach. multidisciplinary rehabilitation has been developed for patients with rheumatoid arthritis (de buck. this is an outpatient clinic - based intervention where medical and psychosocial specialists combine their expertise in advising the patient and his or her occupational physician on aspects of work. this focuses on the employee and supervisor and aims to improve their ability to solve work - related problems with the help of a mediator. more research is needed on what kind of rehabilitation method best suits a particular employee and circumstances. the extent to which employers are willing to accommodate the workplace to employees with a chronic disease or handicap also needs research. we may conclude that empowering employees with a chronic disease with help of a group training programme is feasible and highly valued. for that reason, it should be offered in occupational health care or other health care settings.	purposeemployees with a chronic disease may experience work - related problems that contribute to the risk of job loss. we developed a group - based intervention programme aimed at clarifying problems, making these a subject of discussion at work, and realizing solutions. this process evaluation investigates the intervention s feasibility and the satisfaction of 64 participants in eight groups.methodsdata were collected through process evaluation forms and self - report questionnaires.resultsthe recruitment of participants was time - consuming. highly educated women working in the service sector were overrepresented. the programme was administered as planned, although components were sometimes only discussed briefly, due to lack of time. satisfaction with the overall programme among participants was high ; it was perceived as effective and there were only three dropouts. in particular, the focus on feelings and thoughts about having a chronic disease was highly valued, as were the exchange of experiences and role - playing directed at more assertive communication.conclusionsa vocational rehabilitation programme aimed at job retention is feasible and is perceived to be effective. such a programme should address psychosocial aspects of working with a chronic disease beside practical problems. the recruitment of participants is time - consuming. cooperation with outpatient clinics is necessary in order to reach all groups of employees with a chronic disease that might benefit from job retention programmes. trial registration : isrctn77240155
t lymphocytes predominantly express delayed rectifier k - channels (kv1.3) in their plasma membranes [13 ]. using selective channel inhibitors, patch - clamp studies revealed that the channels generate the k - diffusion potential across the plasma membranes and play crucial roles in facilitating calcium influx necessary to trigger the lymphocyte activation and proliferation [36 ]. previous studies demonstrated the involvement of inflammatory leukocytes, such as t lymphocytes, macrophages, and mast cells, in the pathogenesis of renal diseases, such as glomerulonephritis, chronic kidney disease (ckd), or tubulointerstitial fibrosis [711 ]. since lymphocytes are actually activated and serum cytokine levels are known to be elevated in patients with advanced - stage renal diseases [13, 14 ], kv1.3-channels expressed in lymphocytes would contribute to the progression of the diseases. regarding the molecular mechanisms by which lymphocytes are activated, the rise in the intracellular calcium concentration stimulates the phosphatase calcineurin activity, which then dephosphorylates nuclear factor of activated t cells (nfat), enabling it to accumulate in the nucleus and bind to the promoter of the gene encoding interleukin 2 (il-2) [6, 15 ] (figure 1). therefore, pharmacological targeting of calcineurin has been the main mechanism by which drugs, such as cyclosporine and tacrolimus, exert their immunosuppressive effects. however, recent studies have also revealed that selective inhibition of lymphocyte kv1.3-channels also represses lymphocyte activity and thus suppresses cellular immunity. recent patch - clamp studies, including ours, have shown that commonly used drugs, such as calcium channel blockers (ccbs) [18, 19 ], macrolide antibiotics, and hmg - coa reductase inhibitors, effectively suppress the kv1.3-channel currents in lymphocytes [20, 21 ]. such studies suggested the therapeutic efficacy of these drugs for the treatment of renal diseases, in which chronic inflammation or the overstimulation of cellular immunity is responsible for the pathogenesis. by summarizing the previous and recent findings obtained from studies in the relevant fields, this review provides an overview of the pathological roles of lymphocyte kv1.3-channels in renal diseases. based on the recent in vitro and in vivo evidence that revealed the pharmacological properties of the channels, this review also focuses on the novel therapeutic implications of targeting the channels for the treatment of renal diseases. previous studies have described several laboratory models of renal diseases, including ligation of the renal artery branches or unilateral ureter [23, 24 ], ablation of renal mass by surgery [25, 26 ], toxic nephritis [27, 28 ], and immunologically induced nephritis [29, 30 ]. in the development of glomerulonephritis, inflammatory leukocytes are initially recruited from the bone marrow and infiltrate into the renal interstitium to produce proinflammatory cytokines. therefore, the kidneys from rat models with toxic or immunologically induced nephritis were characterized by the massive infiltration of t - lymphocytes or macrophages [9, 2730 ]. on the other hand, in rat models with 5/6 nephrectomy (subtotal nephrectomy), the injured kidneys were mainly characterized by severe glomerulosclerosis, which was primarily caused by the renal hemodynamic changes, such as the increased glomerular pressure and the protein overload [31, 32 ]. however, with the increase in the serum creatinine, the kidneys from these subtotally nephrectomized rats were additionally characterized by diffuse interstitial fibrosis with the involvement of leukocyte infiltration [7, 8, 33 ]. in rats with subtotal nephrectomy followed by longer recovery periods, serum creatinine and blood urea nitrogen levels were markedly elevated, indicating advanced chronic renal failure (crf) [11, 34 ]. in crf rat kidneys with 8-week recovery period, the cortical interstitium was expanded with fibroedema (figure 2(a)(b) versus figure 2(a)(a)) and there was some infiltration of small round cells among spindle - shaped fibroblasts (figure 2(a)(e) versus figure 2(a)(d)). at 14 weeks, in addition to diffuse fibrosis in the cortical and medullary interstitium (figure 2(a)(c)), the numbers of small round cells were dramatically increased in the cortical interstitium (figure 2(a)(f)). since the cortical expression of cd3 and ed-1, surface markers for t lymphocytes and macrophages, was markedly elevated, they were regarded as inflammatory leukocytes, such as t lymphocytes and macrophages. by immunohistochemistry, the cd3- or ed-1-positive small round cells were actually costained with ki-67, a marker of cellular proliferation (figures 2(b)(a) and 2(b)(b)). the findings indicated that t lymphocytes and macrophages were proliferating prominently within the cortical interstitium of advanced crf rat kidneys. recently, liu. demonstrated that cd4 t - lymphocytes, especially th2 cells, contributed to the progression of renal fibrosis in a rat model of unilateral ureteral obstruction (uuo). in the development of the tubulointerstitial fibrosis in crf rat kidneys, previous studies demonstrated that the inflammatory leukocytes were initially recruited from the bone marrow and infiltrated into the renal interstitium to trigger the proliferation of fibroblasts. in this context, using the rat model of advanced crf, the recent studies further demonstrated that the infiltrated leukocytes proliferated in situ in the cortical interstitium, and thus dramatically increased their numbers [11, 34 ]. a variety of ion channels that are expressed in t lymphocytes include voltage - dependent k - channels (kv), ca - activated k - channels (kca), ca release - activated ca - channels (crac), mg - inhibited ca - permeable current (mic) channels, and swelling - activated cl channels (clswell). among them, human t lymphocytes predominantly express kv1.3-channels in their plasma membranes [1, 2 ]. in patch - clamp studies using thymus - derived murine lymphocytes (thymocytes), stepwise changes in the membrane potential evoked membrane currents characteristic to kv - channels [18, 20, 21, 38, 39 ]. since margatoxin, a selective inhibitor of kv1.3-channels, almost totally abolished the currents in lymphocytes (figure 3(a)), these membrane currents were identified as kv1.3-channel currents. the physiological roles of kv1.3-channels in lymphocytes have been identified by several in vitro studies. patch - clamp studies revealed that these channels generate the k - diffusion potential across the plasma membranes and thus play a role in regulating the resting membrane potential and controlling the cell volume [40, 41 ]. the opening of kv1.3-channels also brings about the membrane hyperpolarization and generates a driving force for the ca influx [4, 5, 37 ]. consequently, it stimulates the following ca signaling pathway necessary to trigger the lymphocyte activation and proliferation. peripheral lymphocytes are activated and the serum cytokine levels are known to be elevated in patients with end - stage renal disease [1214 ]. based on a previous patch - clamp study, the conductance of voltage - dependent k - channels in lymphocytes was increased in such patients and the activity of the channels was strongly associated with the severity of renal dysfunction. in experimental animal models of renal diseases, such as renal allograft rejection and glomerulonephritis, immunosuppression by the blockade of lymphocyte kv1.3-channels actually prevented or ameliorated the progression of the diseases. by using selective kv1.3-channel inhibitors, such as shk and psora-4, therapeutically [9, 10 ], these studies demonstrated the contribution of the channels to the pathogenesis of renal diseases. in previous studies, the overexpression of kv1.3-channnels was demonstrated in cells under various pathologic conditions, such as cancer [43, 44 ], ischemic heart disease, and autoimmune disorders [46, 47 ]. in autoimmune disorders, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, the inhibition of the kv1.3-channel modulated the calcium influx patterns in t lymphocytes and thus exerted immunosuppressive effects [4850 ]. recently, using a rat model with advanced crf, we demonstrated for the first time that kv1.3-channels were overexpressed in proliferating leukocytes within fibrotic kidneys. in our study, margatoxin, one of the most potent inhibitors of kv1.3-channels which almost totally inhibits the channel currents in lymphocytes (figure 3(a)), decreased the number of proliferating lymphocytes and ameliorated the progression of renal fibrosis. these findings indicated that the overexpression of kv1.3-channels in kidney lymphocytes promoted their cellular proliferation in advanced ckd. as previously demonstrated in cancer cells, the membrane hyperpolarization induced by the overexpression of the channels is thought to trigger the cell cycle progression in lymphocytes [52, 53 ]. since the cytokines produced by lymphocytes stimulate the activity of fibroblasts to produce collagen, the proliferating lymphocytes in the interstitium would promote the progression of renal fibrosis in advanced crf, contributing to the rapid deterioration of renal function. in addition to their cardiovascular effects on hypertension and ischemic heart disease, ccbs are also known to exert immunosuppressive properties in humans [55, 56 ]. according to several in vitro studies, ccbs, including nifedipine, verapamil, and diltiazem, recently, using human peripheral leukocytes, matsumori. demonstrated that ccbs also suppress the production of proinflammatory cytokines, such as il-1, tumor necrosis factor (tnf-), and interferon (ifn-) [59, 60 ]. according to their studies, 1,4-dihydropyridine (dhp) ccbs, including nifedipine, amlodipine, and benidipine, which are highly lipophilic compared to the other types of ccbs [61, 62 ], exert relatively stronger immunomodulatory effects. among them, benidipine is one of the most lipophilic and longest acting dhp ccbs [63, 64 ]. in our patch - clamp study using murine thymocytes, benidipine almost totally and irreversibly inhibited the kv1.3-channel currents (figure 3(b)), indicating its usefulness as a potent kv1.3-channel inhibitor. although the effects of benidipine on cytokine production have not yet been directly examined [59, 60 ], the marked inhibition of the channel currents by this drug strongly suggested its higher immunosuppressive potency than the other ccbs. moreover, the persistent effect of benidipine in decreasing the channel currents may suggest its longer duration of action in thymocytes, as previously demonstrated in cardiomyocytes and isolated coronary arteries. in our recent study, the overexpression of kv1.3-channels in kidney lymphocytes was strongly associated with their in situ proliferation in advanced crf rat kidneys, and the channel inhibition by margatoxin actually decreased the number of infiltrating leukocytes and slowed the progression of renal fibrosis. based on one of our patch - clamp studies, since benidipine was also highly potent as a kv1.3-channel inhibitor, the therapeutic effects of this drug on ckd were examined in our most recent study. previous studies have shown the therapeutic efficacy of antihypertensive drugs, such as ccbs and angiotensin converting enzyme inhibitors (aceis), for the prevention of glomerulosclerosis, since these drugs hemodynamically ameliorate glomerular hypertension. however, it is not well known how these pharmacological approaches slow the progression of tubulointerstitial fibrosis independently of their effects on glomerulosclerosis. in advanced crf rat kidneys with benidipine treatment, the size of the cortical interstitium, which included the areas of fibrosis, edema, and the inflammatory leukocyte infiltration, was smaller (figure 4(a)(b) versus figure 4(a)(a)), and the number of proliferating leukocytes was much reduced (figure 4(b)(b) versus figure 4(b)(a)) together with a significant decrease in the proinflammatory cytokine expression (figure 4(c)). in these kidneys, masson 's trichrome staining and the immunohistochemistry for fibrosis markers, such as collagen iii, demonstrated less staining in the cortical interstitium (figures 5(a)(b) and 5(a)(d) versus figures 5(a)(a) and 5(a)(c)). however, in the glomeruli, the amount of periodic acid schiff positive material was a comparable with that in vehicle - treated crf rat kidneys (figure 5(b)(b) versus figure 5(b)(a)). these results indicated that benidipine ameliorated the progression of renal fibrosis without affecting the deterioration of glomerulosclerosis. benidipine, which blocks l- and t - types of calcium channels in the renal vasculature, is known to dilate both afferent and efferent arterioles of the glomeruli and to reduce glomerular hypertension. in our recent study, however, benidipine did not decrease the severe proteinuria nor did it improve the systemic hypertension in advanced crf rats. therefore, factors other than reducing the glomerular capillary pressure may also be involved in its pharmacological effects of ameliorating the renal injury. recently, benidipine was shown to reduce the circulating levels of inflammatory cytokines or proteins, such as il-6 and high mobility group box-1 (hmgb-1), in patients with chronic kidney disease. since il-6 and hmgb-1 accelerate the inflammation of systemic organs by promoting lymphocyte activation and proliferation [14, 70 ] ; such effects of benidipine may have therapeutic potential for slowing the progression of renal fibrosis in advanced crf. grgic. demonstrated the therapeutic efficacy of blocking the intermediate - conductance ca - activated k - channels (kca3.1) for renal fibrosis, since fibroblasts overexpressed the channels under a pathologic condition. in a separate animal study, they also demonstrated the prophylactic efficacy of blocking lymphocyte kv1.3-channels to prevent renal allograft rejection. as an extension of these studies, our studies further suggested that targeting the kv1.3-channels overexpressed in leukocytes would also be useful for the treatment of renal fibrosis in advanced crf [11, 34 ]. in our series of patch - clamp studies, in addition to ccbs, such as benidipine and nifedipine, macrolide antibiotics and hmg - coa reductase inhibitors (statins) also effectively suppressed lymphocyte kv1.3-channel currents [20, 21 ] (table 1). according to separate in vitro studies, these drugs exerted immunomodulatory properties besides their anti - inflammatory, antimicrobial, and anticholesterol effects [56, 7175 ]. since lymphocyte kv1.3-channels trigger calcium influx, which is necessary for il-2 synthesis and since channel blockade by highly selective inhibitors, including margatoxin, shk - dap, and pap-1, actually repressed the immune response in lymphocytes [38, 7678 ] (table 2), the suppressive effects of nsaids, macrolide antibiotics, and statins on the channel currents were considered to contribute to their immunomodulatory properties. additionally, as previously detected by the whole - cell patch - clamp technique, the amplitude of the peak channel currents was deeply associated with the activation of the channel currents. therefore, the stronger suppression of the peak currents by macrolide antibiotics or statins may indicate their higher immunosuppressive potency. in this regard, besides the use of the previously developed selective blockers for the channels [7880 ], the use of ccbs, macrolide antibiotics, or statins could also potentially be useful as antifibrotic agents in patients with advanced ckd (figure 6). the most likely side effects of the kv1.3-channel inhibition include epileptic seizures or enteric twitches, since the channels are also expressed in the synapses of both central and enteric nervous systems [81, 82 ]. in normal rat kidneys, kv1.3-channels are expressed in some proximal tubules and predominantly in the basolateral membranes of the inner medullary collecting duct cells. in these cells, the channels regulated the cellular or total body fluid volume by maintaining the driving force for na reabsorption. therefore, the channel inhibition may also affect such tubular functions and the urinary k secretion, which was actually demonstrated in previous studies using isolated collecting ducts. however, compared to the highly selective inhibitors, which were originally derived from venom, scorpion, or sea anemone peptide toxins [49, 79, 85, 86 ], the drugs, such as ccbs, macrolide antibiotics, or statins, could be used more safely, since they have been employed in a common clinical practice for a longer period of time. in a physiological condition, kv1.3-channels expressed in t lymphocytes play crucial roles in the initiation of the immune response. in renal diseases, such as ckd, acute glomerulonephritis, and renal allograft rejection, the channels contribute to the pathogenesis of the diseases. in rat kidneys with advanced crf, the overexpression of the channels in lymphocytes facilitated the progression of tubulointerstitial fibrosis by promoting lymphocyte proliferation, suggesting that the channel could be a potent therapeutic target for advanced - stage ckd. benidipine, one of the most commonly used ccbs, which also strongly and persistently inhibits the lymphocyte kv1.3-channel currents, suppressed the proliferation of kidney lymphocytes and actually ameliorated the progression of renal fibrosis. since other drugs, such as nsaids, macrolide antibiotics, and statins, also effectively suppress the channel currents in lymphocytes, they may be useful for treating or preventing renal diseases. chronic inflammatory diseases are a category of diseases, in which chronic inflammation or the overstimulation of cellular immunity is responsible for the pathogenesis. besides infectious diseases and autoimmune disorders, a number of diseases, such as cancer, neuroinflammatory diseases, and metabolic disorders, nowadays fall into this category. recently, in addition to renal diseases, the involvement of lymphocyte kv1.3-channels has also been demonstrated in the development of these chronic inflammatory diseases [4345, 4850, 52, 8891 ]. therefore, our future tasks would include revealing the as yet unknown significance of the channels in the pathogenesis of these diseases and revealing novel therapeutic applications based on targeting these channels.	delayed rectifier k+-channels (kv1.3) are predominantly expressed in t lymphocytes. based on patch - clamp studies, the channels play crucial roles in facilitating the calcium influx necessary to trigger lymphocyte activation and proliferation. using selective channel inhibitors in experimental animal models, in vivo studies then revealed the clinically relevant relationship between the channel expression and the pathogenesis of autoimmune diseases. in renal diseases, in which chronic inflammation or the overstimulation of cellular immunity is responsible for the pathogenesis, the overexpression of kv1.3-channels in lymphocytes promotes their cellular proliferation and thus contributes to the progression of tubulointerstitial fibrosis. we recently demonstrated that benidipine, a potent dihydropyridine calcium channel blocker, which also strongly and persistently inhibits the lymphocyte kv1.3-channel currents, suppressed the proliferation of kidney lymphocytes and actually ameliorated the progression of renal fibrosis. based on the recent in vitro evidence that revealed the pharmacological properties of the channels, the most recent studies have revealed novel therapeutic implications of targeting the lymphocyte kv1.3-channels for the treatment of renal diseases.
chronic periodontitis is one of the most common inflammatory diseases that lead to destruction of the periodontal ligaments and loss of the adjacent bone and teeth (1). with the progression of periodontitis, bacteria and their endotoxins (lipopolysaccharide) and other products enter the periodontal tissue and blood circulation causing, systemic or local inflammatory reactions in the host. also, periodontal disease causes th1 reaction and releases cytokines such as tumor necrosis factor- (tnf-) and interleukin-1 (il-1), which can increase the risk of coronary heart disease (2). reported that serum and gingival cervical fluid (gcf) levels of tnf-, il-1 and il-6 seem to be relating factors between periodontal disease and high serum lipid levels (3). several studies have shown that there is a relationship between periodontitis and high serum lipid levels (4 - 9), while other studies have found no such relationship (10 - 14). losche., cutler., and moeintaghavi. concluded that plasma levels of lipids in individuals with periodontitis were significantly higher than healthy periodontal subjects (4, 7, 8). hamissi. on the other hand, reported no significant difference between levels of total cholesterol (chl), triglycerides (tg), high - density lipoprotein (hdl) and low - density lipoprotein (ldl) cholesterol in periodontitis subjects compared with controls (14). according to the controversy between different studies, this study was performed to determine the relationship between chronic periodontitis and blood serum lipid levels. this case - control study was conducted at the school of dentistry, ahvaz jundishapur university of medical sciences, ahvaz, iran, during march 2011. the study protocol was approved by the ethics committee (number : 216/36/ mp / d). from the patients that referred to the periodontics department, 100 subjects with chronic periodontitis that had at least one periodontal pocket with 4 mm depth in every quadrant and showed bone destruction in their radiography, were selected on the basis of the simple random sampling method, and then based on the exclusion criteria 45 subjects were excluded from the case group. also, 45 periodontally healthy subjects that did not meet the exclusion criteria were selected for the control group by the simple random sampling method, from 100 individuals referred to the oral medicine department. according to previous studies and the statistical formula, in total 90 subjects (45 cases and 45 controls) were selected to participate in the study ; the significance of the study was set at = 0.05 and the power was set at 80%. these two groups were matched for confounding factors such as gender, age and number of teeth. one periodontist performed all periodontal examinations in a dental chair using a dental mirror and a world health organization (who) periodontal probe according to the who instructions. clinical oral examinations included assessment of the periodontal pockets in all teeth except the third molars. dental and medical history was determined on the basis of information obtained from the health interviews and examinations. exclusion criteria were periodontal treatment in the last six months, diseases such as cancer, cardiovascular disease and diabetes mellitus or any endocrine disease, hypertension, use of alcohol, tobacco smoking, drug treatment for hyperlipidemia and excessive obesity (bmi 30 kg / m). body mass index (bmi) was calculated as body weight (kg) divided by height (m) (15). all subjects were referred to the central laboratory of golestan hospital where fasting blood samples were collected to determine serum lipid levels, using routine enzymatic methods (autoanalyzer, bt 3000, italy), which were calibrated daily by the operator. the pathological cut - off points according to the laboratory s recommendations were as follows : total cholesterol (chl) > 220 mg / dl, triglycerides (tg) > 200 mg / dl, low density lipoproteins (ldl) > 178 mg / dl and high density lipoproteins (hdl) 160 mg / dl and hdl 178 mg / dl and hdl < 29 mg / dl was considered pathological by the laboratory ; therefore less patients were in this range. confirmed a positive relationship between deep periodontal pockets and high total cholesterol and ldl levels, using the index community periodontal index of treatment needs (cpitn) (5, 6). reported similar results to this study, yet the odds ratio for high tg and chl in this study was lower than that reported by moeintaghavi (6.7 for tg and 5.9 for cholesterol) (8, 9). machado. concluded that mean levels of cholesterol and tg were higher in patients with periodontitis yet the difference was not significant (10). saxlin. carried out a cross - sectional study on 1297 subjects and concluded that serum lipid levels were not associated with an increased likelihood of periodontal infection among normal weight subjects, yet were associated with the presence of deepened periodontal pockets (4 mm or more) among obese subjects (11). sridhar. rejected the relationship between periodontal disease, coronary heart disease and serum lipid levels (12).. carried out a case - control study where the case and control groups were matched by, age, gender, diet, weight, height, health condition and number of teeth. they concluded that serum lipid levels in the case group were higher than the control group yet this difference was not significant. they claimed that the difference between their results and other studies was due to samples size and the severity of periodontitis in the case group (14). stated that the relationship between hyperlipidemia and periodontal infection could be due to polymorphonuclear leucocytes (pmn - cells) dysfunction (17). it has been shown that acute infections can interrupt lipid metabolism, and there is a significant rise in plasma tg during gram - negative bacterial infections (18). it seems that these changes are due to cytokines such as tnf-, il- 1 and il-6 which are produced in large amounts in infectious periodontal tissues (19 - 21). these cytokines increase the mobilization of lipids from the liver and adipose tissue (22), and increase the binding of ldl to endothelium and smooth muscles (23). it has been reported that oxidative modification of ldl leads to an increase in cholesterol accumulation because modified ldl is very susceptible to macrophage uptake (24). on the other hand, several studies have reported the possible role of hyperlipidemia in periodontitis progression (25). it has been shown that hyperlipidemia causes an increase in white blood cell (wbc) activity (26, 27), which helps the progression of periodontal disease in adults (28, 29). this study was carried out on non - smoking subjects thus confounding relationships with the toxic effects of smoking were omitted. subjects with bmi 30 were also excluded because many studies report a relationship between obesity and periodontal infections (30 - 32). the case and control groups were matched by age, because older age is a risk factor for hyperlipidemia. this study showed a relationship between periodontitis and hyperlipidemia yet whether alterations in lipid metabolism are the cause or result of periodontitis can not be determined. therefore we suggest that cohort studies with more samples should be carried out to better understand this relationship. this study, similar to some other previous reports, proves a relationship between periodontal disease and hyperlipidemia. therefore, it is recommended that patients with heart disease should strongly consider periodontal treatment.	background : periodontitis is a local chronic inflammatory condition of the supporting structures of the teeth resulting from a dental plaque biofilm attached to teeth surfaces. recent studies have indicated that this oral disease may have effects on systemic health.objectives:the aim of the present study was to evaluate the association between periodontitis and hyperlipidemia.patients and methods : this case - control study was conducted in iran during march 2011. in this case - control study, levels of serum lipids in 45 subjects with periodontitis were measured and compared with 45 age, gender and body mass index (bmi) matched controls. data were analyzed using student t - test and chi - square test with p < 0.05 as the limit of significance.results:mean values of total cholesterol (chl) (periodontitis group = 218.11 29.77, control group = 162.31 48.24) and triglycerides (tg) (periodontitis group = 209.77 44.30, control group = 125.60 44.16) were significantly higher in the periodontitis group (p < 0.001). high - density lipoprotein (hdl) and low - density lipoprotein (ldl) cholesterol levels were higher in the case group, but this difference was not statistically significant. frequency of pathological values of chl and tg were significantly higher in cases compared with the controls (p = 0.002 and p = 0.015, respectively).conclusions : this study indicates that hyperlipidemia may be associated with periodontal disease in healthy individuals ; yet whether periodontitis causes an increase in levels of plasma lipids or whether hyperlipidemia is a risk factor for periodontal infection and cardiovascular disease, it needs further investigations.
gap junctions are cell cell connections that ensure harmonious integration, regulation and equalization of metabolic events and signaling in tissues and organs. their role in the coordination of cell behavior is vividly illustrated in the heart, where gap junctions in the intercalated discs provide pathways that allow direct intercellular electrical communication essential for synchronous contraction of component myocytes and for generating waves of rhythmic contractions observed in arteries. gap junctions are constructed of paired connexin hemichannels (cxhcs), each composed of six protein subunits arranged around a central pore, and occur at adhesive areas where plasma membranes touch. hcs are continuously recruited from surrounding unapposed plasma membrane areas and subsequently dock head to head with partners from adjacent cells and attach to the rims of preexisting gap junction plaques. the operational area and size of gap junction plaques where intercellular communication occurs are regulated by a balanced internalization and degradation of the dodecameric cx channel units (laird 2006 ; goodenough and paul 2009). many approaches have been applied to study the structure and function of gap junctions and their constituent cxhcs (harris and locke 2009). this account deals with the development of peptides that correspond to specific short cx sequences that block widely the operation of gap junctions and cxhcs. the consequences ensuing from the actions of these peptides on cx channels are allowing research to move forward into the realms of translational innovation across many fronts, especially in addressing the pathological consequences of ischemic stress that induces the channels to become leaky. peptidomimetic approaches are complemented by gene knockout and antisense sirna approaches to study cx - based communication. the development and application of short peptides mimicking sequences in various protein domains of cxs, especially cx43, initially focused on the two loops projecting outside the cell membrane ; but peptides corresponding to sequences in the cytoplasmic intracellular loop and carboxyl tail are now finding application. over the last 25 years, mimetic peptides have become important tools in elucidating a panoply of roles for gap junctions and their constituent cxhcs in a wide range of cells, tissues and organs. these are summarized in tables 1, 2 and 3.table 1examples of the use of gap26 and -27 mimetic peptides in studying the functions of gap junctions and connexin hemichannels in tissues / organs, cell layers and slicestest modelpeptideeffectsreferencearteriesgap26/27block rhythmic contractionschaytor. (1999)arteriesgap26/27block ehf signalingchaytor. (2005)kidneygap27blocks renal vasodilatationde vriese. (a gap27 acting on cx40 channels (srpteknvfiv) has been used on vascular tissues where this cx is expressedtable 2examples of use of gap 26 and 27 mimetic peptides on various cells in culturetest model / cellspeptideeffectreferencesskin fibroblasts, keratinocytesgap27increases migration in diabeteswright. (2012)blood brain barrier endotheliumgap27inhibits atp release and permeability of endotheliumde bock. (2010)bladder cancergap26/27inhibit atp releasede vuyst. (2006)t lymphocytes1,848blocks gj dockingmendoza - naranjo. (2009)vdcflsrptekt peptide 5 derived from extracellular loop 2 of cxhc43sequence of the cx mimetic peptide not disclosedtable 3effects of various intracellular cx mimetic and other short and mainly cx43 peptides on gap junctions and hemichannelstest modelpeptideeffectreferencebrain synapsescarboxyl tailprevents cx36/gj formationflores. (2011)heartcarboxyl tailmay open gap junctionslewandowski. (2008)heartr, any amino acidmay open gap junctionsverma. (unpublished)corneal endothelial and c6 glioma cellstat - l2blocks cxhc but not gap junctionsponsaerts. (2012)mdckct9 peptide carboxyl tailblocks ca oscillations by removing high ca closurede bock. (2012)gap24 : a cx32 gap20 homologue ghgdplhleevk (from intracellular loop)peptide rprpddleigap20 eikkfkyg examples of the use of gap26 and -27 mimetic peptides in studying the functions of gap junctions and connexin hemichannels in tissues / organs, cell layers and slices gap26, vcydksfpishvr ; gap27, srptektifi gap27 analogue. see table 2 for sequence. a gap27 acting on cx40 channels (srpteknvfiv) has been used on vascular tissues where this cx is expressed examples of use of gap 26 and 27 mimetic peptides on various cells in culture vdcflsrptekt peptide 5 derived from extracellular loop 2 of cxhc43 sequence of the cx mimetic peptide not disclosed effects of various intracellular cx mimetic and other short and mainly cx43 peptides on gap junctions and hemichannels gap24 : a cx32 gap20 homologue ghgdplhleevk (from intracellular loop) following the deduction of the complete amino acid sequences especially of cx32 and cx43, two of the 20 members of the cx family of proteins, a number of short mimetic peptides were chemically synthesized and coupled to immunogenic carriers to generate antibodies to target specific domains and epitopes. the two highly conserved extracellular loops of cx have proven to be poorly immunogenic, and it has been difficult to generate antibodies to these domains. nevertheless, antibodies to both extracellular loop domains have been used in studies of a wide range of functions underwritten by cx channels. these include (1) the topographical arrangement of cx proteins in the membrane (zimmer. 1987), (2) the roles of gap junctions in the development of mouse embryos (becker. 1995), (3) ca wave signaling across cell layers connected by gap junctions (boitano. 1998), (4) subcellular assembly of gap junctions (rahman. 1993), (5) coordination of ca transients in beating cardiac myocytes (verma. 2009b), (6) cx43 as a candidate component of the immunological synapse (mendoza - naranjo. 2011), (7) the functional importance of the two exposed extracellular loops (goodenough. 1988), (8) cxhc organization in polarized cells (clair. 2008) and (9) tracking conformational changes as cx traffic from the golgi apparatus to gap junctions (sosinsky. antibodies to peptides from intracellular regions have been used extensively as diagnostic immunological / analytical tools and are widely available from commercial sources. although reagents such as heptanol, octanol, oleamide, lithium ions, quinine derivatives, carbenoxolone, fenamates, anandamide, oleamide, triarylmethanes and glycyrrhetinic acid inhibited gap junctional communication (herve and dhein 2010 ; juszczak and swiergiel 2009 ; bodendiek and raman 2010), there remained a need for more specific reagents with a known mechanism of action. from early on, it became evident that cx mimetic peptides used to generate the antibodies to gap junctions might prove useful as chemical tools to manipulate channel operation ; it was argued that the utility of antibodies was restricted by their size and limited penetration across the cell membrane and into intercellular regions where gap junctions are located. such drawbacks would be overcome by using small mimetic peptides that could penetrate into intercellular junctions, disrupt the docking and/or operation of hemichannels and, thus, target the gap junction (fig. peptides bind to extracellular loop regions one and two, respectively, of cxhc, causing closure of channels within minutes. at later time intervals (30 min or longer) and depending on factors such as cell confluency, tissue, organ, tissue slice origin and thickness and conditions of perfusion of various organs, peptides permeate into intercellular spaces in gap junctions, causing disruption and diminished cell coupling. cxhcs with attached peptide move laterally toward the rims of gap junction plaques as they assemble and are then internalized mechanism of action of gap26 and -27 mimetic peptides. peptides bind to extracellular loop regions one and two, respectively, of cxhc, causing closure of channels within minutes. at later time intervals (30 min or longer) and depending on factors such as cell confluency, tissue, organ, tissue slice origin and thickness and conditions of perfusion of various organs, peptides permeate into intercellular spaces in gap junctions, causing disruption and diminished cell coupling. cxhcs with attached peptide move laterally toward the rims of gap junction plaques as they assemble and are then internalized two studies using model systems marked the beginning of the exploration of cx mimetic peptides as tools to study gap junction functions. the first took advantage of the contractile behavior displayed by embryonic chick heart myoballs and known to require coordination provided by intercellular communication via gap junctions. the effects of a series of 15 cx peptides, corresponding to short sequences mainly in intra- and extramembrane amino acid regions of the tetraspan membrane protein in delaying gap junction functions, were determined (warner. 1995). a parallel study that used six dodecapeptide cx mimetics to interrupt communication across gap junctions generated in xenopus oocytes transfected with rna to cx32 (dahl. 1994) likewise pointed to the potential of using short peptides to tamper with cx - dependent intercellular communication. warner. (1995) pinpointed motifs that included short sequence motifs, srptek in extracellular loop 1 and shvr in extracellular loop 2, as likely potent peptides for use in disrupting cell communication. these motifs were later incorporated into gap26 and -27 mimetic peptides and their close homologues (see tables 1, 2, 3). kwak and jongsma (1999) used dye coupling and dual patch - clamp approaches to study the inhibition of cx channels using peptide mimetics from the second extracellular loop of cx43 and cx40. an extensive literature has since built up around the study of cx - dependent cell communication processes, especially with peptides mimicking sequences in cx43 and, to a lesser extent, cx40 and cx37, which are widely expressed in the vascular system as well as with cx32 (de bock. gap26 and -27 have emerged as mimetic peptide tools that have entered the literature in studies that explore the operation and function of cx channels in several settings (tables 1, 2). as discussed below, the blockage of direct cell coupling across gap junctions (evans and boitano 2001) is now likely to be a secondary event that follows initial interaction of the peptides with cxhcs. recent work is increasingly focused on the translational and therapeutic possibilities offered by the action of the mimetic peptides, especially in averting or reversing tissue damage in ischemia and inflammation. the view that cxhcs possess functions in their own right and are able to operate in different modes from gap junctions has now become generally accepted (goodenough and paul 2003 ; bennett. cxhcs were detected in xenopus oocytes (ebihara and steiner 1993), a test bed to study gap junction expression and function and where the channels were observed to open in low - ca media. hc opening was also detected in vertebrate retinal dendrites (malchow. 1993). these early studies appeared against the background view that cxhcs sustained in open configuration in membranes would lead to potentially catastrophic cellular outcomes by allowing transmembrane escape from cells of small intracellular signaling molecules, e.g., atp and glutamate, and would result in a collapse or dissipation of ionic gradients. the possible importance of cxhcs operating under normal physiological situations in cells and tissues was critically evaluated (spray. collateral evidence for the functional reality of cxhcs began to appear later for roles in pathology with, e.g., the demonstration that leaky mutated cxhcs in the ear were linked to deafness (stong. 2006 ; scott and kelsell 2011) and a mutation in the intracellular loop of cx43 that decreased single - channel conductance and is linked to neurological disturbances in oculodentodigital dysplasia (lai. reconstituted hcs were used to investigate the influence of ca and atomic force microscopy, to study hc pore opening (thimm. it is now generally accepted that cxhcs open under environmental circumstances considered to be stressful to cells such as volume or osmotic changes ; oxidative, metabolic and mechanical stresses ; and especially hypoxia / ischemia. if not contained, leaky cxhcs can lead to apoptosis and cell death (saez. 2010 ; decrock. 2009a, b). binding of mimetic peptides such as extracellular loop peptides gap26 and -27 to the extracellular face inhibited functions associated with cxhcs ; these are also regulated by membrane depolarization, phosphorylation of several sites on the carboxyl tail of cx43 (solan and lampe 2009), s - nitrosylation (retamal. 2006) and sumoylation of lysine residues in the intracellular loop (kjenseth. the particle receptor hypothesis (see below) explained the mechanics of gating in those cxs with extended cytoplasmic tails such as cx43, cx40 and cx45 (delmar. recent evidence suggests that mimetic peptide perturbation of intracellular domains, especially the interactive cytoplasmic loop (cl) and the carboxyl tail (ct), also influences hc functions (ponsaerts. cxhc gating is also conditioned by signaling cascades operating in subplasmalemmal environs (fig. several kinases may also be involved ; pkc closes hc, while p38mapk and calmodulin kinases result in hc opening. the cytoplasmic ca concentration ([ca]i) is also an important modulator ; below 500 nm [ca ] calmodulin is a key ca binding protein and specific binding sites are present on cx. an increase in intracellular ca causes hc opening, while hc activation is lost at higher concentrations. arachidonic acid stimulates hc opening, and amino acid metabolites generated by pla2 activation may contribute to this. ba sites on the exposed extramembrane regions of cx43 where the mimetic peptide sequences originated. b proposed intramolecular mechanism of cxhc gating involving interaction of the carboxyl tail with the intracellular loop. c binding of a nonapeptide mimetic derived from l2, the cx43 intracellular loop region to a site on the carboxyl terminus regulates the closure of cxhc and leads to blockage of the channel a several intracellular signals and events influence cxhc functions. several kinases may also be involved ; pkc closes hc, while p38mapk and calmodulin kinases result in hc opening. the cytoplasmic ca concentration ([ca]i) is also an important modulator ; below 500 nm [ca ] calmodulin is a key ca binding protein and specific binding sites are present on cx. an increase in intracellular ca causes hc opening, while hc activation is lost at higher concentrations. arachidonic acid stimulates hc opening, and amino acid metabolites generated by pla2 activation may contribute to this. ba sites on the exposed extramembrane regions of cx43 where the mimetic peptide sequences originated. b proposed intramolecular mechanism of cxhc gating involving interaction of the carboxyl tail with the intracellular loop. c binding of a nonapeptide mimetic derived from l2, the cx43 intracellular loop region to a site on the carboxyl terminus regulates the closure of cxhc and leads to blockage of the channel besides structural differences, with hcs being asymmetrical and gap junctions being symmetrical double channels, there are other important differences between the functions of these weakly selective channels. gap junction channels allow the cytoplasms of cells to be linked directly, whereas open cxhcs provide channels connecting the cell s external environment with the cytoplasmic mileu beneath the plasma membrane. in a cell - signaling context, changes in cxhc open probability provide a mechanism for paracrine intercellular communication by allowing small molecules such as atp and k to exit and ca to enter cells. 2000 ; kang. 2008), a function shared with pannexin channels. gap junction channels exist normally in open mode, and an elevation in intracellular ca leads to closure (generally 5002,000 nm). in contrast, cxhcs open at 500 nm (de vuyst. 2006, 2009, 2011). differences in ca sensitivity of the two channel types may relate to their complementary roles in regulating intracellular ca oscillations and the intercellular propagation of ca waves (verma. the various intracellular processes that influence the gating of cx43hcs are shown in fig. gap junctions and cxhcs respond differently to lipopolysaccharide and basic fibroblast growth factor, a consequence of their involvement in releasing atp (de vuyst. 2007) ; their channel functions also respond differently to many growth factors (schalper. it is therefore not surprising that gap26 and -27, derived from cx43 sequences, have found extensive use. a modified gap27 peptide (table 1) incorporating a sequence mimicking that in cx40 has also proven useful in vascular tissues and other tissues where both cxs are present (chaytor. 1999 ; wright. early studies showed that inhibition by mimetic peptides was largely reversible as assessed by intercellular transfer of small fluorescent reporter dyes of varying size and by measuring the extent of ca wave propagation over multiple rows of cells (boitano and evans 2000). as the operational presence of cxhcs became evident, inhibition by mimetic peptides of atp release in a wide range of tissues and cells (tables 1, 2), of glutamate in astrocytes (yang. 2009) and astrocytes / microglia (orellana. 2011) and of ion trafficking, especially ca entry, were observed. uptake of reporter dyes across cxhcs became a reliable and routine method to demonstrate open or leaky cxhcs (li. a range of other effects were also noted on prolonged exposure (20 min or longer) to the mimetic peptides such as smooth muscle contraction in endothelium - denuded arteries (chaytor. 1997 ; hutcheson. 1999) and immunoglobulin production by lymphocytes (oviedo - orta. 2001). subsequently, as the presence of cxhcs widened, it became increasingly clear that the primary action of gap26 and -27 is in fact directed at cxhcs. nevertheless, a combination of direct signaling across gap junctions and paracrine signaling acting through metabotropic purinergic receptors following release of atp (fig. 2) provides scope for complementary intercellular interplay involving both routes in several settings (anselmi. initial experiments showing inhibition by mimetic peptides of cxhcs were carried out mainly using cultured cells (table 2), followed by numerous examples in various tissues and organs, cell layers and thin tissue slices (table 1). 2011) has led to studies that attempt to unravel the potential functions in the realms of adhesion and cell movement. enhancement of migration of dermal fibroblasts after treatment with gap27 pointed to nonjunctional roles for cxhcs in cell movement and extending, e.g., to assessing their potential translational application in addressing their efficacy in accelerating wound healing in diabetes and in a range of micro-/macrovascular diseases (wright. gap27 increased migration of human keratinocytes and dermal fibroblasts, and the efficacy of the mimetic peptide was different in euglycemia and hyperglycemia (wright. gene array approaches indicated that gap27 induced upregulation of genes involved in extracellular matrix remodeling and cell adhesion. exposure of cells to gap27 may have effects on cx43 phosphorylation, especially of serine 368 ; phosphorylation of this amino acid decreases cx43 channel activity (solan and lampe 2009) and is a key process in the gating of cx43 channels. many of the effects of the mimetic peptides were seen after long - term exposures. cx43 has been detected in the inner mitochondrial membrane of heart cells and has been implicated in cardiac preconditioning (boengler. 2009 ; rottlaender. 2012). the opening of mitochondrial cxhc influences k fluxes in processes that are linked to cardioprotection. the functions of cx43hc at this location are open to study using mimetic peptides developed to cx43 cytoplasmic sequences (verma. to further develop and refine the actions of cx mimetics, it is important to gain insight into the mechanisms by which they block cx channels. a major advance was the demonstration that the primary action of gap26 and -27 was likely to be on cxhcs prior to blockage of gap junctions. with the acceptance that cxhcs and gap junctions were targets but in different time frames, the effects of the peptides on both were examined using electrophysiological approaches. these had already proven useful in studying electrical coupling across gap junctions in cell pairs and the electrical properties of cxhcs (harris 2001 ; contreras. 2003 ; saez. 2005). using a voltage - clamp approach in nonconfluent and confluent hela cells expressing cx43, gap26 inhibited macroscopic currents through cxhcs in 23 min. in contrast, electrical coupling in cell pairs was delayed and completely inhibited after 30 min or more (desplantez. 2012), indicating direct action of gap26 on an exposed extracellular loop, as previously suggested by studies of cxhcs inserted into lipid bilayers (liu. the extended time course of action on gap junctional electrical coupling suggests that longer diffusion pathways into plasma membrane junctional domains lead ultimately to blockage of gap junctional coupling (fig. 1). the changes in conductance and voltage gating of cxhcs by gap26 suggested interaction of the peptide at a consensus sequence in the first extracellular loop that may contribute to the inner wall of the pore (sosinsky and nicholson 2005), resulting in a decrease in channel diameter in the extracellular vestibule. (2012) also proposed that, of the two voltage gates controlling hc, the slow gate is the more likely to be influenced by the binding of gap26. (unpublished) examined the mechanism of action of gap26 and -27 in hela cells stably transfected with cx43 and in pig ventricular myocytes endogenously expressing cx43 using a voltage - clamp approach. an important outcome of this study with cardiac translational implications was that unitary current activity was promoted by a moderate elevation of cytoplasmic ca, an event observed in cardiac arrhythmogenesis. gap26 and -27 have been used mainly to inhibit cx43 channels, but actions on other cx channels have also been studied. for example, wright. (2009) carried out an extensive study of the effects of gap26 and -27 on dye coupling in cells expressing several human cxs and found that gap27 had broader cx specificity than gap26. in some situations where multiple cxs are expressed, as in skin, broad specificity can be an advantage. the precise protein domains to which gap26 and -27 mimetic peptides attach and any conformational changes induced in cx channels remain to be determined. fluorescently labeled gap26 and -27 are poorly soluble ; but evidence points to attachment and retention at cell exteriors, and problems of image resolution could not answer the issue of whether attachment of a mimetic peptide ligand to a channel receptor sequence can lead to peptide internalization (evans, unpublished work). voltage - clamp approaches have been useful in deciphering the inhibitory action of these peptides on cx channels and go a long way toward resolving questions concerning the specificity of mimetic peptide inhibitory effects. cx mimetics have been claimed as effective inhibitors due to steric pore block (wang. steric block may indeed occur, but recent data indicate that it only occurs when peptides are used in the range of 1 mm and above (wang. the examples shown in tables 1 and 2 used mainly protein concentrations of 100250 m. in the vast majority of these studies, investigators have used scrambled peptides or short peptides derived from regions believed not to be directly involved in channel operation (often internal sequences) as controls and, in each instance, they pointed to a high sequence dependence of mimetic peptide action in blocking gap junctional coupling and, more recently, atp release or dye entry across cxhcs. pannexins, of which three are identified compared to around 22 in the cx family of proteins, also form oligomeric channels with a similar tetraspan topographical arrangement in the membrane to cxhcs. however, these two protein families share no amino acid homology. it follows that they are not expected to generate gap junction - like structures and, indeed, are found at higher levels inside cells (dhondt. in contrast, a recent report claims that they can form gap junctions and act as ca leak channels in the endoplasmic reticulum (ishikawa. pannexins are relatively unaffected by changes in cytoplasmic ca levels and, unlike cx, are calmodulin - insensitive. pannexins are especially abundant in neural tissues. the action of gap26 and -27 on pannexin channel currents has not yet been rigorously tested. it is worth noting that high sequence diversity occurs in the intracellular loop of cx proteins ; the carboxyl tail of larger cx proteins also varies in length, sequence and the extent of posttranslational modification. small peptides generally have little structural organization but may assume some on binding to a target that then becomes subject to conformational change. studies of the interaction of calmodulin with mimetic peptides from the intracellular loop of cx43 demonstrate a way forward toward functionally dissecting this key region of cxs (myllykoski. a calmodulin binding region in cx43 was located to amino acid residues 136158 (zhou., further knowledge of the structural organization of cx43hc is awaited along the lines available on cxhc26 (maeda. table 3 lists the growing number of mimetic peptides derived from the cl and ct that act on cxhcs and gap junctions. many of these peptides are not able to cross the plasma membrane. to gain access to their sites of action, many of these mimetics need to be attached first to trojan cell - penetrating peptides that contain a membrane - translocation motif (jarver and langel 2006). intracellular loop mimetic peptides with a positive charge due to several lysine residues stand out as candidates, for they can, depending on length, gain access to intracellular targets without need for attachment of a membrane - penetrating peptide or by microinjection, as discussed below. indeed, peptides of around 1 kda or less may be able to access the cytoplasm via open cxhcs. in a cardiac context, blockage by gap26 and -27 of gap junctional coupling between myocytes negated their candidacy for use, especially in therapeutic approaches to address cardiac arrhythmia, for the outcomes of peptide treatment were likely to compound the pathology. consequently, studies in heart focused on nonmimetic hexapeptides shown to be prorhythmic, especially zp123 derived from aap10 and later renamed rotigaptide (hagen. this hexapeptide promoted changes to cx phosphorylation, increased gap junctional coupling (clarke. 2006) and showed promise in animal models, where it prevented ventricular tachycardia in myocardial ischemia (xing. cellular studies showed that despite its prorhythmic action on gap junctions, zp123 also led to the opening of cxhcs, causing atp release. in a cardiac myocyte ischemia model, gap26 blocked the release of atp, whereas zp123 enhanced its release across cx43hc (clarke. other antiarrhythmic peptides such as gap134, a prorhythmic dipeptide were also developed (hennan. 2009). in the meantime, encouraging outcomes in addressing ischemic cardiomyopathy using gap26 have appeared. these results can be explained by the direct action of this mimetic peptide on cxhc rendered open under hypoxic / ischemic conditions. (2010) showed that following myocardial ischemic insult, gap26 helped to confer protection through its binding to and blockage of cx43hc. vascular leak and retinal ganglion cell death were reduced by application of a close homologue of gap27 in an atrial ischemia model where cx43hc expression was increased (danesh - meyer. the same group also reported that infusion of gap27 in a large animal model with cerebral injury delayed the onset of ischemic injury and suggested that gap27 homologue peptides reduced inflammation. for example, mimetic peptide treatment reduced the spread of damage after traumatic spinal cord injury where cx43hc plays a critical role (ocarroll. such studies combine to show that gap26 and -27 mimetic peptides acting on cxhc - related functions, especially in situations of tissue injury, show therapeutic potential. in brain, inhibition of inflammation - induced activation of cx43hc in astroglial cells was attenuated by treatment with gap26 and -27, suggesting that the channels play a critical role in instigating neuronal death and pointing to a neuroprotective role for these mimetic peptides (froger. interaction of the mimetic peptides with cxhcs is also likely to modify intracellular signaling cascades in subplasma membrane environments (fig. the action of gap26 and -27 on cxhcs and later at gap junctions encouraged the view that pathological outcomes could be fine - tuned if mimetic peptides were to become available that confined their blocking action on cxhcs resident in the plasma membrane. attention had already focused on the intracellular loop region implicated in explaining gap junction gating in cardiac cells and involving intramolecular interaction of the carboxyl tail region with the intracellular loop l2 region (a region in cx43 incorporating amino acids 119144) and described as the particle receptor hypothesis (delmar. the role of the l2 domain as a key molecular determinant of cx43 function originated from the acidification - related closure of gap junctions (delmar. 2004), and the particle - receptor hypothesis has been proposed to explain cx43 closure during acidification, with the ct moving as a flexible gating particle that binds to the l2 site at the intracellular vestibule and leading to closure of the gap junction pore. because of the similarity to the inactivation of k channels, this mechanism has also been called the ball and chain model of gap junction closure. a 34-amino acid nonmimetic peptide (rxp - e) and its derivatives were developed to target the cx43ct (shibayama. 2006 ; lewandowski. when coupled to cell - penetrating peptides, these composite peptides successfully restored gap junctional coupling and impulse propagation in cultured neonatal rat ventricular cardiomyocytes. although the ability of l2 peptide to bind to the cx43ct region has been intensively studied, less is known about how it is influenced by ph. the interaction of a cytoplasmic loop domain with the c - terminal region is an important requisite for the opening of cx43hc in response to stimuli such as lowering of extracellular ca or increasing intracellular ca. in contrast, cx43 gap junctions behave in the opposite manner and are closed by intramolecular loop one way to suppress cxhc operation in response to high intracellular ca is to activate the actomyosin contractile system, a process that appears to physically dislodge the ct from the cl region (ponsaerts. importantly, the selective myosin11-atpase inhibitor blebbistatin restores cx43hc activity when cells are exposed to high intracellular ca (ponsaerts. the proteins involved in this mechanism linking cx43hc and the actomyosin contractile system have not been identified, but a likely target is the ct region of cx43hc, for cx43 lacking the ct is inactive. even in the absence of a functional actomyosin cytoskeleton, loop tail interactions in cx43hc can be disrupted using mimetic peptides from the l2 region, and coupling the peptides to a tat cell - penetrating sequence inhibits cxhc opening. an important interactive target of l2-region peptides is the last 10 amino acids of the carboxyl tail (ct10). direct binding between the l2 domain and ct10 has been demonstrated by surface plasmon resonance. thus, tat - l2 allows exploitation of the opposite regulation of gap junctions and cx43hc and can provide a route to selectively inhibit cx43hc while maintaining gap junctional communication. the importance of the 10amino acid terminal carboxyl domain has been studied in detail also using electrophysiological approaches in xenopus oocytes where the tat - l2 and tat - ct10 peptide constructs have been crucial in analyzing and deciphering intramolecular loop tail interactions (ponsaerts. these studies illustrate how mimetic peptide approaches for selectively studying the physiological functions of cx43hc can complement knockdown / knockout approaches in, e.g., processes leading to cell death (decrock. 2011) and in brain functions in the basolateral amygdala, where cx mimetic peptides such as gap27 demonstrate that cx43hc activities are implicated in amnesia (stehberg. 2012). a short nonmimetic peptide (designated rxp - e) that bound to the carboxyl tail of cx43, derived from heart lysates and studied in animal and cellular models, prevented action potential block in the heart (lewandowski. 2008). a series of nonmimetic peptides with a motif designated rxp and containing a predominance of basic amino acid increased the mean open time of gap junction channels ; these peptides were proposed as potential functional regulators in ischemia - induced arrhythmias (shibayama. 2006 ; verma. currently, the application of novel short peptides mimicking sequences in the l2 region of cx43 (delmar. 2004) where a putative calmodulin binding site is identified (zhou. one major aim is to design mimetic peptides that confine their blocking actions to cxhcs with no interference on gap junction functionality, thus avoiding pro - arrhythmic consequences and allowing the bound mimetic peptide to arrest, e.g., the loss of vital cell metabolites via cxhcs in cardiomyocytes subject to hypoxia or ischemia perfusion injury. a potential tool for more selective inhibition of hcs without associated inhibition of gap junctions is a synthetic mimetic peptide corresponding to the l2 region that, when delivered into the cell by a whole - cell recording pipette, prevented gap junctional opening to the subconductance state at high transjunctional voltage and increased the channel open time (ponsaerts. 2010). also, l2 peptide linked to the tat membrane translocation motif (tat - l2) inhibited cx43hc activation, further suggesting that prevention of interaction of ct and ct suppresses hc opening. consequently, the particle receptor hypothesis described above has an opposite outcome for hcs compared to gap junctions. this raises questions concerning why this is so, for the composite proteins of the channels are identical but the ca sensitivity of the cx43hc and gap junctions differs. it now appears that intracellular interactive partners may also be different at the cytoplasmic aspects of junctional and nonjunctional regions of the plasma membrane. one possible reason for these differences is that hcs interact with the actomyosin contractile system, causing dynamic loop tail interactions that control hcs that differ from those occurring at gap junctions (ponsaerts. new mimetic peptides derived from the carboxyl tail of cx43 are also being assessed. here, peptide mimetic design has to deal with a region where the ct interacts with cytoskeletal elements (herve. this region also incorporates several phosphorylation sites that condition channel gating (solan and lampe 2009). in the heart, the cytoskeletal adaptor protein ankyrin - g interacts with cx43 and is a likely key intercalated disc complex in the pathophysiology of arrhythmias (sato. 2011). a short cx43-ct peptide incorporating the last nine amino acids and linked to a cell permeabilization sequence inhibited pathological changes at gap junctions that are related to ventricular arrhythmias (oquinn. 2011). this peptide disrupted the interaction between the pdz domain of zo1 and cx43, thus accelerating assembly of gap junctions from a precursor pool of hcs (hunter. 2005 ; rhett and gourdie 2012). the peptide also enhanced pkc - epsilon - associated phosphorylation at the cx43-s368 site, an effect that is activated in ischemic preconditioning and can reduce cardiac injury (ek - vitorin and burt 2012 ; srisakuldee. 2009). a similar peptide with the same sequence named ct9 prevented high ca induced closure of cx43hc (de bock., a 15amino acid mimetic peptide derived from a sequence in the ct of cx36 modified gap junction conductance in goldfish electrical synapses, and this peptide was injected intradendritically (flores. 2012). peptides that mimic short sequences in extramembrane domains of cx proteins show huge potential in addressing cx - based communicationopathies by pharmacological means. the functions attributed to cx, especially cx43, continue to expand and include not only roles in intercellular cell communication effected by cxhc and gap junctions but also cell adhesion, cell migration and mitochondrial inner membrane channel functions. it is likely that the range of cx mimetics and their applications will increase, for they provide activation mechanisms for manipulation of intercellular signaling and communication. furthermore, the peptides can also help cells protect themselves from events emanating from leaky channels, events that may ultimately lead to apoptosis. intriguingly, in the brain the gap26 and -27 mimetic peptides that act mainly on astrocyte cx channels have been shown to affect depression (anhedonia), epileptiform activity, memory consolidation and amnesia, further emphasizing the importance of cx - mediated communication and signaling in a wide range of settings.	gap junctions are key components underpinning multicellularity. they provide cell to cell channel pathways that enable direct intercellular communication and cellular coordination in tissues and organs. the channels are constructed of a family of connexin (cx) membrane proteins. they oligomerize inside the cell, generating hemichannels (connexons) composed of six subunits arranged around a central channel. after transfer to the plasma membrane, arrays of cx hemichannels (cxhcs) interact and couple with partners in neighboring attached cells to generate gap junctions. cx channels have been studied using a range of technical approaches. short peptides corresponding to sequences in the extra- and intracellular regions of cxs were used first to generate epitope - specific antibodies that helped studies on the organization and functions of gap junctions. subsequently, the peptides themselves, especially gap26 and -27, mimetic peptides derived from each of the two extracellular loops of connexin43 (cx43), a widely distributed cx, have been extensively applied to block cx channels and probe the biology of cell communication. the development of a further series of short peptides mimicking sequences in the intracellular loop, especially the extremity of the intracellular carboxyl tail of cx43, followed. the primary inhibitory action of the peptidomimetics occurs at cxhcs located at unapposed regions of the cell s plasma membrane, followed by inhibition of cell coupling occurring across gap junctions. cxhcs respond to a range of environmental conditions by increasing their open probability. peptidomimetics provide a way to block the actions of cxhcs with some selectivity. furthermore, they are increasingly applied to address the pathological consequences of a range of environmental stresses that are thought to influence cx channel operation. cx peptidomimetics show promise as candidates in developing new therapeutic approaches for containing and reversing damage inflicted on cxhcs, especially in hypoxia and ischemia in the heart and in brain functions.
inverted papilloma is a true epithelial neoplasm characterized by hyperplastic epithelium inverting into the underlying connective tissue. the first description of this entity was made by ward in1854. this lesion originates from the lateral nasal cavity wall or a paranasal sinus, usually the antrum and appears as a soft, pink or brown, polypoid or nodular growth. possible causes include allergy, chronic sinusitis, occupational exposure to dusts and aerosols, tobacco, and viral infections. the characteristic features of inverted papilloma are the presence of associated nasal polyps, its tendency to recur, destructive potential, and malignant transformation. recurrences after conservative surgical excision have occurred in 75% of cases and transformation to malignancy, usually scc, in 324% of cases. as a result, close postoperative follow - up is necessary for early detection of recurrences and malignant transformations. a 41-year - old woman presented to the department of oral and maxillofacial surgery, faculty of dentistry, tabriz university of medical sciences, tabriz, iran, with a chief complaint of a nodule on the hard palate. the patient had a history of diabetes mellitus and clinical intraoral examination revealed a nodule measuring 0.5 0.7 cm with soft consistency. on radiographic studies no changes were seen. excisional biopsy was performed and histological findings showed multiple islands of epithelium extending into the underlying connective tissue, suggestive of inverted papilloma with mild dysplasia. the patient returned 5 months later with a rapidly growing lobulated mass on the hard palate and maxillary alveolar ridge [figure 1 ]. a lobulated mass on the hard palate and maxillary alveolar ridge surface ulceration was evident in some parts. computed tomography (ct) scan of this massive lesion did not show sinus involvement and bone lysis [figure 2 ]. palatal biopsy showed epithelial proliferation with multiple inverting islands of epithelium extending into the underlying connective tissue. varying degrees of cellular and nuclear pleomorphism and individual cell keratinization were seen [figure 3 ]. the patient was sent for chemotherapy before surgery and the treatment has not come to an end yet. scan did not show sinus involvement and bone lysis varying degrees of cellular and nuclear pleomorphism and individual cell keratinization were demonstrated inverted papilloma (schneiderian papilloma) is considered the most common benign sinonasal tumor and it is characterized by aggressive local invasion, high recurrence rate, and transformation into malignancy. the name inverted is justified by the endophilic growth pattern of the superficial epithelium. inverted papilloma originates from the nasal cavity lateral wall, and it secondarily affects the maxillary, ethmoidal, frontal, and sphenoidal sinuses. the incidence ranges from 0.5 to 4% of all primary nasal tumors and it is prevalent in the fifth and sixth decades of life. the symptoms include nasal obstruction, hyposmia, frontal headache, epistaxis, and rhinorrhea (buchwald., 1989 ; 1995b). ct scan is considered the investigation of choice (lund and lloyd, 1984) but the size of an inverted papilloma may be exaggerated by ct (lund, 2000). with mri the tendency to overestimate the size of ip is less than that with ct (oikawa., 2003). treatment is complete surgical excision, but in cases associated with scc the lesion is treated as a malignancy by performing more radical surgery with or without radiotherapy and chemotherapy. recurrences are usually noted within 2 years of surgery and smoking is associated with increased risk of recurrence. patients with carcinoma in ip are older than those ip patients without carcinoma and they are predominantly males. (2003) reported two cases of nasal inverted papillomas in 60- and 73-year - old males with malignant transformation. in the case we presented inverted papilloma had originated from the hard palate without sinonasal involvment, which is quite different from what we know about the origin of inverted papilloma in the literature. in this case, the tumor was limited to the palate ; therefore, complete resection was achieved by an intraoral surgery.	the inverted papilloma is a unilateral sinonasal benign tumor which is characterized by aggressive local invasion, high recurrence rate, and transformation into malignancy. the etiology of inverted papilloma is still unknown. possible causes include allergy, chronic sinusitis, occupational exposure to dusts and aerosols, tobacco, and viral infections. treatment is complete surgical excision and close postoperative follow - up is necessary. here we report a case of inverted papilloma arising from the hard palate with malignant transformation in a 41-year - old female. clinical and histological features and treatment are discussed with the review of literature.
this module was developed keeping in mind the need for awareness of these issues for medical graduates as well as the areas that need to be strengthened as determined by the analysis of the questionnaire answers. the medication safety module was developed with four themes (sub - modules) as mentioned in tables 1 and 2 : knowledge about themes in medication safety (premodule) knowledge about topics in medication safety (premodule) adverse effects risks and meaning of adverse events, need for pharmacovigilance, clinical trials and relevance, medicine safety profile, drug interactions, genetic differences and risks in alternative systems of medicinesmedicines management medication errors, high risk medicines, toxicity of medicines, zero order kinetics and relevance, medicine misuse, storage issues and expiry issues, lasa (look alike sound alike)quality of medicines generics, innovators, bioequivalence, regulatory needs, counterfeit medicines, substandard medicines, process for recognition and reportingrational use of medicines rational use principles, irrational use consequences and factors, essential medicines list, formularies, standard treatment guidelines. adverse effects risks and meaning of adverse events, need for pharmacovigilance, clinical trials and relevance, medicine safety profile, drug interactions, genetic differences and risks in alternative systems of medicines medicines management medication errors, high risk medicines, toxicity of medicines, zero order kinetics and relevance, medicine misuse, storage issues and expiry issues, lasa (look alike sound alike) quality of medicines generics, innovators, bioequivalence, regulatory needs, counterfeit medicines, substandard medicines, process for recognition and reporting rational use of medicines rational use principles, irrational use consequences and factors, essential medicines list, formularies, standard treatment guidelines. each sub - module was taught in a 30 minute period with a cumulative time of two hours spent on the entire module. all the topics were framed based on what would be important to improve the safety profile of patients while on medicines, to improve the safety responsibility of the img and to encourage an integrated style of thinking and clinical management. the variables assessed were knowledge about adverse effects, quality of medicines, medication management, and rational use [table 1 ]. a questionnaire with 20 questions (maximum score of 20) was administered based on the variables mentioned before and after administering the module. each theme was represented by five questions (maximum score of five per theme). within each theme with a confidence level of 95% and power of 80%, assuming a gain of at least 20% knowledge between pre - and post test, the sample size estimated was 96. all medical students attending pharmacology in phase 2 of medical curriculum were considered for participation in the study. among those who consented, 88 students were finally included from the class since the others were not able to attend the initial session of the study. after students initially answered the questionnaire, analysis was done. after recognizing the areas of weakness and strengths, themes or sub - modules (dealing with above variables) subsequently, after a period of one month, the questionnaire was re - administered and then analyzed. an overall mean score was used for assessment of overall knowledge and mean scores also calculated for individual themes (sub - modules). all information regarding the purpose and process of study were explained through an information sheet and subsequently those who agreed were asked to sign an informed consent form. this module was developed keeping in mind the need for awareness of these issues for medical graduates as well as the areas that need to be strengthened as determined by the analysis of the questionnaire answers. the medication safety module was developed with four themes (sub - modules) as mentioned in tables 1 and 2 : knowledge about themes in medication safety (premodule) knowledge about topics in medication safety (premodule) adverse effects risks and meaning of adverse events, need for pharmacovigilance, clinical trials and relevance, medicine safety profile, drug interactions, genetic differences and risks in alternative systems of medicinesmedicines management medication errors, high risk medicines, toxicity of medicines, zero order kinetics and relevance, medicine misuse, storage issues and expiry issues, lasa (look alike sound alike)quality of medicines generics, innovators, bioequivalence, regulatory needs, counterfeit medicines, substandard medicines, process for recognition and reportingrational use of medicines rational use principles, irrational use consequences and factors, essential medicines list, formularies, standard treatment guidelines. adverse effects risks and meaning of adverse events, need for pharmacovigilance, clinical trials and relevance, medicine safety profile, drug interactions, genetic differences and risks in alternative systems of medicines medicines management medication errors, high risk medicines, toxicity of medicines, zero order kinetics and relevance, medicine misuse, storage issues and expiry issues, lasa (look alike sound alike) quality of medicines generics, innovators, bioequivalence, regulatory needs, counterfeit medicines, substandard medicines, process for recognition and reporting rational use of medicines rational use principles, irrational use consequences and factors, essential medicines list, formularies, standard treatment guidelines. each sub - module was taught in a 30 minute period with a cumulative time of two hours spent on the entire module. all the topics were framed based on what would be important to improve the safety profile of patients while on medicines, to improve the safety responsibility of the img and to encourage an integrated style of thinking and clinical management. the variables assessed were knowledge about adverse effects, quality of medicines, medication management, and rational use [table 1 ]. a questionnaire with 20 questions (maximum score of 20) was administered based on the variables mentioned before and after administering the module. each theme was represented by five questions (maximum score of five per theme). within each theme with a confidence level of 95% and power of 80%, assuming a gain of at least 20% knowledge between pre - and post test, the sample size estimated was 96. all medical students attending pharmacology in phase 2 of medical curriculum were considered for participation in the study. among those who consented, 88 students were finally included from the class since the others were not able to attend the initial session of the study. after students initially answered the questionnaire, analysis was done. after recognizing the areas of weakness and strengths, themes or sub - modules (dealing with above variables) were developed for content. subsequently, after a period of one month, the questionnaire was re - administered and then analyzed. an overall mean score was used for assessment of overall knowledge and mean scores also calculated for individual themes (sub - modules). all information regarding the purpose and process of study were explained through an information sheet and subsequently those who agreed were asked to sign an informed consent form. there were a total of 88 students (mean age 20.8 years) who participated of which 70 were female. the overall mean score for knowledge about medication safety (before educational module) was 9.52 (sd = 2.41) out of a maximum score of 20 (47.6%). the mean score across the themes appear to be similar though the weakest theme was quality of medications. the overall mean score for knowledge about medication safety after the educational module was 12.24 (sd = 2.23). the difference of 2.72 between pre and post module was statistically significant (p < 0.001). inspite of the huge burden due to medication errors and adverse effects of medicines, there has been relatively less emphasis on this area within medical education from a concept, approach and practical relevance point of view. a separate education module dealing with various medication safety issues deserves the space and time considering the potential harm that medicine can cause to patients. in our study, we found that overall knowledge about medication safety was only 47%. in actual practice this could translate into a situation where there could be a potential catastrophe with a medicine in every second patient. as students move into other subjects, the chance to revise pharmacology the student is therefore faced with a prospect of very little pharmacological knowledge and in particular about safety aspects. to ensure patient safety, the gap in knowledge has to be bridged with a safety module before their first prescription in internship. our study [table 1 ] showed that all of the themes in the safety module had a knowledge score between 2.26 to 2.55 (out of a maximum of 5). if one assumes that at least 50% knowledge is needed, based on the score of university exams, only the rational use theme appears to cross this cut off. however, even that score (2.55/5) is just above 50% which is not optimal. table 2 shows the correct response scores and percentage for each topic within a theme. the highest score was for pharmacovigilance possibly due to the recent spur in pharmacovigilance activity within hospitals as well as some emphasis within lectures on this topic. however it is important to note the need for doctors to be aware of these topics. firstly, doctors may need to instruct their own patients about how to store medicines, temperature and humidity issues etc. secondly, as leaders of the medical community and hospitals, storage concerns of medicines need to be looked into. lasa is an issue that is now on the list of compliance issues for hospital accreditation. also, mistakes in dispensing, administering or prescribing due to look alike or sound alike medications are often fatal. clinical trials have been again within the realm of the pharmaceutical industry and b pharm curriculum. it is important to understand that all new medications are born from clinical trials and many adverse effects may not be apparent during trials. a potential future practitioner therefore should be acquainted with these issues in order to make a judgment call on whether to use a new medicine or not. an increased score of approximately 28.7% demonstrates a reasonable level of effectiveness of the module. even though this is a statistically significant increase, it would have been ideal to have a much higher increase as some of the issues taught in the module could save lives of patients and ensure an overall better health profile. there are a number of factors which could have influenced a relatively low increase in score, factors such as lack of preparedness (contrary to an actual exam), co and extracurricular activities leading to lack of reading) and so on. however, it is important to remember that an increase in even this much knowledge, could potentially translate into reducing harm for patients in real life and possibly saving lives. the difference in scores between themes or topics was not analyzed since the number of questions representing each was not adequate for such an analysis. the study was done only at one point of time and on one set of students i.e., phase 2 in the mbbs curriculum. the post module washout period before questionnaire was re - administered was arbitrarily taken as one month to prevent recall bias. this would be important in the long run so that good habits and approach for safe prescribing could be instilled in the future indian medical graduate. in our study, we found that overall knowledge about medication safety was only 47%. in actual practice this could translate into a situation where there could be a potential catastrophe with a medicine in every second patient. as students move into other subjects, the chance to revise pharmacology the student is therefore faced with a prospect of very little pharmacological knowledge and in particular about safety aspects. to ensure patient safety, the gap in knowledge has to be bridged with a safety module before their first prescription in internship. our study [table 1 ] showed that all of the themes in the safety module had a knowledge score between 2.26 to 2.55 (out of a maximum of 5). if one assumes that at least 50% knowledge is needed, based on the score of university exams, only the rational use theme appears to cross this cut off. however, even that score (2.55/5) is just above 50% which is not optimal. table 2 shows the correct response scores and percentage for each topic within a theme. the highest score was for pharmacovigilance possibly due to the recent spur in pharmacovigilance activity within hospitals as well as some emphasis within lectures on this topic. however it is important to note the need for doctors to be aware of these topics. firstly, doctors may need to instruct their own patients about how to store medicines, temperature and humidity issues etc. secondly, as leaders of the medical community and hospitals, storage concerns of medicines need to be looked into. lasa is an issue that is now on the list of compliance issues for hospital accreditation. also, mistakes in dispensing, administering or prescribing due to look alike or sound alike medications are often fatal. clinical trials have been again within the realm of the pharmaceutical industry and b pharm curriculum. it is important to understand that all new medications are born from clinical trials and many adverse effects may not be apparent during trials. a potential future practitioner therefore should be acquainted with these issues in order to make a judgment call on whether to use a new medicine or not. an increased score of approximately 28.7% demonstrates a reasonable level of effectiveness of the module. even though this is a statistically significant increase, it would have been ideal to have a much higher increase as some of the issues taught in the module could save lives of patients and ensure an overall better health profile. there are a number of factors which could have influenced a relatively low increase in score, factors such as lack of preparedness (contrary to an actual exam), co and extracurricular activities leading to lack of reading) and so on. however, it is important to remember that an increase in even this much knowledge, could potentially translate into reducing harm for patients in real life and possibly saving lives. the difference in scores between themes or topics was not analyzed since the number of questions representing each was not adequate for such an analysis. the study was done only at one point of time and on one set of students i.e., phase 2 in the mbbs curriculum. the post module washout period before questionnaire was re - administered was arbitrarily taken as one month to prevent recall bias. this would be important in the long run so that good habits and approach for safe prescribing could be instilled in the future indian medical graduate. the module developed for medication safety is effective in improving knowledge to a certain extent. what is needed additionally is change in approach and attitude and a fundamental understanding of the need to use medicines appropriately in order to minimize the risk to the patient. towards this, the module will hopefully provide an initial platform and can spur a positive approach in improving the safety profile with regard to medicine use. it is also hoped that the findings of this study will encourage policy makers in both medical education and healthcare practice to the need for specific learning modules in the area of medication safety within the medical curriculum and also continuing medical education programs.	objective : a rising number of medicines and minimal emphasis on rational prescribing in the medical curriculum may compromise medication safety. there is no focused module in the curriculum dealing with factors affecting safety such as quality, medicines management, rational use, and approach to adverse effects. creating awareness of these issues would hopefully plant a seed of safe prescribing and encourage pharmacovigilance. a study was therefore done to determine the need for such a module.method:a quasi - experimental pre - post module study. medical students (n = 88) completing pharmacology term were recruited after informed consent. a questionnaire containing 20 questions on various themes was administered and scored. subsequently a module was developed and relevant safety themes taught to the students. after one month, the questionnaire was re-administered.results:the pre module score was 9.52/20. knowledge about the various themes, adverse effects, medication management, quality issues and rational use were similar though poor knowledge was evident in specific areas such as clinical trials, look alike - sound alike medicines (lasa) and medicine storage. the post module score was 12.24/20. the improvement of score was statistically significant suggesting the effectiveness of the module.conclusion:the relatively poor knowledge and improvement with a specific educational module emphasizes the need of such a module within the medical curriculum to encourage safe use of medicines by indian medical graduates (img). it is hoped that the policy makers in medical education will introduce such a module within the medical curriculum.
the source population for the current study was the entire population of denmark aged 15 to 90 years at risk for suicide between january 1st, 1994 and december 31st, 2006. participants had to reside in denmark for the entire calendar year before suicide (or the match date in control participants) to allow us to collect complete data on social variables. the primary criterion for identification as a case in the study we matched up to 30 controls from a 25% representative sample of the danish population to suicide cases on gender, date of birth, and calendar time, yielding 199,306 controls. if 30 or fewer matched controls were available, we selected all available controls. if more than 30 controls matched a case, then we randomly selected 30 from those available. the cause - of - death register contains information on cause and date of death, and was used to identify suicide cases.7 the danish psychiatric central register contains dates on outpatient and inpatient psychiatric treatment in denmark, as well as validated diagnoses, and was used to obtain data on adjustment disorder and psychiatric covariate diagnoses.8 finally, we used the civil registration system for control participant selection,9 and we used the integrated database for labour market research to obtain data on social variables.7 we linked these registries using the civil registration number, which is assigned to all citizens and residents of denmark. appendix 1 lists the variables involved in the current analyses and their international classification of disease codes.10,11 suicide is classified as a cause of death in denmark as follows. this inquest involves collecting information about the death itself and the deceased from the deceased s physician, family and friends, and the person(s) who discovered the body. information is collected about place of death, method of dying, and presence of a suicide note. if the cause of death is still uncertain following this inquest, an autopsy is conducted. the final determination of death from suicide is made by the independent forensic doctor based on information from all of these sources. this process has been described in more detail elsewhere.12 we included only psychiatric diagnoses that occurred before the first adjustment disorder diagnosis as potential confounders and modifiers, as it is possible that these disorders are on the causal pathway between adjustment disorder and suicide. in addition, we used only primary diagnoses to identify both adjustment disorder and psychiatric covariates. psychiatic covariates included depression, substance abuse, and anxiety disorders. for demographic covariates, we assessed marital status, income, and geographic location. marital status was recorded as of november in the calendar year before suicide date or match date for control participants. income was recorded in the calendar year before suicide date or match date for control participants, and was categorized into quartiles within strata of sex and age groups. we calculated the frequency and proportion of cases and controls within categories of the exposure and covariates. we used conditional logistic regression used to estimate the association between adjustment disorder diagnosis and completed suicide. as risk set sampling was used to sample control participants, the odds ratios obtained from these conditional logistic regression analyses provide unbiased estimates of the corresponding rate ratios.13 we used change - in - estimate methods as a model building strategy to select confounders from the candidate psychiatric and demographic covariates listed above.14 to address potential misclassification of psychiatric disorders, we conducted a bias analysis to assess the impact of residual confounding by depression, substance abuse, and anxiety disorders.15 we calculated the corrected estimates using a sensitivity of 50%, 70%, and 90% and assumed that misclassification was nondifferential and independent for cases and controls because the data were recorded prospectively. in addition, we assumed that specificity would be perfect, given the extensive process for psychiatric disorder validation that occurs in denmark. finally, because cases may have been treated in a psychiatric facility more than controls before committing suicide, and therefore would have more accurate psychiatric disorder confounder classification, we conducted a bias analysis to examine differential imperfect sensitivity of confounder classification among cases and controls. for this analysis, we used a sensitivity for cases of 0.92 (the minimum sensitivity that resulted in nonnegative cells in the corrected tables) and a sensitivity for controls of 0.5. we used sas software (version 9.1.3 ; sas institute, cary, nc) to conduct all analyses. the source population for the current study was the entire population of denmark aged 15 to 90 years at risk for suicide between january 1st, 1994 and december 31st, 2006. participants had to reside in denmark for the entire calendar year before suicide (or the match date in control participants) to allow us to collect complete data on social variables. the primary criterion for identification as a case in the study we matched up to 30 controls from a 25% representative sample of the danish population to suicide cases on gender, date of birth, and calendar time, yielding 199,306 controls. if 30 or fewer matched controls were available, we selected all available controls. if more than 30 controls matched a case, then we randomly selected 30 from those available. the cause - of - death register contains information on cause and date of death, and was used to identify suicide cases.7 the danish psychiatric central register contains dates on outpatient and inpatient psychiatric treatment in denmark, as well as validated diagnoses, and was used to obtain data on adjustment disorder and psychiatric covariate diagnoses.8 finally, we used the civil registration system for control participant selection,9 and we used the integrated database for labour market research to obtain data on social variables.7 we linked these registries using the civil registration number, which is assigned to all citizens and residents of denmark. appendix 1 lists the variables involved in the current analyses and their international classification of disease codes.10,11 suicide is classified as a cause of death in denmark as follows. after any unnatural death, independent forensic medical doctors conduct an inquest. this inquest involves collecting information about the death itself and the deceased from the deceased s physician, family and friends, and the person(s) who discovered the body. information is collected about place of death, method of dying, and presence of a suicide note. if the cause of death is still uncertain following this inquest, an autopsy is conducted. the final determination of death from suicide is made by the independent forensic doctor based on information from all of these sources. this process has been described in more detail elsewhere.12 we included only psychiatric diagnoses that occurred before the first adjustment disorder diagnosis as potential confounders and modifiers, as it is possible that these disorders are on the causal pathway between adjustment disorder and suicide. in addition, we used only primary diagnoses to identify both adjustment disorder and psychiatric covariates. marital status was recorded as of november in the calendar year before suicide date or match date for control participants. income was recorded in the calendar year before suicide date or match date for control participants, and was categorized into quartiles within strata of sex and age groups. we calculated the frequency and proportion of cases and controls within categories of the exposure and covariates. we used conditional logistic regression used to estimate the association between adjustment disorder diagnosis and completed suicide. as risk set sampling was used to sample control participants, the odds ratios obtained from these conditional logistic regression analyses provide unbiased estimates of the corresponding rate ratios.13 we used change - in - estimate methods as a model building strategy to select confounders from the candidate psychiatric and demographic covariates listed above.14 to address potential misclassification of psychiatric disorders, we conducted a bias analysis to assess the impact of residual confounding by depression, substance abuse, and anxiety disorders.15 we calculated the corrected estimates using a sensitivity of 50%, 70%, and 90% and assumed that misclassification was nondifferential and independent for cases and controls because the data were recorded prospectively. in addition, we assumed that specificity would be perfect, given the extensive process for psychiatric disorder validation that occurs in denmark. finally, because cases may have been treated in a psychiatric facility more than controls before committing suicide, and therefore would have more accurate psychiatric disorder confounder classification, we conducted a bias analysis to examine differential imperfect sensitivity of confounder classification among cases and controls. for this analysis, we used a sensitivity for cases of 0.92 (the minimum sensitivity that resulted in nonnegative cells in the corrected tables) and a sensitivity for controls of 0.5. we used sas software (version 9.1.3 ; sas institute, cary, nc) to conduct all analyses. suicide cases were primarily male (71%), single (58%), and in the lowest income quartile (42%). the initial conditional logistic regression analysis, adjusting for the control - to - case matching on gender, date of birth, and calendar time, revealed that those diagnosed with adjustment disorder had 19 times the rate of suicide of people without this preceding diagnosis (95% confidence interval [ci ] : 1721). using the change - in - estimate model building method, we identified depression, marital status, and income as confounders of this association. those with adjustment disorder had 12 times the rate of suicide as those without a preceding adjustment disorder, when controlling for these variables in addition to the matched factors (95% ci : 1114). we conducted a bias analysis to evaluate whether residual confounding by psychiatric covariates would affect the observed associations between adjustment disorder and suicide, assuming a valid bias model and the accuracy of the values assigned to the bias parameters. at all levels of sensitivity tested, an association between adjustment disorder and suicide remained. finally, we conducted a bias analysis to explore differential misclassification of psychiatric covariates among cases and controls, given that cases may have been treated in a psychiatric facility more than controls before committing suicide, and may therefore have a more accurately recorded psychiatric history (table 3). assuming a valid bias model, and accurate values assigned to parameters, it does not appear from these results that differential sensitivity of classification of psychiatric disorder confounders among cases and controls would have a significant impact on the results. consistent with our hypotheses, we found that adjustment disorder was associated with an increased rate of completed suicide, controlling for history of depression, income, marital status, and the matched factors. possible mechanisms that could account for this observed association include subsyndromal psychiatric symptoms, particularly depressed mood, which are associated with suicidal ideation among adolescents and with adjustment disorder.16 in addition, stressful events that may result in an adjustment disorder diagnosis, which were not assessed in the current study, are associated with suicidal behavior among adolescents and young adults.17 the current investigation has limitations worth noting. first, we restricted psychiatric covariates to the period before adjustment disorder diagnosis and only used primary diagnoses from the psychiatric central register (and not secondary diagnoses). it is possible that there is residual confounding of our observed adjustment disorder and suicide associations due to underascertainment of comorbid psychiatric illness. because of this concern, we conducted a bias analysis, and found that even with a sensitivity of confounder classification of 50%, the associations between adjustment disorder and suicide could not be explained by this type of misclassification. second, it is possible that cases were treated in a psychiatric facility more frequently before committing suicide, and therefore would have better psychiatric confounder documentation than controls., it appears that this differential misclassification would not have had a substantial impact on the results of the current study. finally, we only included psychiatric diagnoses that were obtained at a psychiatric setting in denmark. it is possible to be diagnosed with a psychiatric disorder at a somatic hospital in denmark, and therefore the exposed participants in the current study may represent more severe cases of adjustment disorder, which may in turn be more strongly associated with suicide. we examined the number of adjustment disorder cases with a primary discharge code from somatic hospitals in the same time period, and there were too few to conduct a separate set of analyses on this group to see if the pattern of results was different from those presented here. despite these limitations, the current study is the first to report on the relation between adjustment disorder and completed suicide. research to date has focused on the association between adjustment disorder and suicidal ideation or suicidal attempts. the current study examined the association between adjustment disorder and completed suicide using a population - based sample and prospectively collected data. the results of this study highlight completed suicide as an important potential sequelae of adjustment disorder diagnosis	adjustment disorder is a diagnosis given following a significant psychosocial stressor from which an individual has difficulty recovering. the individual s reaction to this event must exceed what would be observed among similar people experiencing the same stressor. adjustment disorder is associated with suicidal ideation and suicide attempt. however the association between adjustment disorder and completed suicide has yet to be examined. the current study is a population - based case control study examining this association in the population of denmark aged 15 to 90 years. all suicides in denmark from 1994 to 2006 were included, resulting in 9,612 cases. for each case, up to 30 controls were matched on gender, exact date of birth, and calendar time, yielding 199,306 controls. adjustment disorder diagnosis was found in 7.6% of suicide cases and 0.52% of controls. conditional logistic regression analyses revealed that those diagnosed with adjustment disorder had 12 times the rate of suicide as those without an adjustment disorder diagnosis, after controlling for history of depression diagnosis, marital status, income, and the matched factors.
benign prostatic hyperplasia (bph) is the most common urological problem associated with aging in men. one - quarter of men in their 50s, one - third in their 60s, and half of men older than 80 have bph. bph is characterized by hyperplasia of prostatic stromal and epithelial cells, and it manifests as a severe obstruction in urinary flow with discomfort and pain. the pathogenesis of bph is not completely understood ; however, the most significant risk factors for the development of bph are androgen level and aging. growth factors and their receptors, including members of the fibroblast growth factor (fgf) family, insulin - like growth factor (igf) family, epithelial growth factor (egf) family, and transforming growth factor (tgfb), which regulate the growth of prostatic stromal and epithelial cells, are also involved in the pathogenesis of bph [35 ]. genetic strategies have been used over the past few decades to investigate bph. in particular, studies have shown that polymorphisms of growth factors and their receptor genes are associated with bph. indeed, previous studies have reported that a codon 10 polymorphism in tgfb1 was associated with the development of bph in japanese and iranian populations, suggesting the importance of the tgf pathway in the development of prostatic diseases. mullan. reported a significant association of a codon 10 polymorphism in tgfb1 with treatment for bph, and an association of a ca - repeat polymorphismin egfr with international prostate symptom score (ipss) in bph. moreover, a ca - repeat polymorphism of 19-allele in igf1 appears to increase the risk of bph with a gene dosage effect in the japanese population. however, despite a widespread consensus on the involvement of growth factors in prostatic growth, attempts to address growth factor gene polymorphisms in patients with bph have been limited. fgfs are involved in multiple biological processes such as differentiation, motility, and proliferation and mediate their cellular responses by activating a family of four receptor tyrosine kinases, fgfr1 through fgfr4. are abundantly expressed in the prostate stroma and/or epithelium [1114 ], and fgfr4 has relatively low expression in the epithelium. in the prostate, fgfrs play an important role in prostate organogenesis, and perturbations in fgfr expression have been potently implicated in prostatic disease. previous studies have reported that overexpression of fgfrs is important in the development of bph [1618 ]. indeed, fgfr1 was observed to be increased in the stroma of bph patients compared to that from normal prostates [16, 17 ], and increased expression of fgfr2 has been detected in bph. furthermore, although no studies have reported changes in expression of fgfr4 in bph, a genetic study showed a significant association between fgfr4 polymorphisms and bph in a japanese population. in light of these findings, we postulated that fgfrs may be involved in the pathogenesis of bph. in this study, we investigated the genetic associations between bph and the fgfr genes (fgfr1 and fgfr2) in korean bph patients by analyzing single nucleotide polymorphisms (snps) of the fgfr genes. a total of 233 male patients with bph (mean age standard deviation, 65.77 9.46 years) and 213 male control subjects (mean age, 61.89 8.26 years) were enrolled. all patients with bph were from kyung hee university hospital between january 2002 and december 2008, and in all patients, lower urinary tract symptoms were quantified using ipss. uroflowmetry was performed to measure peak urinary flow rate (qmax) for all patients. serum prostate - specific antigen (psa) level was measured in all bph patients. patients with serum psa level more than 4 ng / ml underwent transrectal ultrasound - guided prostate biopsy to rule out prostate cancer. patients with prostate cancer, neurogenic bladder, urinary tract infection, uncontrolled diabetes mellitus, or cardiovascular disease were excluded. the clinical characteristics of bph patients are summarized in table 1. to determine the relationship between polymorphisms of fgfr genes and clinical phenotypes, bph patients were divided into subgroups according to prostate volume (0.05). differences in genotype distributions and allele frequencies for the four snps between bph and control were analyzed. as shown in table 2, rs1047100 of fgfr2 was associated with bph in the additive (ga versus aa versus gg ; p = 0.024, or = 1.92, 95% ci = 1.073.43) and dominant models (ga / aa versus gg ; p = 0.034, or = 1.89, 95% ci = 1.043.45). allele frequency analysis also showed an association between rs1047100 and bph (p = 0.029, or = 1.85, 95% ci = 1.063.23). we further analyzed the associations between snps and the clinical phenotypes of bph (prostate volume, psa level, ipss, and qmax). in analysis according to small or large prostate volume (< 30 or 30 ml), we found that rs1047100 of fgfr2 was significantly associated with prostate volume in the additive (ga versus aa versus gg ; p = 0.016, or = 0.43, 95% ci = 0.210.87) and dominant models (ga / aa versus gg ; p = 0.010, or = 0.38, 95% ci = 0.180.81) (table 3). the frequency of genotypes containing the a allele was lower in bph patients with large prostate volume (ga = 9.6%, aa = 0.8%), compared to those with small prostate volume (ga = 21.5%, aa = 0.9%). although allele frequency analysis also revealed that rs1047100 was associated with prostate volume (p = 0.021, or = 0.45, 95% ci = 0.230.89), this result was not significant after bonferroni correction. we also found a significant association between rs13317 of fgfr1 and ipss in an analysis according to low or high ipss (< 20 or 20). as shown in table 4, rs13317 of fgfr1 was associated with ipss in the additive (tc versus cc versus tt, p = 0.0022, or = 0.50, 95% ci = 0.320.79) and dominant models (tc / cc versus tt, p = 0.005, or = 0.43, 95% ci = 0.230.78). allele frequency analysis also revealed that rs13317 of fgfr1 was associated with ipss (p = 0.005, or = 0.55, 95% ci = 0.360.84). in particular, the frequency of the c allele was decreased in bph patients with high ipss (30.3%) compared to those with low ipss (44.2%). these results remained significant after bonferroni correction. in analysis according to other clinical phenotypes (psa level and qmax), we were not able to find any association of polymorphisms in the fgfr genes (data not shown). we examined the association of polymorphisms of fgfr1 and fgfr2 with bph and its clinical phenotypes in a korean population. however, in analysis according to clinical phenotypes, we found associations between rs1047100 of fgfr2 and prostate volume and between rs13317 of fgfr1 and ipss. bph is a progressive disease found in many men, and numerous factors, including androgen level and aging, have been linked with the risk of bph progression [2224 ]. prostate volume is the most extensively studied risk factor for bph progression [25, 26 ]. indeed, it was reported that men with prostate volume 30 ml were more likely to suffer moderate - to - severe symptoms (3.5-fold increase), decreased flow rates (2.5-fold increase), and acute urinary retention (3- to 4-fold increase), compared to men with prostate volume < 30 ml. reduced urinary flow, increased ipss, and increased psa have been also suggested as predictors of bph progression. clinical study demonstrated that men with prostate volume 31 ml, psa 1.6 ng / ml, or qmax < 10.6 ml / sec at baseline had a significantly increased risk of overall clinical progression of bph. although ipss and psa are simple bph diagnostic factors used in the primary care setting, a previous study showed a high correlation between bph diagnosed by simple tests (medical history, ipss, digital rectal examination (dre), and psa) and that diagnosed by a full battery of tests including ultrasonographic assessment of residual and prostatic volume, and uroflowmetry. thus, these factors may also be useful as predictors of bph progression. in our study, although polymorphisms of fgfr1 and fgfr2 were not associated with psa level or qmax in bph patients, rs1047100 of fgfr2 and rs13317 of fgfr1 were associated with prostate volume and ipss, respectively. these results indicated that polymorphisms of fgfr1 and fgfr2 may be related to the severity of bph and implicated in the progression rather than the incidence of bph. in particular, we found that the frequency of genotype containing the minor allele, a, of rs1047100 in fgfr2 was lower in bph patients with large prostate volume than in those with small prostate volume. in addition, the frequency of the minor allele, c, of rs13317 in fgfr1 was significantly decreased in bph patients with high ipss. these finding indicated that patients with genotypes containing the a allele of rs1047100 or the c allele of rs13317 may be protected from severe progression of bph. previous studies reported that fgfrs were abundantly expressed in the normal prostate [1114 ], and that the expression of fgfr1 and fgfr2 was elevated in prostates of bph patients [1618 ]. to our knowledge, there are no reports indicating that the expression of fgfr1 or fgfr2 is upregulated or downregulated according to the alleles of rs1047100 and rs13317. however, rs13317 could be involved in regulating expression considering that it is located in the 3utr which modifies stability and transport of mrna as well as translation efficiency, and whose snps are well known to increase the efficiency of 3 end processing. in addition, although rs1047100 is synonymous snp, recent studies reported that synonymous snps play an important role in protein activities and specificities without influencing amino acid sequences [33, 34 ]. thus, we postulated that fgfr1 and fgfr2 might be overexpressed in bph but that the expression of fgfr1 and fgfr2 might be relatively low in bph patients with genotypes containing the a allele of rs1047100 or the c allele of rs13317 compared to bph patients without those. furthermore, a previous study showed that rs13317 of fgfr1, which plays a role in wound healing and is a positive regulator of skeletal formation, was associated with delayed bone healing after bone fracture and, in particular, that the frequency of the c allele of rs13317 in fgfr1 was increased in individuals with delayed bone healing compared to those with uneventful healing. however, in that study, no significant associations were observed between fgf polymorphisms and delayed bone healing. thus, they suggested the possibility that specific alterations in the receptor, despite fgfs functions, may be involved in triggering the pathologic process during fracture healing. given this report, we also postulated that rs13317 may affect the activity of fgfr1. thus, fgfr1 activity may be relatively decreased in bph patients with the c allele of rs13317, resulting in lower and slower progression of bph in those individuals. further studies are needed to determine how fgfr1 and fgfr2 polymorphisms affect their expression and/or activity in bph progression. the major limitation of our study was the small sample size used for comparison within bph subgroups. however, this is the first genetic study on the relationship between snps of fgfr1 and fgfr2, and bph. our results revealed associations between fgfr1 and fgfr2, and the clinical phenotypes of bph. in conclusion, we found that polymorphisms of fgfr1 and fgfr2 were not associated with bph. however, clinical analysis revealed that a fgfr2 polymorphism was associated with prostate volume and a fgfr1 polymorphism was associated with ipss in bph patients. these results suggest that fgfr1 and fgfr2 may be related to bph severity and progression.	fibroblast growth factors (fgfs) and their receptors (fgfrs) have been implicated in prostate growth and are overexpressed in benign prostatic hyperplasia (bph). in this study, we investigated whether single nucleotide polymorphisms (snps) of the fgfr genes (fgfr1 and fgfr2) were associated with bph and its clinical phenotypes in a population of korean men. we genotyped four snps in the exons of fgfr1 and fgfr2 (rs13317 in fgfr1 ; rs755793, rs1047100, and rs3135831 in fgfr2) using direct sequencing in 218 bph patients and 213 control subjects. no snps of fgfr1 or fgfr2 genes were associated with bph. however, analysis according to clinical phenotypes showed that rs1047100 of fgfr2 was associated with prostate volume in bph in the dominant model (ga / aa versus gg, p = 0.010). in addition, a significant association was observed between rs13317 of fgfr1 and international prostate symptom score (ipss) in the additive (tc versus cc versus tt, p = 0.0022) and dominant models (tc / cc versus tt, p = 0.005). allele frequency analysis also showed significant association between rs13317 and ipss (p = 0.005). these results suggested that fgfr genes could be related to progression of bph.
haemoglobin (hb) abnormalities are the most frequent genetic disease, affecting approximately 7 per cent of the world population1. today, abnormal hbs are generally discovered during a systematic study performed within programmes for prevention of thalassaemias or sickle cell disease. in several regions (india, turkey, irak, iran, gaza strip, saudi arabia, cyprus, etc.), these are found during a premarital screening. in other regions, like west europeans countries, the research for the main hb disorders is often limited to populations at risk. it is done either as preconceptional or neonatal screening programmes. in all cases, besides the study of the hb, a first essential test that need to be performed is a complete blood cell count (cbc) looking mostly for anaemia, microcytosis and, hypochromia. if necessary, it should be completed by a control of the blood iron status. several high throughput methods, now available, such as cation - exchange high performance chromatography (ce - hplc)23 or more recently capillary electrophoresis (ce)4 are generally employed. nevertheless, electrophoretic studies [cellulose - acetate electrophoresis or isoelectric focusing (ief) ] are still performed in many laboratories5. in some developing countries, the naked eye single tube red cell osmotic fragility test (nestroft), a cost effective, rapid and reliable screening test for detection of -thalassaemia trait, is largely used as a first approach6. the investigation of a patient presenting with a haematological disorder such as a chronic haemolytic anaemia, an unexplained polycythaemia or a permanent cyanosis is another, completely different condition, leading to the discovery of an abnormal hb. in this review, we will discuss (i) the methods used for characterization of haemoglobin disorders ; (ii) the problems linked to diagnosis of thalassaemic trait ; (iii) the strategy for detection of common hb variants ; and (iv) the difficulties in identification of rare variants. electrophoresis, a test based on the migration of electrically charged molecules under an applied electric field, occupies one of the most important places in the history of abnormal hb detection. hb s [6 glu > val ], the first abnormal hb described, was discovered in 1949 by pauling using moving boundary electrophoresis7. zone electrophoresis performed on cellulose acetate strips (cae) is still used in many clinical laboratories. this technique has a resolution lower than that of ief, but because of its simplicity remains among the more popular methods used in hb screening. in this technique, hb s, which has an additional positive charge compared to hb a migrates more slowly5. in ief, a ph gradient is established by carrier ampholytes submitted to an electric current and the hb molecules migrate across this gradient until they reach the position where their net charge is zero (isoelectric point)8. 1. this technique allows separating molecules with isoelectric points differing only by 0.02 ph unit. hb d - punjab [121 glu > gln ], not detectable from hb s by cae, could easily be recognized by this technique. the disadvantage of this method is its relatively high cost and the requirement for a well - experienced laboratory staff. isoelectric focusing analysis of red blood cells lysates containing different hb variants, technical details are available in ref. line 1 : thalassaemia trait ; line 2 : normal adult ; line 3 : homozygous thal ; line 4 : hba / hb korle bu ; line 5 : hba / hbd - punjab ; line 6 : hba / hbo - arab ; line 7 : normal newborn ; line 8 : hba / hbe ; line 9 : hba / hbc ; line 10 : hba / hbs ; line 11 : hbs / hbc. electrophoresis of hbs on agar at acidic ph has been introduced some forty years ago9. it is not primarily sensitive to the charge of the mutated residue but to structural modifications of positively charged regions of the hb molecule interacting with the agaropectin contained in the gel10. this property is of special interest to distinguish hb s from other variants displaying mobility close to that of hb s on cae or ief. this technique shares several advantages with ce - hplc : it is a high resolution method associated with semi - automation, on - line detection and direct quantification of normal and abnormal hb fractions. the capillarys system (sebia, evry, france) is equipped with eight capillaries in parallel, allowing multiple and simultaneous sample analysis. cation - exchange hplc is considered as the method of choice to quantify the various normal and abnormal hb fractions13. this is the method of reference for measuring hb a1c for monitoring of diabetes mellitus. it is also of general use for measurement of the levels of hb a2, hb f and of several abnormal hbs23. cation - exchange hplc and ce are the two methods recommended for detection of thalassaemic traits necessary for genetic counselling since these are the ones allowing a precise quantification of hb a2, hb f and abnormal hb fractions. to make the diagnosis of a thalassaemic trait or quantify the abnormal hb fraction, the use of densitometry scanning of electrophoretic pattern is a too imprecise method to be used today. the first one is an adaptation of ief which allows the simultaneous study of about 90 samples on a single plate, using a central cathode and two anodes14. at a glance, any band with abnormal mobility is detected as well as the absence of the normal hb a band. this method is considered as a first line test and requires a confirmation by a second one, usually ce - hplc. cation - exchange - hplc equipment devoted to neonatal screening have been developed which, in a 2.5 min programme per sample, allow quantitative detection of hb s and of the most common hb variants15. this approach by ce - hplc is also suitable for neonatal diagnosis of thalassaemic patients16. characterization of rare hb variants responsible for a haematological disorder requires a completely different approach. the presence of a variant is usually obtained by ief, which shows either a band with a clearly different isoelectric point or an abnormal aspect of the hb a band suggesting that it contains two fractions (fig. 1). cation - exchange hplc or ce may confirm the presence of this abnormal component or reveal an abnormal hb, not detected by ief. for example, reversed - phase high - performance liquid chromatography (rp - hplc) reveals differences in hydrophobicity allowing discrimination between variants displaying identical charges. this approach also indicates which globin chain is affected18. neonatal diagnosis for hb s and hb d - punjab. analysis of rbc lysates obtained from a dried blood spot obtained during the neonatal screening programme done in our laboratory. (a) isoelectric focusing : hb s and hb d - punjab have close isoelectric points and the resolution between the two bands is poor. the absence of hba and thickening of hbs band help to diagnose. (b) cation - exchange hplc shows two abnormal peaks : one is eluted in s window and the other in the d window. (c) capillary electrophoresis : (above) : newborn heterozygous for hb d - punjab and (below) : newborn heterozygous for hb s. the two hbs have different migrations. in well - equipped laboratories of industrialized countries mass spectrometry methods may be used to determine the structural abnormalities. electrospray mass spectrometry may reveal a globin chain with an abnormal mass and, from the mass difference observed, the type of amino acid exchanged could often be deduced. methods such as maldi - tof or mass - mass spectrometry in which the globin chains are cleaved in small fragments will show a single fragment, or a family of fragments from which the exact substitution could be determined1920. today, the facility for doing a dna sequence analysis and its relatively low cost changed completely the approach for characterization of some rare hb variants associated with a severe pathological phenotype not detected by conventional methods. there are many different pcr - based techniques that can be used to diagnose the globin gene mutations, including dot - blot analysis, reverse dot - blot analysis, the amplification refractory mutation system (arms), high resolution melting (hrm), gap - pcr and restriction endonuclease analysis2122. the key element in the diagnosis of a -thalassaemia trait is the presence of non - iron deficient microcytic hypochromia anaemia. the screening of thalassaemia trait in the areas with limited laboratory facilities is often done by nestroft test6. despite its sensitivity and rapidity, in around one out of four cases of iron deficiency anaemia the best approach to screen the thalassaemia traits is, therefore, determining the hb a2 level. the large majority of -thalassaemia carriers present with a high hb a2 level, and this often accompanied with a moderate increase in hb f level25. hb a2 level is correctly measured by ce - hplc but it is necessary to keep in mind that the normal value depends upon the experimental procedures used26. from one chromatographic system to another slight differences may be observed and values within the normal range in a given system may well fall out of the normal range in another system. thus the normal values of the laboratory where the test is performed should always be specified. the most frequent problem is the co - existence of an iron deficiency which may even normalize the hb a2 level requiring a novel hb assay after iron deficiency treatment. in -thalassaemia carriers presenting with a normal hb a2 level the thalassaemic alleles are not exceptional affecting at least more than one per cent of general population27. the most frequent thalassaemic variant is hb yialoussa [27 ala > ser ] which is quite frequent in mediterranean region28. other - and one should add the two hb a2 fractions to obtain the total hb a2 level. in some cases the abnormal hb a2 fraction may only be visualized by using another analytical test. conversely, falsely increased levels of hb a2 may result from the co - existence of a variant with electrophoretic or chromatographic properties close to that of hb a2. as a rule, this situation has to be verified when a level of hb a2 higher than 8 per cent is observed. the presence of a thalassaemia will generally be ruled out by considering haematological data showing absence of microcytosis and hypochromia. nevertheless, two common variants, which elute on ce - hplc in the hb a2 window, are really thalassaemic hbs. the first one is hb e, frequent in populations from south east asia, and the second is hb lepore. both variants are easy to distinguish by their electrophoretic behaviour. since many other thalassaemic defects are present in the same populations as hb e, a large variety of clinical phenotype associated with hb e is observed29. these range from very mild ones, the simple heterozygous state for hb e, to severe ones, which associate hb e to thalassaemia. another important element for the diagnosis of a -thalassaemia trait is the presence of hb f. in the past, the classical way to determine its percentage was to perform a resistance to alkali denaturation test. as other kinetic techniques, it needs to be performed under rigorous experimental conditions with a precise respect of the timing of the reaction30. this method measures together hb f and its acetylated fraction, which amounts to about 10 per cent of it. now ce - hplc and ce allow a direct measurement of the hb f fractions. depending on the system used, some other hb adducts may elute together with hb f and increase slightly the area of the elution peak. in these systems acetylated hb f elutes generally in another position than hb f and is therefore, not included in the result. thus, in the same sample the percentage of hb f will differ according to the method used. this is not really important since in an adult any value of hb f higher than 1 per cent should be considered as unusual. increased hb f level may indicate a -thalassaemia trait but it could also be the result of a hereditary persistence of foetal hb (hpfh) or of another condition, such as diabetes mellitus, or pregnancy. diagnosis of an thalassaemia will be suspected by haematological indices showing some microcytosis and hypochromia, with a degree depending upon the type of thalassaemia. the hb a2 percentage is theoretically decreased in proportion with the number of defective genes. abnormal hb tetramers are observed in the more severe forms : hb bart 's (4) in newborns, and hb h (4) in adults. the presence of an thal-2 in which only one of the four genes is inefficient is one of the most common genetic modifications observed worldwide. an interaction of this situation with other globin abnormalities should always be considered as a possible modulator factor31. in a carrier of a - thalassaemic trait the co - existence of an -thalassaemia will partially compensate the disequilibrium in globin synthesis ; due to a lesser amount of toxic free globin chain bound to the red cell membrane, the hypochromic anaemia will be much better tolerated. conversely the association of a -thalassaemic trait with additional genes may lead to a -thalassaemia intermedia phenotype32. it is specially encountered in populations from african ancestry, essentially carried on chromosomes with senegal, benin or bantou haplotypes33. in india and arabic countries, hb s has frequently another origin, since in many cases it is found in a haplotype named arabo - indian and its characteristic is to be linked to a relatively high hb f level. in these regions, the hb s allele being much less frequent than in africa, the patients who appear as homozygous are frequently compound heterozygous for hb s and a thalassaemia. the allele in homozygous status is mostly found in some tribal populations of india34. if ief is still used in many laboratories, it becomes the rule to be accomplished by ce - hplc or ce. since hb s shares its electrophoretic and chromatographic properties with several variants it is necessary, in the absence of familial history of hb s, to do a confirmatory test. in the past, the solubility test was considered as the golden standard since hb s is the only abnormal hb, which is insoluble at high concentrations when deoxygenated. this test is now commercially difficult to get, and the large majority of the clinical laboratories do not want to prepare their own reagents. a good compromise is to perform ce - hplc and ce, or acid agar electrophoresis. only in some exceptional cases in whom another mutation is associated in cis with that of hb s the heterozygous carriers can also present a severe pathologic phenotype. examples are provided by the heterozygous carriers of hb s - antilles [6 glu > val and 23val > ile]35 or hb s - oman [6glu > val and 121glu > lys]36 in whom the second mutation enhances sickling. recently, an unstable allele () associating in cis the allele to a mutation causing instability of the haemoglobin was described37. a few common variants, which lead to sickle cell anaemia when associated to hb s have to be imperatively characterized, e.g. hb d - punjab and o - arab. both affect position 121 (gh4) where the glu is replaced by a gln in the first case, and by a lys in the second one. this residue is located at the outside of the hb tetramer in a region, which participates in the building of the hb s fiber during sickling. these two mutations facilitate polymerization and, as a result, compound heterozygotes may be, clinically, as severe as homozygous hb s. hb d - punjab can not be easily recognized from hb s by using conventional cae. for this method the same is true for hb o - arab that may be confused with hb e or hb c. in contrast, ce - hplc or ce allows an easy identification of these two variants. since hb d - punjab is the only variant with hb d mobility, which leads to a severe disease when associated with hbs, its identity must be recognized. other variants such as hb d - iran, hb korle - bu are completely harmless. 3 shows ce - hplc elution profiles of carriers of hb o - arab in various different compound heterozygous states. in the heterozygous or homozygous forms, hb d - punjab or o - arab have no clinical consequences but these need to be identified in programmes for prevention of sickle cell anaemia. the individuals compound heterozygous for one of these abnormalities and a -thalassaemia allele are haematologically identical to a simple -thalassaemia carrier. ce - hplc : analysis of haemolysates from patients presenting with different associations involving hb o - arab. (a) compound heterozygous hb o - arab/thalassaemia. presence of hb s in homozygous or compound heterozygous with hb variants, involved in sickling, modifies the rbc density distribution which can be studied easily using phthalate density gradient3839. 4 illustrates the different profiles that could explain the clinical heterogeneity of patients presenting with sickle cell anaemia associated with different genotypes. it has to be pointed out that this simple and low cost technique may be helpful to diagnose different or -thalassaemia associated to other hb variants. red blood cell density profiles of patients with hb s associated to various genotypes (technical details are available in ref. the percentage of cells denser than the phthalate index (y axis) is plotted against the density of the phthalate mixtures (x axis) used expressed in g / ml. (a) homozygous hb s. dense cells and young cells (reticulocytes) are observed at the right and left side of the distribution, respectively. (c) compound heterozygous hb s / hb c. (d) homozygous hb s associated to an thal-2. the curve is shifted to the left when hb s is associated to a thalassaemic allele. the first one, which originates from the region of upper volta is frequent in africa, while the second one is common in south east asia40. since, both result from the replacement of a glutamic acid by a lysine these variants share some electrophoretical properties and it is difficult to distinguish these by cae. the diagnosis between hb c and hb e can be established by ce - hplc and ce, which show a different profile for these two variants. in thailand several cases, initially considered as hb e, were characterized finally as hb c using above mentioned techniques41. hb c and hb e both lead to some degree of microcytosis. as a consequence of low expression of allele, hb e is associated with hypochromia, whereas hbc with hyperchromia because of some dehydration of the red blood cell. regarding the high frequency of hb s and hb c in the population originated from africa, compound heterozygotes are not rare. and due to increasing intracellular concentration of total hb (high mchc linked to hb c) the compound heterozygotes hb s / hb c sickle. considering their low mchc, the rare cases of hb s / hb e have a mild degree of clinical symptoms. the distribution of rbcs of heterozygous individuals of hb e and hb c can be also easily distinguished using phthalate density gradient. among the rare hb variants some may have a high local incidence as hb c, hb s, hb e or the various thalassaemic defects because these bring some protective effect against malaria. this is likely the case of these unstable chain variants which display in the heterozygous state some mild thalassaemic features. among those the most frequent is perhaps hb constant spring resulting from a mutation of the termination codon, leading to a low expressed elongated unstable chain42. many variants belonging to this group are now known and some have been reported in asian populations43. among the other rare variants the first group is made by those variants which are without any clinical effects and are fortuitously found during screening programmes. the only reason that leads to characterize these is to eliminate a problem of possible interaction with hb s or with a thalassaemic syndrome. these will be found because of a different electrophoretic or chromatographic behaviour. by comparing their properties in a battery of tests (ief, ce - hplc, rp - hplc, globin chain electrophoresis, electrospray mass measurement) it is often possible to identify these without need for a complete protein sequence analysis or dna sequencing. a dominant phenotype of haemolytic anaemia is always observed in the case of unstable hbs. historically, the concept of this group of hbs arose some 50 years ago from the study of patients suffering from chronic non - spherocytic haemolytic anaemia. their haemolysate displayed a normal electrophoretic picture, but when incubated at 50c revealed the presence of a hb component, which precipitated faster than hb a44. blood smears, either spontaneously or after incubation with an oxidant dye, showed inclusion bodies, made from hb precipitates. later on it was demonstrated that these hb variants displayed structural modifications in some specific regions of the hb molecule such as the haem pocket or the 11 interface. other variants having together electrophoretic abnormalities and instability were found later on45. from a biological and clinical point of view, each of the mutations responsible for an unstable hb affects only a limited number of individuals in a few families, usually unrelated. de novo mutations are frequently observed since these mutations are always a cause of disease. these do not bring any selective advantage and are, thus, eliminated in the forthcoming generations. in industrialized countries the founder effects are usually identified since any patient suffering from haemolytic anaemia will be investigated until the exact aetiology of the disease will be explained. today, detection of hb mutants is usually easily performed by automatic methods of protein study (ce - hplc and ce). this led to the discovery of an impressive list of variants which are reported on the hbvar database (http://globin.cse.psu.edu/hbvar/menu.html)45. conversely, those found during the course of a haematological disorder bring frequently the aetiological answer for the disease. often unusual clinical presentations may be explained by the interaction of several hb abnormalities and their identification may require further investigations.	haemoglobin (hb) abnormalities though quite frequent, are generally detected in populations during surveys and programmes run for prevention of hb disorders. several methods are now available for detection of hb abnormalities. in this review, the following are discussed : (i) the methods used for characterization of haemoglobin disorders ; (ii) the problems linked to diagnosis of thalassaemic trait ; (iii) the strategy for detection of common hb variants ; and (iv) the difficulties in identification of rare variants. the differences between developing and industrialized countries for the strategies employed in the diagnosis of abnormal haemoglobins are considered. we mention the limits and pitfalls for each approach and the necessity to characterize the abnormalities using at least two different methods. the recommended strategy is to use a combination of cation - exchange high performance chromatography (ce - hplc), capillary electrophoresis (ce) and when possible isoelectric focusing (ief). difficult cases may demand further investigations requiring specialized protein and/or molecular biology techniques.
following replantation and revascularisation of digits, functional outcome is often not satisfactory, so secondary surgical procedures are necessary to improve the final result. few reports have focused on the order in which secondary procedures should be performed ; this is often left to the surgeons to decide on the basis of their experience. wang reviewed 1227 cases of upper limb replantation published between 1977 and 2000 and designed a decision procedure for determining the order and appropriateness of performing a variety of secondary procedures including soft tissue stabilisation, bone synthesis, sensory restoration, joint flexibilisation and tendon reconstruction [figure 1 ]. however, the utility of such decision procedure has not been corroborated by further reports in the clinical setting. the decision - theoretic approach to functional enhancement after upper limb replantation published by wang. we tested the hypothesis that using wang 's decision procedure to determine the order in which secondary procedures should be performed facilitated improvement of function and avoided surgical complications. in the case sensitivity, range of motion (rom) and patient - reported outcomes were the main measures of outcome. the patient was a 20-year - old male who had suffered a saw injury at flexor tendon zone ii of his dominant hand. unfortunately, 6 days after revascularisation of the second to fourth fingers and replantation of the thumb, an infection of the wound manifested. all the venous grafts for the arterial bypasses to the fingers were embedded in pus and failed, as did the tenorrhaphies and neurorrhaphies. although cultures gave negative results, the wound was insufficiently clean to accept new bypasses. in addition, the index finger was barely vascularised ; a wait - and - see policy was adopted towards this digit. three weeks after debridement and empiric systemic antibiotics, all wounds had healed, and all fingers survived. however, the index finger was pale, cold and presented very slow capillary refill (figure unavailable). the patient scored 65 in the disabilities of the arm, hand and shoulder (dash) questionnaire. following the decision procedure described by wang, first, the thoracodorsal bundle was transferred microsurgically to the index finger to enhance index vascular viability. in brief, the thoracodorsal artery and vein were dissected for 15 cm and a muscle cuff 2 cm wide 3 cm long was preserved at its tip to monitor the permeability of the bundle. the subscapular artery and one vein were anastomosed to the radial vessels laterally at the anatomic snuffbox, and the bundle was tunnelled subcutaneously to the radial side of the index finger [figure 2 ]. in addition, grafts from the superficial peroneal nerve were used to reconstruct gaps in the collateral nerves of the index and ring fingers. after healing was complete, the index finger showed good vascularisation [figure 3 ]. the patient was then referred for therapy that focused on oedema absorption and passive joint mobilisation. the technique described by hussl. was used to transfer the thoracodorsal bundle to the radial artery at the anatomic snuffbox. the bundle was accompanied by a muscle cuff, which was implanted and skin - grafted at the middle phalanx. in the same operation, we used the superficial peroneal nerve to reconstruct the proper digital nerve post - operative appearance of the index finger after late revascularisation. note that the thumb was replanted, the second to fourth fingers survived the injury and the tip of the fifth finger was not replanted eight weeks later the metacarpophalangeal (mcp) joints in all fingers were fully supple. however, under radiological examination, the third mcp joint showed signs of erosion [figure 4a ]. following wang 's decision procedure, surgical exploration of the joint showed denuded and scarred cartilage [figure 4b and c ]. the defect was reconstructed by transposing a full - thickness mcp joint from the fifth digit with the middle mcp joint [figures 4d and 5 ]. this transposition included the fat volar to the mcp joint, the volar plate and the joint and the intrinsic tendons to the extensor hood. the joint was vascularised based on the ulnar collateral artery of the little finger [figure 5 ]. a metacarpal ray step - cut was synthesised using 2.3 mm compression screws (medartis ag, basel, switzerland). the proximal phalanx was stabilised using two 1 mm kirschner wires [figure 6 ]. two dorsal veins at the level of the joint were transected before transposition and then anastomosed to the veins of the dorsum of the hand [figure 7 ]. an island of skin was left intact on the dorsum of the joint for monitoring. after 8 weeks of therapy, the transposed joint showed bone union and a full passive rom [figure 6 ]. (a) erosion of the metacarpophalangeal joint of the index finger a few weeks after the injury. (b) the joint was removed, and the clinical exploration (c) showed clear degeneration. (d) the full - thickness defect was reconstructed by transferring the fifth metacarpophalangeal joint to the third ray the fifth metacarpophalangeal joint was transferred to the third finger based on the ulnar collateral artery under the subcutaneous fat of the palm (volar view). the proximal phalanx was synthesised using kirschner wires, and the metacarpal bone was fixed with two compression screws radiological appearance after vascularised joint transfer the dorsum of the transferred joint included two veins that were anastomosed to the veins of the dorsum of the hand (dorsal view). the extensor tendon was repaired using non - absorbable mattress sutures once all the remaining joints were healthy, the fingers had been neurotised and there was stable soft tissue coverage, the patient was scheduled for two - stage reconstruction of the flexor tendons from the second to fourth fingers., amstelveen, the netherlands) were placed finger - to - wrist under a2 and a4 pulley annular tendon - graft reconstructions [figure 8 ]. when the scar was removed, a 1 cm 1 cm volar defect developed in the index finger, which the latissimus dorsi cuff was rotated to cover. three months later, after passive mobilisation of all fingers, all joints were fully supple, so the patient was scheduled for the second stage of flexor tendon reconstruction. tendon grafts of extensor digitorum longus (second to fourth slips) were anchored to the distal phalanges using 40 mitek anchors (depuy mitek inc., leeds, uk), and pulvertaft 40 repairs were performed at the wrist. all grafts were sutured under tension to maintain 80 of mcp, 85 of proximal interphalangeal (pip) and 15 of distal interphalangeal joint flexion. passive flexion and protected extension were encouraged at the pip joint after the first post - operative day. annular pulley reconstructions in all fingers the patient was allowed to stretch the tenorrhaphies progressively only after 3 weeks, followed by assisted active flexion for 9 weeks, after which the patient was allowed to flex against resistance. as a final step, a cosmetic revision was performed 5 months later to remove the redundant muscle island from the thoracodorsal vessels, increase the first web space through skin z plasties and diminish soft tissue redundancy on the ulnar side of the palm by fat and skin removal. sensitivity returned to s3 for the index finger, and s4 for the third and fourth fingers and the thumb, using the measures specified by the medical research council scale. the patient was able to return to work with an almost complete active rom, for all long fingers [video 1 ]. successful replantation or revascularisation of digits usually requires secondary procedures to improve their function. to improve the function of salvaged fingers, our patient required secondary revascularisation, nerve reconstruction, joint reconstruction, two - stage flexor tendon reconstruction and skin revision. this algorithm followed a decision procedure, and our patient showed a notable functional improvement by reducing the dash score 45 points from his initial score, which is three times the minimum reduction in the score that is required to identify patients whose condition has improved. wang 's decision procedure provides a stepwise approach for the reconstruction of replanted or devascularised fingers. for its development the decision procedure has an anatomical basis in that patients need soft tissue reconstruction first, regardless of what procedures may be needed later. the use of flaps is preferred because only they allow the tendon gliding. then, nerve reconstruction is needed, not only to enable sensitivity in the finger but also to prevent complications such as silicone rod extrusion that arises as a result ofthe rehabilitation. afterwards, the finger must be completely supple, which occurs if the joints are healthy. only after that can the most frequently needed procedure, which is the reconstruction of tendons, be performed. however, in our case, a decision procedure, aided in designing a surgical plan for the patient. despite its theoretical appeal, wang 's decision procedure 's, clinical effectiveness remains unknown because secondary surgery is not performed very often in such cases. finger stiffening is common after zone ii tendon injury and often necessitates secondary reconstruction of joints and tendons. the lack of vascular bundles, low sensitivity and need for a2 and a4 pulley reconstructions in the context of two - stage tendon reconstruction, followed by aggressive hand therapy, may pose a risk as silicone implant infection and extrusion, or graft rupture. in addition, arthrolysis and tenolysis often require scar removal and carry out a risk of devascularisation of revascularised or replanted digits. thoracodorsal vessel implantation was proposed to treat cold intolerance because it is held to improve the vascularisation of severed digits. grafts from the superficial peroneal nerve were used to restore sensation before silicone rod implantation. the superficial peroneal nerve was harvested easily in the supine position, and the proximal stump remained subfascially deep in the leg, minimising the risk of painful neuroma formation. in our case, revascularisation and neurotization allowed safe scar removal, tenoarthrolysis, silicone rod implantation and tendon grafting in an otherwise barely vascularised finger. otherwise tendon reconstruction may be unsuitable in poorly vascularised and stiff fingers because tenorrhaphies do not heal or glide smoothly in scarred beds or across stiff joints. in addition, alloplastic tendon spacers may be subject to infection or extrusion when implanted in insensate digits. although arthrolysis is often required after digit revascularisation or replantation, the need to reconstruct a joint completely is rare. osteochondral grafts and microvascular metatarsophalangeal joint transfers have been used to reconstruct the pip joint with varying degrees of success, and their use was challenged in a systematic review. osteochondral grafts are better used in acute joint injuries because the rom they provide is significantly greater than that achieved after their application in chronic injuries. we did not consider silicone arthroplasty of the mcp because of the presence of major injury to the flexor and extensor tendons. classically, amputated fingers have served as tissue banks for the reconstruction of other severed fingers in the acute setting. in contrast, in our case, the stump of an amputated finger was used as a tissue source for reconstruction in the late post - operative period. a healthy fifth mcp joint was transferred to the third ray followed by venous repairs. we chose to preserve the ulnar collateral artery and only repair the two dorsal veins because there were plenty of recipient veins available in the dorsum. the joint showed complete extension and a full rom just a few weeks after surgery. other interventions included two - stage tendon reconstruction and the revision of skin scars, both of which were uneventful. the replanted thumb required no other revision, except for z - plasty of the skin in the first web space. the functional status of the patient, indicated by the difference between pre- and post - operative dash questionnaire scores improved notably. the success of replantation should be measured by functional outcome. in this case report, we described the use of a decision - theoretic approach to the secondary reconstruction of revascularised fingers with a complicated post - operative course that required the use of both established and less frequently used techniques. it proved safe for our patient, who demonstrated an improvement in patient - reported disabilities, gained almost complete restoration of rom, and was able to return to work. our experience using this decision procedure in a multistaged case was excellent, but to confirm its efficacy in practice, further evaluation in the clinical setting is required.	secondary surgical procedures can improve the function of revascularised and replanted digits. we describe the case of a patient who underwent multidigit revascularisation and replantation following a saw injury at flexor tendon zone ii. to achieve maximal functional improvement after finger revascularisation, we performed secondary surgical procedures in an order that was determined by following a reconstructive decision procedure that covered late revascularisation, nerve reconstruction, pedicled vascularised joint transfer, staged flexor tendon reconstruction and skin revision. performing the procedures in this manner ensured overall safety. the patient 's disabilities of the arm, hand and shoulder questionnaire score improved by 45 points, and the patient was able to return to work with an almost complete range of motion.
early diagnosis and treatment of these disorders can slow progression to end - stage renal disease leading to lowering mortality and morbidity as well as reducing the overall costs. therefore, selection of cost - effective methods of diagnosing and screening for early chronic kidney diseases is needed to identify and manage subjects with this risky condition. in this regard, following the presence of microalbuminuria as a sensitive marker especially in high - risk groups such as diabetics or hypertensive patients not only can provide valuable information on the stage of renal insufficiency but also can be cost - beneficial and prevent other diagnostic and therapeutic costs. assessment and screening for microalbuminuria is a confirmed useful tool for identifying subjects at risk for renal insufficiency and its progression. according to the this fact that the presence of microalbuminuria can be an alerting finding of increased risk for nephropathy and even cardiovascular disorders, a proper method for diagnosing and screening microalbuminuria should be considered to minimize the risk for these life - threatening events and related mortality and morbidity [46 ]. however, it is now controversy with regard to the type of urine test with acceptable cost - effectiveness and cost - beneficial to be used for evaluating microalbuminuria. the development of gold standard tests such as radioimmunoassay, immune - electrophoresis, and immune - turbidimetry method has provided the possibility for the measurement of even low concentrations of albumin in urine and its urinary secretion before the appearance of clinical warning manifestations. however, because of its high cost and also inaccessibility to all segments of society particularly to residents in rural states, using this procedure repeatedly and also at different time points for accurate and definitive diagnosis is not applicable [8, 9 ]. in this regard, benefits and diagnostic accuracy of scheduling a single assessment of microalbuminuria and its cost - effectiveness in our country as a developing society remained unclear. hence, we aimed to assess the cost - benefit of a single assessment of microalbuminuria especially in different subgroups at risk for developing renal failure. the current cross - sectional survey was a part of a large comprehensive population - based cohort named the isfahan healthy heart program (ihhp) with the aim to identify subjects at risk for cardiovascular diseases, to control of cardiovascular risk profile, and to modify lifestyle. in this study, a total of 126 consecutive persons were enrolled and interviewed at baseline for assessment of demographic parameters as well as hypertension state that was categorized hypertensive state if resting systolic blood pressure was 140 mm hg and/or diastolic 90 mm hg, or the subject was treated with antihypertensive medications. because it was a part of a great population - based screening study named ihhp, we considered the consecutive persons who were scheduled for concurrent assessment of hypertension state and urine analysis. thus, only those without complete data on hypertension status, those who did not agree with participation in the study (agreeing with blood pressure measuring or urinary examination) were not included into the study. the different stages of the test were performed by the trained and experienced personnel who were blinded to the purposes of the study. in this the blood pressure was measured four times using a mercury sphygmomanometer, with an appropriate size cuff, and the mean of these measurements was considered as criteria for defining hypertension. during the measurements, the participant remained seated for 10 minutes with the arm comfortably placed at the level of the heart. in this study, those with the history or any evidences of diabetes mellitus, acute inflammatory disorders, trauma, major surgery, febrile disorders, connective tissue disorder, cerebrovascular accident, recent myocardial infarction, cardiac arrhythmias, chronic pulmonary diseases, malignancies, or heavy physical activities a day before the study begins were all excluded. the study protocol, which complies with the principles of good clinical practice and the declaration of helsinki, has been approved by the relevant ethics committee at the participating center. albuminuria was assessed by collection of two fasting random urine specimens on arrival to the clinic as well as one week later in the morning. urine creatinine was measured by the picric acid method, and urine albumin content was measured by a sensitive, nephelometric technique (pars azmoon kits, iran). the urinary albumin / creatinine ratio (uacr) and all other laboratory values were determined in a central laboratory within 24 hours after obtaining the urine samples. microalbuminuria was defined as uacr from 30 to 300 mg / g in two consecutive portions taken a least 1 week apart. according to the definition of microalbuminuria as albumin exceeding 30 mg / g in two consecutive samples, the benefits of one measurement of this parameter were assessed compared to the diagnosis on two first consecutive measurements as the gold standard. kidney damage was also defined as uacr > or = 200 mg / g. results were reported as mean standard deviation (sd) for quantitative variables and percentages for categorical variables. we evaluated specificity, sensitivity, and positive (ppv) and negative (npv) predictive values of measurement of microalbuminuria in a first - morning urine sample in comparison with the measurements in two timed urine collections as the gold. a receiver operating characteristic (roc) curve was used to identify values of both two methods for measurement of microalbuminuria to discriminate normal from damaged kidney conditions. for the statistical analysis, the statistical software spss version 19.0 for windows (spss inc., in overall and based on the diagnosis of microalbuminuria as uacr from 30 to 300 mg / g in two consecutive portions, 17 out of 126 participants suffered from microalbuminuria that, among them, 12 subjects were also diagnosed as microalbuminuria once assessing this factor with a sensitivity of 70.6%, a specificity of 100%, a ppv of 100%, a npv of 95.6%, and an accuracy of 96.0% (table 1). the measured sensitivity, specificity, pvv, npv, and accuracy in hypertensive patients (n = 88) were 73.3%, 100%, 100%, 94.8%, and 95.5%, respectively. also, these rates in nonhypertensive groups were 50.0%, 100%, 100%, 97.3%, and 97.4%, respectively. according to the roc curve analysis (figure 1), once measurement of uacr had a high value for discriminating defected from normal renal function state (c = 0.989, 95% ci : 0.9671.000). urinary albumin concentration in a once measurement had also high discriminative value for diagnosis of damaged kidney (c = 0.995, 95% ci : 0.9821.000), while measurement of urine creatinine level had no acceptable value for discriminating damaged kidney function from normal function state (c = 0.638, 95% ci : 0.2870.989). several studies have demonstrated cost - effectiveness of microalbuminuria screening in high - risk populations leading optimization of patients ' care as well as future implementation of ckd screening programs [8, 9, 13 ]. however, the limitations due to availability of instruments and also high costs for repeated measurement of biochemical markers in rural areas in most developing countries do not permit to the test in successive time periods. the purpose of this study was to answer the question whether a single testing for microalbuminuria results in a reliable conclusion leading to costs saving. according to our observation, the once testing rather frequent testing leads to high diagnostic sensitivity, specificity, and accuracy as well as high predictive values in total population and also in hypertensive subgroups. on the other hand, the use of tests at different consecutive time points can be confidently replaced by the once testing with the partially same diagnostic value. under the existing tariffs on testing microalbuminuria, the cost per test is estimated to be the equivalent of ten thousands of rials that can be reduced by half using one testing. our purpose was not directly estimating and calculating the cost of urine analysis and comparing this cost between the single and repeated analyses. in fact, we attempted to point that because of high obtained accuracy by conducting single urine albumin analysis, it is not needed to repeat analyses and in this regard, this approach can certainly result in lower spending. in our study, the diagnostic accuracy of the first - morning test for microalbuminuria was cost - benefcial for hypertensive patients as well as the total population. in fact, this once test can be beneficially used to screen microalbuminuria in both hypertensive and normotensive patients with the minimum cost. it has been previously shown that screening recommendations can be extended to include persons with hypertensive or diabetic patients [8, 13 ]. this is important because the bulk of the costs in these risk subgroups are spent for treatment and prevention of related complications. thus, the use of one testing can be even more cost - benefcial in hypertensive patients with the same diagnostic accuracy. besides assessing the diagnostic accuracy of the one testing uacr for detection of microalbuminuria, we test diagnostic performance of one estimating urine albumin level and also urine creatinine level for assessment of kidney damage. according to the obtained discriminative values, the value of uacr and urine albumin level determination was equal, but measurement of urine creatinine level alone was notably low for this aim. however, it is useful to compare the amount of albumin in the sample against its concentration of creatinine. the uacr has been shown to be convenient, cost - effective, and efficient in screening patients for microalbuminuria when compared with 24-hour collections [14, 15 ]. it seems that because uacr assessed the condition of urine albumin excretion based on the ability of kidneys to clear creatinine, the use of uacr marker is preferred to estimate urine albumin level alone for assessing severity of kidney damage. the main limitation of the study was considering a partially small sample size leading to difficulties in high - power estimation of diagnostic accuracy (leading to a wide confidence interval especially for normotensive group) as well as diagnostic power measured by the roc analysis. in another portion, considering other baseline variables especially socioeconomic level can help to explain more the affordability of the single testing. in addition, by considering different levels and severity of hypertension, assessment of affordability according to hypertension state can be achieved with more precision. in conclusion, our results showed that the detection of both urinary albumin concentration and also estimation of uacr in single urine sample is accurate and cost - benefit procedure to identify microalbuminuric subjects regardless of hypertension state.	background. the purpose of this study was to answer the question whether a single testing for microalbuminuria results in a reliable conclusion leading costs saving. methods. this current cross - sectional study included a total of 126 consecutive persons. microalbuminuria was assessed by collection of two fasting random urine specimens on arrival to the clinic as well as one week later in the morning. results. in overall, 17 out of 126 participants suffered from microalbuminuria that, among them, 12 subjects were also diagnosed as microalbuminuria once assessing this factor with a sensitivity of 70.6%, a specificity of 100%, a ppv of 100%, a npv of 95.6%, and an accuracy of 96.0%. the measured sensitivity, specificity, pvv, npv, and accuracy in hypertensive patients were 73.3%, 100%, 100%, 94.8%, and 95.5%, respectively. also, these rates in nonhypertensive groups were 50.0%, 100%, 100%, 97.3%, and 97.4%, respectively. according to the roc curve analysis, a single measurement of uacr had a high value for discriminating defected from normal renal function state (c = 0.989). urinary albumin concentration in a single measurement had also high discriminative value for diagnosis of damaged kidney (c = 0.995). conclusion. the single testing of both uacr and urine albumin level rather frequent testing leads to high diagnostic sensitivity, specificity, and accuracy as well as high predictive values in total population and also in hypertensive subgroups.
balya (~strength promoting activity) is an action which is considered as one of the important concept as per charaka. due to this reason a group of 10 drugs is described for balya activity. in general any action that increases bala (~ strength) of the individual is seen as in other words that which is favorable and lead to the increase of bala or strength is considered as hence it is quite necessary to understand the concept of bala as per ayurveda. thus to understand the action balya it is necessary to know the concept of bala in ayurveda. even though, bala stands for the strength of the body, the word is being used in different contexts to denote various aspects accordingly. all the aspects related to balya action in - fact do have either functionality or even a direct or indirect beneficial effect on the strength of the body. thus, the evaluation of an activity of the drug with respect to a complex action like balya, it is very important to have a cluster approach of many investigations, such that a total and a precise evaluation of actions like balya are possible. the paper tries to provide a profound review of the action and also gives possible common investigations to be adopted for the evaluation of balya action clinically. the word bala has been used invariably for strength of mind body and its components. however, the term is also used as a synonym for those components of the body which have the function of providing strength, protection, nourishment and stability to the other parts of the body. hence, the word bala is synonymously used for normal kapha dosha (~the factor primarily responsible for strengthening and lubricating the body) and ojus (~bioenergy or body component that helps in sustaining of life). it also stands for immunity and immune system in normal state. according to sushruta, the factor due to which one obtains the nourishment and stability of mamsa dhatu (~the muscular tissues of the body), ability to perform various tasks efficiently, good complexion, clearness and pleasantness of voice along with clear and efficient working of all the organs either external like jnyaanendriya (sense organs) or karmendriya (organs with motor functions) and internal like manas (mind), aatma (spirit), etc. in sushruta the word bala refers to ojus also which has a function of maintaining the bala of the body. in - fact many authors of ayurveda are of the opinion that bala should be referred to ojus. the word bala also denotes the normal kapha dosha as it has the function of providing bala for the body normally. further charaka also classifies bala as three types namely sahaja (natural), kalaja (seasonal and age) and yuktikrita (act of intelligence). among the three, the yuktikrita type is explained as the bala that is developed by activities specially performed for increasing the same. these interventions include various exercises, yoga, food articles and drugs having rasayana (~a general tonic that nourishes all the tissues of the body) and vajikarana (~an action that involves nourishment to the shukra primarily) properties. according to chakrapani, the food includes the articles like meat, ghee, etc. and activities includes rational practice of rest and exercise while yoga refers to various medicinal preparations that are used in the treatment as rasayana and vajikarana., bala is considered as effect of the food, medicine or activities which are having the action of balya. on the whole the term bala refers to three contexts as per the various ayurvedic references as illustrated in the figure 1. the action balya is defined as balaaya hitam balyam which refers to substances those are beneficial and tend to increase bala of an individual. as per the review of bala concept, the balya action possesses different dimensions which are to be assessed to ascertain complete action with respect to a given drug. hence, it is important to have a review of the action that is necessary to understand the modalities of its assessment. emergence of concepts of immunity, bioenergy and nutritional supplements in the contemporary science have given an opportunity for scientific validation of concept of bala and related balya action. the action balya can be regrouped on the basis of three categories of bala as shown in figure 2. schematic diagram of dimensions of balya action and its effects the first group balya action consists of nourishing factors responsible for providing necessary nutrients to ensure stability of certain tissues and organs. they include substances that possess actions such as bulk promoters, hematinics, stomachic, intestinal tonics, nervine tonics, cardiac tonics and vascular tonics. the effects of such drugs are usually on a certain part ensuring its stability and nutrition. it may not have a generalized effect or that may have secondary action on other parts. they have a primary effect of providing enough energy to ensure generalized stability of the body and strengthening it by protecting against any intrusion by various types of pathogens. they include substances that possess actions like generalized tonics, anti - periodic, food substances with high nutritional value, nutritional supplements and activities that help in increasing the bioenergy of the individual like yoga. the third group of action regarded as balya consists of those which provide necessary supplementation for the sustenance of normal kapha dosha. they are usually considered as secretions or fluids that are responsible for the protection of body 's vital organs, especially the brain, heart, joints, etc. as most of these fluids mainly contain proteins they are usually supplemented with nutritional supplements. they include substances like general tonics, medicated ghee, medicated oils, cardio tonics and therapies like oil massages. the balya actions with of various substances, either medicaments or dietary substances are clearly mentioned in ayurvedic classics. the assessment of this action is done by examining bala of the individual with respect to the method of balya action. the quantification of exercise is subjective and needs objective approach to obtain the proper scheme for the comprehensive evaluation of the action. on the basis of the observations regarding bala, the drugs that are grouped under the heading of balya in ayurveda can be reclassified into three headings namely : that increases the mamsa dhatu or muscular tissues of the bodyimmunomodulators and bioenergythat which enhances lubrication in the body. that increases the mamsa dhatu or muscular tissues of the body immunomodulators and bioenergy that which enhances lubrication in the body. the balya action of drug may have at least of the the three above listed effects. hence a comprehensive plan with all the three major dimensions is required to evaluate a balya drug. in light of the present known scientific laboratory investigation methods, those which are helpful in the evaluation of the above three dimensions of the drug action are enlisted in table 1. the methods for the evaluation of the three dimensions of balya karma the tmt or the bruce treadmill test is a standardized procedure used to evaluate the cardiovascular fitness of athletes or anyone else for that matter. it is one of the methods for the assessment of volume of maximum oxygen (vo2 max) which refers to the maximum amount of oxygen that an individual can utilize during intense or maximal exercise. it is one of the factors that can determine a person 's capacity to perform sustained exercise and is linked to aerobic endurance. it is also used to estimate the metabolic equivalent of task (met) with respect to an individual. the met, or simply metabolic equivalent, is a physiological concept expressing the energy cost of physical activities as multiples of resting metabolic rate. the immunoassay is a biochemical test that measures the presence or concentration of a macromolecule in a solution through the use of an antibody or immunoglobulin. there are several tests under immunoassay out of which enzyme - linked immunosorbent assay (elisa), memory lymphocyte immunostimulation assay (melisa) and radioimmunoassay would be useful. again the necessary immunological tests that are necessary to specify the antibodies generated can also be used as per the requirement. the comprehensive metabolic panel consists of 14 laboratory tests namely serum glucose, serum calcium, blood urea nitrogen (bun), serum creatinine, serum electrolyte (4 tests - sodium level, potassium level, chloride level, carbon dioxide level), serum total protein, serum albumin, serum bilirubin, serum alkaline phosphatase, serum aspartate amino transferase (ast) or sgot and serum alanine amino transferase (alt) or sgpt. among the tests the main organs that are being screened is kidney and liver. the additional tests that depict the protein, electrolytes and glucose while provide the necessary data to assess the basic metabolic condition and health status of the body. even investigations involving major fluids of the body like synovial fluid, peritoneal fluid, pericardial fluid and meningeal fluid like joint fluid analysis will be much more helpful in evaluation. upon exhaustive review about the action balya it was found that the action holds a broader base and understanding which covers almost every system of the body. according to the context where the word bala is used, the action balya also gets applied as per the definition. hence the action balya also gets a broader meaning and application like a type of hridya, an action that results in an increase of ojus, etc. each of these were considered and briefed enough such that the exact nature of action with respect to balya of ayurveda can be determined. for this purpose generalized evaluation regarding the methods mentioned in the assessment of balya action revealed that the method to analyze the same is examination of vyayaama shakti. this is possible only when the person performs a good physical activity. among the various tests that are available today, the tmt or treadmill stress test stands to be one of the most suitable ones for the purpose of assessing the magnitude of physical activity of the individual. the tmt test is a method that renders the report on the condition of three major systems of the body namely musculoskeletal system, circulatory system and respiratory system. these three systems are either directly or indirectly involved with all the 7 dhatus (~tissues) of the body. especially the musculoskeletal system is predominantly involved with mamsa (~muscular tissue) and asthi (~bone tissue) ; circulatory system is predominantly involved with rasa (~nutritional fluids), rakta (~blood cells), majja (~bone marrow) and indirectly with shukra (~semen) and respiratory system is predominantly associated indirectly with all the dhatus (~tissues) as it is the major supplier of oxygen which is responsible for almost all the chemical processes that are executed in the body. thus, along with the reading of vo2 max intake, it also gives the idea on the condition of the metabolic status of the body too through the reading of met. a clear portrait of health status with the three major systems is established with the test interpreting the readings pertaining to blood pressure, the cardiac output and the respiratory rate during different levels of stress along with the main readings. in the present context the measurement of vo2 max will be useful to measure the capacity of the individual to sustain physical stress or vyayamashakti. it also gives an idea of the individuals bioenergy in terms of met which is useful to determine the second dimension of balya action with regards to bioenergy. other effects those are resultant of balya action are also to be assessed for complete review of the action. laboratory investigations suggesting immunological condition and the quantitative changes as a result of drug effect could be immunoassay. there are various tests under immunoassay out of which elisa, melisa and radioimmunoassay could be useful to explain general status of the immune system in the body. these tests in turn can be interpreted as the ability of the body to face and counter the infiltration of various disease pathogens. thus, the tests are helpful in resolving the second dimension of the balya action with respect to vyadhikshamatwa (~immune system) of the body. even the fluid balance and the proteins present in the protective fluids of vital organs can be generally evaluated with the help of the complementary metabolic panel which has 14 investigations covering the functions of liver, kidney, plasma proteins. the comprehensive metabolic panel comprises of those tests that are necessary that depict the functions of two vital organs namely the liver (serum total protein, serum albumin, serum bilirubin, serum alkaline phosphatase, serum ast or sgot and serum alt or sgpt) and kidney (bun and serum creatinine). the two vital organs in - fact give the inference on the general condition of the body. this is because ; the organ liver is one which is most important for metabolism as every nutrient material is screened by it upon absorption from the gut. the kidney on the other hand is the one that is mostly used pathway for the excretion of toxic materials or by - products of metabolism or bio - wastes that have resulted in the process of a series of chemical reactions in the body. thus, any abnormality in the tests regarding these two organs usually depicts the pathological condition in the body usually resulting due to metabolism. it also helps in inferring the general condition of the body. along with these the other tests together will help in ascertaining the changes in the general condition of the body. thus giving further insight in the point where the drug has acted such that there is a notable improvement in the stamina, general health and well - being of the individual. thus the tests of the panel will be useful in ascertaining the condition of various parts of the body thoroughly in general such that an inferential outcome can be evaluated with respect to various fluids that protect the vital organs of the body. thus the tests help in the resolution of the third dimension of the balya action. further investigations into the individual fluids like synovial fluid analysis can be undertaken if necessary such that the aspect of drug action can be still clear. but these tests are not recommended as it will result in deep interventions that may have adverse effects. thus, the present modern laboratory investigations as enlisted will be useful in giving a complete portrait of the balya action with respect to an individual drug along with possible differentiation further. balya action mentioned in ayurveda is a complex one, which unfolds many activities of the drug on deep analysis. thus a comprehensive protocol involving many testing mechanisms is necessary for the evaluation and ascertainment of an activity of ayurveda like balya. even though the physical activity is the main parameter for the evaluation of bala, the other forms of bala is not to be ignored while evaluating the activity of balya. hence it can be concluded that the combined approach of evaluation of the physical activity and bioenergy by tmt stress test, immunity by immunological assay and inference of condition of protective or lubricating fluids of the body by the complementary metabolic panel is necessary for ascertaining balya action of ayurveda.	medicinal and dietary substances have been used for various purposes including nutritional from time immemorial. various activities such as immunomodulator, bulk promoting, nutritional, etc. that enhance strength, immunity, bulk of the body resulted by the use of medicinal or dietary substances are termed in total as balya in ayurveda. the term balya originally stands for all those actions that enhance the bala. the word bala refers to the strength and ability of the body or part of the body to cope up with various physical stressors. this term bala refers to various body components and functions as per the science of ayurveda. even in the presence of modern scientific knowledge regarding body strength, ayurveda concepts add varied dimensions to provide detailed explanation of balya term. the balya action is critically analyzed and discussed in this review.
frequent or chronic tension - type headache (tth) in children is a prevalent and debilitating condition for the child, often leading to medication overuse.1,2 in its infancy, research in children with tth is limited, though findings from adults highlight the areas that need to be examined. the pathophysiological factors underlying tth are of high interest, as new knowledge will aid in updating prevention and treatment activities. for example, research in adolescents links impaired function of the neck and shoulder muscles with tth.3,4 the rate of force development (rfd), as a component of strength activation in muscles, was likewise found impaired in painful muscles, in adult chronic pain research5 and could be hypothesized to be affected in children with headache. together with muscular deficits, increased pericranial tenderness is a symptom of interest pointing to hypersensibility of the peripheral or central nervous system linked with chronic headache.6,7 interesting research results from the longitudinal nord - trondelag health study (hunt) i and ii also show that low physical activity is a risk factor for developing non - migraine headache.8 the first step involved in exploring related parameters and their association with chronic headache in children is to examine the repeatability of current test methods. repeatability represents fundamental knowledge essential to interpreting clinical trial results and refers to examining changes and the variability of the mean between two identical repeated test occasions. the repeatability of isometric strength testing has been found acceptable in research with adolescents 17 years of age with and without tth,9 and the repeatability showed high values with intraclass correlation coefficients (icc) (95% confidence interval [ci ]) for neck flexion / extension in healthy subjects was 0.99/0.98 (0.970.99) and in subjects with tth was 0.99/0.99 (0.980.99). likewise was the repeatability of isometric strength measurements in adults with neck pain compared with healthy controls,1012 and in studies of healthy adults found acceptable.1315 rfd was also examined and found reliable in adults.5,16 earlier studies by bendtsen validated use of the total tenderness scoring system (tts) to examine pericranial muscle tenderness.17,18 the first to examine the repeatability of the tts in children was the study by soee, and it found the correlation coefficient between the two test days was acceptable (r = 0.81).19 confirmation of the repeatability of these measurements in both healthy and children ill with tth is warranted. the aim of the present study was to determine the test - retest repeatability of isometric maximal voluntary contraction (mvc) and force steadiness (fs) of neck flexion and extension from a neutral position, and of isometric mvc and rfd of dominant unilateral shoulder abduction, in healthy children 9 to 18 years of age. another objective of this study was to determine the test - retest repeatability of the tts and of a submaximal cycle ergometer test for predicting maximal oxygen uptake (vo2 max). to the best of our knowledge, the present study is the first to examine these combined measurements in children 9 to 18 years of age this study is a part of a larger case - control study examining the associations between headache and physical capacity in children with primary headache compared with healthy controls. forty - one healthy children accepted participation in the case - control study and out of these, a convenience sample of 28 children consented to participate in two rounds of testing. two girls failed to follow the schedule and one boy did not show up for the second test date. thus, only 25 of the children (ten boys and 15 girls) participated in the test - retest design within a 1-week interval. none of the children suffered from frequent episodic or chronic tth, migraine, or any other diseases, and all of them attended mainstream schools. the children were recruited after may 1, 2010 from primary or secondary schools in copenhagen and its metropolitan area, based on the guidelines and with approval of the danish national committee on biomedical research ethics. sealed envelopes containing detailed information were distributed to the children by their teachers at school, notifying them about the conditions involved in participating in a medical study, the right to confidentiality, and the right to freely withdraw from the study at any time. in order for the children to participate, their parents had to actively contact the researcher and provide written informed consent and the children had to provide oral informed consent. the study followed the ethical principles of the world medical association 2008 declaration of helsinki20 and the united nations 1989 convention on the rights of the child.21 the study was also approved by the danish data protection agency and registered with clinicaltrials.gov (identifier : h-3 - 2009 - 081). the strength measurement device was a vishay nobel, type kis-2, max. 2 kn, computerized force transducer (vishay precision group, malvern, pa, usa) wall mounted on a custom - built adjustable stand (figure 1). the signals from the force transducer were generally sampled with a frequency of 100 hz and low pass filtered with a cutoff frequency of 10 hz. for the fs measurement, the signal was low pass filtered with a cutoff frequency of 5 hz and the standard deviation (sd) was calculated. the rfd was calculated as the maximum slope (n 10 s) for a 0.1-second linear fit to the force signal. the bicycle, recalibrated on each test day, was a calibrated monark ergomedic 939e pc (monark exercise ab, stockholm, sweden). the measurements were conducted at the danish headache center, glostrup hospital, copenhagen, denmark. an experienced research lab technician did all the testing and was blinded to the history of the child. the test - retest was carried out on the same weekday and time within a 1-week interval. general joint mobility was screened using the revised beighton score,22 which is a bilateral examination in a standing position, of five joint movements that includes the fifth metacarpophalangeal joint, the thumb, elbow, knee, and hands flat on the foor with stretched knees. a goniometer (smith & nephew rolyan inc, london, uk, a441 - 1) was used to measure the passive bilateral dorsiflexion of the fifth metacarpophalangeal joint and the passive bilateral hyperextension of the elbow and knee. tenderness by palpation was scored in a sitting position, in a chair with an adjustable neck and leg support, using the validated tts,18 with a four - point scale scored by the lab technician as follows : 0 = denial of tenderness, no visible reaction ; 1 = verbal report of discomfort or mild pain, no visible reaction ; 2 = verbal report of moderate pain, with or without visible reaction ; and 3 = verbal report of marked pain and visible expression of discomfort. the palpation was conducted with small rotational movements, and pressure was maintained for 4 to 5 seconds. the palpation pressure was initially controlled by a palpometer17 on an arbitrary scale 80200 arbitrary units (au), reaching a standardized pressure of 120 au.18 the palpometer was the original instrument prepared at the danish headache center. a total score was calculated from palpation of seven bilateral sites (masseter, frontalis, temporalis, processus mastoideus, occipital insertion, trapezius, and sternocleidomastoideus). the addition of scores is recognized worldwide as a ratio scale and an important tenderness measure used in headache and muscle - related pain research.23,24 the child was positioned on a chair with the upper and lower trunk fixed with belts, in order to avoid associative work of the trunk muscles,14 while the arms hung relaxed at the child s sides. the child s position was corrected until an upright and symmetrical posture was obtained, with the neck in a neutral position in relation to the sagittal, frontal, and rotational axis. for neck extension measures, the padded force transducer was positioned with the lower edge corresponding with the protuberantia occipitalis. for neck flexion measures, the lower edge of the pad corresponded with the line between the child s eyebrows (figure 1a and b). for isometric mvc of neck extension and neck flexion, the child was instructed to build up pressure for a few seconds to reach a maximum amount of tension, to hold the tension briefly, and then to release it. if the force of the third trial exceeded 5%, for fs in neck extension and flexion, the child was instructed, by means of a computer screen, to build up tension (10 seconds) according to a line (30% of mvc) and then to hold that tension at the line for 25 seconds (figure 1c). for shoulder measurements, the child was asked to lie on a mattress on his or her back in a supine position. the dominant shoulder was then positioned in external rotation and abduction, while the elbow was stretched, to position the back of the wrist at the pad. the child was instructed to press the wrist of the dominant arm against the pad as quickly (rfd) and as forcefully (mvc) as possible. for cycle ergometer testing, the seat height was adjusted to allow the child s knee to be slightly flexed when the plantar region of the foot was in its lowest position. heart rate was monitored using an adjustable polar pulse belt (proterapi a / s, brndby, denmark) and recorded continuously on a computer. the child cycled 6 to 7 minutes, with a cadence of 60 repetitions per minute and at a resistance starting at 50 watts. if the child was over the age of 14 or appeared to be in the later stages of puberty, the start resistance was estimated based on the status of the child s general level of fitness and raised accordingly to 75 watts. power was added until the child s pulse exceeded 120 beats / min. in order to obtain a submaximal steady - state pulse, time was added (maximum, 7 minutes) according to original test procedures.25 vo2 max was predicted from a steady - state submaximal pulse, using the strand nomogram.26 descriptive statistics of age and anthropometric measures of the study group and test values were presented as means and sds. three trials of strength measurements were chosen for data analysis because the results from the third and fourth trial showed a trend of learning and building up more power. for the mvc and rfd tests, the largest force of three trials was chosen as the peak value. for fs, the minimum of the measured sd was calculated as the peak value. the test - retest repeatability was examined using lexell and downham s approach27 and divided into four steps. in step one, the icc was calculated using a one - way random analysis of variance (anova) for simple replication. icc values > 0.75 represent excellent reliability, and values between 0.4 and 0.75 represent fair to good reliability.27 step two involved calculating changes in the mean for peak data by studying the indices, in order to detect systematic changes. the mean differences (test 2 test 1) were calculated together with the sd of the differences, the standard error of the mean, and the 95% ci. if the 95% ci did not include zero, this indicated a significant systematic change in the mean between the two test occasions, thus suggesting either a learning effect or a fatigue effect. the distributional assumption of the differences was examined by plots and the kolmogorov - smirnov test. in step three, a visual plot and calculation were used ad modum bland and altman28,29 in order to visualize the level of agreement between the measurements. according to bland and altman, the plot can be used for measurements taken with different devices or for repeated measures. the mean difference (test 2 test 1) was organized as a y - axis reference line, with two additional lines at 2 times the sd as the limits of agreement. a random scatter of points above and below the reference line was considered acceptable as a measure of repeatability. if scatter points were clustered or repeated identically, systematic errors, lack of agreement or distribution transformation should be considered. data that was not significantly normally distributed was transformed using a lognormal transformation for further examination. the last step,27 step four, involved quantifying the variability of the measurements by (a) the method error (me) defined by me = sddiff 2 where the sddiff refers to the standard deviation of the mean difference between test 2 and test 1. (b) the coefficient of variation (cv%) defined by cv% = (me / mean) 100, where the mean is the mean of all data from test 2 + test 1.27 the me and cv% make it possible to determine the typical variation between two test occasions and means that an improvement after an intervention smaller than the variation does not indicate a clinically important improvement. differences between the two test occasions in tenderness for individual muscles were analyzed using a wilcoxon rank test. a significance level was set at p 0.05, while spss 17.0 (spss inc, chicago, il, usa) and 19.0 (ibm, armonk, ny, usa) were used for statistical analysis. this study is a part of a larger case - control study examining the associations between headache and physical capacity in children with primary headache compared with healthy controls. forty - one healthy children accepted participation in the case - control study and out of these, a convenience sample of 28 children consented to participate in two rounds of testing. two girls failed to follow the schedule and one boy did not show up for the second test date. thus, only 25 of the children (ten boys and 15 girls) participated in the test - retest design within a 1-week interval. none of the children suffered from frequent episodic or chronic tth, migraine, or any other diseases, and all of them attended mainstream schools. the children were recruited after may 1, 2010 from primary or secondary schools in copenhagen and its metropolitan area, based on the guidelines and with approval of the danish national committee on biomedical research ethics. sealed envelopes containing detailed information were distributed to the children by their teachers at school, notifying them about the conditions involved in participating in a medical study, the right to confidentiality, and the right to freely withdraw from the study at any time. in order for the children to participate, their parents had to actively contact the researcher and provide written informed consent and the children had to provide oral informed consent. the study followed the ethical principles of the world medical association 2008 declaration of helsinki20 and the united nations 1989 convention on the rights of the child.21 the study was also approved by the danish data protection agency and registered with clinicaltrials.gov (identifier : h-3 - 2009 - 081). the strength measurement device was a vishay nobel, type kis-2, max. 2 kn, computerized force transducer (vishay precision group, malvern, pa, usa) wall mounted on a custom - built adjustable stand (figure 1). the signals from the force transducer were generally sampled with a frequency of 100 hz and low pass filtered with a cutoff frequency of 10 hz. for the fs measurement, the signal was low pass filtered with a cutoff frequency of 5 hz and the standard deviation (sd) was calculated. the rfd was calculated as the maximum slope (n 10 s) for a 0.1-second linear fit to the force signal. the bicycle, recalibrated on each test day, was a calibrated monark ergomedic 939e pc (monark exercise ab, stockholm, sweden). the measurements were conducted at the danish headache center, glostrup hospital, copenhagen, denmark. an experienced research lab technician did all the testing and was blinded to the history of the child. the test - retest was carried out on the same weekday and time within a 1-week interval. general joint mobility was screened using the revised beighton score,22 which is a bilateral examination in a standing position, of five joint movements that includes the fifth metacarpophalangeal joint, the thumb, elbow, knee, and hands flat on the foor with stretched knees. a goniometer (smith & nephew rolyan inc, london, uk, a441 - 1) was used to measure the passive bilateral dorsiflexion of the fifth metacarpophalangeal joint and the passive bilateral hyperextension of the elbow and knee. tenderness by palpation was scored in a sitting position, in a chair with an adjustable neck and leg support, using the validated tts,18 with a four - point scale scored by the lab technician as follows : 0 = denial of tenderness, no visible reaction ; 1 = verbal report of discomfort or mild pain, no visible reaction ; 2 = verbal report of moderate pain, with or without visible reaction ; and 3 = verbal report of marked pain and visible expression of discomfort. the palpation was conducted with small rotational movements, and pressure was maintained for 4 to 5 seconds. the palpation pressure was initially controlled by a palpometer17 on an arbitrary scale 80200 arbitrary units (au), reaching a standardized pressure of 120 au.18 the palpometer was the original instrument prepared at the danish headache center. a total score was calculated from palpation of seven bilateral sites (masseter, frontalis, temporalis, processus mastoideus, occipital insertion, trapezius, and sternocleidomastoideus). the addition of scores is recognized worldwide as a ratio scale and an important tenderness measure used in headache and muscle - related pain research.23,24 the child was positioned on a chair with the upper and lower trunk fixed with belts, in order to avoid associative work of the trunk muscles,14 while the arms hung relaxed at the child s sides. the child s position was corrected until an upright and symmetrical posture was obtained, with the neck in a neutral position in relation to the sagittal, frontal, and rotational axis. for neck extension measures, the padded force transducer was positioned with the lower edge corresponding with the protuberantia occipitalis. for neck flexion measures, the lower edge of the pad corresponded with the line between the child s eyebrows (figure 1a and b). for isometric mvc of neck extension and neck flexion, the child was instructed to build up pressure for a few seconds to reach a maximum amount of tension, to hold the tension briefly, and then to release it. if the force of the third trial exceeded 5%, for fs in neck extension and flexion, the child was instructed, by means of a computer screen, to build up tension (10 seconds) according to a line (30% of mvc) and then to hold that tension at the line for 25 seconds (figure 1c). for shoulder measurements, the child was asked to lie on a mattress on his or her back in a supine position. the dominant shoulder was then positioned in external rotation and abduction, while the elbow was stretched, to position the back of the wrist at the pad. the child was instructed to press the wrist of the dominant arm against the pad as quickly (rfd) and as forcefully (mvc) as possible. for cycle ergometer testing, the seat height was adjusted to allow the child s knee to be slightly flexed when the plantar region of the foot was in its lowest position. heart rate was monitored using an adjustable polar pulse belt (proterapi a / s, brndby, denmark) and recorded continuously on a computer. the child cycled 6 to 7 minutes, with a cadence of 60 repetitions per minute and at a resistance starting at 50 watts. if the child was over the age of 14 or appeared to be in the later stages of puberty, the start resistance was estimated based on the status of the child s general level of fitness and raised accordingly to 75 watts. power was added until the child s pulse exceeded 120 beats / min. in order to obtain a submaximal steady - state pulse, time was added (maximum, 7 minutes) according to original test procedures.25 vo2 max was predicted from a steady - state submaximal pulse, using the strand nomogram.26 descriptive statistics of age and anthropometric measures of the study group and test values were presented as means and sds. three trials of strength measurements were chosen for data analysis because the results from the third and fourth trial showed a trend of learning and building up more power. for the mvc and rfd tests, the largest force of three trials was chosen as the peak value. for fs, the minimum of the measured sd was calculated as the peak value. the test - retest repeatability was examined using lexell and downham s approach27 and divided into four steps. in step one, the icc was calculated using a one - way random analysis of variance (anova) for simple replication. icc values > 0.75 represent excellent reliability, and values between 0.4 and 0.75 represent fair to good reliability.27 step two involved calculating changes in the mean for peak data by studying the indices, in order to detect systematic changes. the mean differences (test 2 test 1) were calculated together with the sd of the differences, the standard error of the mean, and the 95% ci. if the 95% ci did not include zero, this indicated a significant systematic change in the mean between the two test occasions, thus suggesting either a learning effect or a fatigue effect. the distributional assumption of the differences was examined by plots and the kolmogorov - smirnov test. in step three, a visual plot and calculation were used ad modum bland and altman28,29 in order to visualize the level of agreement between the measurements. according to bland and altman, the plot can be used for measurements taken with different devices or for repeated measures. the mean difference (test 2 test 1) was organized as a y - axis reference line, with two additional lines at 2 times the sd as the limits of agreement. a random scatter of points above and below the reference line was considered acceptable as a measure of repeatability. if scatter points were clustered or repeated identically, systematic errors, lack of agreement or distribution transformation should be considered. data that was not significantly normally distributed was transformed using a lognormal transformation for further examination. the last step,27 step four, involved quantifying the variability of the measurements by (a) the method error (me) defined by me = sddiff 2 where the sddiff refers to the standard deviation of the mean difference between test 2 and test 1. (b) the coefficient of variation (cv%) defined by cv% = (me / mean) 100, where the mean is the mean of all data from test 2 + test 1.27 the me and cv% make it possible to determine the typical variation between two test occasions and means that an improvement after an intervention smaller than the variation does not indicate a clinically important improvement. differences between the two test occasions in tenderness for individual muscles were analyzed using a wilcoxon rank test. a significance level was set at p 0.05, while spss 17.0 (spss inc, chicago, il, usa) and 19.0 (ibm, armonk, ny, usa) were used for statistical analysis. twenty - five children with a mean age 13.7 years (sd 1.8) completed the study and cooperated well during the test procedures. none of the children were obese, and two children reached the hypermobility cutoff of 5/9. the anova analysis revealed a test - retest repeatability of the peak maximal voluntary strength measurements for neck extension with an icc of 0.91 (95% ci : 0.810.96) ; for neck flexion, with icc 0.91 (95% ci : 0.820.96) ; and for shoulder abduction, with icc 0.97 (95% ci : 0.920.98). the rfd showed an icc of 0.79 (95% ci : 0.580.90), while the tts likewise, showed repeatability, with an icc of 0.87 (95% ci : 0.740.94). the prediction of vo2 max showed repeatability with the icc 0.80 (95% ci : 0.600.91). the fs measurements, however, showed only fair - to - good repeatability, with the lowest icc in the direction of neck flexion the mean icc was 0.39 (95% ci : 0.010.67), while the peak icc was 0.67 (95% ci : 0.390.84). the indices of change in the mean between the two test occasions (table 4) show a systematic change in the fs measurements, especially in the direction of flexion. the bland - altman plots reveal a similar picture in that the plots of mvc and rfd show acceptable agreement. the plots of tts and the prediction of vo2 max show a random scatter of points above and below the mean difference line, thus showing good agreement. the fs measurements, however, show, in keeping with data from tables 3 and 4, an unequal spread, especially regarding the direction of flexion, thus illustrating a trend of systematic error. no significant changes were found in tts for individual muscles between the two test occasions. quantifying the size of the variability by me and cv%, as shown in table 5, in this study, 25 healthy children 9 to 18 years of age were tested twice, by a single trained and experienced, blinded research lab technician, for isometric strength measurements in the neck and shoulder, pericranial tenderness, and prediction of vo2 max using a cycle ergometer test and the strand nomogram. this agreed with earlier findings in adults by andersen.30 the mean and peak mvc isometric strength of the neck also showed repeatability without systematic changes and an acceptable agreement between measurements. a finnish study using a comparable device system has previously shown repeatable mean and peak force measurements of adolescents with and without headache,9 in both extension and flexion directions. in our study, the peak forces seemed slightly more stable, thus indicating that this should be an area of focus in future research. the test of maximal voluntary contraction is an unusual situation for the participant and means a learning effect might have existed during the trial. as a result, considering the highest value as the maximal performance supported is relevant in the present study. our study comprised a 1-week interval between two measurements, and in contrast to comparable studies where the time between measurements was considerably shorter, issues involving fatigue or increased results after repetitive learning are less obvious.9,10 our results reveal that the fs measurements, especially regarding neck flexion, showed systematic changes between the two test occasions. a study by ylinen showed that in adults, the extension measures are more reliable than flexion measures. the researchers concluded that this phenomenon might be due to the fact that the extension muscle force is stronger than that of flexion and thus more stable. oleary,31 who examined the steadiness of cervical flexion at 20% and 50% of mvc in 20 adults in a supine position, found that at 20% the measures had a perfect reliability by repeat, while at 50% in a small group of eight healthy controls, the measures demonstrated very poor reliability. as children are reportedly less stable than adults,32 a trend of systematic changes likely due to fatigue is, to some degree, understandable with a fs measurement at 30% of mvc. as a result, we recommend that further study in children in this area use resistance at 20% and 30% of mvc. the tenderness and the aerobic power tests show acceptable repeatability and agreement between measurements. in accordance with the tts and following procedures validated by bendtsen,18 we used the same calibrated palpometer, the same experienced lab technician, and the same lab for both sets of tests. in the early original study, the intraobserver reliability was particularly consistent, and the pressure of palpation was stable if the pressure was initially controlled by the palpometer. the degree of tenderness is usually reported as an aggregate of the scores. in keeping with the recommendations of bendtsen,17,18 our studies are based on two assumptions : first, the observer is assumed to have the ability to evaluate pain during palpation using both verbal and behavioral scoring ; second, the assumption is made that a ordinal scale score, summed to a total score (tts), is considered as a ratio scale. bendtsen found a positive linear relationship between tts and pain intensity scores during palpation of the trapezius muscle and concluded that the four - point tenderness scale, and thus the aggregate sum, can be considered a ratio scale.17,18 as a result, it can be concluded that repeated recording of tts is of good quality for examining pericranial tenderness in children. however, upon close examination of the quantified variability, the tts revealed a large me and corresponding cv%, which is an issue that must be taken into consideration when interpreting clinical improvements in trials.. one main limitation of this study is that it did not involve children suffering from frequent or chronic tth. future testing on children with headache, based on the method presented here, would increase our knowledge even further. as a result, families with ill children must be encouraged to seek treatment at the headache clinic, and the burden of test - retest procedures on families must be taken into consideration. despite current drawbacks, the present study provides fundamental support for future studies examining underlying pain mechanisms in children with tth. one main limitation of this study is that it did not involve children suffering from frequent or chronic tth. future testing on children with headache, based on the method presented here, would increase our knowledge even further. as a result, families with ill children must be encouraged to seek treatment at the headache clinic, and the burden of test - retest procedures on families must be taken into consideration. despite current drawbacks, the present study provides fundamental support for future studies examining underlying pain mechanisms in children with tth. in conclusion, the measurements of strength capacity, aerobic power and pericranial tenderness show an acceptable repeatability, suitable for research in children. our study demonstrates that two of the examined test procedures, the tts and the submaximal aerobic power test, both of which only require a limited amount of equipment, can have a positive impact on clinical practice. moreover, the procedures are easily learned, and the test situation is comfortable for the participant. results from future research in children will hopefully provide additional knowledge about children s muscle capacities and their possible relation to headache and other pain disorders. this knowledge, in turn, can have profound implications for the choice of treatment and the interpretation of intervention results.	backgroundfrequent or chronic tension - type headache in children is a prevalent and debilitating condition for the child, often leading to medication overuse. to explore the relationship between physical factors and tension - type headache in children, the quality of repeated measures was examined. the aim of the present study was to determine the test - retest repeatability of parameters determining isometric neck and shoulder strength and stability, aerobic power, and pericranial tenderness in children.methodstwenty-five healthy children, 9 to 18 years of age, participated in test - retest procedures within a 1-week interval. a computerized padded force transducer was used for testing. the tests included the isometric maximal voluntary contraction and force steadiness of neck flexion and extension, and the isometric maximal voluntary contraction and rate of force of the dominant shoulder. pericranial tenderness was recorded by means of standardized manual palpation, and a submaximal cycle ergometer test predicted maximal oxygen uptake (vo2 max). the measurements were evaluated in steps, using the intraclass correlation coefficient (icc) ; changes in the mean between the two test occasions ; the levels of agreement, visualized in bland - altman plots ; and by quantifying the variability.resultsthe results showed an acceptable test - retest repeatability of isometric maximal voluntary contraction (icc 0.900.97). the force steadiness measurements revealed a trend of systematic changes in the direction of neck flexion and need further examination in both healthy and ill children. the rate of force development, total tenderness score, and prediction of vo2 max showed repeatability, with icc 0.800.87.conclusionthe measurements of strength capacity, aerobic power, and tenderness provide acceptable repeatability, suitable for research in children.
crohn 's disease (cd) is a chronic ibd that can involve any part of the gastrointestinal tract. nearly 80% of patients with cd require surgical treatment for complications or failure of medical management.1 however, patients with cd tend to have a high incidence of postoperative complications, including anastomotic leakages, fistulas, bowel obstructions, and wound infections,2 these complications are directly related to mortality rates. one study reported that using infliximab decreased cd recurrence after surgery.3 however, the effect of infliximab therapy on postoperative complications of cd, such as fistulas, is unknown. we present the case of a patient with cd who was treated with infliximab for a postoperative fistula a 31-year - old man with cd was admitted to the hospital because of severe abdominal pain and diarrhea. he had been diagnosed with cd 10 years previously, but he was lost to follow - up, during which he experienced repeated exacerbations and remissions. the patient had taken mesalazine 3,000 mg / day and azathioprine 50 mg / day and had started infliximab one year prior to admission. with infliximab, the patient experienced immediate relief of diarrhea and abdominal pain, but obstructive symptoms had gradually worsened. he complained of abdominal pain, increased borborygmus, decreased oral intake, and a decrease in body weight. abdominal ct scan and small bowel series revealed an ileocolic fistula, multiple ulcers, and severe stricture of the small bowel (fig. vital signs (blood pressure, 120/80 mmhg ; pulse rate, 80 beats / min ; respiratory rate, 20 beats / min ; body temperature, 36.9) were normal. he has a height of 171 cm, a weight of 53 kg, and a bmi of 18.1 kg / m. the patient had a chronically ill appearance with pale conjunctiva, mild tenderness in the lower abdomen, and accentuated bowel sounds. laboratory testing revealed anemia with a hemoglobin level of 8.7 g / dl, leukocyte count of 3,970/mm, and platelet count of 265,000/mm. serum chemistry tests were consistent with severe malnutrition : ast / alt 53/29 iu / l, bun / creatinine 15/0.64 mg / dl, total protein / albumin 4.3/2.2 g / dl, and total cholesterol 88 mg / dl. esr and crp level were 35 mm / h and 2.1 mg / dl, respectively. both anti - saccharomyces cerevisiae - antibodies iga and igg were negative, and the patient 's cdai was about 261 points. he had been experiencing approximately 25 liquid stools per week, severe abdominal pain, and a very poor sense of well - being. to improve the patient 's condition, wide resection of involved bowel and colostomy would be needed ; however, because of a psychological reaction to personal disfigurement and the need to relearn bowel habits, he strongly refused colostomy. therefore, we decided to perform a small and large bowel segmental resection and primary repair. gross findings during surgery included an abscess connected to the terminal ileum, bladder adhesion, a jejuno - sigmoid fistula, and huge inflammatory masses. an ileocecectomy, jejunal resection, and sigmoid colon resection were performed, followed by primary repair of the jejunum, rectum, and sigmoid colon (fig. however, one week postoperatively, the patient developed a high fever and abdominal pain. the drainage fluid was dark yellow and highly viscous but gradually cleared after continuous use of antibiotics. however, despite no further evidence of infection (white blood cell count 4,270/mm and crp 0.3 mg / dl), approximately 30 - 50 cc of fluid issued from every drain daily, even one month after the operation. the patient was started on 5 mg / kg of infliximab to treat the postoperative fistula and prevent a recurrence. after infliximab therapy was initiated, drainage decreased dramatically to less than 10 cc a day. in follow - up abdominal ct scans, the abscess in the abdominal cavity and the postoperative fistula disappeared (fig. the percutaneous drainages were consecutively removed, the patient began receiving oral nutrition, and the intravenous antibiotics were stopped. three months after surgery, the patient looked healthy and reported passing stools once or twice a day. his body weight increased by 8 kg, bmi increased to 20.9 kg / m, and his cdai decreased to 60. in follow - up laboratory exams, the patient 's hemoglobin level was 12.7 g / dl, total protein and albumin were 6.9/3.9 g / dl, and total cholesterol was 130 mg / dl. even though advances have been made in the medical care of patients with cd, about 80% of patients with cd undergo several operations in their lifetimes.4 however, these patients have a high rate of complications after surgery (6 - 45%) due to the presence of abscesses in the abdominal cavity, obstruction, malnutrition, and chronic steroid use. they usually require multiple anastomoses, which results in higher mortality rates (0.5 - 5.5%) due to complications as compared to patients without cd.5 - 7 complications from surgery include wound infections, anastomotic leakages, fistulas, anastomotic site strictures, and sepsis in the pelvis. fistulas are relatively frequent postoperative complications in patients with cd, but the underlying mechanism of fistula formation has not yet been determined. in patients with cd, fistulas are generally related to deep mucosal ulcers characterized by transmural inflammation. to repair the mucosal defect, epithelial cells migrate from the ulcer wall into the ulcer base. for faster migration, epithelial cells undergo epithelial - mesenchymal cell transition and develop a mesenchymal cell - like phenotype with loose cell - to - cell contacts that finally penetrate into deep layers of the bowel wall.8 lamina propria fibroblasts in the mucosa of patients with cd have reduced migratory potential and might therefore, not be able to migrate successfully to the tissue defect. however, different mechanisms may be involved in each fistula type, and each fistula type may respond differently to infliximab treatment. for example, internal fistulas usually occur immediately proximal to the stricture and wall thickening, causing high intraluminal pressure with distension and increased wall tension of the prestenotic bowel segments. fistulas formed by these mechanisms may react less to anti - inflammatory agents due to fixed fibrotic tissues and muscular hyperplasia.9 infliximab is a widely used monoclonal antibody against tumor necrosis factor that seems to be effective for treating fistulas in patients with cd. there is some dispute as to whether infliximab use preoperatively increases the risk of postoperative complications. reported that infliximab use within 3 months prior to surgery is associated with increased postoperative sepsis, abscess, and readmissions in patients with cd.10 marchal. found no significant difference between patients who received infliximab before surgery and those who did not, based on the occurrence of complications and the period of hospitalization.11 the effect of infliximab on postoperative fistulas in patients with cd has not been studied sufficiently endoscopic recurrence rates after surgery have been reported to be 65 - 90% in the first 12 months, 80 - 100% in the first 3 years, and 20 - 25% every year after ileocecal resection.12 some studies have reported that using infliximab after surgery was effective in preventing cd recurrence.13 - 16 sorrentino. performed a prospective cohort study of 12 consecutive patients who were treated immediately after surgery with maintenance infliximab (5 mg / kg). three years after surgery, none of the patients had clinical or endoscopic evidence of cd recurrence. however, discontinuation of infliximab resulted in endoscopic recurrence after 4 months in 10 of the 12 patients (83%).13 furthermore, regueiro. reported a one - year, randomized, double - blind, placebo - controlled trial of 24 patients who had undergone ileocolonic resection and found that the endoscopic recurrence rate of the patient group managed with 5 mg / kg of infliximab (9.1%) was significantly lower than that of the placebo group (84.6%).14 moreover, the histological recurrence rate in the infliximab group (27.3%) was lower than that in the placebo group (84.6%). however, no large - scale, randomized, controlled studies that have followed patients for an extended period have been reported. our case of successful treatment of a patient with cd with infliximab to resolve a postoperative fistula indicates that this is a promising treatment option for postoperative fistulas. in korea although the incidence is still low compared to western countries, the mean annual incidence rate of cd per 100,000 inhabitants was 0.53 in seoul for a 20-year period.16 the number of operations performed is expected to increase in the future. therefore, additional clinical studies on the role of infliximab in treating postoperative fistulas in patients with cd are required.	nearly 80% of patients with crohn 's disease (cd) require surgical treatment for complications or failure of medical management. we managed a 31-year - old man with cd who presented with a post - operative fistula. the patient had undergone surgery due to multiple strictures and a fistula. however, a new fistula developed that connected to the intraperitoneal abscess. intravenous antibiotics were started and multiple percutaneous drainage tubes were inserted to treat the abdominal abscess. however, the amount of drainage was consistently high, even one month after the operation. to treat the postoperative fistula, 5 mg / kg of infliximab was started, and the amount of drainage decreased dramatically to less than 10 cc a day. some studies have reported that infliximab decreases the recurrence of cd after surgery. the effect of infliximab on post - operative fistulas in patients with cd has not been sufficiently studied. our results indicated that the use of infliximab to treat post - operative fistula should be explored further in future clinical studies.
literature has identified and defined evening - fever syndrome (efs) as the thermal capacitance effect of overheated buildings that manifests in neonatal hyperthermia usually during the mid afternoon and early evening periods of sunny days. efs affects all newborns, especially the premature escalating morbidity, prolonging hospitalisation period and impoverishing overall outcome. the effect of efs has been reported from many parts of nigeria with extreme cases occurring within the savannah regions of the middle - belt and the desert northern regions of the country. at the federal medical centre nguru (fmc - nguru) located within this zone, efs is a significant contributor to neonatal morbidity resulting from soaring local ambient temperature that goes as high as 47c at times. in a recent publication amadi. this implicated the climate, environment, and civil structures among other reasons for excessive room warming that instigates efs. this suggested that efs is a condition of time - dependent neonatal hyperthermia that can be clinically diagnosed, typically being associated with the following constituents : it notably occurs on high ambient days, often in excess of 37cit readily affects neonates within room / nursery building of which walls, windows, and/or ceiling are directly exposed to high intensity of rays of sunlightneonates acquire high body temperature during early postmeridiem (p.m.) perhaps due to heat transfer from the energised surroundings but regain thermal stability much later in the evening when the surroundings have cooled down. it notably occurs on high ambient days, often in excess of 37c it readily affects neonates within room / nursery building of which walls, windows, and/or ceiling are directly exposed to high intensity of rays of sunlight neonates acquire high body temperature during early postmeridiem (p.m.) perhaps due to heat transfer from the energised surroundings but regain thermal stability much later in the evening when the surroundings have cooled down. apart from occasional planting of trees within residential compounds in nguru town and the raising of concrete flooring by up to 1 ft above ground level as seen from the structures at fmc - nguru, there are no other building standards that technically suggest adaptation to this extreme high ambient temperature. as a result the interiors of buildings including the special care baby unit (scbu) of this hospital often get higher than 40c of room temperature. this is significantly high enough to trigger hyperthermal crisis for the immature neonate even for a short period of exposure [4, 5 ]. neonatal thermal stability, defined as the condition of maintaining the body temperature of a given neonate within the physiologically allowable range of 36.5c to 37.4c, is a crucial factor for the survivability of any newborn. the scbus of nigerian hospitals often respond to this extreme condition of environmental high temperature with all kinds of desperate clinically inefficient measures. some clinicians often suspect a disease process responding with doses of unnecessary antibiotics and complicating situations and from which many neonates might have lost their lives. in addition, nurses resort to the desperate practice of opening incubator access windows / doors, turning - on fans, turning - on air conditioners or entirely switching - off the incubator. at the fmc - nguru babies there are down - sides to these desperate measures : opening port - holes compromises the microenvironment as a mini - isolation unit for the delicate premature baby [6, 7 ] ; fans and air conditioners are subject to adequacy of power supply, frequent breakdown, and threat of hypothermia for cot babies ; incubator switch - off often results in sudden hypothermia during environmental thermal recovery at sunset ; baby bathing has momentary effect only and often demands unaffordable high volume of nursing time as this has to be repeated several times during the day. this hence results in frustration for the few nurses that would attempt to thermally stabilise any neonate. we hypothesise that any measures that can achieve naturally cooled nursery environment would eliminate the uncontrollable excessive warming of neonates due to efs. the relatively poor setting of tropical africa would constitute complications due to factors that are naturally difficult to change or be sustainably managed. these involve the sun and sunlight intensity as the source of heat generation, the relocation of the hospital complex to a more appropriate site, provision of uninterruptable air conditioning units, or supply of specially designed adaptable incubators to replace a host of the climate - stricken standard models. in the face of these seeming difficulties it became imperative to seek to investigate how design or building structural factors could be optimized for a probable permanent solution [9, 10 ]. therefore, the aims and objectives of this study were (1) to develop miniaturised laboratories with distinct unconventional building factors that are capable of ameliorating the warming effect of the sun in a nursery and (2) to validate these by carrying out a comparative analysis of room warming and neonatal thermal stability of babies being nursed in the laboratories. the project was divided into two segments involving (1) design and construction of two different nursery laboratories and (2) validation of the new constructs through controlled comparative analyses of outcomes. ethical approval for the execution of the project was obtained from the medical ethics committee of the federal medical centre nguru, nigeria. patient - informed consents for the validation aspect of the research were also obtained from carers, in most cases the mothers, after carefully explaining the project and how their babies might be minimally involved in the use of data collected during normal nursing / clinical procedures whilst treating them. it was more difficult recruiting from illiterate carers who were more in number than the educated ones. a total of 97 neonates were recruited, 33 for the wet season months (september - october 2012) analyses and 64 for the dry season months (march - april 2013) analyses. average admission - weight was 1966 g (range : 1050 g2500 g). average gestation age of the sample population could not be accurately quantified as most cases lacked antenatal information. literature reveals that nursery warming due to extreme sunlight intensity comes through capacitance effect of nursery walls that are directly heated by the sun amongst other mediums. the laboratory design therefore incorporated a double - wall with in - between air space for all sides that were exposed to direct rays of sunlight. these were flooring, roof / ceiling height and materials, window size and positioning, window blind materials, and room lighting. the first (lab-1) represented the construction of a purpose built nursery structure that targeted the total elimination of efs. this integrated all possible constraints that were capable of minimising the transmission and concentration of sun - heat within the interior of the structure. the second laboratory (lab-2) represented a possible renovation that could be done to an existing nursery structure so that the warming of the interior by the sun would be considerably reduced. lab-2 was designed to avoid expensive or extensive constructions that could be either unaffordable or lengthily disruptive to the activities of an existing nursery. this was implemented by the renovation of an existing office space at the special care baby unit (scbu) complex of fmc - nguru. the double - wall for the side exposed to sunlight was provided by adding an inner wall on that side. an arbitrary air space of 6 cm was provided in - between the walls and vented into the roof space through the ceiling. this created escape route for heated air within the space and was expected to enhance the reduction of heat transfer to the inner wall. lab-2 was finished to a rectangular floor area of 5.9 m, the original ceiling height of 3 m, and one door and one window. the design of lab-1 extensively integrated other measures that could contribute to further heat reduction on the inner wall and the interior of the structure as explained below.prior to design, a simple thermometric investigation revealed that a significant temperature difference existed between the sandy ground level surface of the site and the bottom of an excavation 90 cm below the surface. it was therefore considered a good idea to take advantage of this and tap the coldness for the room. the floor was therefore designed as a 5 cm thick concrete mixture that was set at 120 cm below ground level. the surrounding single wall continued until ground level where this transformed externally into the double wall as described for lab-2. inner walls and floor were finished in smooth ceramic tiles.the floor - to - ceiling height was 380 cm, that is, 80 cm more than the standard height of rooms in the hospital complex. this was to achieve a good height for warmer air that tends to raise the room temperature at the height of the cots and incubators. a roof extractor fan that automatically covered its outlet whilst switched - off was provided to maintain the escape of heated air from the room and vented air within the roof space as shown in figure 1.a controlled valve - operated heat exchanger was integrated in the design. this was a system of flowing water made to traverse the interior surface of the inner wall, behind the tiling. the copper - piped water entered building from the left end via a manual valve, crisscrossed the entire upper half of the inner wall above ground level, and exited through the right end (figure 2(a)). water was supplied from an insulated overhead tank to ensure adequate pressure head and emptied into the same underground tank from which the overhead tank received supply (figure 2(b)). the underground water storage served a purpose of recooling the water before recycling to the overhead tank. heat exchanger was operated during data collection on few prescribed days only for the investigation of any possible contribution such application might offer towards the cooling of lab-1. the coolness pattern of lab-1 relative to heat exchanger days was evaluated by the quantification of average temperature rise of the water from the system outlet as a measure of heat extraction from the lab interior.two opposite wall windows were integrated such that these were indirectly facing each other to ensure better air circulation. cotton material window blinds were affixed and operated when sun - heat intensity through the windows became very intense. electric lighting of the interior and medical examination lamps were provided by low wattage led system installations to minimise heat generation by these applications. prior to design, a simple thermometric investigation revealed that a significant temperature difference existed between the sandy ground level surface of the site and the bottom of an excavation 90 cm below the surface. it was therefore considered a good idea to take advantage of this and tap the coldness for the room. the floor was therefore designed as a 5 cm thick concrete mixture that was set at 120 cm below ground level. the surrounding single wall continued until ground level where this transformed externally into the double wall as described for lab-2. the floor - to - ceiling height was 380 cm, that is, 80 cm more than the standard height of rooms in the hospital complex. this was to achieve a good height for warmer air that tends to raise the room temperature at the height of the cots and incubators. a roof extractor fan that automatically covered its outlet whilst switched - off was provided to maintain the escape of heated air from the room and vented air within the roof space as shown in figure 1. this was a system of flowing water made to traverse the interior surface of the inner wall, behind the tiling. the copper - piped water entered building from the left end via a manual valve, crisscrossed the entire upper half of the inner wall above ground level, and exited through the right end (figure 2(a)). water was supplied from an insulated overhead tank to ensure adequate pressure head and emptied into the same underground tank from which the overhead tank received supply (figure 2(b)). the underground water storage served a purpose of recooling the water before recycling to the overhead tank. heat exchanger was operated during data collection on few prescribed days only for the investigation of any possible contribution such application might offer towards the cooling of lab-1. the coolness pattern of lab-1 relative to heat exchanger days was evaluated by the quantification of average temperature rise of the water from the system outlet as a measure of heat extraction from the lab interior. two opposite wall windows were integrated such that these were indirectly facing each other to ensure better air circulation. cotton material window blinds were affixed and operated when sun - heat intensity through the windows became very intense. electric lighting of the interior and medical examination lamps were provided by low wattage led system installations to minimise heat generation by these applications. lab-1 was finished to a rectangular floor area of 8.3 m, roof height of 3.8 m, and one door and two windows. the existing nursery section for babies born within the fmc - nguru facility (in - born unit) was applied in the study as the control ward while lab-1 and lab-2 were the test - wards. this has a rectangular floor area of 70.5 m, room height of 3 m, and two opposite french doors and six windows arranged for effective crossventilation with window - blinding shutters. electric power supply to all three wards was provided via two assembles of 3.5 kva inverter the power - banks ensured uninterrupted power supply to the experimental wards all through the periods of data collection. these acted as ready backups during unexpected power failures as normally witnessed in nigerian cities. environmental temperature, humidity, and other meteorological data within all three wards and the outside of the complex were automatically collected and stored every 30 minutes. this was achieved by the application of three assembles of watson 's w-8681 weather station. babies from consenting carers / parents were randomly assigned to available cots and incubators in all three wards. neonates were nursed and vital signs data were collected by nurses at the usual practice times of the day. nurses were posted to the wards on rota basis and the fmc - nguru standard neonatal nursing techniques and procedures particularly remained unaltered in the three wards. this also involved the practice that required any hyperthermic baby to be water - bathed (sponged) if body temperature climbed up to 37.9c. exclusion / inclusion criteria were carried out as follows : it included all preterm neonates during special care nursing.it included all full - term neonates below 2500 g birth weight but was limited to half of the neonatal period (first 2 weeks of life) as healthy older neonates might be capable of significant auto thermal regulation.it excluded any neonates undergoing treatment for a well - established disease or disease process. it included all full - term neonates below 2500 g birth weight but was limited to half of the neonatal period (first 2 weeks of life) as healthy older neonates might be capable of significant auto thermal regulation. it excluded any neonates undergoing treatment for a well - established disease or disease process. experimental setup and data collection continued for over 24 months, covering every month of the year for up to two times. data for the latest 12 months were applied for the comparative analyses of outcomes from the three wards in relation to the impacting environmental conditions of the outside surrounding. the collected data was applied to study impact of extreme hot days in the laboratories and the control ward. this was treated as an extreme hot day if the day 's peak temperature exceeded 36c. the entire data covering the 12 months of the year was carefully examined, extreme hot days were identified, and their data were extracted into a file and were applied for further processing as described below.plots of temperature against time - of - the - day were produced in superimposition for all three wards and outdoor temperature. these were produced for each extreme hot day to study the daily pattern of heat gain or loss in each ward in relation to changes in out - wind temperature.records of relative - humidity (rh) against time - of - the - day were studied to assess the suitability of the wards for effective neonatal nursing without extra room humidification. this was also used to determine how the wards responded to changes in outdoor rh.the daily peak temperatures for each of the four environments (i.e., the 2 labs, the control ward and outdoor environments) for all extreme hot days were identified and their averages for each environment quantified.the averages of the daily relative humidity were quantified for each of the four environments.the behaviour of incubators in all three wards on the extreme hot days under assessment was evaluated from an incubator data collection chart to determine how environmental overheat affected them.all the patients ' temperature chart folders for the period of study were recalled. patients admitted during the two hottest months of the dry season (march and april) and the two relatively hot months of the wet season (september and october) were extracted and applied to obtain the total number of baby water - sponging events for each of the three wards being studied.the effect of the heat exchanger was evaluated by activating this on other specific days primarily to test the exchanger only. temperature of water at laboratory inlet and outlet were measured and noted at 12 noon, 3 pm, and 6 pm on each day. data was applied to quantify the averages of inlet and outlet water temperatures during the specified times of the day. this would enable the evaluation of the most cost - effective time of operating such a device to improve heat extraction from the ward. plots of temperature against time - of - the - day were produced in superimposition for all three wards and outdoor temperature. these were produced for each extreme hot day to study the daily pattern of heat gain or loss in each ward in relation to changes in out - wind temperature. records of relative - humidity (rh) against time - of - the - day were studied to assess the suitability of the wards for effective neonatal nursing without extra room humidification. this was also used to determine how the wards responded to changes in outdoor rh. the daily peak temperatures for each of the four environments (i.e., the 2 labs, the control ward and outdoor environments) for all extreme hot days were identified and their averages for each environment quantified. the averages of the daily relative humidity were quantified for each of the four environments. the behaviour of incubators in all three wards on the extreme hot days under assessment was evaluated from an incubator data collection chart to determine how environmental overheat affected them. patients admitted during the two hottest months of the dry season (march and april) and the two relatively hot months of the wet season (september and october) were extracted and applied to obtain the total number of baby water - sponging events for each of the three wards being studied. the effect of the heat exchanger was evaluated by activating this on other specific days primarily to test the exchanger only. temperature of water at laboratory inlet and outlet were measured and noted at 12 noon, 3 pm, and 6 pm on each day. data was applied to quantify the averages of inlet and outlet water temperatures during the specified times of the day. this would enable the evaluation of the most cost - effective time of operating such a device to improve heat extraction from the ward. the working floor areas of the control ward and labs were not equal in dimension to enable a direct building cost comparison. however, engineers were commissioned to design and produce bill - of - quantities for 2 equal size structures of floor area of 16.6 m, each based on the construction details of the control ward and lab-1, respectively. they also separately quantified the cost of adding double - wall to the two sides of the control ward patterned design. all quantifications were based on the prevailing costs of building materials and labour within and around the town of nguru nigeria. this revealed that the sum of 4.69 million naira (about us$29,000) was required to complete the project based on the control, including all fittings and air conditioning. lab-1 patterned counterpart was estimated to cost 4.93 million naira (about us$31,000) including entire heat exchanger assembly. extra double wall on two sides for lab-2 pattern would require ngn317,000 (about us$2,000). superimposed daily plots of environmental warming for 24 hours of the day typically showed that ward peak temperatures were always lower than the out - wind peak temperature during the day times. these also revealed that lab-1 peak temperatures were always the lowest and rarely exceeded 33.5c even when out - wind peak soars up to 46c (figures 3 and 4). on the contrary, the control ward always registered the hottest peak temperatures of the day amongst the three tested wards. this was typically lower than the out - wind peak temperatures, often within 3c. on the average, lab-1 was constantly much cooler than out - wind peak - temperature often with margins of up to 11c (figure 4). when the four overall hottest days of the study period (each registering over 44c peak temperature) were separately examined as shown in figure 5, lab-1 remained stably cool at 32.9 0.5c ; range : 32.1c to 33.2c across the four days. figure 6 shows a chart of the overall average peak temperatures of the entire extreme hot days for the four environments during the extreme hot days of dry season months of march / april and wet season months of september / october with their average relative - humidity (rh). typically, all the incubators in lab-1 maintained set - point values at all times without overheating during the extreme hot days. the incubators of lab-2 overshot set - point on few occasions while those of the control ward were constantly overheated beyond set - point values. figure 7 shows the typical performance of three incubators set at the same operational set - point value of 32.2c in the three respective wards as captured on october 17, 2012. figure 8 shows the outcome of baby water - sponging events as a result of efs - induced hyperthermia in the 3 wards for the months of march, april, september, and october. on the overall impact on the wellness of neonates, 70% (representing 28) of babies in the control ward were water - sponged 63 times during the wet season months of september and october. during the dry season months of march and april, 100% (representing all 16) lab-1 folders revealed only 2 water - sponging events on 1 of 13 participating babies during the dry season months of march and april. there was no reported case of water sponging in lab-1 all through the wet season study period. we observed that preterm and low - birth - weight (lbw) babies were quicker to succumb to efs attack as compared to full term and bigger neonates. in addition to excessive high body temperatures, attack was found to be associated with relative increase in heart - rate (hr) most of the time. respiration - rate (rr) was also affected as this was faster than normal in some patient but slower than normal in other patients during efs attack. water sponging would bring about sudden quench of fever for a short period of time. this improved rr in some patients for the short time but this did not necessarily slow down hr during this period. figure 9 is a graphical display of the signs of skin - temperature, hr, and rr in a typical case of comparing physiological events in the control ward and lab-1 during a 5-day period from the very hot month of april 2013. the two babies being compared were water - sponged several times during the day due to efs attack whilst they remained in the control ward. however when one of them was transferred to lab-1 on april 11 just before midnight, this baby recovered and never had any need for water sponging again for the rest of the comparing period unlike its counterpart that remained in the control ward (figure 9). there was observable difference in the relative coolness of lab-1 with the heat exchanger operation, especially just before the sun began to set at 6 pm. average inlet / outlet temperatures of water through the exchanger are shown in table 1. the present study has sought to create and validate a method of building a sustainable weather - proof neonatal nursing environment that can curb morbidity due to tropical evening - fever syndrome (efs). at study inception we hypothesised that a naturally cooled nursing environment with minimised heat - impact of the sun would guarantee effective incubator performance in maintaining desired incubation set - points without externally influenced overheating. we also hoped that such thermal - friendly environment would guarantee a more stable nursing of open - cot babies within the environment with lower frequency of hyperthermia due to efs. an earlier study demonstrated that neonates readily tend to assume body temperatures similar to their environmental room temperatures when exposed to these. this is consequential to the classical procedures of maintaining effective neonatal warming in a cold environment using appropriately designed wrapping suits for full - term neonates and adequately controlled incubator microenvironments for preterm neonates [12, 13 ]. preterm neonates exhibit higher ratio of skin - surface - area to body - mass as compared to full - term neonates. this delimits the preterm neonate 's ability to autoregulate its body temperature when exposed to a room temperature outside the physiological range. therefore, efs can fundamentally set in when environmental room temperature exceeds the acceptable physiological body upper limit of 37.4c. the present study created lab-1 and lab-2, testing their respective abilities to maintain room temperatures that were well below this physiological limit even with prevailing extreme heat intensity from the sun. our lab-1 results have shown that by carefully integrating some factors as considered in this study, a naturally cooled environment could be guaranteed. lab-2 was relatively warmer than lab-1 because this lacked the additional cooling effect the underground floor / walls provided in lab-1. the control ward 's room temperatures readily exceeded this physiological upper limit as can be seen from figure 6. this consequently led to a very frequent manifestation of efs, incubator overheating, and general un - wellness of babies in this ward. in general, the room temperatures of all the wards during the early hours of the day were far below the hypothermic threshold of 36.5c and at times as low as 26c. this condition would normally require the incubators to operate at high set - points to supply extra heating for the preterm neonates while cot - babies were given adequate extra wrapping. the room temperatures of our control ward changed very rapidly with the appearance of the sun and its rising intensity as the day progressed into the afternoon. this rapid increase of room temperature resulted in additional uncontrollable heating that would soon overheat the cot - babies, the incubator, and its occupant. in most cases the attending nurses were unaware of the hyperthermic babies until the next vital - signs check that happened only once every 4 hours (6 am, 10 am, 2 pm, 6 pm, and 10 pm), with 2 am being verily inconsistent. the only exception to this was in cases where the uncomfortable babies reserved any energy to cry or show signs of unrest or the incubators produce overheat alarm. in extreme cases complete switch - off of incubator heating would not remove the problem as the sun singularly supplied enough energy to warm the nursery beyond 38c. this results in more and more babies being given the ultimate solution of temporary comfort of water sponging. we do not yet know of the consequences of such sudden quench of high fever on the neonates as this has not been studied. however, such practice of sudden temperature crash from hyperthermia to hypothermia and a quick buildup back to hyperthermia without any obvious thermal stability creates a condition of neonatal thermal shock that could readily initiate apnoeic attacks. this extreme complication was however not observed in the test wards due to their relative coolness and more sluggish rate of temperature increases as compared to the control ward. lab-1 maintained an average temperature - gain rate of 0.75c / hr to average maximum peak of 33 1c as compared to the control ward at average rate of 1.63c / hr to average maximum peak of 39 1c. this situation guaranteed a more stable and relatively cooled lab-1 enabling the incubators to solely control their neonatal microenvironments, maintaining their operational set - points for the entire day. the operational effectiveness of the new lab designs in maintaining such coolness in a life - size nursery unit becomes more believable when lab-1 relative disadvantages are considered. lab-1, for example, was only a small room less than 12% of the area size of the control, with only 2 windows and 1 door. hence, this lacked the airy advantage that could possibly aid the cooling of the control. during the period of this experimentation, nurses reported instances when incessant hyperthermia would have led doctors to suspect a disease process and hence commenced antibiotic treatment as a matter of procedural practice. instead, some of the nonparticipating patients were temporarily moved to lab-1 (one at a time due to lack of cot space) and the patients quickly recovered, similar to the example in figure 9. this clearly demonstrated no presence of disease for which the use of antibiotic would have led to complications that could be capable of claiming the life of such baby. lab-1 clearly eliminated efs but still required well - trained nurses for effective dynamic regulation of the incubator to determine the appropriate set - point that could stabilise baby within the safe zone of 36.5c37.4c. baby could become hypothermic if lower - than - required incubator set - point was maintained. this explained why baby control - lab-1 showed signs of hypothermia during the first few hours in lab-1 (figure 9). this revealed that the control ward was overheated far above the physiologically acceptable neonatal upper limit temperature of 37.4c. this explains why so many water - sponging events were recorded in the control on the one hand (figure 8). on the other hand, the temperature peak values of the 2 labs never rose up to this physiological limit, resulting in the relative neonatal stability as depicted by figure 8. we can therefore conclude that neonatal efs can be reduced by correcting an existing scbu building using the specifications of lab-2 or where the funds could be afforded, eradicated by the construction of entire new structure with the specifications of lab-1. the cost of erecting lab-1 patterned structure might be slightly higher than that of the control ward based on projected bill - of - quantity ; however, this could be quickly offset by the running cost of the air - conditioning units in control ward during operation. at experimented heat extraction factor of 2.2c / unit - time the heat exchanger time of operation would be between 3 pm and 4 pm for best result. further study would investigate a possible link of disease appearance to the presence of efs. we do not understand why efs resulted in extremely low rr sign in some patients and extremely high in others whilst hr remained slightly high. (2009) noted that tachycardia is a sign of fever ; this is consistent with the high rr in our observation. however, the cases of lower rr might as well be associated with the thermal - shock effect explained earlier and hence a gradual approach of apnoea. a foreseeable limitation to the application of the present proposals (i.e., lab-1) is in the event of flooding. since such nursery design is likely going to be the lowest floor in the hospital complex, adequate allowance to prevent any possible occurrence of flooding must be incorporated in the design. adequate precautions should be taken to avoid development of wall cavities that could lead to stagnation of leaking water as this could become breeding grounds for mosquitos and dampness with possible consequences of malaria or other infections.	neonatal thermal stabilisation can become challenging when uncontrollable factors result in excessive body temperature. hyperthermia can rapidly slow down baby 's progress and response to treatment. high sunlight intensity in tropical countries such as nigeria manifests in incessant high neonatal temperatures towards early evenings. the ugly consequences of this neonatal evening - fever syndrome (efs) can only be eradicated by the development of a controlled weatherproof nursery environment. two laboratories and a control ward ' were applied. lab-2 was a renovation of an existing room in a manner that could correct an existing nursery. lab-1 was an entirely new building idea. the laboratories were assessed based on comparative ability to maintain environmental coolness and neonatal thermal stability during hot days. data collection continued for 12 full calendar months. on average, at evaluated out - wind peak temperature of 43c (range : 41c46c), the control - ward peak was at 39c, lab-2 peak at 36c, and lab-1 peak at 33c. all incubators in the control overheated during the hot periods but there was no overheating in lab-1. forty - four (86%) of sampled babies were fever - quenched by water sponging 131 times in the control whilst only one baby received same treatment in lab-1. nursery designs patterned after lab-1 can significantly reduce efs - induced neonatal morbidity.
a sample of 30 hiv - infected individuals from a integrated counselling and testing centre (ictc) in vadodara, gujarat, india were selected to participate in individual face - to - face in - depth interviews after pilot testing. hiv - infected patients were eligible to participate in the study if they were : (1) english - or gujarati - speaking ; (2) 18 years or older ; and (3) diagnosed with hiv for at least 3 months. all interviews were conducted in a private area by trained, bilingual staff after taking written informed consent. each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide. two note - takers were present at each interview to document the dialogue verbatim and take notes, including non - verbal communication. two focus groups were convened, one with seven medical doctors and the other with eight non - medical providers. hiv service providers were eligible to participate if they were : (1) gujarati - speaking ; (2) 18 years or over ; and (3) had worked with hiv - positive patients for more than 1 year as either a medical doctor, a non - governmental organization staff member, integrated counselling and testing counselor, or peer educator for hiv - positive persons. each of the focus groups lasted about 2 hours and was audio - recorded with prior permission of the participants. the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation. the entire research protocol was reviewed and approved by institutional review board (irb) of both the university of north carolina at chapel hill, usa and the medical college, baroda, india. the in - depth interviews and focus group guides were developed by research teams in india and the u.s. in addition, a community advisory board (cab), comprising experts from the field of social work, medical providers, and hiv consultants was convened to discuss and refine the content of the qualitative instruments. the in - depth interview guide was designed to assess topics pertaining to hiv disclosure processes, perceived barriers and facilitators to disclosure, and hiv - associated stigma and discrimination related to disclosure among hiv - positive individuals. the following topics were explored with the hiv service providers : (1) secondary prevention services provided to plwha ; (2) the advice they gave to plwha regarding whom to disclose to and why ; and (3) the consequences that they perceived their clients faced after disclosure. extensive notes of in - depth interviews were recorded in gujarati, and translated into english. the focus group discussions were transcribed verbatim in gujarati from the audio - recordings and then the transcripts were translated into english. in the early phase of coding, we based initial themes on topics covered in the interview / focus group discussion (fgd) guides. in the later phases, in - depth interview and fgd transcripts were read several times and content analyzed, respectively using the technique of open coding to discover conceptual patterns, or themes, in the text. research staff in india and u.s. coded the 8 of the 30 interview transcripts independently and then met to discuss the codes and both the fgds, and refined the code definitions. once all of the interviews were coded, a reliability exercise was conducted for the in - depth interviews. based on the reliability exercise, over 90% of the codes matched with the codebook developed by the study staff. in all, two codebooks were generated for the interview and fgds respectively. all interviews and fgds were coded using maxqda software, 2007 (berlin, germany). once the interview and fgd codebooks were generated, a triangulation technique was used to analyze which themes from the hiv - positive individuals and from hiv service providers respective perspectives supported as well as which contradicted one another. a sample of 30 hiv - infected individuals from a integrated counselling and testing centre (ictc) in vadodara, gujarat, india were selected to participate in individual face - to - face in - depth interviews after pilot testing. hiv - infected patients were eligible to participate in the study if they were : (1) english - or gujarati - speaking ; (2) 18 years or older ; and (3) diagnosed with hiv for at least 3 months. all interviews were conducted in a private area by trained, bilingual staff after taking written informed consent. each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide. two note - takers were present at each interview to document the dialogue verbatim and take notes, including non - verbal communication. two focus groups were convened, one with seven medical doctors and the other with eight non - medical providers. hiv service providers were eligible to participate if they were : (1) gujarati - speaking ; (2) 18 years or over ; and (3) had worked with hiv - positive patients for more than 1 year as either a medical doctor, a non - governmental organization staff member, integrated counselling and testing counselor, or peer educator for hiv - positive persons. each of the focus groups lasted about 2 hours and was audio - recorded with prior permission of the participants. the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation. the entire research protocol was reviewed and approved by institutional review board (irb) of both the university of north carolina at chapel hill, usa and the medical college, baroda, india. a sample of 30 hiv - infected individuals from a integrated counselling and testing centre (ictc) in vadodara, gujarat, india were selected to participate in individual face - to - face in - depth interviews after pilot testing. hiv - infected patients were eligible to participate in the study if they were : (1) english - or gujarati - speaking ; (2) 18 years or older ; and (3) diagnosed with hiv for at least 3 months. all interviews were conducted in a private area by trained, bilingual staff after taking written informed consent. each in - depth interview lasted approximately 2 h and followed a culturally sensitive semi - structured interview guide. two note - takers were present at each interview to document the dialogue verbatim and take notes, including non - verbal communication. two focus groups were convened, one with seven medical doctors and the other with eight non - medical providers. hiv service providers were eligible to participate if they were : (1) gujarati - speaking ; (2) 18 years or over ; and (3) had worked with hiv - positive patients for more than 1 year as either a medical doctor, a non - governmental organization staff member, integrated counselling and testing counselor, or peer educator for hiv - positive persons. each of the focus groups lasted about 2 hours and was audio - recorded with prior permission of the participants. the medical doctors were given 2500 rupees and non - medical providers were given 1500 rupees for their participation. the entire research protocol was reviewed and approved by institutional review board (irb) of both the university of north carolina at chapel hill, usa and the medical college, baroda, india. the in - depth interviews and focus group guides were developed by research teams in india and the u.s. in addition, a community advisory board (cab), comprising experts from the field of social work, medical providers, and hiv consultants was convened to discuss and refine the content of the qualitative instruments. the in - depth interview guide was designed to assess topics pertaining to hiv disclosure processes, perceived barriers and facilitators to disclosure, and hiv - associated stigma and discrimination related to disclosure among hiv - positive individuals. the following topics were explored with the hiv service providers : (1) secondary prevention services provided to plwha ; (2) the advice they gave to plwha regarding whom to disclose to and why ; and (3) the consequences that they perceived their clients faced after disclosure. extensive notes of in - depth interviews were recorded in gujarati, and translated into english. the focus group discussions were transcribed verbatim in gujarati from the audio - recordings and then the transcripts were translated into english. in the early phase of coding, we based initial themes on topics covered in the interview / focus group discussion (fgd) guides. in the later phases, in - depth interview and fgd transcripts were read several times and content analyzed, respectively using the technique of open coding to discover conceptual patterns, or themes, in the text. research staff in india and u.s. coded the 8 of the 30 interview transcripts independently and then met to discuss the codes and both the fgds, and refined the code definitions. once all of the interviews were coded, a reliability exercise was conducted for the in - depth interviews. based on the reliability exercise, over 90% of the codes matched with the codebook developed by the study staff. in all, two codebooks were generated for the interview and fgds respectively. all interviews and fgds were coded using maxqda software, 2007 (berlin, germany). once the interview and fgd codebooks were generated, a triangulation technique was used to analyze which themes from the hiv - positive individuals and from hiv service providers respective perspectives supported as well as which contradicted one another. there were 30 hiv - infected patients, equal number of men and women, who participated in the study. their demographic details have been represented in table 1. among the total patients, majority (both men and women) were from the younger age groups of 20 - 30 years and 31 - 40 years as compared to the older age groups of 41 - 50 years and 51 - 60 years. about three - fourth of the patients were married, a couple of female patients were widowed, a couple of patients (equal number of men and women) were divorced, few patients (more women than men) were separated from their spouse and a couple of patients (equal number of men and women) were unmarried at the time of the study. demographic details of plwha attending ssg hospital, vadodara the highest level of education among the patients was up to graduation which was found among a couple of female patients. in case of the male patients, less than half of them were educated up to 10 standard and about one - third were educated up to 12 standard. there were equal number of men and women among one - fifth of the patients who were illiterate. as regards employment, more than half of the patients (more men than women) were employed at the time of the study. among the patients selected for the study from vadodara district, less than half were from urban areas, one - third were from urban slums one - fifth were from rural areas and a couple of patients were from tribal areas. stigma associated with hiv was described as a barrier to disclosure because participants perceived hiv - positive status to have a negative impact on plwha and their children. the potential for hiv stigma to limit children 's marriage prospects was particularly worrisome and cited as a reason not to disclose one 's serostatus to people other than a spouse. a widowed female participant aged 30 years said, it would have adverse effects on my son ; the school might create problems for him. people may break relations and behave badly with me and my family as they consider hiv as a bad disease. in addition, a 33-year - old widow and a mother of three children, anxious about their future said, i fear that if i disclose my hiv positive status to everyone, it might affect my daughters marriage prospects. participants perceived that many of their friends and family held misconceptions about hiv which would lead to increased stigma and mistreatment after disclosure. a married female aged 28 years said, it would create a lot of problems for us, even if there is a slight indication in the society. people believe that one is infected by hiv only because of illicit relations. a separated female participant aged 42 years narrated, i had not disclosed my positive status to relatives and neighbors, but my husband revealed my positive status in his family and neighborhood. now, all my in - laws have cut off their relations with me and my son and neighbors discriminate against us. a female medical service provider stated plwha should not disclose their hiv status to the community due to hiv - related discrimination. the desire to protect others from the pain of knowing about their illness motivated some participants not to disclose. this concern seemed particularly salient in decisions about disclosing to parents, particularly if the parents were old or in poor health, or because they feared that the reality would be too painful and agonizing for their parents. i have not revealed my hiv positive status to my mother as i know that she would not be able to bear the reality. hiv service providers pointed out that in some cases, hiv - positive male participants withheld disclosure to their prospective spouse for fear of rejection, in turn passing infection on to her after marriage. a remarried male participant, aged 44 years, who had not disclosed his serostatus to his wife, discussed his dilemma saying, i have never practiced sex without using condoms saying i do nt want children. but my wife (who is unaware of my serostatus) fights a lot with me as she wants children. there were instances where family members disclosed participants hiv - positive status to others without their consent or will or in an, upset and disappointed by his ex - wife 's disclosure of his hiv status said, she told her brother, who in turn told her parents and then my father came to know. in half of the participants, physical incapacity and health problems were cited as reasons why their hiv - positive status was disclosed involuntarily to family members. a married male participant aged 44 years who disclosed his hiv - positive status due to his ill health, stated i have only told my mother as i was feeling weak and i could not even walk. medical providers stated that it was important for hiv - positive patients to disclose to their families, to protect caregivers from the potential risk of hiv infection. a married hiv - infected female participant aged 32 years, described how her and my husband 's condition did not improve at all, so the doctor asked to get the hiv test done. my report also came positive. while involuntary disclosure was a common thread among the participants, some discussed experiences of voluntary disclosure. voluntary disclosure occurred when participants felt morally obligated to divulge their serostatus to a spouse so s / he could get tested for hiv. a divorced male participant aged 30 years, who voluntarily disclosed his hiv - positive status to his wife stated, after reading the report, i thought this should not be kept a secret. the next day i took my wife and 11-year - old daughter for the test. another reason participants gave for voluntarily disclosing their hiv - positive status was to obtain emotional and physical support from family. an example is this explanation from a married female participant aged 26 years, who gave her reason for the decision to disclose her and her husband 's hiv - positive status to their parents, we told this to our parents only as they care for us. there are many consequences of disclosure [figure 1 ], but a couple of salient themes are presented below. a severe consequence of disclosure was family disruption leading to separation and divorce. a female participant aged 28 years, my mother - in law told me not to keep the child because i had hiv, the child could get it from me. but the doctors told us to take medicines and assured us with a 99% guarantee that the child would be hiv - negative and asked us to decide whether we want to keep the child or not. a divorced female participant aged 28 years expressed her grief, the doctor told me to get my cd4 count tested. so the doctor told me to abort the child as my cd4 count was low. consequences of disclosure some married female participants living in joint families, talked about being blamed by their mothers - in - law for either placing their son at risk for hiv or for being the source of their son 's hiv infection. my mother - in - law held me responsible for her son 's hiv infection. other women became aware of their serostatus only after being remarried and getting tested for hiv when they became pregnant. a divorced female participant aged 28 years, said with a lot of emotion, my first husband died due to hiv. my in - laws never told me about his hiv positive status even after his death. during my second marriage, i came to know about my being hiv positive when i was pregnant again. i advised him that whenever you get married, you either marry a positive person like yourself or inform the person about your positive status beforehand. after a few months, that same person came for getting his wife 's hiv test done. his wife was found positive ; he had not informed his wife about his hiv - positive status before marriage. although majority of patients described the barriers of disclosure, some hiv service providers and patients described the benefits. a female non - medical service provider explained, we try to explain to the patients that if they disclose their status to their families, they would take better care of them. if you tell any member of your family, who is close to you, then it would be better for you. medical service providers believed that hiv - positive patients would benefit from treatment by telling their providers about their seropositive status when going for any kind of treatment. this would also allow doctors to screen for tuberculosis, one of the most common opportunistic infections among plwha in india. a married female participant said, those persons in the neighborhood to whom i have disclosed my status, are good. they say that this disease does not spread by talking to an infected person or touching, so there is no harm in even eating food prepared by me. there were 30 hiv - infected patients, equal number of men and women, who participated in the study. their demographic details have been represented in table 1. among the total patients, majority (both men and women) were from the younger age groups of 20 - 30 years and 31 - 40 years as compared to the older age groups of 41 - 50 years and 51 - 60 years. about three - fourth of the patients were married, a couple of female patients were widowed, a couple of patients (equal number of men and women) were divorced, few patients (more women than men) were separated from their spouse and a couple of patients (equal number of men and women) were unmarried at the time of the study. demographic details of plwha attending ssg hospital, vadodara the highest level of education among the patients was up to graduation which was found among a couple of female patients. in case of the male patients, less than half of them were educated up to 10 standard and about one - third were educated up to 12 standard. there were equal number of men and women among one - fifth of the patients who were illiterate. as regards employment, more than half of the patients (more men than women) were employed at the time of the study. among the patients selected for the study from vadodara district, less than half were from urban areas, one - third were from urban slums one - fifth were from rural areas and a couple of patients were from tribal areas. stigma associated with hiv was described as a barrier to disclosure because participants perceived hiv - positive status to have a negative impact on plwha and their children. the potential for hiv stigma to limit children 's marriage prospects was particularly worrisome and cited as a reason not to disclose one 's serostatus to people other than a spouse. a widowed female participant aged 30 years said, it would have adverse effects on my son ; the school might create problems for him. people may break relations and behave badly with me and my family as they consider hiv as a bad disease. in addition, a 33-year - old widow and a mother of three children, anxious about their future said, i fear that if i disclose my hiv positive status to everyone, it might affect my daughters marriage prospects. participants perceived that many of their friends and family held misconceptions about hiv which would lead to increased stigma and mistreatment after disclosure. a married female aged 28 years said, it would create a lot of problems for us, even if there is a slight indication in the society. people believe that one is infected by hiv only because of illicit relations. a separated female participant aged 42 years narrated, i had not disclosed my positive status to relatives and neighbors, but my husband revealed my positive status in his family and neighborhood. now, all my in - laws have cut off their relations with me and my son and neighbors discriminate against us. a female medical service provider stated plwha should not disclose their hiv status to the community due to hiv - related discrimination. the desire to protect others from the pain of knowing about their illness motivated some participants not to disclose. this concern seemed particularly salient in decisions about disclosing to parents, particularly if the parents were old or in poor health, or because they feared that the reality would be too painful and agonizing for their parents. i have not revealed my hiv positive status to my mother as i know that she would not be able to bear the reality. hiv service providers pointed out that in some cases, hiv - positive male participants withheld disclosure to their prospective spouse for fear of rejection, in turn passing infection on to her after marriage. a remarried male participant, aged 44 years, who had not disclosed his serostatus to his wife, discussed his dilemma saying, i have never practiced sex without using condoms saying i do nt want children. but my wife (who is unaware of my serostatus) fights a lot with me as she wants children. there were instances where family members disclosed participants hiv - positive status to others without their consent or will or in an, upset and disappointed by his ex - wife 's disclosure of his hiv status said, she told her brother, who in turn told her parents and then my father came to know. in half of the participants, physical incapacity and health problems were cited as reasons why their hiv - positive status was disclosed involuntarily to family members. a married male participant aged 44 years who disclosed his hiv - positive status due to his ill health, stated i have only told my mother as i was feeling weak and i could not even walk. medical providers stated that it was important for hiv - positive patients to disclose to their families, to protect caregivers from the potential risk of hiv infection. a married hiv - infected female participant aged 32 years, described how her and my husband 's condition did not improve at all, so the doctor asked to get the hiv test done. my report also came positive. while involuntary disclosure was a common thread among the participants, some discussed experiences of voluntary disclosure. voluntary disclosure occurred when participants felt morally obligated to divulge their serostatus to a spouse so s / he could get tested for hiv. a divorced male participant aged 30 years, who voluntarily disclosed his hiv - positive status to his wife stated, the next day i took my wife and 11-year - old daughter for the test. another reason participants gave for voluntarily disclosing their hiv - positive status was to obtain emotional and physical support from family. an example is this explanation from a married female participant aged 26 years, who gave her reason for the decision to disclose her and her husband 's hiv - positive status to their parents, we told this to our parents only as they care for us. there are many consequences of disclosure [figure 1 ], but a couple of salient themes are presented below. a severe consequence of disclosure was family disruption leading to separation and divorce. disclosure also impacted reproductive decision - making. a 21-year - old pregnant female participant, talked about her dilemma, my mother - in law told me not to keep the child because i had hiv, the child could get it from me. but the doctors told us to take medicines and assured us with a 99% guarantee that the child would be hiv - negative and asked us to decide whether we want to keep the child or not. a divorced female participant aged 28 years expressed her grief, the doctor told me to get my cd4 count tested. so the doctor told me to abort the child as my cd4 count was low. consequences of disclosure some married female participants living in joint families, talked about being blamed by their mothers - in - law for either placing their son at risk for hiv or for being the source of their son 's hiv infection. a married female participant, aged 35 years, said, my mother - in - law held me responsible for her son 's hiv infection. other women became aware of their serostatus only after being remarried and getting tested for hiv when they became pregnant. a divorced female participant aged 28 years, said with a lot of emotion, my first husband died due to hiv. my in - laws never told me about his hiv positive status even after his death. during my second marriage, i came to know about my being hiv positive when i was pregnant again. i advised him that whenever you get married, you either marry a positive person like yourself or inform the person about your positive status beforehand. after a few months, that same person came for getting his wife 's hiv test done. his wife was found positive ; he had not informed his wife about his hiv - positive status before marriage. although majority of patients described the barriers of disclosure, some hiv service providers and patients described the benefits. a female non - medical service provider explained, we try to explain to the patients that if they disclose their status to their families, they would take better care of them. if you tell any member of your family, who is close to you, then it would be better for you. medical service providers believed that hiv - positive patients would benefit from treatment by telling their providers about their seropositive status when going for any kind of treatment. this would also allow doctors to screen for tuberculosis, one of the most common opportunistic infections among plwha in india. a married female participant said, those persons in the neighborhood to whom i have disclosed my status, are good. they say that this disease does not spread by talking to an infected person or touching, so there is no harm in even eating food prepared by me. negative reactions influence the behavior of plwha and can undermine hiv disclosure. in india, hiv is perceived by some to be associated with an immoral lifestyle, which may contribute to hiv - related discrimination and stigmatization. such perceptions can negatively affect families, communities, workplaces, schools, and health - care settings. findings from this study revealed several factors which influenced the decision to disclose one 's positive hiv status, including perceived hiv - associated stigma, fear of discrimination, and family breakdown. fear of social stigma prevented hiv - positive participants from disclosing their status immediately to their partners and families which contributed to social isolation. this study also revealed that most of the hiv - positive wives disclosed their status to their husbands, whereas only a few of the hiv - positive husbands revealed their status to their wives. in a cross - sectional study conducted in kolkata, all of the female patients (100%) had disclosed their serostatus to their sexual partners, as compared to 65% of male patients. lack of disclosure and resulting secrecy on the husband 's part contributed to conflict in the relationship. in one case, the husband insisted on condom use to protect his wife, but his wife resisted condom use because of her desire for children, which resulted in intimate partner violence (ipv). the wife 's decision to have children may have been different had she known about her husband 's hiv status, which may have prevented ipv altogether. hiv service providers have an important role, as they may help foster hiv disclosure. in this study, many non - medical service providers explained the benefits of disclosure to their clients including spousal and family support. however, some findings of the present study also suggest discriminatory practices by hiv service providers which undermine the delivery of hiv prevention services including facilitating disclosure. in one qualitative study conducted in southern india, hiv - positive women felt discriminated against by their own health - care providers. one study reported that negative attitudes and discriminatory behaviors by providers towards plwha indicated a need for ethics training. additional and ongoing training in these areas are critical in creating environments supportive of hiv disclosure. our findings also showed that involuntary disclosure was more common than voluntary disclosure among hiv - infected participants. voluntary disclosure occurred when participants felt morally obliged to divulge their serostatus to their spouse or due to obtain emotional support from their family. a similar pattern regarding disclosure emerged from discussions with health service providers except that they felt that patients also disclosed their hiv - positive status to close friends. in western countries, disclosure to friends is more common as they may be more supportive than family members. however, in the present study, fear of discrimination by friends, peers, and colleagues at their workplace led to non - disclosure of hiv - positive status by participants. this study indicated some benefits of disclosure to family including : spousal support, care from other family members, protecting the seronegative spouse from hiv, and avoiding unintended pregnancy. however, although there are several benefits to disclosure, including the prompt hiv testing of sexual partners, it may not be enough given that hiv - related stigma can prevent hiv testing and treatment, which reinforces the notion that that the person who has the illness can be identified as the marked other via testing. addressing hiv - related stigma as a way to increase hiv disclosure remains a critical area for intervention. given the cultural context in india, gender - specific approaches to enhance hiv disclosure need to be considered. there has been a suggestion in previous studies about individualized approaches to post - test counseling, including enhanced support services for hiv - positive women and public education to de - stigmatize hiv - disease, particularly because hiv - positive women are more often at the receiving end of discrimination and hiv - associated stigma when compared to men. in addition, hiv - positive women are looked upon with suspicion and their moral character is questioned, especially when they are tested earlier than their spouse. our study showed similar findings with respect to hiv - positive test results having a disproportionate negative impact on women. couples - based counseling models which focus on both the seropositive and seronegative partner may also be critical in preventing hiv transmission. hiv prevention counseling in integrated counselling and treatment centre must address issues of fear of disclosure, potential negative consequences of disclosure, including stigma and discrimination when disclosing hiv - positive serostatus. training local counselors and other hiv service providers in the identified barriers and facilitators of serostatus disclosure is recommended.	background : human immunodeficiency virus (hiv) disclosure offers important benefits to people living with hiv / aids. however, fear of discrimination, blame, and disruption of family relationships can make disclosure a difficult decision. barriers to hiv disclosure are influenced by the particular culture within which the individuals live. although many studies have assessed such barriers in the u.s., very few studies have explored the factors that facilitate or prevent hiv disclosure in india. understanding these factors is critical to the refinement, development, and implementation of a counseling intervention to facilitate disclosure.materials and methods : to explore these factors, we conducted 30 in - depth interviews in the local language with hiv- positive individuals from the integrated counselling and testing centre in gujarat, india, assessing the experiences, perceived barriers, and facilitators to disclosure. to triangulate the findings, we conducted two focus group discussions with hiv medical and non - medical service providers, respectively.results:perceived hiv - associated stigma, fear of discrimination, and fear of family breakdown acted as barriers to hiv disclosure. most people living with hiv / aids came to know of their hiv status due to poor physical health, spousal hiv - positive status, or a positive hiv test during pregnancy. some wives only learned of their husbands hiv positive status after their husbands died. the focus group participants confirmed similar findings. disclosure had serious implications for individuals living with hiv, such as divorce, maltreatment, ostracism, and decisions regarding child bearing.interpretation and conclusion : the identified barriers and facilitators in the present study can be used to augment training of hiv service providers working in voluntary counseling and testing centers in india.
boxing, which originated in ancient egypt, has evolved over the course of 5,000 years1. wearing gloves, both boxers attack and defend using their fists inside a ring2. a boxing match consists of three rounds of 3 min, with 1 min of rest between rounds3, 4. during each round, judges assess the scores of each boxers according to the number of clean punches made with the knuckle part of the glove against the target region of the opponent5. the target region is defined as any part of the front or sides of the head or body above the belt6. weight categories in boxing are divided into 10 divisions ranging from ~48 to 90 kg or more7. hippocrates (400 bc) recognized two types of human somatotype : habitus phthisicus and habitus apoplecticus. long and thin, and habitus apoplecticus means short and thick. however, it was impossible to define standardized somatotype characteristics, as there were differences according to lifestyles. the heath - carter method determines the somatotype according to the body s size, bone width, and skin thickness10,11,12. this classification suggests that acquired behavior and lifestyles could have an effect on body shape. heath - carter s classification can be applied to elite boxing athletes and used to determine the effect of elite boxing on the somatotype. studies have shown that the adaptation to physical effort as a result of training and the process of selection results in a decrease in somatotype diversity among athletes in similar sports or athletes using similar skills compared with nonathletes13,14,15. however, research on elite boxing athletes has rarely been done. our study, therefore, provides the physical characteristics of elite boxing athletes to establish a reference for their training and rehabilitation. forty - six volunteers who had no physical or psychological conditions provided written informed consent to participate in this study. the participants were asked to complete a questionnaire administered by individual in - depth interviews that took 2030 min per person16. the participants wore only shorts during the measurements, and the measurements were taken by a single person. before the measurements, all the participants rested for 30 min. the weight categories were divided into four divisions : a lightweight class (~49 to ~56 kg), light middleweight class (~60 to ~64 kg), middleweight class (~69 to ~75 kg), and heavyweight class (~81 to 91 kg or more). the somatotypes were classified as ectomorphic, mesomorphic, endomorphic, and central types according to heath - carter s modified somatotype method10,11,12. the girths of the upper arms and the thickest part of the calves were then determined with a tapeline. the breadths of the biepicondylar humerus and biepicondylar femur were measured with a large anthropometer. finally, the triceps brachii, subscapular, suprailiac, and calf skinfold thicknesses were determined with a medical skinfold caliper (jamar, anaheim, ca, usa). the weight categories in the present study were divided into four divisions : the lightweight (a), light middleweight (b), middleweight (c), and heavyweight (d) classes. l class, lightweight class (~49 to ~56 kg) ; l - m class, light middleweight class (~60 to ~64 kg) ; m class, middleweight class (~69 to ~75 kg) ; h class, heavyweight class (~81 to 91 kg or more) ; endo, endomorphy ; meso, mesomorphy ; ecto, ectomorphfig. the somatochart was prepared as described in the subjects and methods (quoted and modified from heath and carter10,11,12.). the somatotypes were classified as endomorphic, mesomorphic, ectomorphic, and balanced types. they were further broken into 13 subcategories : balanced endomorph (ben, the endomorphic component is dominant, and the values of the mesomorphic and ectomorphic components do not differ by more than 0.5), mesomorphic endomorph (men, the endomorphic component is dominant, and the value of the mesomorphic component is higher than that of the ectomorphic component), mesomorph - endomorph (m - en, the values for the mesomorphic and endomorphic components do not differ by more than 0.5, and the value of the ectomorphic component is lower than the other values), endomorphic mesomorph (enm, the mesomorphic component is dominant, and the endomorphic component is higher than the ectomorphic component), balanced mesomorph (bm, the mesomorphic component is dominant, and the values of the endomorphic and ectomorphic components do not differ by more than 0.5), ectomorphic mesomorph (ecm, the mesomorphic component is dominant, and the ectomorphic component is higher than the endomorphic component), mesomorph - ectomorph (m - ec, the values of the mesomorphic and ectomorphic components do not differ by more than 0.5, and the value for the endomorphic component is lower than that of the other values), mesomorphic ectomorph (mec, the ectomorphic component is dominant, and the mesomorphic component is higher than the endomorphic component), balanced ectomorph (bec, the ectomorphic component is dominant, and the values for the endomorphic and mesomorphic components do not differ by more than 0.5), endomorphic ectomorph (enec, the ectomorphic component is dominant, and the endomorphic component is higher than the mesomorphic component), endomorph - ectomorph (en - ec, the values for the endomorphic and ectomorphic components do not differ by more than 0.5, and the value for the mesomorphic component is lower than that of the other values), ectomorphic endomorph (ecen, the endomorphic component is dominant, and the ectomorphic component is higher than the mesomorphic component), and central type (c, the values do not differ by 1 in any of the components)10,11,12, 17, 18 (fig. the formulas for the heath - carter method are as follows : endomorphic component = 0.7182 + 0.1451 sf 0.00068 sf + 0.0000014 sf, where sf = (sum of the triceps brachii, subscapular, and suprailiac skinfold thicknesses) [170.18/height (cm) ]. somatotype analysis was performed, as described in the subjects and methods differences in somatochart among the weight divisions of elite boxing athletes. the weight categories in the present study were divided into four divisions : the lightweight (a), light middleweight (b), middleweight (c), and heavyweight (d) classes. l class, lightweight class (~49 to ~56 kg) ; l - m class, light middleweight class (~60 to ~64 kg) ; m class, middleweight class (~69 to ~75 kg) ; h class, heavyweight class (~81 to 91 kg or more) ; endo, endomorphy ; meso, mesomorphy ; ecto, ectomorph schematic representation of the somatochart for sports physiotherapy. the somatochart was prepared as described in the subjects and methods (quoted and modified from heath and carter10,11,12. mesomorphic component = 0.858 breadth of biepicondylar humerus + 0.601 breadth of biepicondylar femur + 0.188 modified girth of upper arm + 0.161 modified girth of the calf height 0.131 + 4.5, where the modified value is [value (1/10 of the skinfold thickness of the part) ]. the ectomorphic component is calculated as the difference according to the value of the height weight ratio (hwr, hwr = height / weight). if 38.25 < hwr < 40.75, then ectomorphic component = 0.463 hwr 17.63. if hwr 38.25, then ectomorphic component = 0.1. the formulas marked on the somatotype chart are as follows : x = ectomorphic component endomorphic component y = 2 mesomorphic component (endomorphic component + ectomorphic component) statistical analyses were conducted using the sas software (version 6.12) to calculate averages and standard deviations. the data are expressed as means standard error (se) of the measurements. the significance level was set to =0.05 when performing an independent t - test for group comparisons. the protocol for the study was approved by the committee of ethics in research of the university of yongin, in accordance with the terms of resolution 5 - 1 - 20, december 2006. different physical characteristics were observed between the nonathletes and the elite boxing athletes (table 1table 1. characteristics of the nonathletes and elite boxing athletesvariablenonathleteselite boxing athletesage (yr)22.6 0.619.3 0.3gendermale (% /%)23 (100.0/50.0)23 (100.0/50.0)female (% /%)--height (cm)174.5 0.8173.2 1.0weight (kg)69.1 1.470.2 2.4bmi (kg / m)22.7 0.523.3 0.6career (yr)-5.6 0.5training time-5.1 0.3 h / day-30.1 1.9 h / weekweight divisionl (~49 to ~56 kg) (% /%)-6 (26.1/13.0)l - m (~60 to ~64 kg) (% /%)-8 (34.8/17.4)m (~69 to ~75 kg) (% /%)-5 (21.7/10.9)h (~81 to 91 kg or more) (% /%)-4 (17.4/8.7)somatotypeendomorphy (% /%)8 (34.8/17.4)1 (4.3/2.2)mesomorphy (% /%)4 (17.4/8.7)16 (69.6/34.8)ectomorphy (% /%)6 (26.1/13.0)4 (17.4/8.7)central (% /%)5 (21.7/10.9)2 (8.7/4.3)endo c.3.0 0.22.3 0.2meso c.3.0 0.23.7 0.2ecto c.2.6 0.22.3 0.2data are presented as means se. bmi, body mass index ; l, lightweight class ; l - m, light middleweight class ; m, middleweight class ; h, heavyweight class ; endo c., endomorphic component ; meso c., mesomorphic component ; ecto c., ectomorphic component. the endomorphic component values of the boxing athletes were lower than those of the nonathletes. however, the mesomorphic component values in the boxing athletes were higher than in the nonathletes (table 1). on the somatotype chart, the boxing athletes were more likely to be gathered in the central and upper parts (mesomorphic side) than the nonathletes (fig. there were also differences among the weight categories, with the higher weight divisions tending to have higher values of height, weight, and bmi than the lower weight divisions (table 2table 2. differences in somatotype among the weight division of elite boxing athletesvariableweight divisions of elite boxing athleteslightweight(~49 to ~56 kg)light middleweight(~60 to ~64 kg)middleweight(~69 to ~75 kg)heavyweight(~81 to 91 kg or more)number (% /%)6 (100.0/26.1)8 (100.0/34.8)5 (100.0/21.7)4 (100.0/17.4)height (cm)168.10.6171.41.1177.61.2179.01.3weight (kg)58.30.968.01.772.51.989.34.0bmi (kg / cm)20.70.323.20.623.00.527.91.63 somatotypesendo (% /%)---1 (25.0/4.3)meso (% /%)4 (66.7/17.4)6 (75.0/26.1)3 (60.0/13.0)3 (75.0/13.0)ecto (% /%)2 (33.3/8.7)1 (12.5/4.3)1 (20.0/4.3)-central (% /%)-1 (12.5/4.3)1 (20.0/4.3)-endo c.1.80.22.30.32.10.33.50.6meso c.3.20.23.70.23.50.54.50.4ecto c.3.10.22.20.32.60.21.10.313 somatotypestypes (% /%)m - ec 3 (50.0/13.0) bm 3 (37.5/13.0)m - ec 2 (40.0/8.7)enm 2 (50.0/8.7)ecm 1 (16.7/4.3)enm 2 (25.0/8.7)bm 1 (20.0/4.3)bm 1(16.7/4.3)m - en1 (12.5/4.3)enm 1 (20.0/4.3)m - en 2 (50.0/8.7)mec 1 (16.7/4.3)mec 1 (12.5/4.3)cen 1 (20.0/4.3)cen 1 (12.5/4.3)data are presented as means se. bmi, body mass index ; endo c., endomorphic component ; meso c., mesomorphic component ; ecto c., ectomorphic component ; m - ec, mesomorph ectomorph ; ecm, ectomorphic mesomorph ; bm, balanced mesomorph ; mec, mesomorphic ectomorph ; enm, endomorphic mesomorph ; m - en, mesomorph endomorph ; cen, central. # p < 0.05). in addition, the higher weight divisions tended to have higher values for the endomorphic and mesomorphic components and a lower value for the ectomorphic component than the lower weight divisions (table 2). the somatotype chart shows the classifications of the somatotypes, with points shifted left and up for the higher weight divisions (fig. 2). the group of nonathletes consisted of eight endomorphs, four mesomorphs, six ectomorphs, and five central types. however, for the boxing athletes, there were 16 mesomorphic, four ectomorphic, and two central types and only one endomorphic type. subdividing the athletes into 13 somatotypes resulted in five balanced mesomorphs, five endomorphic mesomorphs, five mesomorph - ectomorphs, three mesomorph - endomorphs, two mesomorphic ectomorphs, two central types, and one ectomorphic mesomorph type (figs. 1 and 2, and table 2). bmi, body mass index ; l, lightweight class ; l - m, light middleweight class ; m, middleweight class ; h, heavyweight class ; endo c., endomorphic component ; meso c., mesomorphic component ; ecto c., ectomorphic component. p < 0.05 data are presented as means se. bmi, body mass index ; endo c., endomorphic component ; meso c., mesomorphic component ; ecto c., ectomorphic component ; m - ec, mesomorph ectomorph ; ecm, ectomorphic mesomorph ; bm, balanced mesomorph ; mec, mesomorphic ectomorph ; enm, endomorphic mesomorph ; m - en, mesomorph endomorph ; cen, central. # p < 0.05 in our study, although similar in weight, height, and bmi, the boxing athletes were more muscular than the nonathletes. the endomorphic component values of the boxing athletes were also lower than those of the nonathletes. however, the mesomorphic component values of the boxing athletes were higher than those of the nonathletes. the endomorphic and mesomorphic components of the nonathletes were not different, whereas the boxing athletes had higher mesomorphic component values and lower endomorphic component values. there was a much higher percentage of mesomorph types in the elite boxer group. this result was also seen in other studies, with athletes in other combat sports, such as judo, korean wrestling also called ssireum, wrestling, and jujitsu, tending to have a mesomorphic somatotype17,18,19,20,21. however, the endomorphic component value of soccer players was lower than that in contact combat sports, such as judo and korean wrestling17, 19. boxers have to avoid attacks and powerful hits by their opponent and continue to move their entire body until the end of the match. as they require speed, power, and endurance, they have lower endomorphic component values and higher mesomorphic component values than athletes in other combat sports. even in the same sports, physical characteristics differ according to the weight category. in a study of the somatotype of korean wrestling players, higher weight divisions were shown to have higher values for height, weight, and bmi than lower weight divisions ; there were also different somatotype components between the weight categories17, 19. the same result was found in the present study of elite boxers, with the higher weight divisions having higher values for height, weight, and bmi than the lower weight players. the heavyweight and lightweight classes had higher endomorphic and ectomorphic component values, respectively, than the other weight divisions. however, in each weight category, the mesomorphic component was more dominant than the other components. to summarize our data, boxing athletes have a higher mesomorphic component and a lower endomorphic component for their athletic performance. therefore, we suggest that elite boxing athletes who are injured or training for a match need to be training for fat reduction and muscle power when returning from injury. however, further studies in the area of sports physiotherapy are needed to corroborate the pathologic mechanism of sports injury23,24,25.	[purpose ] the purpose of this study was to show somatotype and physical characteristic differences between elite boxing athletes and non - athletes. [methods ] the somatotypes of 23 elite boxing athletes and 23 nonathletes were measured with the heath - carter method. the subjects were divided into four weight divisions as follows : lightweight, light middleweight, middleweight, and heavyweight class. [results ] the endomorphic component values of the boxing athletes were lower than those of the nonathletes. however, the mesomorphic component values of the boxing athletes were higher than those of the nonathletes. there was no significant difference in the ectomorphic component between the two groups. the higher weight divisions tended to have higher values of height, weight, and bmi than the lower weight divisions. the higher weight divisions also tended to have higher values for the endomorphic and mesomorphic components and a lower value for the ectomorphic component than the lower weight divisions. the group of nonathletes consisted of eight endomorphs, four mesomorphs, six ectomorphs, and five central types. among the boxing athletes, there were 16 mesomorphic, four ectomorphic, and two central types and one endomorphic type. subdividing the athletes into 13 somatotypes resulted in five balanced mesomorphs, five endomorphic mesomorphs, five mesomorph - ectomorphs, three mesomorph - endomorphs, two mesomorphic ectomorphs, two central types, and one ectomorphic mesomorph type. [conclusion ] the data from this study provides in part physical characteristics of elite boxing athletes that can be used to establish a reference for systemic study of sports physiotherapy.
skeletal muscle is the major site for disposal of ingested glucose in lean healthy normal glucose tolerance (ngt) individuals [14 ]. following a meal, approximately one third of ingested glucose is taken up by the liver and the rest by peripheral tissues, primarily skeletal muscle via an insulin dependent mechanism [14 ]. the postprandial hyperglycemia stimulates insulin secretion from the pancreas and the rise in plasma insulin concentration stimulates glucose uptake in skeletal muscle leading to the disposal of ingested glucose [14 ]. in insulin resistance states, such as t2 dm and obesity, insulin - stimulated glucose disposal in skeletal muscle the decreased insulin - stimulated glucose uptake is due to impaired insulin signaling and multiple postreceptor intracellular defects including impaired glucose transport and glucose phosphorylation, and reduced glucose oxidation and glycogen synthesis [710 ] (table 1). although the exact mechanism that leads to the development of insulin resistance in skeletal muscle is not yet fully understood, an increased intramyocellar fat content and fatty acid metabolites have been shown to play a pivotal role in the development of insulin resistance in skeletal muscle [1115 ]. the recent studies have reported the existence of a defect in mitochondrial oxidative phosphorylation in skeletal muscle in insulin resistance states [1620 ] and suggest that this mitochondrial defect contributes to the increased intramyocellar fat content. in this paper we will summarize the evidence that supports the existence of insulin resistance in skeletal muscle, the cellular mechanism(s) that lead to the development of insulin resistance, and the clinical consequences of insulin resistance in skeletal muscle. skeletal muscle utilizes both glucose and free fatty acid (ffa) as fuel sources for energy production. during the postabsorptive state, since the plasma insulin concentration is the principal factor that restrains lipolysis in adipocytes and stimulates glucose uptake in skeletal muscle, during the fasting state, muscle glucose uptake is low and the plasma ffa concentration is elevated. thus, under fasting conditions, ffa serves as the principal fuel source for energy production in skeletal muscle, while the brain exclusively utilizes glucose. following glucose ingestion, the increase in plasma glucose concentration stimulates insulin secretion from the beta cell and the resultant hyperinsulinemia suppresses lipolysis, leading to decline in plasma ffa concentration and subsequent decrease in the rate of lipid oxidation. simultaneously, insulin stimulates glucose uptake in skeletal muscle, and the increased glucose flux into skeletal muscle, together with the activation of key enzymes in glucose metabolism by insulin, leads a marked increase in muscle glucose oxidation. thus, under postprandial conditions, for example, mixed meal, muscle energy metabolism switches from predominant oxidizion of fat during the fasting state, to predominant oxidizion of glucose. the ability of skeletal muscle to switch from fat oxidation during the fasting state to glucose oxidation during the postprandial state has been referred to as metabolic flexibility. after glucose is transported into the myocyte via the glut4 transporter, it is immediately phosphorylated by hexokinase, and the phosphorylated glucose either is converted to, and stored as glycogen, or enters the glycolytic pathway for oxidation. approximately 90% of glucose entering the glycolysis is oxidized and the remaining 10% is released as lactate. at low plasma insulin concentration, for example, fasting state, glycogen synthase, and glucose oxidation however, with increasing plasma insulin concentration, glycogen synthase is activitaed by insulin and glycogen synthesise predominate (~70% of glucose disposal). the term insulin resistance refers to an impairment in insulin action in insulin - target tissues, such as skeletal muscle, adipocytes, and liver. with respect to skeletal muscle, the primary action of insulin is to stimulate glucose uptake and metabolism [14 ]. in lean healthy individuals insulin stimulates glucose uptake into skeletal muscle in a dose - dependent manner, with a half - maximal effect (ec50) at a plasma insulin concentration ~60 u / ml. in insulin resistant states, insulin - stimulated glucose uptake is markedly reduced in skeletal muscle (figure 1). himsworth and kerr, using a combined oral glucose and intravenous tolerance test, were the first to demonstrate that tissue sensitivity to insulin is diminished in t2 dm patients. in 1975, ginsberg., using the insulin suppression test, provided further evidence that the ability of insulin to promote tissue glucose uptake in t2 dm was severely reduced. the most conclusive documentation for increased insulin resistance in skeletal muscle in lean, as well as obese t2 dm subjects, has been provided by defronzo and colleagues [15, 21, 27, 28 ] and butterfield and whichelow. using the gold standard euglycemic hyperinsulinemic clamp technique to quantify insulin - stimulated glucose uptake, they demonstrated that both lean and obese t2 dm subjects have marked decrease (> 50%) in whole body glucose disposal during the insulin clamp. although glucose disposal during the insulin clamp represents insulin - stimulated glucose uptake by all peripheral tissues, the great majority of this glucose uptake take place in skeletal muscle. under euglycemic conditions studies, using the insulin clamp in combination with femoral artery and vein catheterization has shown that approximately 80% of total body glucose uptake occurs in skeletal muscle. in response to a physiologic increase in plasma insulin concentration (80100 u / ml), leg muscle glucose uptake increases progressively in healthy subjects and reaches a platue value of approximately 10 mg / kg leg wtmin. in contrast, during the last hour of the insulin clamp study, the rate of glucose uptake is reduced by ~50% in lean t2 dm subjects (figure 2). thus, the dose response curve relating insulin - stimulated glucose uptake and the plasma insulin concentration shifts to the right with an increase in ec50 (to ~120140 u / ml) in subjects with t2 dm (figure 1). in addition, the onset of insulin action in skeletal muscle in t2 dm subjects is markedly delayed (figure 2). even though the insulin infusion is continued for an additional 60 minutes in subjects with t2 dm to allow insulin these studies indicate that insulin resistance in skeletal muscle in subjects with t2 dm is manefisted, not only by a reduction in the magnitude of insulin action, but also by s delayed onset of insulin action to stimulate glucose uptake. thus, lean healthy offspring of diabetic parents have a significant decrease in insulin - stimulated total body and muscle glucose disposal which is of a similar magnitude to that observed in their diabetic parents [3032 ]. insulin resistance in skeletal muscle also can develop independent of family history of t2 dm. thus, obese ngt subjects without a family history for t2 dm [33, 34 ] and individuals with essential hypertension and ischemic heart disease also have a 3550% decrease in whole body insulin - mediated glucose disposal (figure 3). insulin resistance in skeletal muscle also has been reported in association with the normal ageing process, dyslipidemia (increased plasma triglyceride / decreased hdl cholesterol) [4, 26 ], and in association with many disease states including polycystic ovary syndrome (pcos), chronic kidney failure, heart failure, myotonic dystrophy, and lipodystrophy. in addition, insulin resistance develops in acute severe illnesses such as injury and sepsis, perhaps secondary to the acute inflammatory state that prevails. insulin resistance in skeletal muscle also can develop secondary to pharmacological therapy, for example, glucocorticoids, anti - hiv therapy, and beta blockers. these studies indicate that although insulin resistance in skeletal muscle is a hallmark of t2 dm, muscle insulin resistance and the accompanying insulin resistance syndrome are more widely prevalent and the metabolic and clinical consequences of insulin resistance (e.g., increased cardiovascular risk) affect nondiabetic individuals as well. because the molecular mechanisms that lead to the development of insulin resistance in skeletal muscle have been extensively studied in obese and t2 dm subjects, we will focus the discussion on the molecular / biochemical pathways that cause muscle insulin resistance in these two common metabolic disorders. however, similar molecular defects have been reported in other insulin resistant states. under basal conditions, for example, fasting state, the plasma insulin concentration is low (510 u / ml). thus, glucose uptake that takes place in skeletal muscle during the postabsorptive state is insulin independent. the noninsulin dependent glucose uptake in skeletal muscle also is decreased in insulin resistant individuals. the mechanism(s) responsible for the decrease in noninsulin dependent glucose uptake has (have) yet to be defined. obese ngt subjects display marked skeletal muscle insulin resistance compared to lean age and sex matched individuals [33, 34 ], and the severity of muscle insulin resistance is related to the increase in bmi. studies in experimental animals and in man have demonstrated that the increase in body weight is accompanied with an increase in skeletal muscle insulin resistance [4852 ]. conversely, weight loss in obese ngt individuals improves / reverses insulin resistance in skeletal muscle. collectively, these studies demonstrate a causal role of obesity per se in the development of skeletal muscle insulin resistance. the mechanism via which obesity causes insulin resistance in skeletal muscle is related to the accumulation of fat in the myocytes. muscle biopsy studies [53, 54 ] have demonstrated increased triglyceride content in skeletal muscle of obese ngt subjects compared to lean individuals and an inverse relationship between muscle insulin sensitivity and the intramuscular triglyceride content. although these initial studies documented the importance of increased muscle fat content in the etiology of insulin resistance in obesity, they did not distinguish between the contribution of fat accumulation inside the muscle fiber (intramyocellar, imcl) versus fat accumulation in the muscle outside the muscle cell (extramyocellar, emcl). more recent studies [55, 56 ] using magnetic resonance spectroscopy have demonstrated that although imcl fat content contributes only a small fraction (~1%) to the total muscle fat content, it plays a key role in the development of insulin resistance in skeletal muscle compared to emcl. muscle insulin resistance strongly correlates with imcl, independent of total body fat mass and diabetes status, while the correlation between emcl and insulin ressitance is not significant. obese ngt individuals are characterized by an increase in plasma free fatty acid (ffa) concentration. the important role of elevated plasma ffa in the pathogenesis of insulin resistance is now well established [12, 14, 57 ]. considerable data implicate a causative role for elevated plasma ffa level in insulin resistance in skeletal muscle. the majority of insulin resistant individuals, both diabetic and nondiabetics, are overweight or obese, and both lean and obese insulin resistant individuals manifest multiple disturbances in ffa metabolism. nondiabetic obese and type 2 diabetic individuals are characterized by day - long elevation in the plasma ffa concentration, which fails to suppress normally after ingestion of a mixed meal or an oral glucose load. elevated plasma ffa levels correlate strongly with reduced insulin - stimulated glucose disposal in skeletal muscle. ffas are stored within adipocytes in the form of triglycerides and serve as an important source of energy during fasting. insulin is a potent inhibitor of lipolysis and restrains the release of ffas from adipocytes. in insulin resistant individuals, for example, nondiabetic obese and lean type 2 diabetic subjects, the ability of insulin to inhibit lipolysis and reduce plasma ffa concentration is markedly impaired [21, 59 ], leading to an increased rate of lipolysis and chronic elevation in the plasma ffa concentration. it is well established that chronically elevated plasma ffa levels cause insulin resistance in skeletal muscle [1, 12, 14, 6265 ]. both acute (4 hours) and chronic (4 days) physiologic elevation of plasma ffa concentration in insulin sensitive individuals impairs insulin signaling and causes insulin resistance in skeletal muscle [28, 6268 ]. the inhibition of insulin signaling and induction of skeletal muscle insulin resistance caused by elevation in the plasma ffa concentration is dose dependent. conversely, reduction of plasma ffa concentration in insulin resistant individuals, for example, type 2 diabetic subjects, augments insulin - stimulated glucose disposal in skeletal muscle [6972 ], and the improvement in insulin sensitivity is closely associated with a reduction in intramyocellular facoa concentration. collectively, these results support a causative role of elevated plasma ffa levels in the pathogenesis of insulin resistance in skeletal muscle and have been referred to as lipotoxicity. much evidence supports the hypothesis that the lipotoxic action of ffa on skeletal muscle insulin sensitivity is due to an increase in intramyocellar fat content : (i) in insulin resistant individuals, insulin - stimulated glucose disposal correlates more strongly with intramyocellar fat content than with the extramtocellar fat [55, 56 ] ; (ii) the decrease in insulin resistance brought about by reducing the plasma ffa concentration with acipimox in insulin resistant individuals is associated with a reduction in intracellular facoa content, and the magnitude of decrease in insulin resistance strongly correlates with the decrease in facoa content ; (iii) the increase in insulin resistance caused by elevating the plasma ffa concentration in insulin sensitive individuals is associated with an increase in the intramyocellar fat content ; (iv) in animals, a high fat diet causes an increase in intramuscular fat content, which correlates closely both with the severity and temporal development of insulin resistance [75, 76 ] ; (v) in rodents, injection of recombinant adiponectin increases fat oxidation, reduces intamyocellular lipid, and improves insulin sensitivity ; (vi) muscle - specific over expression of lipoprotein lipase leads to the accumulation of fat / facoa in muscle and severe muscle insulin resistance, while the fat content and insulin sensitivity in other tissues, that is, liver, remain unchanged ; (vii) weight loss, induced by bariatric surgery, results in a complete normalization of insulin sensitivity despite the presence of continued obesity (bmi reduced from 49 to 39 kg / m). the normalization of muscle insulin sensitivity is associated with a reduction of muscle fat content to normal despite a persistent increase in total body fat content. these observations collectively indicate that muscle triglyceride (which is metabolic inert) is not directly involved in determining insulin sensitivity but rather represents a marker of imbalance between lipid supply and lipid oxidation in skeletal muscle, while increases in intramyocellar facoa and other lipotoxic metabolites (dag, ceramide) have a more direct role in the development of skeletal muscle insulin resistance. the accumulation of fat inside the muscle fiber could originate from excess supply of ffa to the muscle, a decreased rate of muscle fat oxidation, or some combination of the two. considerable debate exists regarding the mechanism by which lcfas are transported into skeletal muscle [8284 ]. because lcfas are highly hydrophobic molecules and easily can cross the plasma membrane of muscle via passive diffusion, the rate of lcfa influx into muscle fiber is, in part, driven by the concentration gradient of lcfa across the plasma membrane. thus, an increae in plasma ffa concentration is an important regulator of ffa supply to skeletal muscle. indeed, early studies demonstrated the rate of lipolysis was a key determinant of ffa uptake and oxidation in skeletal muscle and documented a linear relationship between the plasma lcfa concentration and their rate of uptake and oxidation in the muscle [8587 ]. more recent studies have reported that under conditions of marked increase in energy demand in the muscle, for example, during exercise, lcfas uptake initially increases linearly then plateaus, suggesting the existence of a facilitated transport mechanism for lcfa into skeletal muscle. three putative fatty acid binding proteins located at the plasma membrane have been identified : (i) the plasma membrane - bound fatty acid binding protein (fabp) ; (ii) fatty acid translocase (cd36) ; (iii) fatty acid transport protein (fatp). there is abundant evidence which indicates that cd36 plays an important role in lcfa uptake into skeletal muscle in rodents and human and its concentration and activity are increased following exercise [8996 ]. however, the transport of lcfa by cd36 into skeletal muscle does not seem to regulate the rate of lcfa oxidation. during conditions of decreased fat oxidation and increased cd36 levels, muscle uptake of lcfa increased, resulting in increased muscle triglyceride synthesis and increased imcl content [97, 98 ]. the cd36 content and lcfa transport capacity have been reported to be increased in obese and t2 dm individuals and correlate with increased muscle triglyceride content, while fat oxidation is decreased. several studies have reported decreased fat oxidation in skeletal muscle, independent of the plasma ffa concentration [100102 ]. in one study that compared muscle fat oxidation across the leg in obese (bmi = 34 kg / m) compared to lean (bmi = 23 kg / m) individuals, the rate of fatty acid oxidation was reduced in the obese group despite similar arterial ffa concentrations. these results suggest the presence of impaired muscle fat oxidation independent of fat supply to skeletal muscle. similar results have been reported in rectus abdominus muscle in morbidly obese individuals and in obese nondiabetic women. studies in experimental animals also have reported a decreased rate of lipid oxidation in skeletal muscle in obese zuker rat. collectively, these studies indicate the existence of an intrensic defect in mitochondrial capacity to oxidize fat in obesity and t2 dm. since the majority of fat oxidation take place in the mitochondria, impaired fat oxidation in insulin resistant individuals suggests the presence of a mitochondrial defect that contributes to the impaired muscle fat oxidation and increased imcl fat content. studies in humans, using molecular, biochemical, and mr spectroscopic techniques, have documented a defect in mitochondrial oxidative phosphorylation in a variety of insulin resistance states. because of its accessibility, most of these studies have been performed in skeletal muscle. in vivo measurement of oxidative phosphorylation with p - nmr has demonstrated impaired atp synthesis in a variety of insulin resistant states. lean normal - glucose - tolerant (ngt), insulin - resistant offspring of t2 dm parents have a 3040% decrease in metabolic flux through the tricarboxylic acid cycle and oxidative phosphorylation in skeletal muscle under basal conditions. a similar impairment in resting flux through oxidative phosphorylation also has been reported in subjects with type 2 diabetes. furthermore, unlike lean insulin - sensitive individuals, both diabetic subjects and ngt insulin - resistant offspring of two diabetic parents fail to increase mitochondrial oxidative phosphorylation flux following insulin stimulation despite a significant increase in glucose disposal in skeletal muscle [105, 106 ]. subjects with t2 dm also have decreased exercise tolerance and impaired recovery of intracellular phosphocreatinine concentration following exercise [107, 108 ], indicating that the mitochondrial defect in oxidative phosphorylation may contribute to the impairment in exercise capacity in insulin - resistant individuals. insulin resistance also is a characteristic feature of the normal aging process and is associated with a decrease in mitochondrial atp synthesis rate and an increase in intramyocellar fat content. lastly, experimental induction of insulin resistance in skeletal muscle, by a physiological increase in plasma ffa concentration in lean healthy insulin - sensitive individuals, is associated with a decrease in oxidative phosphorylation flux in skeletal muscle. collectively, these results indicate that, regardless of its etiology, insulin resistance in skeletal muscle is associated with decreased mitochondrial oxidative phosphorylation. the mitochondrial defect in atp synthesis rate documented in vivo with mrs could be due to a reduction in the number of mitochondria in skeletal muscle with normal function of individual mitochondria, an intrinsic defect in a quantitively normal number of mitochondria, or some combination of the two. all three of these scenarios would result in a reduced in atp synthesis rate measured in vivo with mrs. other important unanswered questions are whether the mitochondrial defect is inherited or acquired and whether this defect is the cause or the effect of the insulin resistance. an acquired defect potentially could be reversed or prevented, while an inherited defect would be permanent. although several lines of evidence support a reduction in mitochondrial number in skeletal muscle in insulin resistant individuals, not all studies have yielded consistent results. electron microscopic studies have revealed a significant reduction (~40%) in mitochondrial density in skeletal muscle in lean ngt offspring of type 2 diabetic parents, in obese nondiabetic individuals and in t2 dm subjects compared to lean insulin - sensitive, age - matched controls [111, 112 ]. the decrease in mitochondrial density in skeletal muscle in insulin - resistant individuals is consistent with decreased expression of pgc-1 gene, the master regulator for mitochondrial biogenesis. diabetic subjects have been reported to have a decrease in muscle fiber oxygen consumption measured ex vivo. however, when oxygen consumption was related to mitochondrial copy number, both diabetic and nondiabetic subjects had similar rates of muscle oxygen consumption, implying a decrease in mitochondrial density without an intrinsic mitochondrial defect in t2 dm subjects. however, other studies have reported contradictory results. thus, studies which have assessed mitochondrial copy number in skeletal muscle in insulin - resistant individuals have reported conflicting results [114116 ]. furthermore, insulin resistance, caused by physiological elevation in plasma ffa concentration in lean healthy individuals, was not associated with a significant change in mitochondrial density in skeletal muscle. several morphological and functional studies support the concept of an intrinsic mitochondrial defect in insulin resistant individuals. em studies have demonstrated a variety of morphological abnormalities in mitochondria in insulin - resistant individuals and these changes were reversable with weight loss and increased physical activity. ex vivo measurement of electron transport chain activity (etc) and mitochondrial atp synthesis in isolated mitochondria [120, 121 ] have demonstrated a decrease in etc activity and atp synthesis in obese insulin - resistant individuals compared to lean insulin - sensitive subjects. since, in these studies [112, 120 ], etc activity and mitochondrial atp synthesis rate were related to the amount of mitochondrial protein and/or citrate synthase activity, these observations indicate the existence of a basic functional defect in mitochondrial activity in skeletal muscle of obese insulin - resistant individuals. biochemical studies have reported a decrease in the activity of a variety of mitochondrial enzymes, also supporting an intrinsic functional mitochondrial defect in type 2 diabetic subjects. an intrinsic mitochondrial defect, with or without a decrease in mitochondrial density, would be expected to result in impaired lipid oxidation, an increase in intramyocellular lipid content, and the development / exacerbation of insulin resistance. additional studies are needed to resolve some of the inconsistencies reviewed above and to explore the biochemical / molecular basis of the mitochondrial defect measured in vivo in skeletal muscle in insulin resistant individuals. an inherited mitochondrial oxidative defect in the absence (but especially in the presence) of enhanced ffa influx to skeletal muscle would be expected to lead to intramyocellular fat accumulation. an inherited defect in mitochondrial oxidative capacity should be present at or shortly after birth and, most importantly, not be reversable. with regard to this, several lines of evidence indicate that the mitochondrial defect associated with insulin resistant is reversible, at least in part : (i) modest weight reduction (~10% of body weight), produced by dieting and increased physical activity, reduces insulin resistance and intramyocellular lipid content and these changes are associated with increased fat oxidation in skeletal muscle. furthermore, the magnitude of increase in fat oxidation following weight loss correlated strongly with the decrease in insulin resistance ; (ii) marked weight reduction following bariatric surgery in morbidly obese, severely insulin - resistant subjects completely normalized insulin sensitivity, and correction of the insulin resistance was accompanied by depletion of intramyocellar fat content. although mitochondrial function was not assessed following weight loss in this latter study, the depletion of intramyocellular fat and normalization of insulin sensitivity suggests a reversible mitochondrial defect in these morbidly obese, insulin - resistant individuals ; (iii) the morphological changes which characterize mitochondria in obese insulin - resistant individuals can be reversed following weight loss and increased physical activity ; (iv) the improvement in insulin sensitivity following exercise is associated with improved muscle oxidative capacity [125, 126 ] and increases in both mitochondrial density and activity [128130 ]. these observations indicate that the mitochondrial defect which accompanies insulin resistance is, at least in part, reversible. the ability to reverse the defect in mitochondrial oxidative function argues against an inherited defect. importantly, a mitochondrial defect similar to that observed in insulin - resistant individuals can be produced in young lean healthy subjects by physiologic elevation of the plasma ffa concentration [110, 131 ]. in addition, the insulin resistance associated with aging in lean subjects with negative family history of diabetes is associated with a decrease in mitochondrial function. thus, normal mitochondrial function in young, insulin - sensitive subjects suggests that the mitochondrial defect and insulin - resistance are acquired as part of the normal ageing process. the evidence reviewed above supports the hypothesis that the mitochondrial defect which accompanies insulin resistance is reversible, at least in part, and is likely to be acquired. regarding the etiology of the acquired defect in mitochondrial function, in vivo mitochondrial atp synthesis rate, measured with p - nmr strongly and inversely correlates with the fasting plasma ffa concentration, suggesting a possible role for elevated plasma ffa / toxic intramyocellar ffa metabolite concentrations in the mitochondrial dysfunction in insulin resistant individuals. with respect to this, we recently have demonstrated that a physiologic increase in facoa concentration inhibits mitochondrial atp synthesis in vitro in mitochondria isolated from skeletal muscle of ngt healthy lean subjects. this observation is consistent with the decrease in oxidative phosphorylation observed following elevation in the plasma ffa concentration in lean healthy individuals, and it indicates that elevated plasma ffa / intramyocellular metabolite concentration can cause an acquired mitochondrial defect in oxidative phosphorylation. the mitochondrial defect in oxidative phosphorylation described in vivo with mrs and ex vivo in muscle fibers and isolated mitochondria could contribute to the increase in intramyocellar ffa metabolite levels observed in obesity and t2 dm and contribute to the insulin resistance. however, one also can imagine another scenario in which the mitochondrial defect results from insulin resistance. if the increase in intramyocellar fat content in insulin resistant individuals was associated with an increase in fat oxidation and excessive production of reactive oxygen species or other toxic metabolites, then the decrease in mitochondrial oxidative phosphprylation could result from the downregulation of mitochondrial function to ameliorate the production of these toxic molecules. reproduction of the mitochondrial defect in oxidative phosphorylation in normal healthy individuals by elevation of the plasma ffa concentration would argue for a causative role of the mitochondrial defect in insulin resistance. however, since muscle insulin resistance also develops following lipid infusion and since the time course of the development of the mitochondrial defect and insulin resistance were not monitored during lipid infusion, one can not completely exclude the possibility that the mitochondrial defect results secondary to insulin resistance. studies in experimental animals and cultured muscle cells have attempted to address the relationship between mitochondrial function and skeletal muscle insulin resistance. over expression of the pgc-1 alpha gene in skeletal muscle in mice in vivo enhanced mitochondrial activity, augmented the expression of multiple proteins involved in fat oxidation and glucose transport, and increased by ~35% insulin - stimulated glucose uptake. similarly, activation of sirt1 with resveratrol in mice resulted in increased mitochondrial activity and protected the animals from diet - induced obesity and insulin resistance. down regulation of mitochondrial function in myotubes with oligomycin, which inhibits mitochondrial atp synthesis, and with ethidium bromide, which impairs mitochondrial dna replication, caused an increase in intracellular fat content, impaired insulin signaling, and decreased insulin - stimulated glucose uptake. however, treatment of the muscle cells with azide, an inhibitor of mitochondrial complex iv, increased basal glucose uptake without affecting insulin - stimulated glucose uptake in myotubes. down regulation of electron transport chain activity in mice by knocking down apoptosis inintiating factor (aif) resulted in enhanced insulin sensitivity and protection the animal from fat - induced insulin resistance. in summary it can be concluded that both decreased mitochondrial fat oxidation and increased ffa influx into skeletal muscle take place during insulin resistance state. if the rate of fat supply exceeds the demand for fat oxidation, the muscle redirects the fat entering the cell toward triglyceride synthesis leading to increased imcl content. in contrast if fat oxidation exceeds the rate of fat supply, all fat entering the muscle will be directed to fat oxidation and no fat will accumulate in the muscle. thus, the imcl content in skeletal muscle reflects the dynamic balance between the demand for fat oxidation and fat supply to the muscle. the cellular events via which insulin initiates its stimulatory effect on glucose metabolism start with binding of the hormone to specific receptors that are present on the muscle cell surface [2, 138140 ]. the binding of insulin activates the insulin receptor and the activated insulin receptor generates second messengers that activate a cascade of phosphorylation - dephosphorylation reactions that eventually result in the stimulation of intracellular glucose metabolism. the first step in glucose metabolism involves activation of the glucose transport system (glut4), leading to glucose influx into muscle cells. the free glucose, which has entered the cell, subsequently is metabolized by a series of enzymatic steps that are under the control of insulin. of these, the most important are glucose phosphorylation (catalyzed by hexokinase ii), glycogen synthase (which controls glycogen synthesis), and phosphofructokinase (pfk) and pyruvate dehydrogenase (pdh) (which regulate glycolysis and glucose oxidation, resp.). the insulin receptor is a glycoprotein comprised of two -subunits and two -subunits linked by disulfide bonds [2, 138140 ]. the two -subunits of the insulin receptor are located at the extracellular surface of the muscle plasma membrane and contain the insulin - binding domain. the -subunits have an extracellular domain, a transmembrane domain, and an intracellular domain that expresses insulin - stimulated kinase activity directed toward its own tyrosine residues. the binding of insulin to the -subunit causes phosphorylation of the -subunit on multiple tyrosine residues. the activation of insulin receptor tyrosine kinase activity is essential for the action of insulin on glucose metabolism. mutagenesis of any of the three major phosphorylation sites (at residues 1158, 1163, and 1162) impairs insulin receptor tyrosine kinase activity, leading to a decrease in the metabolic and growth - promoting effects of insulin [141, 142 ]. following the activation of insulin receptor tyrosine kinase, specific intracellular proteins, of which at least nine have been identified [138, 143 ], become phosphorylated. in skeletal muscle insulin - receptor substrate (irs)-1 serves as the major docking protein and undergoes tyrosine phosphorylation by the activated insulin receptor in regions containing specific amino acid sequence motifs (figure 4). the phosphorylated motifs serve as recognition sites for proteins containing src - homology 2 (sh2) domains. mutation of these specific tyrosine residues in irs-1 severely impairs the ability of insulin to stimulate muscle glycogen synthesis, glucose uptake and oxidation, and other acute metabolic- and growth - promoting effects of insulin. the phosphorylated tyrosine residues of irs-1 mediate an association with the 85-kda regulatory subunit of phosphatidylinositol 3-kinase (pi3k), leading to activation of the enzyme [138140, 143, 144 ]. activation of pi3k by phosphorylated irs-1 leads to activation of protein kinase b / akt which is a central intermediate for many of the metabolic and growth actions of insulin. it has been identified as one of the kinases responsible for the inactivation of glycogen synthase kinase through its phosphorylation, a process that leads to the activation of glycogen synthase. akt also phosphorylates the newly identified akt substrate as160, leading to its redistribution in the cell and activation of rab proteins required for the translocation of the vesicles containing glut4 to the plasma membrane. inhibitors of pi3k impair glucose transport and block the activation of glycogen synthase and hexokinase- (hk-) ii expression [138146 ] the action of insulin to increase protein synthesis and inhibit protein degradation also is mediated by pi3k. other proteins with sh2 domains, including the adapter protein grb2 and shc, also interact with irs-1 and become phosphorylated following exposure to insulin [138140, 143 ]. grb2 and shc link irs-1/irs-2 to the mitogen - activated protein kinase- (mapk-) signaling pathway, which plays an important role in the generation of transcription factors and promotes cell growth, proliferation, and differentiation [138, 143 ]. inhibition of the mapk kinase pathway prevents the stimulation of cell growth by insulin but has no effect on the metabolic actions of the hormone. under anabolic conditions, insulin augments glycogen synthesis by simultaneously activating glycogen synthase and inhibiting glycogen phosphorylase [148, 149 ]. the effect of insulin is mediated through the pi3k pathway, which inactivates kinases such as glycogen synthase kinase-3 and activates phosphatases, particularly pp1. pp1 is believed to be the primary regulator of glycogen metabolism. in skeletal muscle, pp1 associates with a specific glycogen - binding regulatory subunit, causing dephosphorylation (activation) of glycogen synthase. the precise steps that link insulin receptor tyrosine kinase / pi3k activation to stimulation of pp1 have yet to be defined. studies [138, 150 ] have demonstrated convincingly that inhibitors of pi3k inhibit glycogen synthase activity and abolish glycogen synthesis. early studies have demonstrated a modest 2030% reduction in insulin binding to monocytes and adipocytes from type 2 diabetic patients, but this has not been a consistent finding [1, 151154 ]. the decrease in insulin binding is caused by a reduction in the number of insulin receptors without change in insulin receptor affinity. some caution, however, should be used in interpreting these studies because muscle and liver, not adipocytes, are the major tissues responsible for the regulation of glucose homeostasis in vivo, and insulin binding to solubilized receptors obtained from skeletal muscle and liver has been shown to be normal in obese and lean diabetic individuals [152, 153, 155 ]. moreover, a decrease in insulin receptor number can not be demonstrated in over half of type 2 diabetic subjects, and it has been difficult to demonstrate a correlation between reduced insulin binding and the severity of insulin resistance [156158 ]. a variety of defects in insulin receptor internalization and processing have been described in syndromes of severe insulin resistance and diabetes. the insulin receptor gene, however, has been sequenced in a large number of type 2 diabetic patients from diverse ethnic populations and, with very rare exceptions, physiologically significant mutations in the insulin receptor gene have not been observed [159, 160 ]. this excludes a structural gene abnormality in the insulin receptor as a common cause of insulin resistance in skeletal muscle. insulin receptor tyrosine kinase activity has been examined in skeletal muscle from normal - weight and obese diabetic subjects. most [1, 7, 152, 153, 156, 161 ] but not all investigators have found a reduction in tyrosine kinase activity that can not be explained by alterations in insulin receptor number or insulin receptor binding affinity. restoration of normoglycemia by weight loss, however, has been shown to correct the defect in insulin receptor tyrosine kinase activity, suggesting that the defect in tyrosine kinase activity, at least in part, is acquired secondary to some combination of hyperglycemia, distributed intracellular glucose metabolism, hyperinsulinemia, and insulin resistance, all of which improved after weight loss. exposure of cultured fibroblasts to high glucose concentration also has been shown to inhibit insulin receptor tyrosine kinase activity. because insulin receptor tyrosine kinase activity assays are performed in vitro, the results of these assays could provide misleading information with regard to insulin receptor function in vivo. to circumvent this problem, investigators have used the euglycemic hyperinsulinemic clamp with muscle biopsies and antiphosphotyrosine immunoblot analysis to provide a snap shot of the insulin - stimulated tyrosine phosphorylation state of the receptor in vivo. in insulin - resistant obese nondiabetic subjects, in the ngt insulin resistant offspring of two diabetic parents, and in type 2 diabetic subjects, a substantial decrease in insulin receptor tyrosine phosphorylation has been demonstrated. however, when insulin - stimulated insulin receptor tyrosine phosphorylation was examined in normal - glucose - tolerant insulin - resistant individuals (offspring of two diabetic parents) at high risk for developing type 2 diabetes, a normal increase in tyrosine phosphorylation of the insulin receptor was observed. these findings are consistent with the concept that impaired insulin receptor tyrosine kinase activity in type 2 diabetic patients is acquired secondary to hyperglycemia or some other metabolic disturbance. the ability of insulin to activate insulin receptor and irs-1 tyrosine phosphorylation in muscle in obese nondiabetic subjects is modestly reduced ; whereas in type 2 diabetics insulin - stimulated insulin receptor and irs-1 tyrosine phosphorylation are severely impaired. association of the p85 subunit of pi3k with irs-1 and activation of pi3k also are greatly attenuated in obese nondiabetic and type 2 diabetic subjects compared with lean healthy controls [7, 165, 166 ]. the decrease in insulin - stimulated association of the p85 regulatory subunit of pi3k with irs-1 is closely correlated with the reduction in insulin - stimulated muscle glycogen synthase activity and in vivo insulin - stimulated glucose disposal. impaired regulation of pi3k gene expression by insulin also is impaired in skeletal muscle, and similar impairment has been demonstrated in adipose tissue of type 2 diabetic subjects. in animal models of diabetes, an 80%90% decrease in insulin - stimulated irs-1 phosphorylation and pi3k activity has been reported [168, 169 ]. in the insulin - resistant, normal glucose tolerant offspring of two type 2 diabetic parents, irs-1 tyrosine phosphorylation and the association of p85 protein / pi3k activity with irs-1 are markedly decreased despite normal tyrosine phosphorylation of the insulin receptor ; these insulin signaling defects are correlated closely with the severity of insulin resistance measured with the euglycemic insulin clamp technique. in summary, impaired association of pi3k with irs-1 and its subsequent activation are characteristic abnormalities in type 2 diabetics, and these defects are correlated closely with in vivo muscle insulin resistance. the nature of the defect in irs-1 that impairs its ability to activate pi3-kinase does not seem to include a structural defect in irs-1. a common mutation in the irs-1 gene (gly-972-arg) has been associated with type 2 diabetes, insulin resistance, and obesity, but rodent and human studies have suggested increased serine phosphorylation of irs-1 in insulin resistant states [111, 171173 ]. in general, thus, conditions that increase serine phosphorylation of irs-1 impair tyrosine phosphorylation by the insulin receptor and its activation, thus, leading to an impartment in the insulin signaling cascade. an important, yet an answered, question is the identity of the factor(s) responsible for the activation of serine kinases that phosphorylate irs-1. increased activity of protein kinase c (pkc- and has been reported in insulin resistant states and can be induced during lipid infusion [174, 175 ]. furthermore, increased intramyocellar levels of fat metabolites, for example, facoa and dag, which are strong activators of protein kinase c, have been demonstrated following lipid infusion. moreover, prevention of the irs-1 serine phosphorylation by mutating key serine residues in the irs-1 prevents the development of insulin resistance in skeletal muscle caused by high - fat feeding. increased stress kinase activity also has been implicated in contribute to the serine phosphorylation of irs-1 and subsequent impairment tyrosine phosphorylation [177, 178 ]. in summary, increased serine phosphorylation of irs-1 is believed to impair its tyrosine phosphorylation by the insulin receptor and interrupt the insulin signalin cascade, leading to the development of insulin resistance in skeletal muscle. because nitric oxide synthase also is activated by the irs-1/pis - k pathway, impaired insulin signaling leads to reduced nitric oxide generation and endothelial dysfunction, which have implicated in the development of accelerated atherosclerosis in t2 dm individuals. insulin resistance in the pi3k signaling pathway contrasts with an intact stimulation of the mapk pathway by insulin in insulin - resistant type 2 diabetic and obese nondiabetics individuals [7, 165 ]. physiologic hyperinsulinemia increases mitogen - activated protein kinase / extracellular signal - regulated kinase 1 activity (mek-1) and extracellular signal - regulated kinase1/2 phosphorylation activity (erk) similarly in lean healthy subjects, insulin - resistant obese nondiabetic, and type 2 diabetic patients. intact stimulation of the mapk pathway by insulin in the presence of insulin resistance in the pi3k pathway may play an important role in the development of atherosclerosis. if the metabolic (pi3k) pathway is impaired, plasma glucose levels rise, resulting in increased insulin secretion and hyperinsulinemia. because insulin receptor function is normal or only modestly impaired, especially early in the natural history of type 2 diabetes, this leads to excessive stimulation of the mapk (mitogenic) pathway in vascular tissues, with resultant proliferation of vascular smooth muscle cells, increased collagen formation, and increased production of growth factors and inflammatory cytokines [179, 180 ]. activation of the insulin signal transduction system in skeletal muscle stimulates glucose transport through a mechanism that involves translocation of a large intracellular pool of glucose transporters (associated with low - density microsomes) to the plasma membrane and their subsequent activation after insertion into the cell membrane [181, 182 ]. there are five major, different facilitative glucose transporters (gluts) with distinctive tissue distributions [183, 184 ]. glut4, the insulin regulatable transporter, is found in skeletal muscle, has a km of approximately 5 mmol / l, which is close to that of the plasma glucose concentration, and is associated with hk - ii [173, 174 ]. glut4 concentration in the plasma membrane increases markedly after exposure to insulin, and this increase is associated with a reciprocal decline in the intracellular glut4 pool. a recent study has reported a parallel translocation of glut12 from the cytosol to the plasma membrane following insulin stimulation in human skeletal muscle. glut1 is the predominant glucose transporter in the insulin - independent tissues (brain and erythrocytes) but also is found in muscle and probably contributes to muscle glucose uptake during the basal state. glut1 is located primarily in the plasma membrane where its concentration is unchanged following exposure to insulin. thus, it is likely to contribute to muscle glucose uptake during the basal state. mmol / l) and is well suited for its function, which is to mediate basal glucose uptake. it is found in association with hk - i. in insulin resistant individuals with t2 dm, glucose transport activity is severely impaired [156, 181, 182, 187189 ]. muscle tissue from lean and obese type 2 diabetic subjects exhibits normal or increased levels of glut4 mrna expression and normal levels of glut4 protein, thus demonstrating that transcriptional and translational regulation of glut4 is not impaired [190, 191 ]. using a novel triple - tracer technique, the in vivo dose - response curve for the action of insulin on glucose transport in forearm skeletal muscle has been examined in type 2 diabetic subjects, and insulin - stimulated inward muscle glucose transport has been shown to be severely impaired [192, 193 ]. impaired in vivo muscle glucose transport in type 2 because the number of glut4 transporters in the muscle of diabetic subjects is normal, impaired glut4 translocation and decreased intrinsic activity of the glucose transporter must be responsible for the defect in muscle glucose transport. large populations of type 2 diabetics have been screened for mutations in the glut4 gene. such mutations are very uncommon and, when detected, have been of questionable physiologic significance. hexokinase isoenzymes (hk - i iv) catalyze the first committed step of glucose metabolism, the intracellular conversion of free glucose to glucose-6-phosphate (glu-6-p) [183, 184, 186, 198 ]. hk - i, hk - ii, and hk - iii are single - chain peptides that have a very high affinity for glucose and demonstrate product inhibition by glu-6-p. hk - iv, also called glucokinase, has a lower affinity for glucose and is not inhibited by glu-6-p. in human skeletal muscle, hk - ii transcription is regulated by insulin, whereas hk - i mrna and protein levels are not affected by insulin [199, 200 ]. in response to physiologic euglycemic hyperinsulinemia of 2 to 4 hours ' duration, hk - ii cytosolic activity, protein content, and mrna levels increase by 50% to 200% in healthy nondiabetic subjects, and this is associated with the translocation of hexokinase ii from the cytosol to the mitochondria. in forearm muscle, insulin - stimulated glucose transport (measured with the triple - tracer technique) is markedly impaired in lean type 2 diabetics [192, 193 ], but the rate of intracellular glucose phosphorylation is impaired to an even greater extent, resulting in an increase in the free glucose concentration within the intracellular space that is accessible to glucose. these observations indicate that in type 2 diabetic individuals, although both glucose transport and glucose phosphorylation are severely resistant to the action of insulin, impaired glucose phosphorylation (hk - ii) appears to be the rate - limiting step for insulin action. studies using p nuclear magnetic resonance in combination with [1-c ] glucose also have demonstrated that both insulin - stimulated muscle glucose transport and glucose phosphorylation are impaired in type 2 diabetic subjects, but the defect in transport exceeds the defect in phosphorylation. because of methodologic differences, the results of the triple - tracer technique [192, 193 ] and magnetic resonance imaging studies can not be reconciled at present. nonetheless, these studies are consistent in demonstrating that abnormalities in both muscle glucose phosphorylation and glucose transport are well established early in the natural history of type 2 diabetes and can not be explained by glucose toxicity. in healthy nondiabetic subjects, a physiologic increase in the plasma insulin concentration for as little as 2 to 4 hours increases muscle hk - ii activity, gene transcription, and translation. in lean type 2 diabetics, the ability of insulin to augment hk - ii activity and mrna levels are markedly reduced compared with controls. decreased basal muscle hk - ii activity and mrna levels and impaired insulin - stimulated hk - ii activity in type 2 diabetic subjects have been reported by other investigators [200, 202 ]. a decrease in insulin - stimulated muscle hk - ii activity several groups have looked for point mutations in the hk - ii gene in individuals with type 2 diabetes, and, although several nucleotide substitutions have been found, none are close to the glucose and atp binding sites and none have been associated with insulin resistance [203205 ]. thus, an abnormality in the hk - ii gene is unlikely to explain the inherited insulin resistance in common variety type 2 diabetes mellitus. impaired insulin - stimulated glycogen synthesis is a characteristic finding in all insulin - resistant states. obese, igt, and diabetic subjects have severe impairment in insulin - stimulated glycogen synthase that accounts for the majority of the defect in insulin - mediated whole - body glucose disposal [1, 2, 21, 195, 206218 ]. impaired glycogen synthesis also has been documented in the normal - glucose tolerant offspring of two diabetic parents, in the first - degree relatives of type 2 diabetic individuals, and in the normoglycemic twin of a monozygotic twin pair in which the other twin has type 2 diabetes [206, 210, 211 ]. glycogen synthase is the key insulin - regulated enzyme that controls the rate of muscle glycogen synthesis [148, 150, 200, 212214 ]. insulin activates glycogen synthase by stimulating a cascade of phosphorylation - dephosphorylation reactions which ultimately lead to the inhibition of glycogen synthase kinase and activation of pp1 (also called glycogen synthase phosphatase). the regulatory subunit of pp1 has two serine phosphorylation sites, called site 1 and site 2. phosphorylation of site 2 by camp - dependent protein kinase inactivates pp1 ; whereas phosphorylation of site 1 by insulin activates pp1, leading to the stimulation of glycogen synthase. phosphorylation of site 1 of pp1 by insulin in muscle is catalyzed by insulin - stimulated protein kinase (ispk)-1. because of their central role in muscle glycogen formation, the three enzymes, glycogen synthase, pp1, and ispk-1, have been extensively studied in individuals with type 2 diabetes. glycogen synthase exists in an active (dephosphorylated) and an inactive (phosphorylated) form [148150 ]. under basal conditions, total glycogen synthase activity in type 2 diabetic subjects is reduced, and the ability of insulin to activate glycogen synthase is severely impaired [7, 215217 ]. the ability of insulin to stimulate glycogen synthase also is diminished in the normal glucose - tolerant, insulin - resistant relatives of type 2 diabetic individuals. in insulin - resistant nondiabetic and diabetic pima indians, because pp1 dephosphorylates glycogen synthase, leading to its activation, a defect in pp1 appears to play an important role in the muscle insulin resistance of type 2 diabetes mellitus. the effect of insulin on glycogen synthase gene transcription and translation in vivo has been studied extensively. most studies have demonstrated that insulin does not increase glycogen synthase mrna or protein expression in human muscle [201, 220, 221 ]. glycogen synthase mrna and protein levels, however, are decreased in muscle of type 2 diabetic patients, partly explaining the decreased glycogen synthase activity [221, 222 ]. the major abnormality in glycogen synthase regulation in type 2 diabetes is its lack of dephosphorylation and activation by insulin, as a result of insulin receptor signaling abnormalities. the glycogen synthase gene has been the subject of intensive investigation, and dna sequencing has revealed either no mutations or rare nucleotide substitutions that can not explain the defect in insulin - stimulated glycogen synthase activity [223225 ]. the genes encoding the catalytic subunits of pp1 and ispk-1 have been examined in pima indians and danes with type 2 diabetes [226, 227 ]. several silent nucleotide substitutions were found in the pp1 and ispk-1 genes in the danish population, but the mrna levels of both genes were normal in skeletal muscle. no structural gene abnormalities in the catalytic subunit of pp1 were detected in pima indians. thus, neither mutations in the pp1 and ispk-1 genes nor abnormalities in their translation can explain the impaired enzymatic activities of glycogen synthase and pp1 that have been observed in vivo. similarly, there is no evidence that an alteration in glycogen phosphorylase plays any role in the abnormality in glycogen formation in type 2 diabetes. in summary, glycogen synthase activity is severely impaired in type 2 diabetic individuals, and the molecular cause of the defect most likely is related to impaired insulin signal transduction. glucose oxidation accounts for approximately 90% of total glycolytic flux ; whereas anaerobic glycolysis accounts for the other 10%. phosphofructokinase and pyruvate dehydrogenase play pivotal roles in the regulation of glycolysis and glucose oxidation, respectively. in type 2 diabetic individuals, the glycolytic / glucose oxidative pathway has been shown to be impaired. although one study has suggested that pfk activity is modestly reduced in muscle biopsies from type 2 diabetic subjects, most evidence indicates that the activity of pfk is normal [216, 221 ]. insulin has no effect on muscle pfk activity, mrna levels, or protein content in either nondiabetic or diabetic individuals. pdh is a key insulin - regulated enzyme with activity in muscle that is acutely stimulated by insulin. in type 2 diabetic patients, insulin - stimulated pdh activity has been shown to be decreased in human skeletal muscle [231, 232 ]. obesity and type 2 diabetes mellitus are associated with accelerated ffa turnover and oxidation [1, 2, 21, 233 ], which would be expected, according to the randle cycle, to inhibit pdh activity and consequently glucose oxidation. therefore, it is likely that the observed defects in glucose oxidation and pdh activity are acquired secondary to increased ffa oxidation and feedback inhibition of pdh by elevated intracellular levels of acetyl - coa and reduced availability of nad. consistent with this scenario, the rates of basal and insulin - stimulated glucose oxidation are not reduced in the normal glucose - tolerant offspring of two diabetic parents and in the first - degree relatives of type 2 diabetic subjects ; whereas they are decreased in overtly diabetic subjects. in summary, a defect in the insulin signaling cascade at the level of irs-1 is likely the primady defect that leads to insulin resistance in skeletal muscle. other defects in the insulin signaling pathway, for example, diminished insulin binding, when present, are modest and secondary to downregulation of the insulin receptor by chronic hyperinsulinemia. in insulin resistant individuals with overt hyperglycemia, for example, t2 dm, a number of postbinding defects have been demonstrated, including reduced insulin receptor tyrosine kinase activity and altered insulin signal transduction, decreased glucose transport, diminished glucose phosphorylation, and impaired glycogen synthase activity. from the quantitative standpoint, impaired glycogen synthesis represents the major pathway responsible for the insulin resistance and this defect is present long before the onset of overt diabetes, that is, in normal glucose - tolerant, insulin - resistant prediabetic subjects, and in individuals with igt. the impairment in glycogen synthase activation is likely due to a defect in the ability of insulin to phosphorylate irs-1, causing a reduced association of the p85 subunit of pi 3-kinase with irs-1 and decreased activation of the enzyme pi3k. increased intramyocellar fat content and fatty acid metabolites, for example, facoa and dag, are likely to play a pivotal role in the development of insulin resistance in skeletal muscle. through activation of serine / threonine kinases and serine phosphorylate the irs-1, fatty acid metabolites impair irs-1 phosphorylation by the insulin receptor and lead to the defect in insulin signaling in insulin resistant individuals. the cause for the intramycellar accumulation of fat and fat metabolites has yet to be defined. a mitochondrial defect in oxidative however, the contriution of this mitochondrial defect to the intamyocellar fat accumulation is not yet clear.	insulin resistance in skeletal muscle is manifested by decreased insulin - stimulated glucose uptake and results from impaired insulin signaling and multiple post - receptor intracellular defects including impaired glucose transport, glucose phosphorylation, and reduced glucose oxidation and glycogen synthesis. insulin resistance is a core defect in type 2 diabetes, it is also associated with obesity and the metabolic syndrome. dysregulation of fatty acid metabolism plays a pivotal role in the pathogenesis of insulin resistance in skeletal muscle. recent studies have reported a mitochondrial defect in oxidative phosphorylation in skeletal muscle in variety of insulin resistant states. in this review, we summarize the cellular and molecular defects that contribute to the development of insulin resistance in skeletal muscle.
methocel k100 m and ethocel was gifted by colorcon asia pvt. ltd., mumbai, india. all the formulations as shown in table 1 were developed using direct compression method. extended release trimetazidine dihydrochloride tablets were formulated by direct compression method. all the ingredients were weighed and passed through 40#. the drug, diluents and polymer were blended in an octagonal blender for 10 min. then the mixture was lubricated using talc and magnessium stearate in a blender for 5 min. different polymers were used as release retardants such as methocel k100 m, eudragit rspo, ethocel and cellulose acetate. the lubricated mixture was then compressed on a cadmach tabletting machine (cadmach machinery co. pvt. ltd. tablet weight was set at 250 mg with hardness of 5 - 6 kg / cm. formulation development formulations t8 to t11 were developed using wet granulation method [table 2 ]. all the ingredients were weighed and passed through 40#. the mixture of drug, diluent and polymer were blended in planetary mixer for 20 min. the dried granules were finally passed through 20#. the dried granules were lubricated using talc and magnessium stearate in a blender for 5 min. the granules were then compressed using 11 mm round flat faced punches with beveled edges. tablet weight was set at 250 mg with hardness of 5 - 6 kg / cm. all the ingredients were weighed and passed through 40#. drug was dispersed in molten stearic acid and congealed. the mass was then screened through 20#. this was blended in a blender with diluent and methocel k100 m. solution of polyvinyl pyrrolidone was prepared in isopropyl alcohol and was added to form a wet mass. the wet mass was dried in fluidized bed dryer and dry granules were then passed through 20#. dried granules were lubricated using talc and magnessium stearate and compressed on cadmach tabletting machine on 11 mm round flat faced punches with beveled edges. tablet weight was fixed at 375 mg with hardness of 4 - 5 kg / cm. the granules were evaluated for flow properties like carr 's index, hausner 's ratio, density (bulk and tap density), angle of repose and loss on drying. the tablets were evaluated for physical appearance, diameter, thickness, hardness, friability, drug content and in vitro release studies. were carried out using usp type i dissolution apparatus containing 200 ml of ph 1.2 hydrochloric acid buffer for 2 h and phosphate buffer ph 7.2 for remaining 8 h. the apparatus was operated at 100 rpm at a temperature of 370.5. 5 ml aliquots were withdrawn at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10 h intervals and absorbance was recorded using uv spectrophotometry at max of 269 nm. the mechanism of drug release from the matrix tablet was analyzed using korsmeyer - peppas equation where mt / m is the fractional solute release, t is the release time, k is a kinetic constant characterisic of the drug / polymer system, a is the area of the sample, cd is the tracer loading concentration and n is an exponent which characterises the mechanism of release of the tracers. mt / m = kt(1), dmt / adt = ncdkt (2), when the diffusional release exponent (n) is equal to 1 then it characterizes zero order drug release behaviour. in vitro drug release profile of the marketed trimetzidine dihydrochloride sustained release tablets was compared with the drug release profile of test product under similar experimental conditions. the data obtained from this drug release studies was used to determine the similarity factor between marketed and test product. 3, f2 = 50 log { [1+(1/n)(ri ti) ] 100 } (3), where n is number of time points, ri and ti are dissolution of reference and test products at time i. if f2 is greater than 50 then it is considered that 2 products have similar drug release behavior. accelerated stability studies ensures stability of the product under exaggerated conditions of temperature and humidity. the optimized formulation was strip packed and subjected to stability studies at 25/60%rh, 30/65%rh and 40/75%rh at regular intervals for 6 months. the tablets were evaluated for dimension, hardness, friability, drug content and in vitro release. all the formulations as shown in table 1 were developed using direct compression method. extended release trimetazidine dihydrochloride tablets were formulated by direct compression method. all the ingredients were weighed and passed through 40#. the drug, diluents and polymer were blended in an octagonal blender for 10 min. then the mixture was lubricated using talc and magnessium stearate in a blender for 5 min. different polymers were used as release retardants such as methocel k100 m, eudragit rspo, ethocel and cellulose acetate. the lubricated mixture was then compressed on a cadmach tabletting machine (cadmach machinery co. pvt. ltd. tablet weight was set at 250 mg with hardness of 5 - 6 kg / cm. formulation development formulations t8 to t11 were developed using wet granulation method [table 2 ]. all the ingredients were weighed and passed through 40#. the mixture of drug, diluent and polymer were blended in planetary mixer for 20 min. the dried granules were finally passed through 20#. the dried granules were lubricated using talc and magnessium stearate in a blender for 5 min. the granules were then compressed using 11 mm round flat faced punches with beveled edges. tablet weight was set at 250 mg with hardness of 5 - 6 kg / cm. all the ingredients were weighed and passed through 40#. drug was dispersed in molten stearic acid and congealed. the mass was then screened through 20#. this was blended in a blender with diluent and methocel k100 m. solution of polyvinyl pyrrolidone was prepared in isopropyl alcohol and was added to form a wet mass. the wet mass was dried in fluidized bed dryer and dry granules were then passed through 20#. dried granules were lubricated using talc and magnessium stearate and compressed on cadmach tabletting machine on 11 mm round flat faced punches with beveled edges. tablet weight was fixed at 375 mg with hardness of 4 - 5 kg / cm. the granules were evaluated for flow properties like carr 's index, hausner 's ratio, density (bulk and tap density), angle of repose and loss on drying. the tablets were evaluated for physical appearance, diameter, thickness, hardness, friability, drug content and in vitro release studies. were carried out using usp type i dissolution apparatus containing 200 ml of ph 1.2 hydrochloric acid buffer for 2 h and phosphate buffer ph 7.2 for remaining 8 h. the apparatus was operated at 100 rpm at a temperature of 370.5. 5 ml aliquots were withdrawn at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10 h intervals and absorbance was recorded using uv spectrophotometry at max of 269 nm. the mechanism of drug release from the matrix tablet was analyzed using korsmeyer - peppas equation where mt / m is the fractional solute release, t is the release time, k is a kinetic constant characterisic of the drug / polymer system, a is the area of the sample, cd is the tracer loading concentration and n is an exponent which characterises the mechanism of release of the tracers. mt / m = kt(1), dmt / adt = ncdkt (2), when the diffusional release exponent (n) is equal to 1 then it characterizes zero order drug release behaviour. in vitro drug release profile of the marketed trimetzidine dihydrochloride sustained release tablets was compared with the drug release profile of test product under similar experimental conditions. the data obtained from this drug release studies was used to determine the similarity factor between marketed and test product. 3, f2 = 50 log { [1+(1/n)(ri ti) ] 100 } (3), where n is number of time points, ri and ti are dissolution of reference and test products at time i. if f2 is greater than 50 then it is considered that 2 products have similar drug release behavior. accelerated stability studies ensures stability of the product under exaggerated conditions of temperature and humidity. the optimized formulation was strip packed and subjected to stability studies at 25/60%rh, 30/65%rh and 40/75%rh at regular intervals for 6 months. the tablets were evaluated for dimension, hardness, friability, drug content and in vitro release. 1, formulation t1 to t7 did not exhibit drug release as per desired specifications. 2, formulation t8 to t11 was tried in order to increase the cohesion and binding by formation of granules, using solution of polyvinyl alcohol in isopropyl alcohol as binder, dcp as diluent, and different polymers as release retardants. therefore, the method used was melt congealing followed by wet granulation. in order to increase the hydrophobicity of the tablet various wax systems in combination with different polymers stearic acid and methocel k100 m in different ratios were tried as release retardants (formulation t12 to t13). 3). release of trimetazidine dihydrochloride from directly compressed tablets release of trimetazidine dihydrochloride from trimetazidine dihydrochloride tablets formulated by direct compression method : t1 (--) ; t2 (--) ; t3 (--) ; t4(--) ; t5 (--) ; t6 (--) and t7 (--) dissolution of trimetazidine dihydrochloride tablets formulated by wet granulation dissolution profile of trimetazidine dihydrochloride tablets formulated by wet granulation method, t8 (--) ; t9 (--) ; t10 (--) and t11 (--) release profile of marketed formulation and formulations prepared by melt congeling method comparison of release profile of marketed formulation and formulations prepared by melt congelling method. marketed (--) ; t12 (--) and t13 (--) the results of the evaluation of tablets for appearance, tablet weight, diameter, thickness, hardness, friability is given in table 3. the dissolution media used was 200 ml of ph 1.2 hydrochloric acid buffer for 2 h and phosphate buffer ph 7.2 for remaining 8 h. the basket type dissolution apparatus (electrolab) was operated at 100 rpm ; 370.5. formulations prepared using different combinations of polymers were tried and release was observed. trimetazedine dihydrochloride tablets formulated by direct compression failed to give drug release as per desired specifications. the drug being highly water soluble and a hydrophilic material which allows quicker penetration of water in to the pores of the tablet system, resulted in faster drug release. increase in cohesion and binding using pva solution could not help in retardation of drug release. evaluation of the formulation melt congealling followed by wet granulation was thought to be a better process to increase the hydrophobicity of the matrix. batch t12 although gave burst release it was quite close to the desired release profile. further increase in the quantity of stearic acid and methocel k100 m (batch t13) gave desired release profile. increased quantity of stearic acid could coat the drug to a greater extent to give desired release profile. therefore, various wax systems in combination with different polymers were tried in order to increase the hydrophobicity of the system since the drug is highly water soluble. thus tablets were formulated using methocel k100 m, stearic acid, a wax system, dicalcium phosphate and other excipients by melt congealing method. different ratios and combinations were tried to achieve a release of about 90% in 10 h. level of methocel k100 m was varied from 120 mg to 180 mg and stearic acid from 90 mg to 150 mg. the amount of dihydrogen calcium phosphate was also varied from 2.5 mg to 3.75 mg. formulation t13 having methocel k100 m 180 mg and stearic acid 150 mg gave release according to set specifications and was compared with the marketed preparation. the optimized formula concludes that not only high molecular weight polymer but also stearic acid wax system was required to obtain the tablet of our interest. the formulation, which gave release as per desired specifications, was subjected to stability studies. the release exponent (n) indicates the release mechanism of the solute i.e. fickian diffusion, anomalous non - fickian diffusion, zero order time independent release and supercase ii transport from the tablet. the diffusional release exponent (n) with their respective release mechanism and solute release rate is given in table 4. analysis of the diffusional release mechanisms the diffusional release exponent according to korsmeyer peppas eqn. was found to be n = 0.6053, suggesting that the mechanism that leads to the diffusion of trimetazidine dihydrochloride from the sustained release tablets is anomalous (non - fickian, drug diffusion in the hydrated matrix and polymer relaxation) diffusion[1618 ]. the similarity factor f2 is the simple measure for the comparison of two dissolution profiles. it ensures the uniformity of product from batch to batch and help to predict bioavailability for formulation development. the similarity factor analysis between prepared tablets and marketed tablets showed the f2 factor (f2 = 66.81) greater than 50. the f2 factor confirms that the formulated preparation t15 shows similar drug release as per marketed preparation. twice daily er tablets of trimetazidine dihydrochloride containing 30 mg of drug were successfully formulated by melt congealing followed by wet granulation method and have a uniform sustained action with increased patient compliance to overcome the drawbacks of multidosing. tablets which were formulated using drug, stearic acid and methocel k100 m in the ratio of 1:5:6 gave release as per set specifications. optimized formulation was subjected to accelerated stability studies at room temperature 30/65% rh at regular intervals for 6 months. the formulation was found stable with no change in all the parameters and in vitro release.	trimetazidine dihydrochloride, a cellular antiischemic agent indicated in the management and prophylaxis of angina pectoris is given as 20 mg thrice daily in the conventional dosage regimen. the purpose of the present study was to formulate and evaluate twice a day extended release tablets containing 30 mg trimetazidine dihydrochloride. the method developed to formulate these extended release tablets was melt congealing followed by wet granulation which exhibited uniform sustained release action and overcame the drawbacks of multidosing. the formulation was developed with methocel k100 m and stearic acid as release retardant.
inflammation is a major driving force underlying the initiation of coronary plaques, their unstable progression, and eventual rupture. inflammation biomarkers have been extensively investigated in the case of coronary artery disease (cad). current evidence supports that inflammation markers, including cytokines [such as interleukin (il)-10, il-6 ] may play an important role in the risk of developing coronary artery disease, and may correlate with its outcomes., it has multifaceted anti - inflammatory properties which have been shown to play a protective role for atherosclerotic lesion development and progression in animal studies., in the c7e3 anti platelet therapy in unstable refractory angina (capture) study, patients with elevated il-10 levels had a decreased risk of death and nonfatal myocardial infarction during a 6-month follow - up. these data suggest that il-10 may be protective against pro - inflammatory mediators in cad. il-6 can drive production of reactant proteins, including c - reactive protein (crp), and may increase plaque instability by driving expression of matrix metalloproteinases (mmps), tumor necrosis factor alpha (tnf-), and monocyte chemoattractant protein-1 (mcp-1). in the fragmin and fast revascularization during instability in coronary artery disease ii (frisc ii) trial, elevated il-6 (> 5 ng / l) was associated with higher 6- and 12-month mortality, independent of troponin and high - sensitivity crp (hs - crp). however, few clinical researches have evaluated the prognostic performance of il-10 or il-6 levels, in particular, the il-6/il-10 complex on long - term cardiovascular outcomes in patients presented with chest pain. most patients with acute coronary syndrome (acs) exhibit mild to moderate stenosis, which is associated with vulnerable plaques. abrupt rupture of plaques resulting intracoronary thrombosis are the principal etiology of acs. therefore, the present study investigate whether il-10 and il-6 concentrations or the il-6/il-10 ratio have prognostic impact in predicting future cardiovascular events in patients presented with chest pain and mild to moderate stenosis of the coronary artery. patients aged 1880 years hospitalized in beijing an zhen hospital for chest pain from february 2007 to may 2009 and who showed segmental stenosis resulting in > 20% and 2.25 mm, lumen diameter stenosis 70%, left ventricular ejection fraction 2.5 mg / dl (if male) or > 2.0 mg / dl (if female), baseline alanine aminotrasferase or aspartate aminotransferase three times that of normal, heart transplant recipients, patients whose life expectancy was less than three years, and patients who could not comply with our research program. written informed consents for all procedures were obtained from each patient. the present study complied with the declaration of helsinki and was approved by the institutional review board of beijing an zhen hospital, capital medical university, and beijing institute of heart, lung and blood vessel diseases, beijing, china. after evaluating the lesion with cag and qca, demographic features and clinical characteristics fasting blood samples were drawn from the ulnar vein on the morning after the cag procedure and were filled in tubes with ethylene diamine tetraacetic acid (edta) as anticoagulation. plasma samples were immediately separated into multiple aliquots and stored at 80c within 30 min. triglyceride (tg), total cholesterol (tc), low - density lipoprotein cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c), serum creatinine and other routine blood biochemical examination were measured in a biochemical analyzer (hitachi 7600, tokyo). the concentrations of the plasma il-10 and il-6 were determined using commercially available protein arrays (raybiotech, norcross, ga, usa) following the manufacturer 's instructions. all the patients were followed - up by telephone interviews at 1, 3, 6, 12, 18, 24, 36, 48, 60, 72, 84, and 96 months after cag. the primary endpoint [major adverse cardiac events (maces) ] was a composite of all cause death, nonfatal acute myocardial infarction, revascularization, and angina pectoris requiring rehospitalization. angina pectoris requiring rehospitalization was defined as a recurrent episode of ischemic chest pain requiring inpatient hospital admission for further evaluation, according to the symptoms of chest pain, electrocardiographic changes in ischemia, and levels of cardiac enzymes. all endpoints were centrally adjudicated by the independent clinical events committee that had no knowledge of patient data. in the cumulative analysis of endpoints, events were counted only once, whichever occurred first. normally - distributed continuous variables were presented as mean sd, variables with a skewed distribution as the median together with interquartile range (iqr). the relationship between il-10, il-6 and the baseline cardiovascular risk factors was investigated using spearman 's rank correlation statistic. the kaplan - meier method was used to illustrate the timing of events during follow - up in relation to il-10, il-6 and il-6/il-10 and statistical assessment was performed with the log - rank test. the prognostic value of il-10, il-6 and il-6/il-10 was assessed by investigating its relation with the composite outcome in cox proportional - hazards analyses. various cox regression models were performed to evaluate the predictive values of il-10, il-6 and il-6/il-10 on the risk of cardiovascular events. the first model was adjusted for traditional risk factors [including age, gender, body mass index (bmi), hypertension, diabetes, smoking status and ldl - c ]. further adjustment was performed for the hs - crp and white blood count levels (model 3). the proportional - hazards assumptions of all cox proportional - hazards analyses were assessed with schoenfeld 's tests, and no relevant violations were observed. patients aged 1880 years hospitalized in beijing an zhen hospital for chest pain from february 2007 to may 2009 and who showed segmental stenosis resulting in > 20% and 2.25 mm, lumen diameter stenosis 70%, left ventricular ejection fraction 2.5 mg / dl (if male) or > 2.0 mg / dl (if female), baseline alanine aminotrasferase or aspartate aminotransferase three times that of normal, heart transplant recipients, patients whose life expectancy was less than three years, and patients who could not comply with our research program. written informed consents for all procedures were obtained from each patient. the present study complied with the declaration of helsinki and was approved by the institutional review board of beijing an zhen hospital, capital medical university, and beijing institute of heart, lung and blood vessel diseases, beijing, china. after evaluating the lesion with cag and qca, demographic features and clinical characteristics were recorded. fasting blood samples were drawn from the ulnar vein on the morning after the cag procedure and were filled in tubes with ethylene diamine tetraacetic acid (edta) as anticoagulation. plasma samples were immediately separated into multiple aliquots and stored at 80c within 30 min. triglyceride (tg), total cholesterol (tc), low - density lipoprotein cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c), serum creatinine and other routine blood biochemical examination were measured in a biochemical analyzer (hitachi 7600, tokyo). the concentrations of the plasma il-10 and il-6 were determined using commercially available protein arrays (raybiotech, norcross, ga, usa) following the manufacturer 's instructions. all the patients were followed - up by telephone interviews at 1, 3, 6, 12, 18, 24, 36, 48, 60, 72, 84, and 96 months after cag. the primary endpoint [major adverse cardiac events (maces) ] was a composite of all cause death, nonfatal acute myocardial infarction, revascularization, and angina pectoris requiring rehospitalization. angina pectoris requiring rehospitalization was defined as a recurrent episode of ischemic chest pain requiring inpatient hospital admission for further evaluation, according to the symptoms of chest pain, electrocardiographic changes in ischemia, and levels of cardiac enzymes. all endpoints were centrally adjudicated by the independent clinical events committee that had no knowledge of patient data. in the cumulative analysis of endpoints normally - distributed continuous variables were presented as mean sd, variables with a skewed distribution as the median together with interquartile range (iqr). the relationship between il-10, il-6 and the baseline cardiovascular risk factors was investigated using spearman 's rank correlation statistic. the kaplan - meier method was used to illustrate the timing of events during follow - up in relation to il-10, il-6 and il-6/il-10 and statistical assessment was performed with the log - rank test. the prognostic value of il-10, il-6 and il-6/il-10 was assessed by investigating its relation with the composite outcome in cox proportional - hazards analyses. various cox regression models were performed to evaluate the predictive values of il-10, il-6 and il-6/il-10 on the risk of cardiovascular events. the first model was adjusted for traditional risk factors [including age, gender, body mass index (bmi), hypertension, diabetes, smoking status and ldl - c ]. further adjustment was performed for the hs - crp and white blood count levels (model 3). the proportional - hazards assumptions of all cox proportional - hazards analyses were assessed with schoenfeld 's tests, and no relevant violations were observed. a total of 566 patients were recruited between february 2007 and may 2009. of these subjects,, 201 events occurred : 31 patients died, 14 patients suffered a nonfatal myocardial infarction, 57 patients underwent revascularization and 99 patients were readmitted for angina pectoris. as shown in table 1, the median age was 59.1 years and 68.7% of the patients were men. discharge diagnosis of the study population included four types as follows : coronary atherosclerosis in 269 patients, stable angina pectoris in 82 patients, unstable angina pectoris in 193 patients, and acute myocardial infarction in 22 patients. il-10 levels were skewed distributed, with a median of 60.02 ng / l (iqr : however, no significant differences were found in the il-10 concentrations among the different diagnosis groups (p = 0.595). the il-6 levels were also skewed distributed, with a median of 168.49 ng / l (iqr : 94.09 310.75 ng although all subjects in the study population had mild to moderate coronary artery lesions, there were significant differences in the il-6 concentrations among the different diagnosis groups (p 0.05). as shown in the kaplan - meier survival curves, patients with below - median levels of il-10 (log - rank p = 0.006) and above - median levels of il-6/il-10 (log - rank p = 0.012) had a much higher rate of maces during follow - up, whereas il-6 did not demonstrate this association (long - rank p = 0.076) (figures 24). table 5 shows the unadjusted hrs of il-10, il-6 and il-6/il-10 for outcomes (hr = 0.668, 95% ci : 0.5010.892, p = 0.006 ; hr = 1.311, 95% ci : 0.9701.773, p = 0.078 ; hr = 1.483, 95% ci : 1.0862.025, p = 0.013, respectively). even after adjustment for age, gender, bmi, hypertension, diabetes, smoking status and ldl - c in model 1, associations between il-10, il-6/il-10 and long - term maces remained significant (table 5). moreover, in the models including discharge diagnosis (table 5, model 2) and even the value of hs - crp and wbc (table 5, model 3), il-10 remained a strong independent predictor of adverse outcomes (p = 0.013 and 0.046, respectively). however, il-6 showed no statistically significant association with the outcome (the p values for il-6 were 0.118, 0.179 and 0.226 in the three models, respectively) (table 5). bmi : body mass index ; ckmb : creatine kinase mb ; hdl - c : high - density lipoprotein cholesterol ; hs - crp : high - sensitivity c - reactive protein ; il : interleukin ; ldl - c : low - density lipoprotein cholesterol ; tc : total cholesterol. bmi : body mass index ; ckmb : creatine kinase mb ; hdl - c : high - density lipoprotein cholesterol ; hs - crp : high - sensitivity c - reactive protein ; il : interleukin ; ldl - c : low - density lipoprotein cholesterol ; tc : total cholesterol. model 1 adjusted for age, gender, bmi, hypertension, diabetes, smoking status and ldl - c ; model 2 was additionally adjusted for discharge diagnosis ; model 3 further adjusted for traditional risk factors hs - crp and white blood count. bmi : body mass index ; hs - crp : high - sensitivity c - reactive protein ; il : interleukin ; ldl - c : low - density lipoprotein cholesterol ; maces : major adverse cardiovascular events. a total of 566 patients were recruited between february 2007 and may 2009. of these subjects,, 201 events occurred : 31 patients died, 14 patients suffered a nonfatal myocardial infarction, 57 patients underwent revascularization and 99 patients were readmitted for angina pectoris. as shown in table 1, the median age was 59.1 years and 68.7% of the patients were men. discharge diagnosis of the study population included four types as follows : coronary atherosclerosis in 269 patients, stable angina pectoris in 82 patients, unstable angina pectoris in 193 patients, and acute myocardial infarction in 22 patients. il-10 levels were skewed distributed, with a median of 60.02 ng / l (iqr : 35.1496.28 ng / l). however, no significant differences were found in the il-10 concentrations among the different diagnosis groups (p = 0.595). the il-6 levels were also skewed distributed, with a median of 168.49 ng / l (iqr : 94.09 310.75 ng although all subjects in the study population had mild to moderate coronary artery lesions, there were significant differences in the il-6 concentrations among the different diagnosis groups (p 0.05). as shown in the kaplan - meier survival curves, patients with below - median levels of il-10 (log - rank p = 0.006) and above - median levels of il-6/il-10 (log - rank p = 0.012) had a much higher rate of maces during follow - up, whereas il-6 did not demonstrate this association (long - rank p = 0.076) (figures 24). table 5 shows the unadjusted hrs of il-10, il-6 and il-6/il-10 for outcomes (hr = 0.668, 95% ci : 0.5010.892, p = 0.006 ; hr = 1.311, 95% ci : 0.9701.773, p = 0.078 ; hr = 1.483, 95% ci : 1.0862.025, p = 0.013, respectively). even after adjustment for age, gender, bmi, hypertension, diabetes, smoking status and ldl - c in model 1, associations between il-10, il-6/il-10 and long - term maces remained significant (table 5). moreover, in the models including discharge diagnosis (table 5, model 2) and even the value of hs - crp and wbc (table 5, model 3), il-10 remained a strong independent predictor of adverse outcomes (p = 0.013 and 0.046, respectively). however, il-6 showed no statistically significant association with the outcome (the p values for il-6 were 0.118, 0.179 and 0.226 in the three models, respectively) (table 5). bmi : body mass index ; ckmb : creatine kinase mb ; hdl - c : high - density lipoprotein cholesterol ; hs - crp : high - sensitivity c - reactive protein ; il : interleukin ; ldl - c : low - density lipoprotein cholesterol ; tc : total cholesterol. bmi : body mass index ; ckmb : creatine kinase mb ; hdl - c : high - density lipoprotein cholesterol ; hs - crp : high - sensitivity c - reactive protein ; il : interleukin ; ldl - c : low - density lipoprotein cholesterol ; tc : total cholesterol. model 1 adjusted for age, gender, bmi, hypertension, diabetes, smoking status and ldl - c ; model 2 was additionally adjusted for discharge diagnosis ; model 3 further adjusted for traditional risk factors hs - crp and white blood count. bmi : body mass index ; hs - crp : high - sensitivity c - reactive protein ; il : interleukin ; ldl - c : low - density lipoprotein cholesterol ; maces : major adverse cardiovascular events. the present study demonstrates that elevated il-10 levels are associated with a more favorable long - term prognosis in chinese patients with chest pain and mild to moderate coronary artery lesions. when we combined markers as il-6 to il-10 ratios, we observed a strong relationship between them and the development of maces even after adjustment of traditional risk factors. as far as our knowledge, this is the first study demonstrating the role of il-10 levels in chinese patients presented with chest pain and mild to moderate lesions. it is now widely accepted that inflammation plays a fundamental role in the genesis and development of atherosclerosis. in recent years, multiple scientific studies have emphasized the role of pro - inflammatory cytokines in the development of atherosclerosis. nevertheless, there is little evidence available on the potential role of anti - inflammatory cytokines in this process. il-10, which is produced by various inflammatory cells, especially macrophages, has multifaceted anti - inflammatory properties. in vitro and in vivo studies in animals, indeed, numerous recent experimental studies have shown that either systemic or local il-10 gene transfer not only attenuates atherogenesis,, but also affects processes associated with lesion progression. a previous study demonstrated that elevated levels of il-6 were associated with increased risk of future myocardial infarction in apparently healthy men, which support a role for cytokine - mediated inflammation in the early stages of atherogenesis. hu,. found that myocardial infarction patients had significantly higher levels of il-10 and il-6 than healthy controls. however, smith,. demonstrated serum il-10 concentrations were lower in unstable angina patients compared with those who had chronic stable angina (28.4 vs. 14.0 pg / ml, 95% ci : 9.819.0, p < 0.0001) even after adjustment for confounding variables, while il-6 concentrations were higher in the unstable angina group (29.0 vs. 11.4 pg / ml ; 95% ci : 1.012.6, p = 0.04). evaluated plasma circulatory markers in patients with cad and lower levels were found for il-10 in cad patients compared with healthy controls. george,. compared the il-6 and il-10 levels between vulnerable and stable patients, which indicated il-10 may discriminate them and have a protective role against plaque rupture in patients with coronary atherosclerosis. results from the european collaborative project on inflammation and vascular wall remodeling in atherosclerosis - intravascular ultrasound (atheroremo - ivus) study showed lower circulating il-10 was associated with higher plaque burden and large virtual histology - thin cap fibroadenoma lesions, which suggesting a protective role for the cytokine in extent and vulnerability of atherosclerosis. previous studies on the prognostic utility of il-10 in patients with acs have provided conflicting results. malarstig,. evaluated the concentrations of il-10 and found il-10 reflected a pro - inflammatory state in patients with acs. cavusoglu,. measured baseline plasma il-10 levels in 193 well - characterized male patients with acs and followed for 5 years for the development of major adverse cardiovascular events. after controlling for a variety of baseline variables, elevated plasma il-10 levels were proved to be a strong and independent predictor of the composite outcome of death or non - fatal myocardial infarction. however, more evidences are available supporting the protective effect of il-10 on future outcomes. in the c7e3 anti platelet therapy in unstable refractory angina (capture) study, elevated il-10 levels are associated with a decreased risk of death and nonfatal myocardial infarction in patients with acs during a 6-month follow - up. one data show a highly significant inverse independent relationship between il-10 levels and the composite endpoint of all - cause mortality, reinfarction and cerebrovascular events during a 4-year follow - up in patients with an acute myocardial infarction. the results indicate about a 6-fold increased risk of suffering a future event when being below the median level of il-10 in the study population. lower levels of il-10 were observed on admission in patients with unstable angina who subsequently had cardiovascular events during a 3 months follow - up period. a study including 80 patients with non - st elevation acute coronary syndrome (nsteacs) demonstrated that il-6/il-10 ratio was the most important predictor for coronary events during the 12 months follow - up period (p = 0.006). our findings are in keeping with data from animal model studies that suggest il-10 has a protective role in atherosclerosis. although we have not found any significant relationship between il-6 concentrations and adverse outcomes, il-6 concentrations were higher in patients who had events. these results suggest that the risk for development of maces is higher in patients with higher il-6 and lower il-10 concentrations. in particular, the il-10 was the most powerful predictor of the development of maces during the 8-year follow - up period. this finding suggests that anti - inflammatory cytokine il-10 can be used for early risk assessment in patients presented with chest pain and mild to moderate coronary artery lesions. although il-10 is proved to be a good marker for long - term outcomes, it is not routinely available in the clinical practice. undoubtedly, the association between inflammation, coronary plaque instability, and clinical outcomes is extremely complex. although strict inclusion and exclusion criteria have been set, many potential variables can modulate interleukin values which could not be adjusted. apart from the above, almost ten percent of the patients lost to follow - up during the 8-year study period, which could have influenced the results. elevated il-10 levels are associated with a more favorable long - term prognosis in patients with chest pain and mild to moderate coronary artery lesions. although il-10 is proved to be a good marker for long - term outcomes, it is not routinely available in the clinical practice. undoubtedly, the association between inflammation, coronary plaque instability, and clinical outcomes is extremely complex. although strict inclusion and exclusion criteria have been set, many potential variables can modulate interleukin values which could not be adjusted. apart from the above, almost ten percent of the patients lost to follow - up during the 8-year study period, which could have influenced the results. elevated il-10 levels are associated with a more favorable long - term prognosis in patients with chest pain and mild to moderate coronary artery lesions.	backgroundinterleukin (il)-10, il-6 and their ratio (il-6/il-10) play an important role in the risk of developing coronary artery disease, and may correlate with its outcomes. few clinical trials have investigated the prognostic impact of these factors on long - term cardiovascular events in patients presented with chest pain.methodsa prospective study was performed on 566 patients admitted with chest pain and identified mild to moderate coronary artery lesions. il-10, il-6 and il-6/il-10 were measured.resultsa total of 511 patients completed the follow - up. the median follow - up time was 74 months. kaplan - meier analysis demonstrated a clear increase of the incidence of major adverse cardiac events during the follow - up period in patients with below - median levels of il-10 (p = 0.006) and above - median levels of il-6/il-10 (p = 0.012). multivariate cox proportional hazards analysis indicated the il-10 levels to be strong independent predictors after adjustment for underlying confounders.conclusionselevated il-10 levels are associated with a more favorable long - term prognosis in patients with chest pain and mild to moderate coronary artery lesions. il-10 could be used for early risk assessment of long - term prognosis.
deficiencies in quality of diabetes care are found in many countries with different health care systems for both process of care measures (such as physical examination maneuvers and the ordering of tests by physicians) and for outcomes of care (the achievement of treatment targets and prevention of complications). in a large study of american patients, fewer than 20% received the recommended two a1c tests, one lipid profile, and one dilated eye exam in 1 year.1 fewer than half of patients surveyed in italy reported receiving a foot exam over 1 year,2 and only 11% of canadian diabetic seniors with established coronary artery disease were prescribed an antiplatelet agent, a statin, and an angiotensin - converting enzyme inhibitor.3 at least partially as a result of these inadequate processes of care, outcomes for patients are often poor. health surveys found that 41% of diabetic patients had a1c levels above 8.0%, and 18% had levels over 9.5%. only 42% of patients had ldl cholesterol levels below 3.4 mmol / l and fewer than two thirds had blood pressure levels below 140/90 mmhg.4 similar results have been found in countries around the world.58 the proportion of patients meeting glycemic, blood pressure and cholesterol targets did not differ between 19881994 and 19992000,9 and although recent data suggest some small trends toward improvement,10 large numbers of patients remain inadequately treated. to address these gaps between evidence and practice, some have speculated that specialists may deliver better chronic disease care than generalists.11 although many investigators have examined this question in diabetes, few studies accounted for patient clustering within physicians and hence statistical nonindependence. table 1a selection of studies comparing processes of diabetes care between specialists and generalistsstudyprocess measureperformance rate by specialists (%) performance rate by generalists (%) lafata.1measuring lipid profile7770measuring a1c8458eye examination5133all three3516greenfield.12measuring lipid profile4551measuring urine protein5842measuring a1c9079eye examination4134blood glucose self - monitoring8373de berardis.14measuring total cholesterol6666measuring microalbuminuria5331measuring a1c7343eye examination5538foot examination5240shah.15drug escalation in response to poor glycemic control4537schaars.16measuring blood pressure73indicates the performance rate in the whole study population, as the performance rate by group was not reported separately. a selection of studies comparing processes of diabetes care between specialists and generalists indicates the performance rate in the whole study population, as the performance rate by group was not reported separately. a study in american ambulatory care clinics found that patients with shared care between endocrinologists and generalists versus generalist - only care received more a1c and lipid tests, and more dilated eye examinations.1 another smaller study of 1,750 patients across the united states found nonstatistically significant trends favoring specialists for many processes of care.12 an italian group found that several processes were more common for specialists patients compared to general practitioners, including performance of self - monitoring of blood glucose, foot examinations, a1c testing, microalbuminuria testing, and dilated eye exams.2,13,14 a study using large canadian administrative databases showed that specialists escalated glucose - lowering medications for patients with poor glycemic control more often than generalists,15 and a dutch study found that specialized diabetes clinics measured patients blood pressures more often than generalists did.16 thus, specialists often do provide better processes of care than generalists, and yet in most cases, the absolute difference is small compared to the deficiency that even specialist care has compared to the ideal performance level of 90% to 100% at which most of these processes ought to be performed (see table 1). because differences between specialists and generalists for many processes are small, it is not surprising that clinical outcomes have not been shown to markedly differ between them. in the medical outcomes study, which prospectively followed 424 diabetic patients in the 1980s, most clinical, functional status and well - being measures did not differ between physician groups ; only foot ulcer prevalence showed greater improvement for patients followed by endocrinologists, but their patients had a higher prevalence at baseline.17 the pittsburgh epidemiology of diabetes complications study prospectively followed patients with type 1 diabetes for 10 years, and showed that greater time under specialist care during follow - up was associated with a reduced incidence of overt nephropathy, but other micro- and macrovascular complications were not different.18 in italy, despite small differences in processes between specialists and general practitioners, glycemic, blood pressure, and lipid control did not differ.13,14 canadian and german studies have also shown only small differences in glycemic control between specialist and generalist care.6,19 specialist care did not significantly reduce the risk of diabetes - related hospitalizations or emergency department visits.20 a study in american ambulatory care clinics found that patients with shared care between endocrinologists and generalists versus generalist - only care received more a1c and lipid tests, and more dilated eye examinations.1 another smaller study of 1,750 patients across the united states found nonstatistically significant trends favoring specialists for many processes of care.12 an italian group found that several processes were more common for specialists patients compared to general practitioners, including performance of self - monitoring of blood glucose, foot examinations, a1c testing, microalbuminuria testing, and dilated eye exams.2,13,14 a study using large canadian administrative databases showed that specialists escalated glucose - lowering medications for patients with poor glycemic control more often than generalists,15 and a dutch study found that specialized diabetes clinics measured patients blood pressures more often than generalists did.16 thus, specialists often do provide better processes of care than generalists, and yet in most cases, the absolute difference is small compared to the deficiency that even specialist care has compared to the ideal performance level of 90% to 100% at which most of these processes ought to be performed (see table 1). because differences between specialists and generalists for many processes are small, it is not surprising that clinical outcomes have not been shown to markedly differ between them. in the medical outcomes study, which prospectively followed 424 diabetic patients in the 1980s, most clinical, functional status and well - being measures did not differ between physician groups ; only foot ulcer prevalence showed greater improvement for patients followed by endocrinologists, but their patients had a higher prevalence at baseline.17 the pittsburgh epidemiology of diabetes complications study prospectively followed patients with type 1 diabetes for 10 years, and showed that greater time under specialist care during follow - up was associated with a reduced incidence of overt nephropathy, but other micro- and macrovascular complications were not different.18 in italy, despite small differences in processes between specialists and general practitioners, glycemic, blood pressure, and lipid control did not differ.13,14 canadian and german studies have also shown only small differences in glycemic control between specialist and generalist care.6,19 specialist care did not significantly reduce the risk of diabetes - related hospitalizations or emergency department visits.20 these observations indicate that the magnitude of the difference between specialist and generalist care is inconsequential compared to the magnitude of the overall deficiency in quality of care. therefore, research examining differences between types of physicians is asking the wrong question ; instead, research should be identifying methods to improve quality of care, regardless of the specialty of the providers. the cochrane collaboration s effective practice and organisation of care group has developed a conceptual framework for interventions that improve delivery of care (fig. 2).21 several groups have systematically reviewed trials of interventions to improve quality of care, both in general,2125 and for diabetes care in particular.26,27 the interventions that have been evaluated in diabetes care have included educational materials and meetings, local consensus processes, educational outreach visits, audit and feedback programs, reminder systems, changes to the health care team, formal case management programs, and changes in record keeping and communication between professionals. in addition, because diabetes self - management requires the maintenance of a long - term therapeutic alliance among patients, their family members, and their health care providers,28 successful quality improvement interventions could target patients or their families. such interventions that have been evaluated include individual- or group - based diabetes education programs delivered by various health professionals, telephone- and computer - based education programs, and written educational material for patients.2931 notwithstanding the methodological limitations of many of these trials, most interventions have shown limited ability to change the delivery of care, and many areas of the framework for change remain unstudied. furthermore, educational interventions appear not to be additive : multiple interventions do not significantly change behavior beyond what is achieved by a single intervention.25 the enormous gaps that remain in our ability to effectively change the delivery of care underline an urgent need to devise interventions to improve care and to test their effectiveness in heterogeneous real - world populations by using pragmatic trials.32figure 2a conceptual framework for interventions to improve quality of care. adapted from the cochrane collaboration s effective practice and organisation of care group.21 a conceptual framework for interventions to improve quality of care. adapted from the cochrane collaboration s effective practice and organisation of care group.21 however, developing such interventions will not be easy. in 1995, the veterans administration (va) health care system was reengineered by implementing more than 100 interventions to improve quality of care, mostly organizational and educational interventions.33 when compared to the medicare system, many performance indicators (including annual measurement of a1c and semiannual lipid screening for diabetic patients) improved over the next several years.34 however, many clinical measures continued to have room for improvement ; for example, thousands of diabetic patients continued to miss their annual retinal examinations. in decentralized health care systems, such as those available to most patients in most countries, it may be more difficult to apply the comprehensive and resource - intensive interventions that the va could implement, which would then attenuate any improvement in quality of care that could be achieved. nonetheless, a recent study did show that halfway through a decade - long government and professional initiative to improve quality of care in the united kingdom, some improvement in diabetes care was achieved.35 the elusiveness of a magic bullet for practice improvement and the systemic barriers to change in many settings should not dissuade efforts to identify interventions, which will produce small but important advances. from the framework for changing practice (fig. 2), educational interventions to change physician and patient behavior have been studied to some degree, although much more work needs to be done. however, there is a genuine paucity of research examining organizational, financial, and regulatory interventions that change the environment in which care is delivered. some specific organizational changes that may help improve quality of care include coordinated teams composed of multiple professionals, where each element of care is delivered by the provider with the greatest expertise and experience ; information technology to support communication and data transfer within the team of providers and between the team and the patient ; and fostering the organizational culture of all stakeholders (providers, institutions, payers, and regulators) to encourage quality through system improvement.36 financial interventions may include capitation, salary, or pay - for - performance initiatives for primary care physicians,37 while regulatory interventions could include tying licensure to the delivery of quality care. ultimately, interventions to improve ambulatory diabetes management will have to meet several objectives to be successful. they will have to be practicable so that they can be implemented within busy clinical practices. because diabetes is so common, the interventions will need to be affordable to ensure that they can be disseminated widely, and they will have to be generalizable so that they can be used by physicians with different practice styles in different regions and with access to different resources. finally, to ensure high uptake of the interventions, they will likely need to include some incentives to encourage physicians to use them. clinical research continues to find new and better ways of treating diabetes and its complications to reduce the morbidity and mortality for patients with the disease. but the efficacy improvements of these new treatments could be matched by relatively modest increases in the utilization of older, cheaper, and less efficacious predecessor treatments among eligible patients (see fig. 3). while this calculation may be different were the new treatments vastly more effective than their predecessors, governments are choosing to invest resources in a potentially less efficient way of improving health. the u.s. congress, for example, budgets just one penny for the agency for healthcare research and quality to improve quality of care for each dollar budgeted to the national institutes of health to fund the development of new treatments, in addition to the billions spent on drug development by the pharmaceutical industry.38 we argue in this paper that no magic bullet for improving quality of care in diabetes has been found, and that attention focused on specialist care is misplaced because of its limited benefits and the financial and human resources implications of increasing specialist care. however, the search for the multiplicity of system and educational, professional, and patient support strategies that will together achieve improved quality surely warrants more investment than the trifling amount it currently receives. figure 3an example from woolf.38 to contrast the impact on population mortality of improving drug efficacy versus improving drug utilization. in this example, developing a new drug, which requires billions of dollars of public and private sector investment, reduces mortality to the same extent as a relatively modest improvement in the utilization of an existing drug might do. an example from woolf.38 to contrast the impact on population mortality of improving drug efficacy versus improving drug utilization. in this example, developing a new drug, which requires billions of dollars of public and private sector investment, reduces mortality to the same extent as a relatively modest improvement in the utilization of an existing drug might do.	the quality of diabetes care delivered to patients falls below the expectations of practice guidelines and clinical trial evidence. studies in many jurisdictions with varying health care systems have shown that recommended processes of care occur less often than they should ; hence, outcomes of care are inadequate. many studies comparing care between specialists and generalists have found that specialists are more likely to implement processes of care. however, this provides little insight into improving quality of care, as the difference between specialists and generalists in these studies is small compared to the overall deficiency in quality. therefore, future research should instead focus on ways to implement high quality care, regardless of specialty. to date, few methodologically rigorous studies have uncovered interventions that can improve quality of care. the development of such interventions to help all physicians implement better quality care could greatly benefit people with diabetes.
this retrospective review of images and medical records was approved by the our institutional review board. between march 2004 and october 2006, a search of medical records revealed 145 consecutive patients presenting with abnormal nipple discharge. all patients underwent a mammography and us to evaluate the cause of nipple discharge, which was an initial work - up protocol at our institution. us - guided vacuum - assisted removal was performed in 50 patients with an intraductal mass revealed by us. patients with abnormal nipple discharge and an intraductal mass by us that subsequently underwent surgery due to biopsy results of atypia (n = 6) or malignancy (n = 2), and those without follow - up data (n = 4) or a follow - up examination less than 12 months after the treatment (n = 2) were excluded from the study. the 36 remaining patients (age range, 18 - 80 years ; mean age, 46 years) with serous (23 of 36, 64%) or bloody (13 of 36, 36%) nipple discharge constituted the study population. twenty (56%) patients had a negative finding, four (11%) had a mass, two (6%) had microcalcifications, one (3%) had a mass with microcalcifications, and nine (25%) had a focal asymmetry. us examinations were performed by one of eleven radiologists who specialized in breast imaging using a high - resolution sonographic unit equipped with a 10- or 12-mhz linear transducer (voluson 730, kretz, austria ; hdi 5000, advanced technology laboratories, bothell, wa). patients were placed in the supine or supine - oblique position before undergoing the us. radial and antiradial us scans were performed by tracing the discharging duct, lactiferous duct, and trigger points to identify and localize the cause of the nipple discharge. us - dvar was performed using an 11-gauge vacuum - assisted device (mammotome ; ethicon - endosurgery, cincinnati, oh) under the guidance of a high - resolution sonographic unit with the 10- or 12-mhz linear transducers mentioned above. the patient 's skin was sterilized and about 10 - 15 ml of 1% lidocaine hydrochloride was injected into the skin, subcutaneous fat, and tissues around the dilated duct. next, a probe was inserted either through or directly subjacent to the lesion and then manually rotated to sample both the target lesion and adjacent tissues in a clockwise manner. the mean number of core samples obtained was 9.3 (range : 4 - 22). fourteen (39%) procedures were performed by one of three attending radiologists with 2 - 8 years of breast imaging and intervention experience, 22 (61%) procedures were performed by one of eight fellows who conducted their first 5 to 10 biopsies under the supervision of an attending radiologist. pathology results were compared with relevant imaging findings to determine concordances and discordances of the biopsy results. patients with a biopsy finding indicating a malignancy or of a high - risk lesion underwent a surgical excision. if the lesion had a highly suspicious sonographic finding, a surgical excision was recommended, despite a benign pathologic result. if the imaging findings were determined to be concordant with a benign pathology, six, 12 and 24 month follow - ups were recommended. the presence of nipple discharge or a residual lesion, such as solid nodules, were evaluated by follow - up us and clinical records. radiologists who performed the procedures recorded the lesion size (maximal diameter on us), lesion location, final assessment category according to the breast imaging reporting and data system (bi - rads) (12), and the number of core samples taken during the procedure. the presence of abnormal nipple discharge as well as visible residual lesion were recorded on us examination reports during follow - up us. symptom elimination was defined as the absence of nipple discharge on the latest us examination report. we evaluated whether symptom elimination was affected by lesion variables (lesion size, location including distance from the nipple, and bi - rads category), biopsy variables (number of core samples, radiologist experience), the presence of a residual lesion by follow - up us, and histologic findings. the test, fisher 's exact test, and the student 's t - test were performed using spss version 12.0 for windows (spss inc., chicago, il), and results were deemed statistically significant at a two - sided significance level of 5%. this retrospective review of images and medical records was approved by the our institutional review board. between march 2004 and october 2006, a search of medical records revealed 145 consecutive patients presenting with abnormal nipple discharge. all patients underwent a mammography and us to evaluate the cause of nipple discharge, which was an initial work - up protocol at our institution. us - guided vacuum - assisted removal was performed in 50 patients with an intraductal mass revealed by us. patients with abnormal nipple discharge and an intraductal mass by us that subsequently underwent surgery due to biopsy results of atypia (n = 6) or malignancy (n = 2), and those without follow - up data (n = 4) or a follow - up examination less than 12 months after the treatment (n = 2) were excluded from the study. the 36 remaining patients (age range, 18 - 80 years ; mean age, 46 years) with serous (23 of 36, 64%) or bloody (13 of 36, 36%) nipple discharge constituted the study population. twenty (56%) patients had a negative finding, four (11%) had a mass, two (6%) had microcalcifications, one (3%) had a mass with microcalcifications, and nine (25%) had a focal asymmetry. us examinations were performed by one of eleven radiologists who specialized in breast imaging using a high - resolution sonographic unit equipped with a 10- or 12-mhz linear transducer (voluson 730, kretz, austria ; hdi 5000, advanced technology laboratories, bothell, wa). patients were placed in the supine or supine - oblique position before undergoing the us. radial and antiradial us scans were performed by tracing the discharging duct, lactiferous duct, and trigger points to identify and localize the cause of the nipple discharge. us - dvar was performed using an 11-gauge vacuum - assisted device (mammotome ; ethicon - endosurgery, cincinnati, oh) under the guidance of a high - resolution sonographic unit with the 10- or 12-mhz linear transducers mentioned above. the patient 's skin was sterilized and about 10 - 15 ml of 1% lidocaine hydrochloride was injected into the skin, subcutaneous fat, and tissues around the dilated duct. next, a probe was inserted either through or directly subjacent to the lesion and then manually rotated to sample both the target lesion and adjacent tissues in a clockwise manner. the mean number of core samples obtained was 9.3 (range : 4 - 22). fourteen (39%) procedures were performed by one of three attending radiologists with 2 - 8 years of breast imaging and intervention experience, 22 (61%) procedures were performed by one of eight fellows who conducted their first 5 to 10 biopsies under the supervision of an attending radiologist. pathology results were compared with relevant imaging findings to determine concordances and discordances of the biopsy results. patients with a biopsy finding indicating a malignancy or of a high - risk lesion underwent a surgical excision. if the lesion had a highly suspicious sonographic finding, a surgical excision was recommended, despite a benign pathologic result. if the imaging findings were determined to be concordant with a benign pathology, six, 12 and 24 month follow - ups were recommended. the presence of nipple discharge or a residual lesion, such as solid nodules, were evaluated by follow - up us and clinical records. radiologists who performed the procedures recorded the lesion size (maximal diameter on us), lesion location, final assessment category according to the breast imaging reporting and data system (bi - rads) (12), and the number of core samples taken during the procedure. the presence of abnormal nipple discharge as well as visible residual lesion were recorded on us examination reports during follow - up us. symptom elimination was defined as the absence of nipple discharge on the latest us examination report. we evaluated whether symptom elimination was affected by lesion variables (lesion size, location including distance from the nipple, and bi - rads category), biopsy variables (number of core samples, radiologist experience), the presence of a residual lesion by follow - up us, and histologic findings. the test, fisher 's exact test, and the student 's t - test were performed using spss version 12.0 for windows (spss inc., chicago, il), and results were deemed statistically significant at a two - sided significance level of 5%. of the 36 lesions, 69% (25 of 36) were intraductal papillomas, 28% (10 of 36) were attributed to fibrocystic change, whereas 3% (1 of 36) was a fibroadenoma based on the us - dvar histology. subsequent surgical excision was performed in nine patients (mean 8.1 7.2 months after us - dvar, range 1 - 21 months) due to persistent or recurrent nipple discharge (n = 7, 78%), radiologic - pathologic discordance (n = 1, 11%) without nipple discharge, or an increase in the size of another solid nodule (n = 1, 11%). no malignancy was found in either the nine patients who underwent surgical excision or the 27 who underwent a follow - up us (table 1). us - dvar eliminated the nipple discharge in 69% (25 of 36) of the patients (fig. 1), the other 31% (11 of 36) remained symptomatic (fig. the mean follow - up duration was 25 months (range, 12 - 42). one (4%, 1 of 27) patient underwent a final follow - up at 12 months, nine patients (33%, 9 of 27) at 13 - 24 months, and 17 patients (63%, 17 of 27) at 25 - 42 months. at the initial visits after us - dvar, nipple discharge had disappeared in 81% (29 of 36) of patients. however, four of these 29 later experienced symptom recurrence (2 at 6 to 12 months and 2 at 19 months ; 3 intraductal papillomas and 1 fibrocystic change, by surgical excision). the characteristics of groups with eliminated and persistent nipple discharge are summarized in table 2. mean lesion sizes were 1.0 cm and 1.0 cm (p = 0.649), whereas the mean lesion distances from the nipple were 1.0 cm and 1.1 cm, respectively (p = 0.393). no difference was observed between these two groups in terms of bi - rads category (p = 1.000) the number of core samples (p = 0.101), radiologist experience (i.e., attending radiologists versus fellows) (p = 0.467), the presence of a residual lesion on follow - up us (p = 0.328), and histologic results by us - dvar (p = 0.792). of the four lesions with fibrocystic change that underwent surgical excision, one benign papilloma, two atypical papillomas, and one fibrocystic change were revealed (table 1). of the six lesions that showed a fibrocystic change and the one fibroadenoma, which was not excised, no case showed a persisting symptom or symptom recurrence over a mean follow - up of 31 months (range 23 - 42). of the 36 patients with abnormal nipple discharge, us - dvar eliminated nipple discharge in 25 (69%) at a mean follow up of 25 months. at the six month follow - ups, four of the 36 (11%) developed the symptom later, and three of these four patients were found to have intraductal papilloma at surgery. considering the 95 - 97% resolution rate reported in previous studies (10, 11), the rate found in the present study is poorer than expected. first, the key to eliminating nipple discharge is to correctly identify and remove the cause, and in the present study, target lesions within dilated ducts were traced to discharging nipple ducts by radial and antiradial us alone. (13), a certain number of lesions invisible to us, are visualized with a galactography. furthermore, dennis. (10) evaluated symptom causes by repeated ductographic us correlation, and found that this improved correct target lesion identification. in addition, us has been shown to be limited in terms of the detection of small intraductal lesions in peripheral ducts, especially when they are located at 3 cm or more from the nipple (13, 14). second, although we attempted to remove all evidence of intraductal lesions by us - dvar in patients that presented with discharge, there was a substantial probability of residual lesion not visible during procedure (8), which might cause recurring symptoms later. in our study, a follow - up us revealed residual lesions near the dvar site in 30% of patients with recurrent or persistent symptoms. furthermore, us - dvar was performed in the present study by one of eight fellows or three attending radiologists, and symptom recurrence was slightly lower in the group performed by the attending radiologists compared to the fellows (3 of 14 [21% ] versus 8 of 22 [36% ] ; p = 0.467), although the difference was not statistically significant. variability of their experience could have affected the lower resolution rate of us - dvar. in the present study, we also evaluated the factors affecting symptom resolution to identify a subset of patients that could be applied to the us - dvar. however, lesion size, location, bi - rads category, number of core samples, and the presence of a residual lesion by follow - up us were found to be unrelated to symptom resolution. in terms of histologic findings, 69% (25 of 36) of the lesions were papillomas, and the other 31% were fibrocystic change (n = 10) or fibroadenomas (n = 1). of the four cases of fibrocystic change with the remaining symptom, surgical excision revealed one benign papilloma and two atypical papillomas where the cause of discharge might have not been accurately targeted during us - dvar. however, earlier researches reported that 20% of nipple discharge cases were caused by fibrocystic changes or ductectasia (11, 13), and often appeared as intraductal echogenic lesions on us. in the present study, six cases with fibrocystic changes and one case with fibroadenomas in whom symptoms did not recur at the mean follow - up period of 31 months (range 23 - 42), were considered to have been accurately targeted during us - dvar. in terms of the diagnostic accuracy of us - dvar, no malignant lesion was found among the nine lesions treated surgically or among the 27 lesions followed - up. the five lesions with papilloma at biopsy histology were found to be benign papillomas in four cases and atypical papilloma in one case at surgery. recent studies have concluded that benign papillomas detected by core needle biopsy should be surgically excised, because 21 - 31% of the benign papillary lesions were upgraded to high - risk lesions or cancer at surgery (15, 16). however, it remains to be determined whether an us - dvar diagnosis of benign papilloma can obviate the need for surgical excision, which is beyond the scope of this study. first, we did not perform a consecutive case collection and include all symptomatic patients. third, follow - up period is not long enough to evaluate recurring symptoms or establish the benignity. however, to confirm the benignity of the lesion, a 24-month follow - up is required. fourth, we did not perform galactography to identify the cause of abnormal nipple discharge. small peripheral intraductal lesions, which are not visible on us, may cause residual symptoms. to establish the management of nipple discharge with intraductal solitary lesions on us, the proper selection of pure intraductal lesions based on galactography and prospective large group study results comparing surgical excision and vacuum - assisted removal of intraductal lesions would be needed. to conclude, us - guided directional vacuum - assisted removal of a benign intraductal mass eliminated nipple discharge in only 69% of patients and thus, this modality should not be considered as an alternative to surgical excision.	objectiveto evaluate whether the removal of an intraductal mass using an ultrasound (us)-guided directional vacuum - assisted device can eliminate symptoms in patients presenting with abnormal nipple discharge.materials and methodsbetween march 2004 and october 2006, 36 patients who presented with abnormal nipple discharge, underwent us - guided, 11-gauge vacuum - assisted biopsy for a benign intraductal single mass on us. the ability of the procedure to eliminate nipple discharge was evaluated by physical examination during follow - up us. lesion characteristics, biopsy variables, and histologic features were analyzed to identify factors affecting symptom resolution.resultsof the 36 lesions, 25 (69%) were intraductal papillomas, 10 (28%) were fibrocystic changes, and one (3%) was a fibroadenoma. the nipple discharge disappeared in 69% (25 of 36) of the women at a mean follow - up time of 25 months (range 12 - 42 month). there was no difference in the lesion characteristics, biopsy variables, and the histologic features between groups that eliminated the symptom compared those with persistent nipple discharge.conclusionus-guided directional vacuum - assisted removal of an intraductal mass appears to eliminate nipple discharge in only 69% of patients and thus, it should not be considered as an alternative to surgical excision.
the confirmed cases of hge analyzed in this study were identified through active and passive surveillance systems described elsewhere (19). informed consent for participation in the active tick - borne disease surveillance study was obtained from all participants or their parents or guardians, according to a protocol approved by the yale school of medicine human investigation committee. that committee approved a waiver of consent for this analysis. only cases detected in 19972000 in residents of the 12-town area around lyme, connecticut, a confirmed case was defined as illness in a patient who had a seroconversion or 4-fold change in antibody titer between acute- and convalescent - phase serum specimens (by indirect fluorescent antibody or enzyme - linked immunosorbent assay), a positive polymerase chain reaction assay with primer pairs directed to genomic sequences specific to hge, or detection of the specific 44-kda protein band by western blot analysis. a probable case was defined as an illness in a patient with a positive antibody titer from only a single serum sample or a 10. then the disease rate was calculated individually for each census block group on the basis of this larger number of cases and larger population. annualized incidence by census block group was calculated in arcview after the data were exported back into the gis. the technique of producing a smoothed map of disease rates allows for the display of data at a smaller geographic scale while preserving the stability of the estimated disease rates. spatial cluster analysis was performed on the confirmed cases of hge to test whether the cases were distributed randomly over space and, if not, to evaluate any identified spatial disease clusters for statistical significance (31). we applied the spatial scan statistic (31) to test the null hypothesis that the relative risk (rr) of hge was the same between any block group, or collection of block groups, and the remaining block groups. by scanning varied size areas for possible disease clusters without prior assumptions of cluster size or location satscan software, version 2.1 (28), designed specifically to implement this test, imposed a circular window on the map. this window moved over the area and centered on the centroid of each census block group. the area within the circular window varied in size from zero to a maximum radius, never including > 50% of the total population. the satscan software tested for possible clusters within the variable window around the centroid of each block group. cluster analysis was performed with the default maximum spatial cluster size of 10. then the disease rate was calculated individually for each census block group on the basis of this larger number of cases and larger population. annualized incidence by census block group was calculated in arcview after the data were exported back into the gis. the technique of producing a smoothed map of disease rates allows for the display of data at a smaller geographic scale while preserving the stability of the estimated disease rates. spatial cluster analysis was performed on the confirmed cases of hge to test whether the cases were distributed randomly over space and, if not, to evaluate any identified spatial disease clusters for statistical significance (31). we applied the spatial scan statistic (31) to test the null hypothesis that the relative risk (rr) of hge was the same between any block group, or collection of block groups, and the remaining block groups. by scanning varied size areas for possible disease clusters without prior assumptions of cluster size or location satscan software, version 2.1 (28), designed specifically to implement this test, imposed a circular window on the map. this window moved over the area and centered on the centroid of each census block group. the area within the circular window varied in size from zero to a maximum radius, never including > 50% of the total population. the satscan software tested for possible clusters within the variable window around the centroid of each block group. cluster analysis was performed with the default maximum spatial cluster size of < 50% of the population and again with a smaller maximum cluster size of < 25% to look for possible subclusters. for each window of varying position and size, the software tested the risk of hge within and outside the window, with the null hypothesis of equal risk. an additional cluster analysis was conducted by using both confirmed and probable hge cases to address potential inclusion biases in the observed clustering of cases. these biases may have arisen because active surveillance cases were more likely to have provided both acute- and convalescent - phase samples than were cases detected through passive surveillance, and thus, had greater chance of being classified as confirmed cases and being included in analysis. identical methods to those described above were used to perform the cluster analysis on the combined confirmed and probable cases. two hundred forty - five cases of hge were identified through the active and passive surveillance systems in 19972000. a total of 136 confirmed cases of hge were identified ; 128 (94%) of these were geocoded to points in an arcview theme (figure 2a). addresses that were not geocoded consisted of two incomplete ones (street names with no number) and six post office boxes from chester, essex, haddam, lyme, madison, and westbrook. a. confirmed human granulocytic ehrlichiosis (hge) cases identified through active and passive surveillance systems, 19972000 ; b. raw annualized incidence of confirmed hge cases by town, 19972000 ; c. raw annualized incidence of confirmed hge cases by census block group ; d. smoothed annualized incidence of confirmed hge cases by census block group. annualized incidence rates for 19972000 were calculated by town and census block group by using 1990 census data to show the crude distribution of hge in the 12-town area (figure 2b, c). rates by census block group ranged from 0/100,000 to 187/100,000 and demonstrated a high degree of random variation because of the small population size and low case numbers. smoothing provided a clearer picture of the areas of increased risk on a smaller scale than by town. a filter number of 10 provided the most appropriate map of smoothed incidence rates (figure 2d). this filter number decreased random variation and showed an increased risk of contracting hge around the mouth of the connecticut river with risk decreasing to the north and west. using the maximum spatial cluster size of < 50% of the total population, the spatial cluster analysis identified a single cluster that included all census block groups in the towns of lyme, old saybrook, chester, essex, deep river, and westbrook, all but one in the town of old lyme, and one from the town of clinton (figure 3a). the overall rr within the cluster was 1.8, with an observed number of cases of 106 compared with 59 expected cases. this elevated risk within a nonrandom pattern of disease distribution was significant (p=0.001). a. single identified cluster of human granulocytic ehrlichiosis (hge) cases within the 12-town area (maximum cluster size 50% total population), relative risk (rr)=1.8, p=0.001 ; b. two identified clusters of hge cases within the 12-town area (maximum cluster size 25% total population) : primary cluster : rr=2.6, p=0.001, secondary cluster : rr=2.6, p=0.16. to investigate the possibility of smaller clusters, the same analysis was performed with a maximum spatial cluster size of < 25% of the total population. two clusters were identified, one including lyme and old lyme as well as parts of essex, old saybrook, and deep river (figure 3b). this cluster contained 18.2% of the total population and had an overall rr of 2.6 (p=0.001), with 61 cases observed compared with an expected 23 cases. this cluster contained 4.2% of the total population and had an overall rr of 2.6 (p=0.16), with 14 cases observed compared with 5 cases expected. while the primary cluster identified in this analysis was significant and showed a higher overall rr, the larger cluster from the first analysis, as a result of the lack of preselection bias, better represented the areas of increased risk for infection on the basis of the spatial distribution of hge in the 12-town area. cluster analyses were also performed on confirmed and probable case data. by using the < 50% maximal cluster size in the subcluster analysis that used the < 25% cluster size, both subclusters identified with confirmed cases only were found to be statistically significant (p<0.005). the primary subcluster was geographically circumscribed when confirmed and probable cases were included in the analysis. using a gis and spatial statistics, we investigated the spatial distribution of confirmed cases of hge and identified areas of increased risk within an area highly endemic for tick - borne diseases. such diseases have become recognized as serious health threats in the northeast united states in the last 20 years because of increasing prevalence and heightened detection. areas characterized by low residential density and a landscape of recently reforested deciduous forest are strongly associated with the risk for lyme disease (30,32,33). areas of high lyme disease risk have been shown to also have an increased risk for hge (19). furthermore, the analysis demonstrated that combining thorough surveillance information with spatial analysis techniques can increase understanding of the epidemiology of hge within a highly disease - endemic area. the next step, to investigate the underlying causes of increased risk in the identified areas, will be analysis of landscape attributes and identification of the environmental variables characteristic of high - risk areas. the spatial statistics analyses clearly yielded a nonrandom distribution of hge within the 12-town area. spatial filtering (smoothing) identified areas of increased risk centered around the mouth of the connecticut river, primarily on the eastern side of the river, in the towns of lyme and old lyme. increased likelihood of disease was seen on the western side of the river but was not as consistently high as the risk observed in lyme and old lyme. spatial cluster analysis identified a statistically significant cluster (rr=1.8, p=0.001) in the same area, around the mouth of the connecticut river, including the towns of chester, deep river, essex, lyme, old lyme, old saybrook, and westbrook. one census block group in southeastern old lyme was not included in the cluster, and one block group in clinton was included. this cluster analysis was performed by using the default maximum spatial cluster size of < 50% of the total population. using this default method minimizes pre - selection bias of cluster size. however, to investigate the possibility of subclusters, additional cluster analysis based on a maximum spatial cluster size of < 25% of the total population identified two subclusters, one significant (rr=2.6, p=0.001) and the second not significant (rr=2.6, p=0.19). the decrease in risk for hge infection as one moves away from the coast is consistent with the results of nicholson and mather, who described a decreasing lyme disease risk with increasing latitude in rhode island (34). gathering and including vector population data (including population density, distribution, and infection prevalence rates) and environmental variables in the risk analysis of hge in the 12-town area may provide a more comprehensive view of the disease risk. the relationship between lyme disease, i. scapularis vectors, and landscape characteristics has been studied from remotely sensed and field - gathered data (3537), but it is unknown whether these relationships can be applied to other tick - borne diseases, including hge. increased lyme disease risk has been well correlated with increased tick abundance and prevalence of infected ticks (34,35,38). the spatial distribution of lyme disease rates is correlated with widespread tick populations and pathogen prevalence (25). environmental risk factors and landscape characteristics associated with lyme disease have been identified (22,3537). using techniques similar to those used for lyme disease, combined with the results of this study further, discernment of the aspects of the natural history of hge that are not understood, especially pertaining to the reservoir host, may supply additional information that can be used to further refine areas of hge risk. while similar numbers of specimens were submitted for hge testing to both the active and passive surveillance systems, the low rate of convalescent - phase specimen collection and the application of only one diagnostic test in passive surveillance resulted in fewer cases from passive surveillance being confirmed and included in the current analysis. persons who live at the edges of the 12-town area may have been more likely to visit practitioners outside the active surveillance area. these case - patients would have been identified through passive surveillance but would have been less likely to be confirmed. while the practices participating in active surveillance were located throughout the 12-town area and include one practice outside that area, the lower rate of confirmed cases in the passive system may have biased the results toward the center of the surveillance area. however, the similar results obtained from the spatial statistics analysis that used both confirmed and probable cases suggest that this error may not have played a large role in the observed patterns of disease. because of variations in testing throughout the 4 years of surveillance, analysis for temporal clusters was not possible. retrospective testing of banked samples from previous surveillance years or continuing accumulation of surveillance data in years to come will be needed to investigate the temporal as well as spatial spread of hge within the 12-town area. temporal trends, combined with time series analysis of remotely sensed land cover and land - use data, may provide indications of future areas at increased risk for hge. concurrent analysis of the spatial and temporal distributions of other i. scapularis borne diseases in this area, including lyme disease and babesiosis, may clarify the similarities and differences in risk among these common vector - borne infections. our study was based on the assumption that people acquire infection with the agent of hge peridomestically, or near their homes. falco and fish (30) demonstrated that most cases of lyme disease were acquired peridomestically, but no studies have investigated whether hge infections are similarly acquired. while the life cycle similarities of these two pathogens support the assumption that hge transmission dynamics are similar to those of lyme disease, additional research is needed to test this hypothesis. this area of active surveillance was identified previously by its high rates of lyme disease, and the towns to the east were excluded because at the time the study was initiated (1997) those towns had lower rates of lyme disease compared with the 12 study towns. however, in this analysis, understanding of the spatial distribution of hge would be enhanced if the towns to the east of the current study area were also included in the active surveillance, given the high rates of hge in lyme and old lyme. stemming from this analysis, the towns to the east of the original study area (salem and east lyme) were added to the active surveillance area in 2002. the tools and methods described in this study can identify areas where increased surveillance is recommended. human behavior is a strong predictor of tick - borne disease risk, including how people move in their environment, their outdoor activities, and the individual protection they use to prevent tick bites. reforestation in areas previously used for agriculture results in more favorable conditions for tick and reservoir hosts, while the trend towards residential preferences in well - shaded suburban and rural areas exacerbates the tick - human interactions. change in human behavior concurrent with an ecologic transition further increases and alters tick - borne disease risk. local weather variations and the periodicity of weather patterns also play a role in tick - borne disease risk. the combination of these factors results in a high variability of risk even within an area known to be hyperendemic for tick - borne diseases. on the basis of data on peridomestic lyme disease infections (30), prevention strategies are recommended that focus on persons risk at home. in an area in which tick - borne diseases are highly endemic, aiming prevention strategies at areas of highest risk can potentially increase the program s effectiveness. persons at highest risk should be informed of that risk and of the possibilities for risk reduction. funds spent on programs might be better spent on areas where cost - effectiveness can be maximized. at this time, practical prevention advice to prevent tick - borne disease in highly disease - endemic areas is elusive. the tools described in this article, gis and spatial statistics, provide an opportunity to clarify and quantify the health burden from tick - borne disease within a highly endemic area and a foundation to pursue further investigation into the environmental factors resulting in increased disease burden. to implement specific and geographically appropriate risk - reduction programs, the use of such spatial analysis tools should become integral components in the epidemiologic description and risk assessment of tick - borne diseases.	geographic information systems combined with methods of spatial analysis provide powerful new tools for understanding the epidemiology of diseases and for improving disease prevention and control. in this study, the spatial distribution of a newly recognized tick - borne disease, human granulocytic ehrlichiosis (hge), was investigated for nonrandom patterns and clusters in an area known to be endemic for tick - borne diseases. analysis of confirmed cases of hge identified in 19972000 in a 12-town area around lyme, connecticut, showed that hge infections are not distributed randomly. smoothed hge incidence was higher around the mouth of the connecticut river and lower to the north and west. cluster analysis identified one area of increased hge risk (relative risk=1.8, p=0.001). this study demonstrates the utility of geographic information systems and spatial analysis to clarify the epidemiology of hge.
eccrine angiomatous hamartoma, a very rare benign hamartoma, usually presents at birth or during early infancy as solitary nodule or plaque generally over extremities. eccrine angiomatous hamartoma (eah) is characterized by benign proliferation of eccrine glands associated with capillary in close approximation with surrounding dilated capillaries. in most of the cases they are solitary and they may present as angiomatous nodules to erythematous - purpuric plaques with hypertrichosis, localized hyperhidrosis or occasional pain. eighty percent of the cases are reported over the extremities, with a predilection for palms and soles. we report a rare case of an eah on the face below the right lower eyelid. a 20-year - old female patient presented to us with complaints of solitary small red swelling on the face below the right lower eyelid, gradually increasing in size over the past 8 years. occasionally, she noticed throbbing pain and sweating over the lesion, which aggravated during physical exertion or emotional stress. she did not give any history of birth injury or trauma or any other symptoms suggestive of systemic involvement. cutaneous examination revealed solitary, irregular, erythematous, soft, slightly elevated plaque of about 3 2 cm in size having a smooth, moist surface just below the right lower eyelid [figures 1 and 2 ]. on palpation, beaded drops of sweat were observed on the surface of the lesion on mild stroking and physical exertion. no pulsation or bruit was felt over the lesion. based on the history and clinical features, a possibility of eah was considered. this was confirmed histopathologically by the presence of circumscribed foci of thin walled capillary channels surrounding large coils of increased eccrine structures in the dermis [figures 3 and 4 ]. color doppler study of the lesion, in addition, revealed a mild to moderate intensity vascularization in the deep dermis. above clinicopathological features supported our diagnosis of eah. solitary, irregular, erythematous plaque with beaded drops of sweat below the right lower eyelid distant view of the same increased number of eccrine coils with surrounding dilated thin walled capillaries in the dermis (h and e, 100) dilated thin walled capillaries in close approximation with eccrine coils (h and e, 400) eah usually appears at birth or during childhood, although in our case it appeared during adolescence. eah presenting with a blue - colored, ill - defined swelling over the dorsum of left hand in a 26-year - old female has recently been reported. generally, they enlarge very slowly ; more rapid growth has been described during pregnancy and adolescence. the bluish - red or frankly angiomatous smooth nodules or plaques are usually solitary, although multiple lesions have also been described. an unusual presentation of a verrucous lesion over the gluteal region in an elderly person has been reported. lesions mostly favor sites of increased number of eccrine glands like extremities, but there are case reports of occurrence over face and trunk. the lesions are mostly asymptomatic although pain spontaneous or on pressure, may be encountered and might be due to involvement of nerve fibers. they often have overlying hyperhidrosis, as in our case, probably due to stimulation of eccrine glands by the local temperature generated within the angioma. lesional hypertrichosis and the association of eah with cowden 's syndrome, neurofibromatosis, etc. histopathologically, the large secretory eccrine coils are surrounded by thin walled blood vessels and lymphatic channels in the deep dermis with occasional invasion of adipose tissue. the presence of less number of capillary channels in our case helped us to differentiate it from sudoriparous angioma where numerous such vessels are seen. when there are nonspecific clinical findings, common differentials like nevus flammeus, glomus tumor, smooth muscle hamartoma, blue rubber bleb nevus syndrome, angiokeratoma, and macular telangiectatic mastocytosis can readily be differentiated by histopathology. color doppler study, as was done in our case, is not diagnostic but along with magnetic resonance imaging (mri) can delineate the extent of the lesion. immunohistochemical studies to demonstrate s-100 protein, carcinoembryonic antigen (cea) and antifactor viii - related antigens can be helpful, but were not done for institutional nonavailability. although botulinum toxin, lasers, and sclerosants are now being used as treatment options, we referred the patient to plastic surgery outpatient department (opd) for excision and repair. the unusual location of eah in our case and the paucity of its mention in the literature prompted us to report this case.	eccrine angiomatous hamartoma (eah) is a very rare benign neoplastic condition characterized by hamartomatous proliferation of eccrine glands and accompanying blood vessels and lymphatics. these lesions are more often present at birth or appear during early infancy and childhood and present as solitary nodule or plaque with occasional pain and sweating. they are generally present on the extremities, mostly the palms and soles. we report here a case of eah in a 20-year - old female who presented with a solitary angiomatous plaque close to the lower eyelid of the right eye with occasional pain and sweating for its rarity.
coccidioidomycosis is an endemic disease of arid and semi - arid regions of northern and southern america caused by coccidioides posadasii and coccidioides immitis, which can only be separated by dna sequencing. travellers to endemic areas are at risk of developing coccidioidomycoses through inhalation of airborne conidia. a history of travel to endemic areas as well as a high index of suspicion is imperative for timely and accurate diagnosis. in order to differentiate this infection from other systemic mycoses, diagnostic spherules on june 2011, a 41-year - old, otherwise healthy, turkish man travelled totexas for 2 months where he spent his spare time in walking through the forests, and then took 1 month trip by car to grand canyon, las vegas and san francisco during which he fasted in accordance with ramadan, but felt unusually weak and lost weight. on his 80th day in the usa, he felt very ill near niagara falls and laid down on the grass, suffered from chest pain and severe sweating. ten days later, he was admitted to an hospital in texas with coughing, fever, vomiting and loss of appetite (day 0). he was diagnosed with pneumonia and given 400 mg / day moxifloxacin. the next day he returned to batman, turkey and attended university hospital with nausea, vomiting, weakness, sweating, productive coughing and fever (day + 1). serologic assays for igm levels of cmv, herpes type ii, rubella, parvovirus b19, and ebv ebna were all negative. mg / l and increased to 42.5 mg / l (day + 4). sputum, throat, urine cultures and haemoculture as well as stains for acid - fast bacteria and mycobacterial cultures were also negative while moxifloxacin was continued. chest x - rays were normal but chest ct scan revealed a 18 mm nodule on the left upper lobe with suspected histoplasmosis. due to persistent fever up to 40 c and night sweat, patient was switched to imipenem and clarithromycin. he refused pulmonary biopsy and left hospital (day + 11). following the development of a cutaneous lesion of about 1 cm diameter on his neck (day + 38) mg / l and increased to 22.5 mg / l (day + 42). cytology and stains for acid - fast bacteria, as well as fungal and mycobacterial microscopy and cultures of two subsequent bronchoalveolar lavage (bal) samples and a lymph node biopsy specimen were all negative. for his neck lesion, amoxicillin - clavulanic acid, 1000 mg, t.i.d. and a topical fusidic acid cream were started and then he was discharged (day + 46). he attended batman state hospital with severe chest pain, lack of appetite, the persistent lesion on the neck and an additional small papule in the left palm (day + 47). an abdominal ct scan revealed ascites, and the cytology of aspirated peritoneal fluid showed numerous lymphocytes, neutrophils, eosinophils and histiocytes. he was admitted to another hospital and a chest ct scan revealed a nodule of about 18 mm on the left upper lobe and oedema (day + 54). pathological examination of a punch biopsy of his neck lesion revealed pseudoepitheliomatous hyperplasia - like verrucose proliferation and neutrophils in the epidermis. a dense infiltrate of neutrophils, lymphocytes, plasma cells, histiocytes and eosinophils invading epithelia were present in the dermis. pas - positive, round, thick - walled fungal elements were observed in the cytoplasm of the giant cells in neutrophilic abscesses. was prescripted for 6 months. when he was admitted to a university hospital in ankara (day + 164), crp level was 1.74 a chest ct scan revealed additional lymph nodes with an infectious appearance in the left lung. a chest ct scan made in batman (day + 308) showed micronodules in the left lung and left hilar lymphadenopathy of a tree - in - bud appearance with some regression when compared with the ct scans of day + 2. his palm lesion was healed but lung lesions were sustained after having been treated with itz 200 mg / day for 6 months. on follow - up visit (day + 363), because of his persisted neck lesion despite itz treatment, the patient was transferred to a research hospital in istanbul for further evaluation for suspected blastomycosis and its treatment with amphotericin b (amb). when he admitted to the dermatology department (day + 368), he was concious, cooperative and oriented with normal physical examination. he had an erythematous, slightly elevated atrophic lesion on his neck of about 4.3 cm and brightly coloured peripheric minor papules. serology for hbsag, anti hbs, anti hcv and anti - hiv were all negative. neck ultrasonography revealed several lymph nodes having a reactive appearance, smaller than 12 mm in diameter. abdominal magnetic resonance imaging (mri), cranial ct scans and a whole body bone scintigram were normal (day + 373). two subsequent punch biopsies of 5 mm were carried out (days + 368 and + 379) and sent to cerrahpasa medical faculty (cmf), deep mycoses laboratory (dml), with clinical suspicion of north american blastomycosis and the patient was discharged at his own request. when the patient was seen on day+465, his neck lesion was healed and his physical examination and routine clinical laboratory data were unremarkable. on the chest ct scan parenchymal miliary lesions were resolved, lesions defined on the left lung and mediastinal windows showed no significant differences with those of the previous years, but there was a significant reduction of the diameter of the left axillary and mediastinal lymph nodes. nodullary consolidation documented on the left lung altered to granuloma - like configuration (fig. a previous sinoorbital mri was reviewed but revealed no abnormality except right concha hypertrophy and deviation of nasal septum. a t2-weighted mri of the brain showed a few small gliotic foci on the basal surface of the frontal lobe as well as tubinate hypertrophy of the nasal mucosa that blocked the nasal passage. csf direct microscopy and cultures of the csf samples were negative. based on radioscopic data, antifungal treatment was continued and a 3-month follow - up appointment scheduled. when seen on day + 564, authors speculate that the presence of turbinate hypertrophy of the nasal mucosa facilitated the incubation of the pathogen. two subsequent biopsy specimens from neck nodules were submitted to cmf, dml for mycological evaluation (days + 368 and + 379), with a clinical suspicion of north american blastomycosis. direct microscopical examination was carried out using gram, ehrlich ziehl nielsen, giemsa, and methylene blue stained slide preparations of the imprinted tissue samples. specimens were inoculated by embedding onto sabouraud dextrose agar (sda), chocolate agar with sheep blood (5%) plates as primary culture media and incubated at 30 c and 37 c. microscopic morphology of the isolate was examined by staining with lactophenol cotton blue. pas stained histopathological preparations from day + 54 were also requested and examined for microorganisms. the isolate was identified by sequencing of the internal transcribed spacers (its) 1 and 2, and 5.8s domains of rdna. they were compared with the sequences of type and reference strains deposited in genbank. in vitro susceptibility of the isolate against amb, itz, fluconazole (flz), voriconazole (vrz) and posaconazole (psz) tests were carried out according to the manufacturer 's instructions. direct microscopical examination of giemsa stained imprinted tissue slides of biopsy materials revealed the presence of a few, thick - walled, round fungal cells, some with a granular protoplasm (fig. 2), small chains of oval to round cells and a few sporangium - like, large, thick walled, round, dense or empty cells of between 20 and 40 m in size (fig. histopathological sections of the tissue biopsy carried out on day + 54 were sent for mycological re - examination and the organism appeared in tissue in double - encapsulated, empty spherical form. after 45 days incubation, cultures on sda at 30 c and 37 c initially grew glabrous colonies just around the tissue biopsy specimens. fungal morphology seen directly in patient material and glabrous colonial characteristics of the early culture raised the possibility of the presence of a prototheca sp, compatible with his rest near a waterfall. however, after 23 weeks of incubation the colonies were white, floccose, with a tan reverse. microscopic examination of lactophenol cotton blue stained slide preparations made at the early mould phase revealed smooth, hyaline, septate, sterile hyphal structures with some spirals similar to those of dermatophytes. subcultures were submitted to jg at reus for molecular confirmation and the strain was identified as c. immitis by sequencing the rdna its region showing a 99% sequence similarity with the reference strains cbs 113852, cbs 113856 and cbs 113857 (accesion numbers ef186788, ef186789 and ef186790, respectively). the isolate is stored in the collection of university of the centraal bureau voor schimmelcultures, utrecht, the netherlands, at cbs 136242 (fmr 12608). mics (g / ml) were 32 for amb, > 256 for flz, > 32 for itz, 0.64 for vrz, 0.047 for psz. regarding the results of direct microscopical examination of tissue preparations stained by pas and giemsa methods and early glabrous colony morphology, prothothecosis, chlorellosis, coccidioidomycosis or skin dermatophytosis were all initially suspected. culture slides were prepared twice, once at the early mould growth phase and again at maturity. early preparations revealed only thin, hyaline, septate hyphae without conidia, resembling dermatophyte hyphae but mature colonies revealed the presence of typical barrel - shaped arthroconidia. all patient 's cultures were immediately destroyed by autoclaving them together with biological indicators, then placing them in boxes set aside for biohazardous waste. environmental controls were carried out periodically in both the mycology laboratory and inside the incubators. petri dishes containing sda were opened for 3 h and then incubated at 30 c and 37 c. the same fungus grew in cultures, which were autoclaved immediately. all surfaces in the laboratory were disinfected using sodiohyphochlorite (10%) and the cleaning staff wore n95 respirators, disposable protective laboratory coatings, gloves and hair covers. air was decontaminated with pulverising hydrogen peroxide (15 ml / m), allowing overnight contact. the dml was kept closed, signs were posted to alert personnel that this may be a contaminated area that should not be entered and work was transferred to another room. the infectious disease committee of cmf was officially informed immediately. environmental controls and decontamination using hydrogen peroxide were carried out four more times. air sampling and cultures are still currently being continued as a precaution despite culture results having been negative on the third test. antifungal prophylaxis with itz was given at a therapeutic dose to laboratory workers for 3 months and sinonasal and chest mris were taken at the end of the prophylaxis, which showed no abnormalities. two subsequent biopsy specimens from neck nodules were submitted to cmf, dml for mycological evaluation (days + 368 and + 379), with a clinical suspicion of north american blastomycosis. direct microscopical examination was carried out using gram, ehrlich ziehl nielsen, giemsa, and methylene blue stained slide preparations of the imprinted tissue samples. specimens were inoculated by embedding onto sabouraud dextrose agar (sda), chocolate agar with sheep blood (5%) plates as primary culture media and incubated at 30 c and 37 c. microscopic morphology of the isolate was examined by staining with lactophenol cotton blue. pas stained histopathological preparations from day + 54 were also requested and examined for microorganisms. the isolate was identified by sequencing of the internal transcribed spacers (its) 1 and 2, and 5.8s domains of rdna. they were compared with the sequences of type and reference strains deposited in genbank. in vitro susceptibility of the isolate against amb, itz, fluconazole (flz), voriconazole (vrz) and posaconazole (psz) direct microscopical examination of giemsa stained imprinted tissue slides of biopsy materials revealed the presence of a few, thick - walled, round fungal cells, some with a granular protoplasm (fig. 2), small chains of oval to round cells and a few sporangium - like, large, thick walled, round, dense or empty cells of between 20 and 40 m in size (fig. histopathological sections of the tissue biopsy carried out on day + 54 were sent for mycological re - examination and the organism appeared in tissue in double - encapsulated, empty spherical form. after 45 days incubation, cultures on sda at 30 c and 37 c initially grew glabrous colonies just around the tissue biopsy specimens. fungal morphology seen directly in patient material and glabrous colonial characteristics of the early culture raised the possibility of the presence of a prototheca sp, compatible with his rest near a waterfall. however, after 23 weeks of incubation the colonies were white, floccose, with a tan reverse. microscopic examination of lactophenol cotton blue stained slide preparations made at the early mould phase revealed smooth, hyaline, septate, sterile hyphal structures with some spirals similar to those of dermatophytes. when colonies had matured, for about 23 weeks (fig. subcultures were submitted to jg at reus for molecular confirmation and the strain was identified as c. immitis by sequencing the rdna its region showing a 99% sequence similarity with the reference strains cbs 113852, cbs 113856 and cbs 113857 (accesion numbers ef186788, ef186789 and ef186790, respectively). the isolate is stored in the collection of university of the centraal bureau voor schimmelcultures, utrecht, the netherlands, at cbs 136242 (fmr 12608). mics (g / ml) were 32 for amb, > 256 for flz, > 32 for itz, 0.64 for vrz, 0.047 for psz. regarding the results of direct microscopical examination of tissue preparations stained by pas and giemsa methods and early glabrous colony morphology, prothothecosis, chlorellosis, coccidioidomycosis or skin dermatophytosis were all initially suspected. culture slides were prepared twice, once at the early mould growth phase and again at maturity. early preparations revealed only thin, hyaline, septate hyphae without conidia, resembling dermatophyte hyphae but mature colonies revealed the presence of typical barrel - shaped arthroconidia. all patient 's cultures were immediately destroyed by autoclaving them together with biological indicators, then placing them in boxes set aside for biohazardous waste. environmental controls were carried out periodically in both the mycology laboratory and inside the incubators. petri dishes containing sda were opened for 3 h and then incubated at 30 c and 37 c. the same fungus grew in cultures, which were autoclaved immediately. all surfaces in the laboratory were disinfected using sodiohyphochlorite (10%) and the cleaning staff wore n95 respirators, disposable protective laboratory coatings, gloves and hair covers. air was decontaminated with pulverising hydrogen peroxide (15 ml / m), allowing overnight contact. the dml was kept closed, signs were posted to alert personnel that this may be a contaminated area that should not be entered and work was transferred to another room. the infectious disease committee of cmf was officially informed immediately. environmental controls and decontamination using hydrogen peroxide air sampling and cultures are still currently being continued as a precaution despite culture results having been negative on the third test. antifungal prophylaxis with itz was given at a therapeutic dose to laboratory workers for 3 months and sinonasal and chest mris were taken at the end of the prophylaxis, which showed no abnormalities. although travel history is important in diagnosing endemic mycosis particularly in non endemic countries, it is not alway easy. in the present case, the patient was first diagnosed as north american blastomycosis in several separate hospitals based on his travel story and clinical findings dispite negative culture, during nearly 1 year course. diagnosis of coccidioidomycosis is usually determined based on the presence of endospores however microscopical findings are not always typical. environmental algae prototheca and chlorella spp. can be isolated from grass, soil and water and malnutrition is listed among risk factors of algae infection as was in this patient. the two subsequent biopsy specimens revealed randomly distributed atypical thick - walled large yeast - like cells that were not indicative of either coccidioides sp. or an algae infection. multinucleated endosporulating sporangium - like, small, immature spherules seen in giemsa and pas stained tissue preparations, double - encapsulated, empty spherical form seen in histopathological sections and initially glabrous colony morphology on sda lead us to suspect both algal infection and coccidioidomycosis. misleading and unusual immature forms of c. immitis have previously been reported in vivo [25 ]. undiagnostic, thin, hyaline hyphae seen in preparations of this glabrous colony pointed to a possible dermatophyte isolation, albeit from two separate biopsy specimens. although single - celled, barrel - shaped arthroconidia were expected to occur in 47 days on sda at 25 c, the late maturation of the isolates might probably be due to the partial inhibition of the patient 's long - term antifungal treatment before biopsy specimens were obtained and submitted to dml. a positive serological test may have diagnostic value for acute coccidioidomycosis in patients from nonendemic countries, particularly in relation with a travel history, but negative tests should not be used to rule out the disease, and serological tests for c. immitis are uncommon in europe and turkey. they applied both histoplasmin and coccidioidin to 66 patients with pulmonary lesions and 11 of them were positive for both antigens and two of them positive for coccidioidin. today 's changes in travelling behaviour may increasingly cause people to encounter infections with organisms that are uncommon in their own countries. because of the low incidence of coccidioidomycosis on europe, diagnosis of the disease is not well known in practice. we found 19 cases of coccidioidomycosis imported into europe as outlined in table 1 [725 ]. in the data given by the european confederation of medical mycology (ecmm) working group of the survey of coccidioidomycosis in europe, 36 cases were reported from nine countries but limited or no data were provided (http://www.ecmm.eu/node/193). the first report in turkey found spherule - like forms in skin biopsy specimens of facial erythematous papulo - pustular lesions presented by a 53-year - old woman gardener. the strain was inoculated to mice and spherules were observed in visceral organs, although the isolate was not submitted to a reference centre for confirmation. austen. reported a patient from the netherlands who travelled to california in 2008. two weeks after her visit, she travelled to turkey, where she was admitted to an intensive care unit with severe community - acquired pneumonia, but no pathogen was identified. approximately 40% of infected people are asymptomatic or develop flu - like symptoms. in rare instances, individuals develop severe lung disease (e.g., cavitary pneumonia) and in 256) and itz (> 32). since no defined susceptibility breakpoints exist for both flz and itz activity against coccidioides spp., and comparable mic values of the wild type isolate were not available, and the patient 's response to itz gradually decreased, facing such a high mic value may suggest possible acquired resistance to both azoles occured during long term therapy. voriconazole and psz which were appearently more active azoles in vitro were reported as options for salvage treatment of refractory coccidioidomycosis in patients who were unresponsive to the first line antifungal agents. when seen on day + 564, actually, the patient is free of symptoms. all laboratory staff had to have an antifungal prophylaxis for 3 months against the risk of presence of mature disarticulated conidia in the air, although the benefits of such a prophylactic approach have not been proven. laboratory - associated coccidioidomycosis is a documented hazard of working with sporulating cultures of coccidioides spp. attack rates for laboratory exposure is higher than for ambient exposure when large numbers of conidia are inhaled. risk of respiratory infection from exposure to infected tissue or aerosols of infected secretions is low. therefore, biosafety level 3 practices, containment equipment, and facilities were recommended for manupulating sporulating cultures of coccidioides to avoid inhalation of infectious arthroconidia from cultures of the mould form. in conclusion, with increase in international travels, coccidioidomycosis is now emerging as an important infection outside its previously known endemic areas. in non - endemic countries, in spite of the patient 's prior travel history, coccidioidomycosis may not be easy to diagnose if there has been previous empirical antifungal treatment and/or atypical strain morphology. laboratory personnel may be at risk of exposure to fungal cells while working with unexpected fungi during incubation and identification. furthermore, the lack of appropriate biosafety level 3 conditions in laboratories in european countries is a significant risk factor for exposure to coccidioides.	coccidioidomycosis caused by coccidioides immitis or coccidioides posadasii is endemic in arid climate zones in america, travel - related cases have been reported. we report the first documented case of coccidioidomycosis in turkey, overviewing reported cases in europe and underlying difficulties of differential diagnosis outside endemic regions. the patient was an otherwise healthy 41-year - old man who travelled endemic areas. laboratory diagnosis was based on direct microscopy of two subsequent subcutaneous biopsy specimens and culture and confirmed molecularly. laboratory personnel should become aware that biosafety level-3 organisms may become more frequent and widespread.
leptospirosis is a spirochetal disease, which does not classically present as weil syndrome which is rare, but rather as flu - like illness. pulmonary involvement is present in about 20% to 70% of cases and varies in severity. the leptospirosis pulmonary hemorrhage syndrome (lphs) has been increasingly recognized as a severe manifestation of the disease with mortality rates surpassing 50%. a previously healthy 32-year - old man started to develop flu - like symptoms 1 day after returning from a caribbean cruise trip, which stopped in jamaica, mexico, and the cayman islands. his symptoms persisted, and he presented to the hospital after 10 days of fever, chills, nausea, vomiting, myalgia, nonproductive cough, and worsening dyspnea. laboratory workup showed neutrophil - predominant (62%) leukocytosis (24.6 10/l), thrombocytopenia (78 10/l), creatinine level of 5.29 mg / dl, aspartate transaminase of 736 u / l, alanine transaminase of 495 u / l, and markedly elevated levels of direct bilirubin (17.8 mg / dl) and creatine kinase (2426 u / l). he was initially admitted to the regular floor, but 1 day after admission, he developed worsening multiorgan dysfunction and episodic hypotension requiring intensive care unit admission. after 36 h of arrival, the patient developed massive hemoptysis and acute hypoxemic respiratory failure, leading to acute respiratory distress syndrome. he received hemodynamic support with norepinephrine and vasopressin, mechanical ventilation, and renal replacement therapy. there was a high clinical suspicion for severe leptospirosis, based on his history of exposures, and the clinical presentation of liver failure, pulmonary hemorrhage, and neutrophil - predominant leukocytosis in a relevant time frame. leptospirosis indirect hemagglutination assay eventually yielded positive results, and the patient completed a 14-day course of doxycycline and 7 days of ceftriaxone. repeat cxr revealed extensive bilateral pulmonary infiltrates, pao2/fio2 ratio (p / f) remained below 30 mm hg, and plateau pressures remained above 40 cm h2o despite optimized ventilator support, deep sedation, and paralysis. at that point, the decision was made to start veno - venous extracorporeal membrane oxygenation (vv - ecmo) to facilitate gas exchange. patient was cannulated in a femoral - femoral approach, and vv - ecmo was introduced without complications. oxygen saturation prior to vv - ecmo initiation ranged from 74% to 80%, improving to 98% to 100% within minutes after ecmo initiation. consequently, p / f ratio continued to improve dramatically, allowing for a lung - protective ventilation strategy. hemoptysis persisted during vv - ecmo despite an adjusted heparin protocol with a target activated clotting time (act) range of 160 to 180 seconds (standard ecmo regimen dictates an act range : 180 - 220 seconds), requiring the use of a significant amount of blood products. to stop and prevent further alveolar hemorrhage, and based on previous studies on ecmo patients, initiation of continuous aminocaproic acid infusion at 20 ml / h was tried after about 13 days on ecmo for a duration of 6 days, which presumably resulted in improved lung aeration and significantly decreased transfusion requirements. due to persistent thrombocytopenia, he required multiple platelet transfusions to maintain a count of about 60 to 120 10/l. the last unit of platelets was given about 15 days after presentation, around the same time of ecmo discontinuation, which coincides with his clinical improvement. the patient was extubated and discharged home to continue with outpatient rehabilitation after a 40-day hospitalization. he achieved a complete recovery and had evidence of normal pulmonary function tests at 6-month follow - up. the use of ecmo has been increasingly recognized as an alternative for cases in which hypoxemia persists despite the use of high fio2 and positive end - expiratory pressure (peep). using an extracorporeal circuit for gas exchange, ecmo allows a ventilation strategy with lower tidal volumes, fio2, and peep, which in turn decreases the rate of ventilator - associated lung injury. conditions which hinder the use of systemic anticoagulation are considered relative contraindications to the use of ecmo given the increased risk of in - circuit thrombosis. nonetheless, cases describing the use of ecmo in patients with diffuse alveolar hemorrhage (dah) have been reported. furthermore, strategies described to prevent further hemorrhage in patients with active bleeding while on ecmo include the use of fresh - frozen plasma, vitamin k, aprotinin, and recombinant activated factor vii, as well as avoiding the concomitant use of heparin and using only nafamostat mesilate as a regional anticoagulant. aminocaproic acid is a lysine analogue effective in certain bleeding disorders due to its inhibitor effect on plasmin. despite the theoretical risk of in - circuit thrombosis, retrospective studies indicate that its use is safe in patients on ecmo who develop life - threatening hemorrhage. nevertheless, experience with the use of aminocaproic acid in patients with dah who are on ecmo remains limited. to the best of our knowledge, this is the first report of continuous aminocaproic acid infusion along with an adjusted heparin protocol to stop and prevent further alveolar hemorrhage in a patient on ecmo in the setting of lphs. another case report by liao describes a patient with severe leptospirosis complicated by massive pulmonary hemorrhage, who was managed successfully with ecmo for resistant hypoxemic respiratory failure for 6 days ; compared with our patient, the duration of ecmo was much less and there was no apparent need of prothrombotic therapy to help manage the active bleed. whether aminocaproic acid led to the improvement of our patient or unfortunately, besides act, fibrinogen, complete blood counts, and coagulation studies, there was no follow - up of other hematologic laboratory values, such as thromboelastography analysis, to determine the specific role of aminocaproic acid in our patient. however, indirect measures such as symptomatic improvement and a significant decrease in blood product requirement suggest a beneficial effect. possibly not in the setting of a severe infection, such as a patient with leptospirosis and pulmonary hemorrhage, there are several studies on the use of ecmo and aminocaproic acid with and without steroids in stem cell transplant patients with dah. aminocaproic acid has also been more studied in pediatric patients on ecmo complicated by bleeding. prospective studies are needed to confirm the safety and efficacy of aminocaproic acid to stop and prevent further alveolar hemorrhage in adult patients undergoing ecmo. as evidenced by this clinical case, leptospirosis can be life - threatening in its most severe forms, and the most prevalent areas should increase awareness to the general public regarding recreational, occupational, or household exposures that could potentially cause disease transmission.	a 32-year - old man presented with a 10-day history of fever, chills, nausea, vomiting, myalgia, nonproductive cough, and worsening dyspnea after freshwater swimming in the caribbean 1 week prior to presentation. shortly after arrival at the hospital, the patient developed severe respiratory distress with massive hemoptysis. based on serologic workup, he was diagnosed with leptospirosis pulmonary hemorrhage syndrome leading to diffuse alveolar hemorrhage, severe hypoxemic respiratory failure, and multiorgan failure. he received appropriate antibiotic coverage along with hemodynamic support with norepinephrine and vasopressin, mechanical ventilation, and renal replacement therapy in an intensive care unit. introduction of extracorporeal membrane oxygenation was initiated to provide lung - protective ventilation supporting the recovery of his pulmonary function. aminocaproic acid was used to stop and prevent further alveolar hemorrhage. he fully recovered thereafter ; however, it is uncertain whether it was the use of aminocaproic acid that led to the resolution of his disease.
total knee replacement (tkr) is a common and effective treatment for end - stage knee arthritis. as the population ages, the frequency of surgery is increasing proportionally,1 bringing with it the challenge of managing older patients with frequent comorbid diseases and an increased risk of complications. poor postoperative quality of recovery may result in patient and family suffering, a prolonged hospital stay, and a greater demand on health care resources.2,3 both general anesthesia (ga) and regional anesthesia have been used for tkr surgery. peripheral nerve blocks (pnbs) combining lumbar plexus and sciatic nerve blocks can be used as the primary anesthetic for tkr, or combined with general anesthesia to provide postoperative pain relief.46 they form an alternative approach to the central neuraxial blockade for regional anesthesia for knee surgery and have shown improved postoperative recovery.7 this technique has become common in our institution for knee surgery. the quality of recovery is becoming a clinical and research area of increasing significance. it reflects a change in emphasis from hospital - based outcomes to patient - focused outcomes. an early measurement of quality of recovery tends to focus on physiological or nociceptive recovery and readiness for patient discharge ; whereas, longer - term recovery tends to focus on patient satisfaction, a poor metric of quality of recovery in objectively measured domains.8 the postoperative quality of recovery scale (pqrs) is a research tool designed to measure the postoperative quality of recovery in multiple domains and over multiple time periods. recovery domains include physiological, emotive (depression and anxiety), nociceptive (pain and nausea), cognitive, activities of daily living (adl), and overall patient perspective (including satisfaction).8,9 importantly, the pqrs surveys are objective and measure recovery at predetermined time points, rather than relying on subjective patient recall. further, the measurement of recovery in multiple domains facilitates an understanding of how recovery in one area can impact on other recovery domains. the inclusion of cognition is important as poor cognitive recovery is being increasingly recognized as a major morbidity. our aim was to identify whether our technique of combining lumbar plexus and sciatic nerve blocks with propofol sedation could alter the quality of recovery after tkr in elderly patients during the first week after surgery compared to general anesthesia. our hypothesis was that patients who received combined lumbar plexus and sciatic nerve blocks supplemented with propofol sedation would have a higher incidence of recovery than patients who were receiving general anesthesia during the first week after surgery. the study was conducted at the anesthesia and operation center of the chinese people s liberation army general hospital from june the research protocol was approved by the institutional ethical committee of the chinese people s liberation army general hospital. the inclusion criteria were patients who were : 6590 years old ; undergoing elective single tkr surgery ; had met the american society of anesthesiologists physical status i iii ; had a mini - mental score examination (mmse) > 23 ; and had no an allergy to local or general anesthetics. the exclusion criteria included contraindications to nerve blocks (coagulation defects, infection at puncture site), current severe psychiatric disease, alcoholism or drug dependence, severe visual or auditory disorder, and unwillingness to return for study follow - up.10,11 general anesthesia was induced with midazolam (0.0150.03 mg / kg), fentanyl (1.83.5 g / kg), etomidate (0.20.3 mg / kg), and rocuronium (0.40.6 mg / kg), followed by a laryngeal mask airway insertion and mechanical ventilation. anesthesia was maintained with a remifentanil infusion (0.150.30 g / kg / minute) and propofol (target controlled infusion [tci ] 0.62.0 g / ml) with 100% oxygen. infusion rates of propofol and remifentanil varied, according to clinical judgment and a target bispectral index (bis) range of 4060. patients randomized to the peripheral nerve blocks group received midazolam (0.0150.03 mg / kg) and fentanyl (0.82.0 g / kg) titrated to provide conscious sedation before nerve block insertion. after sterile preparation and draping, the nerve blocks were administered using a 21-gauge, 100 mm nerve block needle (stimuplex a, b braun melsungen ag, tochigi - ken, japan) under nerve stimulator guidance. a posterior approach to lumbar plexus block was performed with patients in the lateral decubitus position. the puncture site was 3 cm caudad and 4 cm midline to the spinous process of l4. a sagittal insertion direction was used, and we avoided a medial angulation of the needle. upon contact with transverse process of l5, the needle was retracted and then advanced over the transverse process (2 cm). the nerve stimulator was used to determine muscle activity from the femoral nerve (quadriceps or vastus lateralis). the puncture was also possible at the level of the transverse process of l4, with caudad advancement of the needle under the transverse process. after a quadricep s muscle response had been identified with the nerve stimulator settings at 2 hz frequency and current at 0.30.5 ma, 2530 ml of 0.35% ropivacaine was injected in divided doses.12 a sciatic nerve block was performed in the same position after a twitch of hamstring, soleus, foot, or toes had been elicited using a similar current, and 1525 ml of 0.35% ropivacaine was injected. sensory and motor blocks on the operated limb were evalu ated every 5 minutes after completion of the procedure until the achievement of adequate nerve block to allow surgery. a decision on the adequacy of the pnbs was made at 15 minutes after an insertion of the block. sedation during surgery was provided by propofol (tci 0.31.5 g / ml) with light sleep with easy arousal and a target bis range of 6080. postoperative pain management included intravenous patient - controlled analgesia (sufentanil 1.25 g / hour as background dose and 1.25 g bolus with an 8-minute lockout time), parecoxib (40 mg, every 12 hours), and oral oxycodone, 20 mg, was administered every 6 hours if required. antiemetic tropisetron 2 mg by intravenous infusion before surgery completion and then 0.1 mg / hour intravenously until postoperative pain management finished. to standardize recovery times and conditions, a hemostatic tourniquet was always placed around the thigh and inflated to double the systolic arterial blood pressure. the questions assessing the ability to stand or walk of the adl domain were not measured, due to the confounding effect of the knee surgery during the early recovery period. the pqrs has five assessment tools : orientation name, date, and place (adapted from the mmse). digits forward assesses working memory (adapted from the wechsler adult intelligence scale - lll). digits backward assesses working memory and scanning (adapted from the wechsler adult intelligence scale - lll). patients are given 30 seconds to say as many words as they can, starting with a given letter., we had established that the word generation test was difficult for the elderly chinese patients to comprehend, leading to low baseline scores. previous research found that an assessment of orientation had shown a limited ability to discriminate between individuals.9 accordingly, we made an a priori decision to exclude orientation and word generation in the cognitive domain. the pqrs was measured at 15 minutes (t15) and 40 minutes (t40) after the operation and at 1 day, 3 days, and 7 days postoperatively, (d1, d3, and d7). recovery is defined as return to baseline values or better at each time point. change scores are calculated as the value at each time - baseline value, and the recovery is scored if the changes score 0. further validation of the pqrs, which occurred after the commencement of our study, has resulted in a recommendation to introduce an adjustment factor into the cognitive domain to account for performance variability.13 we used the new scoring proposed for cognitive recovery. in addition, patients were excluded from analysis in the cognitive domain (and all domains recovery) if their baseline scores on the cognitive questions were the adjustment factor, as low scores would automatically be scored as recovered.13 other recovery domains, such as the nociception of emotive domains, use a 3- or 5-point likert scale to measure the patient experience. the questions have both verbal as well as pictorial content to facilitate understanding across a larger age range. consent was obtained by an investigator who did not participate in the surgery nor collect postoperative data. randomization was performed using a computer - generated random numbers list using permuted blocks of size 4, created with statmate v.1.01i software (graphpad software, inc, la jolla, ca, usa), and allocated by an investigator who was not involved in the surgery or the postoperative data collection. the pqrs surveys were conducted by investigators who were not involved in the conduct of surgery or anesthesia and were blinded to allocation. we excluded eight patients who had failed peripheral nerve blocks and required general anesthesia alone ; postoperative data was not collected. the primary outcome was quality of recovery in each domain measured with the pqrs, up to day 7. secondary outcome measures were the consumption of opioid drugs, anesthesia time (defined as the duration of anesthesia or sedative drug administration), intraoperative blood pressure and heart rate, and the occurrence of side effects, such as the complications of pnbs. haenszel (cmh) approach and data from the pqrs study and our preliminary test. the study was powered to detect a moderate effect size (odds ratio = 3), using a cmh test approach for all domains recovery (using nqueryadvisor 7.0, statistical solutions, cork, ireland), which required a minimal sample size of 85 per group. we adopted a conservative approach and increased the sample size to 110 per group to account for potential noncompletion of patients in this elderly cohort. categorical variables were presented as numbers and percentages, and continuous variables are presented as mean and standard deviation. the chi - square test and the student s t - test were both used to investigate differences in patient characteristics and operative details between the two groups. because of the risk of type i error with multiple comparisons, significance was defined as 23 ; and had no an allergy to local or general anesthetics. the exclusion criteria included contraindications to nerve blocks (coagulation defects, infection at puncture site), current severe psychiatric disease, alcoholism or drug dependence, severe visual or auditory disorder, and unwillingness to return for study follow - up.10,11 general anesthesia was induced with midazolam (0.0150.03 mg / kg), fentanyl (1.83.5 g / kg), etomidate (0.20.3 mg / kg), and rocuronium (0.40.6 mg / kg), followed by a laryngeal mask airway insertion and mechanical ventilation. anesthesia was maintained with a remifentanil infusion (0.150.30 g / kg / minute) and propofol (target controlled infusion [tci ] 0.62.0 g / ml) with 100% oxygen. infusion rates of propofol and remifentanil varied, according to clinical judgment and a target bispectral index (bis) range of 4060. patients randomized to the peripheral nerve blocks group received midazolam (0.0150.03 mg / kg) and fentanyl (0.82.0 g / kg) titrated to provide conscious sedation before nerve block insertion. after sterile preparation and draping, the nerve blocks were administered using a 21-gauge, 100 mm nerve block needle (stimuplex a, b braun melsungen ag, tochigi - ken, japan) under nerve stimulator guidance. a posterior approach to lumbar plexus block was performed with patients in the lateral decubitus position. the puncture site was 3 cm caudad and 4 cm midline to the spinous process of l4. a sagittal insertion direction was used, and we avoided a medial angulation of the needle. upon contact with transverse process of l5, the needle was retracted and then advanced over the transverse process (2 cm). the nerve stimulator was used to determine muscle activity from the femoral nerve (quadriceps or vastus lateralis). the puncture was also possible at the level of the transverse process of l4, with caudad advancement of the needle under the transverse process. after a quadricep s muscle response had been identified with the nerve stimulator settings at 2 hz frequency and current at 0.30.5 ma, 2530 ml of 0.35% ropivacaine was injected in divided doses.12 a sciatic nerve block was performed in the same position after a twitch of hamstring, soleus, foot, or toes had been elicited using a similar current, and 1525 ml of 0.35% ropivacaine was injected. sensory and motor blocks on the operated limb were evalu ated every 5 minutes after completion of the procedure until the achievement of adequate nerve block to allow surgery. a decision on the adequacy of the pnbs was made at 15 minutes after an insertion of the block. sedation during surgery was provided by propofol (tci 0.31.5 g / ml) with light sleep with easy arousal and a target bis range of 6080. postoperative pain management included intravenous patient - controlled analgesia (sufentanil 1.25 g / hour as background dose and 1.25 g bolus with an 8-minute lockout time), parecoxib (40 mg, every 12 hours), and oral oxycodone, 20 mg, was administered every 6 hours if required. antiemetic tropisetron 2 mg by intravenous infusion before surgery completion and then 0.1 mg / hour intravenously until postoperative pain management finished. to standardize recovery times and conditions, surgery was performed during the morning operating session. a hemostatic tourniquet was always placed around the thigh and inflated to double the systolic arterial blood pressure. the questions assessing the ability to stand or walk of the adl domain were not measured, due to the confounding effect of the knee surgery during the early recovery period. the pqrs has five assessment tools : orientation name, date, and place (adapted from the mmse). digits forward assesses working memory (adapted from the wechsler adult intelligence scale - lll). digits backward assesses working memory and scanning (adapted from the wechsler adult intelligence scale - lll). patients are given 30 seconds to say as many words as they can, starting with a given letter., we had established that the word generation test was difficult for the elderly chinese patients to comprehend, leading to low baseline scores. previous research found that an assessment of orientation had shown a limited ability to discriminate between individuals.9 accordingly, we made an a priori decision to exclude orientation and word generation in the cognitive domain. the pqrs was measured at 15 minutes (t15) and 40 minutes (t40) after the operation and at 1 day, 3 days, and 7 days postoperatively, (d1, d3, and d7). recovery is defined as return to baseline values or better at each time point. change scores are calculated as the value at each time - baseline value, and the recovery is scored if the changes score 0. further validation of the pqrs, which occurred after the commencement of our study, has resulted in a recommendation to introduce an adjustment factor into the cognitive domain to account for performance variability.13 we used the new scoring proposed for cognitive recovery. in addition, patients were excluded from analysis in the cognitive domain (and all domains recovery) if their baseline scores on the cognitive questions were the adjustment factor, as low scores would automatically be scored as recovered.13 other recovery domains, such as the nociception of emotive domains, use a 3- or 5-point likert scale to measure the patient experience. the questions have both verbal as well as pictorial content to facilitate understanding across a larger age range. consent was obtained by an investigator who did not participate in the surgery nor collect postoperative data. randomization was performed using a computer - generated random numbers list using permuted blocks of size 4, created with statmate v.1.01i software (graphpad software, inc, la jolla, ca, usa), and allocated by an investigator who was not involved in the surgery or the postoperative data collection. the pqrs surveys were conducted by investigators who were not involved in the conduct of surgery or anesthesia and were blinded to allocation. we excluded eight patients who had failed peripheral nerve blocks and required general anesthesia alone ; postoperative data was not collected. the primary outcome was quality of recovery in each domain measured with the pqrs, up to day 7. secondary outcome measures were the consumption of opioid drugs, anesthesia time (defined as the duration of anesthesia or sedative drug administration), intraoperative blood pressure and heart rate, and the occurrence of side effects, such as the complications of pnbs. sample size estimates were performed using the cochran mantel haenszel (cmh) approach and data from the pqrs study and our preliminary test. the study was powered to detect a moderate effect size (odds ratio = 3), using a cmh test approach for all domains recovery (using nqueryadvisor 7.0, statistical solutions, cork, ireland), which required a minimal sample size of 85 per group. we adopted a conservative approach and increased the sample size to 110 per group to account for potential noncompletion of patients in this elderly cohort. categorical variables were presented as numbers and percentages, and continuous variables are presented as mean and standard deviation. the chi - square test and the student s t - test were both used to investigate differences in patient characteristics and operative details between the two groups. because of the risk of type i error with multiple comparisons, significance was defined as < 0.01 for these measurements. repeated measures analysis of variance was used to investigate the differences in the systolic blood pressure and the heart rate. recovery domains of the pqrs were assessed by the cmh test, which assesses a global difference between groups over multiple time periods. for the global tests of repeated measures analysis of variance and cmh, september 2013, a total of 356 consecutive patients aged 65 years old in our hospital were approached to participate in the study. of these, 138 patients failed the inclusion criteria, did not consent to the study, or withdrew voluntarily prior to randomization, leaving 218 patients who completed the enrollment. five patients were discharged < 7 days after surgery and were lost to follow - up. also, 213 patients were successfully completed in the analysis 108 patients in the ga group and 105 patients in the pnbs group (figure 1). the anesthetic induction time of the pnbs group was longer (20.17.4 versus 8.42.5 minutes, p<0.001), but it did not significantly alter total anesthetic time (162.326.8 versus 147.323.1 minutes, p=0.097). the mean bis was higher in the pnbs group (6513.2 versus 539.3, p<0.001). the intraoperative cumulative drug doses of the pnbs group were lower (all p<0.001) than the ga group. the intraoperative systolic blood pressure was higher in the pnbs group (p<0.001) as was the heart rate (p<0.001), and it is shown in figure 2. physiological recovery was higher at t15 and t40, for the pnbs group, but equivalent by day 1 (p<0.001). recovery in the nociceptive domain was higher with pnbs (figure 3, p<0.001), though equivalence was reached by day 3. the recovery profile for nausea and pain is shown in figure 4 and shows improved recovery in both pain and nausea (p<0.001) in the pnbs group, with equivalence reached by day 3. the postoperative sufentanil requirement was lower in the pnbs during 24 hours and 72 hours (all p<0.001, table 2). recovery was improved with pnbs in the emotive domain (p<0.001) with better early recovery shown from both depression (p=0.003) and anxiety (p=0.008) questions (figure 4). the modified adl domain only included the assessment of eating and dressing and was not different between groups (figure 3, p=0.181). the modified cognitive domain was assessed using digits forward, digits back, and word recall questions only. ten patients in the ga group and 12 patients in the pnbs group were excluded from analysis as their baseline scores were too low, being the adjustment factor for the cognitive tests, leaving 98 patients in ga group and 93 patients in pnbs group for analysis in the modified cognitive domain and all domains recovery. recovery for the modified cognitive domain was better for pnbs (figure 3, p<0.001) with equivalence reached by day 7. recovery for all domains requires recovery in each of the physiological, emotive, nociceptive, cognitive, and adl domains where applicable to the time period. the recovery of all domains was better for pnbs group (figure 3, p<0.001) with equivalence reached by day 7. this domain is not a recovery domain as it has no baseline measurement, but it aims to capture the patients subjective assessment of their recovery. satisfaction was high at all of the time points measured (exceeding 85%) and was not different between the groups. this study demonstrated that patients over the age of 65 years undergoing tkr achieved a high proportion of recovery in all domains 1 week after surgery. the pnbs, as the primary anesthetic technique supplemented by propofol sedation and compared to the general anesthesia consisting of remifentanil and propofol, were associated with a faster recovery in all domains other than adl, less fluctuant intraoperative blood pressure and heart rate, and lower postoperative opioid consumption. no differences in recovery were found between day 3 and day 7 for different assessment domains. the measurement of quality of recovery represents a paradigm shift for anesthesia and surgery practice, as it changes the focus on outcomes from hospital - based (morbidity, mortality, and length of stay), to patient - focused outcomes. for anesthesiologists, this represents a concept that the techniques used in the operating room may impact on recovery long after the physiological and pharmacological effects of the anesthetic have diminished. although the use of the pnbs predominantly affects the pain response in the first few hours after surgery, they also reduce the exposure to sedative or general anesthetic drugs, which in turn could have a longer - term impact on recovery. the pqrs is a measurement tool that was purposely designed to look at a broad range of recovery domains in an objective fashion over repeated time periods to identify the impact of surgery and anesthesia on recovery in multiple different aspects or domains of recovery. this included cognition, because it is increasingly being recognized as a major source of morbidity for patients, even though they have physically recovered well. the use of regional anesthesia is common in the people s republic of china, but it is not universally adopted as a preferred technique worldwide. there is always a debate about the risk / benefit ratio of using regional anesthesia. the total ropivacaine dose of nerve blocks in this study did not exceed 4 mg / kg. there were no instances of symptoms consistent with local anesthetic toxicity in the perioperative period. there was one minor neuropraxia which resolved over 3 weeks, but as upper leg tourniquets were used, we can not be totally confident that it was solely due to the nerve block. in our hands, the technique was effective for providing surgical anesthesia. however, general anesthesia is not without risk, and the increased exposure to anesthetic drugs could incur risk, including poorer patient recovery. when comparing techniques, a risk risk analysis is perhaps better than individually assessing risk benefit analysis for individual techniques. our data do not allow us to define the relative risks of the two techniques, other than to state that both techniques were associated with low morbidity. a meta - analysis compared regional to general anesthesia for lower limb joint surgery in 2009.6 strong conclusions were unable to be drawn due to the limited number of randomized studies and the larger variation in endpoints to define full or poor recovery. however, there was a trend toward poorer cognitive outcomes in patients who received only general anesthesia. further, most studies were conducted in a western population, rather than a broad representation of countries, racial groups, and the differences in techniques within these countries. our study adds to the literature in this area by using a multidimensional, objective recovery assessment tool as well as defining recovery in an elderly chinese population. postoperative pain associated with tkr is considerable and requires adequate analgesia.14,15 the single - injection of pnbs provided 1224 hours of analgesia in our study, which is similar to the previous report.16 the pain recovery was markedly different at the 15-minute and the 40-minute time periods between the two groups. though the incidence of recovery for pain decreased from 40 minutes to 1 day in the pnbs group, the pain recovery was still higher than the ga group at 1 day, suggesting the presence of residual analgesia from nerve blocks in a proportion of patients. by day 3 nausea was lower with pnbs in the early time periods only, which may be, in part, due to an improved pain relief as well as the lower consumption of opioid analgesia.17,18 the emotive recovery was faster in both anxiety and depression questions in the pnbs group, although equivalence was achieved by day 3. we can not determine whether this effect was due to improved analgesia or a lower consumption of anesthesia and analgesia medication. further, emotive recovery is a complex area ; it can involve preoperative expectation and the hospital environment, including altered sleep patterns, which may be more pronounced in the elderly.19 the modified adl domain only includes the ability to eat and to dress due to the limitation imposed by surgery. postoperative cognitive recovery is a very important area of research and has been studied extensively. the literature is confusing, however, due to the many different tests and the definitions used to measure cognitive decline. many elderly patients suffer significant deterioration of cognitive function after cardiac surgery20 or noncardiac major surgery,10,21 even after seemingly uneventful procedures. the pqrs assesses recovery, taking a different approach in that it assesses cognitive recovery, rather than the postoperative cognitive deficit (pocd),22,23 although there is likely to be an association with failure to recover and pocd. further, the pqrs does not assess delirium, which is a fluctuating mental state,23 although it is likely that delirious patients will be less likely to show recovery. the definition of cognitive recovery has been amended from the original pqrs publication after the validation of the test accuracy in normal volunteers not undergoing surgery.13 normal volunteers will have variability in cognitive performance based on the time of day. accordingly, an adjustment factor is applied to each test, such that patients can perform a little worse than baseline and still be coded as recovered. the recommendation is that recovery rates exceeding 80% should be considered in a range of very good recovery.13 further, the cognitive recovery in our study is not equivalent to other pqrs publications as we modified the scale a priori. although this is a limitation of our study, the results for the remaining cognitive tests showed a faster cognitive recovery in the pnbs group, with equivalence reached by day 7. we can not determine from our data the exact reason for this, but rather we can identify that the specific anesthesia and analgesia protocol with the pnbs resulted in faster cognitive recovery than the specific general anesthesia and analgesia regimen in our comparative group. we urge caution not to extrapolate our findings to regional anesthesia versus general anesthesia, as there may be marked differences in the various regional anesthesia techniques, and for different combinations of anesthetic drugs. our study only assessed recovery to day 7, and we can not determine if the failure to recovery by day 7 is linked to longer - term recovery. in a study of cardiac surgery patients, royse showed lower pocd at 35 days with propofol versus desflurane, but this was not associated with a difference in pocd at 3 months after surgery.24 more research is required to identify whether failure to recover in the pqrs in the first week after surgery is a harbinger of pocd. when assessing complete recovery (recovery in all domains), the pnbs group recovered faster than the ga group with equivalence reached by day 7. the overall perspective domain is not a recovery domain as there is no baseline measurement. patients always underestimate their cognitive performance compared with objective testing, and this effect was shown in the original pqrs publication,9 emphasizing the importance of objective testing of cognition. this is a consistent finding and emphasizes that satisfaction is a poor discriminator of quality of recovery.8 there are limitations to our study. a number of patients were excluded from the cognitive recovery analysis, because they had very low baseline values. in the pqrs, a tolerance factor was applied, such that the patients can be a little worse and still score as recovered, due to the normal performance variability for cognitive performance. if the baseline score is to the adjustment to the score, then they would automatically score as recovered, and the test would lose discriminant ability to measure poor cognitive recovery in these patients.13 we reduced this risk by first screening with mmse and excluded patients with scores of < 24. however, the mmse is primarily aimed at detecting dementia,25 and so we post hoc applied the recommendation of royse to exclude patients from the cognitive analysis when their baseline scores were to the correction factor for the tests. the modification of the adl and cognitive domains does limit conclusions about recovery to our cohort and is not directly comparable to other pqrs publications. the randomized trial design, however, does allow us to directly compare our two groups. we were concerned that anxiety and concerns about the forthcoming operation can affect the baseline values26 and so conducted most baseline assessments at least 1 day prior to surgery. to reduce the assessor bias, it is not possible, however, to absolutely assure concealment as patients with a successful nerve block may have an absence of pain and leg weakness early after surgery. the anesthetic techniques used in this study, as well as the postperative management including physical therapy, though commonly used in our hospital, may be different to other centers in the western world. we can not comment on whether our techniques could impact on hospital resource use or the ability to fast track patients to discharge. lumbar plexus and sciatic blocks with sedation facilitates faster postoperative recovery than general anesthesia, but not at 1 week, after tkr in patients 65 years or older. this provides proof of concept that the use of regional anesthesia could impact on quality outcomes.	backgroundboth peripheral nerve blocks with sedation or general anesthesia can be used for total knee replacement surgery.objectiveswe compared these anesthetic techniques on the postoperative quality of recovery early in elderly patients.materials and methodsin our study, 213 patients who were 65 years old and undergoing total knee replacement were randomized to peripheral nerve blocks (pnbs) lumbar plexus and sciatic with propofol sedation, or general anesthesia with combined propofol and remifentanil. blocks were performed using nerve stimulation and 0.35% ropivacaine. all patients received postoperative multimodal analgesia. postoperative recovery was assessed at 15 minutes, 40 minutes, 1 day, 3 days, and 7 days after surgery, with the postoperative quality of recovery scale, in physiological, nociceptive, emotive, modified activities of daily living, modified cognitive, and overall patient perspective domains.resultsintraoperative blood pressure and heart rate were more stable with pnbs (p<0.001). the recovery was better with pnbs in physiological (p<0.001), emotive (depression and anxiety) (p<0.001), nociceptive (pain and nausea) (p<0.001), modified cognitive (p<0.001), and all domains recovery (p<0.001), but not in activities of daily living (p=0.181). intraoperative drugs and the postoperative sulfentanil requirement of the pnbs group were lower (all p<0.001). differences were greatest early after surgery with equivalence by 1 week. satisfaction was high and not different between groups (p=0.059).conclusionlumbar plexus and sciatic blocks with sedation facilitates faster postoperative recovery than general anesthesia, but not at 1 week after total knee replacement in patients who were 65 years or older. the trial has been registered at clinicaltrials.gov. (nct01871012).
during many years i have been observing very often some misunderstanding therapeutic ideas from several physicians about the management of patients with lipid disorders mostly in older hypertensive patients. this situation is directly related to an insufficient therapeutic control due to inadequate drug dosages. however, we must not neglected this point because good control is crucial for clinical point of view and for the prognosis of the patients. it is well established the relevance to provide some measures for the prevention and delay of atherosclerosis that is closely related to the well - known cardiovascular risk factors, such as, hypertension, smoking habit, dyslipemia, obesity, diabetes, and many others disorders. on the other hand, severe or fatal cardiovascular events related to the risk factors are also important in order to offer the patients a better management to reduce stroke, artery coronary disease, peripheral arteriopathy, and other vascular diseases in western countries. hypertension, together with smoking habit and high blood lipid levels, represents the most important pathogenic cause for atherosclerosis and subsequent clinical severe diseases. the following ideas are based on my clinical experience during many years on this particular clinical disorder. as part of an overall strategy to stop atherosclerosis, control of hypertension is very important (bp 140/80 mmhg) in all cases using general measures (diet, salt restriction, relief of stress, regular aerobic exercise, and dietary management : caloric restriction, reduction of cholesterol and saturated fats intake) as well as drug therapy when necessary. smoking habit, for example, must be stopped as soon as possible, and current national guidelines recommend having cholesterol level screening in adults. they also propose to obtain a fasting lipid profile (including total cholesterol, triglycerides, ldl cholesterol, and hdl cholesterol) in patients with known vascular diseases and those with several risk factors or elevated total cholesterol levels. unfortunately, three types of mistakes are very common present in clinical practice by many physicians : (1) some colleagues take as normal lipid values clearly high and neglecting the reference values expressed on table 1. at this point, many patients with moderately high cholesterol, triglycerides, ldl - cholesterol and decreased hdl cholesterol with values below ranging between 35 and 40 mg / dl do not receive any treatment ; (2) it is well - known that statin / fibrate combination therapy is not recommended in same patient to treat mixed dyslipemia ; but unfortunaly, this procedure is maintain by physicians in many cases. these associations are not adequate because of their increasing side effects rhabdomyolysis, severe and even fatal in some patients ; (3) in my clinical experience, the most common error that i have observed is the discontinuation of lipid lowering therapy when normal values are reached ; obviously expected, lipid values become again pathologically high after suspension of therapy (statins, fibrates, or ezetimibe) (table 2). nevertheles, thanks to the therapeutic arsenal available today, observed normalization of lipid values in a large percentage of patients. the 4s study demonstrated for the first time that intensive treatment to decrease lipid levels achieved reduced mortality rate in patients with coronary heart disease and cholesterol values between 212 and 309 mg / dl (mean : 260 mg / dl). the care study showed beneficial effects in patients with history of coronary heart disease and total cholesterol of 175 to 240 mg / dl (mean : 211 mg / dl) ; the lipid trial showed an improvement in total cardiovascular mortality rate with cholesterolemia levels below those reported in the 4s study (mean : 220 mg / dl). the afcaps / texcaps study showed the benefit of lipid - lowering treatment of cardiovascular morbidity and mortality rate in primary prevention in individuals with levels between 180 and 264 mg / dl (mean, 221 mg / dl) aged 4575 years, as well as a slight decrease of hdl - c levels in individuals with cholesterol values considered within the normal limits (180264 mg / dl ; mean : 221 mg / dl). the prove - it demonstrated that intensive lipid - lowering therapy versus standard treatment reduced significantly cardiovascular morbidity and mortality rate after acute coronary syndromes. these studies have cleared the way to recommend statins therapy in patients treated for secondary prevention (high risk) and primary prevention with other related risk factors (table 3). our experience demonstrated in hypertensive patients older than 60 as the coadministration of ezetimibe / simvastatin + fenofibrate improved atherogenic lipid profile with mixed hyperlipidemia. another recent study, with participation of my group showed that the association of extended - released niacin and laropiprant (ern / lrdt) + statin significantly improved the lipid profile compared to the run - in dose doubled, and it was generally well tolerated in patients with primary hypercholesterolemia and mixed dyslipemia. the protocol was the following : after a 2- to 6-week run - in statin (simvastatin 10 or 20 mg or atorvastatin 10 mg) period, 1216 patients were randomised equally to one of two treatment groups in a double - blind fashion : group 1 received ern / lrpt (1 g) plus the run - in statin dose and advanced to ern / lrpt (2 g) after 4 weeks for an additional 8 weeks, with no adjustments to the run - in statin dose ; group 2 received simvastatin or atorvastatin at twice their run - in statin dose and remained on this stable dose for 12 weeks. some of the molecules under study (clinical trials phase ii or iii) in which our unit is participating actively are expressed in merck pipeline : cardiovascular : mk-0736, mk 6621 (vernakalant), atherosclerosis mk-0524-a (tredaptive), mk-0524b, and mk-0859 (anacetrapib). in summary, treatment and control of arteriosclerotic vascular disease in hypertensive patients needs the overall treatment of the risk factors ; in particular, in patients with dyslipemia, it is necessary to normalize blood pressure, to reduce ldl - c values to normal levels by the inhibition of endogenous and exogenous cholesterol, as well as to decrease triglycerides, and increase hdl - c levels, so that it is essential to maintain dietetic measures (reduction of salt and unsaturated fat intakes) and continuous pharmacologic treatment in most of the patients.	lipid disorders are a common clinical challenge in the western countries. in patients with dyslipemia (total cholesterol > 200 mg / dl, hdl cholesterol 130 mg / dl and triglycerides > 150 mg / dl) it is mandatory to normalize blood pressure (< 130/80 mmhg) as well to reduce ldl - c values to normal levels by using drugs to inhibit of endogenous and exogenous cholesterol, to decrease triglycerides, and increases hdl - c up to normal range. it is also essential to maintain for this purpose suitable dietetic measures (reduction of unsatured fats and salt intakes<2.5 g / daily) and without interruption, to support pharmacologic treatment in most of the patients.
meat and meat products are a good source of amino acids and their proteins are considered of high biological quality. after consumption of meat, free amino acids are rapidly absorbed, while proteins are easily hydrolysed into peptides and amino acids, which in turn are also absorbed. apart from their nutritional importance, amino acids also influence meat palatability and flavour, through the generation of volatile compounds by maillard reactions and strecker degradations [36 ]. during the processing of dry - cured meat products, such as dry - cured ham or loin, there happens an increase in free amino acid content as a consequence of proteolytic activity [4, 7 ] ; indeed, the amount of most amino acids increases with processing time and with higher processing temperatures [3, 8, 9 ]. glutamic acid and phenylalanine have been found to be the major amino acids in fresh meat, while in dry - cured products glutamic acid, arginine, and lysine have shown the highest levels. traditionally, the most common method to analyze free amino acids in food matrices has been reverse phase - high performance liquid chromatography (rp - hplc) with a precolumn derivatization step. gas chromatography coupled with mass spectrometry (gc - ms) can be also used as an alternative method, especially when sample amounts are limited and high sensitivity is required. in addition gc presents higher resolution and speed of analysis and lower instrumental cost than hplc. when gc was first used for the analysis of amino acids, its main drawback was the time consuming and tedious derivatization steps (esterification + acylation) required. then, the simultaneous silylation of the amino and carboxyl groups in a single step, first using bis(trimethylsilyl)trifluoroacetamide (bstfa) and later with n - methyl - n-(tert - butyldimethylsilyl)trifluoroacetamide (mtbstfa), was developed. have demonstrated the suitability of using mtbstfa for the determination of free amino acids in different animal source foods. most extraction methods for amino acids in food products involve using perchloric acid or hydrochloric acid (hcl) diluted in water or in ethanol [9, 10, 12, 1620 ]. other solvents for the extraction of amino acids have also been described in the scientific literature, such as ethanol, and solvent mixtures, such as water / acetonitrile (50 : 50, v / v) or 0.1% (v / v) formic acid in 20% (v / v) methanol. after mixing the sample with the solvent, the homogenization step is essential for the amino acids to be extracted. for this purpose, different techniques have been used, that is, stirring, ultraturrax [16, 17 ], stomacher [10, 17 ], omni mixer, rotary mixer at 50c, vortex, and a heating block at 40c with stirring. then, centrifugation is usually carried out, followed by the collection of the supernatant and its filtration through glass wool [9, 10 ], nylon membrane, or whatman 42 paper. some authors clean up the supernatant through a cartridge [12, 21 ] and others do not specify the filtration procedure [9, 20 ]. the search for new and accurate methods for amino acid analysis in meat and meat products is challenging. the development of derivatization and chromatographic procedures has been thoroughly studied, while less attention has been paid to the extraction methods. recently, jimnez - martn. described a gc - ms method for the determination of free amino acids in animal source food. in this methodology, the sample is homogenized with hcl 0.1 m by using a stomacher. the application of this gc - ms method for the determination of amino acids in meat and meat products constitutes an important reduction in time and solvents in the separation and detection procedures in comparison to rp - hplc with diode array detector method [3, 4, 8, 9 ]. however the extraction protocol is time consuming and requires a large amount of sample and solvent, which makes it frequently not suitable for routine analysis. the present work is focused on the homogenization step for the amino acid extraction from meat samples, with the main goal of reducing sample amount, solvent volume, and extraction time. recently, segura and lopez - bote developed a new procedure to extract intramuscular fat from pork based on homogenizing the samples using a mixer mill, which allowed minimizing the sample amount, the solvent use, and the analysis time, which are important advantages for routine analysis. the mixer mill is a compact versatile bench - top unit, which has been developed specially for homogenizing small amounts of sample quickly and efficiently by impact and friction. the inertia of the grinding balls causes them to impact with high energy on the sample material at the rounded ends of the grinding jars and pulverizes it. also, the movement of the grinding jars combined with the movement of the balls results in the intensive mixing of the sample. thus, the objective of this study was to evaluate the use of the mixer mill as homogenization tool in the extraction of free amino acids from meat samples, in order to analyze a large number of samples in the shortest time, minimizing sample amount and solvent volume. this study was developed with two different meat samples, fresh pork loin and dry - cured ham. subsequently, the moisture content of the products was determined according to the method of the association of official analytical chemists (moisture reference 935.29). the rest of the ground samples were stored at 80c until free amino acid analysis. hydrochloric acid (hcl), 37% extra pure, was used for the amino acid extraction (scharlau, barcelona, spain). acetonitrile of hplc - gradient grade (panreac, barcelona, spain) and dichloromethane (merck, darmstadt, germany) were used for the amino acid deproteinization and derivatization procedures. standard amino acids (sigma - aldrich) purchased for preparing the standard solutions were alanine, glycine, valine, leucine, isoleucine, proline, methionine, serine, threonine, phenylalanine, aspartic acid, hydroxyproline, cysteine, glutamic acid, arginine, asparagine, lysine, glutamine, histidine, tyrosine, tryptophan, and cystine. dl - norleucine (sigma - aldrich) was used as internal standard (is). they mainly differ in the homogenization procedure, carried out with stomacher (s) or mixer mill (m). both methods used hcl 0.1 m as solvent extraction and the sample : solvent ratio was 1 : 7.5. the free amino acid content of fresh loin (n = 6) and dry - cured ham (n = 6) was analysed by using the two extraction methods. samples (2 g) were weighed, mixed with hcl 0.1 m (15 ml), and subsequently homogenized in a stomacher (stomacher 400, lab - blender, barcelona, spain) for 4 min, as described by jimnez - martn.. from the stomacher bag, 2 ml was transferred to a safe - lock micro test tube and centrifuged (10000 rpm) (eppendorf centrifuges, model 5810r) for 15 minutes at 4c. ground samples (0.2 g) were homogenized with hcl 0.1 m (1.5 ml) and three stainless steel balls (2 mm of diameter) in the mixer mill (mm400, retsch technology, haan, germany) during 2 min and centrifuged (10000 rpm, 15 min, 4c). the sample, 250 l of acetonitrile was mixed with 100 l of the extract in safe - lock micro test tube and centrifuged at 10000 rpm for 3 min. 100 l of the supernatant was transferred to heat - resistant tubes and 100 l of is solution (5 g ml) was added. the residual water was removed adding 50 l of dichloromethane to the dried samples and again evaporated under nitrogen. finally, 50 l of mtbstfa and 50 l of acetonitrile were added to the dried tubes, which were shaken and subsequently incubated at 100c for 60 min to induce the derivatization reaction to occur. then, tubes where stored at refrigeration and analyzed by gc - ms within the next 24 hours. the chromatographic analysis was carried out in a gc equipment 5890 series ii (hewlett - packard, barcelona, spain) coupled to a mass selective detector (msd) electron impact (ei), model 5973 (agilent, barcelona, spain). a 1 l portion of the derivatized extract was injected in splitless mode onto the column., 1.05 m, hp-5 (hewlett - packard), being a 5% phenyl - methyl polysiloxane bonded phase fused silica capillary column. column head pressure was 12.8 psi, resulting in a flow of 1.2 ml / min at 280c. the oven program was as follows : 170c for 5 min, 4c / min ramp to 200c, held at 200c for 3 min, 4c / min ramp to 290c, held at 290c for 1 min, 20c / min ramp to a final temperature of 325c, and held for 15 min. the transfer line to the mass spectrometer program was as follows : 280c for 35 min, 10c / min ramp to 320c. free amino acids were identified both by their retention time and by comparison of their characteristic m / z ions with those published in the literature [9, 10 ]. the quantification was carried out in the selected ion monitoring (sim) mode. table 1 shows retention time (rt), ions selected in sim mode, and the selected ion for quantification of each amino acid in this study. a calibration curve (quantification ion aa peak area / quantification ion is peak area versus aa amount / is amount) was constructed, obtaining r values of 0.9999. the final results, expressed in microgram per 100 gram sample dry weight, take into account the moisture content and the exact weight of the sample. a standard calibration solution containing 200 g ml for (0.5 g of each amino acid was dissolved in 250 ml of hcl 0.1 m). from this solution, seven decreasing dilutions were made (150, 100, 50, 25, 10, 5, and 1 g ml). a stock solution of is at 5 g ml was prepared in 0.1 m hcl. quality control of the gc - ms analysis was performed through the routine analysis of procedural blanks and quality control standards and samples to ensure the absence of contaminants and the possible carryover between samples and to assess the quality of the results. limit of detection (lod) and quantification (loq) based on a signal / noise ratio of 3 : 1 and 10 : 1, respectively, were determined using aqueous standard solutions (n = 5) with the following equations : lod = 3sd / b and loq = 10sd / b, where, for each free amino acid, sd is the standard deviation of the average of the signal obtained for the calibration solution of lowest concentration (0.1 mg/100 ml) and b is the slope of the analytical curve calculated with the calibration solutions. for calculating the relative standard deviation (rsd) run - to - run, five replicate analyses of samples were done. in these determinations, aa, loin and dry - cured ham samples were spiked with appropriate amounts of aa (7.540 g) each and were extracted using s and m methods. moreover, the recovery was also calculated in unspiked samples, using the aqueous standard solutions. the effect of the extraction method on total chromatographic area as well as on the content of each detected amino acid was analysed by the student 's t - test for independent samples. linear regression analysis was carried out in order to compare the response of the different homogenization tools. the spss package (v 18.0) the chromatographic areas of each free amino acid detected in fresh loin and dry - cured ham samples homogenized by using s and m are shown in figure 1. most aa showed no statistical differences in fresh loin between s and m, whereas in dry - cured hams chromatographic areas of free amino acids were significantly higher (p 0.90, except for tryptophan (r = 0.837). most amino acids showed a poor linearity above 150 g ml ; thus, curve point at this concentration or higher was avoided. lod and loq of the analytical procedure ranged from 3.8106.610 g l to 1.3102.210 g l, respectively, for alanine, glycine, valine, leucine, isoleucine, proline, methionine, serine, threonine, phenylalanine, aspartic acid, histidine, and tyrosine. for glutamine, asparagine, lysine, glutamic acid, tryptophan, and cysteine these values hydroxyproline and cysteine had higher values for lod (0.38 and 1.27 g l, resp.) and loq (0.98 and 2.98 g l, resp.). in fact, previous studies using rp - hplc - dad for analyzing amino acids from dry - cured hams did not allow the detection of hydroxyproline and cysteine. adequate precision was achieved with a rsd of 2.1520.15% for run - to - run. table 5 shows the recovery of aa in aqueous standard solution and in spiked samples (loin and dry - cured ham) extracted by using both s and m extraction methods. in aqueous standard solution all aa showed high recoveries (94.49105.75%), indicating the accuracy of the chromatographic procedure. in the samples, most aa showed higher recoveries when using the m method for the extraction in comparison to the s one, especially in dry - cured ham. this result points out the suitability of the mixer mill for the extraction of aa and it is in concordance with other studies in aa from meat samples. the mixer mill is an appropriate tool for the homogenization step in the extraction procedure of free amino acids from meat samples, especially in samples with high free amino acids content. in addition, this technique notably reduces sample amount and solvent volume as well as analysis time. thus, it could be an adequate option for routine analysis of free aa in meat and meat products.	this study evaluated the use of a mixer mill as the homogenization tool for the extraction of free amino acids in meat samples, with the main goal of analyzing a large number of samples in the shortest time and minimizing sample amount and solvent volume. ground samples (0.2 g) were mixed with 1.5 ml hcl 0.1 m and homogenized in the mixer mill. the final biphasic system was separated by centrifugation. the supernatant was deproteinized, derivatized and analyzed by gas chromatography. this procedure showed a high extracting ability, especially in samples with high free amino acid content (recovery = 88.73104.94%). it also showed a low limit of detection and quantification (3.8 1046.6 104 g l1 and 1.3 1032.2 102 g l1, resp.) for most amino acids, an adequate precision (2.1520.15% for run - to - run), and a linear response for all amino acids (r 2 = 0.7410.998) in the range of 1100 g ml1. moreover, it takes less time and requires lower amount of sample and solvent than conventional techniques. thus, this is a cost and time efficient tool for homogenizing in the extraction procedure of free amino acids from meat samples, being an adequate option for routine analysis.
vitiligo is an acquired depigmentary condition caused by inactivation or destruction of melanocytes in epidermis and hair follicle. vitiligo is a common pigmentary disorder seen in our country.(1) it is an idiopathic, acquired, circumscribed hypomelanotic / demelanotic skin disorder, characterized by milky white patches of different sizes and shapes and affects 1 - 2% of the world population.(2) the incidence of vitiligo is found to be 0.25 - 2.5% in india. gujarat and rajasthan states have highest prevalence ~8.8%.(2) widespread prejudices, ignorance, taboos, lack of scientific appraisal, and confusion of vitiligo with leprosy all make it a social embarrassment for the patients.(3) this disorder does not result in restriction of capacity to work or expectancy of life, but it causes cosmetic disfigurement leading to psychological trauma to the patients.(1) since gujarat shows high prevalence of vitiligo in india, the present study was conducted to know the various clinical patterns, positive family history, and to find out the various skin disorders associated with it at rural setup near anand, gujarat. all new cases of idiopathic depigmented clinically diagnosed as vitiligo attended in outpatient department of dermatology, shree krishna hospital, karamsad and matar camp, gujarat over 1 year period were included in this study after consent of the patients. all depigmented patches observed since birth as well as acquired depigmented patches due to infections, physical trauma, chemical injury, burns, nutritional deficiency, inflammatory dermatosis, and drugs were excluded. specific emphasis was given on age of onset, duration of disease, site of onset, most common site, most common type of vitiligo, precipitating factors, presence of leukotrichia, koebner 's phenomenon, family history, and any other cutaneous or systemic illness. apart from routine blood and urine examination, blood sugar and thyroid function test were done whenever necessary. according to area of body part involvement, they were classified into vitiligo vulgaris, acrofacial, segmental, universal, and mucosal. a total of 1,010 patients were included in the study. among these 578 (57.3%) were female and 432 (42.7%) were male. the age at onset was found to be in the 2 decade of life in 274 (27.0%) patients, while majority of patients belonged to the age group between 21 and 30 years - 251 (24.8%) [table 2 ]. mean duration of disease presentation was 1 - 5 years 559 (55.5%) [table 3 ]. a positive family history was present in 204 (20.4 %) and 24 (2.4%) patients had more than one family member affected [table 4 ]. trauma 80 (8%) was the most common precipitating factor followed by itching 63 (6.3%), friction 25 (2.5%), and stress 15 (1.5%). mixed precipating factors were present in 40 (4.01%) patients and 787 (77.9%) patients had no precipitating factor. out of the total, 923 (91.3%) patients were vegetarian and 87 (8.6%) patients were on mixed diet. progressive disease was present in 618 (61.1%) patients ; however, spontaneous regression was found in 72 (7.2%) patients. initial site of lesion in majority of patients was lower limb (417, 41.5%) followed by scalp (254, 25.2%), face (204, 20.2%), upper limb (119, 11.7%), and trunk (16, 1.5%). most common site affected was lower limb (758, 75.4%), followed by upper limb (688, 68.3%), face (503, 50.0%), trunk (458, 45.7%), and genitals (67, 6.7%) [table 5 ]. vitiligo vulgaris (580, 57.8%) was the commonest morphological pattern, other pattern seen were acrofacial (277, 27.6%), segmental (69, 6.6%), universal (69, 6.9%), and mucosal (15, 1.5%) [table 6 ]. majority of patients had bilateral distribution (721, 71.6%) and 613 (61.3%) patients had less than 25% body surface area involvement. the associated cutaneous diseases noted in our study were alopecia areata (19, 1.9%), lichen planus (7, 0.7%), and eczema (10, 1.0%).[table 7 ] associated dermatological condition the untiring efforts of scientists, over a period of many years, have failed to lift up the curtain of ignorance till today and as we know, the etiology of vitiligo is still an enigma. various theories of origin, genetic, toxic, neurogenic, and autoimmune have been proposed by different workers, yet none is definite.(4) the prevalence of vitiligo is high in india, varying in range of 0.46 - 8.8%. the different ethnic backgrounds of population residing in different geographic region with different environmental condition may contribute to the wide variation in prevalence of vitiligo in india.(5) the female to male ratio in our study was 1.5:1. most of the other reports shown that males and females were affected with almost equal frequency,(2) which was different from that reported by handa and kaur, koranne.,(6) and khaitan.,(7) showed, male was more commonly affected than female. a few studies show slightly higher prevalence in female population.(35) the number of female vitiligo patients were found to be higher than male because women notice the change in appearance and approach the doctors sooner than men and of the social stigma in the community, young females tend to report earlier due to matrimonial anxiety. in our study mean age of onset was 2 decade of life, consistent with the most reports from india and the west.(268) this shows that the disease starts at a younger age in the indian population. however, one study from denmark showed the age of onset to be between 40 and 60 years.(9) genetic factors play an important role in manifestation of vitiligo. though various studies indicate involvement of genetic factors, the patterns are not consistent with single locus mendelian transmission, but appear to be polygenic. one hypothesis postulates that recessive alleles at multiple unlinked autosomal loci interact epistatically in pathogenesis of vitiligo.(10) in our study, 204 (22.4%) patients had positive family history of vitiligo, 24 (2.4%) patients had more than one family member affected. familial occurrence has been reported to vary from 5 to 30% in different studies.(2111213) human leucocyte antigen (hla) type significantly related to family history and early onset of vitiligo.(14) positive family history is considered to be a poor prognostic factor.(2) in our study, lower extremities was the commonly involved site in majority (75.4%) of cases which support the finding of other studies.(2121415) next commonest sites was upper limb followed by face, trunk, and scalp. most common site of onset was lower extremity this finding also similar with study by shajil.(2) however, gao., showed face was the most common site of onset.(17) one study by karelson., of 155 adult patients shows most common site of onset was upper limb.(18) majority (60.9%) of the patients had progressive vitiligo at the time of presentation. vitiligo vulgaris (57.4%) was most common type observed in our study followed by acrofacial, segmental, universal, and mucosal vitiligo which is similar with other studies.(2361319) this indicates that the process of depigmentation, either immune - mediated or toxic may occur simultaneously or subsequently at various unrelated distant sites. koebner phenomenon was observed in 208 (20.8%) patients, while in other study it was between 5 and 16%.(71819) trauma was most common precipitating factor (8%) in our study followed by itching, friction, and stress. some studies shows higher prevalence.(1720) vitiligo is associated with many systemic as well as cutaneous disorders. in our study most common was diabetes mellitus was found to be 1.7% in our study, whereas the reported value was 1.18 - 2%.(27) hypertension were found in 1.7% of patients. thyroid disease was seen in seven (0.70%) patients, which was reported to be 0.94% in study done by shajil.,(2) and 7.8% by martis.,(3) most common thyroid disease associated with vitiligo is hypothyroidism and autoimmune thyroiditis. vitiligo occurs most commonly in the 3 decade with a female preponderance and should be entitled to more than ordinary consideration. the data suggest that local epidemiological behavior of vitiligo need not be the same across different regions.	introduction : vitiligo is an acquired depigmentary condition caused by inactivation or destruction of melanocytes in epidermis and hair follicle. worldwide incidence of 1% has been reported ; similar to various dermatological clinics in india. widespread prejudice, ignorance, taboos, lack of scientific appraisal, and confusion of vitiligo with leprosy makes it an immense psychological stress.aim:to know the clinical profile of vitiligo patient with associated cofactors.materials and methods : total 1,010 patients of vitiligo attended in outpatient department at shree krishna hospital (skh) and matar camp, gujarat over 1 year period from august 2011 to july 2012 were included in this study. detail history and clinical examination of patients were done.results:out of 1,010 patients 57.3% were females and 42.7 % were males. most cases developed vitiligo by 2nd decade of life. progressive course was found in 60.9 % of patients. vitiligo vulgaris (57.8%) was most common morphological type. most common site of onset (41.5%) and involvement (75.7%) was lower limb. family history was present in 20.4%.conclusions : vitiligo constitutes important dermatological disease especially in india. the data suggest that local epidemiological behavior of vitiligo need not be the same across different regions. vitiligo differs substantially in various clinical aspects.
molecular biologists often identify specific genes important in crop growth or stress tolerance. increasing the expression of such genes is now a popular approach for genetic improvement of crops. but natural selection among the wild ancestors of crops is unlikely to have missed simple genetic improvements that would consistently have enhanced individual fitness under past conditions (denison. any constitutive increase in expression of a gene for drought tolerance, for example, would almost certainly regenerate a trait repeatedly rejected by past natural selection, presumably due to tradeoffs. increased expression of a gene that is beneficial under drought might, for example, decrease growth under well - watered conditions. drought - tolerant maize cultivar, for which yield data were reported only under a particular drought treatment (nelson. natural selection could have rejected this option for other reasons, of course, such as tradeoffs with competitiveness. a genotype that is radically different from anything existing today may never have been tested by natural selection, so we can not assume it was rejected due to tradeoffs. but producing such genotypes is likely to require more - complex genetic changes than the evolution of c4 photosynthesis, which natural selection has achieved repeatedly (kellogg 1999). unfortunately, our present scientific ability to predict all of the field - level consequences of such complex genetic changes is even more limited than our technical ability to implement them. genetic improvement of crop yield potential by humans has usually involved tradeoffs rejected by past natural selection but acceptable to us (denison 2009), although these tradeoffs have not always been obvious. a crop grown at a new latitude may need different photoperiod responses to complete seed development before winter. irrigation or fertilizer use can create new opportunities for genetic improvements linked to tradeoffs between root acquisition of water versus phosphorus (ho. increasing atmospheric co2 may call for changes in stomatal behavior or in nitrogen allocation to chlorophyll versus rubisco. a particularly important class of tradeoff was identified by colin donald (1968). he hypothesized that there can be negative relationships between the competitive ability of cultivars and their capacity for yield in pure culture. he described an ideotype design for wheat plants with low competitiveness against neighbors given good weed control, these would mostly be fellow wheat plants but high community productivity. consistent with donald 's ideas, past increases in crop yield potential have often been linked to decreased competitiveness, where competitiveness was in conflict with pure - culture yield (reynolds. natural selection, driven by competition among plants, led to taller plants than was optimal for total seed production by a plant community. greater investment in stems increased height and individual competitiveness for light, at the expense of collective production of seeds. humans reversed past natural selection and selected for shorter, higher - yielding genotypes. when genotypes were grown separately, shorter rice plants that allocated more resources to grain had higher yield than taller ones that allocated more resources to stems. in competition, however, the shorter genotype rapidly disappeared (jennings and de jesus 1968). moving beyond stem height, donald (1968) suggested many other improvements linked to competitiveness - versus - yield tradeoffs. ideotype have not necessarily emphasized such tradeoffs (rasmusson 1987), genetic increases in yield potential over decades have often coincided with donald 's suggestions. for example, higher - yielding wheat cultivars tend to have fewer stems per plant (austin., we extend donald 's (1968) ideas regarding the benefits of erect leaves. when the sun is overhead, crops with more - erect leaves spread the available sunlight over more leaf area. because photosynthesis tends to light - saturation, spreading the same amount of light over more leaf area can result in higher overall canopy photosynthesis. manipulation of leaf angle in soybean (kokubun 1988) and even in maize (pendleton. 1968) showed a yield benefit from erect leaves, even though light - saturation is less likely in c4 maize. interactions with nitrogen storage in leaves may also be important (sinclair and sheehy 1999). least favorable pattern of leaf inclination, which they attributed to earlier natural selection for aggressiveness. horizontal leaves may use light less efficiently, but they can shade neighboring competitors. indeed, erect - leaf cultivars are usually less competitive with weeds (tanner. we hypothesized that solar tracking, or diaheliotropism, in which leaves turn to face the sun (bonnet 1754), might have similar effects on canopy photosynthesis and competitiveness, throughout the day, as horizontal leaves do when the sun is overhead. ehleringer and forseth (1980) suggested that, if increased heat load is not a problem, then tracking in sparse canopies (e.g. isolated seedlings) would increase net photosynthesis by increasing total light interception. in dense canopies, however, they noted that solar tracking by upper leaves would reduce light available to lower leaves. we evaluated the effects of solar tracking on canopy photosynthesis, using an experiment and a computer model. the ideal comparison would be between two genotypes differing only in tracking, but these were not available. we therefore simulated this comparison, using rotation in the horizontal plane to temporarily change leaf orientation. we then compared light interception and net canopy co2 exchange in normal tracking mode to that with tracking disrupted. we argue below that this approach will overestimate the benefits of tracking, partly due to edge effects. therefore, we also simulated the effects of tracking, without edge effects, using a computer model. the effects of tracking on canopy photosynthesis were expected to depend on how sunlight and nitrogen are distributed with depth in the canopy. if tracking reduces light reaching lower leaves, then those leaves may lose more of their photosynthetic capacity (hodgkinson 1974), perhaps by transferring nitrogen or other resources to the upper leaves, than if they were shaded less (mooney and gulmon 1982). both the experiments and the model attempted to address possible longer - term effects of tracking on differences in leaf photosynthetic capacity with depth, in addition to the immediate effects of tracking on canopy photosynthesis. we measured the effects of solar tracking on light interception and net canopy photosynthesis in sparse and dense canopies of alfalfa, medicago sativa cv. weevlchek. plants were grown outdoors in tightly packed 8 8 arrays of 15-cm diameter pots in beckley, west virginia. plants were watered daily, fertilized weekly with a nutrient solution, and cut back to 5 cm height at intervals of 1532 days, as needed to prevent lodging. to test the effects of solar tracking during growth on maintenance of photosynthetic capacity in lower leaves, one 8 8 array of pots was turned twice daily during weeks of growth, to reduce the effects of tracking on shading (and photosynthetic capacity) of lower leaves. a control group was never turned, but was allowed to track normally each day, presumably shading lower leaves more than in the disrupted (turned - during - growth) treatment. canopy photosynthesis and light interception by plants from both treatments were measured separately, as weather allowed, using a flow - through chamber fitted with a clear acrylic top (fig. measurements were collected from a constructed alfalfa community consisting of 40 pots in tight hexagonal packing, approximately 0.8 m in total area, pots, taken preferentially but not exclusively from inner rows of the 8 8 continuous - tracking or turned - daily growth arrays to reduce edge effects, were placed in the chamber in the same orientation as during growth. co2 concentrations at the chamber output and input (supplemented so that concentration inside the chamber approximated ambient co2) were measured with an infrared gas analyzer. photosynthetically active radiation (par) beneath the canopy was measured with a line quantum sensor at soil surface height and compared with ambient par measured simultaneously with a spot quantum sensor outside the chamber. a slurry of water and ice in the lower, opaque, double wall of the chamber prevented condensation on the transparent top and maintained constant (1c) chamber air temperature within an experiment. all measurements were made on cloudless mornings, as diffuse light during cloudy weather would reduce the effects of solar tracking. solar elevation angles midway through experiments ranged from 40 to 44. leaf area index [lai, ratio of leaf area to land area (watson 1958) ] was estimated by destructive sampling of leaf area from three randomly selected pots, using a leaf - area meter, after each experiment. closing the lid on the photosynthesis chamber. about 45 min after closing the chamber, and then twice at 15-min intervals, the table supporting the plants in the chamber was rotated 180 in the horizontal plane. net photosynthesis rate was calculated as the difference in co2 concentration between inlet and outlet air, times the air flow rate through the chamber, divided by the total area occupied by the pots. to avoid confounding solar - tracking effects with turning - induced transients (see below), data for each 15-min period at a given orientation were fitted to the equation p(t) = a+btexp(t/), where p(t) is the co2 concentration difference (input minus outlet, proportional to photosynthesis rate) at time t, and a, b, and are constants. steady - state photosynthesis was calculated from the linear term at the end of each 15-min turn. the effect of solar tracking on net canopy photosynthesis was calculated as a ratio : the average of the two steady - state values in the tracking orientation divided by steady - state photosynthesis at the intervening turned orientation [applying this procedure to a nontracking control (half of plants placed in chamber in reversed orientation) gave an original : turned ratio of 1.004 0.012 ; mean sd, n = 3 ]. the relationship between lai and tracking effects on canopy photosynthesis (tracking : turned ratio) was determined by linear and second - order - polynomial regression. we used a published mechanistic computer simulation model, alfalfa (denison and loomis 1988), to simulate photosynthesis and light interception in the turning experiments described above, plus full - season simulations to predict the effect of solar tracking on annual forage production under field conditions. the model simulates growth of a field of identical alfalfa plants, simulating light interception, photosynthesis, transpiration, respiration, and growth of leaves, stems, and roots, with a 1-h time - step. fortran source code is available from the author in electronic form, as is a 73-page user manual which also includes the source code (37 pages). it is not possible to prove that such a complex model is even approximately accurate under all conditions, but testing during model validation found that ten of twelve harvest yields at two locations (ranging from 1.5 to 6 t / ha) were predicted within 10%, while seasonal yields in an irrigation experiment at a third location (ranging from 6 to 22 t / ha) were predicted with r = 0.99 (denison and loomis 1988). for the present paper, the light - interception and photosynthesis subroutine is particularly important, as only its predictions were compared to our measurements. (1967) simulates a multilayer canopy, with the number of layers and total leaf area increasing as the simulated crop grows taller. the fraction of solar radiation penetrating through each layer (i.e. not absorbed by leaves) was calculated from the cosine of incidence for the solar beam on leaves, cos(a), and the solar elevation, h, as e (duncan. 1967), light intercepted within a layer was assumed to be divided equally over leaf area in that layer. the nonlinear response of photosynthesis to the absorbed light was based on published data for alfalfa (sheehy. the model also includes temperature and water - stress effects on photosynthesis, but these would not affect the relative photosynthesis in tracking and disrupted orientations. to compare model output with our experiments, we first generated virtual alfalfa canopies with a range of lai values, by letting the model simulate growth until each target lai was reached. we then restarted the model using the state variables (stem height, size of individual leaves, etc.) corresponding to each target lai and simulated net canopy photosynthesis and light interception using solar elevations similar to that in the experiments, with cos(a) = 0.5 or 0.6 for nontracking and tracking orientations, respectively. these values were chosen to match overall canopy light penetration to actual measurements. to simulate the effects of light distribution in the canopy affecting leaf photosynthetic capacity, potential (light - saturated) photosynthesis was assumed to decrease linearly with physiological age (to zero at day 35), reducing the simulated photosynthesis of lower leaves. as a best - case alternative, we also ran the simulations with no decrease in photosynthetic capacity with leaf age. full - season simulations to estimate the effects of tracking on seasonal forage yield used the same cultivar- and location - specific parameters used in model validation (denison and loomis 1988), except that leaves in all layers were assumed to maintain cos(a) of either 0.6 (tracking) or 0.5 (nontracking), as explained above. six cuttings were simulated, which is typical for the california locations where the model was developed and validated. we measured the effects of solar tracking on light interception and net canopy photosynthesis in sparse and dense canopies of alfalfa, medicago sativa cv. weevlchek. plants were grown outdoors in tightly packed 8 8 arrays of 15-cm diameter pots in beckley, west virginia. plants were watered daily, fertilized weekly with a nutrient solution, and cut back to 5 cm height at intervals of 1532 days, as needed to prevent lodging. to test the effects of solar tracking during growth on maintenance of photosynthetic capacity in lower leaves, one 8 8 array of pots was turned twice daily during weeks of growth, to reduce the effects of tracking on shading (and photosynthetic capacity) of lower leaves. a control group was never turned, but was allowed to track normally each day, presumably shading lower leaves more than in the disrupted (turned - during - growth) treatment. canopy photosynthesis and light interception by plants from both treatments were measured separately, as weather allowed, using a flow - through chamber fitted with a clear acrylic top (fig. measurements were collected from a constructed alfalfa community consisting of 40 pots in tight hexagonal packing, approximately 0.8 m in total area, pots, taken preferentially but not exclusively from inner rows of the 8 8 continuous - tracking or turned - daily growth arrays to reduce edge effects, were placed in the chamber in the same orientation as during growth. co2 concentrations at the chamber output and input (supplemented so that concentration inside the chamber approximated ambient co2) were measured with an infrared gas analyzer. photosynthetically active radiation (par) beneath the canopy was measured with a line quantum sensor at soil surface height and compared with ambient par measured simultaneously with a spot quantum sensor outside the chamber. a slurry of water and ice in the lower, opaque, double wall of the chamber prevented condensation on the transparent top and maintained constant (1c) chamber air temperature within an experiment. all measurements were made on cloudless mornings, as diffuse light during cloudy weather would reduce the effects of solar tracking. solar elevation angles midway through experiments ranged from 40 to 44. leaf area index [lai, ratio of leaf area to land area (watson 1958) ] was estimated by destructive sampling of leaf area from three randomly selected pots, using a leaf - area meter, after each experiment. closing the lid on the photosynthesis chamber. about 45 min after closing the chamber, and then twice at 15-min intervals, the table supporting the plants in the chamber was rotated 180 in the horizontal plane. net photosynthesis rate was calculated as the difference in co2 concentration between inlet and outlet air, times the air flow rate through the chamber, divided by the total area occupied by the pots. to avoid confounding solar - tracking effects with turning - induced transients (see below), data for each 15-min period at a given orientation were fitted to the equation p(t) = a+btexp(t/), where p(t) is the co2 concentration difference (input minus outlet, proportional to photosynthesis rate) at time t, and a, b, and are constants. steady - state photosynthesis was calculated from the linear term at the end of each 15-min turn. the effect of solar tracking on net canopy photosynthesis was calculated as a ratio : the average of the two steady - state values in the tracking orientation divided by steady - state photosynthesis at the intervening turned orientation [applying this procedure to a nontracking control (half of plants placed in chamber in reversed orientation) gave an original : turned ratio of 1.004 0.012 ; mean sd, n = 3 ]. the relationship between lai and tracking effects on canopy photosynthesis (tracking : turned ratio) was determined by linear and second - order - polynomial regression. we used a published mechanistic computer simulation model, alfalfa (denison and loomis 1988), to simulate photosynthesis and light interception in the turning experiments described above, plus full - season simulations to predict the effect of solar tracking on annual forage production under field conditions. the model simulates growth of a field of identical alfalfa plants, simulating light interception, photosynthesis, transpiration, respiration, and growth of leaves, stems, and roots, with a 1-h time - step. fortran source code is available from the author in electronic form, as is a 73-page user manual which also includes the source code (37 pages). it is not possible to prove that such a complex model is even approximately accurate under all conditions, but testing during model validation found that ten of twelve harvest yields at two locations (ranging from 1.5 to 6 t / ha) were predicted within 10%, while seasonal yields in an irrigation experiment at a third location (ranging from 6 to 22 t / ha) were predicted with r = 0.99 (denison and loomis 1988). for the present paper, the light - interception and photosynthesis subroutine is particularly important, as only its predictions were compared to our measurements. (1967) simulates a multilayer canopy, with the number of layers and total leaf area increasing as the simulated crop grows taller. the fraction of solar radiation penetrating through each layer (i.e. not absorbed by leaves) was calculated from the cosine of incidence for the solar beam on leaves, cos(a), and the solar elevation, h, as e (duncan. 1967), light intercepted within a layer was assumed to be divided equally over leaf area in that layer. the nonlinear response of photosynthesis to the absorbed light was based on published data for alfalfa (sheehy. the model also includes temperature and water - stress effects on photosynthesis, but these would not affect the relative photosynthesis in tracking and disrupted orientations. to compare model output with our experiments, we first generated virtual alfalfa canopies with a range of lai values, by letting the model simulate growth until each target lai was reached. we then restarted the model using the state variables (stem height, size of individual leaves, etc.) corresponding to each target lai and simulated net canopy photosynthesis and light interception using solar elevations similar to that in the experiments, with cos(a) = 0.5 or 0.6 for nontracking and tracking orientations, respectively. these values were chosen to match overall canopy light penetration to actual measurements. to simulate the effects of light distribution in the canopy affecting leaf photosynthetic capacity, potential (light - saturated) photosynthesis was assumed to decrease linearly with physiological age (to zero at day 35), reducing the simulated photosynthesis of lower leaves. as a best - case alternative, we also ran the simulations with no decrease in photosynthetic capacity with leaf age. full - season simulations to estimate the effects of tracking on seasonal forage yield used the same cultivar- and location - specific parameters used in model validation (denison and loomis 1988), except that leaves in all layers were assumed to maintain cos(a) of either 0.6 (tracking) or 0.5 (nontracking), as explained above. six cuttings were simulated, which is typical for the california locations where the model was developed and validated. disrupting solar tracking, by turning plants 180 out of their normal tracking orientation, usually increased light penetration through the canopy, as exemplified by fig. 2a for a canopy with lai of 6.3. of 15 experiments, only the one at the lowest lai failed to show increased light penetration with disruption of solar tracking and tracking effects increased with lai (fig. light - penetration results were similar for plants that had or had not been turned twice daily during growth (filled vs. open circles in fig. either to or from the original tracking orientation) resulted in a transient decrease in photosynthesis. shading half of the chamber with an opaque cover, then moving the cover to shade the other half, gave qualitatively similar transient decreases in photosynthesis (data not shown). this phenomenon may result from an induction requirement for photosynthesis in shaded leaves moving into sunflecks (pearcy. (a) photosynthetically active radiation reaching the soil surface beneath solar - tracking alfalfa plants, with solar - tracking disrupted by 180 rotation from minutes 1530. (b) net co2 uptake for the same plant community, with fitted curves used to estimate steady - state photosynthesis at each orientation. (a) effects of tracking (ratio of tracking : turned) on light reaching soil surface below constructed canopies of alfalfa plants differing in lai (leaf - area index ; ratio of leaf area to ground area). plants had been grown for weeks with (solid circles) or without (open circles) twice - daily turning to reduce shading of lower leaves by solar - tracking upper leaves during growth. (b) effects of solar tracking on net canopy photosynthesis (ratio of tracking : turned). lines are regression for plants that had been turned during growth (solid) or not turned during growth (dotted), and model predictions, with (short dash) and without (long dash) decreased leaf photosynthetic capacity with age. the effects of tracking on canopy photosynthesis varied with lai (fig. 3b is the ratio of photosynthesis in the natural tracking orientation, divided by photosynthesis of the same plants in the turned (tracking - disrupted) orientation, with each ratio calculated from data similar to fig. open circles show tracking : turned photosynthesis ratios for plants that were never turned during growth. closed circles show tracking : turned photosynthesis ratios for plants that had been turned twice per day, over weeks of growth, to disrupt solar tracking and allow the greater illumination of lower leaves expected in a hypothetical nontracking cultivar. the most important results were qualitatively similar for plants from both pretreatments (turned or not turned during growth) : direct effects of tracking on photosynthesis were small but positive for sparse (low - lai) canopies, becoming small but negative as lai increased (fig. 3b). for plants turned during growth (filled circles), the second - order - polynomial term was not significant. the maximum canopy - photosynthesis benefit for solar tracking (extrapolated to lai = 0) was about 3% and tracking reduced canopy photosynthesis above lai = 4.5. for plants not turned during growth, there was considerably more scatter, so that two measurements with similar lai often differed greatly. the second - order term in the regression shown was statistically significant (p < 0.05) with r = 0.71. 3b) generally showed a greater photosynthetic cost of tracking at high lai than was found experimentally. this was particularly true when it was assumed (lower, long - dash line) that lower leaves lost none of their original photosynthetic capacity with age and depth in the canopy. in the season - long simulation, the model predicted about 5% higher seasonal forage yield, 27 712 kg / ha, when we assumed less tracking, cos(a) = 0.5, relative to 26 468 kg / ha with the greater tracking, cos(a) = 0.6, more representative of existing cultivars. the discussion of experimental results will focus on plants that were turned twice daily during growth, because their relationship between lai and tracking effects on photosynthesis was more consistent than for plants not turned during growth (fig. plants not turned during growth may have had increased pot to pot variability in the photosynthetic capacity of lower leaves due to greater light penetration along the edge of the synthetic canopy relative to pots in the interior of the canopy (edge effects) as the plants grew undisturbed. by temporarily disrupting solar tracking, twice - daily turning during growth increased light penetration deeper into the canopy, which presumably helped to maintain photosynthetic capacity in lower leaves and reduced pot to pot variability in this characteristic. since some photosynthesis measurements included more edge - grown plants than others, the measured photosynthetic opportunity cost of tracking was more variable in experiments using plants that were not turned during growth. 3b), shading during growth in the not - turned pretreatment may have reduced the photosynthetic capacity of lower leaves well below what it would be in a nontracking cultivar, eliminating some potential photosynthetic benefit of disrupting solar tracking. under our well - watered conditions, solar tracking in alfalfa was found to increase light interception, relative to the disrupted leaf orientation achieved by turning in the horizontal plane. this would not necessarily be true for plants under drought conditions (berg and hsiao 1986). diaheliotropism increased net canopy photosynthesis at low lai in our experiment, but only by a few percent. for lai around 1, model and experiment agreed on a photosynthetic benefit of about 2% from tracking (fig. model and experiment also agreed that this benefit decreased with increasing lai, but there were quantitative discrepancies between model and experiment at high lai. when a model disagrees with reality, the model is clearly wrong. in this case, however, our artificial community of alfalfa plants in pots is itself a model system. in contrast to the field situation, a significant fraction of the light reaching our lower leaves came from the side, rather than down through the canopy. this extra light means that lower leaves would experience less tracking - induced - shading cost than if they were completely surrounded by other plants. it is therefore likely that, at high lai, the experiments somewhat underestimated the photosynthetic cost of solar tracking. it is possible, therefore that the computer model gave a more accurate picture of tracking effects than the experiments with the model plant community. this hypothesis could be tested using even smaller model plant communities (20 pots rather than 40, say), which would be predicted to give similar results at low lai, but with little or no cost of tracking at high lai. the whole - season simulations suggested a 5% photosynthetic cost to solar tracking. in these simulations, benefits at low lai were outweighed by costs at high lai, even with six cuttings per year. alfalfa cut fewer times per year could spend less time in the low - lai state typical of early regrowth, so the net photosynthetic cost of solar tracking could be even greater. together, model and experiment show that the photosynthetic benefits of solar tracking are low for alfalfa and that tracking at high lai is more likely to reduce canopy photosynthesis than to increase it. heliotropism is a complex behavior dependent on many genes, so loss - of - function mutations that eliminate tracking must have arisen repeatedly over the course of evolution. the evolutionary persistence of solar tracking, despite its apparent photosynthetic cost at high lai, therefore requires explanation. one possibility is that photosynthetic benefits of tracking by seedlings or other low - lai (e.g. recently grazed) plants are sufficient to maintain diaheliotropism, despite its photosynthetic costs once plants grow larger and start shading their own lower leaves. this would be analogous to antagonistic pleiotropy maintaining alleles for early reproduction at the expense of longevity (williams 1957). travis and reed (1983) reported that cos(a) changes with depth in the canopy, but unfortunately the equation they used to calculate cos(a) from their leaf - angle data is incorrect (comstock and mahall 1985). an alternative hypothesis is that solar tracking even in dense canopies makes positive contributions to lifetime fitness. at lai = 5, a diaheliotropic plant might reduce light available to seedlings growing in its shade to 80% of that below a nontracking plant (fig. 3a), reducing their photosynthesis nearly 20%, while reducing its own photosynthesis by less than 1% (fig. sacrificing any fitness to injure another member of the same species would be an example of spite (hamilton 1970). however, a perennial plant may receive direct, albeit delayed, fitness benefits from suppressing potential competitors, in which case solar tracking would not be an example of spite. furthermore, the shaded competitors could often be members of other species, even in agriculture. our experiments used monospecific stands of alfalfa, but real alfalfa fields usually include weeds. alfalfa plants that track the sun are more competitive against fellow alfalfa plants, which may reduce potential yield by 5% or more, but tracking plants are also more competitive against weeds. a nontracking alfalfa cultivar, or one with reduced tracking at high lai, might be useful only in fields with excellent weed control.	despite the optimism of some molecular biologists, natural selection among the wild ancestors of crops is unlikely to have missed simple genetic improvements that would consistently have enhanced individual fitness. tradeoff - free opportunities for further improvement of crop traits like photosynthetic efficiency or drought tolerance may therefore be elusive. opportunities linked to acceptable tradeoffs may be abundant, however. tradeoffs between individual competitiveness and the collective productivity of plant communities (e.g. those linked to height) have been key to past increases in yield potential. solar tracking by leaves could involve such tradeoffs, if photosynthetic benefits to tracking leaves are outweighed by increased shading of leaves lower in the canopy. this hypothesis was tested using rotation in the horizontal plane to disrupt solar tracking in alfalfa. in sparse canopies, solar tracking increased net canopy photosynthesis, but rarely by more than 3%. as leaf area increased, solar tracking tended to decrease net canopy photosynthesis, despite edge effects in our 1-m2 artificial communities, which probably exaggerated net photosynthetic benefits of tracking. computer modeling suggested that the season - long effects of solar tracking on community productivity can be negative. solar tracking may have persisted, nonetheless, because individuals whose leaves track the sun increase shading of competitors.
a total of 97 participants participated in the study, 54 native left - to - right readers and 43 right - to - left readers (table 1). all participants were fluent in english, and seventy - five of the participants, including the 43 right - to - left readers, were bilingual. of the native left - to - right readers, 30 were male (m = 25.8 years, sd = 5.5 years), and of the native right - to - left readers 20 were male (m = 25.2 years, sd = 5.3 years). all procedures received ethical approval from the university of saskatchewan behavioural research ethics board and all participants were recruited from the university of saskatchewan through posters. all participants were paid for their participation in the study.table 1participant s native languageltor reading languagesnumber of participantsrtol reading languagesnumber of participantsenglish22urdu19hindi14persian12chinese6arabic8bengali3aramaic1korean2hebrew1malayalam2pashto1chichewa1punjabi1oriya1ukrainian1russian1vietnamese1the native languages of left - to - right (ltor) readers and the corresponding number of participants, as well as the native languages of right - to - left (rtol) readers and the corresponding number of participants participant s native language the native languages of left - to - right (ltor) readers and the corresponding number of participants, as well as the native languages of right - to - left (rtol) readers and the corresponding number of participants participants simultaneously viewed two greyscales stimuli (fig. 1) on a 17 inch crt monitor. the horizontal midlines of the stimuli were aligned with the middle of the screen with a vertical distance of 100 pixels between the upper and lower stimulus. two reversed luminance gradients that changed in brightness from one end to the other were outlined by a thin black outline and shown against a grey background. the stimuli measured 79 pixels high and changed in brightness over 80 increments, creating stimuli changing from black at one end to white at the other. the vertical position of the pixels within each increment was randomized to create a smooth change in brightness and create slight differences in the stimuli. the rectangles were presented as mirror reversals one on top of the other, but were equiluminant at a global level. the stimuli were presented in six different lengths : 320, 400, 480, 560, 640, and 720 pixels.fig. 1sample stimulus pairs with opposite orientations from the greyscales task. both a and b are 400 pixels long, but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left. a left response results from the participant choosing the stimulus with the darker feature on the left, irrespective of whether the stimulus is on the top or bottom sample stimulus pairs with opposite orientations from the greyscales task. both a and b are 400 pixels long, but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left. a left response results from the participant choosing the stimulus with the darker feature on the left, irrespective of whether the stimulus is on the top or bottom the task consisted of 96 trials that were presented in a pseudo - randomized order. the combinations of length (320, 400, 480, 560, 640, and 720 pixels) and stimulus orientation (upper stimulus dark on left / lower stimulus dark on right and vice versa) were repeated four times. participants were seated 500 mm from the computer with the center of the monitor located along their midline. participants were asked to determine which stimulus appeared darker overall, and responses were made using the numbers 8 and 2 on the number pad. a simple button press was used to limit the amount of motor movement evoked by the task (mccourt and olafson 1997). the participants responses were categorized based on which stimulus they selected as having the darker feature on the left or the right irrespective of whether it was on the top or bottom. a leftward response was indicated when the participant chose the stimulus with the darker feature on the left, irrespective of whether the stimulus was situated on the top or bottom, whereas a rightward response was indicated when the participant chose the stimulus with the darker feature on the right, irrespective of whether the stimulus was situated on the top or bottom. the response bias was calculated by subtracting the number of leftward responses from the number of rightward responses and could range from 96 to + 96 ; hence a negative score indicated a leftward bias. participants were initially seated in a windowless room with overhead lighting and gave written consent to participate in the study. prior to completing the greyscales task, participants completed a demographic questionnaire addressing sex, age, native language, visual or hearing impairments, and handedness and footedness (elias. a total of 97 participants participated in the study, 54 native left - to - right readers and 43 right - to - left readers (table 1). all participants were fluent in english, and seventy - five of the participants, including the 43 right - to - left readers, were bilingual. of the native left - to - right readers, 30 were male (m = 25.8 years, sd = 5.5 years), and of the native right - to - left readers 20 were male (m = 25.2 years, sd = 5.3 years). all procedures received ethical approval from the university of saskatchewan behavioural research ethics board and all participants were recruited from the university of saskatchewan through posters. all participants were paid for their participation in the study.table 1participant s native languageltor reading languagesnumber of participantsrtol reading languagesnumber of participantsenglish22urdu19hindi14persian12chinese6arabic8bengali3aramaic1korean2hebrew1malayalam2pashto1chichewa1punjabi1oriya1ukrainian1russian1vietnamese1the native languages of left - to - right (ltor) readers and the corresponding number of participants, as well as the native languages of right - to - left (rtol) readers and the corresponding number of participants participant s native language the native languages of left - to - right (ltor) readers and the corresponding number of participants, as well as the native languages of right - to - left (rtol) readers and the corresponding number of participants participants simultaneously viewed two greyscales stimuli (fig. 1) on a 17 inch crt monitor. the horizontal midlines of the stimuli were aligned with the middle of the screen with a vertical distance of 100 pixels between the upper and lower stimulus. two reversed luminance gradients that changed in brightness from one end to the other were outlined by a thin black outline and shown against a grey background. the stimuli measured 79 pixels high and changed in brightness over 80 increments, creating stimuli changing from black at one end to white at the other. the vertical position of the pixels within each increment was randomized to create a smooth change in brightness and create slight differences in the stimuli. the rectangles were presented as mirror reversals one on top of the other, but were equiluminant at a global level. the stimuli were presented in six different lengths : 320, 400, 480, 560, 640, and 720 pixels.fig. 1sample stimulus pairs with opposite orientations from the greyscales task. both a and b are 400 pixels long, but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left. a left response results from the participant choosing the stimulus with the darker feature on the left, irrespective of whether the stimulus is on the top or bottom sample stimulus pairs with opposite orientations from the greyscales task. both a and b are 400 pixels long, but stimuli a is positioned with the upper stimulus dark on left and lower stimulus dark on right where as stimuli b is positioned with the upper stimulus dark on the right and lower stimulus dark on the left. a left response results from the participant choosing the stimulus with the darker feature on the left, irrespective of whether the stimulus is on the top or bottom the task consisted of 96 trials that were presented in a pseudo - randomized order. the combinations of length (320, 400, 480, 560, 640, and 720 pixels) and stimulus orientation (upper stimulus dark on left / lower stimulus dark on right and vice versa) were repeated four times. participants were seated 500 mm from the computer with the center of the monitor located along their midline. participants were asked to determine which stimulus appeared darker overall, and responses were made using the numbers 8 and 2 on the number pad. a simple button press was used to limit the amount of motor movement evoked by the task (mccourt and olafson 1997). the participants responses were categorized based on which stimulus they selected as having the darker feature on the left or the right irrespective of whether it was on the top or bottom. a leftward response was indicated when the participant chose the stimulus with the darker feature on the left, irrespective of whether the stimulus was situated on the top or bottom, whereas a rightward response was indicated when the participant chose the stimulus with the darker feature on the right, irrespective of whether the stimulus was situated on the top or bottom. the response bias was calculated by subtracting the number of leftward responses from the number of rightward responses and could range from 96 to + 96 ; hence a negative score indicated a leftward bias. participants were initially seated in a windowless room with overhead lighting and gave written consent to participate in the study. prior to completing the greyscales task, participants completed a demographic questionnaire addressing sex, age, native language, visual or hearing impairments, and handedness and footedness (elias. the response bias was analyzed with a 2 (reading direction [left - to - right, right - to - left ]) 2 (sex [male, female ]) repeated - measures anova. there was a significant effect for reading direction, f(1, 93) = 8.489, p =.004, 2 =.0894, but no significant effect of sex f(1, 93) =.026, p =.872, 2 =.0003, and no interaction between the two factors, f(1, 93) =.049, p =.825, 2 =.0005. the native left - to - right readers demonstrated a larger response bias (m = 34.6, sd = 37.67) compared to the right - to - left readers (m =.99, sd = 42.06) (fig. 2). one - sample t tests, compared to the test - value of zero, were conducted to evaluate whether a significant side bias was observed ; left - to - right readers demonstrated a leftward response bias, t(53) = 5.097, p <.001, 95 % ci 48.28, 21.46, whereas right - to - left readers failed to demonstrate a significant bias t(42) =.109, p =.914, 95 % ci 19.50, 17.45.fig. 2the mean response bias for the greyscales task demonstrated by native left - to - right (ltor) and native right - to - left (rtol) readers. a negative score indicates a preference for the darkest edge of the equiluminant gradient stimulus pair to be located on the left. error bars represent standard error of the mean the mean response bias for the greyscales task demonstrated by native left - to - right (ltor) and native right - to - left (rtol) readers. a negative score indicates a preference for the darkest edge of the equiluminant gradient stimulus pair to be located on the left. error bars represent standard error of the mean additionally, the mean reaction time for left and right responses was analyzed with a 2 (reading direction [left - to - right, right - to - left ]) 2 (response choice [right, left ]) mixed - measures anova (fig. there was no significant difference between the reaction time of left - to - right and right - to - left readers responses, f(1,95) =.079, p =.779, 2 =.0008, and no interaction between the two factors, f(1, 95) =.091, p =.763, 2 =.0009, however there was a significant difference between the reaction time for left and right choices, f(1, 95) = 4.029, p =.048, 2 =.0424, with leftward responses (m = 2755.98, sd = 1736.18) occurring faster than rightward responses (m = 2939.12, sd = 1841.54).fig. 3the mean reaction times for left - to - right (ltor) and right - to - left (rtol) readers left and right response choices. error bars represent standard error of the mean the mean reaction times for left - to - right (ltor) and right - to - left (rtol) readers left and right response choices. in accord with a converging body of research examining left - to - right and right - to - left readers performance on a variety of visuospatial tasks, the reading groups demonstrated different bias strengths for the greyscales task. consistent with previous research employing the greyscales task, native left - to - right readers in the present study demonstrated a statistically significant leftward bias (mattingley. however, inconsistent with nicholls and roberts (2002) findings, native right - to - left readers failed to demonstrate a significant leftward bias.. the sample of hebrew readers examined by nicholls and roberts (2002) may have had increased exposure to left - to - right scanning order, leading to their null results. hebrew children have been found to display weaker right - to - left tendencies compared to arabic children (kugelmass and lieblich 1979), potentially resulting from the left - to - right notation of arithmetic, music, and writing of some individual letters in hebrew (vaid and singh 1989). increased exposure to left - to - right notation, especially if hebrew was not nicholls and roberts (2002) participants native language, would have influenced the participants scanning tendencies and eye movement exploration, increasing the likelihood that a leftward bias will be observed (abed 1991 ; chokron and imbert 1993). the fact that participants with contrasting native reading directions performed differently on the greyscales task, demonstrates that scanning tendencies influence eye movement exploration and orientation of attention. eye tracking experiments examining left - to - right readers have found reliable initial saccades at the beginning of visual exploration to the left side of a image followed by a longer and weaker bias to the right (dickinson and intraub 2009 ; foulsham. 2014), even in the presence of a right - sided target (nuthmann and matthias 2013). however, these initial saccades to the left during scene viewing and line bisection task can be manipulated by the shape of a gaze - contingent window (foulsham. limited research has examined right - to - left readers visual exploration with eye tracking, however, smith (2013) explored eye movements of right - to - left and left - to - right readers during a target finding task and observed that left - to - right readers identified targets the quickest when located in the upper left quadrant, whereas right - to - left readers had near identical identification times for targets located in the upper - right and upper - left quadrants. thus native scanning habits and manipulation of viewing contingencies appear to influence initial fixation and location of attention. the influence of scanning habits on attention asymmetries observed in the current study is consistent with literature examining the influence of scanning habits on visuospatial tasks. left - to - right and right - to - left readers have demonstrated significantly different perceptual biases in line bisection tasks (chokron. 1997 ; chokron and imbert 1993 ; chokron and de agostini 1995) ; aesthetic preference tasks (chokron and de agostini 2000 ; friedrich. 2014 ; ishii. 2011 ; nachson. 1999 ; perez gonzalez 2012), and lighting tasks (smith and elias 2013). in addition to natural scanning habits developed from reading direction, manipulation of scanning direction during line bisection tasks (brodie and pettigrew 1996), and face perception (butler and harvey 2006) have also been found to influence the direction and magnitude of the perceptual bias. visuospatial tasks are perceptual, however, the strategies used to complete visuospatial tasks influence orientation of attention across the visual field (thomas. 2014). pesudoneglect is often explained by the preferential activation of the right hemisphere that distributes attention to the left visual field and increases the salience of features located in the left hemispace the activation - orienting hypothesis (kinsbourne 1970). manipulating orientation of attention with cues during visuospatial tasks systematically biases perception leading participants to commonly overestimate portions of space to which their attention is directed, and thus spatial distribution of attention and attentional asymmetries have been proposed to underlie and influence perceptual biases (milner. furthermore, orientation influences perceptual biases in right unilateral spatial neglect patients, as cues located on the left of visual spatial tasks annul rightward displacement (nichelli. 1989). specifically, the typical leftward response bias observed during the greyscales task is reversed by attentional cues, and the leftward response bias is insensitive to changes in the presentation of the stimuli (nicholls. 2004 ; nicholls. these converging results have lead researchers to suggest that the greyscales task is a robust and sensitive measure to attentional biases, which appear, based on the findings in the current experiment, influenced by scanning habits and reading direction. the underlying neurological mechanisms responsible for the increased salience of features located in the left hemispace is currently debated (de schotten. however, right - to - left scanning habits developed from reading direction appears to interact with spatial attention that is oriented to the left hemispace, resulting in no perceptual bias. thus left - to - right scanning habits developed from reading direction strengthen the lateralized bias whereas right - to - left scanning habits weaken the bias. this pattern is evident in the current study as both reading groups responded faster to leftward choices resulting from increased attention to the left hemifield, but the salience of stimulus with the darkest feature located on the left was weakened when participants native language read from right - to - left. hence, the results of the current study lead us to argue that the strength of perceptual biases is influenced by behavioural biases, such as scanning habits, and neural and anatomical asymmetries in the right parietal and frontal cortices (szczepanski and kastner 2013 ; de schotten. 2011). to further isolate the influence of reading direction and scanning habits on perceptual biases researchers should examine homogenous sample groups to ensure that cultural differences are not contributing to the different response biases found. ideally, research examining left - to - right and right - to - left reading groups should examine groups that have extralinguistic (e.g. context of usage, direction of arithmetic and music) and linguistic similarities (e.g. phonology and grammar), as well as a common geographical location and cultural foundation to minimize the confounding effects of cultural influences on reading direction. based on our findings, examining two samples with similar cultural values, but who also differ in reading direction understanding how perceptual asymmetries manifest, regardless of their underlying cause, is essential when studying behaviour as a marker of cognitive processes. patients with right unilateral hemispheric damage have been found to demonstrate a stronger rightward bias during the greyscales task (mattingley. 2004), leading researchers to suggest that the greyscales task is a highly sensitive and efficient tool for assessing pathological perceptual and attentional biases (mattingley. 1994). however, the altered response bias for right - to - left readers compared to left - to - right readers questions the strength and reliability of the leftward bias displayed by healthy participants, as the leftward bias is not consistent and is affected by learned cultural factors such as reading direction. thus, clinicians should exhibit caution when using abnormal performance on the greyscales task to aid diagnosis of neglect, specifically when the patient s native language reads from right - to - left. furthermore, research should examine right - to - left reading populations to determine the normal range of bias scores for right - to - left readers to increase the clinical utility of the greyscales task.	reliable leftward attentional and perceptual biases demonstrated in a variety of visuospatial tasks have been found to deviate from the left in research examining the influence of scanning habits. the aim of the current research was to examine the influence of native script direction on pseudoneglect during the greyscales task in a representative sample of native right - to - left readers. fifty - four native left - to - right readers and 43 right - to - left readers completed the greyscales task, which required judging the darker of two left right mirrored brightness gradients. native left - to - right readers demonstrated a left response bias on the greyscales task, whereas right - to - left readers failed to demonstrate a bias, however, both groups responded more quickly when making leftward choices. the research suggests that the strength of attentional biases are influenced by behavioural biases, such as scanning habits, and neural and anatomical asymmetries in the right parietal and frontal cortices. thus, to improve the clinical utility of the greyscales task for diagnosing neglect, right - to - left readers should be examined to fully understand the normal range of biases displayed by neurologically healthy individuals.
the relationship between malocclusion and facial form has been a focus of orthodontists since early 20 century. dental arch width and facial form are important factors for determining success and stability of orthodontic treatment. arch form is the position and relationship of teeth to each other in all three dimensions. according to hawley, ideal arch width was based on an equilateral triangle with a base representing the inter - condylar width. the lower anterior teeth were arranged on an arc of a circle with a radius determined by the combined width of the lower incisors and canines, with the premolars and molars aligned with the second and third molars toward the center. facial morphology has long been accepted to be the result of each person 's genotype and its phenotypic expression. it is also commonly believed that there is interaction between the functional capacity and the size of masticatory muscles and craniofacial form. those with long face have excessive vertical facial growth which is usually associated with an anterior open bite, increased sella - nasion mandibular plane (sn - mp) angle, increased gonial angle, and increased maxillary / mandibular plane angle. the short face types have reduced vertical growth that is accompanied by a deep overbite, reduced facial heights, and reduced sn - mp angle. between the two types noted that the mean maxillary and mandibular arch circumferences and mandibular inter - molar width were greater in subjects with low mp - sn angle. in their study, the subjects were adolescents without discussion of gender and ethnicity. using posteroanterior cephalograms, christie found that adult brachyfacial males, when compared with standard males had greater maxillary and mandibular widths. no difference, however, was found in arch widths of brachyfacial versus standard females. in terms of difference in arch width between males and females, wei evaluated posteroanterior cephalograms of chinese adults. correct identification of a patients arch form is an important aspect of achieving a stable, functional and aesthetic orthodontic treatment result ; failure to preserve the arch form might increase the probability of relapse. improper arch wire changes can result in periodontal breakdown, recurrence of crowding of buccal segments, or increased crowding of labial segments particularly when inter - canine width and inter - molar width have been expanded. the original arch form for straight wire appliance was determined based on the mean dental arch form of orthodontically untreated normal occlusal samples of us population. most of the orthodontic arch wires are designed in the usa and have been distributed all over the world without much research. therefore, even with latest orthodontic appliances, education in the biological diversity of our patients and reasonable technical training for arch wire fabrication and adjustment are still essential in advanced orthodontic programs. one genetic factor contributing to the dental arch form and facial type is the patient 's ethnic background. the purpose of this study is to investigate whether dental arch widths are correlated with vertical facial types (mp - sn angle) and whether there are any differences in arch widths between untreated adult males and females in south indian population. a total of 180 untreated patients above 18 years old were employed in the study. the samples employed in the study consisted of two groups, males and females, of 90 patients each. each group was further divided into low angle [figures 3a, 4a ], average angle, [figures 3b, 4b ] and high angle [figures 3c, 4c ]. subjects with a full dentition were included in the study (except third molars). the exclusion criteria were previous orthodontic treatment, edentulous spaces, history of trauma, significant cuspal wear, extensive restorations or prosthetics, anterior and posterior cross bites, and severe crowding (> 9 mm) or spacing (> 9 mm). the lateral cephalograms were traced individually and sella nasion pointa sna, sella nasion pointb snb, pointa nasion pointb anb, and sn - mp were measured. the dental arch width was measured on the dental cast using a digital calliper accurate to 0.001 mm. maxillary study model (arch width measurements) tooth size - arch length discrepancy on study model low - angle male : casts, lateral cephalogram, photographs average - angle male : casts, lateral cephalogram, photographs high - angle male : casts, lateral cephalogram, photographs low - angle female : casts, lateral cephalogram, photographs average - angle female : casts, lateral cephalogram, photographs high - angle female : casts, lateral cephalogram, photographs the following maxillary and mandibular dimensions were measured [figure 1 ]. inter - canine width (buccal cusp tip and widest labial aspect),first and second inter - premolar width (buccal cusp tip and widest labial aspect),first inter - molar widths (mesiobuccal cusp, central fossa, widest buccal, and narrowest lingual aspect). inter - canine width (buccal cusp tip and widest labial aspect), first and second inter - premolar width (buccal cusp tip and widest labial aspect), first inter - molar widths (mesiobuccal cusp, central fossa, widest buccal, and narrowest lingual aspect). tooth size - arch length discrepancy was calculated by first determining the arch length available and then subtracting the sum of mesiodistal widths of teeth anterior to first molar from it [figure 2 ]. descriptive statistics, including mean and standard deviation (sd) were calculated for all measurements. a student 's two - tailed t - test was used to determine whether the differences in measurements between male and female groups were significant. analysis of variance anova test was carried out to show that inter - arch width varies significantly with different levels of mp - sn. regression analyses were carried out to predict arch widths with known values of mp - sn. -coefficients were taken to check whether the relation between inter - arch widths and mp - sn is inverse or direct. in order to evaluate intraexaminer error, lateral cephalograms and models of 15 males and 15 females were re - measured after 4 weeks, and their mean differences were assessed with paired t - test and compared with pearson 's correlation coefficients. tooth size - arch length discrepancy was calculated by first determining the arch length available and then subtracting the sum of mesiodistal widths of teeth anterior to first molar from it [figure 2 ]. descriptive statistics, including mean and standard deviation (sd) were calculated for all measurements. a student 's two - tailed t - test was used to determine whether the differences in measurements between male and female groups were significant. analysis of variance anova test was carried out to show that inter - arch width varies significantly with different levels of mp - sn. regression analyses were carried out to predict arch widths with known values of mp - sn. -coefficients were taken to check whether the relation between inter - arch widths and mp - sn is inverse or direct. in order to evaluate intraexaminer error, lateral cephalograms and models of 15 males and 15 females were re - measured after 4 weeks, and their mean differences were assessed with paired t - test and compared with pearson 's correlation coefficients. anova statistics is done to compare the arch widths of the samples at different levels of mp - sn. the result showed that the arch width significantly varies at different levels of mp - sn (p < 0.05). the mean arch widths at maxillary canine (cusp tip) for males are 31.72, 32.66, and 33.35, respectively, for high, average, and low mp - sn values. anova statistics (p < 0.05) shows that the arch width at inter - canine cusp tip varies significantly at different levels of mp - sn. similar statistical results were obtained in all dental arch width measurements on maxillary and mandibular arches in both males and females. regression analysis was used to predict arch widths, with known values of mp - sn (independent variable). -coefficients for arch widths were calculated and results show that as mp - sn increases, the dental arch widths decreases. the -coefficient for arch width for males at maxillary inter - canine at cusp tip is 0.848. it indicates that as mp - sn increases, the dental arch width at canine cusp tip decreases significantly and for every 10 increase in mpsn, the inter - canine width decreased by 0.848 mm. the r value of 0.720 for males at maxillary canine cusp tip shows that 72% variation of inter - canine width can be explained by mp - sn. similar statistical results were obtained in all dental arch width measurements on maxillary and mandibular arches in both males and females. t- test was applied to find the difference in arch widths of male and female samples. average arch width of maxillary inter - canine (from cusp tip) for males is 32.67 mm, whereas for females, 31.77 mm. student 's t- statistics (p < 0.05) shows that the arch width measurements of female samples in the maxillary canine cusp tip were significantly less when compared to male samples. the results were similar for all the inter - arch measurements in maxillary and mandibular arches. t- test was done to check the intra examiner error in both maxillary and mandibular inter - arch measurements and mp - sn values on lateral cephalogram. since all the p values were greater than 0.05, there is no significant difference in the first and second measurements. it is an essential criterion for each orthodontist to understand the relationship between vertical facial height and dental arch width for proper diagnosis and treatment planning. large variations are found in the vertical dimension and these affect the clinician 's approach to successful diagnosis, treatment planning, and mechanics. errors in the evaluation of patient 's facial type can lead to undesirable and sometimes irreversible consequences during orthodontic treatment. this study aims to evaluate the inter - relationship between vertical facial height and inter - arch widths in untreated south indian adult males and females. it also compares the dental arch widths between male and female samples and to find out whether there is a difference between them or not. in this study, subjects without previous orthodontic treatment were only included because prior treatment might have influenced the vertical development of the dentoalveolar process or the dimensions of mid - face structures. additionally, care was taken that all samples were of south indian origin to avoid any major ethnic difference in craniofacial morphology. in order to have a greater distribution of the facial patterns, 180 samples were taken and divided into three groups : high angle, average angle, and low angle. assessment of high, average, and low - angle groups allows estimation of its relation to dental arch widths. for each patient, standardized lateral cephalogram and study models were taken and confirmed that none of the exclusion criteria were present. the measurements to assess vertical facial height were done from the lateral cephalogram and study models were used to measure the dental arch widths in both upper and lower arches. after the initial tracing of anatomical landmarks, sn - mp angle was traced and it was used as a measurement for vertical facial morphology. ten dental arch width measurements were taken from both maxillary and mandibular study models (inter - canine cusp tip and most buccal, first premolar buccal cusp tip and most buccal, second premolar buccal cusp tip and most buccal, first molar mesiobuccal cusp tip, central fossa, most buccal, and most lingual / palatal). these measurements have been taken as a standard for dental arch width analysis by many investigators.[1113 ] in order to exclude intra examiner error, lateral cephalograms and models of 15 males and 15 females were selected randomly and re - measured after 4 weeks by the same examiner. the results showed that, in maxillary and mandibular arches, there was a statistically significant inverse relationship between vertical facial height and dental arch widths among the maxillary canines, first premolars, second premolars, and first molars in male and female samples [tables 1 and 2 ]. the mean arch widths of maxillary inter - canine at cusp tip for males are 31.72, 32.66, and 33.35 mm in high-, average-, and low - angled samples, respectively. the mean inter - canine arch widths from cusp tip for males in mandibular arch were 23.15, 23.82, and 25.10 mm for high-, average-, and low - angled samples, respectively. in females, the inter - arch widths at canine cusp tip in maxilla were 31.11, 31.77, and 32.29 mm, and in mandibular arch 23.07, 23.65, and 24.26 mm for high-, average-, and low - angled samples, respectively. the statistical analysis [tables 3 and 4 ] shows that inter - canine arch width at cusp tip decreases significantly (p < 0.05) with increase in mp - sn angle. all the 10 measurements in maxillary and mandibular arches showed similar significant difference (p < 0.05) in the arch widths among high - angled, average - angled, and low - angled samples. regression analysis was used to predict arch widths, with known values of mp - sn [tables 3 and 4 ]. -coefficients for arch widths were calculated and results showed that as mp - sn increases, the dental arch widths decreases. the -coefficient for arch width for males at maxillary inter - canine at cusp tip is 0.848. it indicates that as mp - sn increases, the dental arch width at canine cusp tip decreases significantly and for every 10 increase in mp - sn, the inter - canine width will be decreased by 0.848 mm. the r2 value of 0.720 for males at maxillary canine cusp tip shows that 72% variation of inter - canine width can be explained by mp - sn. similarly, the -coefficient values of all the 10 measurements in maxillary and mandibular arches showed that the dental arch widths decreased significantly with the increase in vertical facial height and using regression analysis, we can predict the arch widths at all the 10 sites in both the arches with known values of mp - sn. similar statistical results were obtained in all dental arch width measurements on maxillary and mandibular arches in both males and females. the prediction of inter - arch width helps us in situations such as cross bites, ectopically positioned teeth, transpositions, scissors bite, impacted teeth, missing teeth, etc., where we can not determine exact inter - arch widths and fabricate customized arch wires for the patient. comparison of arch width based on different levels of mp - sn for males comparison of arch widths based on different levels of mp - sn for females linear regression analysis for males linear regression analysis for females inter - arch width measurements showed that there is significant difference in arch widths among males and females in untreated south indian adult population [table 5 ]. mean inter - arch width for maxillary canine from cusp tip is 32.67 mm for males and 31.77 mm for females and from most buccal it is 38.02 and 36.63 mm for males and females, respectively. the statistical analysis shows that (p < 0.05) the arch width at inter - canine region is significantly greater for males compared to females in maxillary arch. similar observations were found in all 10 dental arch width measurements in maxillary and mandibular arches. this observation is in accordance with the observations in caucasians, where the arch width measurements were larger for males compared to females. wei evaluated posteroanterior cephalograms of chinese adults and noted gender differences in maxillary and mandibular inter - canine widths. gross. observed that boys displayed larger arch width than girls and given that this is due to the fact that boys tend to be physically larger than girls. increase in arch width during growth was found more in males than females and this can be a reason for males having broader arch than females. difference in arch widths among males and females in untreated south indian adult population musculature has been considered as the possible link in this close relationship between the transverse dimension and vertical facial morphology. a number of studies[1719 ] have illustrated the influence of masticatory muscles on craniofacial growth. the general consensus is that individuals with strong or thick mandibular elevator muscles tend to exhibit wider transverse head dimensions. this in turn may cause an introduction of sutural growth and bone apposition which then results in increased transverse growth of the jaws and bone bases for the dental arches. spronsen. found that long - faced subjects have significantly smaller masseter and medial pterygoid muscles than normal subjects. they found that individuals with thick masseter had a vertically shorter facial pattern and individuals with thin masseter have a long face. these results are in agreement with previous studies done by weijis., kiliardis and kalebo, benington., and raadsheer.[2123 ] proffit. have proved that the mean bite force is greater for short face, normal in average face, and low in high - angle subjects. the mechanical stress brought about by occlusal bite forces and volume of certain masticatory muscles have found that mean bite force values were significantly higher in males than in females. the increased bite force might be a reason for the increased arch width in males when compared to females. the direction of mandibular growth is influenced by the tongue base position, as the anterior tongue pressure might influence on the rotation of mandibular corpus. high - angle subjects had a larger tongue gap than those with normal and low angles and the tongue position may be parallel to downward and backward rotation of mandible. this indicated the relationship between tongue base position and long face syndrome because increased tongue base position correlated with an increased lower anterior facial height. because of the lowered positioning of tongue, the balance between the tongue and buccinators muscle (buccinators mechanism) might be disturbed and this can be a reason for the arch constriction in maxilla. mandible also constricts along with maxilla since maxillary and mandibular arches are mutual counterparts according to enlows counterpart principle. functional matrix theory also suggests that width of palatal complex is influenced by location of tongue. the data from this study showed an inverse relationship between mp - sn angle and dental arch widths with a strong correlation. it seems the mp - sn angle might be only one of the contributing factors. hence, the prediction of dental arch width is generalized and can be influenced by other factors. moreover, in agreement with eroz.29 and forster, the results demonstrated that the male arch widths were significantly greater than female arch widths. when comparing the arch width of south indian populations with the observations of forster. in caucasian population, the inter - arch widths of south indian population are narrower than the caucasian population. christie already proved that the caucasians with normal occlusion tend to be more brachyfacial than dolichofacial. proved that japanese population has been found to be more retrognathic with a greater vertical direction of facial growth than caucasians. the relationships between the vertical facial morphology and dental arch widths in untreated south indian adults have an inverse relationship as in caucasian population. hence, irrespective of ethnicity and race of the population group, sn - mp and inter - arch widths can be used as a valuable tool in assessing the vertical and transverse craniofacial and dentoalveolar morphology. the variation of arch widths between caucasians and south indians and between males and females highlights the variations of arch widths according to race, ethnicity, and gender and also the importance of using customized arch wires according to pre - treatment arch form and width for every patient during orthodontic treatment. within the parameters of the study, following conclusions are made : relationship between dental arch width and vertical facial pattern is determined by the steepness of mandibular plane in untreated south indian adult population.the relationship was found to be an inverse relation in both males and females of untreated south indian adults, as mp - sn angle increased, the dental arch widths tended to decrease.a generalized prediction was done for the dental arch widths with a given sn-mp.the dental arch widths of males were found to be wider than females among untreated south indian adults.since dental arch width is associated with gender, vertical facial morphology, and population groups, during orthodontic treatment, it is suggested to use individualized arch wires according to each patient 's pre - treatment arch form and widths. relationship between dental arch width and vertical facial pattern is determined by the steepness of mandibular plane in untreated south indian adult population. the relationship was found to be an inverse relation in both males and females of untreated south indian adults, as mp - sn angle increased, the dental arch widths tended to decrease. a generalized prediction was done for the dental arch widths with a given sn - mp. the dental arch widths of males were found to be wider than females among untreated south indian adults. since dental arch width is associated with gender, vertical facial morphology, and population groups, during orthodontic treatment, it is suggested to use individualized arch wires according to each patient 's pre - treatment arch form and widths.	background : anterior cranial base can be taken as a reference line (sn) to determine the steepness of mandibular plane. subjects with high mandibular plane angle tend to have a long face and one with low mp - sn angle has a shorter face.objective:this study was done to investigate if dental arch widths correlated with vertical facial types and if there are any differences in arch widths between untreated male and female adults in south indian population.materials and methods : lateral cephalogram and dental casts were obtained from 180 untreated south indian adults (90 males and 90 females) above 18 year old with no cross bite, minimal crowding and spacing. the angle between the anterior cranial base and the mandibular plane was measured on lateral cephalogram of each patient. dental casts were used to obtain comprehensive dental measurements including maxillary and mandibular inter canine, inter premolar and inter molar widths, as well as amount of crowding or spacing.results:the results showed that male arch widths were significantly larger than those of females (p < 0.05) and there was a significant decrease in inter arch width as the mp - sn angle increased in untreated adult south indian population. the results obtained in our study when compared with studies done in other population groups showed that there is difference in inter arch widths according to ethnicity and race.conclusion:it was concluded that the dental arch width is associated with gender, race and vertical facial morphology. thus using individualized arch wires according to each patient 's pre treatment arch form and width is suggested during orthodontic treatment.
about 300 million people live with malaria due to infection by plasmodium falciparum, which causes over 500000 deaths per year, predominantly in infants. although advances have been made to limit and treat infections, many strains have developed resistance to current drugs and threaten individuals in developing countries where malaria is an epidemic.plasmodium falciparum macrophage migration inhibitory factor (pfmif) is a homologue of the human protein that induces inflammation, is expressed in all stages of the parasite s life cycle, and disrupts the emergence of an appropriate memory t - cell response. the structures of pfmif and the human mif (hmif) homotrimer resemble two microbial enzymes, 4-oxalocrotonate tautomerase (4-ot) and 5-carboxymethyl-2-hydroxymucanate (chm) isomerase, that use an n - terminal proline located between subunits as a catalytic base. proline is also present at the n - terminus of pfmif, is conserved among mif cdnas from caenorhabditis elegans to humans, and has an unusually low pka of 5.6 in hmif that is consistent with a function as a catalytic base. extracellular mif functions by binding its receptor cd74 and recruiting the signaling subunit cd44 to the mif the chemokine receptors cxcr2 and cxcr4, either alone or in a complex with cd74, also are activated by mif leading to a variety of biological responses. given the upstream role of mif in immunity, it is not surprising that various parasites encode their own mif that binds to human cd74, dysregulating the immune response and promoting parasite survival. although a link among the enzymatic site, cd74 binding, and mif biology is controversial, certain small molecule ligands that bind to the catalytic site form a mif complex that functions as a cd74 antagonist, whereas other mif small molecule complexes have no effect on cd74 binding. virtual screening and optimization has been successful in identifying ligands that bind to the active site for human mif. in a recent study, the structure of p. falciparum mif was used for virtual screening of the enzymatic site with 2.1 million compounds to identify various inhibitors with low micromolar ki values and used in structure activity relationships (sar) to develop more potent inhibitors. in this study, we cocrystallized pfmif with two compounds identified from the virtual screen / optimization study. these are the first pfmif inhibitor complexes reported and may be used for development of novel therapeutics to treat malaria. the compounds in this study are 3-[(2-methyl-6-phenylpyridin-4-yl)oxy]phenol (1) and 4-(3-methoxy-5-methylphenoxy)-2-(4-methoxyphenyl)-6-methylpyridine (2) and have ki values for inhibition of the 4-hydroxyphenylpyruvate tautomerase activity of 39 8 and 38 9 nm, respectively. inhibitor structures with models of hmif complexes reveals why these two compounds are selective for pfmif versus hmif. finally, we present data indicating that one of these two inhibitors complexed to mif is a potent antagonist of human cd74. cocrystallographic studies of pfmif complexed to either compounds 1 or 2 (supporting information (si), table s1) were performed to characterize the binding interactions between the small molecules and pfmif and elucidate the selectivity over hmif. in contrast with the published apo - pfmif structure, the protein in this study did not contain a poly - his tail at the c - terminal site. both pfmif inhibitor complexes were crystallized in a different space group (i222) than apo - pfmif (p213). x - ray data were collected to 3.02 and 2.87 for pfmif complexed to compounds 1 and 2, and the structures were solved and refined to a rwork / rfree of 0.24/0.32 and 0.28/0.34, respectively. comparison of the structures of the apo - pfmif and pfmif inhibitors indicated there were no global conformational changes. the root - mean - square (rms) deviation between the apo - pfmif and pfmif complexed to compounds 1 or 2 was 0.73 and 0.71, respectively. these regions include the 60s loop and various c - terminal residues among the three subunits. protein sequence alignment of the apo - pfmif and pfmif inhibitor complexes with unmodelled residues (due to the lack of electron density) highlights that there are far more c - terminal residues missing in the apo - pfmif than the complexed structures (si, figure s1). the c - terminal region has roles in the integrity of the active site and the stability of the trimer, as -strands from the c - terminal region intertwine with a -sheet from an adjacent subunit and contribute to the seven - stranded -sheet core of each monomer. the truncation of the c - terminal region in hmif significantly decreases its catalytic activity and lowers the thermal denaturation of the trimer. the thermostability of pfmif was measured by temperature - dependent circular dichroism (cd) (1) to examine the association between missing electron density for the c - terminal region (that indicates flexibility of this region) and reduction of pfmif tautomerase specific activity that is 20% of hmif, and (2) to determine whether stability of the trimer in complex with inhibitors is increased (si, figure s2a, b). it was surprising to find that the melting temperature (tm) was 89 c for apo - pfmif, 15 c greater than the corresponding tm of hmif. analysis of pfmif to identify potential interactions responsible for this enhanced thermostability revealed interactions along the 3-fold axis as well as complementary electrostatic subunit there are three residues located along the solvent channel coincident with the 3-fold axis that form hydrogen bond interactions. the side chain carbonyl oxygen of gln45 makes a hydrogen bond with the side chain nitrogen of asn41 from an adjacent subunit. the side chain amide nitrogen of the same gln45 makes a hydrogen bond with a carboxylate oxygen of asp43 of the same adjacent subunit (figure 1a, b). at the equivalent position of the hmif structure, the trimer is stabilized by a hydrogen bond between the residue his40 of one subunit and gln45 of another (si, figure s3a, b). subunit interface indicates a significant complementarity in electrostatic interactions (figure 1c, d). in contrast, the subunit subunit interface of hmif is hydrophobic (si, figure s3c, d). it is difficult to quantitate the energies of these interactions, particularly for the buried complementary electrostatic potential at the subunit interface where the dielectric constant is low. however, these interactions are likely to increase thermostability of pfmif relative to hmif. thermostability is correlated to resistance to proteolysis, suggesting that pfmif has evolved to preserve its structure and function in vivo to protect plasmodium falciparum from the host immune response. (a) hydrogen bonding interactions in the opening of the solvent channel increase the stability of the trimer. (b) gln45 makes two intersubunit hydrogen bonds with asn41 and asp43 for each monomer. the rotation used was down the 3-fold axis to observe the electrostatic potential subunit subunit interface. subunit interface shows strong electrostatic complementarity (dotted circles) between the two monomers. the positive and negative potentials are shown in blue and red, respectively. the possibility that the compounds could fit into the initial electron density in two orientations the compounds were fit into electron density in each orientation, refined, and analyzed. in all instances, there were either clashes with active site residues or poor fitting with the opposite orientation (si, figure s4). figure 2 displays the final coordinates of each compound with the corresponding electron density. interactions between the pfmif active site and the two inhibitors were examined using pymol and ligplus and calculated with contact. analysis of the interactions of compounds 1 and 2 with the active sites revealed two common features. in both pfmif complexes, the hydroxyphenyl ring of 1 and the 3-methoxy-5-methylphenyl ring of 2 are buried into the active site pocket, forming aromatic and van der waals interactions. although ether oxygens are not known for forming strong hydrogen bonds, we note that the biaryl ether oxygen of each compound forms a close contact with the nitrogen of pro1 (figure 3). the distance between the nitrogen of pro1 and the biaryl ether oxygen of compound 1 is 2.4 (figure 3a). the inhibitor also forms a number of aromatic and van der waals interactions with residues pro1, tyr37, met39, ser64, and asn106 of chain a and phe50, phe59, and tyr96 of the adjacent subunit (chain c). in the pfmif2 complex, the inhibitor was found in two active sites in the same orientation but with different conformations (figure 3b). in the active site between chains a and b, the distance of the biaryl ether oxygen of compound 2 with pro1 is 2.3 (figure 3c), with the 3-methoxy-5-methylphenyl ring having aromatic and van der waals interactions with pro1, tyr37, met39, ser64, asn106, phe107, and ala108 of chain b and phe50, tyr57, glu98, phe99, and arg100 of chain a. in the conformation of the active site that is located between chains b and c, the distance between the biaryl ether oxygen and the pro1 nitrogen 2.6. in this conformation, the 3-methoxy-5-methylphenyl ring makes aromatic and van der waals interactions with pro1, met39, ser64, and ala108 of chain c and phe50 and tyr57 of chain b (figure 3d). a ligplot shows the interactions between active site residues and inhibitors (si, figure s5). fc maps contoured at 3.0. (a) compounds 1 and (b, c) 2 in the electron density at active sites of pfmif. 2 is in the same orientation but with different conformations at the two sites of pfmif. the inhibitors and pfmif are shown with yellow and cyan carbon atoms, respectively. blue and red atoms are nitrogen and oxygen atoms, respectively. interaction profile of 1 and 2 in the active sites of pfmif. differences in the displayed residues for each active site are due to different interactions and to missing atoms (due to the absence of electron density) for some residues. the orientations of the compounds are based on superposition of each subunit and arranged with pro-1 on the left of the compounds. the compound is stabilized by an interaction between the biaryl ether oxygen of 1 and the nitrogen of pro1 and aromatic aromatic and van der waals interactions of 1 with active site residues. (b) the two orientations of 2 in the active site of pfmif based on superposition of the protein atoms from the two subunits. (c, d) interactions between the active site residues and 2 in its two orientations. similar to pfmif1 interactions, 2 forms an interaction with nitrogen of pro1 and a number of aromatic and van der waals interactions with active site residues. the red dotted lines show the interaction between the biaryl ether and pfmif. for (a, c, d), yellow, red, and blue spheres are overlaid on the carbon, oxygen, and nitrogen atoms of each inhibitor, respectively. comparison of the crystallographic structures with the docking - based models from the virtual screen shows significantly different conformations and interactions for the molecules in the active site (si, figure s6a this is not surprising given the versatility to accept different conformations of a ligand in the active site of hmif and the dynamics of mif as determined by nmr when some ligands bind to the active site. this is also evident from the two conformations of compound 2 in separate pfmif active sites. another complication of the original docking / optimization in the virtual screen was an imperfect target model created by using the c - terminal region of plasmodium berghei mif structure to compensate for the missing electron density for this region in the apo - pfmif structure. using the crystal structures of pfmif inhibitor complexes, we repeated docking of 1 and 2, focusing only in the active sites that are occupied by the inhibitors. the new docking - based models were significantly improved, with one orientation of compound 2 having significant superposition with the compound in the crystal structure (si, figure s6d biochemical experiments verified the selectivity of the two compounds for pfmif over hmif. to understand the structural basis of selectivity, the structures of hmif (pdb entry 3djh) and the pfmif complexes were superimposed using the secondary structure method (ssm). the rms deviation of superimposed c atoms was 2.23 and 2.25 for hmif on pfmif complexed to compound 1 and 2, respectively. the fairly high rms values are due to the low sequence identity (29%) between the two proteins (si, figure s7a). comparison between modeled complexes of hmif with both compounds reveals two common clashes, one with ile64 and the second with tyr95 from an adjacent subunit. compound 1 in hmif also clashes with met2 and met101 (si, figure s7b). the two methionine residues met2 and met101 that clash with compound 1 do not interfere with compound 2. instead, val106 and phe113 clash with compound 2 in the site between subunits a and b, and phe113 clashes with compound 2 between chains b and c (si, figure s7c, d). some mif response effects of compounds 1 and 2 on binding of pfmif to the human scd74 revealed that compound 2 is more effective than 1 on inhibiting pfmif receptor interactions (figure 4). at the highest concentration for compound 2, 75% of the pfmif compound 2 in contrast, compound 1 prevents binding of about 25% of pfmif at its highest concentration. consistent with the lack of inhibition of hmif by compounds 1 and 2, neither compound is found to inhibit the interaction of hmif with scd74. an emerging hypothesis related to disruption of mif cd74 emanates from a small structure activity study of inhibitors of the hookworm - inducing parasite ancylostoma ceylanicum macrophage migration inhibitory factor (acemif). in that study, it appeared that chemical moieties that protruded from the active site into the solvent were more effective at disrupting the acemif one of the conformations of compound 2 has a chemical moiety outside the active site (si, figure s8), consistent with the hypothesis that mif inhibitors with chemical groups outside the active site are more effective at disrupting mif receptor interactions. the structures from this study can be used to increase affinity to pfmif, increase inhibition of pfmif interactions with human cd74, and optimize for absorption, distribution, metabolism, and excretion (adme) properties that are necessary for a therapeutic. effect of inhibitors 1 and 2 on (a) pfmif or (b) human mif binding to the human ectodomain cd74 receptor (scd74 = cd746114232). measured values are relative to diluent (dmso) control for each concentration of inhibitor.	we report the crystal structures of two inhibitors of plasmodium falciparum macrophage migration inhibitory factor (pfmif) with nanomolar ki s, analyze their interactions with the active site of pfmif, and provide explanations regarding their selectivity of pfmif versus human mif. these inhibitors were also found to selectively inhibit interactions between pfmif and the human mif receptor cd74. the results of this study provide the framework for the development of new therapeutics that target pfmif.
ablative surgical therapy is frequently adopted for the control of malignancies and other abnormal growths in oral cavity. the consequences of postsurgical effect can be serious as it disturbs both form and function of normal stomatognathic system and also the facial contour. postsurgical maxillary defects predispose the patients to hypernasal speech and fluid leakage through the nose, including possibility if aspiration. as a critical member of the team, primary objective of prosthodontist should be to preserve and restore the function of speech and swallow. prosthetic rehabilitation with obturator prosthesis is a predictable intervention to recreate an anatomic barrier between the cavities and to restore functional capabilities of speech, oral food intake, and deglutition. rehabilitation of maxillary defect with obturator has been well defined as comprehensive treatment care to achieve optimal success in improving posttreatment quality of life. the degree of extension of obturator in the defect part depends on factors such as configuration of defect, character of lining tissue and functional requirements for prosthesis retention, support, and stability. in large defects, obturator is aggressively extended vertically to engage the surgical defect and horizontally to engage the undercuts at the expense of its size and weight. increased weight of obturator makes the prosthesis uncomfortable as well as nonretentive for the patient jeopardizing its function. to reduce the weight of prosthesis, according to wu and schaaf, hollowing of the obturator significantly reduces prosthesis weight from 6.55% to 33.06% depending on the size of the defect. this case report describes the fabrication of one piece closed hollow bulb definitive cast partial obturator prosthesis for a patient with acquired maxillary defect (aramany 's class i) on the left side. a 21-year - old male patient was referred to the department of prosthodontics with chief complaints of difficulty in eating, speaking, and deformity of upper midface due to surgical removal of lesion. the patient had a history of squamous cell carcinoma on the left maxilla for which hemimaxillectomy of the same side was performed. on intraoral examination, defect was aramany 's class i extending till floor of the orbit associated with depressed cheek, nasolabial fold, and lack of lip support [figure 1 ]. preoperative intraoral photograph of the patient the patient was initially rehabilitated with interim obturator for 6 months and then planned for one piece closed hollow bulb definitive cast partial obturator prosthesis. maxillary and mandibular primary impressions were made with irreversible hydrocolloid (zelgan, dentsply) and poured in type 3 gypsum product (kalabhai, kaldent) to obtain diagnostic castsdiagnostic casts were surveyed, and a cast partial framework was planned with the following components : embrasure clasps in relation to 14, 15 ; occlusal rest on 16 and 17, cingulum rest on 13, modified complete palatal type of major connector extended till palatal surfaces of teeth. distal surface of 11 was also prepared to act as a guiding planemouth preparation was done before making final impression with polyvinylsiloxane impression material (affinis, coltene whaledent) and master cast was then poured in die stone (kalabhai, ultrastone). wax pattern was adapted on refractory cast and casting of metal framework was carried out. trial of finished and polished framework and needed adjustments were done [figure 2]autopolymerizing resin was attached with framework on defect side so that impression of the defect part would be made more precisely. the impression of the defect was made with low fusing impression compound (dpi, mumbai) relined with polyvinylsiloxane (light body consistency) (affinis, coltene whaledent) [figure 3 ]. cast was poured by altered cast techniqueautopolymerizing resin attached to the framework was removed with bur and wax sheet was adapted on the roof of defect in the cast and size of balloon was approximated by adapting in the defect portion of castthe curing of the prosthesis was done in two parts : first bulb portion and then remaining part of the prosthesis containing teeth. bulb portion of the prosthesis was made hollow by sandwiching water filled balloon between two layers of heat cure resin [figure 4a ] and curing was completed using short curing cycle [figure 4b ]. balloon was not removed later on as it would have no affect on prosthesis functionbulb portion was held in situ during adjustments with retentive clasps of cast partial framework and adjusted in the patient 's mouth by disclosing pastejaw relations were recorded and try in done over the cured bulb portion which was found to be satisfactory. curing of remaining part of the prosthesis containing teeth was done over the bulb portionfinal prosthesis was adjusted in the patient 's mouth and occlusal adjustments were done to make passive contacts on defect side [figure 5a and b ]. maxillary and mandibular primary impressions were made with irreversible hydrocolloid (zelgan, dentsply) and poured in type 3 gypsum product (kalabhai, kaldent) to obtain diagnostic casts diagnostic casts were surveyed, and a cast partial framework was planned with the following components : embrasure clasps in relation to 14, 15 ; occlusal rest on 16 and 17, cingulum rest on 13, modified complete palatal type of major connector extended till palatal surfaces of teeth. distal surface of 11 was also prepared to act as a guiding plane mouth preparation was done before making final impression with polyvinylsiloxane impression material (affinis, coltene whaledent) and master cast was then poured in die stone (kalabhai, ultrastone). wax pattern was adapted on refractory cast and casting of metal framework was carried out. trial of finished and polished framework and needed adjustments were done [figure 2 ] autopolymerizing resin was attached with framework on defect side so that impression of the defect part would be made more precisely. the impression of the defect was made with low fusing impression compound (dpi, mumbai) relined with polyvinylsiloxane (light body consistency) (affinis, coltene whaledent) [figure 3 ]. cast was poured by altered cast technique autopolymerizing resin attached to the framework was removed with bur and wax sheet was adapted on the roof of defect in the cast and size of balloon was approximated by adapting in the defect portion of cast the curing of the prosthesis was done in two parts : first bulb portion and then remaining part of the prosthesis containing teeth. bulb portion of the prosthesis was made hollow by sandwiching water filled balloon between two layers of heat cure resin [figure 4a ] and curing was completed using short curing cycle [figure 4b ]. balloon was not removed later on as it would have no affect on prosthesis function bulb portion was held in situ during adjustments with retentive clasps of cast partial framework and adjusted in the patient 's mouth by disclosing paste jaw relations were recorded and try in done over the cured bulb portion which was found to be satisfactory. curing of remaining part of the prosthesis containing teeth was done over the bulb portion final prosthesis was adjusted in the patient 's mouth and occlusal adjustments were done to make passive contacts on defect side [figure 5a and b ]. cast partial metal framework definitive impression of defect portion (a) fabrication of bulb portion by balloon. (b) final acrylic bulb portion of the prosthesis (a) intraoral frontal view. maxillary and mandibular primary impressions were made with irreversible hydrocolloid (zelgan, dentsply) and poured in type 3 gypsum product (kalabhai, kaldent) to obtain diagnostic castsdiagnostic casts were surveyed, and a cast partial framework was planned with the following components : embrasure clasps in relation to 14, 15 ; occlusal rest on 16 and 17, cingulum rest on 13, modified complete palatal type of major connector extended till palatal surfaces of teeth. distal surface of 11 was also prepared to act as a guiding planemouth preparation was done before making final impression with polyvinylsiloxane impression material (affinis, coltene whaledent) and master cast was then poured in die stone (kalabhai, ultrastone). wax pattern was adapted on refractory cast and casting of metal framework was carried out. trial of finished and polished framework and needed adjustments were done [figure 2]autopolymerizing resin was attached with framework on defect side so that impression of the defect part would be made more precisely. the impression of the defect was made with low fusing impression compound (dpi, mumbai) relined with polyvinylsiloxane (light body consistency) (affinis, coltene whaledent) [figure 3 ]. cast was poured by altered cast techniqueautopolymerizing resin attached to the framework was removed with bur and wax sheet was adapted on the roof of defect in the cast and size of balloon was approximated by adapting in the defect portion of castthe curing of the prosthesis was done in two parts : first bulb portion and then remaining part of the prosthesis containing teeth. bulb portion of the prosthesis was made hollow by sandwiching water filled balloon between two layers of heat cure resin [figure 4a ] and curing was completed using short curing cycle [figure 4b ]. balloon was not removed later on as it would have no affect on prosthesis functionbulb portion was held in situ during adjustments with retentive clasps of cast partial framework and adjusted in the patient 's mouth by disclosing pastejaw relations were recorded and try in done over the cured bulb portion which was found to be satisfactory. curing of remaining part of the prosthesis containing teeth was done over the bulb portionfinal prosthesis was adjusted in the patient 's mouth and occlusal adjustments were done to make passive contacts on defect side [figure 5a and b ]. maxillary and mandibular primary impressions were made with irreversible hydrocolloid (zelgan, dentsply) and poured in type 3 gypsum product (kalabhai, kaldent) to obtain diagnostic casts diagnostic casts were surveyed, and a cast partial framework was planned with the following components : embrasure clasps in relation to 14, 15 ; occlusal rest on 16 and 17, cingulum rest on 13, modified complete palatal type of major connector extended till palatal surfaces of teeth. distal surface of 11 was also prepared to act as a guiding plane mouth preparation was done before making final impression with polyvinylsiloxane impression material (affinis, coltene whaledent) and master cast was then poured in die stone (kalabhai, ultrastone). wax pattern was adapted on refractory cast and casting of metal framework was carried out. trial of finished and polished framework and needed adjustments were done [figure 2 ] autopolymerizing resin was attached with framework on defect side so that impression of the defect part would be made more precisely. the impression of the defect was made with low fusing impression compound (dpi, mumbai) relined with polyvinylsiloxane (light body consistency) (affinis, coltene whaledent) [figure 3 ]. cast was poured by altered cast technique autopolymerizing resin attached to the framework was removed with bur and wax sheet was adapted on the roof of defect in the cast and size of balloon was approximated by adapting in the defect portion of cast the curing of the prosthesis was done in two parts : first bulb portion and then remaining part of the prosthesis containing teeth. bulb portion of the prosthesis was made hollow by sandwiching water filled balloon between two layers of heat cure resin [figure 4a ] and curing was completed using short curing cycle [figure 4b ]. balloon was not removed later on as it would have no affect on prosthesis function bulb portion was held in situ during adjustments with retentive clasps of cast partial framework and adjusted in the patient 's mouth by disclosing paste jaw relations were recorded and try in done over the cured bulb portion which was found to be satisfactory. curing of remaining part of the prosthesis containing teeth was done over the bulb portion final prosthesis was adjusted in the patient 's mouth and occlusal adjustments were done to make passive contacts on defect side [figure 5a and b ]. prosthesis was functionally and esthetically pleasing [figure 6a and b ]. cast partial metal framework definitive impression of defect portion (a) fabrication of bulb portion by balloon. (b) final acrylic bulb portion of the prosthesis (a) intraoral frontal view. maxillectomy patients suffer from functional as well as facial deformity so role of a prosthodontist comes into play to restore function and esthetics. the primary goal of the treatment of maxillectomy defect is to give a prosthetic obturation which closes the defect and separates the oral cavity from the sinonasal cavities. the size and location of the defects influence the degree of impairment and difficulty in prosthetic rehabilitation. maxillary obturator prosthesis is the most accepted treatment modality than surgical reconstruction due to ease of fabrication and maintenance. hollow bulb provides advantages such as reduction in weight, increased retention, and making prosthesis comfortable. closed hollow bulb obturators provide the advantage of preventing fluid and food accumulation, reducing airway space, and allow for maximum extension. various materials are used for making obturators such as heat cure resin and light cure resin but fabricating the prosthesis with metal provides durability, biocompatibility, and longevity of the prosthesis. different methods have been developed for making obturators hollow, but most of the techniques described are time - consuming and complex. materials such as sugar and ice were used to create the hollow space inside the processed resin. processing the two halves of the obturator separately followed by joining them with an autopolymerizing resin was also well explained in the previous literature. two - step processing technique, using preformed plastic shapes or plaster matrix were tried by some authors. the acrylic resin shim and a polyurethane foam were incorporated into the defect area during packing to create hollow space by some authors. patil and patil presented a method of hollow obturator by inserting a preshaped wax bolus while packing of heat cured material. elangovan and loibi presented a method of fabrication of hollow bulb obturator by the use of attachments. although the method was simple the cost of treatment would be high with the use of attachments. the patient presented here had a well - healed defect so definitive hollow bulb obturator prosthesis was planned for rehabilitation. cast partial framework was planned for prosthesis as it increases longevity and durability of the prosthesis. the technique described in this case report was superior to other methods because of its simplicity and economical from patient 's perspective. moreover, most of the methods presented in literature used autopolymerizing resin material which leaches out with use, but on the contrary, in the presented method only heat cure resin material was used which aids in novelty of this approach. in the presented case report technique described was a variation of some previously described methods but the ability of this technique to provide durable cast partial denture along with one piece closed hollow bulb obturator justifies its novelty. the ease of fabrication reduced time and reduced cost were the added advantages of this technique. the obturator provided to the patient increased function by providing better masticatory efficiency, phonetics by adding resonance, and also improved the esthetics. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed.	in maxillary defects, role of a prosthodontist is to rehabilitate the intra- and extra - oral structures and to endow with the normal function of mastication, speech, deglutition, and esthetics. malignancies are common in the oral region and are treated usually through surgical intervention. surgical intervention creates anatomical defects creating communication between oral and nasal cavity. patients pose difficulties while performing normal functions such as swallowing and speaking, due to this communication. to overcome the problems encountered by the patient, obturators are fabricated. the main problem with rehabilitation of large defect is the weight of prosthesis ; the prosthesis becomes very bulky and nonretentive due to its weight. hollow obturators are fabricated to conquer this tribulation by different techniques. this case report describes a simplified method of fabrication of a definitive hollow bulb obturator for rehabilitation of a maxillary defect (aramany 's class i) by insertion of balloon.
perinatal depression is considered the most underdiagnosed pregnancy complication in the united states with more than 400,000 infants born to mothers who are depressed each year.1 the pregnancy and postpartum phases of the perinatal period are considered periods of high risk.8 perinatal depression, defined here as minor or major episodes of depression occurring during pregnancy or the first postpartum year, affects an estimated 7% to 20% of women with rates as high as 35% to 40% among low - income and minority women.9,10 depressive symptoms occurring during pregnancy often persist after delivery, suggesting an important role of antenatal symptoms in predicting postpartum depression. approximately half of postpartum depression cases have onsets during pregnancy.11 recognizing insufficient evidence demonstrative of a clear difference between postpartum and pregnancy depressive episodes, the american psychiatric association recently proposed the term peripartum onset as a specifier for major depressive episodes that occur during pregnancy and/or in the postpartum period.12 understanding risk factors for perinatal depression is essential for healthcare providers to more easily identify women at risk for developing this condition. furthermore, antenatal period depression is the most significant risk factor for postpartum depression development.13 depression and anxiety are highly comorbid in nonpregnant samples, with nearly 60% of women diagnosed as depressed also meeting diagnostic criteria for an anxiety disorder.14 a recent systematic review, which examined the relation between maternal anxiety and depression in 11 studies, found anxiety to be one of the strongest associations with antenatal depressive symptoms.15 depression disproportionately affects low - income women. research suggests that poverty is a powerful predictor of depression irrespective of race / ethnicity.16 research has found that nearly 40% of mothers participating in head start, a program serving predominantly low - income preschool children and their families, may experience depression.17 other studies have concluded that race and ethnicity are risk factors for perinatal depression. this is particularly concerning, as research also demonstrates that ethnic minority patients are less likely to obtain care for depression than white patients and are less likely to receive clinically appropriate treatment when they do access care.18 stress is another established risk factor for perinatal depression. stress can be conceptualized in many ways, and the majority of studies that have examined the relation between stress and depression have conceptualized stress as stressful life events (divorce, serious illness, death in the family) or daily hassles (work hassles, time pressures, financial strain).15 a significant body of research has examined life stress as a predictor of perinatal depression, with most studies finding positive associations. a sizeable body of research supports a significant relation between social support and perinatal depression. there is a moderate correlation between social support and perinatal depression across many studies when assessing social support available to perinatal women as support from any source.15 however, perinatal women who report the absence of a supportive partner have been found to be at greatest risk for perinatal depression across multiple studies.15 it is important to mention that many studies do not adequately control for important confounders of this relation (income, education, socioeconomic status, relationship violence). extant research has found that between 3% and 9% of women experience ipv during pregnancy, though there are well - established risk factors associated with higher rates of abuse during pregnancy, including young age, low socioeconomic status, single marital status, and minority race / ethnicity.19 research conducted in low - income perinatal women found that up to 85% of women experiencing ipv during pregnancy also screened positive for depressive symptomatology on the edinburgh postnatal depression scale (epds) ; these women were at significantly increased risk for suicidal ideation.20 - 22 additional risk factors for perinatal depression include pregnancy complications.23 numerous studies have examined the relation between cesarean delivery and postpartum depression, with the majority of studies concluding mode of delivery is not a significant predictor ; however, research has demonstrated that women with a strong preference for vaginal delivery but who require a cesarean delivery are at increased risk for postpartum depression in the early postpartum period.24 - 26 further, neonatal factors may be associated with an increased risk for depressive symptoms, for example, infant colic and inconsolable crying are associated with maternal depressive symptoms.27 because perinatal depression can have serious consequences regarding maternal morbidity and mortality (as well as adverse neonatal and early childhood outcomes), there is significant interest in improving its detection to increase appropriate treatment modalities.28 screening for depression has been widely supported as a component of routine obstetric care.29 in 2015, the american congress of obstetricians and gynecologists (acog) released a committee opinion recommending that clinicians screen patients for depression and anxiety symptoms at least once during the perinatal period using a validated tool.30 acog 's guidelines also highlight the importance of coupling screening with appropriate follow - up and treatment as indicated.30 recognizing the negative effects of perinatal depression on early childhood outcomes and the critical role pediatric healthcare providers play in providing care, the american academy of pediatrics recommends that pediatric providers routinely screen mothers for depression during 1-, 2-, and 4-month well - child visits.1 the epds is the most commonly utilized instrument to screen for perinatal depression. this self - rated, 10-item instrument asks a woman to answer questions based on how she has felt in the previous 7 days (i have felt sad or miserable ; i have been able to laugh and see the funny side of things ; things have been getting on top of me) and choose one of four possible responses.31 the epds does not focus on some of the somatic symptoms (changes in appetite, sleeping difficulties, energy level) that are more common among perinatal women in the absence of a mental disorder. its psychometric properties are the most established of any depression screening tool and it has been validated in diverse perinatal populations.32 recognizing the importance of universal screening for perinatal depression, the national institute for health and clinical excellence recommends the use of two case - finding questions for depressive symptoms in perinatal women : during the past month, have you often been bothered by feeling down, depressed or hopeless ? ; and during the past month, have you often been bothered by little interest or pleasure in doing things ? if a positive response is given to either question, the recommended follow - up question states : is this something you feel you need or want help with ? these two case - finding questions have been validated in diverse primary care settings, including perinatal women, and are endorsed by united states and canadian bodies for depression screening in adults.33,34 screening for perinatal depression is viewed as acceptable by most perinatal women, and the majority of perinatal women are in favor of psychosocial screenings, such as depression and ipv.35 importantly, women are more likely to accept depression screening if they understand the purpose of the screening, interpretation of results, and treatment options available.35 these findings support providers of perinatal women using a patient - centered approach to screening. in addition to paper and pencil screening, healthcare providers should consider physiological disturbances, which may contribute to depressive symptoms. research has demonstrated an increased depression risk in perinatal women with concurrent anemia, thyroid dysfunction, and fatigue.36 screening for anemia, particularly among women with excessive blood loss during their deliveries, is an important consideration and easily remedied. in addition, iron deficiency anemia may alter thyroid hormone metabolism, so thyroid function should be assessed in cases of suspected anemia as well as in women with a history of thyroid disease or who exhibit symptoms of thyroid disease. in women with a history of thyroid disease or with symptoms suggestive of thyroid disease regardless of the instrument used to screen for depression, it is important for healthcare providers to be aware that a positive screening result is not diagnostic for depression. the epds was developed specifically to screen for symptoms of possible perinatal depression. screening techniques aim to identify women at risk for perinatal depression and in need of further diagnostic assessment. depressive symptoms in the perinatal period range from maternity blues (commonly seen in the postpartum period) to major depressive disorder and postpartum psychosis.1 approximately 50% to 80% of new mothers experience maternity blues within the first few days after delivery.1 symptoms may include crying, sadness, and mood swings ; however, these symptoms most often resolve within 1 to 2 weeks. major depressive disorder (mdd) is defined by one or more major depressive episodes (mde) along with the lifetime absence of mania and hypomania.12 an mde is constituted by a patient 's report of at least five of nine symptoms present during the same 2-week period. specific to mdd in the perinatal period, the diagnostic and statistical manual of mental disorders, fifth edition (dsm-5) expanded the previous dsm - iv qualifier with postnatal onset to include with peripartum onset.12 in addition to mdd onsets occurring within 4 weeks after delivery, the dsm-5 now includes onsets over the course of the entire pregnancy. this change recognizes that a high prevalence of postpartum depressive episodes has an onset in pregnancy and persist (often becoming more severe) throughout the postpartum period.37 further, this revision dispels the previously held belief that the pregnancy timeframe was protective against the development of mdd and acknowledges the importance of healthcare providers in addressing mental health throughout the perinatal period. although generally accepted that pregnancy is not a protective period with respect to new onset or relapse of mdd, there continues to be a fair amount of controversy regarding how best to treat perinatal depression. regardless of the ongoing debate, medication use during pregnancy has become relatively common with a 2- to 4-fold increase in medications for mdd over the past decade.38 this increase is in spite of a fairly stable prevalence of psychiatric illness.39 recent research supports that nearly 13% of all pregnant women are taking an antidepressant during pregnancy with selective serotonin reuptake inhibitors (ssris) and selective serotonin norepinephrine reuptake inhibitors (snris) being the most frequently prescribed antidepressants.39,40 currently, the research literature on the safety of antidepressant use during pregnancy and breastfeeding has yielded mixed results, making clinical recommendations difficult. research examining the adverse reactions of ssri exposure during pregnancy on the developing fetus has found an increased risk of various congenital malformations (congenital heart defects, craniosynostosis) ; however, inconsistencies between study results remain. limitations in study interpretation include small sample sizes (low statistical power), failure to adequately control for maternal depression effect, and failure to control for other important confounders (smoking, folic acid intake, alcohol use).41 - 44 nonetheless, the healthcare provider must carefully discuss treatment options both pharmacologic and nonpharmacologic with each woman so an informed decision can be made. many healthcare providers initiate ssris if pharmacologic treatment is warranted, as they are generally well tolerated.38 of the ssri medications, fluoxetine and sertraline have more data regarding safety than the newer ssris (escitalopram) and snris.38,42 the risk of pharmacologic therapy is largely focused on the infant 's exposure to the antidepressant via breast milk in the postpartum period.45 the majority of research examining the safety of breastfeeding with antidepressant use has found low rates of adverse reactions in infants exposed to antidepressants, including ssris.45 as a general recommendation, breastfeeding mothers on drug therapy for perinatal depression should be instructed to take their medication immediately after breastfeeding. this helps minimize the amount of antidepressant present in the breast milk while maximizing its clearance prior to the next feeding.45 a growing number of nonpharmacologic interventions are aimed at mitigating the effects of perinatal depression, including individual cognitive behavioral therapy (cbt), interpersonal psychotherapy (ipt), and group therapy.46,47 ipt is a time - limited therapy with demonstrated benefit for treating mdd. ipt is designed to improve depressive symptoms by assisting patients in navigating changes in their personal relationships. ipt focuses on issues, such as role change, social support, relationships, and life stress.47 cbt has been used extensively in patients, including those with both depressive and anxiety disorders. cbt aims to reduce depressive symptoms by targeting and modifying negative patterns of thinking and behavior. cbt is typically provided by a clinical psychologist over a predetermined number of sessions ; however, in practice, the length of therapy can vary (between 4 to 20 sessions).46 a recent meta - analysis provides strong support for incorporating these interventions into the treatment plan for perinatal depression, with all interventions examined demonstrating greater benefit on depressive symptoms as compared to control conditions.46 furthermore, results demonstrated ipt to be more beneficial than group psychotherapy with regards to change in depressive symptoms pre- and postintervention. interventions, including an interpersonal therapy component, had greater effects compared to interventions including a cbt component. further studies are needed that examine manual - based ipt and cbt, administered with high fidelity, to firmly establish one modality as more effective than the other. comparisons across these types of interventions have been difficult in part due to differences in intervention fidelity. this research is particularly vital given the rather low acceptability of pharmacologic therapy in this population coupled with a preference for nonpharmacologic interventions.48 similar to other screening (behavioral, psychosocial) initiatives in clinical practice, perceived barriers to implementation exist on the part of the healthcare provider, including a lack of time, inadequate training to diagnose or counsel, insufficient mental health referral sources, and lack of reimbursement.49 continuing to omit these critical screenings counters policies established to increase access to basic and quality care particularly for low - income communities. the new focus on comprehensive care and wellness is purported to be best delivered through the patient - centered medical home (pcmh) model, featured prominently in the affordable care act.50 the pcmh model is particularly salient for low - income and underserved women. a lack of health insurance and inadequate social support often render this population particularly vulnerable to fragmented care.51 however, the pcmh model can provide continuous, patient - centered care, assisting perinatal women with coordination of care through linkages with key community agencies.50 these linkages could be particularly helpful for women without adequate support as well as those women who had pregnancy complications that may be exacerbating depressive symptoms. social media provides an ideal platform to link mothers who may report difficulties breastfeeding or infants with colic, for example, with support groups or resources in their communities. additionally, national websites, such as postpartum support international (www.postpartum.net), office on women 's health (www.womenshealth.gov/mental-health/illnesses/postpartum-depression.html), and postpartum progress (www.postpartumprogress.com) offer an array of resources for antenatal, perinatal, and postpartum depression, including links to local resources. perinatal depression is an important public health issue with well - documented consequences for mothers, children, and families. healthcare providers caring for perinatal women and their children are uniquely situated to address perinatal depression given women are more likely to access healthcare during this time. providers caring for underserved women and families are well positioned to improve maternal and early childhood outcomes for families at greatest risk of healthcare disparities. efforts to expand access to care and facilitate the coordination of care in the perinatal period may serve to improve perinatal outcomes.	abstract : perinatal depression is a common condition with significant adverse maternal, fetal, neonatal, and early childhood outcomes. the perinatal period is an opportune time to screen, diagnose, and treat depression. improved recognition of perinatal depression, particularly among low - income women, can lead to improved perinatal health outcomes.
transmission of medical images aiming at medical consultation, diagnosis, and treatment, or for training purposes demands highly reliable and high - speed communication systems. since, typically, medical images contain large amounts of crucial clinical data, no visible encoding error is tolerated in the clinically important regions. hence, advanced methodologies for gaining distortion - less reconstructed images, in fairly acceptable transmission rate, make it possible to transmit a medical image over a noisy channel. however, even by utilizing current high - speed broadband connections, the transmission of medical images has not been entirely successful ; although using more parity bits enables a higher protection of the data against channel noise, the transmission time and the complexity of the channel coding considerably increase. by prioritizing the image regions based on their clinical information contents in one hand, and establishing a variable parity length with regard to the state of the communication channel on the other hand a higher data speed and immunity against noise can be achieved. the concept of image coding utilizing discrete wavelet transform (dwt) and embedded zerotree wavelet (ezw) has been initially proposed in and various methods have been developed based on this idea [28 ]. in, an effective scheme for image compression has been proposed where the spatial - spectral features of the image have been taken into account in order to show that wavelet transform is particularly well suited for progressive transmission. in, the concepts of dwt, ezw, progressive image transmission, and roi have been utilized. these techniques have been effectively employed in our work. in, a method for progressive image transmission using ezw is presented where a number of constraints are imposed to provide variable bitrates for each frequency band. in, a compression method using linked significant tree (lst) has been utilized to encode all the significant coefficients in order to reduce the number of symbols and increase the compression efficiency. the authors in have developed an image coding algorithm that incorporates the features of zerotree and zero - block based algorithms. the main contribution in this recent algorithm is partitioning the wavelet - transformed image into coefficient blocks and to generate roots in top - most subband by using a block tree. in, another progressive image transmission method has been proposed based on a quadtree segmentation procedure in order to provide fairly good quality transmitted images while keeping the computational cost low. the authors in have developed strategies to exploit the wavelet coefficients in different subbands for designing different vector quantization (vq) coding to achieve a fast and efficient progressive transmission. shannon 's information theory states that the performance of transmission schemes can be optimized in source and channel coding separately. however, the result holds with infinite block size, infinite coding complexity, and stationary channels. hence, joint source - channel coding (jscc) scheme attracts the interest of many. the jscc scheme consists of a quantizer, an entropy, and channel coders to meet the target source rate, to achieve the required robustness in channel coding, and to find an optimal bit allocation between source and channel coding systems. several methods have focused on designing adaptive joint source - channel coding (jscc) schemes and introducing the properties of unequal error protection (uep) and rate allocation to achieve efficient transmission [920 ]. in, authors surveyed several algorithms in jscc system design and fast source - channel bit allocation for various transmission channels. they also provided a local search strategy to improve the initial low - complexity and rate - optimal scheme to achieve a distortion - optimal solution. an image transmission system was proposed in connection with jpeg2000 [10, 11 ]. the jscc and uep schemes in this approach try to optimize the rate - distortion function to achieve an efficient transmission. jpeg2000 provides high compression efficiency and introduces various features not included in other compression algorithms. this method has also adopted a standard component for medical image compression by digital imaging and communications in medicine (dicom). jpeg2000 adopts embedded block coding with optimal truncation (ebcot) as a source coder. the ebcot algorithm has high compression efficiency with a high complexity price, which needs a powerful central processing unit (cpu) for support. the compression part of the proposed scheme is similar to that of jpeg2000 's. however, unlike jpeg2000 's our scheme provides variable parity length for an adaptive channel coding, which replaces ebcot with ezw for compression and progressive image transmission. in, a framework for solving the end - to - end problem of progressive transmission of images over noisy channels has been presented which allows finding the optimal length of parity codes for each fixed length package to have minimum distortion in the decoded images. in, a joint source - channel decoder - based method for data transmission over noisy channels has been introduced. the authors in have used the maximum a posteriori (map) method to design a joint source - channel coding system. an adaptive source - channel coding scheme based on subband coding has been used in. in this approach, a suitable source and channel coding rates for each subblock has been proposed to minimize the total distortion. in, a low - complexity technique for the transmission of medical images has been proposed whereby the channel noise information has been effectively exploited. the main idea in is to segment the bitstream into consecutive subblocks of variable lengths and consider a tradeoff between the levels of source and channel coding systems. the authors in proposed a jscc method for transmission of images over fading channels and demonstrated the application of rate - compatible low - density parity - check (rc - ldpc) codes constructed by the progressive edge - growth algorithm, and used the uep to protect the images. in, a parametric methodology in progressive source - channel coding for rate allocation has been developed. the channel code rate is chosen based on the properties of source coder and the conditions of channel. the uep strategies for efficient progressive transmission are proposed in. under the condition of a target transmission rate, the jscc algorithm computes a uep scheme that maximizes the number of corrected bits over a noisy channel. in, authors have used a concatenation of rate - compatible punctured convolution code and cyclic redundancy check (crc) code to form a uep scheme and find the optimal rate allocation solutions for progressive image transmission. many communication systems allow two - way communication implying that the signals are back from receiver to transmitter to adjust the system parameters and obtain better system performance. the authors in [2023 ] have utilized the concept called hybrid automatic repeat request (harq) to ask for retransmission of erroneously received data and tradeoff allocation between the source and channel codes according to a rate - distortion optimization policy. many researches on tradeoff allocation bits between source codes and channel codes assume that the noise - level in the channel is known in advance. therefore, the feedback signal is figured out based on the known noise levels and the constraints set by the user. in the proposed algorithm, this has been modified since the parity lengths change according to the noise level in the received data whereby the amount of detected incorrect data is used to predict the conditions of the practical transmission channel. for medical image transmission, the quality of the reconstructed images (especially in the roi) should be acceptable. this can be set as the constraint for the quantizer and the compression algorithm in advance. therefore, the quality of the reconstructed image is only affected by the channel state and the proximity to the image roi. the feedback signal in the proposed scheme updates the parity length without the need for retransmission of the data or adding any extra overhead. the principal idea behind all these methods is that in a progressive transmission framework, the receiver reconstructs the transmitted image at various bit rates, which makes the fast and reliable retrieval of large images possible. in other words, the quality of the reconstructed image totally depends upon the volume of the received data, and the images can be reconstructed in any bitrate. furthermore, the image subblocks are coded separately. due to the high sensitivity to transmission noise, progressive transmission of images over noisy channels has to be accompanied by an appropriate channel coding, or a joint source - channel coding scheme. the noise in the current communication systems can be due to the electronic components, fading, doppler shift for mobile systems, bad weather, interferences, attenuations, and so forth. the reed - solomon (rs) codes utilized here are block - based error correcting codes and are widely used for channel coding. the rs(p, q) codes correct the symbol error and not the bit error ; lengths in terms of symbols. in this paper, we propose a novel interactive and adaptive joint source - channel coding with feedback algorithm for progressive transmission of medical images. this approach benefits from the idea of the jscc, roi, uep, and feedback technique together as follows.the conventional rs channel coding has been used.the variability of the parity code corresponds to both the proximity to the center of roi and the state of the practical transmission channel at the same time ; this makes an efficient source - channel coding possible.the selectivity of the roi is totally interactive and can be defined by the user in the receiver. this makes the method favorable to be used by clinicians who require fast access to the roi in the image.an algorithm for detection of the blocks in error is developed to detect and recover the corrupted data, estimate the noise level in the practical transmission channel, and feedback the information of the noisy channel to the transmitter to control the error rate in the reconstructed images in the subsequent transmission. the variability of the parity code corresponds to both the proximity to the center of roi and the state of the practical transmission channel at the same time ; this makes an efficient source - channel coding possible. the selectivity of the roi is totally interactive and can be defined by the user in the receiver. this makes the method favorable to be used by clinicians who require fast access to the roi in the image. an algorithm for detection of the blocks in error is developed to detect and recover the corrupted data, estimate the noise level in the practical transmission channel, and feedback the information of the noisy channel to the transmitter to control the error rate in the reconstructed images in the subsequent transmission. by utilizing our flexible system, a minimum distortion of the transmitted images in a fairly shorter transmission time is achieved. as the main contribution of this research, we adaptively control the lengths of parity code streams simultaneously with respect to the selected region (i.e., longer lengths correspond to the areas closer to the center of roi) and the amount of corrupted received data in the receiver. the system block diagram is shown in figure 1., we provide the details of roi selection. in section 2, application of rs channel coding in a variable - parity length scheme will be explained. in section 4, an algorithm for detection of the blocks in error is developed to evaluate the amount of incorrect received data in the receiver. simulation results are subsequently reported in section 5 followed by concluding remarks in section 6. wireless transmission of medical images involves construction of an effective joint source - channel coding to not only preserve the diagnostic information but also to enable progressive streaming of the data from the host to the receiver. ezw is a simple, efficient, and flexible compression algorithm for low bitrate image coding. the properties of dwt and ezw allow us to code and compress the data blocks individually and also compress it at any bitrate. therefore, based on progressive encoding, we can compress a block into a bitstream with increasing accuracy. traditionally, the input images are decomposed into many subblocks each to be coded, compressed, and transmitted individually. therefore, the input image is segmented into a number of subblocks firstly. and then wavelet transform (wt) decomposes each subblock into different time - frequency components. as it is detailed in, ezw codes the image into streams of six symbols, namely, p, n, z, t, 0, and 1. in mathematical terms, considering the image amplitude at location (x, y) is denoted by (x, y), and tn is the threshold in nth iteration, the definitions of the symbols are given in table 1. ezw suits progressive data transmission since it enables hierarchical encoding and decoding. in the proposed system, we chose a 3-level haar wavelet transform (hwt) to perform the dwt for each subblock due to its simplicity and being faster and easier to implement in comparison with other dwt methods. the coefficients in the lowest frequency subbands show the background information of the subblocks. the coefficients in the higher frequency subbands represent the details and edges. after computing the hwt, we compress the coded data according to a variable thresholding mechanism governed by the ezw. hence, a suitable approach is to use a variable threshold and transmit only those coefficients to the decoder that are larger than the threshold. the first step in the ezw algorithm is to determine the initial threshold level t0 and then repeatedly lowering the threshold by half at a time until the threshold has become smaller than the smallest coefficient to be transmitted ; or the iteration is stopped by request. the initial threshold t0 is set as follows:(1)t0=2n, n = log2max(\|(x, y)\|), where max() refers to the maximum value. the final threshold level determines the length of the bitstream output through the ezw process, the compression ratio of the input images, and the resolution of the reconstructed image. the length of the output bitstream mi is related to the number of times the threshold is halved as (2)mi=k=0ntib(t0i2k), where b(t) is the output bitstream of ezw based on the threshold t. t0i is the initial threshold in the ith subblock, and nti is the number of times the threshold is halved in the ith subblock. therefore, potentially, we can achieve any resolution in the reconstructed images through setting the initial threshold, and the number of times the threshold is halved. as an example, figure 2 shows the three regions of roi, r1, r2 centered at point (x, y). in figure 2, the resolution in area r2 is the lowest and that of roi is the highest. therefore, the quality of the reconstructed subblocks and consequently the compression rate depends on the size of the embedded zerotree. based on the assigned parameters for ezw, the data in each subblock would be compressed with different rates depending on the location of the subblock. often, the physician is only interested in a particular part of the image. therefore, the system is designed in such a way to enable changing the location and size of the roi without any emphasis on the other regions. in this example, the roi, r1, and r2 may be defined as for roi : (3)(ix)2+(jy)2ra, for r1 : (4)rarb, where x and y are the coordinates of the center of roi assigned by the physician using a mouse click, thereafter both values are sent back to the host. the values of ra and rb are the radius of roi and r1, and i and j express the x y image pixels ' coordinates. in the successive progressive stages, the values of ra and rb gradually expand in the next progressive transmission to create the reconstructed image of higher quality. in our proposed algorithm, then, the reconstruction is progressively continued regarding the highest priority of roi. for roi : (3)(ix)2+(jy)2ra, for r1 : (4)rarb, the progressive bitstream is heavily affected by the channel noise ; a single bit error may make the reconstruction of the image impossible. the adaptive joint source - channel coding and the developed blind technique for evaluation of errors in the receiver are the effective ways to address this problem. the channel coding variables can be adjusted by using the feedback information about the channel state and the image region content. the rs code is a subset of bch codes ; they are linear block codes and are efficient for bursty - type transmission channels. the rs codes are constructed by considering a polynomial for the input information and then use the polynomial coefficients instead of the original data for transmission. in an rs(p, q) code, p = 2 1 is the number of symbols in a codeword, and m is the number of bits in each symbol. in the proposed algorithm, the data in different image regions, as denoted in figure 2 for three regions, are protected by variable length parity codes as for the uep. the data in the roi is treated as the most important information and protected by longer length parity codes. figure 3 illustrates the channel coding strategy and figure 4 shows the receiver. the overall channel code length remains fixed and the length of message k, qk ; k = 1, 2,, n, and the parity length c are variable. for rs codes, (255 qn)/2 indicates the error - correction capability of rs coder. here, the rs codes, rs(255, q), have 255 symbols in length. according to the uep, the ratio of parity to overall code length for the n regions should follow (6)croi > cr1>cr2>crn1, where croi, the length of parity code, is for the roi and so on. furthermore, the length of parity code is also affected by the noise in the channel, that is, cregion~(r, n), where r is the distance from the center of roi, and n expresses the noise in the practical transmission channel. an adaptive variable parity allocation requires the error between the transmitted image i(x, y) and the received image i^(x, y) to be minimized under the desired conditions. suppose that the error is defined as (7)=i(x, y)i^(x, y)2, where \|\|\|\|2 denotes the i2-norm. generally, we wish to have the optimum parity length such that (8)copt = minc subject to t, where t is an acceptable error level in the receiver. according to the above discussion, the parity length can be defined as (9)c = g(r, sn)=f(r, ber), where s / n and ber stand, respectively, for signal - to - noise ratio and bit - error rate (the ber here represents the noise situation in the channel and does not refer to the output bit - error rate). the functions g and f are generally nonlinear functions, which can be defined empirically based on a number of trials. from figure 5, f~(0 r) for a fixed ber, where r is measured with respect to the number of pixels, and from figure 6, it can be concluded that f~(ber + 0) for fixed proximity distance r. in more general applications, a more accurate and possibly complicated function may be adopted. therefore, a reasonably accurate function can be modeled as (10)f(r, ber)=(0r)(ber+0), or (11)f(r, ber)=3ber2rber1r+0, where is are constant coefficients and can be easily found based on figures 5 and 6. embedded coding has many advantages, especially for progressive image / video transmission, because the reconstructed images can be decoded at any bitrate. however, it is highly sensitive to transmission noise and frequently collapses if even a single transmitted information bit is incorrectly decoded. in most cases, the receiver sends back a signal called harq to the transmitter requesting for retransmission under the new constraints. here, the channel noise level has to be somehow estimated in the receiver. the estimation process is blind since there is no a priori information about the channel. in some practical cases however, a test image can be transmitted occasionally and evaluated in the receiver. in the proposed scheme, this algorithm detects the address in which the number of symbols for each subblock is indicated. the algorithm reassigns the number of symbols to each subblock according to a built - in decision making criterion (policy) when the number of symbols within the received data is determined incorrectly by the receiver. since the header information provides the number of symbols per each data package, an extra check can be carried out to ensure that the header is divisible by 4 and is not greater than 64 (to address each pixel) for the 8 8 subblocks. for more accuracy, the algorithm checks the next address of the number of symbols to ensure that the current data is correct. if successive error data is detected, the algorithm is able to determine the number of subblocks and reassign the number of symbols for each subblock in the set of detected incorrect data. although, the proposed algorithm does not have capability of recovery of the current corrupted package, but it can conjecture an appropriate number to replace the corrupted data based on the built - in decision making criterion (policy) to avoid the reconstructed image to collapse. this adjusts the system for the transmission of the subsequent package and prevents propagation of the error. this not only enhances the quality of the reconstructed image but also provides a feedback for the adjustment of parity length. the performance of the system in different noise - levels is evaluated using the peak signal - to - noise ratio (psnr) defined as (12)psnr(db)=10 log 10a2mse, where a is the maximum image amplitude, and mse is defined as (13)mse=i^i22i22, where i^ represents the reconstructed subblock of the image, and i is the subblock of the original input image. finally, the parallel structure of the channel coding and decoding scheme is very suitable in hardware implementation of the system. a number of parallel boards can be easily used in order to speed up the overall process. in this work, the proposed system is simulated in both binary symmetric channel (bsc) and flat - fading rayleigh channel. the bsc is the simplest channel model, only zeros and ones are conveyed in the channel. but the wireless mobile communication channels are often considered to be with flat - fading rayleigh noise. in this paper, we simulated both bsc and flat - fading channel models and tested the performance of the proposed techniques against both models. the noise in the simulated channel has been considered in such a way to set a ber of 0.01 in the received end. for when the errors are uniformly distributed, the average parity length is 42 for a 255 length code length. this recovers the roi perfectly when either bsc or rayleigh channel is considered and the channel noise is equivalent to ber = 0.01. the developed algorithm has been tested for a number of images, two of which are analyzed here. the first image is a 150 123 pixels color dental implant image, and the second image is a 508 512 pixels monochrome x - ray bone image. each noisy channel involves a binary symmetric channel (bsc) and flat - fading rayleigh channel with a certain ber. in this simulation, the error bits are generated by randomly inverting a certain percentage of bits in the ezw bitstream. to verify the effectiveness of the system, the image regions are progressively transmitted through four stages of p1, p2, p3, and p4. during p1, the background image is transmitted. p2 and p3 are the second and third stages, both for transmission of roi, r1, and r2. p4 is the fourth stage mainly to transmit the details of the roi (and the rest of the image if necessary). in the approach presented here, firstly, the user (physician) specifies the address of the transmitted medical image in the transmitter within the dialogue - based software. after receiving the low - resolution background image, the user identifies the center of roi by a mouse and the radius of roi by entering a value within the dialogue - based software. then, the algorithm adjusts the length of the parity codes initially proportional to the proximity of the image regions to the center of roi as c0, c0 2, c0 4 for roi, r1, and r2, respectively. accordingly, the receiver detects and counts the packages in error by estimating the status of the channel. the parity lengths remain the same if the distortion in the reconstructed image is acceptable. otherwise, the codes will be adjusted automatically. typically, the ratio of parity code to the overall code length is larger for the clinically higher priority areas, that is, the areas closer to the center of roi as stated in (6). figure 5 indicates the ratios of parity length and the overall codeword according to the experimental results. table 2 gives an example of the percentage of information for the regions r1, r2, and r2 for fixed proximity levels of ra and rb, as in figure 9. table 3 indicates the average compression ratios for various regions before the channel coding during the four successive transmission stages. the compression ratio is defined as the ratio between the data volume of the coded data and the original data. however, by changing one of roi coordinates, or ra and rb, data in tables 2 and 3 are also changed. in figure 6, the parity lengths are found by averaging the results of 10 trials under various noise levels. these are estimated by the algorithm developed for detection of the blocks in error in the receiver. data in roi is the most important data in the overall image ; therefore, the length of parity codes is longer than that in r1 and r2. in the proposed system, the codeword length of rs codes is 255, and the number of error bit is generated at random. figure 7 shows the frequency of the set of parity lengths in 10 trials for when the channel noise is set by ber = 0.003 equivalent to the occurrence of 7 errors. as long as the error in the receiver remains higher than a threshold th, the length of parity increases. consequently, in these cases, the parameters in (10) and (11) are approximately 0 = 0.08, = 2 10, 0 = 5, = 5 10 and accordingly 0 = 00 = 0.4, 1 = 0 = 10, 2 = = 1, and 3 = 0 = 4 10. figure 8 shows the psnrs for successive transmission of four stages under various noise - level conditions. figure 9(b) is progressively reconstructed image after stage p2 in which the roi, r1, and r2 are reconstructed. at this stage, figure 9(c) represents the reconstructed image at stage p3 during which the regions roi, r1, and r2 are reconstructed. the same procedure can be followed for encoding and transmission of any other medical image. however, the coordinates of the center of roi as well as the size of roi maybe adjusted according to the requirement by the user. for example, in figure 10, the roi is selected in the corner. figure 11 demonstrates that a fixed - size parity code is not suitable for an efficient transmission system. however, the system has been modified based on the proposed method in sections 2 and 4 to allow variable lengths of parity. figures 12 and 13 show no error in the roi stating that the overall system has been remarkably improved. in figure 14, another example of a decoded image (a 508 512 monochrome x - ray bone image) is given, and the variable length parity has been examined. however, the transmission can continue until the last stage during which a complete error - free image is reconstructed. in this paper, we presented a new adaptive source - channel coding with feedback for the progressive transmission of medical images. the system is adaptive to both image content and channel specifications. however, this application is merely for wireless channels (generally narrowband). the capability of data error detection and correction with automatic adjustment, low image transmission time, and efficient communication are the key features in this proposed system. therefore, the length of parity codes can be adjusted automatically based on the location of the image subblocks and the practical characteristics of the communication channel to provide an adequate data protection. the overall code length for the channel encoder / decoder is fixed. a wide range of fluctuations in the channel characteristics (mainly noise level) can be tolerated in the system. the algorithm of detection of subblock in errors can detect the packages in error in the receiver and feed it back to the transmitter for adjustment of the parity length. the proposed system has also been tested for the communication channels with different capacities and noise levels. the presented results verify the effectiveness of the system in terms of both adaptivity and flexibility of interaction. a matlab - based tcp / ip connection has been established to demonstrate the proposed interactive and adaptive progressive transmission system. although some theoretical results are comparable to those of other new techniques such as the uep or rate allocation approaches, this contribution provides a practical, flexible, and interactive method which suits medical applications.	a novel adaptive source - channel coding with feedback for progressive transmission of medical images is proposed here. in the source coding part, the transmission starts from the region of interest (roi). the parity length in the channel code varies with respect to both the proximity of the image subblock to the roi and the channel noise, which is iteratively estimated in the receiver. the overall transmitted data can be controlled by the user (clinician). in the case of medical data transmission, it is vital to keep the distortion level under control as in most of the cases certain clinically important regions have to be transmitted without any visible error. the proposed system significantly reduces the transmission time and error. moreover, the system is very user friendly since the selection of the roi, its size, overall code rate, and a number of test features such as noise level can be set by the users in both ends. a matlab - based tcp / ip connection has been established to demonstrate the proposed interactive and adaptive progressive transmission system. the proposed system is simulated for both binary symmetric channel (bsc) and rayleigh channel. the experimental results verify the effectiveness of the design.
a sigmoid - shaped interventricular septum (sis) is generally considered a normal part of the aging process and is of little clinical significance. however, certain patients with sis may experience clinical symptoms such as dyspnea upon effort and syncope. in patients with hypertrophic obstructive cardiomyopathy (hocm), narrowing of the left ventricular outflow tract (lvot) generates a left ventricular (lv) pressure gradient, resulting in reduced cardiac output and subsequent syncope. in these patients, the vasovagal reflex, i.e., the bezold by contrast, the mechanisms of syncope in patients with sis without left ventricular hypertrophy (lvh),, have yet to be fully elucidated. here, we report two patients with sis who presented with recurrent syncope. we were successful in clarifying the mechanisms of syncope and effects of beta - blockers in these cases. an 81-year - old man with no history of heart disease was admitted to our hospital for the treatment of recurrent episodes of syncope after drinking. he experienced the first episode at age 75. on admission, his blood pressure (bp) was 144/81 mmhg and the heart rate (hr) was 56 beats / min. physical examination was otherwise unremarkable except for a grade 2/6 systolic ejection murmur at the apex area. a 12-lead electrocardiogram revealed normal sinus rhythm and left axis deviation without lvh, st segment depression, and t - wave inversion. two - dimensional transthoracic echocardiography (2de) revealed an sis protruding into the lv (fig. color doppler echocardiography revealed turbulent systolic flow at the lvot with a pressure gradient of 8 mmhg, determined by continuous - wave doppler (fig. the reliability of the doppler measurement of the pressure gradient at the lvot was validated by a board certified fellow of the japan society of ultrasonics in medicine (jsum). m - mode echocardiography demonstrated systolic anterior movement of the mitral chordae tendineae. to investigate the cause of syncope after drinking, a head - up tilt test (hut) was performed with 25 g of alcohol loading. at 16 min of a 60-degree hut, the lvot pressure gradient increased from 8 to 176 mmhg, and m - mode echocardiography demonstrated mid - systolic hemiclosure of the aortic valve that was not noted when the patient was in the supine position (fig. 1c). at 18 min of hut, the patient experienced presyncopal symptoms with a decrease in both bp and hr (from 138/74 to 83/46 mmhg and from 52 to 46 beats / min, respectively). hut with 0.3 mg nitroglycerine (ng) provocation (hut+ng) also induced hypotension and bradycardia with presyncopal symptoms following an increase in the lvot pressure gradient. we started the patient on bisoprolol (2.5 mg / day), and his syncopal episodes have not recurred since. a 66-year - old man presented with recurrent syncope after drinking. on admission, his bp and hr were 123/82 mmhg and 65 beats / min, respectively. physical examination was unremarkable except for a grade 2/6 systolic ejection murmur at the fourth intercostal space on the left sternal border. an electrocardiogram revealed sinus rhythm (62 beats / min) and left axis deviation without lvh and st - t changes. his chest x - ray was normal, and 2de showed sis with normal systolic function (ejection fraction, 74%). color doppler echocardiography revealed turbulent systolic flow at the lvot with a peak pressure gradient of 8 mmhg, determined by continuous - wave doppler. m - mode echocardiography demonstrated systolic anterior movement of the mitral chordae tendineae. after excluding other possible causes of syncope with non - invasive tests, hut 2). no symptoms occurred, and both bp and hr were not significantly decreased during the baseline tilt (60) or during hut+ng. however, presyncopal symptoms developed in association with abrupt hypotension and baradycardia at 2.5 min of hut with an isoproterenol infusion (0.015 g / kg / min). a task force monitor (cn systems, graz, austria) revealed that at the time of presyncope, total peripheral vascular resistance (tpr) decreased (fig. a diagnosis of vasovagal reflex was made based on the hut with an isoproterenol infusion. an isoproterenol provocation test was also performed in the supine position. at 1 min and 46 s after initiating the isoproterenol infusion (0.02 g / kg / min), hypotension and sinus bradycardia were induced along with ventricular escape beats and presyncopal symptoms (fig., the peak pressure gradient at the lvot was increased from 8 to 59 mmhg with a decrease in tpr. we started the patient on bisoprolol (2.5 mg / day), and his syncopal episodes have not recurred since. sis is a morphological characteristic of the basal interventricular septum that protrudes into the lv cavity. this cardiac malformation has generally been considered a natural part of aging without pathophysiological or clinical significance,. however, several studies demonstrated that sis could be associated with clinical symptoms such as dyspnea on effort and syncope, as seen in patients with hocm. syncope in patients with sis might be attributable to an increase in the lvot pressure gradient, particularly when vasodilators or inotropic drugs are administered,. in patients with sis, recurrent syncope and dyspnea on effort are caused by a decrease in cardiac output through lvot obstruction, as in patients with hocm. class ia antiarrhythmic drugs such as cibenzoline as well as -blockers have been effective in treating these symptoms,,,. however, in patients with sis, vasovagal reflex could be an important cause of syncope. in case 1, a decrease in cardiac venous return caused by the vasodilatory effects of alcohol or ng reduced cardiac output, thereby enhancing sympathetic nerve activity. an increase in lv pressure concomitant with increased lv contraction can induce vasovagal reflex via activation of the lv mechanoreceptor. in case 2, presyncopal symptoms with hypotension and bradycardia developed during hut with an isoproterenol infusion (fig. a decrease in cardiac venous return caused by a 60-degree hut reduced lv volume, resulting in increased sympathetic nerve activity. isoproterenol further enhanced lv contraction and lvot obstruction, and vasovagal reflex was similarly induced via activation of the lv mechanoreceptor as in case 1. because high - frequency power was increased at the time of presyncope in case 2, isoproterenol induced not only lvot obstruction but also vasovagal reflex, resulting in hypotension and bradycardia. the narrowing of the lvot along with an increase in the left ventricular pressure gradient might be related to clinical symptoms in patients with sis, as seen in hocm patients. it is well known that the negative inotropic action of -blockers decreases the lvot pressure gradient and can prevent syncope in patients with hocm. however, the efficacy of -blockers for the prevention of syncope has not been fully clarified in patients with sis. the administration of cibenzoline in addition to atenolol was recently reported to decrease the lv pressure gradient, thereby relieving clinical symptoms in patients with sis. in the present study, we showed that the vasovagal reflex played a major role in the induction of syncope. we further showed that bisoprolol could prevent syncope by decreasing the lv pressure gradient in two patients with sis.	sigmoid - shaped interventricular septum (sis) is not uncommon in elderly patients and is considered a normal part of the aging process. however, several patients have been reported to have clinical symptoms due to the narrowing of the left ventricular outflow tract (lvot). two patients with sis presented with recurrent episodes of syncope after drinking or taking sublingual nitroglycerin (ng). in both patients, a head - up tilt test involving provocation with alcohol, ng, or isoproterenol induced the vasovagal reflex along with an increase in the pressure gradient between the apex and lvot. the patients experienced no further episodes of syncope after initiating bisoprolol treatment. in patients with sis, induction of the vasovagal reflex via an increase in left ventricular (lv) pressure due to lvot obstruction concomitant with increased lv construction is a potentially important cause of syncope, which may be effectively prevented by beta - blockers.
the principal cause of myocardial infarction in children is kawasaki disease (kd) (1). in kd patients, myocardial dysfunction due to coronary disorders is the most important complication during long - term follow - up (1). several studies have been undertaken to find a noninvasive method for the identification of systolic myocardial dysfunction in kd patients at the early stage of coronary insufficiency (2 - 5). echocardiography has been employed as a noninvasive method in some studies for early diagnosis (1). speckle - tracking echocardiography is a new method in echocardiographic imaging and is suggested for the evaluation of systolic myocardial dysfunction analysis. speckle - tracking - derived parameters are considered as valid indices for the detection of sub - clinical myocardial systolic dysfunction secondary to coronary insufficiency (6). a few studies conducted on longitudinal and circumferential markers have introduced myocardial strain (s) and strain rate (sr) as the early markers of myocardial dysfunction in patients suffering from kd (1, 7). while some of these studies reported a reduction in s and sr in kd patients, others reported no difference in sr between control groups and kd patients (1, 7, 8). note that all of these studies investigated only s and sr as the markers of systolic dysfunction in kd patients (1, 7, 9). therefore, we sought to assess changes in myocardial time to peak, view point (vp) velocity, longitudinal velocity, global s, and sr, in addition to s and sr, as a noninvasive method for the early diagnosis of the effect of kd on coronary artery insufficiency during the acute phase in kd patients. thirty - two patients diagnosed with kd and 19 control subjects were recruited in this study. participants with a history of hypertension, diabetes, coronary artery disease, myocardial infarction, stroke, congenital heart disease, acquired valvular disease, dysrhythmia, or congestive heart failure were excluded. patients in the acute phase of the disease were enrolled before the administration of intravenous immunoglobulin. demographic characteristics and clinical findings, including systemic blood pressure (systolic and diastolic) and body weight, were recorded in a specially designed questionnaire. was obtained from the parents of all the participants after they had been provided with a thorough explanation about the purpose of the study. the control subjects were selected from healthy children who were referred to the cardiovascular clinic for innocent murmur and had normal echocardiography. echocardiographic evaluations were performed by the same operator, using a mylab60 echo machine (esaote, s.p.a mylab60, florence, italy, class 2a). echocardiography was done with a multi - frequency phased array 122 (3 - 8 mhz) transducer for patients under 1 year old and a phased array 230 (1.6 - 4 mhz) transducer for patients older than 1 year. video clips of the two and three - chamber views were recorded as two - dimensional harmonic image cine - loops. these records were analyzed by xstrain software license (esaote, florence, italy, class 2a) and stored on the computer for off - line analysis. based on the algorithm of optical flow analysis, xstrain software was utilized to calculate longitudinal myocardial vp velocity, longitudinal velocity, s, sr, time to peak systolic strain (tps), and time to peak strain rate (tpsr) from the digitized two - dimensional video clips. initially, the frame at the beginning of systole, when the endocardial border is better visualized, was chosen. after the manual determination of the 3 main points on the mitral annulus and the apex of the endocardial border of the left ventricle in the first patient, the aided heart segmentation (ahs) tool was employed for the rest of the patients to identify the best border. in other words, in the first patient, each point had to be selected and manually placed, whereas in the other patients, the ahs tool suggested which myocardial points (landmarks) should be tracked (1, 6). after the semi - automatic identification of the points by the ahs mode, the points were checked by a skilled operator and the inaccurate points were corrected for the confirmation of the best border. longitudinal velocity, vp velocity, s, and sr were calculated for each point. thirteen equidistant points were tracked in the apical four, two, and three - chamber views (figure 1) to draw 1 subdivision in 6 equidistant segments (each including apical, mid, and basal segments), allowing the use of an 18-segment model. the points positioned in a semi - automatic way (ahs mode) were selected from the septal side of the mitral annulus in the apical four - chamber view (figure 1), from the inferior wall of the mitral annulus in the apical two - chamber view, and from the points starting from the posterior wall to the anteroseptal wall in the three - chamber view. once the procedure was completed, a view point represented by a triangle was shown in the selected cycle. vp velocity was calculated based on the movement of the wall edge from this point. longitudinal velocity, vp velocity, tps, tpsr, s, and sr images were semi - automatically obtained using xstrain software. for intra - observer variability, all the variables for all the segments were measured in 8 subjects by one skilled operator at two - week intervals. mean and standard deviation were reported for all the quantitative echocardiographic data, and the pearson correlation test was used to test intra - observer variability. subsequently, precision was reported with the pearson correlation coefficient (r) and with c bias, which is a bias correction factor for accuracy measurement. the mann - whitney u test was utilized to compare the differences between the groups. repeated measure of analysis of variances was applied to compare the changes of s, sr, tps and tpsr across different levels of left ventricular myocardium (basal, mid and apical) between the kd patients and controls. comparisons were done while the variables were adjusted for age ; because of the importance of the effect of age in the changes of statistical significance, it was set at a p value smaller than 0.05. thirty - two patients diagnosed with kd and 19 control subjects were recruited in this study. participants with a history of hypertension, diabetes, coronary artery disease, myocardial infarction, stroke, congenital heart disease, acquired valvular disease, dysrhythmia, or congestive heart failure were excluded. patients in the acute phase of the disease were enrolled before the administration of intravenous immunoglobulin. demographic characteristics and clinical findings, including systemic blood pressure (systolic and diastolic) and body weight, were recorded in a specially designed questionnaire. was obtained from the parents of all the participants after they had been provided with a thorough explanation about the purpose of the study. the control subjects were selected from healthy children who were referred to the cardiovascular clinic for innocent murmur and had normal echocardiography. echocardiographic evaluations were performed by the same operator, using a mylab60 echo machine (esaote, s.p.a mylab60, florence, italy, class 2a). echocardiography was done with a multi - frequency phased array 122 (3 - 8 mhz) transducer for patients under 1 year old and a phased array 230 (1.6 - 4 mhz) transducer for patients older than 1 year. video clips of the two and three - chamber views were recorded as two - dimensional harmonic image cine - loops. these records were analyzed by xstrain software license (esaote, florence, italy, class 2a) and stored on the computer for off - line analysis. based on the algorithm of optical flow analysis, xstrain software was utilized to calculate longitudinal myocardial vp velocity, longitudinal velocity, s, sr, time to peak systolic strain (tps), and time to peak strain rate (tpsr) from the digitized two - dimensional video clips. initially, the frame at the beginning of systole, when the endocardial border is better visualized, was chosen. after the manual determination of the 3 main points on the mitral annulus and the apex of the endocardial border of the left ventricle in the first patient, the aided heart segmentation (ahs) tool was employed for the rest of the patients to identify the best border. in other words, in the first patient, each point had to be selected and manually placed, whereas in the other patients, the ahs tool suggested which myocardial points (landmarks) should be tracked (1, 6). the frame of the ahs mode was drawn based upon maximum likelihood neighboring points. after the semi - automatic identification of the points by the ahs mode, the points were checked by a skilled operator and the inaccurate points were corrected for the confirmation of the best border. longitudinal velocity, vp velocity, s, and sr were calculated for each point. thirteen equidistant points were tracked in the apical four, two, and three - chamber views (figure 1) to draw 1 subdivision in 6 equidistant segments (each including apical, mid, and basal segments), allowing the use of an 18-segment model. the points positioned in a semi - automatic way (ahs mode) were selected from the septal side of the mitral annulus in the apical four - chamber view (figure 1), from the inferior wall of the mitral annulus in the apical two - chamber view, and from the points starting from the posterior wall to the anteroseptal wall in the three - chamber view. once the procedure was completed, a view point represented by a triangle was shown in the selected cycle. vp velocity was calculated based on the movement of the wall edge from this point. longitudinal velocity, vp velocity, tps, tpsr, s, and sr images were semi - automatically obtained using xstrain software. for intra - observer variability, all the variables for all the segments were measured in 8 subjects by one skilled operator at two - week intervals. mean and standard deviation were reported for all the quantitative echocardiographic data, and the pearson correlation test was used to test intra - observer variability. subsequently, precision was reported with the pearson correlation coefficient (r) and with c bias, which is a bias correction factor for accuracy measurement. the mann - whitney u test was utilized to compare the differences between the groups. repeated measure of analysis of variances was applied to compare the changes of s, sr, tps and tpsr across different levels of left ventricular myocardium (basal, mid and apical) between the kd patients and controls. comparisons were done while the variables were adjusted for age ; because of the importance of the effect of age in the changes of statistical significance, it was set at a p value smaller than 0.05. of a total of 35 kd patients examined by echocardiography, 32 patients had complete echocardiographic data, and of 21 control subjects undergoing echocardiographic examination, 2 were eliminated from our study due low quality imaging. most of the kd patients were male (21 ; 65.5%), and there were no significant differences between the two groups regarding gender and age (table 1). the mean systolic and diastolic blood pressures and ejection fraction were not different between the groups, and nor was there any significant difference between the two groups in terms of body weight (p = 0.40). abbreviation : kd, kawasaki disease. intra - observer variability for c bias was r = 0.90 (95% confidence interval : 0.87 - 0.93 ; p < 0.01), and the bias correction factor (accuracy) was 0.94. in the four - chamber view, there were significant differences in s between kd patients and control group in the basal lateral, mid - wall lateral, basal septal, and mid - wall septal views, as can be seen in table 2. the sr in the basal lateral, basal septal, apical septal, and mid - wall septal views was lower in the kd patients than in the control group (1.75 0.46 vs. 2.21 0.56, 1.73 0.47 vs. 2.09 0.54, 1.53 0.29 vs. 1.86 0.43, and 1.48 0.34 vs. 1.72 0.26, respectively). the apical lateral and apical septal vp velocity markers were significantly higher in the kd patients, while there was no significant difference between the kd patients and the control subjects in long velocity indices. abbreviations : kd, kawasaki disease, vp, view point. compared to the controls, the kd patients had a significantly lower mean s and sr in the basal and mid - wall anterior and basal inferior (table 3). the mean s and sr of the kd patients were significantly reduced in the basal and mid - wall anteroseptal and the basal, mid - wall, and apical posterior in the three - chamber view. no significant differences were seen between the two groups in vp velocity and longitudinal velocity markers. abbreviations : kd, kawasaki disease, vp, view point. in overall, in univariate analysis mean global s and sr was significantly reduced in the kd patients (17.03 1.28 vs. 20.22 2.14% and 1.66 0.16 vs. 1.97 0.25 1/second, respectively) (table 5), while there was no significant difference between the kd patients and the control subjects in global mean time to peak systolic indices. using repeated measure of analysis of variances, we observed that s and sr decreased from base to apical level in both groups (figure 2). the change in the pattern of age adjusted mean s and sr across levels was different between the groups (p < 0.001 for both parameters). as it is shown in figure 2 (a and b) reduction of s and sr was sharply in the controls, while this the change in age adjusted mean time to peak strain from basal to apical level in kd patients was also significantly different from that in controls (p = 0.001) (figure 2 c), showing that in controls mean time to peak strain decreased from base to apical level, whereas it remained unchanged across the levels in kd patients. mean time to peak systolic strain rate, adjusted for age, has almost similar change across levels in both groups (p = 0.060) (figure 2 d). of a total of 35 kd patients examined by echocardiography, 32 patients had complete echocardiographic data, and of 21 control subjects undergoing echocardiographic examination, 2 were eliminated from our study due low quality imaging. most of the kd patients were male (21 ; 65.5%), and there were no significant differences between the two groups regarding gender and age (table 1). the mean systolic and diastolic blood pressures and ejection fraction were not different between the groups, and nor was there any significant difference between the two groups in terms of body weight (p = 0.40). abbreviation : kd, kawasaki disease. intra - observer variability for c bias was r = 0.90 (95% confidence interval : 0.87 - 0.93 ; p < 0.01), and the bias correction factor (accuracy) was 0.94. in the four - chamber view, there were significant differences in s between kd patients and control group in the basal lateral, mid - wall lateral, basal septal, and mid - wall septal views, as can be seen in table 2. the sr in the basal lateral, basal septal, apical septal, and mid - wall septal views was lower in the kd patients than in the control group (1.75 0.46 vs. 2.21 0.56, 1.73 0.47 vs. 2.09 0.54, 1.53 0.29 vs. 1.86 0.43, and 1.48 0.34 vs. 1.72 0.26, respectively). the apical lateral and apical septal vp velocity markers were significantly higher in the kd patients, while there was no significant difference between the kd patients and the control subjects in long velocity indices. abbreviations : kd, kawasaki disease, vp, view point. compared to the controls, the kd patients had a significantly lower mean s and sr in the basal and mid - wall anterior and basal inferior (table 3). the mean s and sr of the kd patients were significantly reduced in the basal and mid - wall anteroseptal and the basal, mid - wall, and apical posterior in the three - chamber view. no significant differences were seen between the two groups in vp velocity and longitudinal velocity markers. abbreviations : kd, kawasaki disease, vp, view point. in overall, in univariate analysis mean global s and sr was significantly reduced in the kd patients (17.03 1.28 vs. 20.22 2.14% and 1.66 0.16 vs. 1.97 0.25 1/second, respectively) (table 5), while there was no significant difference between the kd patients and the control subjects in global mean time to peak systolic indices. using repeated measure of analysis of variances, we observed that s and sr decreased from base to apical level in both groups (figure 2). the change in the pattern of age adjusted mean s and sr across levels was different between the groups (p < 0.001 for both parameters). as it is shown in figure 2 (a and b) reduction of s and sr was sharply in the controls, while this the change in age adjusted mean time to peak strain from basal to apical level in kd patients was also significantly different from that in controls (p = 0.001) (figure 2 c), showing that in controls mean time to peak strain decreased from base to apical level, whereas it remained unchanged across the levels in kd patients. mean time to peak systolic strain rate, adjusted for age, has almost similar change across levels in both groups (p = 0.060) (figure 2 d). in the four - chamber view, there were significant differences in s between kd patients and control group in the basal lateral, mid - wall lateral, basal septal, and mid - wall septal views, as can be seen in table 2. the sr in the basal lateral, basal septal, apical septal, and mid - wall septal views was lower in the kd patients than in the control group (1.75 0.46 vs. 2.21 0.56, 1.73 0.47 vs. 2.09 0.54, 1.53 0.29 vs. 1.86 0.43, and 1.48 0.34 vs. 1.72 0.26, respectively). the apical lateral and apical septal vp velocity markers were significantly higher in the kd patients, while there was no significant difference between the kd patients and the control subjects in long velocity indices. compared to the controls, the kd patients had a significantly lower mean s and sr in the basal and mid - wall anterior and basal inferior (table 3). the mean s and sr of the kd patients were significantly reduced in the basal and mid - wall anteroseptal and the basal, mid - wall, and apical posterior in the three - chamber view. no significant differences were seen between the two groups in vp velocity and longitudinal velocity markers. in overall, in univariate analysis mean global s and sr was significantly reduced in the kd patients (17.03 1.28 vs. 20.22 2.14% and 1.66 0.16 vs. 1.97 0.25 1/second, respectively) (table 5), while there was no significant difference between the kd patients and the control subjects in global mean time to peak systolic indices. using repeated measure of analysis of variances, we observed that s and sr decreased from base to apical level in both groups (figure 2). the change in the pattern of age adjusted mean s and sr across levels was different between the groups (p < 0.001 for both parameters). as it is shown in figure 2 (a and b) reduction of s and sr was sharply in the controls, while this the change in age adjusted mean time to peak strain from basal to apical level in kd patients was also significantly different from that in controls (p = 0.001) (figure 2 c), showing that in controls mean time to peak strain decreased from base to apical level, whereas it remained unchanged across the levels in kd patients. mean time to peak systolic strain rate, adjusted for age, has almost similar change across levels in both groups (p = 0.060) (figure 2 d). echocardiographic methods are employed for detecting myocardial dysfunction due to coronary insufficiency in kd patients in early stages (1, 9, 10). as a noninvasive tool, two - dimensional speckle - tracking echocardiography has been used similarly for both children and adults. this technique describes peak systolic and time to peak systolic s, sr, vp velocity, and longitudinal velocity in 6 views (11 - 13). the existing literature contains some information on two - dimensional speckle - tracking indices such as s and sr for the diagnosis of coronary insufficiency in early stages in kd patients. in this study, we demonstrated that there were reduced s and sr in the kd patients in the four, two, and three - chamber views compared to the controls. we investigated systolic function as a superior marker of coronary insufficiency, although in one study, both diastolic and systolic function indicators were studied (1). reported systolic sr under exercise as an indicator of myocyte dysfunction in kd patients (1). in the present study, we observed that global s and sr decreased from basal to apical levels in both kd and normal groups. in line with our findings, tierney. demonstrated that there was impaired relaxation in kd patients in the early phase of the disease and also in advanced kd patients with coronary artery disease during the disease course (14). yu. compared patients in the acute phase of kd with normal controls (10). we also included kd patients in the acute phase of the disease and confirmed that longitudinal basal and mid - wall s in two views were lower in the kd patients, which chimes in with the results of the yu. nonetheless, in contrast to our study, the authors of the study in question did not observe significant difference between the control and patient groups regarding systolic sr (10). used velocity vector imaging to determine s and sr as systolic dysfunction indicators and showed a reduction in s and sr in kd patients in the acute phase compared with a control group, especially in patients who suffered from coronary artery disease (7). investigated only s and sr as markers of myocardial dysfunction, whereas we studied longitudinal velocity and vp velocity and the time to peak values of these indices, as systolic dysfunction indicators, in addition to s and sr, and showed that vp velocity was higher in the kd patients in some views. the present study has some limitations, first and foremost among which is the unavailability of some echocardiographic techniques, resulting in our inability to perform circumferential measurements. these measurements were studied only during the acute phase of the disease because we could not follow up the patients. we believe that a comparison of the echocardiographic variables of interest in this study between the acute phase and later follow - up may provide more conclusive data. another limitation in this study is that we could not exclude patients with subclinical myocarditis due to the fact that this condition is undetectable by usual methods and that it requires invasive methods for diagnosis. speckle - tracking echocardiography is a new method for the evaluation of systolic ventricular dysfunction. in this study, we used this method in a large sample size and showed that s and sr decreased in the kd patients with different pattern than the controls. however, in contrast to other studies, we demonstrated a drop in s and sr from base to apex in both control and kd groups. reduced global s and sr may be used as early indicators for systolic ventricular dysfunction in kd patients. further studies are needed to compare s and sr obtained in the acute phase of kd with that in later follow - up.	background : evaluation of myocardial function by speckle - tracking echocardiography is a new method for the early diagnosis of systolic dysfunction.objectives:we aimed to determine myocardial speckle - tracking echocardiography indices in kawasaki disease (kd) patients and compare them with the same indices in control subjects.patients and methods : thirty - two patients (65.5% males) with kd and 19 control subjects with normal echocardiography participated in this study. after their demographic characteristics and clinical findings were recorded, all the participants underwent transthoracic echocardiography. strain (s), strain rate (sr), time to peak strain (tps), and strain rate (tpsr), longitudinal velocity and view point velocity images in the two, three, and four - chamber views were semi - automatically obtained via speckle - tracking echocardiography.results:among the patients, twenty - four cases (75%) were younger than 4 years. mean global s and sr was significantly reduced in the kd patients compared to controls (17.03 1.28 vs. 20.22 2.14% and 1.66 0.16 vs. 1.97 0.25 1/second, respectively), while there were no significant differences regarding mean tps, tpsr, longitudinal velocity and view point velocity. using repeated measure of analysis of variances, we observed that s and sr decreased from base to apical level in both groups. the change in the pattern of age adjusted mean s and sr across levels was significantly different between the groups (p < 0.001 for both parameters).conclusions : we showed changes in s and sr assessed in kd patients versus control subjects in the acute phase of kd. however, we suggest that further studies be undertaken to compare s and sr in the acute phase and thereafter in kd patients.
for appropriate stability of the shoulder complex, interaction and static - dynamic balance between various joints and muscles around the shoulders are required. the scapulothoracic joints increase the range of motion of the arms and provide stability for the movement of the upper limb1. for this reason, some clinicians proposed that patients with a protracted scapular posture needed appropriate scapular retraction exercises2. the retractors of the scapulothoracic joints, target muscles of such exercises, are composed of the middle trapezius, the rhomboids, and the lower trapezius3. in addition, the glenohumeral joint has a high degree of motion in the upper extremity and therefore has the possibility of various potential damages4. therefore, most exercises devised to prevent and treat shoulder injuries have been considered strengthening the rotator cuff (rc) muscles important5, 6. in particular, the infraspinatus (is), one of the rc muscles, stabilizes the shoulder joints during the shoulder external rotation and acts as the prime mover of movement3, 5. much research on exercises and therapeutic approaches effective for shoulder joint stabilization has been performed. lee.7 reported that during a push - up plus exercise, the neutral position, internal rotation position, and external rotation position of the hand differently affected the stabilization of the scapulothoracic, and push - up plus during external rotation was more effective for strengthening the serratus anterior. jang and oh8 verified changes in the muscle activity ratios of the is and posterior deltoid muscle while performing shoulder external rotation according to shoulder flexion at 45, 90, and 135. many studies verified the effects of postural changes of the shoulder joints, differences in the base of support, and changes in the angles of the upper limbs on shoulder joint stability or changes in muscle activity. however, in the clinical field, diverse connected methods of treating the shoulders and upper extremity have been effectively applied. among the muscles of the upper extremities, the middle trapezius, rhomboid major (rm), triceps brachii, and finger extensors were proposed as connective muscles identified in the upper extremity posterior surface9. in particular, in treatment methods to improve the upper extremities of cerebral palsy patients, the dynamic stability of the shoulder joints, the proximal part, has been improved through postural adjustments of the hands, the distal part10, 11. however, evidence - based research on such novel treatment methods is very insufficient. accordingly, the purpose of this study is to examine the effects of the extension of the fingers, the distal part of the upper extremities, on the activities of the is and the rm, shoulder stabilization muscles of the proximal part. the subjects were 14 adult males who had not experienced orthopedic and neurological damage for the past six months. those who were equipped with appropriate muscle strength and range of motion to perform the exercises required in this experiment and those who did not show winged scapula symptoms and had no pain in the hands and shoulder joints were selected. the mean age of subjects was 23.78 0.80 years, their mean height was 177.35 5.37 cm, and their mean weight was 75.57 11.46 kg. the protocol for this study was approved by the ethics committee of catholic university of daegu. korea), a surface emg, was used, and surface electrodes were attached to the is and the rm. the collected emg signals were filtered and rectified using a telescan program (version 3.06), and then an integral average emg was calculated to be used for analysis. for the normalization of each data, the rvc method whose standard was the condition of finger relaxation was used. the subjects comfortably lowered the bilateral upper extremities, faced their palms toward the front, and abducted and maintained the shoulder joints at 60. a measurement was taken once for five seconds with the finger relaxed and a measurement was taken again for five seconds with the finger extended. all tasks were repeated three times and one resting period for one minute between each task was provided. a paired t - test was used to examine muscle activity of the muscles around the shoulders during finger relaxation and extension. statistical analysis was performed using spss, version 20 for windows (spss inc., is right (is.r.), is left (is.l.), rm right (rm.r.) and rm left (rm.l.) the right and left muscle activities of the is were statistically significantly higher than when they were relaxed (p<0.05), but the muscle activity of the rm was not statistically significant (table 1table 1.comparing muscle activities of shoulder muscles with respect to finger extensionmuscle % rvcis. r.is. l.rm. r.rm. l.extension/relaxation206.2225.0179.8178.8rvc (%) (213.3)(297.6)(185.6)(162.7) significant : p<0.05. is. much research on the methods to activate muscles related to the scapulothoracic joint or the glenohumeral joint aimed at dynamic - static stabilization of the shoulder complex has been performed2, 5, 7, 8. these studies have focused on the activity of certain muscles and upper extremity movements or angle changes. however, some researchers recently proposed that the muscle activity of various relevant muscles be evaluated in a global view when muscle activity is assessed while performing exercises9, 12. in the clinical field, as well, treatment should not be made in a local view, and diverse treatment methods of connecting the proximal and distal parts in consideration of connected muscle activity have been applied. for example, to improve the abnormal posture of the neck and shoulders, an effective intervention is applied by utilizing the actions of the elbow and hands, the proximal part, as well as the activation of the surrounding relevant muscles to improve the abnormal posture of the neck and the shoulders9,10,11,12. this study intended to approach the theoretical basis for such novel treatment methods using the connected activity of the proximal and distal muscles used in the clinical field. the result was that the extension of the fingers, the distal part of the upper extremities, increased the muscle activity of the is and rm, the shoulder stabilization muscles of the proximal part. among them, the activity of the is statistically significantly increased. such a result shows that only the actions of finger extensors improve the activity of is. in previous studies, such a connected action of the upper extremity muscles was explained with myofascial continuity, which was shown by most research using anatomical studies with cadavers13, 14. however, as a process to perform actual exercises, this study maintained the shoulders and the upper extremities in a proper posture and applied only finger extension as a variable. to verify changes in the muscle activity of delicate movements, such a research method is considered a very sophisticated procedure to verify the connectivity of diverse muscles. selective strengthening of the is has been considered important in the rehabilitation of the shoulder joints8, 15, 16. ha.16 reported that increasing the shoulder flexion angle promoted emg of the is. jang and oh8 reported that the action of the is was very crucial to shoulder stability in the external rotation of the shoulder joints, and performing external rotation with the shoulder flexion at 45 was more efficient. however, such research presented a result confined to the shoulder joints, and the effects of the activity of the muscle connected with other muscles of the upper extremities and the action of the hands were not considered. therefore, based on the result of this study, it is thought to be more efficient to consider the direction and action of the hands in exercises devised for shoulder complex stability. in particular, in exercises to stimulate the activity of the is, finger extension will be a useful method to increase the activity of the muscles. first, the number of subjects was small and they were confined to adult males ; therefore, it is difficult to generalize this study result. second, research was confined to the is and rm with high relevance to finger movement. future research is considered necessary on changes in activity of diverse muscles around the shoulders according to finger movement.	[purpose ] this study aims to examine the effects of the extension of the fingers (distal upper limb) on the activity of the shoulder muscles (proximal upper limb). [subjects and methods ] this study involved 14 healthy male adults with no musculoskeletal disorder or pain related to the shoulders and hands. the subjects in a sitting posture abducted the angle of the shoulder joints at 60 and had their palms in the front direction. electromyography (emg) was comparatively analyzed to look at the activities of the infraspinatus (is) and rhomboid major (rm) when the fingers were extended and relaxed. [results ] the activity of the is was statistically significantly higher when the fingers were extended than when they were relaxed. [conclusion ] according to the result of this study, finger extension is considered to affect the muscles for connected shoulder joint stability.
idiopathic scoliosis is the most common form of scoliosis and its incidence in the total nubmer of scoliosis is from 67% to 90% depending on types of reasrch and the observed age groups. it is present in 2 4% of children aged from 10 to 16 years of age (4), but more than 50% at persons older than 60 years (3). the causes of its origin still are not celar, and probably they are many. more frequent occurance in some families are leading to genetic factors (5), gender distribution also was nt clearly defined. in infatile period both sexes are equaly affected, while in the juvenile and adolescent period girls have bigger tendencies for illness, and at the age of 10 years that ratio is 6:1 (3). during etiology research of idiopathic scoliosis disorder of connective tissues, abnormal biomechnical forces as wel as neuophysiological predisposition was determined. some research are blaming sudden dynamic development of adolescents and asymmetrical tensions that are there created within the musculo - skeletal system (7), or a problematic posture as a factor in enhacing the period of rapid development of the body (8). idiopathic scoliosis is rare in early childhoods and its prevalence is growing in the school age from 2% to 4% as the child getting older. from that reason and beacuse of the risk in specific development stages idiopathic scoliosis can be classified into three groups. scoliosis occured in early childhood (infantile) before the age of three, scoliosis occuring in juventile child age (from 3 to 9 years) and scoliosis that occurs in adolescent age (from 9 to 18 years) (1). adolescent idiophatic scoliosis (ais) is not a disease primary caused due to poor posture, but poor posture may potentiate the deterioration of the existing scoliosis. this is very important in the period of rapid growth beauce epidemiological studies suggest that ais is a leading orthopedic problem of children in school period. due the ill micsonduct, enviromental factors can be potentiate factor in ais developing. changes in intervertebral discs and other parts of vertebra s indicate to the ineffective synthetic response to a pathological enviroment (9). if we considered all these emphasizing factors, we set the goal of this study to deteremine the impact of behavioral and enviromental factors on developing and progression of ais in children at school age. these factors are the one that make difference in living in rural or urban area. in example it is a bigger mobility of osteomuscular structure at children in rural area, longer stay outside, a different diet, alcoholism, smoking, other commitments, habits and interests, etc. adolescent students from two different elementary school s from urban area in epe are the research sample and two elementary school s in rural area in gradaac. they were selected from bigger reasearch sample which included all age groups in elementary school from 6 to 15 years old (adolescent age). the cases with scoliosis as a result of congenital disorder or neuromuscular disease are did not take in consideration. scoliosis is defined as a lateral curvature of spine from 10 and more degrees with vertebral rotation. in addition to the notice of presence or abscence of scoliosis, body height, weight were measured, also bmi (body mass index) and the relationships between this values and occurrence of ais were calculate. research sample is separated of 421 or 68,1% pupils of adolescent age. analysing the gender distribution with x test we established that there is no statistical significant difference in reasrch sample by gender in rural and urban school. there is a statistically significant difference in the average age of subjects (t(410)=23.511 ; p<0.001), we have selected cohort of pupils of 14 years old which was consist of 184 students both sexes, where is no statistically significant difference in gender distribution by area of living. ina cohort made of fourteen years old kids there is no statistically significant differences in average body weight among subjects classified by place of residence (t(99)=-1.834 ; p=0.07), but there is statistically significant difference in average bmi (t(87)=-5.203 ; p<0.001). analyzig data on the presence of the scoliosis in relation to place of residence we found a statistically significant difference (fisher s exact test p<0.001). scoliosis is more common in urban school students. analyzing the results of the research sample (cohort of adolescents) we found that scoliosis is much more common among urban school children. while testing this diff erence we found that it was statistically significant (x(1)=32.127 ; p<0.001). analyzing the correlation between the appearance of scoliosis with the size bmi in the research sample, the pearson correlation coefficient shows a significant positive correlation (p. corr = 0.176 ; p<0.001). analyzing the diff erence between the scoliosis appearance by gender in research sample we found a statistically signifi ca nt diff erence. scoliosis is two time more oft en at girls then boys (x(1)=4.863 ; p=0.027). the intention of this study is to show the impact of enviroment and lifestyle as a potential factors in expression of ais. as we mentioned above idiopathic adolescent scoliosis is not a habit disease, it is not caused by carrying heavy school bags on one shoulder, or bad posture and neither the lack of calcium (13). many reasrch are agree in one that the rapid pubertal growth can be conneceted with assimetric growth of the spine and the occurance of this type of scoliosis (14, 15, 16). we assumed that such, vulnerable conditions some secondary factor can exist which can potentiate and have impact on expresion and progression of ais. th erefore, we look for this factors in diversity of enviormental factors and habits in diff erent living enviroments, urban and rural. many reasrch are showing that osteomuscular mobility, and esspecially mobility of spine and the dominant body posture body can cause many changes which can potentiate scoliosis development. shultz (1984) was talking about poor postural control of spine with ais as biomechanical factor of her progression, neuromuscular mechanism s are te most important (17). tinazci (2009) is pointing to the enviormental eff ects on physical activity at children in urban and rural area (18). reyes (2003) is showing differencies in development at school children of rural and urban area, school children in urban area are in average more more taller and heavier than the children in rural area (19). in our reasrch we did nt measured specific parameters of the physical activity of children, we havent conduct any survey about diet and other habits, but we assumed there are significant differencies between the children in urban and rural area, on a tradition base, earlier research and the usual habits of life. school children in rural area have more physical activity, they are more activly involved in activites helping their parents, and less time they are spending in front of the television and game consol, in total children in rural area have healthier profile (20, 21). there are differencies in osteomuscular development of children in urban and rural area what is shown in our reasrch. in our reaserch children from rural area have higher bmi, and smaller averrage high then the kids form urban elementary schools. it is established that the apperance of scoliosis is more often in school children from urban area, two times more often in girls and it is more oft en at children with higher bmi. there are numerous theories about the ais etiophatogenesis, from those that assume genetic heritage, abnormalites of nervous system, abnormal bone growth, hormonal and metabolical disorders to the biomechanical, and enviromental factors that are result of lifestyle habits (22). our reasrch has shown that incidence of scoliosis at school children is more often in urban area, as it brings need for further research of the factors which are different in living in urban and rural area and which can be cause or reason of scoliosis expresion.	introduction : idiopathic scoliosis is a significant health problem which occurs in 2%4% school kids in adolescent age. reasons of occurrence are not quite clear, there are many theories, but probably it is multifactor disease. among the theories that are mentioned some of them included environmental and behavioral factors.aim:research the impact of some environmental and behavioral factor on development and progression of idiopathic scoliosis in school kids.methodology:research was conducted on 421 pupil in adolescent age, where 120 pupils was from urban schools and 301 pupil from rural schools. environmental factors and habits like the bigger osteomuscular structure mobility at kids from rural schools, longer outdoor time spending, different nutrition, alcoholism and smoking, different obligations, etc. factors which can be cause of scoliosis development.results:in this research we assumed that different environmental and behavioral factors of school kids, which exist in rural and urban areas, can develop to different expression of scoliosis in these areas. in our research we proved that the scoliosis occurrence is more often in urban areas than in rural (fisher s exact test p<0.001).conclusion : it is necessary to research all factors of lifestyle individually, which are different between the urban and rural kids.
the finndiane study is an ongoing, nationwide, prospective multicenter study seeking for clinical, genetic, biochemical, and environmental risk factors for diabetes complications, with emphasis on diabetic nephropathy. participating study centers comprise diabetes and renal outpatient clinics at all five university central hospitals, all 16 central hospitals, the majority (n = 27) of all regional hospitals, and 31 major primary health care centers in finland. patients with type 1 diabetes (icd-10 code e10) were recruited at routine outpatient visits. based on medical records, the attending physician completed a standardized check - list regarding diabetes complications and medication. total leisure - time physical activity was assessed by a separate questionnaire as previously described (6), and low physical activity was defined as 30 measurements : 1.11 versus 0.87 (p = 0.001). this indicates greater variation with increasing number of measurements but with conserved differences in sd between progressors and nonprogressors. there was no correlation between follow - up time and sd of serial a1c (pearson r = 0.006, p = 0.789). kaplan - meier survival curves with quartiles of sd of a1c revealed a more clear association between the entire distribution of variability for progression in renal status (fig. 1b), for which only the highest quartile of sd diverged. in fig. 2a and b, survival curves are given for patients with mean and sd of serial a1c above and below the population medians. 2a), the highest incidence was observed when both mean and sd were above the medians and conversely the lowest incidence when both were below the medians. interestingly, patients above median for mean and below for sd had similar incidence for progression in renal status as patients below median for mean and above for sd, suggesting a distinct and equally important effect of both absolute value and variability of a1c in the risk of progression in renal disease. for cvd events, however, 2b) as all corresponding lines were superimposed, probably because of the fact that mean serial a1c did not differ between patients with and without a cvd event and all lines contain a prerequisite regarding the mean serial a1c. a : kaplan - meier survival curves for any progression in renal status (defined as any increase in albuminuria level or progression to esrd) by quartiles of sd of serially measured a1c values. b : kaplan - meier survival curves for a cvd event (coronary event, stroke, peripheral vascular event) by quartiles of sd of serially measured a1c values. a : kaplan - meier survival curves for any progression in renal status (defined as any increase in albuminuria level or progression to esrd) according to mean and sd of serial a1c above and below the population median. b : kaplan - meier survival curves for a cvd event (coronary event, stroke, peripheral vascular event) according to mean and sd of serial a1c above and below the population median. to depict which baseline patient characteristics were associated with the variability of a1c during follow - up, we examined quartiles of sd of serial a1c. we found that a higher variability was associated with younger age, lower age at onset of diabetes, shorter duration of diabetes, lower insulin sensitivity, dyslipidemia, higher baseline a1c, both current and ever smoking, lower socioeconomic class, and lower leisure - time physical activity (table 3). for the physical activity, we included in a separate analysis only patients with normal uaer, which did not change the results (table 3). in patients with normal uaer, the associations for insulin dose (p 30 measurements : 1.11 versus 0.87 (p = 0.001). this indicates greater variation with increasing number of measurements but with conserved differences in sd between progressors and nonprogressors. there was no correlation between follow - up time and sd of serial a1c (pearson r = 0.006, p = 0.789). kaplan - meier survival curves with quartiles of sd of a1c revealed a more clear association between the entire distribution of variability for progression in renal status (fig. 1b), for which only the highest quartile of sd diverged. in fig. 2a and b, survival curves are given for patients with mean and sd of serial a1c above and below the population medians. 2a), the highest incidence was observed when both mean and sd were above the medians and conversely the lowest incidence when both were below the medians. interestingly, patients above median for mean and below for sd had similar incidence for progression in renal status as patients below median for mean and above for sd, suggesting a distinct and equally important effect of both absolute value and variability of a1c in the risk of progression in renal disease. 2b) as all corresponding lines were superimposed, probably because of the fact that mean serial a1c did not differ between patients with and without a cvd event and all lines contain a prerequisite regarding the mean serial a1c. a : kaplan - meier survival curves for any progression in renal status (defined as any increase in albuminuria level or progression to esrd) by quartiles of sd of serially measured a1c values. b : kaplan - meier survival curves for a cvd event (coronary event, stroke, peripheral vascular event) by quartiles of sd of serially measured a1c values. a : kaplan - meier survival curves for any progression in renal status (defined as any increase in albuminuria level or progression to esrd) according to mean and sd of serial a1c above and below the population median. b : kaplan - meier survival curves for a cvd event (coronary event, stroke, peripheral vascular event) according to mean and sd of serial a1c above and below the population median. to depict which baseline patient characteristics were associated with the variability of a1c during follow - up, we examined quartiles of sd of serial a1c. we found that a higher variability was associated with younger age, lower age at onset of diabetes, shorter duration of diabetes, lower insulin sensitivity, dyslipidemia, higher baseline a1c, both current and ever smoking, lower socioeconomic class, and lower leisure - time physical activity (table 3). for the physical activity, we included in a separate analysis only patients with normal uaer, which did not change the results (table 3). in patients with normal uaer, the associations for insulin dose (p < 0.001), current smoking (p 0.001) with quartiles of sd were similar as for all patients, suggesting that as for physical activity, the associations were not solely because of collinearity with diabetes complications. finally, because there was a clear association between age and a1c variability, we also adjusted the p values for age (table 3) ; however, the abovementioned associations did not change except for age at onset and duration of diabetes, which were no longer associated with a1c variability. baseline patient characteristics according to quartiles of a1c variability defined as intrapersonal sd of serial a1c measurements during follow - up values are expressed as means sd, percent, or median (interquartile range). patients with available data on physical activity. because of several potential confounding factors for the association between the variability of serial a1c and the progression of renal disease and cvd events, we used cox regression as a multivariate analysis. as covariates, we used duration of diabetes, sex, systolic blood pressure, total cholesterol, ever smoking, intrapersonal mean of serial a1c measurements, and number (ln - transformed) of a1c measurements. for incident cvd events, we additionally adjusted for the presence of diabetic nephropathy and cvd events at baseline. as shown in table 4, sd of serial a1c measurements predicted progression in renal status even independently of mean serial a1c. moreover, sd was also an independent predictor of cvd events, whereas mean serial a1c was not. cox regression models for progression in renal status (defined as any increase in albuminuria level or progression to esrd) and a cvd event (coronary event, stroke, peripheral vascular event) during a median follow - up of 5.7 years diabetic nephropathy : macroalbuminuria and/or esrd. serial a1c represents all available a1c measurements from baseline to follow - up, for which intrapersonal mean and sd were calculated. in this study, we show that the variability of a1c predicts the development and progression of incipient and overt renal disease in patients with type 1 diabetes. these data support a recent analysis from the dcct intervention where a1c variability predicted the development of diabetic nephropathy and retinopathy (5). we add to that study by showing the same finding for nephropathy in an observational cohort and further covering the entire spectrum of renal disease (from microalbuminuria to esrd) in diabetes. however, the most interesting novel finding was that the a1c variability also predicted cvd events even though the mean a1c during follow - up was similar in patients with and without a cvd event. this association was independent of nephropathy status at baseline, indicating that the association was not entirely driven by renal disease, which is a strong cvd risk factor in patients with type 1 diabetes (10). we further show that a variable a1c profile is associated with a cluster of baseline factors such as lower insulin sensitivity, dyslipidemia, lower socioeconomic status, physical inactivity, and smoking. in the present longitudinal analysis, this is in line with the large body of evidence showing that lower a1c translates into reduced risk of diabetic nephropathy and retinopathy in patients with type 1 diabetes. before the landmark study dcct (1), this was also indicated by the steno study (11), the oslo study (12), and the stockholm diabetes intervention study (13). regarding glycemic control and cvd the picture is, however, less clear. in type 2 diabetes, the apparent lack of benefit of improved glycemia on cvd outcomes at least in advanced diabetes has recently been debated (14). in type 1 diabetes, there is evidence in some (15,16) but not in all epidemiological studies (1720), as well as meta - analyses (21,22), for an independent relationship between a1c and development of cvd or cvd mortality. in the extended dcct follow - up (edic), there was a delayed benefit of intensive treatment on incident cvd (2). there is further a report that the change in two a1c values (baseline minus follow - up) predicts incident coronary artery disease in type 1 diabetes, even though the baseline a1c was not in itself predictive (23). in the present study, baseline a1c was slightly higher (0.2% unit) in patients that suffered a cvd event during follow - up, but the mean serial a1c during follow - up did not differ between patients with and without a cvd event during follow - up. thus, the role of the level of a1c for cvd is somewhat unclear also in our study but certainly seems less important than is the case for diabetic nephropathy. in addition to the actual level of a1c, it has been postulated that variation in the glycemic profile also could contribute to risk of diabetes complications because glucose peaks seem to cause detrimental in vitro cellular effects (2427). furthermore, glycemic excursions measured by continuous glucose measurement (cgms) correlate with oxidative stress in patients with type 2 diabetes (3) but not type 1 diabetes (28). oxidative stress has been considered fundamental in the pathogenesis of diabetes complications (29). furthermore, experimentally induced oscillating blood glucose may also impair endothelial function, thus possibly linking glucose fluctuations also to cvd (30). for epidemiological studies, the cgms is often impractical and other measures of glycemic variability have therefore been used. in type 2 diabetes, there are some reports that variability of fasting plasma glucose predicts retinopathy (31) as well as cardiovascular and total mortality (32). in these studies, however, the patients had widely differing antihyperglycemic treatment modalities ranging from diet to insulin injections, which might have affected the variability. in type 1 diabetes, the variability of repeated measurements of blood glucose values has been investigated in the publicly available dcct dataset by comparing the quarterly one - day measured pre- and postprandial (breakfast, lunch, and supper) and bedtime glucose values (4). the study by kilpatrick. found no evidence of an effect of variation in blood glucose on the risk of diabetic nephropathy or retinopathy. recently kilpatrick. (5), however, reported that the variability of a1c in the dcct independently predicted the incidence of diabetic nephropathy and retinopathy even after correction for the absolute level of a1c. because the dcct was an intervention study, the investigators excluded a1c measurements from the first 6 months of the study to decrease the risk of artificial variation in a1c because of a new insulin treatment regimen. the risk of bias by the intervention itself, however, is still possible, even though the main finding was seen in the conventionally treated nonintervened patient group, which supports an effect independent of the intervention. our present study was performed as a longitudinal, observational study without intervention, and the results regarding the development of nephropathy are very similar to the dcct data (5). it is likely that the a1c variability reflects other processes compared with the short - term glycemic variability observed by cgms monitoring and multiple blood glucose measurements. this may explain the difference in the predictive value of the variability in blood glucose (4) and the a1c (5) for nephropathy and retinopathy in the dcct cohort. the relationship between short - term and long - term glycemic variability needs to be further clarified. in our study, the finding that a1c variability, but not mean serial a1c, was associated with cvd events might indicate that patients more severely affected by diabetes and its complications have a more variable a1c profile than patients with less complicated disease. nevertheless, the association was still evident when controlling for the presence of diabetic nephropathy and cvd events at baseline (table 4). for renal complications, those patients with a normal uaer at baseline but who progressed to microalbuminuria had higher a1c variability than those who remained with normal uaer, suggesting that a1c variability might not just be secondary to the presence of more severe diabetes complications. the mechanisms behind the association between a1c variability and the risk of diabetes complications are at this stage merely speculative and can not be determined in this observational study. however, several baseline factors indicative of a disadvantageous lifestyle and a low socioeconomic class were associated with a more variable a1c, which might indicate a suboptimal management of diabetes. a1c variability was also associated with insulin resistance, which in itself has been implicated in the pathogenesis of diabetes complications (33). it is also possible that the intrapersonal variation in a1c is secondary to variation in insulin sensitivity, which in turn may be because of infections or other intermittent factors that decrease insulin sensitivity. in fact, we recently showed that serum lipopolysaccharide activity, a marker of gram - negative bacterial infections, is associated with progression of diabetic nephropathy (34). it is further possible that fluctuating long - term glycemic control causes homeostatic imbalance, for example, through intracellular sorbitol accumulation. it was suggested that the normoglycemic re - entry phenomenon (35) might tie a1c variability and risk of retinopathy (5). this phenomenon refers to an initial, but not sustained, worsening of retinopathy when lowering a1c in patients already affected by retinopathy. a further potential mechanism is induction of growth factors, for instance igf-1 and vascular endothelial growth factor, by a changing glycemic environment (36,37), which may contribute to development of diabetes complications. an important remark at this stage, however, is that the available data on a1c variability and risk of diabetes complications can not confirm a direct causal relationship because the association might be because of collinearity with other causal factors. the strengths of this study are the large number of well - characterized patients and the prospective study design. the serial a1c values were collected from available laboratory records as a part of the patient 's routine clinical follow - up. thus, there were no prespecified intervals between a1c measurements, and the number of measurements per individual patient varied, which was, however, statistically adjusted for. another potential limitation is the use of a1c measured at the local study centers, not in a central laboratory. however, this should not affect the intrapersonal variability of a1c because the measurements were performed at the same center. in conclusion, we show in an observational setting that a larger variability of a1c predicts not only incident microalbuminuria and progression of established renal disease but also cvd events in patients with type 1 diabetes.	objectiverecent data from the diabetes control and complications trial (dcct) indicated that a1c variability is associated with the risk of diabetes microvascular complications. however, these results might have been influenced by the interventional study design. therefore, we investigated the longitudinal associations between a1c variability and diabetes complications in patients with type 1 diabetes in the observational finnish diabetic nephropathy (finndiane) study.research design and methodsa total of 2,107 patients in the finndiane study had complete data on renal status and serial measurements of a1c from baseline to follow - up (median 5.7 years), and 1,845 patients had similar data on cardiovascular disease (cvd) events. intrapersonal sd of serially measured a1c was considered a measure of variability.resultsduring follow - up, 10.2% progressed to a higher albuminuria level or to end - stage renal disease, whereas 8.6% had a cvd event. the sd of serial a1c was 1.01 versus 0.75 (p < 0.001) for renal status and 0.87 versus 0.79 (p = 0.023) for cvd in progressors versus nonprogressors, respectively. in a cox regression model, sd of serial a1c was independently associated with progression of renal disease (hazard ratio 1.92 [95% ci 1.492.47 ]) and of a cvd event (1.98 [1.392.82 ]) even when adjusting for mean a1c and traditional risk factors. interestingly for cvd, mean serial a1c itself was not predictive even though sd of a1c was.conclusionsin patients with type 1 diabetes, a1c variability was not only predictive of incident microalbuminuria and progression of renal disease but also of incident cvd events.
gastrointestinal stromal tumours (gists) arise from the interstitial cells of cajal, specialized cells of the autonomic nervous system located within the wall of the gastrointestinal tract and responsible for paced contractions of the intestinal musculature the most common location of gists is in the stomach and accounts for around two - thirds of them, followed up by the small intestine, colon and rectum, and rarely oesophagous or appendix. diagnosis is histological and characterized by a high expression of a c - kit proto - oncogene (cd117, tyrosine kinase receptor), and cd34 on immunohistochemistry. the prediction of malignancy remains difficult and relies on a combination of several criteria that include mitotic activity count, tumour size, presence of necrosis and invasive growth. surgery is the treatment of choice of non - metastatic gists when a complete r0 resection (clear margins) can be achieved with low morbidity, and routine lymphadenectomy is not required as those lesions rarely metastasize to the nearby lymph nodes. with the development of stapling devices, laparoscopic wedge resection of small gastric lesions has become the preferred surgical technique as it significantly reduces post - operative pain and morbidity, shortens hospital length of stay, while maintaining adequate overall long - term clinical outcome. recently published series have demonstrated similar results with gastric gists larger than 5 cm in diameter, when performed by experienced surgeons [68 ]. having said that, caution should be taken when considering laparoscopic excision of locally advanced lesions, where the need for major gastrectomy with or without en bloc resection of neighbouring organ might be necessary. in those circumstances it would be more reasonable to favour a conventional open approach that could increase the probability of complete r0 resection. we present the two cases of locally advanced gastric gists who did benefit from a 2-stages approach and where delayed laparoscopic r0 resection could be successfully achieved, without complication. case one, at the time an 80 years old female patient classified asa class iii with significant cardiopulmonary disease was investigated for anaemia. abdominal ct - scan revealed a large heterogeneous gastric mass measuring 13 13.5 cm, arising from the greater curvature (fig. 1) and with classical features of gist on endoscopic ultrasound (eus). there was no evidence of distant metastases on positron emission tomography (pet) scan and fine needle aspiration (fna) biopsy of the lesion confirmed the diagnosis. she received neoadjuvant imatimib 400 mg per day, as a single daily dosing for a total of 6 months and a significant 70% reduction of the mass was achieved as confirmed on repeat ct - scan imaging confirmed (fig. the patient eventually underwent laparoscopic wedge excision of the gastric wall encompassing the residual gist (fig. 3). although she had an uncomplicated post - operative recovery the patient was discharged home 4 weeks later as she required significant physio mobilisation before being safely discharged home. histopathology results demonstrated an excellent response to imatimib with almost complete tumour necrosis and excision was complete (r0). imatimib was reintroduced at one month following her surgery, but was interrupted 6 months later due to undesired side effects, mainly fluid retention, nausea and loss of appetite. she is today over 5 years post surgery with still no evidence of local recurrence or distant metastases on repeat imaging. case two, a 50 years old male patient when he was diagnosed with a locally advanced non - metastatic 14.5 11.4 cm gastric gist abutting (loss of fat plane) the transverse colon. unrelated, he also had a large cervical lump occupying most of his right thyroid lobe and corresponding to a benign follicular lesion on fna. other relevant problem included presence of central obesity and a 30 pack - years smoking history. the patient received a 6-month neoadjuvant treatment of imatimib 400 mg single daily dose leading to partial tumour response (around 50%) as demonstrated on progressive ct - scan imaging, but showing clear evidence of central necrosis of the gist. incidentally, towards the end of his medical treatment he was found to have a left testicular mass that was replacing most of his testis on us. tumour markers were all negative and there was no evidence of loco - regional or distant metastases on ct - scan. the patient underwent elective laparoscopic excision of the gastric gist including partial wedge excision of the greater curvature (fig. en - bloc colonic resection was unnecessary as the transverse colon and its mesentery were free of tumour, with clear margins (r0) on histopathology. the testicular lesion corresponded to a 4 cm seminoma confined to the testis and the surgical margins where also free of tumour (pt1n0m0). the patient went home on the fourth post - operative day and subsequently received a single dose of adjuvant carboplatin for his seminoma. imatinib was also recommenced following his surgery for a total of 20 months. of interest, he underwent right thyroid lobectomy four months following his initial laparoscopic procedure and was diagnosed three months later with a metastatic seminoma, for which he received three supplementary cycles of second - line chemotherapy. the patient is currently still on complete remission from his gastric gist and metastatic seminoma 3 1/2 years later. surgery is the only potentially curative option for gastric gists. unfortunately, a small proportion of them are locally advanced measuring > 10 cm in size or borderline resectable at initial diagnosis, meaning that en bloc removal of neighbouring organ might be necessary to obtain r0 tumour resection. in those situations, minimally invasive laparoscopic approach is generally unsuitable and conventional open surgery is preferred, which is unfortunate especially in high - risk patients where complication rates are expected to be higher. those two cases are good illustrations of large gastric gists successfully treated laparoscopically following neoadjuvant chemotherapy, while initially unfavourable for such minimally invasive approach. indeed, while still controversial the currently available kit - selective tyrosine kinase inhibitor, imatinib (glivec, formerly known as sti571, novartis pharma ag, basel, switzerland) may be considered as a first line of treatment in order to achieve substantial tumour chemoreduction and improve success rate of delayed curative - intent surgery, as well as overall survival. one can extrapolate that tumour down - staging of potentially excisable locally advanced gist lesions, using neoadjuvant imatinib - based chemotherapy may ultimately allow reconsideration to a minimally invasive approach. an appealing concept that unfortunately will necessitate a large randomized multicentre study with long - term follow up to prove its validity over conventional open surgery. in the mean time, only anecdotal observational case series may offer a partial answer. in summary, patients with locally advanced non - metastatic gastric gists should be offered first - line neoadjuvant imatinib - based cytoreductive chemotherapy as an alternative to radical debulking surgery, as a substantial proportion of them will experience significant tumour shrinkage and therefore eventually benefit from a much less invasive laparoscopic approach. written informed consent was obtained from each of the two patients included in this case series for publication of this case series and accompanying images. a copy of the written consent is available for review by the editor - in - chief of this journal on request.	highlightslaparoscopic resection of locally advanced gastrointestinal stromal tumours (gists) is rarely offered to patients.first-line cytoreductive imatinib represents an interesting alternative to radical debulking surgery.significant downstaging of the primary tumour with neoadjuvant imatinib can increase the success of a delayed laparoscopic approach.neoadjuvant imatininb may soon become the preferred medical option in such situation.
laparoscopic inguinal hernioplasty is being performed more frequently everywhere, now that several well - designed prospective and randomized studies have shown that it produces results that compare favorably to the open conventional approaches with the added and well - known advantages of this type of mini - invasive surgery. however, like any other surgical procedure, it has complications, some of which appear to be unique to this technical modality like intestinal obstruction related to tapp. a 65-year - old white male was seen in our office with the main complaint of bilateral inguinal hernias diagnosed elsewhere. he had had an appendectomy 15 years before. he recently complained of pain in the right side and was referred to our office by his brother, who had been operated with the same diagnosis using laparoscopy in 1993. the rest of the clinical examination was unremarkable, and a chest film and electrocardiogram and pre - operative laboratory workup were within normal limits. accordingly, on january 22, he was admitted to the spanish hospital in mxico city and, under general endotracheal anesthesia, a 2 cm lower umbilical incision was made. under direct vision the anterior fascia of the rectus muscle was incised and its fibers retracted. the preperitoneal space was dissected and a balloon trocar was inserted and advanced to the level of the pubis. during the dissection of the preperitoneal space, it was observed that the peritoneum was torn at the site of the previous appendectomy incision, and the procedure was converted to a transperitoneal approach. two 10 mm trocars were placed in each flank under direct vision and transillumination to avoid injury to the epigastric vessels. the right peritoneal incision was extended and a large indirect hernia with an accompanying direct defect were seen. a lipoma was excised, and the peritoneal sac was dissected free up to the level of the umbilicus. a 15 7 cm polypropylene mesh was introduced, unrolled and properly placed and anchored using a 5 mm tacker (origin medical systems, inc., menlo park, california) in cooper 's ligament, the upper medial corner of the hesselbach 's triangle, and in the lateral upper aspect above the level of the anterior and superior iliac spine. the peritoneal defect was closed with a running suture of 00 pds (polydioxanone ethicon, ltd, u.k.), (figure 1). a similar procedure was carried out in the left side for a smaller indirect hernia. the patient was discharged eight hours later with normal and stable vital signs, tolerating well a liquid diet. the next morning he requested pain medication at home and took the analgesic prescribed (ketorolac p.o. 10 mg), and soon after that he presented nausea and several episodes of forceful vomiting. he was seen and examined at the emergency room at which time he was found to have normal vital signs, mild dehydration and abdominal distention with hyperactive bowel sounds without rebound. he was hospitalized, a nasogastric tube was inserted, intravenous fluids and a second generation cephalosporin was given iv. he failed to improve overnight, having a large ng aspirate, and he developed inguino - scrotal swelling for which reason he was taken to the operating room and re - operated using general endotracheal anesthesia. upon introduction of the 10 mm laparoscope, it was obvious that 30 - 35 cm of small intestine loops were herniated through a right peritoneal suture defect (figure 2). with the aid of two 10 mm babcock - type of forceps, all the loops of small bowel were carefully extracted from the defect and none of them showed signs of severe ischemia nor perforation. the preperitoneal defect was irrigated and 10 ml of normal saline containing 1 g of kanamycin were left in. the peritoneal defect was closed using interrupted figure of x stitches of polydioxanone until a water - tight closure was achieved (figure 3). the postoperative period was uneventful ; he passed flatus in the second postoperative day, tolerated a liquid diet and was discharged on the 3rd postoperative day on oral antibiotics for an additional 10 days. he has remained asymptomatic to this date, having assisted to the office several times and is satisfied with the result of the operation. closure of the defect with interrupted figure of x stitches using 00 polydioxanone. at least nine prospective studies have compared the frequency of complications between laparoscopic and open inguinal hernia repair. overall, the complication index is higher for the open procedures (22% vs 18%), though there is not statistical significance., in their multicentric prospective and randomized study, also found a higher morbidity for the open techniques (20.3% vs 20.1%) even though in this report the use of extra trocars, rupture of instruments, and conversion from tapp to tep techniques were considered complications. fortunately, most of the complications following laparoscopic hernia repair are minor (seromas, hematomas, pneumoscrotum, orchitis, etc.) although there are some serious complications of the operation like nerve entrapment and intestinal obstruction. the latter is rare and may be caused by herniation of an intestinal loop through a port incision (usually bigger that 5 mm) when the wound has not been closed correctly. we found at least 15 cases reported in the literature of intestinal obstruction due to herniation of the intestines or adhesions at the site of peritoneal closure after a transabdominal preperitoneal inguinal repair (tapp). in five of these patients reoperation was done laparoscopically, whereas in three it was done by laparotomy and in seven cases the approach was not specified. one patient with a staple peritoneal closure developed an internal herniation, was re - operated by laparoscopy and presented a second intestinal obstruction in the same place that had been closed again with the staples and had to be re - operated for the second time by laparotomy. herein reported - the 420th consecutive laparoscopic inguinal repair in our group since 1992 - it is possible to speculate that the vomiting crisis that our patient suffered soon after the analgesic ingestion could have caused a sudden increase of the intra - abdominal pressure leading to herniation of intestinal loops through the peritoneal inguinal closure. in general, the authors agree that staple closure can be defective if a large gap is left between them and that this may lead to the shower - curtain effect, which also occurred in this patient who had a running stitch type of closure. our group previously repaired peritoneal defects with a running - suture or figure of x interrupted stitches with 00 pds (polydioxanone) and, as shown in figure 1, the closure seemed to be adequate, initially. after the presentation of this complication, our current opinion is that when tapp is indicated, a safer closure should be done with interrupted figure of x stitches, leaving a space of not more than 0.5 cm between each. the procedure of choice at the present time is to carry out a tep procedure whenever it is possible, in which this complication is avoided. there are, though, indications for a tapp procedure with advantages, and a reduced number of tep procedures will be converted. so, it is necessary to keep in mind the possible complications of the former method to prevent them and treat them correctly once they appear. finally, we think that the approach of choice in a reoperation of this type of case is laparoscopy because the lysis of adhesions and the internal herniation can be solved readily with this method. if needed, the umbilical trocar incision can be lengthened 3 or 4 cms to assist the procedure in order to exteriorize necrotic intestinal loops to perform the resection / anastomosis procedure and introduce them back into the abdominal cavity. alternatively, if deemed necessary, a formal laparotomy can be done once the diagnosis has been established. even though laparoscopic inguinal repair has gained popularity due to its results and advantages, it should not be forgotten that, as in any other surgical procedure, complications may appear and some of them can be serious or even lethal. a water - tight peritoneal closure in the tapp procedure, using interrupted stitches with an adequate closure of the rest of trocar wounds, should reduce the risk of postoperative intestinal obstruction, considerably.	a case is presented of a male patient that presented with intestinal obstruction in the early postoperative period of a transabdominal preperitoneal inguinal repair (tapp) that was diagnosed and repaired successfully using laparoscopy. whenever a total extraperitoneal procedure (tep) can not be performed, the peritoneal closure of the tapp should be done water - tight using interrupted stitches of absorbable monofilament sutures.
globalization and the advances in modern information and communication technologies (ict) are changing the practice of health care and policy making. in the globalized economies of the 21 century, health systems will have to respond to the need of increasingly mobile citizens, patients and providers. at the same time the increased use of ict is enabling health systems to systematize, process and integrate multiple data silos from different settings and at various levels. to meet these challenges effectively, data interoperability within and across heterogeneous health systems, however, is often hampered by differences in terminological inconsistencies and the lack of a common language, particularly when multiple communities of practice from different countries are involved. discuss the functionality and ontological requirements for icf in achieving semantic interoperability of e - health information systems. most solution attempts for interoperability to date have only focused on technical exchange of data in common formats. automated health information exchange and aggregation is a very complex task which depends on many crucial prerequisites. the overall architecture of the health information system has to be defined clearly at macro and micro levels in terms of its building blocks and their characteristics. the taxonomic and conceptual features of the icf make it an important architectural element in the overall design of e - health information systems. to use the icf in a digital environment ontological modeling is also required for linking assessment instruments and clinical terminologies (e.g. snomed) to the icf. to achieve semantic interoperability of e - health systems a carefully elaborated overall health information system architecture has to be established. as a content standard, the icf can play a pivotal role for meaningful and automated compilation and exchange of health information across sectors and levels. in order to fulfill this role	introductionglobalization and the advances in modern information and communication technologies (ict) are changing the practice of health care and policy making. in the globalized economies of the 21 century, health systems will have to respond to the need of increasingly mobile citizens, patients and providers. at the same time the increased use of ict is enabling health systems to systematize, process and integrate multiple data silos from different settings and at various levels. to meet these challenges effectively, the creation of an interoperable, global e - health information infrastructure is critical. data interoperability within and across heterogeneous health systems, however, is often hampered by differences in terminological inconsistencies and the lack of a common language, particularly when multiple communities of practice from different countries are involved.aimdiscuss the functionality and ontological requirements for icf in achieving semantic interoperability of e - health information systems.resultsmost solution attempts for interoperability to date have only focused on technical exchange of data in common formats. automated health information exchange and aggregation is a very complex task which depends on many crucial prerequisites. the overall architecture of the health information system has to be defined clearly at macro and micro levels in terms of its building blocks and their characteristics. the taxonomic and conceptual features of the icf make it an important architectural element in the overall design of e - health information systems. to use the icf in a digital environment the classification needs to be formalized and modeled using ontological principles and description logic. ontological modeling is also required for linking assessment instruments and clinical terminologies (e.g. snomed) to the icf.conclusionsto achieve semantic interoperability of e - health systems a carefully elaborated overall health information system architecture has to be established. as a content standard, the icf can play a pivotal role for meaningful and automated compilation and exchange of health information across sectors and levels. in order to fulfill this role a icf ontology needs to be developed.
surface active agents (saas, surfactants) are a group of compounds with specific chemical composition of their molecules (one part soluble in polar medium : hydrophilic and second in nonpolar medium : hydrophobic). the main classification of surfactants is based on charge of hydrophilic part of their molecules : cationic, anionic, and nonionic compounds. occurrence of polar and nonpolar parts in the saas molecules gives them special properties against different medium. another property of the saas is ability to association in solution and formation of micelles. during the process of formulation micelles surface active agents are adsorbed at boundary phases to remove hydrophobic parts from water to reduce energy of system. due to the specific chemical structure of surfactants molecules they are applied in different areas of human activity. during formulation of households or industrial products compounds from the group of surfactants are used because their presence leads to improving efficiency of the following processes : wetting / waterproofing, de- or foaming, de- or emulsification, dispersion or flocculation of solids particles in liquid phases, solubilization of non-/sparingly soluble reagents in solvents, increase or decrease of viscosity of solution phases. wetting / waterproofing, de- or emulsification, dispersion or flocculation of solids particles in liquid phases, solubilization of non-/sparingly soluble reagents in solvents, increase or decrease of viscosity of solution phases. in table 1 the expected growth of production of that class of compounds is forecasted as 2.8% annually to 2012. approximately 65% of total production corresponds to compounds classified as anionic surfactants, second and third places in global production corresponds to nonionic and cationic compounds, respectively. after use, compounds from the group of surfactants are emitted to various elements of the environment (gas, liquid, and solid phases), where they can undergo numerous physical and chemical processes. therefore, the specific properties of those chemical compounds cause increasing their mobility and unrestrained circulation in the environment. those processes might significantly contribute to disrupting the water cycle within various ecosystems ; hence, it is essential to obtain answers to what levels of concentrations surfactants are present in the environment. investigation of environmental fate of surface active agents can help increase level of knowledge about pollutants migration pathways and better protect living organisms or different ecosystems. in recent years the interest of saa has increased and scientists have started to estimate the possible effects of those compounds on the environmental balance [5, 6 ]. in this paper, basic information about surface active agents (classification, their properties, and areas of use) and their fate after discharging to wastewater treatment plants are mentioned. the brief review of sorption and degradation processes of surfactants in water systems is presented. moreover, the analytical protocols used for determining total concentration of surfactants or individual analytes from particular group of saa in environmental samples are described. this work contains overview of contamination of different ecosystems caused by surfactants (also research data of levels of saa in atmospheric deposition samples collected in urban and nonurban areas). possible impact of compounds from the group of saa on biotic and abiotic elements of the environment (especially as a result of their occurrence in atmospheric waters) is presented. surface active agents are one of the most common applied compounds in industrial, agricultural, and household activities and after use a huge number of surfactants (and/or their degradation products) are discarded to wastewater - treatment plants (wwtp). in nonurban areas (there is no wwtp) wastewaters that contained various classes of surface active agents are discarded directly to surface waters and they might be dispersed into different elements of environment. in wastewater treatment plants compounds from group of saas are completely or partially removed by a combination of different processes (mainly by sorption and aerobically biodegradation) and their degradation pathways were investigated [7, 8 ]. chemical compounds from this group are degraded during secondary treatment processes and under optimized conditions about 9095% of initial saas concentration contained in influent streams can be eliminated (depending on efficiency of wwtps). in should be noticed that considerable part of pollutants is removed as sewage sludge (from 15% to more than 90%) [10, 11 ]. moreover, some surface active agents can be transformed to more toxic degradation products (e.g., degradation products of compounds from the group of alkylphenol ethoxylates (apeo)). after appropriate processes in wwtp effluents and sewage sludge, in which different types of surfactants or their degradation products (several g / l or g / kg) can occur, are discharged into surface waters or used as fertilizer in agricultural areas, respectively. such practices lead to emission of surfactants and their metabolites into different parts of the environment (soils, ground waters, surface waters, and living organisms) [9, 12 ]. in recent years the amount of literature data concerning on surfactants occurrence and their concentration in environmental samples has been markedly increased. if compounds from the group of saas are present in water ecosystems, they can also undergo sorption and aerobic / anaerobic degradation processes. sorption processes inhibit also degradation of chemical compounds because their bioavailability can be reduced. in table 2 research allows observing relationship between higher salinity of water samples and higher sorption percentages for compounds from the group of las onto suspended solids (as calcium and magnesium salts). generally, the higher concentrations of less polar compounds from the group of surfactants (e.g., c13las, np, and npe1 - 2o) were observed in sediment or suspended solid samples. the higher concentrations of more polar compounds (e.g., c10las, short - chain spc, and npec) were observed in the dissolved form [1315 ]. sorption process is relative to hydrophobic nature of compounds : more polar anionic saas were estimated in the dissolved phases;less polar cationic and nonionic saas were estimated in the particulate phases (their transport will be associated with suspended solids). more polar anionic saas were estimated in the dissolved phases ; less polar cationic and nonionic saas were estimated in the particulate phases (their transport will be associated with suspended solids). also other environmental factors like ph, salinity, carbon, or clay content of the particulate phase can have influence on sorption processes [8, 16 ]. degradation of surface active agents caused by microbiological organisms (biodegradation) is the primary transformation taking place in different ecosystems to reduce impact on living organisms. biodegradation is an important process not only in wastewater treatment plants but also in the environment. during this process microorganisms are able to utilize surfactants as substrates to produce energy and nutrients or cometabolize them by microbial metabolic reactions. there are many factors (e.g., chemical structure of analytes, physiochemical parameters of ecosystems like temperature, light, and salinity) that affect the efficiency of biodegradation of compounds from the group of surfactant in the environment. most of compounds from this group can be rapidly degraded by microorganisms in ecosystems in the presence of oxygen (according to the current legislation), while some of them (e.g., las, dtdmac) may be persistent under anaerobic conditions [8, 15, 17 ]. in table 3 the degradation pathway for alkyl trimethyl or dimethyl ammonium compounds (tmac and dmac) is initialed by n - dealkylation and followed by n - demethylation reaction (trimethylamine, dimethylamine, and methylamine were identified as the intermediates of alkyl trimethyl ammonium salts in activated sludge). the length of alkyl chain has an important role in the fate and biological effects of these compounds in the environment. the aerobic biodegradability of compounds from the group of qacs decreases with the number of nonmethyl alkyl groups (e.g., r4n < r3men < r2me2n < rme3n < me4n ; me methyl group) and substitue them with a benzyl group [20, 21 ]. the biodegradation was not observed for ditallow dimethyl ammonium chloride (dtdmac) in anaerobic screening assay and this compound was replaced by diethyl ester dimethyl ammonium chloride (deedmac). analytes from the group of las contained longer alkyl chains and benzene group in external position is more susceptible to biological degradation. mono- and dicarboxylic sulfophenyl acids (spc), las biodegradation intermediates having an alkyl chain length of 4 to 13, are formed during the following stages : -oxidation of the alkyl chain terminal carbon, successive -oxidation, and further desuphonation [7, 2326 ]. for example, it can be observed that in surface waters primary degradation (compounds that lost their chemical structure and properties) of compounds from the group of las is completed after 4 days ; the average half - life of some analytes is 1024 h and 5690% of mineralization can be finished from 7 days up to 30 days. compounds from the group of fatty alcohol sulphates (as) undergo rapidly primary and ultimate biodegradation process under aerobic and anaerobic conditions [7, 8, 21 ]. the degradation process involves the enzymatic cleavage of the sulphate ester bonds to give mixture of inorganic and organic compounds (sulphate and fatty alcohol). during further stages, alcohol is oxidized to aldehyde and next to fatty acid (-oxidation pathway). biodegradation of compounds from the group of sds was reported in antarctic coastal waters with half lives of 160 to 460 h. removal efficiency in wwtp of homologues from the group of sas was estimated from 64 to more that 99% (due to a combination of degradation in the active sludge unit (84%) and sorption processes onto sludge (16%)). the degradation process is depending on length of alkyl chain in sas homologues due to decreasing solubility for longer chains in polar medium (shorter alkyl chain = higher degradation percentage). in addition, long alkyl chain of sas compounds is characterized by a about three times higher tendency to sorption onto sludge surfaces compared with short chain compounds [29, 30 ]. for other anionic surfactant like aes removal efficiency from influent water degradation process of nonionic surfactants was investigated mainly using compounds from the group of nonylphenol ethoxylates. their primary biodegradation is relatively fast (from 4 to 24 days) and their mineralization is typically from 50 to 80% [3234 ]. degradability of npeo compounds with lower molecular weight is easier than that with higher molecular weight. it was confirmed that during biodegradation process the ethoxylated chain becomes progressive shorter as a result of hydrolysis and next oxidations reactions, leading to short - chain npes with one or two ethoxylate groups [32, 36 ]. besides, oxidation reaction of the ethoxylated chain can occur more often than hydrolysis (npecs are the most often found metabolites : 6998%) and npec compounds can be degraded to npe2c [3335 ]. alkylphenol diethoxycarboxylates can be also formed as a consequence of -oxidation and later, -oxidation reaction of the alkyl chain. in different types of environmental samples were detected metabolites of npeo compounds (np, npe1 - 2o, and npec) [8, 37, 38 ]. degradation of compounds from the group of alcohol polyethoxylates (aeo) was also investigated. biodegradation processes can be more efficient for analytes with shorter alkyl and/or ethoxylated chains. in this process fatty acids and polyethylene glycols (peg : slower biodegradation with production of carboxylic acids) can be formed as a consequence of central cleavage of molecule and undergo -oxidation and later, -oxidation reaction of the alkyl chain [39, 40 ]. branched compounds from the group of aeo are characterized by slower degradation and it involves -oxidation and successive, -oxidation reactions of the alkyl chain. therefore, surfactants occurred in water systems can be degraded easily (half - lives from hours to few days) depending on their properties and environmental parameters. compounds from the group of saa can undergo such processes like attachment to suspended solids and accumulation in sediments. in conditions with lack of oxygen (starting in depth of few cm) surfactants can be only degraded by anaerobic pathways. generally, in anaerobic condition processes are slower or they are not observed (e.g., dtdmac) and pollutants occurred longer in sediment [8, 13 ]. however, during laboratory experiments acceptable degradation percent of las has been observed with use of anoxic marine sediments (up to 79% in 165 days). the following stages of anaerobic degradation pathway for las have been reported : initial reaction metabolites (generated via the addition of fumarate) ; their biotransformation into sulfophenyl carboxylic acids, and progressive degradation by -oxidation reactions [4143 ]. for different reasons, it is important to detect, identify, and monitor levels of surfactants in aquatic environments. prior analytics of pollutants from the group of saas in environmental samples pose new challenges. the searching for new tools and new sources for obtaining information about degree of pollution different environmental compartments is dictated by toxicological considerations, the desire to increase the accuracy of the description of the environment, and the study and protection of aquatic ecosystems balance. it is imperative to have the appropriate analytical tools (standard analytical methodologies or their modification) in order to monitor the presence of surface active agents in various environmental samples. the determination of saas in such samples causes a lot of problems, mainly because of [1, 6]the complex composition of environmental samples interfering components which increased or reduced the identified levels of analytes, the low concentrations of individual surfactants in such samples, the diverse chemical structures of surfactants moreover commercially available surfactants are mixtures containing twenty or more individual compounds, the amphiphilic nature of surfactants (a consequence of their chemical structure),the limited availability of commercial standard solutions of surfactants (also isotope - labelled analytes). the complex composition of environmental samples interfering components which increased or reduced the identified levels of analytes, the low concentrations of individual surfactants in such samples, the diverse chemical structures of surfactants moreover commercially available surfactants are mixtures containing twenty or more individual compounds, the amphiphilic nature of surfactants (a consequence of their chemical structure), the limited availability of commercial standard solutions of surfactants (also isotope - labelled analytes). the complex and frequently variable matrix composition of environmental samples and the trace levels of saas mean that suitable isolation and/or preconcentration techniques have to be applied at the sample preparation stage. as a consequence of amphiphilic nature of surfactants molecules during preparation stage an internal standard has to be added to the sample before the solvent extraction (for estimating the losses of analytes), which problematic because of lack of available commercial standards. moreover, the analytical methodologies enabling the determination of a wide range of saas present at different levels in environmental samples should be validated against certified reference materials. nowadays, only liquid reference materials are available on the market ; they can be used to validate methodologies for determining total contents of ionic (cationic or anionic) and nonionic surfactants. on the other hand, there are no reference materials suitable for validating entire analytical procedures. those problems have influence on quality control and quality assurance of measurement results and they might cause difficulties in obtainment of the reliable analytical information [44, 45 ]. currently, despite an increase in the amount of information about the concentration of compounds of the group of saas in environmental samples, the knowledge about degree of pollution caused by surfactants is too low and it is still impossible to determine how they can affect the diversity of ecosystems. the determination of total concentration of ionic and nonionic surfactants in different types of liquid / solid samples can be carried with use of standard analytical methodologies (including liquid - liquid extraction / solid - liquid extraction or soxhlet extraction, resp.). cationic surfactants can be determined as sum of substances that form ion pairs with disulfine blue (disb) and isolated with use of appropriated extraction technique. total concentration of compounds from the group of anionic surfactants can be evaluated as substances which react with methylene blue (mb). at final determination stage spectrophotometrical technique can be employed to measure total concentration of anionic surfactants in solvent extract. the total concentration of nonionic surfactants can be determined with use of the same analytical technique as before but with the application of different reagents. for determining the individual surfactants belonging to different classes of chemical compounds should be applied modification of the available analytical procedures with use of isolation or / and preconcentration techniques at sample preparation stage (e.g., liquid - liquid extraction ; solid phase extraction ; solid phase microextraction) and chromatographic techniques coupled with different detection systems (e.g., gc - ms or hplc - cd, hplc - ms) at the final determination stage. intensive researches in this direction are carried out in a few research centers located only in spain, germany, usa, china, and so forth. gas chromatography (gc) is limited to volatile analytes and this requirement meets only low molecular mass nonionic surfactants (contain low number of ethoxylated groups). this technique is suitable during determination of concentration of other nonionic and anionic after derivatization processes with specific agents. in the literature data presently, high - performance liquid chromatography (hplc) is the most often used analytical technique during analysis of surface active agents from all classes compounds (their homologs, oligomers, and isomers) in environmental samples. in most cases, derivatization processes of analytes are not necessary, because lc technique is suitable for determining level of low - volatility analytes with large molecules. it gives the possibility of excluding this operation from analytical procedures and simplifies them due to the green analytical chemistry (gac) concept. an undoubted advantage of hplc technique is that surfactants levels can be measured in a very short time. for determining the concentration of individual analytes from group of surfactants in appropriate prepared solvent extracts the following types of detectors can be used : fluorescence (fld), ultraviolet (uv), conductometric (cd), mass spectrometry (ms) or their combinations [1, 6 ]. mass spectrometer is universal detector, which can be used in qualitative and quantitative determination of wide range of trace analytes in single analysis. but this detection technique has several disadvantages (high cost of equipment and its operation ; highly qualified staff ; high purity of reagents). according to these drawbacks for example, ion chromatographer coupled to conductometric detector can be applied to determinate individual ionic (cationic and anionic) surfactants in solvent extracts, for example. to investigate other groups of saa compounds (with chromophores groups in their molecules) in appropriate extracts can be involved ion chromatographer coupled to uv detector (compounds from the group of las data under preparation by our research group). other analytical tool used to determine surface active agents coupled to ic equipment is evaporative light scattering detectors (els). this device can detect almost all relatively nonvolatile analytes and is insensitive for gradient condition of analysis [5052 ]. analysis of the literature data confirmed that surface active agents are presented in various elements of the environment, but researchers focused mainly on determination of levels of anionic and nonionic analytes. thus, due to the very limited literature data on the determination of cationic saas, which are characterized by higher toxicity to living organisms and undergo sorption onto solid surfaces. additionally, commercial available surfactants are mixtures of various homologues and/or isomers and their determination in environmental samples becomes more problematic. those aspects indicate the need to develop new methodologies with the use of more selective and specific analytical tools. the ranges of concentrations or mean values of compounds from different groups of surfactants determined in solid and liquid environmental samples are presented in table 4. easily, it can be noticed that there is lack of research describing the problems of determining surface active agents (both total concentration or contents of individual analytes) in aerosol samples, atmospheric precipitation, and atmospheric deposits samples (e.g., dew, hoarfrost, and fog). similarly the process of saas transformation in the snowpack deposited on the ground as well as the transfer of the deposited saas to surface waters has not been satisfactory recognized yet with hardly any publications on this topic. studies on degree of environmental contamination caused by surfactants should be analyzed in mentioned type of samples, because atmospheric deposition is considered as a major source of various pollutants. accordingly, for the first time the total concentration of various groups of saas has been determined in atmospheric deposits samples collected during different seasons on polish territory (table 4section atmospheric waters). to fulfill this research aim, next step in investigation of surfactants fate in the environment should be determination of their individual levels in collected samples with the use of appropriate analytical protocols. it should be noticed that surface active agents also can be derived from natural sources, particularly from phytoplankton activities. the presence of those organisms can be observed in the surface of the euphotic zone, where chemical exchange with the atmosphere is possible. such processes may have significant influence on the boundary between the hydrosphere and the atmosphere (this aspect will be presented in further part of paper) [53, 54 ]. a fraction of surfactant from influent stream can be emitted to the environment via wastewater treatment plant effluent discharge into surface waters. in aquatic ecosystems amount of compounds from the group of saas can be reduced by different processes (dilution, bio- and photodegradation, and sorption to suspended solids and to sediments. application of sewage sludge (containing surfactant and other pollutants) on agricultural lands as fertilizers has an impact on terrestrial living organism and plants. cationic saas, because of positive charge of their molecules, are strongly sorbed to the negatively charged solid surfaces of sludge, soil, sediments, metals, plastics, and cell membranes (susceptibility to accumulation and bioaccumulation). cationic surface active agents exhibit specific properties that may prevent (or retard) the growth or cause mortality of microorganisms. these properties allows for appling them as disinfectants and antiseptic agents in different products. on the other hand, the occurrence of cationic surfactants in aquatic ecosystems is very dangereous for aquatic organisms or, in case of humans, may cause irritation or burns to the skin, eyes and respiratory system [8, 56 ]. anionic compounds can be accumulated in aquatic organisms and interact with their cell membranes, proteins, and enzymes. it causes the disturbances of their biological functions, cell lysis, or even death. additionally, decreasing surface tension properties of anionic surfactants makes easier migration processes of other toxic pollutants into living organisms [5759 ]. the most often applied group of nonionic surfactants (nonylphenol ethoxylates and octylphenol ethoxylates) undergoes a quick degradation in wastewater treatment plants into short - chain alkylphenol ethoxylates and carboxylated derivatives. moreover, it should be noticed that some products of apeo degradation are dangerous also due to their estrogenic properties. the cationic and nonionic surfactants can have influence on sorption processes and transport of some pharmaceutical compounds in living organisms, potentially reducing the rate of their migration through the subsurface. some compounds from the group of surfactants have a low biodegradability or their degradation products are more toxic than initial compounds or even become endocrine disruptor compounds (edc). the toxicity properties of compounds from the group of surfactants can be used to estimate their environmental risks. in table 5 the literature data of toxicity for different classes of surfactants to different test organisms are collected. chemical compounds (edc) that occurred in the environment can present disruption properties of normal functioning of hormonal system in living organism. in 1938 for the first time data about recognition of estrogenic properties of p - n - alkylphenol were published. further research data shows that compounds from op, np, npneo (n = 2, 9, 40), and np1ec have estrogenic activities against different living organisms [6367 ]. in investigation of endocrine disruption effect mixtures of cationic, anionic, nonionic surfactants and some of their degradation products parent compounds have no estrogenic properties for tested organism but for their degradation products (op, np, np1ec, np2ec, and np2eo) were observed positive results. in previous section of article information about sorption and degradation processes of surfactants in wastewater treatment plants or in the environment is presented. herein, other important aspects of impact of compounds from the group of surfactants on different part of abiotic environment will be reviewed. so far, an atmospheric input of compounds from the group of surfactants to aquatic environment has not been considered yet. however, evaporation processes of semivolatile compounds (anionic and nonionic surfactants or product of their degradation) from surface waters, soils and vegetation have been recognized as a significant source for such contaminants in the atmosphere. compounds from the group of saas are able to interact on processes that occurred at different interfaces. they can also have influence on processes between air and water interfaces (e.g., suppression of evaporation processes, modification of the surface temperature field during free surface flows, inflection on gas transport and reduction of momentum transport from air to water, and damping of surface waves). scientists believe that there are two mechanisms of surfactant amount reduction on a water surface. first, rain drop can split into many small drops covered with surface active agents. these drops can be transported away from the surface of water by moving air masses. secondly, bubbles can be formed during rain drop impacts and they will move to water surface, burst, and lead to transporting pollutants to the air [7074 ]. moreover, the occurrence of surfactants in abiotic environment might disturb equilibrium of different compartments. those compounds can also increase the solubility of organic compounds in the aqueous phase (increasing mobility of toxic agents in different ecosystems). in the environment this specific system can be observed as a thin boundary between the water basin (e.g., ocean) and the atmosphere, named as sea - surface microlayer (sml). the formation of sml is complicated and it is unknown which physical and chemical processes have influence on migration of chemical compounds in this boundary. for several times occurrence of surface active agents in sml has been proved. research in this area has shown that saas (consist of low - molecular - weight carbonyl compounds) can be also generated in microlayer by microorganisms. in surface water those compounds are produced photochemically from the degradation of refractory dissolved organic matter (e.g., humic substances) and they are taken up quickly by microorganisms [7577 ]. accumulation of surfactants at surface of water can be toxic for marine and freshwater organisms. some of compounds from the group of surfactants can lead to chronic toxicity or estrogenic responses towards aquatic species [54, 7882 ]. at the sea surface surfactants play a role in the recycling and long - range transport of pollutants via marine aerosols. heavy metals (as pollutants from crustal and urban sources) enriched on the sea surface were found to interact with the surface - active organic matter and become transferred into marine spray [8385 ]. the early studies of surfactants in rain water and atmospheric aerosols found that their concentrations were too low to have any effect upon the cloud physical process at high dilution. more concentrated surface active substances or in different mediums may influence the state of the gas - liquid interfaces of atmospheric particles and droplets. models of cloud formation based on laboratory research suggest that organic compounds significantly provide a decries in surface tension (determining droplet population). surface tension, which is one of the factors that controls the vapor pressure of small droplets, is the consequence of intermolecular attractive forces tending to minimize the surface area of the liquid. some compounds from the group of surfactants found in tropospheric they might participate in generation of more cloud water due to the reduction of surface tension in a droplet and behave like cloud condensation nuclei (ccn) in the atmosphere. the amount of ccn increases the albedo effect and influences climate change in certain areas (due to relation with cooling effect in the atmosphere) [85, 8890 ]. as a result of the intensification of certain types of human activity an upward trend due to their widespread use and freely migration between phases, surfactants and products of their degradation have been detected at various concentrations in different part of abiotic and biotic environment. the occurrence of surface active agents was confirmed in atmospheric precipitation and deposits, surface waters, sediments, soils, and living organisms. compounds from the group of surfactants have been detected in samples (abiotic : air, snow, lake water, and sediment ; biotic : marine and terrestrial organisms) from the areas of residence and economic exploitation by humans and from remote regions like the antarctic as well. the global source refers to long - range atmospheric transportation of pollutants (e.g., anionic surfactants) from lower latitude area, but their transport pathways are not well understood. thus, it is widely accepted that saas play an important role of anthropogenic pollutant emission, having versatile environmental consequences. on the other hand, review of the literature also shows that we are far from understanding the environmental fate of surfactants. there is lack of data on composition, properties, and behavior of the organic material (especially surfactants) in the atmosphere or other environmental compartments [1, 92 ]. the analysis of atmospheric precipitation and deposits is one of the important aspects of the assessment of the degree of environmental pollution caused by surfactants. both atmospheric precipitation and deposits compounds released into the atmosphere are present in the gaseous phases, in the aerosol phases, or are adsorbed on surface of suspended particles. two major deposition mechanisms of those pollutants are distinguished, that is, wet (with rain and snow) or dry removal. deposition is controlled by the distribution of chemical compounds between different phases and their physicochemical properties. on the other hand, the strong dependence was observed between saas concentration and deposition on the existing emission background and meteorological conditions, particularly wind direction, sunlight (photodegradation), rain / snowfall, and so forth. therefore, it is essential to keep control of the content of those compounds in specific environmental samples (e.g., atmospheric precipitation and deposits) and identify parameters that may affect content of surfactants in the different ecosystems. compounds from the group of surfactants are widely applied in different areas of human activities and after use they are discharging to wastewater treatment plant. after appropriated processes (sorption, degradation), they can be emitted to surface waters with effluent streams. in aquatic ecosystems surfactants and their degradation products compounds from the group of saas can also affect living organisms and abiotic parts of the environment. moreover, they can interact with different interfaces (water - air, soil / sediment - water) and change natural processes in those systems. during last decades in environmental samples different types of surfactant were determined using analytical protocols depending on what information is required (total concentration of surfactants or individual analytes from appropriated group of saas compounds). but it is still imperative to develop new analytical procedure for investigation analytes from the group of saas that occurred in ecosystems to make them easier, less cost - consuming, and more safe for biotic and abiotic elements of the environment. research data confirmed that they are able to spread between waters, soils, atmosphere, and living organisms from different geographic regions. but we are far from understanding migration pathways and their behavior of those pollutants, their impact on different ecosystems and living organisms. there is a need to investigate those processes in environment to protect abiotic and biotic part of the environment.	due to the specific structure of surfactants molecules they are applied in different areas of human activity (industry, household). after using and discharging from wastewater treatment plants as effluent stream, surface active agents (saas) are emitted to various elements of the environment (atmosphere, waters, and solid phases), where they can undergo numerous physic - chemical processes (e.g., sorption, degradation) and freely migrate. additionally, saas present in the environment can be accumulated in living organisms (bioaccumulation), what can have a negative effect on biotic elements of ecosystems (e.g., toxicity, disturbance of endocrine equilibrium). they also cause increaseing solubility of organic pollutants in aqueous phase, their migration, and accumulation in different environmental compartments. moreover, surfactants found in aerosols can affect formation and development of clouds, which is associated with cooling effect in the atmosphere and climate changes. the environmental fate of saas is still unknown and recognition of this problem will contribute to protection of living organisms as well as preservation of quality and balance of various ecosystems. this work contains basic information about surfactants and overview of pollution of different ecosystems caused by them (their classification and properties, areas of use, their presence, and behavior in the environment).
male c57bl/6-ins2/j (hereafter ins2) mice and their wild - type (wt) littermates were purchased at 3 weeks of age from jackson laboratory (bar harbor, me). mice (n = 16/group) were studied over a period of 8 to 13 weeks of untreated type 1 diabetes. a separate group of ins2 and wt mice (n = 3/group) were used for the 1 week of type 1 diabetes regeneration study. ins2 mice become spontaneously diabetic at 4 weeks of age because of a heterozygous mutation in the ins2 gene (26). exact onset of diabetes was determined by monitoring blood glucose as previously described (12). the ins2 mice were chosen instead of the commonly used streptozotocin - induced diabetic rodent model because of known growth - arresting effects of streptozotocin on skeletal muscle (27). blood glucose and body mass were measured biweekly (fed state : 12001400 h) in the 8-week experimental groups. blood samples were collected at 2, 4, and 6 weeks of diabetes for analysis of metabolites and hormones. all animal experiments were approved by the mcmaster and york university animal care committees in accordance with canadian council for animal care guidelines. skeletal muscle injury was induced with an intramuscular injection of 10 m cardiotoxin (ctx ; latoxan, france) as previously described (28). injuries were generated in the left tibialis anterior (ta) and quadriceps muscles of both ins2 and wt mice at 1 and 8 weeks of diabetes. the 1-week group was harvested at 10 days postinjury, whereas the 8-week group was subdivided into four recovery time points : 5, 10, 21, and 35 days. after the specified regeneration period, animals were killed and blood was collected from the thoracic cavity after heart excision. injured and uninjured ta muscles were coated in optimum cutting temperature embedding compound and frozen in isopentane cooled by liquid nitrogen, and injured quadriceps muscles were snap - frozen and stored at 80c. to determine if elevations in circulating pai-1 were contributing to impaired skeletal muscle regeneration in the diabetic animals, pai-039, an orally effective inhibitor of active pai-1 (29), an additional group of wt and ins2 mice (n = 4) were treated via oral gavage with vehicle (2% tween-80 and 0.5% methylcellulose in sterile h2o) or vehicle plus pai-039 (2 mg / kg ; axon medchem, the netherlands), respectively. on the day of ctx injury (at 8 weeks of diabetes), mice were treated with vehicle or vehicle plus pai-039 at 1100 h, received ctx injury to the ta at 1200 h, and received pai-039 treatment again at 1500 h. pai-039 treatment was continued twice daily (1100 and 1500 h) throughout the 5-day regeneration period, at which point the animals were killed and tissues were dissected and stored as described above. those treatment time points were chosen to best attenuate the peak of pai-1 activity because of its circadian expression pattern (30). wt mice treated with vehicle (wt + vehicle) demonstrated no significant difference from untreated wt in active urokinase plasminogen activator (upa), active matrix metalloproteinase (mmp)-9, collagen levels, and myh3 ; therefore, these two groups were pooled for comparison with ins2 mice and ins2 mice treated with pai-039 (ins2+pai-039) as illustrated in fig. 3. heparinized blood plasma was analyzed for insulin and total pai-1 (madpk-71 k ; millipore, billerica, ma) at all collection time points. plasma was also analyzed for nonesterified fatty acids with the use of a colorimetric assay (wako diagnostics, richmond, va). snap - frozen quadriceps or ta samples were homogenized, analyzed for protein concentration, electrophoretically separated on acrylamide gels, and transferred to polyvinylidene fluoride membranes as previously described (28). primary antibodies included myh3 (hybridoma bank f1.652), gapdh (abcam 8245, loading control ; cambridge, ma), myogenin (hybridoma bank f5d), mmp9 (abcam 38898), and upa (abcam 28230). upa was analyzed for unbound (active) upa and upa bound to pai-1 as a measure of pai-1 activity in the muscle (31). active upa is found at 48 kda, and inactive (pai-1-bound) upa is found at 93 kda. appropriate horseradish peroxidase - conjugated secondary antibodies were used and visualized with the addition of chemiluminescent reagent (amersham, piscataway, nj). images were acquired with a fusion fx7 imager (vilber lourmat, eberhardzell, germany) and analyzed with imagej. eight - micron skeletal muscle cross - sections were mounted on glass slides and stained as described below. eosin staining was used to determine the average fiber area of uninjured and injured ta. three images spaced evenly throughout the ta (1 mm apart) were used for analysis where 25 fibers per image were analyzed for area (75 total fibers per ta). we have previously demonstrated that in this muscle, quantification of this number of fibers provides a representative analysis of fiber area (12,32). to stain for collagen content, sections were immersed in picrosirius red solution (0.1% w / v direct red 80 [sigma 365548 ; st. louis, mo ] in a saturated aqueous solution of picric acid [sigma p6744 ]) for 1 h. sections were briefly rinsed in two changes of acidified dh20 (0.5% glacial acetic acid), dehydrated, cleared, and mounted. sections were fixed with ice - cold 2% paraformaldehyde, blocked with 10% normal goat serum/1.5% bsa, followed by mouse igg block (bmk 2202 ; vector laboratories inc., burlingame, ca), and incubated with 1:1 dilution of anti - myh3 overnight at 4c. alexa 488 anti - mouse secondary antibody (a-11001 ; invitrogen, carlsbad, ca) was used for detection, and 4,6-diamidino-2-phenylindole was used to identify nuclei. melville, ny) were analyzed using nis elements software (nikon, inc., analysis included determination of collagen positive area and myh3 positive area using signal threshold settings as the detection method. for all experiments, the appropriate t test or two - way anova with bonferroni post hoc analysis was performed between ins2 and wt groups. two - way anova was run on datasets with dependent variables measured over time, and one - tailed t tests were carried out on data with only single comparisons. one - tailed t tests were justified for these comparisons because differences in a specific direction were hypothesized a priori on the basis of our data and previous reports (2125). asterisks denote significant differences identified by t test or bonferroni post hoc test in pairwise comparisons, and significant main effect of diabetes or significant interaction between diabetes and time is listed in figs. ten days after injury, ta muscle of the 1-week diabetic ins2 mice demonstrates a significant loss of (a) mass (t test : p = 0.015 ; n = 3) and (b) myofiber cross - sectional area (t test : p = 0.014 ; n = 3) compared with wt, indicating impaired regeneration after injury at only 7 days of type 1 diabetes. c : hematoxylin eosin staining of ta injured at 8 weeks of type 1 diabetes demonstrates loss of (d) muscle mass (n = 16) and (e) myofiber area (n = 16) compared with wt beginning at 10 days postinjury. the uninjured time point in both panels is the contralateral ta to the 5-day post - ctx muscle and is included to illustrate the decrease in muscle mass and myofiber area associated with the impaired growth of skeletal muscle in the type 1 diabetic state. a main effect of diabetes (main effect : p < 0.001) is observed in both mass and fiber area with the asterisk () denoting specific differences between wt and ins2 as defined by post hoc analysis. note that the type 1 diabetic muscle does not return to wt mass / fiber size at later time points, but continues to lag in regeneration. because early expression of myogenic proteins is critical to the early stages of regeneration, (f) myogenin (main effect : p = 0.062, interaction : p = 0.017 ; n = 8) and (g) embryonic myosin heavy chain (myh3) expression (main effect : p = 0.117, interaction : p = 0.009 ; n = 8) were determined in quadriceps muscle and demonstrate significantly increased expression at 5 days postinjury in wt but not ins2 (labeled ins2, f and g). h : immunofluorescent staining of injured ta with anti - myh3 confirms (i) the lack of myh3 positive fibers in ins2 compared with wt (main effect : p = 0.013 ; interaction : p < 0.001 ; n = 8) at 5 days postinjury. differences between groups at specific time points identified by bonferroni post hoc analysis after 2-way anova (d g, i). (a high - quality digital representation of this figure is available in the online issue.) male c57bl/6-ins2/j (hereafter ins2) mice and their wild - type (wt) littermates were purchased at 3 weeks of age from jackson laboratory (bar harbor, me). mice (n = 16/group) were studied over a period of 8 to 13 weeks of untreated type 1 diabetes. a separate group of ins2 and wt mice (n = 3/group) were used for the 1 week of type 1 diabetes regeneration study. ins2 mice become spontaneously diabetic at 4 weeks of age because of a heterozygous mutation in the ins2 gene (26). exact onset of diabetes was determined by monitoring blood glucose as previously described (12). the ins2 mice were chosen instead of the commonly used streptozotocin - induced diabetic rodent model because of known growth - arresting effects of streptozotocin on skeletal muscle (27). blood glucose and body mass were measured biweekly (fed state : 12001400 h) in the 8-week experimental groups. blood samples were collected at 2, 4, and 6 weeks of diabetes for analysis of metabolites and hormones. all animal experiments were approved by the mcmaster and york university animal care committees in accordance with canadian council for animal care guidelines. skeletal muscle injury was induced with an intramuscular injection of 10 m cardiotoxin (ctx ; latoxan, france) as previously described (28). injuries were generated in the left tibialis anterior (ta) and quadriceps muscles of both ins2 and wt mice at 1 and 8 weeks of diabetes. the 1-week group was harvested at 10 days postinjury, whereas the 8-week group was subdivided into four recovery time points : 5, 10, 21, and 35 days. after the specified regeneration period, animals were killed and blood was collected from the thoracic cavity after heart excision. injured and uninjured ta muscles were coated in optimum cutting temperature embedding compound and frozen in isopentane cooled by liquid nitrogen, and injured quadriceps muscles were snap - frozen and stored at 80c. to determine if elevations in circulating pai-1 were contributing to impaired skeletal muscle regeneration in the diabetic animals, pai-039, an orally effective inhibitor of active pai-1 (29), was administered throughout the regenerative process. an additional group of wt and ins2 mice (n = 4) were treated via oral gavage with vehicle (2% tween-80 and 0.5% methylcellulose in sterile h2o) or vehicle plus pai-039 (2 mg / kg ; axon medchem, the netherlands), respectively. on the day of ctx injury (at 8 weeks of diabetes), mice were treated with vehicle or vehicle plus pai-039 at 1100 h, received ctx injury to the ta at 1200 h, and received pai-039 treatment again at 1500 h. pai-039 treatment was continued twice daily (1100 and 1500 h) throughout the 5-day regeneration period, at which point the animals were killed and tissues were dissected and stored as described above. those treatment time points were chosen to best attenuate the peak of pai-1 activity because of its circadian expression pattern (30). wt mice treated with vehicle (wt + vehicle) demonstrated no significant difference from untreated wt in active urokinase plasminogen activator (upa), active matrix metalloproteinase (mmp)-9, collagen levels, and myh3 ; therefore, these two groups were pooled for comparison with ins2 mice and ins2 mice treated with pai-039 (ins2+pai-039) as illustrated in fig. heparinized blood plasma was analyzed for insulin and total pai-1 (madpk-71 k ; millipore, billerica, ma) at all collection time points. plasma was also analyzed for nonesterified fatty acids with the use of a colorimetric assay (wako diagnostics, richmond, va). snap - frozen quadriceps or ta samples were homogenized, analyzed for protein concentration, electrophoretically separated on acrylamide gels, and transferred to polyvinylidene fluoride membranes as previously described (28). primary antibodies included myh3 (hybridoma bank f1.652), gapdh (abcam 8245, loading control ; cambridge, ma), myogenin (hybridoma bank f5d), mmp9 (abcam 38898), and upa (abcam 28230). upa was analyzed for unbound (active) upa and upa bound to pai-1 as a measure of pai-1 activity in the muscle (31). active upa is found at 48 kda, and inactive (pai-1-bound) upa is found at 93 kda. appropriate horseradish peroxidase - conjugated secondary antibodies were used and visualized with the addition of chemiluminescent reagent (amersham, piscataway, nj). images were acquired with a fusion fx7 imager (vilber lourmat, eberhardzell, germany) and analyzed with imagej. eight - micron skeletal muscle cross - sections were mounted on glass slides and stained as described below. eosin staining was used to determine the average fiber area of uninjured and injured ta. three images spaced evenly throughout the ta (1 mm apart) were used for analysis where 25 fibers per image were analyzed for area (75 total fibers per ta). we have previously demonstrated that in this muscle, quantification of this number of fibers provides a representative analysis of fiber area (12,32). to stain for collagen content, sections were immersed in picrosirius red solution (0.1% w / v direct red 80 [sigma 365548 ; st. louis, mo ] in a saturated aqueous solution of picric acid [sigma p6744 ]) for 1 h. sections were briefly rinsed in two changes of acidified dh20 (0.5% glacial acetic acid), dehydrated, cleared, and mounted. sections were fixed with ice - cold 2% paraformaldehyde, blocked with 10% normal goat serum/1.5% bsa, followed by mouse igg block (bmk 2202 ; vector laboratories inc., burlingame, ca), and incubated with 1:1 dilution of anti - myh3 overnight at 4c. alexa 488 anti - mouse secondary antibody (a-11001 ; invitrogen, carlsbad, ca) was used for detection, and 4,6-diamidino-2-phenylindole was used to identify nuclei. hematoxylin eosin staining was used to determine the average fiber area of uninjured and injured ta. three images spaced evenly throughout the ta (1 mm apart) were used for analysis where 25 fibers per image were analyzed for area (75 total fibers per ta). we have previously demonstrated that in this muscle, quantification of this number of fibers provides a representative analysis of fiber area (12,32). to stain for collagen content, sections were immersed in picrosirius red solution (0.1% w / v direct red 80 [sigma 365548 ; st. louis, mo ] in a saturated aqueous solution of picric acid [sigma p6744 ]) for 1 h. sections were briefly rinsed in two changes of acidified dh20 (0.5% glacial acetic acid), dehydrated, cleared, and mounted. sections were fixed with ice - cold 2% paraformaldehyde, blocked with 10% normal goat serum/1.5% bsa, followed by mouse igg block (bmk 2202 ; vector laboratories inc., burlingame, ca), and incubated with 1:1 dilution of anti - myh3 overnight at 4c. alexa 488 anti - mouse secondary antibody (a-11001 ; invitrogen, carlsbad, ca) was used for detection, and 4,6-diamidino-2-phenylindole was used to identify nuclei. melville, ny) were analyzed using nis elements software (nikon, inc., analysis included determination of collagen positive area and myh3 positive area using signal threshold settings as the detection method. for all experiments, the appropriate t test or two - way anova with bonferroni post hoc analysis was performed between ins2 and wt groups. two - way anova was run on datasets with dependent variables measured over time, and one - tailed t tests were carried out on data with only single comparisons. one - tailed t tests were justified for these comparisons because differences in a specific direction were hypothesized a priori on the basis of our data and previous reports (2125). asterisks denote significant differences identified by t test or bonferroni post hoc test in pairwise comparisons, and significant main effect of diabetes or significant interaction between diabetes and time is listed in figs. ten days after injury, ta muscle of the 1-week diabetic ins2 mice demonstrates a significant loss of (a) mass (t test : p = 0.015 ; n = 3) and (b) myofiber cross - sectional area (t test : p = 0.014 ; n = 3) compared with wt, indicating impaired regeneration after injury at only 7 days of type 1 diabetes. c : hematoxylin eosin staining of ta injured at 8 weeks of type 1 diabetes demonstrates loss of (d) muscle mass (n = 16) and (e) myofiber area (n = 16) compared with wt beginning at 10 days postinjury. the uninjured time point in both panels is the contralateral ta to the 5-day post - ctx muscle and is included to illustrate the decrease in muscle mass and myofiber area associated with the impaired growth of skeletal muscle in the type 1 diabetic state. a main effect of diabetes (main effect : p < 0.001) is observed in both mass and fiber area with the asterisk () denoting specific differences between wt and ins2 as defined by post hoc analysis. note that the type 1 diabetic muscle does not return to wt mass / fiber size at later time points, but continues to lag in regeneration. because early expression of myogenic proteins is critical to the early stages of regeneration, (f) myogenin (main effect : p = 0.062, interaction : p = 0.017 ; n = 8) and (g) embryonic myosin heavy chain (myh3) expression (main effect : p = 0.117, interaction : p = 0.009 ; n = 8) were determined in quadriceps muscle and demonstrate significantly increased expression at 5 days postinjury in wt but not ins2 (labeled ins2, f and g). h : immunofluorescent staining of injured ta with anti - myh3 confirms (i) the lack of myh3 positive fibers in ins2 compared with wt (main effect : p = 0.013 ; interaction : p < 0.001 ; n = 8) at 5 days postinjury. differences between groups at specific time points identified by bonferroni post hoc analysis after 2-way anova (d g, i). (a high - quality digital representation of this figure is available in the online issue.) ins2 mice spontaneously developed type 1 diabetes (hypoinsulinemia / hyperglycemia) at 4 weeks of age, which was maintained throughout the study period (tables 1 and 2) compared with wt littermates. relative to wt mice, ins2 mice also displayed decreased body mass gain and developed hyperlipidemia by 6 weeks of untreated diabetes (tables 1 and 2), consistent with previous findings (33). characteristics of wt and diabetic mice at 8 weeks of diabetes (13 weeks old) biweekly data for the long - term (8 weeks) type 1 diabetic and wt groups (n = 16). two - factor anova was run to determine the main effects of diabetes, time, and interaction ; p values for diabetes main effect and interaction are listed next to the respective data. characteristics of wt and diabetic mice during skeletal muscle regeneration after a period of untreated type 1 diabetes data for the long - term (8 weeks + variable time post - ctx ; n = 16 [4 per time point ]) and short - term (1 week + 10 days post - ctx ; n = 3) type 1 diabetic and wt groups. two - factor anova was run to determine the main effects of diabetes, time, and interaction ; p values for diabetes main effect and interaction are listed next to the respective data. for single comparison of short - term groups, t test revealed no significant difference. histologic assessment of the uninjured ta served as an index for the effects of type 1 diabetes on skeletal muscle growth. we found that uninjured ins2 muscles displayed no impairment in myofiber cross - sectional area within the first 17 days of type 1 diabetes (table 1), whereas a significant reduction (12%) in myofiber cross - sectional area accrual occurred by 8 weeks of type 1 diabetes that did not significantly worsen with increasing disease duration (up to 13 weeks, table 2). this suggests that impaired growth, rather than progressive atrophy, is responsible for the reduced myofiber area observed in ins2, at least until such time as significant neuropathic complications develop (34). in response to muscle damage, ins2 muscles exposed to the type 1 diabetes environment for 1 week before injury demonstrated a 21% decrement in myofiber cross - sectional area at the 10-day regeneration mark (fig. this novel finding suggests that even short - term exposure to type 1 diabetes has profound effects on skeletal muscle s ability to repair after damage. the regenerative capacity was also impaired at 8 weeks of disease progression because muscle masses and myofiber cross - sectional areas of regenerating ins2 muscles were significantly less than wt muscles from 10 days of regeneration onward (fig. e). loss of mass and myofiber area was significant even when expressed as a percentage of either the uninjured, contralateral ta or body mass (supplementary fig. 1), confirming that the poor regeneration in type 1 diabetes extends beyond the reduced growth rate. these changes in overall mass and myofiber area were preceded by alterations in the protein expression of markers of the regenerative process, myogenin and embryonic myosin heavy chain (myh3). myogenin is a myogenic regulatory factor that is expressed during early time points in regeneration and is important for cell - cycle exit of myoblasts and consequent terminal differentiation (35,36). western blot analysis showed suppressed expression of myogenin in ins2 muscle compared with wt muscle at 5 days postinjury (fig. myh3, a developmental myosin isoform, is expressed transiently during skeletal muscle regeneration (37) and used as a reference point to assess the process of differentiation (38). similar to myogenin, the protein expression of myh3 was reduced in ins2 muscle at 5 days of regeneration. both western blot and immunofluorescent neither group displayed expression of myh3 at 21 or 35 days after regeneration, suggesting that the impairments in the regenerative process are within the early phases after injury with diabetes development (10 days), and after this time, maturation proceeds, albeit delayed. skeletal muscle regeneration is a complex process that is heavily dependent, particularly during the early phase, on the optimal functioning of the fibrinolytic system (2125). we speculated that impairments in type 1 diabetes muscle regeneration may be due, at least in part, to elevated pai-1 preventing the activation of upa and its downstream effectors, the mmps (e.g., mmp9). consequently, suppression of the fibrinolytic process would result in attenuation of ecm remodeling, thus creating barriers for infiltration of immune cells and efficient activation and invasion of the myogenic stem cells that are responsible for the formation of new myofibers (14). we observed total pai-1 levels to be more than twofold higher in ins2 mice than in wt mice within 2 weeks of hyperglycemia in the former group, with values remaining elevated with diabetes throughout the experimental period (fig. 2a and b). moreover, regenerating ins2 muscle displayed elevations in collagen content at 5 and 10 days of regeneration compared with wt muscle (fig. active upa levels at 5 days of regeneration were decreased by 35% in ins2 muscle compared with wt muscle (wt : 5978 1362 vs. ins2 : 3873 1241 ; p = 0.15). although this decrease in active upa was not statistically significant, active mmp9, the mmp associated with ecm remodeling in skeletal muscle (39,40), was significantly elevated at 5 days of regeneration in wt but not ins2 muscle, with levels between the two groups similar by 10 days postinjury (fig. type 1 diabetes causes elevated pai-1, suppresses mmp9 activation, and increases collagen content during early regeneration time points. significantly elevated pai-1 levels in ins2 mice compared with wt mice were found in blood plasma collected (a) throughout type 1 diabetes progression (main effect : p < 0.001 ; n = 16) and (b) after ctx injury (main effect : p < 0.001 ; n = 16). this led to the hypothesis that collagen would be elevated in the ins2 mice because of suppression of the fibrinolytic pathway. c : picrosirius red staining of injured ta sections revealed increased collagen (red color), which was statistically significant (d, main effect : p = 0.001, interaction : p = 0.004 ; n = 16). e : mmp9, an important protease in skeletal muscle collagen cleavage, was also found to be significantly repressed in ins2 mice (labeled ins2, e) at the 5-day time point (n = 8). f : the short - term ins2 mice also exhibited increased collagen at 10 days postinjury (t test : p = 0.047, n = 3). differences between groups at specific time points identified by bonferroni post hoc analysis after 2-way anova (a, b, d, e). a e : white bars / circles represent wt, and black bars / squares represent ins2. (a high - quality digital representation of this figure is available in the online issue.) with pai-1 elevated within 2 weeks of type 1 diabetes onset (fig. 2a), we hypothesized that the deficit in regeneration observed in ins2 mice diabetic for 1 week before ctx injury would also display defective ecm remodeling. as hypothesized, collagen content in regenerating ins2 mice, diabetic for a total of 17 days, was significantly elevated (fig. 2f), consistent with a role of pai-1 in the impaired regeneration. by having identified that increased pai-1 levels in ins2 mice are associated with early impairments in muscle regeneration, we then determined if these deficits could be restored with pharmacologic inhibition of pai-1, even in the absence of insulin therapy. twice - daily oral dosing of pai-039 (tiplaxtinin), a pharmacologic inhibitor of pai-1 (29), effectively increased the amount of active (free) upa in the injured muscle of 8-week diabetic ins2 mice compared with untreated ins2 mice (fig. 3a) and increased the ratio of active to inactive upa (upa / pai-1-upa) (ins2 : 1.22 0.13 au vs. ins2+pai-039 : 1.74 0.14 au ; p = 0.02). wt mice treated with vehicle alone demonstrated no significant change in active upa levels (wt : 5978 1362 au vs. wt + vehicle : 7007 778 au ; p = 0.27) or upa / pai-1-upa (wt : 1.40 0.18 au vs. wt + vehicle : 1.28 0.09 au ; p = 0.29). the downstream effect of elevated active upa levels, resultant from pai-039 treatment, was an increase in active mmp9 (fig. 3b) and a normalization of collagen content in the regenerating ins2 muscles to the levels observed in wt mice (fig. 3c and d). the recovery of the fibrinolytic pathway with pai-039 in ins2 mice restored not only normal ecm remodeling but also myh3 expression to levels similar to wt regenerating muscles (fig. injured ta fiber area demonstrated no significant difference between groups (wt + vehicle : 461 29 m vs. ins2+pai-039 : 396 32 m ; p = 0.19), whereas ta mass exhibited a small but significant difference (wt + vehicle : 0.039 0.002 g vs. ins2+pai-039 : 0.031 0.002 g ; p < 0.05). similarly, no difference was noted in fiber area at 5 days postinjury between wt and ins2 mice (fig. 1e), with a small decrease in muscle mass at that time point (fig. the reasons underlying the apparent discrepancy between fiber area and muscle mass in the diabetic mice is unknown ; however, it could be speculated that differences in fibrosis, inflammatory response, or lipid content within the muscles of the various groups could contribute to these observations. pharmacologic treatment against pai-1 improves fibrinolytic pathway activity, collagen degradation, and regeneration at 5 days post - ctx injury in type 1 diabetes. a : treatment with pai-039 caused an increase in free upa in ins2 compared with untreated ins2 (t test : p = 0.004 ; n = 4). b : similarly, active mmp9 was elevated in pai-039treated ins2 (t test : p = 0.025 ; n = 4). these findings are characteristic of restored fibrinolytic pathway activity, which presumably led to the (c and d) reduced collagen levels (t test : p < 0.001 ; n = 4) and (e and f) increased myh3-positive area (t test : p = 0.011 ; n = 4) observed in pai-039treated ins2 compared with untreated ins2 (labeled ins2, c and e). data are presented relative to the mean of the wt and wt + vehicle pooled data. (a high - quality digital representation of this figure is available in the online issue.) to rule out the possibility that pai-039 treatment improves glycemic or insulinemic levels, thus improving the diabetic environment in ways other than affecting pai-1 activity, whole - blood glucose and plasma insulin levels were measured. blood glucose concentrations remained severely elevated in the ins2 mice treated with pai-039 (wt + vehicle : 8.9 mmol / l ; p < 0.05), whereas insulin levels remained low (wt + vehicle : 916 74 pg / ml vs. ins2+pai-039 : 146 29 pg / ml ; p < 0.05). our results indicate that the type 1 diabetic environment negatively affects the health of skeletal muscle, as defined by impaired growth and poor regenerative capacity. the deficits in regenerative capacity occur rapidly with exposure to type 1 diabetes (within 2 weeks) and, as we demonstrated, are consistent with elevated pai-1 and ineffective ecm remodeling. maintaining a healthy muscle mass in the type 1 diabetic population unfortunately, many studies demonstrate impairments in skeletal muscle health (e.g., impaired morphology, decreased strength, and metabolic capacity) observed early in patients with type 1 diabetes who are receiving insulin therapy, changes that may precede other diabetes complications (13). the results presented support these previous findings as we demonstrate that repair from muscle damage is significantly blunted in the diabetic state with as little as 7 days of uncontrolled type 1 diabetes before muscle injury. furthermore, we also demonstrate that pai-1 is significantly elevated within the first 2 weeks of type 1 diabetes onset and that inhibition of this hormone restores the regenerative capacity of type 1 diabetic mice, irrespective of the hypoinsulinemia. although skeletal muscle is capable of maintaining basic function in the face of extreme stressors, this does not equate to a healthy muscle mass that is functioning optimally. we and others have demonstrated that although basic indices of muscle function may not be significantly impaired, dramatic changes are occurring within the muscle demonstrating compromised health (11,12,41). if we heed lessons from other metabolic disease states (e.g., obesity), as muscle health diminishes, disease severity increases. for example, the muscle wasting that occurs with obesity (sarcopenic obesity) is a serious complication resulting in the expedition of complications within other tissues (42). given the importance of skeletal muscle to whole - body fuel metabolism, ensuring that skeletal muscle health is maintained in metabolic disease states is obviously of critical importance. type 1 diabetes onset most often occurs during childhood / adolescence, and previous studies have shown that atrophic stimuli (e.g., hindlimb casting) placed on young, growing muscle result in a rapid and irreversible remodeling process, ultimately leading to a failure to achieve its full potential of adult muscle mass (13). we were interested to determine if type 1 diabetes may prove to be one of these atrophic environments. the present findings illustrate that growing skeletal muscle exposed to type 1 diabetes will display a failure to accrue muscle mass / fiber area, and these findings are not the product of progressive atrophy resultant from prolonged type 1 diabetes exposure or neuropathic complications, because no change in muscle mass or fiber area was observed once into adulthood (a further 5 weeks of uncontrolled type 1 diabetes). although we investigated the uncontrolled diabetic state in a rodent model of diabetes, it is worth considering that there are two situations in which pediatric type 1 diabetic populations may be under this stress : 1) before diagnosis (which may last a period of months) and 2) after diagnosis when glycemic control is difficult and suboptimal (43). consistent with our results, findings in newly diagnosed juvenile type 1 diabetic humans demonstrate a reduced myofiber area compared with healthy age - matched control subjects (10). repair from muscle damage consists of multiple, overlapping stages (14). after the initial injury, an inflammatory phase ensues to remove damaged cells and debris. it is during this early phase that remodeling of the ecm begins, with a dramatic increase in ecm proteins, particularly collagen, which is needed as the structural integrity of the muscle is compromised while damaged muscle fibers undergo phagocytosis. as repair continues, so does ecm remodeling, with the excess of ecm proteins undergoing degradation as nascent myofibers form and mature. activity of the fibrinolytic system (pai-1, upa, mmps) is critical during this ecm remodeling period (2125). newly formed myofibers will initially express immature myosin heavy chain isoforms, such as myh3 (embryonic myosin heavy chain). as maturation continues, the muscle fibers will increase in size, returning to a preinjury state while replacing the immature contractile proteins with mature isoforms. we show in this study that in response to muscle damage, ecm remodeling is impaired in the type 1 diabetic state, and this is a direct result of elevated pai-1. the increase in pai-1 observed in regenerating muscle of type 1 diabetic mice decreased active upa and mmp9 levels, thereby attenuating ecm turnover (as noted by elevated intramuscular collagen) during the first 10 days postinjury. restoration of the fibrinolytic system in type 1 diabetic mice via pharmacologic inhibition of pai-1 restored active mmp9 expression, returned collagen levels to normative values, and ultimately allowed for nascent muscle fiber growth to occur. consistent with these findings, mice deficient in upa exhibit impaired muscle regeneration, whereas pai-1deficient mice exhibit augmented muscle repair (22,44). it is worth noting that, in the current study, pai-039 treatment in ins2 mice resulted in active upa levels that significantly exceeded that measured in wt mice muscles. although this may be considered supraphysiologic, tissues were collected 1 h after pai-039 administration on the day of harvest, a time when the drug effects were at their peak. with a pai-039 half - life of 4.1 h (29), it can be speculated that more time was spent below this elevated level than within it. although the evidence to date suggests that insulin administration does not restore function of the fibrinolytic pathway (because insulin therapy does not reduce pai-1 levels in human pediatric populations) (4), and thus would not improve collagen degradation and de novo myofiber formation, insulin might facilitate the rate of myofiber growth (later stages of regeneration) by modestly improving protein turnover (58). future studies are needed to clearly define if pai-039 treatment, in combination with standard insulin therapy, provides a more optimal regeneration environment. furthermore, we administered pai-039 just before the injury ; however, future studies should consider the clinically important issue of treatment after injury, in terms of both effectiveness and timeframe. a final clinical consideration of pai-039 treatment is the long - term effects of its administration if it were to be given prophylactically or for prolonged periods of time. to date one study administered pai-039 for 42 days through addition to the rodent chow and found an acute protection against radiation - induced intestinal injury but noted no adverse effects of drug treatment (45), whereas a second study provided a 2-month administration of pai-039 to angii / salt treated mice (46). in the latter study, pai-039 was effective in decreasing aortic remodeling with no effect of pai-039 alone on serum amyloid a levels (an index of systemic inflammation). although elevated pai-1 has been linked to other complications usually associated with diabetes, such as coronary artery disease and nephropathy (1820), it is somewhat surprising that this is the first time that a clinically relevant inhibition of pai-1 (through pharmacologic means) has been used to treat a diabetes complication. in fact, to the best of our knowledge, only three other studies have investigated mitigating diabetes complications by altering pai-1 levels. these studies used streptozotocin - induced diabetes in pai-1deficient rodents to investigate the role of pai-1 in mediating the effects of type 1 diabetes on renal morphology and function (18,47,48). collectively, these authors found that elevated pai-1 was contributing to diabetic nephropathy (increased glomerular ecm, decreased glomerular filtration rate) and that amelioration of these symptoms occurred in the pai-1deficient background. given the demonstrated (present study) (18,47,48) and proposed (19,20) linkage of pai-1 with diabetes complications and the fact that pai-1 is elevated in populations with pediatric type 1 diabetes regardless of the level of glycemic control (4,49,50), we propose that aggressive therapeutic approaches, including intensive insulin and pai-1 inhibitor strategies, warrant further investigation for the treatment of young type 1 diabetic patients. this will not only ensure optimal accrual and maintenance of a healthy skeletal muscle mass but also will reduce the onset and progression of other diabetes complications. clearly, future studies are needed to definitively demonstrate the causative role of pai-1 in the impaired muscle regeneration of patients with diabetes, and these studies may also prove valuable in developing therapeutic strategies to ensure the most effective management of other diabetes complications.	objectivetype 1 diabetes leads to impairments in growth, function, and regenerative capacity of skeletal muscle ; however, the underlying mechanisms have not been clearly defined.research design and methodswith the use of ins2wt / c96y mice (model of adolescent - onset type 1 diabetes), muscle regeneration was characterized in terms of muscle mass, myofiber size (cross - sectional area), and protein expression. blood plasma was analyzed for glucose, nonesterified fatty acids, insulin, and plasminogen activator inhibitor-1 (pai-1). pai-039, an effective inhibitor of pai-1, was orally administered to determine if pai-1 was attenuating muscle regeneration in ins2wt / c96y mice.resultsins2wt/c96y mice exposed to 1 or 8 weeks of untreated type 1 diabetes before chemically induced muscle injury display significant impairments in their regenerative capacity as demonstrated by decreased muscle mass, myofiber cross - sectional area, myogenin, and myh3 expression. pai-1, a physiologic inhibitor of the fibrinolytic system and primary contributor to other diabetes complications, was more than twofold increased within 2 weeks of diabetes onset and remained elevated throughout the experimental period. consistent with increased circulating pai-1, regenerating muscles of diabetic mice exhibited excessive collagen levels at 5 and 10 days postinjury with concomitant decreases in active urokinase plasminogen activator and matrix metalloproteinase-9. pharmacologic inhibition of pai-1 with orally administered pai-039 rescued the early regenerative impairments in noninsulin - treated ins2wt / c96y mice.conclusionstaken together, these data illustrate that the pharmacologic inhibition of elevated pai-1 restores the early impairments in skeletal muscle repair observed in type 1 diabetes and suggests that early interventional studies targeting pai-1 may be warranted to ensure optimal growth and repair in adolescent diabetic skeletal muscle.
the online version of this article (doi:10.1007/s11605 - 009 - 1079 - 0) contains supplementary material, which is available to authorized users. laparoscopic cholecystectomy is currently the gold standard for the treatment of symptomatic gallstones.1,2 besides the numerous clinical advantages of laparoscopy over an open approach, recent surveys have demonstrated that patients clearly favor cosmetically superior approaches to the abdominal cavity.3 less invasive and cosmetically superior approaches such as single incision laparoscopy (sil) and natural orifice translumenal endoscopic surgery (notes) have been gaining popularity. while pure notes will result in cosmetically ideal outcomes, this method is still under development and is currently only performed at specialized centers.4,5 while we at ucsd have been actively involved in leading the surgical community towards the safe and proficient development of natural orifice techniques, we realize that there is a need to investigate all approaches which may improve the outcomes of surgical patients. within the past year, single incision laparoscopic surgery has been described for a great variety of procedures with rapidly growing numbers.612 single incision laparoscopic surgery is performed with slightly modified but still conventional rigid laparoscopic instruments, and results in less scaring when compared to conventional laparoscopy. still, single incision laparoscopic surgery requires an abdominal wall incision, usually in the umbilical area, and can lead to all the common problems of conventional laparoscopy particularly pain and hernia formation.1 because of the increased size of the umbilical incision during single incision laparoscopy, the possibility of a growing rate of incisional hernias may be anticipated. additionally, placing a 15 mm or larger incision in the umbilical area might lead to an umbilical deformation depending on technique, original shape, size, and other factors. finally, many publications in the plastic surgery literature indicate the importance of the integrity of the umbilicus for the overall physical appearance.1315 supra - pubic single incision surgery is a novel method for minimal invasive access procedures that preserves the native umbilicus, results in a more discrete scar, and has the potential to reduce the incidence of postoperative hernias. extensive experience with single incision laparoscopic surgery prior to the first human case was gathered in the porcine model and human cadavers using different methods for access (trocars, open approach, and rigid and flexible instrumentation). the initial patient was listed under the institute s institutional review board protocol for single incision laparoscopy. possible advantages and complications of this new investigational method, and the possibility of conversion to conventional (laparoscopic or open) surgery were discussed with the patient and appropriate informed consent was given. a subcutaneous tunnel was formed in an open fashion in a cephalad direction of about 5 cm in length and the anterior rectus sheath visualized. next a 5-mm optiview optical bladeless trocar (endopath xcel, ethicon endo - surgery inc., cincinnati, oh, usa) with an introduced utilizing a 0-degree, 5-mm camera was guided through the subcutaneous tunnel followed by a stepwise passage through the abdominal wall under constant direct visualization. next, two additional 5-mm low profile trocars (ethicon endo - surgery inc., cincinnati, oh, usa), were inserted in a similar fashion, one on each side of the first trocar (fig. 1). a conventional 5-mm rigid laparoscopic grasper was inserted through the left low profile trocar to retract the gallbladder at the fundus. dissection of calot s triangle was achieved with a harmonic scalpel (ethicon endo - surgery inc., flexible laparoscopic instruments were used for dissection when necessary (real hand, novare systems inc., cupertino, ca, usa). after obtaining a clear critical view, the cystic duct was secured using two distal hem - o - lock clips (weck closure systems, research triangle park, nc, usa) and one proximal titanium clip (ethicon endo - surgery inc., cincinnati, oh, usa). then, the gallbladder was dissected free of the liver bed using alternating retraction from the left - sided grasper and the harmonic scalpel. an endo - loop was then introduced through the right trocar and placed around the infundibulum of the gallbladder. all trocars were removed and the three adjacent trocar incisions were joined to facilitate removal of the gallbladder. the abdominal wall and the subcutaneous tunnel were closed in layers under direct visualization with interrupted 2 - 0 vicryl sutures. the skin incision was closed in a subcuticular fashion (fig. 2). the patient was a 30-year - old female (bmi = 24) with symptomatic uncomplicated gallstone disease and no previous abdominal surgery. gallbladder retraction was sufficient, and could provide adequate exposure by alternating the grasping site between the infundibulum and fundus of the gallbladder as needed. visualization was as good as conventional laparoscopic view, and all critical structures were visualized without difficulty. evaluation 3 weeks after the operation showed an uncomplicated course and a clean scar within the re - growing pubic hair (fig. we describe a new surgical technique for single incision laparoscopic surgery applied in a patient. supra - pubic single incision cholecystectomy was feasible in this case using a combination of conventional rigid and articulated laparoscopic instruments. this new method delivers a linear scar that can be hidden within the supra - pubic hairline and might therefore appear cosmetically superior when compared to scars after conventional laparoscopy and single incision transumbilical laparoscopy. in addition, the rate of incisional hernias for port sites which avoid the midline is significantly smaller when compared with those placed in the midline.16 after skin incision access through the abdominal wall can be chosen independent of the location of the skin incision, by creating a subcutaneous tunnel. therefore, perforation through the rectal muscle instead of the linea alba can be performed easily in order to potentially avoid incisional hernias. a limitation of this technique seems to lie in the entrapment of trocars in the subcutaneous tunnel and the entry into the abdominal cavity. parallelism of trocars and limited range of motion of trocars lead to a loss of triangulation, a reduced field of surgery and intermittent conflict of instruments and camera on the outside. however, the use of articulated instruments was helpful to overcome some of these limitations by creating additional degrees of freedom. still, the access to the abdominal cavity seems to represent the most critical challenge during this new method. inserting a trocar through a preformed tunnel to enter the abdominal cavity in an angle smaller than 90 is more difficult than entering in the usual vertical fashion. incidental injuries might occur easier despite the visual control obtained by the camera inside the port. additionally, while using optical trocars for laparoscopic access has been reported in great numbers in the upper abdomen1719, only few reports can be found about their use in the lower abdomen and safety in that anatomical region might be reduced.19 despite this uncertainty, a foley catheter should be positioned during any supra - pubic access case to minimize the risks of any inadvertent bladder injuries. also, flexible endoscopes might facilitate the passage through the subcutaneous tunnel and could be applied especially for longer and/or curved tunnels or be used in combination with laparoscopic instruments. in that sense, multiple tunnels can be placed to enter the abdominal cavity through a single skin incision. different types of instruments could potentially be inserted to cover various aspects of abdominal surgery such as retraction, vision, dissection, and assistance. in that sense, this initial case only represents the feasible variation of a new surgical method and further research will be conducted to advance this technique. this new technique potentially offers less visible scaring (concealable in pubic hair) while maintaining an access through a single incision at a stable part the abdominal wall in order to reduce the risks for incisional hernias. whether this approach turns out to be superior to conventional laparoscopy, single incision laparoscopy and notes regarding clinical parameters remains subject to more substantial research.	introductionsurgery is moving towards less invasive and cosmetically superior approaches such as single incision laparoscopy (sil). while trans - umbilical sil is gaining popularity, incisions may lead to post - operative deformations of the umbilicus and the possibility of an increased rate of incisional hernias. access within the pubic hairline allows preservation of the umbilicus and results in a scar which is concealed within the pubic hair.methodssupra-pubic single incision cholecystectomy was performed in a 30-year - old patient with symptomatic gallstones. a 2.5-cm transverse incision was placed within the pubic hairline and a subcutaneous tunnel was formed. three 5-mm ports were introduced into the tunnel and perforated the anterior rectus sheath superior to the skin incision. the surgical procedure was then undertaken with conventional laparoscopic instrumentation. the adjacent 5-mm incisions were merged for gallbladder removal. the entry site was closed under direct vision.resultsthe above procedure was technically feasible and without complication. operative time was 45 min, and the patient was discharged 5 h post-operatively.conclusionssupra-pubic single incision laparoscopic cholecystectomy may offer a more cosmetically appealing result than standard umbilical access. the operation can be performed by surgeons skilled in single incision techniques with good result.electronic supplementary materialthe online version of this article (doi:10.1007/s11605 - 009 - 1079 - 0) contains supplementary material, which is available to authorized users.
juvenile idiopathic arthritis (jia) is the most common chronic arthritis in children worldwide. it is a heterogeneous inflammatory disease and defined as arthritis persisting 6 weeks or longer with onset before the age of 16 years with no identifiable etiology. the term juvenile idiopathic arthritis has replaced the older terms juvenile rheumatoid arthritis (jra) and juvenile chronic arthritis (jca) proposed by the international league of associations for rheumatology (ilar) in the late 1990s. the ilar classified jia according to the pattern of the arthritis in the first 6 months after onset of the disease into 8 subtypes known as persistent oligoarticular, extended oligoarticular, polyarticular rheumatoid factor- (rf-) negative, polyarticular rf - positive, enthesitis - related arthritis (era), psoriatic arthritis, systemic, and undifferentiated arthritis. the hallmark of jia is joint inflammation and the presence of synovitis which causes synovial tissue thickening and accumulation of synovial fluid. this is manifested clinically as joint swelling, morning stiffness, joint pain, tenderness, and functional disability. joint involvement can be mild and self - limiting and it can be more severe causing joint destruction, severe disability, and loss of joint function. these include uveitis, fever, skin rash, hepatomegaly, splenomegaly, lymphadenopathy, and serositis. there are no diagnostic laboratory investigations for jia but some laboratory findings can help to exclude other causes of arthritis and help in jia classification. aim of jia management is to control active symptoms and to prevent chronic complications in order to improve quality of life which requires comprehensive multidisciplinary team care including rheumatologists, rehabilitation specialists, occupational therapists, physical therapists, social workers, nurses, podiatrists, dieticians, psychologists or psychiatrists, and orthopedic surgeons. there is no curative treatment for jia but early and aggressive management has led to prolonged remission and improved outcome. various studies from different parts of the world showed differences in jia characteristics including incidence, prevalence, age of onset, gender, and frequency of jia subtypes [27 ]. there is paucity of published studies describing jia and its characteristics in saudi arabia and its neighboring countries. most of these studies were hospital - based [814 ]. in this present study, we retrospectively reviewed medical records of a cohort of 82 jia children who are followed up in pediatric rheumatology department at king abdulaziz university hospital (kauh) ; our objective is to present the main characteristics of jia in children followed up in our center to know further about the pattern of the disease in saudi arabia and to compare these characteristics with those from other parts of the world. this is an observational retrospective study carried out at pediatric rheumatology department in king abdulaziz university hospital (kauh), a tertiary center, jeddah, saudi arabia. our study was approved by local ethical committee of pediatric department and by the university research ethics committee. all the medical records of the children who were diagnosed to have juvenile idiopathic arthritis (arthritis in one or more joints lasting 2 weeks or more with no identifiable cause in those who are less than 16 years of age) from january 2007 to january 2015 were included. the diagnosis of jia was done by a pediatric rheumatologist and jia classification was according to ilar classification criteria (table 1). only children who had duration of follow - up of 6 months or more were included in order to know more details about jia characteristics. data were collected about number of patients of each jia subtype, gender, age at disease onset, and duration of follow - up. the percentage of each jia subtype was calculated and the age of disease onset and duration of follow - up were expressed as the mean standard deviation (sd) ; these data are presented in table 2 and figure 1. main clinical data were collected about large joints involvement (knee, ankle, elbow, shoulder, and wrist) or small joints involvement and presence of uveitis during the course of the disease. the presence of fever, skin rash, hepatosplenomegaly, and lymphadenopathy at diagnosis was also recorded. these clinical findings were diagnosed by a pediatric rheumatologist while uveitis was diagnosed by ophthalmologist by slit lamp examination. initial data at diagnosis included white blood cells (leukocytosis defined as wbc > 11 10/l), hemoglobin level (anemia defined as hb 450 10/l), esr > 20 mm / hr, elevated crp > 3 mg / l and positivity of anti - nuclear antibodies (ana), rheumatoid factor (rf), and hlab27. ana was done by elisa technique and ana titre of 1:80 or more was considered positive. positivity of ana and rf is considered only if two samples were positive in at least three months apart. review of anti - rheumatic pharmacologic treatments used during the study period included (nonsteroidal anti - inflammatory drugs (nsaids), intra - articular corticosteroids (iac) or systemic corticosteroids, methotrexate (mtx), and biologic agents) was done. number and percentage of those who were treated with nsaids alone, nsaids and iac, nsaids, iac and mtx, nsaids and systemic steroids, nsaids and mtx, nsaids, mtx and systemic steroids, nsaids, mtx and biologics, nsaids, mtx, and biologics and iac and those who had joint surgery in each jia subtype were calculated and presented in table 5. other disease - modifying antirheumatic drugs (dmards) like leflunomide, cyclosporine, and azathioprine were infrequently used and they were not included. total patients reviewed in this study were 85 ; 3 patients were excluded because of insufficient data due to irregular follow - up (2 oligoarticular jia, 1 polyarticular jia). table 2 shows general characteristics of the patients : systemic onset jia was the most common jia subtype 36.5% (30 patients) followed by polyarticular jia subtype 29.26% (24 patients) and then oligoarticular jia subtype 28% (23 patients). no unclassified patients jia were diagnosed. of our 82 patients, 51 (62.2%) were females and female gender was predominant in all jia subtypes except in era. female - to - male ratio was 1.64 : 1 (51/31). the range of age at onset of symptoms was between 8 months and 14.5 years and the mean age at jia onset was 7.11 3.65 years. the duration of follow - up was from 6 months to 8 years ; mean was 2.67 1.68 years. table 3 shows main clinical features of our patients ; large joints were affected in most of patients 92.68% (76/82 patients) while small joints were affected in 36.6% (30/82 patients). fever was present in all systemic onset jia subtype and in 41.46% of the total number (34/82 patients). uveitis was diagnosed during the course of the disease in 7 patients (8.53%) and in 5 patients (21.7%) of oligoarticular jia subtype. the most common finding at presentation was anemia in 49/82 patients (59.75%), followed by elevated esr in 45/82 patients (54.87%) and then elevated crp and leukocytosis in 33/82 patients (40.24%), while thrombocytosis was found in 31/82 patients (37.8%). anemia, leukocytosis, thrombocytosis, elevated esr, and crp were present mostly in sojia. positive ana was present in 30/82 patients (36.58%) and mostly in oligoarticular jia 12/23 patients (52.17%) and polyarticular jia 11/24 patients (45.83%). table 5 shows the antirheumatic pharmacologic treatment received by our patients during the course of the disease. nsaids as the only treating agent were used in 9/82 patients (10.97%) while nsaids, methotrexate, systemic steroids, and biologics were received by 19/82 patients (23.17%) at different and varying length of time. biologic agents were given to 31/82 patients (37.8%) ; of these patients, 15 belonged to sojia. biologic agents were mainly in decreasing order of frequency : etanercept (enbrel), adalimumab (humira), abatacept (orencia), rituximab (mabthera), and tocilizumab (actemra). joint surgery, other than intra - articular corticosteroid injection, was needed in 3 patients only : one case of oligoarticular jia, rf - negative polyarticular jia, and rf - positive polyarticular jia. this observational study was carried out in pediatric department at king abdulaziz university hospital, which is a tertiary care hospital in jeddah, western coast of saudi arabia. we aimed at this study to present the clinical features of children with jia followed up in our center as knowing the characteristics of this disease in our community is essential to provide a better planning for medical care. to our best of knowledge, there is only one hospital - based similar study describing the pattern of jia in saudi children which was published in 1997 in which arthritis classification was not according to the currently used ilar classification system for jia. the prevalence of jia is variable among different parts of world and different ethnic groups [2, 15 ]. unlike most of previous similar studies where oligoarticular jia was found to be the most common, mainly in europe, usa, canada, south america, and turkey [3, 1618 ], the frequency of jia subtypes in our cohort showed that systemic onset jia was the most common subtype : 30/82 (36.5%). this is similar to a previous tertiary hospital - based study done in central province of saudi arabia which reviewed clinical features of 115 patients with jra in which the percentage of sojia was found to constitute 44% of juvenile rheumatoid arthritis cases. in a large population - based study in japan which included 540 children, sojia constituted 54% of jia cases. our study differs also from two studies from neighboring countries (kuwait and oman) in which the most predominant jia subtype was polyarticular jia [11, 12 ]. polyarticular jia was also the predominant jia subtype in some other cohorts from south africa, bangladesh, and pakistan and in a multinational study done by the paediatric rheumatology international trials organization (printo) which included patients from western and eastern europe and latin america [4, 1921 ]. era was found to be the predominant in other studies [6, 22 ]. since our study was done in a tertiary center, we believe that this may affect our results as some cases of mild jia especially oligoarticular may be treated by general pediatricians or orthopedic doctors and may not be referred to a higher center. we believe also that a single center study may not reflect the correct distribution of jia subtypes in our country which needs multicenter- or a population - based study. it is well known that females are generally more commonly affected than males by jia with variable gender distribution among jia subtypes. apart from the only male child with era, female number was more in all jia subtypes. few studies showed an overall equal or higher male - to - female ratio [4, 6, 15, 25, 26 ] ; this higher male percentage was possibly related to a higher era in one series [3, 25 ]. the mean age of onset of jia symptoms in our patients was 7.11 3.65 years (range : 8 months14.5 years), which is not largely different from most studies [9, 14, 25, 27, 28 ]. however it is higher than the mean age in a large study which included 3167 patients from europe and latin america where mean age of onset of jia symptoms was 5.8 years but lower than a mean age of 9.5 years recorded in a cohort from taiwan of 195 patients. it is well known that jia involves large joints except hip more often than small joints ; our data showed that 92.68% (76/82) had large joint involvement either alone or associated with small joints. fever in sojia is considered one of the diagnostic features according to ilar criteria and it may be the main presenting symptom. as reported in other studies, all our sojia patients had fever [8, 2931 ]. among all our jia children, fever was present in 34/82 (41.46%) patients which is higher than other similar studies [3, 29 ]. which is probably due to the predominance of sojia in our cohort however a higher percent (67.6%) of fever in jra was reported in one series. uveitis is a well - known extra - articular manifestation that affects children with jia and it occurs more commonly in oligoarticular subtype. it may start before the onset arthritis or later during the course of the disease. among all our jia patients 7/82 (8.53%) the overall rate is approximately close to what was reported in spain and taiwan. however there is significant variability in frequency of uveitis in jia ; in a series of 214 jia children in india, only 2.2% were found to have uveitis while a rate of 29.65% was reported in another series of 172 patients. anemia was the most common abnormal laboratory investigation in our patients 49/82 (59.75%) and it was recorded in 80% of sojia patients. anemia was considered as hemoglobin level less than 110 g / l which is according to who definition of anemia a 2sd below the mean for the majority of our patients [35, 36 ]. the prevalence of anemia in our children may be increased as most of cases were sojia in which the anemia is more common than other jia subtypes. furthermore, at presentation, it was not known if this anemia was due to jia alone or due to other common causes as the frequency of anemia in children in our population was 22.3% and 30% in two local studies [37, 38 ]. however, although in other studies the cutoff point for low hemoglobin was lower, there were no major differences in the frequency of anemia [3, 10, 14 ]. being slightly higher than a local study (115 patients) and studies from uk (572 patients), turkey (634 patients), and taiwan (195 patients), the frequency of ana positivity in our children was 36.58% [3, 9, 22, 39 ] although higher and more lower frequencies were reported [4, 39 ]. the aim of multidisciplinary jia management is to control active symptoms, achieve remission, prevent joint damage, and preserve joints function to prevent disability as well as maintaining normal growth which may require early and aggressive management. pharmacologic therapy of jia has major advances over the last two decades since the introduction of biologic agents which are being increasingly used and these agents may be used as initial therapy. we usually start with nsaids for 4 to 6 weeks followed by dmards, most commonly methotrexate in case of no adequate response to nsaids. iac are used to relieve joint inflammation and systemic steroids are usually used for a short time with the lowest effective dose and are tapered once we get the desired response. in case of failure of methotrexate, a trial of another dmard or biologic therapy is introduced. in our study, all patients were started on nsaids either alone or combined with other antirheumatic agents but they were the only used agent in 9/82 patients (11%). jia was found to be the most common indication for biologic treatment among pediatric rheumatologic conditions in a national study. as shown in table 5, 31/82 patients (37.8%) received biologic treatment during their course of the diseases. a major limitation of our study is being a retrospective record - based in nature and a single center - based with a relatively small sample size. however our study can be a starting point for further future nationwide multicenter - based study. although a population - based rather than a single center study will give more details about jia characteristics in saudi arabia, our study can be the start point to a nationwide multicenter study.	introduction. juvenile idiopathic arthritis (jia) is the most common chronic arthritis in children. our aim is to describe demographic, clinical, and laboratory characteristics and treatment of jia patients followed up in pediatric rheumatology clinic in a tertiary center in saudi arabia. methods. medical records of all patients who are followed up between january 2007 and january 2015 were retrospectively reviewed. data were collected about demographic, clinical, and laboratory features and treatment. results. total patients were 82, males were 31 (37.8%), and mean age of jia onset was 7.1 3.6 yr. mean follow - up duration was 2.671.6 yr. systemic onset jia (sojia) was the commonest (36.5%), followed by polyarticular in 29.2% and oligoarticular in 28%. large and small joints are involved in 76 (92%) and 30 (36.6%), respectively. main extra - articular feature was fever in 34 (41.4%). uveitis was diagnosed in 7 (8.5%) and in 5 (21.7%) of oligoarticular jia. anemia was found in 49 (59.7%), high esr in 45 (54.8%), and leukocytosis and thrombocytosis in 33 (40.2%). positive ana was found in 30 (36.5%) mainly in oligoarticular subtype as 12 (52%) patients (out of 23) had this positive test. 9 patients (10.9%) required nsaids only, 6 patients (7.3%) required nsaids and intra - articular steroids only, and 19 (23%) required nsaids, methotrexate, steroids, and biologics. conclusion. sojia is the most common jia subtype in our study. a population based rather than a single center study will give more details about jia characteristics in saudi arabia
the conjugation length (coherence size) in materials and molecules has attracted much attention for a long time.(1) from the material point of view, the exciton radius in bulk system, which corresponds to the coherence size, is shortened by reducing the system size. the conjugation length is in parallel with the system size for a small molecule but is saturated to one value with increasing the system size.(2) the saturation of the conjugation length affects the various physical properties. for example, a red shift in absorption peak is typically observed with increasing the system size.(3) unlike the usual red shift, we previously found a system that showed a blue shift with increasing system size.(4) the system is a derivative of diarylethene (dae). one of the reported examples of the blue shift in absorption spectra with increasing the system size was found in h - aggregate systems.(6) in that case, a large transition dipole appears at the highest energy of the split excited states and transition dipoles of other low - lying excited states disappear due to the cancellation of the transition dipole of each unit. this is due to the special molecular arrangement ; i.e., the transition dipole of each unit is perpendicular to the direction of the increase of the system. in the derivative of diarylethene thus, the blue shift observed in our system is not interpreted by the mechanism found in h - aggregate. it is one of the promising candidates for a molecular switch due to its thermal stability of two isomers, i.e., open - ring isomer 1a and closed - ring isomer 1b (scheme 1) and their fatigue - resistance.(7) to use it as a molecular switch, however, it is crucial to understand the conjugation length of the derivatives. the series of molecules which showed the blue shift in the absorption spectra are two nitronyl nitoroxides (nn) attached closed - ring isomer of dae through bridge phenyl unit (2b, 3b, and 4b in charts 1). interestingly, the red shift was observed for the series of molecules 2b, 5b, and 6b (charts 1) where the thiophene unit instead of the phenyl unit was used as the bridge between dae and nn.(8) (this series is denoted as dae-(thio)-nn.) these dae-(phe)-nn and dae-(thio)-nn have very small singlettriplet energy gap. in contrast, as is easily expected for the system without nn, the red shift was observed for the series of molecules 1b, 7b, and 8b (charts 2),(4) which is denoted as dae-(phe). to understand the mechanism of the blue shift in the absorption spectra of two nitronyl nitroxides attached diaryl - ethenes with increasing the system size, we synthesized the molecules 9b and 10b (dae-(thio) shown in charts 2) and compared the effect of phenyl units to that of thiophene units on the absorption spectrum. we further performed calculations by density functional theory(9) (dft) and time dependent dft(10) (tddft) with the broken symmetry method(11) for singlet biradical ground and excited states. the obtained excitation energies reproduce the blue shift of the absorption spectrum of dae-(phe)-nn and the red shifts of the absorption spectrum of dae-(phe), dae-(thio), and dae-(thio)-nn. to understand those shifts in the absorption spectrum, we analyzed the orbital energies as well as the dependence on the dihedral angle between the dae and the phenyl (thiophene) unit. we found that the experimental absorption around 2.12.4 ev is primarily due to the transition between the highest occupied molecular orbital (homo) and the lowest unoccupied molecular orbital (lumo). the energy gaps between homo and lumo correlate with the excitation energies. by analyzing these energy gaps and orbitals, we attribute the blue shift in absorption spectrum to the suppression of a large -conjugation between dae and nn by inserting the phenyl unit. molecules 9a and 10a (scheme 2) were prepared from 1a,2-bis(6-iodo-2-methyl-1-benzothiophene-3-yl)hexafluorocyclopentene(4) using suzuki coupling with 2-thiopheneboronic acid or 2-(2,2-bithiophen-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane. h nmr spectra were recorded on a jeol jmn - a500 instrument and mass spectra were obtained by a thermofisher scientific exactive apci mass spectrometer. purification was carried out using a hplc (hitachi lc system lachrom) with a semipreparative column (wako wakosil 5sil 10 l250 mm). photoirradiation was carried out using a ushio 500 w super high - pressure mercury lamp by passing the light through a combination of band - pass filter (atg uv - d33s) and monochromator (ritsu mc-20 l). h nmr (tms, 500 mhz, cdcl3) : 1.54 (s, 3.9h, antiparallel), 2.21 (s, 2.1h, parallel), 7.07.7 (m, 10h), 7.82 (s, 0.7h, parallel), 7.91 (s, 1.3h, antiparallel). apci hrms (m / z) [m + h ] calcd for c31h19f6s4 : 633.0268. found : 633.0271. h nmr (tms, 500 mhz, cdcl3) : 1.56 (s, 3.7h, antiparallel), 2.25 (s, 2.3h, parallel), 7.07.7 (m, 14h), 7.84 (s, 0.8h, parallel), 7.93 (s, 1.2h, antiparallel). apci hrms (m / z) [m + h ] calcd for c39h23f6s6 : 797.0023. found : 797.0001. the ground state structures were optimized by restricted dft (for 1b and 7b10b) and unrestricted dft (udft) (for 2b6b) with the b3lyp(12) exchangecorrelation functional. the excited states were calculated by the tddft (for 1b and 7b10b) and unrestricted tddft (tdudft) (for 2b6b) with the b3lyp exchangecorrelation functional. throughout these calculations, the 6 - 31g(d) basis set was used and c2 symmetry was imposed. as we have previously examined, the imposed symmetry does not affect the results.(13) the nn is a stable radical. it was observed from epr that dae - nn (2b6b) has a very small singlettriplet energy gap. the open - shell singlet biradical state was obtained at the broken - symemtry(11) (bs-)udft level. the energy corrections by spin - projection for those results are unnecessary due to the small exchange interaction between two radical spins even for 2b (j = 0.5 10 ev), which was obtained by epr.(5a) to further confirm our conclusion, we performed additional calculations by two long - range corrected functionals, i.e., cam - b3lyp(14) and lc - blyp.(15) for the analysis of molecular orbital energies, we adopted kohnsham orbital energies as approximate molecular orbital energies. to compare the effect of phenyl units to that of thiophene units on the shift of absorption spectrum, the absorption spectra of 9 and 10 were obtained (figure 1). the new absorption peaks found in the photostationary state upon irradiation with uv light are due to the absorption of product of photoreaction, i.e., the closed - ring isomer 9b and 10b for figure 1a, b, respectively. the absorption peaks are at 559 nm (2.22 ev) and 578 nm (2.15 ev) for 9b and 10b, respectively. these results together with our previous experimental results for 1b8b are summarized in figure 2. both dae-(phe) and dae-(thio) showed red shifts, but the shift was much larger in dae-(thio). in both cases, the energy of the absorption peak seems to converge to one value with increasing the number of bridge unit. the experimental shifts were nicely reproduced by the computational excitation energies (figure 2), although all the excitation energies were underestimated. to confirm that these results were not affected by the deficiency of b3lyp exchangecorrelation functional, the long - range corrected functionals (cam - b3lyp and lc - blyp) were used to optimize the geometry and to obtain the excited states (figures s1 and s2, supporting information). those functionals significantly overestimated the excitation energies, however, the blue shifts were reproduced. (a) absorption spectra of 9 in ethyl acetate : open - ring isomer 9a (dotted line) and in the photostationary state upon irradiation with 313 nm light (solid line). (b) absorption spectra of 10 in ethyl acetate : open - ring isomer 10a (dotted line) and in the photostationary state upon irradiation with 365 nm light (solid line) the bridge units are (a) phenyl units and (b) thiophene units. solid lines with filled squares : experimental excitation energies for dae-(phe)-nn in (a) and dae-(thio)-nn in (b). long dashed lines with filled circles : experimental excitation energies for dae-(phe) in (a) and dae-(thio) in (b). dotted lines with open squares : computational excitation energies for dae-(phe)-nn in (a) and dae-(thio)-nn in (b). short dashed lines with open circles : computational excitation energies for dae-(phe) in (a) and dae-(thio) in (b). to understand the mechanism of the blue shift, we further calculated the potential energy surface by imposing a constraint on the dihedral angle between the dae and phenyl unit of the dae - phe - nn (figure 3) and between the dae and thiophene unit of the dae - thio - nn (figure 4). other degrees of freedom in the structures were fully optimized. due to the imposed c2 symmetry, thus, we need to note that the energy barriers (at around 90) between two local minima shown in figures 3 and 4 are about twice the actual energy barrier for the rotation of one dihedral angle between dae and phenyl (thiophene) unit. dihedral angle dependence of the ground state potential energy (open circles) for 3b (dae - phe - nn). dihedral angle dependence of the ground state potential energy (open circles) for 5b (dae - thio - nn). the dihedral angle between the dae and the bridge at the ground state depends on the bridge. the dihedral angle between dae and the phenyl (thiophene) unit at the ground state is 35 (22). the difference between the two dihedral angles is due to the steric hindrance between two hydrogen atoms. the same is the reason for the difference of the dihedral angle between two phenyl (thiophene) units. according to the reported crystal structure, the angle between two phenyl rings of biphenyl is around 10,(17) while two thiophene rings of 2,2-bithiophene are coplanar.(18) the -conjugation is thus less extended in dae-(phe)-nn than in dae-(thio)-nn. the larger dihedral angle between dae and phenyl unit suppresses the orbital mixing between dae and phenyl unit. actually, in the case without nn unit for both, the similar dihedral angle dependence is found in dae-(phe) and dae-(thio) ; larger red shift for dae-(thio) than for dae-(phe). the similar results were reported for many different systems.(19) however, the blue shift is found in the absorption spectra of dae-(phe)-nn, which can not be explained only by the dihedral angles. the kohnsham orbital energies near homo and lumo are shown in figures 5 and 6. the nn attached series is shown in figure 5 and the series without nn is shown in figure 6. even though unrestricted dft calculation was performed for the nn - attached series, due to the small exchange interaction energy between two spins localized on each nn, and orbital energies thus, only orbital energies are shown in figure 5 and closed shell kohnsham ones in figure 6. the somos are localized on the nn and the orbital energies are found near 5 ev, which are quite similar to the orbital energies of the homo. the somo is localized on the left nn as shown in figure 7c whereas the somo is symmetrically localized on the right the homo (figure 7d) and the lumo (figure 7b) are quite symmetric. since the somo orbitals are quite similar due to the localization on nn, only the homo and lumo are shown for the dae-(phe)-nn (figure 8) and the dae-(thio)-nn (figure 9). the value k = 0 at the center is for 2b (dae - nn). the left (right) hand side is for the number of phenyl (thiophene) units, i.e., from left to right : 4b, 3b, 2b, 5b, and 6b. each pair of thick solid lines is homo (lower line) and lumo (upper line) and is responsible for the lowest energy absorption peak. the orbital shown by the dashed line is somo and is localized on one of two nns. from left to right : 8b, 7b, 1b, 9b, and 10b. kohnsham spin orbitals for (a) lumo+1, (b) lumo, (c) somo, and (d) homo of 2b (dae - nn). kohnsham spin orbitals for (a) lumo and (b) homo of 3b (dae - phe - nn) and (c) lumo and (d) homo of 4b (dae-(phe)-nn). kohnsham spin orbitals for (a) lumo and (b) homo of 5b (dae - thio - nn) and (c) lumo and (d) homo of 6b (dae-(thio)-nn). the absorption around 2.12.4 ev is mainly due to the homolumo transition the decrease of the energy gaps, which correlates with the red shift of the absorption spectrum in the dae-(phe) and in the dae-(thio), is found in figure 6. the change of orbital energies with increasing number of phenyl or thiophene units in figure 6 is rather simple ; i.e., the homo energy increases and the lumo energy decreases. in contrast, the change of the homo and the lumo energies are rather complicated in figure 5, but the change of the energy gaps are correlated with the shifts of the absorption spectrum as well. the comparison between the homo (lumo) energies of dae (k = 0 in figure 5) and dae - nn (k = 0 in figure 6) reveals that the homo (lumo) energy of dae - nn is lifted (lowered) by the large orbital mixing between dae and nn in comparison with the homo (lumo) energy of dae. inserting the phenyl unit between dae and nn rather suppresses the -conjugation, which results in the increase of the energy gap in the dae-(phe)-nn and consequently the blue shift of the absorption spectra. as for the dae-(thio)-nn, therefore, the energy gap in the dae-(thio)-nn is reduced with the increasing number of thiophene units and we found the red shift of the absorption spectrum. the explanation of the blue shift of absorption spectra by the extent of the mixing of the kohnsham orbitals between dae and nn is further supported by figures 79. the homo and lumo of dae - nn extend over the dae and nn in figure 7. increasing the number of phenyl unit rather confines both homo and lumo near the dae (figure 8). in contrast, the homo of dae-(thio)-nn still extends over the dae and nn through thiophene unit (figure 9). the large orbital mixing between dae and nn found for the homo and the lumo of dae - nn is due to the following reasons. (i) the mixing takes place between the homo (lumo) of dae and the -homo (-lumo) of nn. the orbital energies of the -homo and the -lumo of nn are 6.20 and 0.16 ev, respectively. those energies are closely located to the orbital energies of the homo (5.47 ev) and lumo (2.72 ev) of dae. in comparison, the orbital energies of the homo and lumo of thiophene (benzene) are 6.33 (6.70) ev and 0.20 (0.10) ev, respectively. the small energy difference between the homo of dae and the -homo of nn induces a large orbital mixing between them. (ii) the dihedral angle between dae and nn is about 3, which is much smaller than those between dae and phenyl unit of dae - phe and between dae and thiophene unit in dae - thio. the small dihedral angle between dae and nn increases the mixing of the -orbitals between them. the extent of the mixing of the orbitals between dae and nn through the bridge is controlled by two factors. (i) as we have seen in figures 3 and 4, the dihedral angles between dae and the phenyl unit as well as between phenyl units are larger than those between dae and the thiophene unit and between thiophene units, which contributes to suppressing the mixing of the orbitals between dae and nn. (ii) in addition, the homo energy of benzene is further away from that of dae in comparison with that of thiophene. the larger orbital energy difference between dae and benzene than that between dae and thiophene contributes to suppressing the mixing of the orbitals between dae and nn. in terms of the orbital energy, the red shifts of absorption spectra in dae-(thio) this is because the difference between the homo energy of dae and that of thiophene is much smaller than the difference between the lumo energy of dae and that of thiophene. since the homo energy of benzene is lower than that of thiophene, the destabilization of the homo is smaller in dae-(phe) than in dae-(thio). the homo energy in dae-(phe)-nn does not change with increasing k but the lumo is destabilized, which causes the blue shift in the absorption spectrum. this is due to the fact that the homo is destabilized and the lumo is stabilized by attaching nn to dae due to the large orbital mixing between dae and nn. since the conjugation length is short for the phenyl group as the bridge, the orbital mixing between dae and nn is reduced by inserting the bridge. thus, the change of the orbital energy is determined by the trade - off between the reduction of orbital mixing between dae and nn and the additional orbital mixing between phenyl unit and dae. in dae-(thio)-nn s case, the reduction of orbital mixing between dae and nn is small and orbital mixing between thiophene and dae is large. consequently, the homo of dae-(thio)-nn is destabilized with increasing k. on the basis of these understandings, the shift in absorption spectrum in figure 2 is interpreted as follows. the shift of absorption spectrum in dae-(phe) is small (0.11 ev) and saturates around k = 2. this indicates that the conjugation length is rather short in dae-(phe) and saturates around k = 2. due to the large orbital mixing between dae and nn, the absorption peak of dae shifted toward red (0.18 ev) by attaching nn. due to the short conjugation length of dae-(phe), the effect of nn is reduced in dae-(phe)-nn with increasing k. consequently, around k = 2 where the conjugation length saturates, the absorption peak position of dae-(phe)-nn is similar to that of dae-(phe). in contrast, the shift of the absorption spectrum in dae-(thio) from k = 0 to k = 2 is larger than that from dae to dae - nn. moreover, the red shift of the absorption spectrum in dae-(thio) with increasing k does not reach the saturation at k = 2. this is consistent with the long conjugated length of oligothiophenes.(20) naturally, we observed the red shift of the absorption spectrum of dae-(thio)-nn with increasing k. one of the interesting issues about the biradical system is the effect of the quinoid structure. in our calculations, only near the nn attached bonds (about two bonds from the nn) is the effect of the quinoid - like bond lengths shown. the explanation of the blue shift in the absorption spectrum of dae-(phe)-nn with increasing k, however, does not require the quinoid effect as we have seen here. the blue shift of the absorption spectrum in dae-(phe)-nn with increasing k is explained by the orbital mixing ; the large -orbital mixing between dae and nn is rather suppressed by the bridge phenyl units. the dae-(thio)-nn showed the red shift of absorption spectrum because of the extended -conjugation in dae-(thio)-nn. this is partly due to the smaller dihedral angles between dae and thiophene unit as well as between thiophene units in comparison with those in dae-(phe)-nn. additionally, the orbital energy of homo of dae is closer to the homo energy of thiophene than that of benzene. these two effects contribute to the larger mixing of the -orbitals through thiophene and results in the extended -conjugation in dae-(thio)-nn than in dae-(phe)-nn.	unusual blue shift of the absorption maxima of two nitronyl nitroxide attached diarylethene through phenyl units (dae - phe - nn) with increasing number of phenyl units is examined by time dependent density functional theory (tddft). the extended -conjugation between nitronyl nitroxide and diarylethene is rather suppressed by the bridge phenyl units. in comparison, the red shift found in two nitronyl nitroxide attached diarylethenes through thiophene units (dae - thio - nn) with increasing number of thiophene units is due to the longer -conjugation induced by smaller dihedral angles between diarylethene and bridge and between bridges.
we included all patients with ggs - positive blood cultures who had been treated from april 1998 through august 2004 at national taiwan university hospital, a 2,000-bed teaching hospital in northern taiwan. we recorded demographic parameters, underlying illness, clinical diagnosis, and outcome for each patient. recurrence of bacteremia was defined as repeated positive blood culture after complete treatment (at least 14 days) of previous bacteremia. differentiation of ggs was based on colony size, hemolytic reaction, voges - proskauer reaction, and -glucuronidase activity. all -hemolytic streptococci, whether large or small colonies, were tested for lancefield group by using an agglutination kit (streptex ; murex biotech ltd., equisimilis was performed for all ggs isolates as described (7). for identification of s. canis, a probable isolate was identified by a negative -glucuronidase result and further confirmed with the 16srna method as described (8). susceptibilities of these isolates were tested by using the broth microdilution method as defined by the clinical and laboratory standards institute (formerly national committee for clinical laboratory standards) (9). to determine the similarity of isolates in cases of recurrence, we used pulsed - field gel electrophoresis (pfge) as described (10). the emm typing of isolates in cases of recurrence were also determined as described (11). the first 160 bases sequenced by emmseq2 that had > 95% identity were defined as having the same genotype (11). during the study period, 106 episodes of ggs bacteremia in 92 patients had been recorded ; 56 episodes occurred during the first half of the study period (before june 2001) and 50 episodes during the second half. equisimilis for 99 episodes, s. anginosus for 5, and s. canis for 2. bacteremia recurred for 9 patients (1 had 4 episodes, and 3 had 3 episodes) ; bacteremia was nosocomial for 7 patients and polymicrobial for 5. all 3 patients who died had a diagnosis of the primary bacteremia caused by s. dysgalactiae subsp. includes 2 patients with psoas muscle abscess, 1 with epidural abscess, and 1 with deep neck infection. among the 9 patients with recurrent bacteremia, the causative agent was s. dysgalactiae subsp. pfge performed with all 13 available isolates from recurrent cases showed that 10 were identical to that of the initial episode, including 1 in a patient with recurrence of s. canis bacteremia. the clinical characteristics of the patients and emm typing results are shown in table 2 ; pfge results are shown in the figure. the underlying diseases of patients with recurrent episodes included genital cancer (4 [44.4% ] patients) and history of cellulitis (6 [66.7% ]), each of which was significantly correlated with the likelihood of recurrence (p<0.01 for each). further analysis showed that a previous history of cellulitis was significantly correlated with female sex (p = 0.01), genital cancer (p<0.01), tissue edema (p = 0.02), heart disease (p = 0.04), and post coronary artery bypass graft (p = 0.03). pulsed - field gel electrophoresis profiles of all isolates from patients with recurrent group g streptococcal bacteremia. isolates b1 and b2, streptococcus canis ; other isolates, s. dysgalactiae subsp. bacteremia caused by -hemolytic s. anginosus with group g antigen was identified for 5 patients, none of whom had cellulitis, compared with 48 (55.8%) of the 86 patients with s. dysgalactiae subsp. polymicrobial bacteremia and nosocomial bacteremia were found in a higher percentage of patients with s. anginosus (60% and 40.0%, respectively) than of patients with s. dysgalactiae subsp. equisimilis bacteremia (4.7% and 5.8%, respectively) ; p<0.01 and p = 0.02, respectively. the 1 patient with s. canis bacteremia was a 33-year - old man with no history of dog bite. he had alcohol - associated liver cirrhosis of child c (severe) classification and leg edema. susceptibility testing showed decreased susceptibility to only macrolides (susceptibity rates : azithromycin 67.4%, clarithromycin 73.9%), clindamycin (87.0%), and quinupristin - dalfopristin (33.7%) (appendix table). we documented 5 cases of primary bacteremia caused by -hemolytic group g s. anginosus and unintentionally documented recurrence of s. canis bacteremia. our finding of 5 -hemolytic s. anginosus isolates and 1 s. canis isolate in patients with ggs bacteremia in this study differs from findings of previous studies (2,3). serotype determination was performed for all -hemolytic streptococci isolated in our hospital, whether colonies were large or small, which might have led to the detection of more streptococcal isolates with g antigen. the pcr method developed in our hospital and used in this study could effectively differentiate s. anginosus from s. dysgalactiae subsp. information about clinical infection with s. milleri with group g antigen is limited (4). in a previous study of ggs bacteremia, cohen - poradosu. reported that 6 of 84 patients had recurrence of bacteremia (3). risk factors were similar to those previously reported for non group a streptococcal cellulitis (13). pfge of these isolates showed that a high percentage of recurrence was caused by identical strains. although cohen - poradosu. reported that emm type stg840 was the most common strain (3), we found emm type stg485 to be most common. for years in taiwan, although we did not test for erythromycin resistance, we found some resistance even to new macrolides. since restriction of macrolide use in taiwan, a linear relationship has been noted between the decline in erythromycin use and the decline in erythromycin resistance in s. pyogenes (15). our study, however, found no decline in macrolide resistance from first half of the study period (27.1%) to the second half (37.0%). in summary, the mortality rate for patients with ggs bacteremia was relatively low (< 10%), but resistance to quinupristin - dalfopristin was extremely high. in vitro susceptibilities of 92 isolates of group g streptococcus, april 1998-august 2004, taiwan	a retrospective observational study in taiwan, 19982004, identified 92 patients with group g streptococcal bacteremia ; 86 had streptococcus dysgalactiae subspecies equisimilis. the most common diagnosis was cellulitis (48 cases), followed by primary bacteremia (34 cases). infection recurred in 9 patients. mortality rate was low (3.3%) ; resistance to quinupristin - dalfopristin was high.
nowadays, more than 1.4 billion adults, 20 and older, were overweight. of these over 200 million men and nearly 300 million women were obese. based on the world health organization reports, obesity is increasing more rapidly in developing countries than developed countries. these risk factors consequently lead to cardiovascular diseases as the most common cause of death in the world. obesity also is an important risk factor for cardiovascular diseases, diabetes type 2 (dm ii), osteoarthritis, hyperlipidemia, apnea, and etc., although studies in the second half of the 20 century about the etiology, pathophysiology and treatment of obesity increased our knowledge in this field of science, but effective methods for treatment and prevention of obesity remains unknown. studies show that the prevalence of obesity has being elevated in iran in the recent years due to increasing urbanization. in controlling weight gain carbohydrate and fat are two most effective component of diet in obese individuals in different societies ; carbohydrate in iran and fat in most asian countries and in the americans and europeans. this agent inhibits -glucosidase activity in the small intestine and pancreas and causes reduced glucose release from the disaccharides and oligosaccharides. acarbose has been known as an overweight medication in some studies with mechanisms including appetite reduction and fat or calorie malnutrition especially in long time use. however, some studies reported non - significant weight loss using this drug in non - asians. on the other hand, acarbose consumption alone will not lead to low blood sugar and hypoglycemia. there are some drugs with confirmed effect on weight loss world widely with the high cost for consumption in iran. the cost of acarbose is less than the other anti - obesity drugs and procedures. high controversy about the effect of acarbose on the weight loss in different countries indicated a well - designed study in the iranian patients with the high carbohydrate diet. this investigation is designed to evaluate the effect of acarbose on weight loss in non - diabetics overweight or obese in iran. from all obese patients referred to the endocrinology clinic of afzalipour hospital and office for treatment from november 2009 to april 2010, 66 non - diabetic subjects were recruited to this study. inclusion criteria were age equal or more than 18 years, willingness to participate in the study, body mass index (bmi) (weight (kg)/height (m)) between 25 and 35, no evidence of cardiovascular disease, diabetes, gastrointestinal disease (inflammatory bowel disease [ibd ], intestinal ulcers, bowel obstruction), liver disease and renal disease, no history of medication for lowering weight, women should not be pregnant or milking. the exclusion criteria were no desire to continue the treatment, development of renal, gastrointestinal and liver disease during treatment and beginning to utilize antihypertensive drugs such as blocker and thiazides. all patients gave written consensus when they were included to the study. at the beginning of study, the baseline characteristics of all selected subjects including height, weight, total cholesterol, and triglyceride (tg) and fasting plasma glucose (fpg) were recorded. the study protocol was approved by the ethics committee of kerman university of medical sciences (k/88/106) and this trial was registered on the iranian registry of clinical trials (irct) website (the registration number of the trial is irct i d : irct201011151774n3). a double - blind randomized, parallel group, placebo - controlled design was used. block randomization was performed centrally by a computer program with minimization for height, weight and bmi according to the study center. after baseline assessment, the eligible patients were allocated by computer generated random numbers to 1 of 2 treatment groups and followed - up at 1 week. the letter a and b was selected for two groups ; physician referred the patient to nurse. the nurse had several envelope the contains the letter a or b in a random manner (for example aabb / abab/ etc.,). the letter a or b belongs to patient in this way and the drug gives to patient by another nurse. treatment group was given acarbose pills (100 mg) and other group took lactose contained placebo (100 mg). acarbose and placebo were packed in sachets of identical appearance ; patients and trial nurses and physician and statistician were all unaware of the study - group assignment until after analysis. acarbose pills (100 mg) and lactose contained placebo (100 mg) were prepared from exir co. and were similar in shape and packing. patients were asked to take medicines in the first 2 weeks with 1 of a tablet daily and from 3 week with 3 tablets daily (to reduce gastrointestinal side - effects). patients in the placebo and treatment groups had nutrition consultation for reducing 500 - 800 kcal at first and the every 45 days. a training exercise cd was given to each person and they planned to exercise at least h daily. subjects were evaluated every month for adverse effects, weight loss, and bmi for 5 months. once every 3 months and a baseline blood sample for fpg, tg, and cholesterol was taken from patients. the analyses were performed by paired t - test and repeated measured anova and also they were adjusted for baseline bmi. from all obese patients referred to the endocrinology clinic of afzalipour hospital and office for treatment from november 2009 to april 2010, 66 non - diabetic subjects were recruited to this study. inclusion criteria were age equal or more than 18 years, willingness to participate in the study, body mass index (bmi) (weight (kg)/height (m)) between 25 and 35, no evidence of cardiovascular disease, diabetes, gastrointestinal disease (inflammatory bowel disease [ibd ], intestinal ulcers, bowel obstruction), liver disease and renal disease, no history of medication for lowering weight, women should not be pregnant or milking. the exclusion criteria were no desire to continue the treatment, development of renal, gastrointestinal and liver disease during treatment and beginning to utilize antihypertensive drugs such as blocker and thiazides. all patients gave written consensus when they were included to the study. at the beginning of study, the baseline characteristics of all selected subjects including height, weight, total cholesterol, and triglyceride (tg) and fasting plasma glucose (fpg) were recorded. the study protocol was approved by the ethics committee of kerman university of medical sciences (k/88/106) and this trial was registered on the iranian registry of clinical trials (irct) website (the registration number of the trial is irct i d : irct201011151774n3). a double - blind randomized, parallel group, placebo - controlled design was used. block randomization was performed centrally by a computer program with minimization for height, weight and bmi according to the study center. after baseline assessment, the eligible patients were allocated by computer generated random numbers to 1 of 2 treatment groups and followed - up at 1 week. the letter a and b was selected for two groups ; physician referred the patient to nurse. the nurse had several envelope the contains the letter a or b in a random manner (for example aabb / abab/ etc. the letter a or b belongs to patient in this way and the drug gives to patient by another nurse. treatment group was given acarbose pills (100 mg) and other group took lactose contained placebo (100 mg). acarbose and placebo were packed in sachets of identical appearance ; patients and trial nurses and physician and statistician were all unaware of the study - group assignment until after analysis. acarbose pills (100 mg) and lactose contained placebo (100 mg) were prepared from exir co. and were similar in shape and packing. patients were asked to take medicines in the first 2 weeks with 1 of a tablet daily and from 3 week with 3 tablets daily (to reduce gastrointestinal side - effects). patients in the placebo and treatment groups had nutrition consultation for reducing 500 - 800 kcal at first and the every 45 days. a training exercise cd was given to each person and they planned to exercise at least h daily. subjects were evaluated every month for adverse effects, weight loss, and bmi for 5 months. once every 3 months and a baseline blood sample for fpg, tg, and cholesterol was taken from patients. the analyses were performed by paired t - test and repeated measured anova and also they were adjusted for baseline bmi. from 66 enrolled patients, 33 were assigned to treatment and 33 to placebo group. of these patients, 21 (63%) in the placebo group and 19 (58%) in the acarbose group the most common reason for not continuing study intervention was the occurrence of side - effects. the study fellow chart ; enrollment and outcomes the subjects consisted of 10 men (25%) and 30 women (75%) with a mean age of 31 8.8 years. the mean weight was 77.8 10.1 kg and bmi was 30.1 2.6 kg / m. as shown in table 1, there was no significant difference in the baseline characteristics of two groups. baseline characteristics of the study population as could be seen in figure 2 the bmi reduced in both groups during the study, but this reduction was higher in the acarbose group (p < 0.001). body mass index in each treatment in the treatment and placebo groups difference of bmi changes in each 1 month interval in two groups (meansd, kg / m) body mass index changes during intervals in the treatment and placebo group this study showed that acarbose can reduce weight in over weight and obese non - diabetic people ; this effect is continuous and progressive. considering the high prevalence of overweight and obesity in society and the importance of this as one of the major risk factors for chronic diseases such as severe cardiovascular diseases, diabetes and cancer, the requirement for therapy is obvious. acarbose is an anti - hyperglycemic agent in dm ii and lower the weight of many diabetic patients reportedly. there is controversy about the effect of acarbose on weight loss in different societies with different diet. in this study, the effect of this drug was investigated on the iranians patients as a society with the high carbohydrate utilization in the meals. as shown in the results, the patients treated with acarbose have a higher rate of weight loss after 2 months. the results of a study by hauner. to investigate the effect of acarbose on stability after low calorie diet induced weight loss confirmed the finding of our study. in that double - blind controlled clinical trial, patients with a bmi between 32 and 38 kg / m were treated with the low calorie diet for 10 - 16 weeks and then 26 weeks with acarbose. however, an investigation on 2002 in taiwan and several asian countries showed the acarbose has higher effect on reducing hemoglobin a1c and post - prandial glucose in dm ii patients than sulfonylurease. furthermore, acarbose was shown to be well - tolerated and safe but did not significantly lower the bmi than other treatment. in that study, the dm ii patients did not get low calorie diet in contrast to the present study in non - diabetic patients with low calorie diet. in another trial by rachmani. although, their diet was very different and low calorie diet was not administered for the subjects. first, the sample size was too small to compare the sex difference in acarbose induced weight loss. consequently combination of acarbose administration with the low calorie diet seems to be an effective treatment for lowering weight in the overweight and obese patients with the high carbohydrate utilization.	background : high prevalence of obesity and the importance of this issue as a risk factor for chronic diseases such as severe cardiovascular diseases, diabetes, and cancer necessitate the need for treatment. the aim of this study was the evaluation of acarbose effect on the weight loss in non diabetic overweight or obese patients in kerman.materials and methods : a double blind randomized controlled clinical trial was performed on 66 patients with the body mass index (bmi) between 25 and 35 kg / m2. patients were divided in treatment and control groups using the randomization. the treatment group took 100 mg acarbose 3 times a day for 20 weeks in combination with the low calorie diet and exercise. control group was given placebo, low calorie diet, and exercise. bmi was measured after 20 weeks. the analyses were carried out using t - test and repeated measured anova.results:patients in acarbose and placebo group had a non significant difference in bmi at baseline. reducing in weight was considered in every month in both groups, but this reduction was higher in the treatment group. at the 5th month, the difference of bmi in the treatment group was significantly lower than the placebo group (2.31 0.6 vs. 0.76 0.6 kg / m2, p < 0.001).conclusion : acarbose, especially in combination with the low calorie diet and exercise, seems to lose weight effectively in obese and overweight patients in communities that have a high carbohydrate intake (like persian diet).
we identified all hospitalizations carrying a diagnosis of mrsa, clostridium difficile associated disease (cdad), vancomycin - resistant enterococcus (vre), pseudomonas aeruginosa, and candida infections for 20002005 from the national inpatient sample data. these data are available on the healthcare costs and utilization project net [hcupnet ] website, administered by the agency for healthcare research and quality (8). we used the corresponding diagnosis codes from the international classification of diseases, 9th revision, clinical modification (appendix table). because few reports of vancomycin - resistant staphylococcus aureus exist (9) we limited hospitalizations in which candida organisms had been identified to deep - seated infections, including candidiasis of the lung, disseminated candidiasis, candidal endocarditis, meningitis, esophagitis, and enteritis. the numbers of discharges per year for infections associated with each organism and in aggregate were stratified by census region. we obtained regional estimates of all us hospitalizations in the corresponding years from the hcupnet (8), and censal and intercensal data on the us population for 20002005 from the us census bureau. because large numbers would predispose the study to type i error, we did not perform formal significance testing ; rather, we focused on clinical and policy - relevant trends. the overall volume of resistant infections increased by 89.6% from year 2000 through 2005 (table). as a proportion of the total volume growth, the increases across regions were comparable. the southern region had the highest raw volume of resistant infections for the study period (2000, 37.3% ; 2005, 39.1%). the west had the smallest contribution in 2000 (19.0%) and 2005 (19.5%). however, the northeast had the highest relative incidence per 1,000 hospitalizations with 14.00 in year 2000 ; its incidence of 19.98 in 2005, however, was lower than that in the south, 20.76/1,000 (table). regional disparities in the population - based incidence of hospitalizations with resistant organisms also occurred (table). thus, the incidence in the northeast was not only the highest for 5 of the 6 years examined, but compared to that seen in the lowest - incidence region, the west, was higher by as much as 41.9% in 2003. while the northeast led other regions in the incidence of cdad hospitalizations over the entire period examined (figure, panel a), the south exhibited the highest population incidence of mrsa and pseudomonas hospitalizations. although temporal patterns of regional population incidence varied somewhat for hospitalizations in which vre and candida spp. infections were diagnosed, by year 2005 the northeast emerged as the region with the highest incidence of vre, while the south had the highest incidence of candida spp. the lowest incidence of vre hospitalizations was consistently seen in the southern region in each of the studied years. the incidence of hospitalizations with pseudomonal infections remained relatively stable regionally over time (figure, panels b, c, d). population incidence of component resistant infections in the united states, by census region, 20002005. a) clostridium difficile associated disease ; b) methicillin - resistant staphylococcus aureus ; c) vancomycin - resistant enterococcus ; d) pseudomonas aeruginosa ; e) candida spp. we have demonstrated a substantial rise in the absolute number, incidence, and geographic variations across the united states in hospitalizations in which infections have been caused by pathogens exhibiting antimicrobial resistance. the northeast consistently outpaced the other regions in the aggregate volume of resistant infections in 5 of the 6 years examined. for individual infections, a region s having a relatively high incidence of 1 organism does not guarantee it will have a high incidence of another organism, as illustrated by the reversal of the regional incidence patterns for mrsa and cdad, for example. most troubling, however, is the general finding of a ubiquitous, substantial, and continuing increase in the incidence of hospitalizations with resistant infections. a notable pattern in our study is that the regions with the higher incidence of cdad (northeast and midwest) also exhibited higher incidence of vre in at least half of the study period, consistent with the observation that infection with cdad can facilitate transmission of vre (10,11). since both pathogens share similar risk factors, why this pattern should be present is biologically unclear (12,13), although a recent report noted a similar pattern of concomitant increases in mrsa and decreases in vre incidence between 1999 through 2005 (14). lastly, we noted that, although substantially discrepant regionally, the incidence of hospitalizations with p. aeruginosa infections, consistent with others observations, has remained relatively stable over the 6-year period (15). we can not illuminate the reasons for the patterns of infection incidences we have uncovered. further studies should encompass much more granular geographic data to confirm our findings and raise hypotheses to explain them. the most important limitation of our study is that case ascertainment was performed by using administrative coding, rather than clinical and microbiologic data, and we were unable to verify diagnostic accuracy either across time or geographic areas ; therefore, the observed increases may be partially due to increased awareness of resistance. however, administrative coding has been used to track the epidemiology of both mrsa and cdad (1,2). furthermore, temporal trends in case volume are similar to trends reported from clinical studies. at least a proportion of the case - patients we identified likely had overlapping infections with multiple organisms. nevertheless, the aggregate number of infections that we have described has implications for hospital resource use because persons with multiple infections likely require more care than those with a single pathogen. finally, we were unable to differentiate between community - acquired and nosocomial infections. in summary, we have demonstrated a notable increase in the incidence of hospitalizations with resistant organisms in the united states. regional variations in the incidence may yield clues for future research efforts to ascertain what modifiable risk factors drive decreases in the incidence of these deadly infections. the nearly 1 million annual hospitalizations in 2005 with resistant infections and their relentless upward trajectory in the united states are undesirable and unsustainable. aggressive and coordinated efforts to reduce inappropriate use of antibimicrobial agents in humans and livestock and to encourage development of novel therapeutics are urgently needed to stem this public health hazard in the united states and throughout the world.	from 2000 through 2005, hospitalizations with resistant infections (methicillin - resistant staphylococcus aureus, clostridium difficile associated disease, vancomycin - resistant enterococcus, pseudomonas aeruginosa, and candida infection) nearly doubled, from 499,702 to 947,393. regional variations noted in the aggregate and by individual infection may help clarify modifiable risk factors driving these infections.
thyroid nodules and thyroid cancer are more common in women [1, 2 ]. the incidence rate of thyroid cancer (tc) is increasing, which could be due to better surveillance techniques. nevertheless, other factors are probably involved because the incidence rates of tumors of all sizes increased. as the increase in incidence of tc appears to be higher in women, sex hormones might have an etiological role. reproductive factors, as history of uterine fibroids, greater number of live births, greater number of reproductive years, and greater estimated number of ovulatory cycles, were associated with an increased risk of tc. also, during pregnancy, thyroid nodules were shown to increase in number and size [7, 8 ], and tc has been shown to affect one in a thousand pregnant women, being the second most common type of cancer in pregnancy. several studies have shown thyroid cells growth in response to estrogen [1013 ], as reviewed recently, and some data suggest a decrease in differentiated function by this hormone in thyroid cells [11, 15 ]. progesterone might have a protective effect on the thyroid because the use of oral contraceptives and menopausal hormone therapy, with the combination of estrogen and progesterone, were not associated with thyroid cancer risk, but irregular menstrual bleeding, usually due to anovulatory cycles, has been associated with it. although progesterone could exert its effects through genomic or nongenomic mechanisms [17, 18 ], its primary action is to regulate the expression of target genes through binding directly to its cognate sequence in the genes, called progesterone responsive elements. as pr have been variably described in thyroid follicular cells [2023 ], the aim of the present study was to evaluate the effects of progesterone on gene expression in a model of normal human thyroid cells in primary culture. tissue samples were obtained from patients submitted to thyroidectomy as part of treatment for differentiated tc. after evaluation by two pathologists, normal tissue that would have been discharged the project was submitted to, and approved by, the research ethics committee of the hospital de clnicas de porto alegre, porto alegre, rs, brazil (cep : 09 - 454), which waived a written informed consent. in accordance with the resolution hcpa 02/97, based on the resolution cns 196/96 of the national health council, brazil, and the guideline 9 of the international ethical guidelines for biomedical research involving human subjects (cioms, who, geneva, 1993), there was no need to obtain informed consent of the patients, because only after the surgical procedure the researchers would know if a tissue sample would be available, they would not know the identity nor had access to the files of the patients, and the tissue samples included would have been discharged by the pathologists. the authors signed the statement of commitment to use of data, which states that no data that could identify patients would be used and also that their anonymity would be preserved in any publication of the results. briefly, thyroid tissue was cut in fragments of about 1 mm and digested by 3 mg / ml type i collagenase (gibco, grand island, ny, usa). the suspension of cells was sequentially filtered through nylon meshes (250, 150, and 60 m pore size) and the filtered fraction, containing epithelial thyroid cells, was resuspended and seeded in a 35 mm petri dish at a density of 1 10 cells / cm. thyroid cells were cultured initially in ham 's f-12 coon 's modification medium supplemented with 10% fetal bovine serum, 10 g / ml insulin, 5 g / ml transferrin, 1 mu / ml tsh, and 100 u / ml kanamycin at 37c with 5% co2 ; after two changes, the concentration of serum was decreased to 5% (3h medium). five or 7 days later, cells were deprived of tsh for 48 hours (3h medium minus tsh : 2h medium) ; subsequently, they were treated with 2h medium and progesterone, mifepristone, or vehicle ethanol, as follows : group 1 : 2h medium ; group 2 : 2h medium + 20 u / ml tsh ; group 3 : 2h medium + 20 u / ml tsh + 10 nm progesterone, and group 4 : 2h medium + 20 u / ml tsh + 10 nm progesterone + 100 nm mifepristone, for 4 h or 48 h, before total rna isolation. total rna was isolated using the trizol reagent (invitrogen life technologies, carlsbad, ca, usa). concentration and purity were assessed by the nanodrop nd-1000 spectrophotometer (nanodrop technologies, rockland, de, usa) and stored at 80c. 1 g total rna was transcribed into cdna by superscript iii reverse transcriptase (invitrogen life technologies) and stored at 20c. expression of ki-67, thyroglobulin tg, thyroperoxidase tpo, and sodium - iodide symporter nis genes was assessed by quantitative reverse transcriptase polymerase chain reaction (rt - qpcr) on applied biosystems stepone real - time pcr system using kit platinum sybr green qpcr supermix - udg (invitrogen life technologies). the primers used for the specific amplification of tg, tpo, and nis were described previously. the sequences of the primers for progesterone receptor were 5-acccgccctatctcaactacc-3 and 5-aggacaccataatgacagcct-3. annealing temperature of 60c was used for amplification ; dissociation curves were performed by running a gradient of 6095c to confirm the specificity of the pcr amplification. all samples were amplified in triplicate, and cdna standard curves were constructed using the threshold cycles with five successive 10-fold dilution points of a pool of cdna samples. rt - qpcr experiments were performed in duplicate and repeated in three (nis gene) or four (ki-67, tg, and tpo genes) independent experiments, with cells isolated from different patients. differences in the means of the treatment groups were evaluated with the generalized estimating equation (gee). after gee, post hoc bonferroni test was used to identify which means were different. all statistical analyses were performed with spss software, version 18.0 (spss, chicago, il), and considered significant when p was less than 0.05. thyroid follicular cells viability and differentiation were assessed by characteristic morphology and by staining tpo protein in immunocytochemistry. as showed in figure 1(a), the thyroid follicular cells isolated in our model present a cuboid shape, with the presence of many vacuoles around the nucleus as observed in previous studies of primary culture of follicular cells thyroid. also, as other epithelioid cells, these cells grow as islands and form a monolayer with extensive cell - to - cell contact. in figure 1(b), tpo protein staining, characteristic of thyroid follicular cells, is shown. the specific thyroid genes expression (tg, tpo, and nis) tsh increased significantly the nis gene expression (2.08 times) after 4 h ; it also increased significantly the tg (2.39 times), tpo (1.58 times), and ki-67 (1.87 times) genes expression, after 48 h, as shown in figure 2. to evaluate the effect of progesterone treatment on the expression of nis, gene expression of progesterone receptor was present in all thyroid cells cultures used in this study (data not shown). when thyroid cells were treated with 20 ui / ml tsh plus 10 nm progesterone, the mean expression of nis mrna after 4 h, and the mean expression of tg, tpo, and ki-67 mrna after 48 h, increased, respectively, by 1.75, 1.77, 1.68, and 1.95 times, when compared to the group treated with tsh (figure 3). in the present study, for the first time, an effect of progesterone on thyroid cells was demonstrated. genes involved in the differentiated protein expression of thyroid cells were upregulated by progesterone, as well as ki-67, a marker for growth ; these effects were abolished by mifepristone. isolated human follicular cells derived from normal thyroid tissue have been studied previously [27, 28 ], with high functional correspondence with the original follicular cells. in our model, tg, tpo, and nis mrna were detected even when the cells were deprived of tsh for 48 h, which could be due to other components of the medium and its supplements, such as insulin, or constitutive activation of thyroid cells. it is known that thyroid cell regulation varies in different species, and, sometimes, the cells can have different phenotypes and responses to tsh [29, 30 ]. in our model, normal human thyroid follicular cells in primary culture were shown to be differentiated and responsive to tsh. the response of gene expression to tsh has been shown to vary with the gene and time after treatment. as no direct effect of progesterone on thyroid cells has been described until now, we have no other studies to compare our results with. as progesterone increased the expression of genes involved in growth and differentiated function of thyroid cells, its effect on the thyroid gland could be protective. observed an increase in free t4 levels in progesterone - treated women, and a tendency towards lower tsh levels during progesterone treatment, in a randomized controlled progesterone trial. our results suggest that progesterone effects on human thyroid cell occur via a transcriptional level through its nuclear receptor, since a specific antagonist, mifepristone, abolished it. the ability of mifepristone to abolish progesterone effect in thyroid cells suggests that these receptors are functional in these cells, although its concentration in normal thyroid has been described as low or undetectable [22, 23 ]. in a recent study pr expression was seen in 75.8% of patients with papillary thyroid cancer. in our study, pr gene expression was identified in all cell cultures. as mifepristone blocks also the action of glucocorticoids and some synthetic progestogens, as medroxyprogesterone, are ligands for the glucocorticoids receptor (gr), the effects described in the present study could have been mediated by these receptors. nevertheless, the ability of progesterone to bind to gr is disputable. although the model used in the present study has contributed to the understanding of thyroid cell growth regulation, the increase in gene expression does not always result in a proportional increase in functional proteins. besides, our monolayer model does not reproduce the complex organization of thyrocytes in the follicle, so functional and growth studies are necessary to better understand a possible role of progesterone in thyroid cells. this work provides evidences that progesterone has a direct effect on thyroid cells, through its receptor, upregulating genes involved in thyroid function and growth.	thyroid cancer and thyroid nodules are more prevalent in women than men, so female sex hormones may have an etiological role in these conditions. there are no data about direct effects of progesterone on thyroid cells, so the aim of the present study was to evaluate progesterone effects in the sodium - iodide symporter nis, thyroglobulin tg, thyroperoxidase tpo, and ki-67 genes expression, in normal thyroid follicular cells, derived from human tissue. nis, tg, tpo, and ki-67 mrna expression increased significantly after tsh 20 ui / ml, respectively : 2.08 times, p < 0.0001 ; 2.39 times, p = 0.01 ; 1.58 times, p = 0.0003 ; and 1.87 times, p < 0.0001. in thyroid cells treated with 20 ui / ml tsh plus 10 nm progesterone, rna expression of nis, tg, and ki-67 genes increased, respectively : 1.78 times, p < 0.0001 ; 1.75 times, p = 0.037 ; and 1.95 times, p < 0.0001, and tpo mrna expression also increased, though not significantly (1.77 times, p = 0.069). these effects were abolished by mifepristone, an antagonist of progesterone receptor, suggesting that genes involved in thyroid cell function and proliferation are upregulated by progesterone. this work provides evidence that progesterone has a direct effect on thyroid cells, upregulating genes involved in thyroid function and growth.
percutaneous coronary intervention (pci) is the first line treatment for the management of coronary artery disease in hospitals with cardiac catheterization, clopidogrel and aspirin combination therapy has been approved to be effective and safe in multiple clinical trials such as clopidogrel versus aspirin in patients at risk of ischemic event, cure and credo. however, clopidogrel response variability and its correlation with antiplatelet efficiency and the incidence of adverse cardiac events have been reported in patients with coronary heart disease (chd). the poor respondents will have high on - treatment platelet reactivity (htpr) which might have a higher risk for thrombotic events whereas the risk for bleeding events might be raised in the high respondents with nonhigh on - treatment platelet reactivity (nhtpr) after the admission of clopidogrel. unfortunately, the mechanism by which factors contribute to htpr and to what extent htpr is associated with adverse cardiac events, especially in chinese population with acute coronary syndrome (acs), are currently poorly understood. diverse factors that affect the effectiveness of antiplatelet therapy in patients with coronary artery disease may influence the outcomes of antiplatelet therapy, such as drug - drug interaction, genetic factors or drug interaction combined with genetic factors, concomitant diseases, and patient compliance, among which genetic factors are receiving more and more attention. clopidogrel is a prodrug requiring two major biotransformation steps, cytochrome p-450 (cyp) isoenzymes, transforms prodrug an active metabolite that binds irreversibly to the platelet adenosine 5-diphosphate (adp) receptor p2y12. several functional polymorphisms have been found with genes encoding cyp isoforms which involve in clopidogrel metabolic activation upstream of p2y12 (e.g., cyp2c19) but their influence on the pharmacodynamic response to clopidogrel has not been systematically investigated, especially in chinese people with acs. furthermore, to identify patients with abnormal responses to antiplatelet drugs via platelet function testing is critical to clinical outcomes. there are some platelet function tests, such as light transmittance aggregometry (lta), thrombelastography (teg), vasodilator - stimulated phosphoprotein assay, and platelet function analyzer (pfa-100, plateletworks, and verifynow system). conventional lta was once the gold standard of platelet function testing that has been used widely due to low medical cost, however, this is a time - consuming technique which requires specially trained staffs to conduct the tests, large amount of specially prepared platelet - rich plasma with poor reproducibility. it is a dynamic test for the process of blood coagulation and fibrinolysis by measuring the physical properties of the thrombus with whole blood. this study aimed to investigate the correlation between htpr rate using teg test and the incidence of major adverse cardiovascular event (mace) rate in chinese acs patients ; then to determine the contribution of cyp2c19 polymorphisms by testing the residual platelet reactivity after clopidogrel administration in chinese acs patients. from june 2014 to january 2015, we screened 510 patients presented in ed to rule out acs, among which 116 patients (aged 1880 years) were diagnosed with acs by quantitative coronary angiography and enrolled into this study with patient consent per human subjects study protocol approved by peking university ethics committee. the exclusion criteria included contraindications to clopidogrel, aspirin, heparin, and contrast agents as well as quantitative coronary angiography ; severe cardiac dysfunction and left ventricular ejection fraction (%) of 30% or less ; severe renal with glomerular filtration rate 25 mlmin1.73 m or hepatic function failure with aspartate aminotransferase and alanine transaminase 80 u / l ; serious infection, systemic immune system diseases, malignant tumor, hematologic diseases ; pregnancy, lactation, and long - term use of contraceptive agents ; history of bleeding or cerebrovascular disease - related accident within the last 3-month ; major operation within the last 1-month and the use of a glycoprotein iib / iiia antagonist before the procedure. all patients were administered, 75 mg / d clopidogrel, with a loading dose of 300 mg clopidogrel in addition to the other available medical therapy, including aspirin, statins, -blockers, angiotensin - converting enzyme inhibitor (acei) or angiotensin receptor blockers (arb), or calcium channel blockers. after teg testing, dropped heparin - anticoagulated whole blood was collected by the hemostasis laboratory. blood sample genomic dna was isolated using the dna extractor axyprep-96 (axygen scientific inc., single nucleotide polymorphisms cyp2c19 2 (681 g > a, rs4244285), cyp2c19 3 (636 g > a, rs4986893), and cyp2c19 17 (806c > t, rs12248560) were identified by ligase detection reaction - real time polymerase chain reactions analysis system (perkin - elmer gene amp pcr systems 9600, perkin - elmer shanghai inc., shanghai, china), and genescan672 and genemapper (abi3730 dna analyzer, applied biosystems, california, usa) of the system were used to analyze the cyp2c19 alleles. four milliliter of citrated whole blood and 4 ml of heparin - anticoagulated whole blood were drawn at least 3 days after the administration of the first clopidogrel maintenance dose. these samples were sent to the hemostasis laboratory and thromboelastography was performed within 3 h after blood collection according to the manufacture 's instruction (teg 5000, haemoscope corporation, heamoscope, illinois, usa) by trained technicians of the hospital. teg utilizes four channels to detect the effects of apt activity via the arachidonic acid and adp pathways. a teg pir of less than 30% after the maintenance dose of clopidogrel reached steady state was defined as htpr, while patients with nhtpr had pir of at least 30% according to the instruction and previous study. the patients were followed up at 1-, 3-month after the coronary angiography, respectively, and the clinical outcomes after the patients discharged were recorded. the primary endpoint was mace, which were a composite of cardiac death, nonfatal myocardial infarction (mi), and target lesion revascularization (tlr). cardiac death was defined as any mortality caused by cardiac diseases. a five - fold increase in creatine kinase and troponin levels, over the upper normal limit plus the occurrence of ischemic symptoms, was defined as nonfatal mi. tlr was defined as ischemia driven pci or coronary artery bypass graft on the target lesion. secondary endpoints included definite stent thrombosis, unstable angina (ua), nonfatal stroke, and major bleeding. definite stent thrombosis was defined according to the definition of the academic research consortium with angiographic evidence of thrombotic stent occlusion. ua was defined as the occurrence of ischemic symptoms in 1-month requiring a hospital stay but without an increase in creatine kinase and troponin levels. nonfatal stroke was defined as ischemic stroke caused by thrombosis or hypotension and hemorrhagic stroke because of intracranial or subarachnoid hemorrhage. major bleeding was defined as intracranial bleeding or clinically overt bleeding associated with a decrease in hemoglobin of 5 g / dl according to the thrombolysis in myocardial infarction criteria. the data were expressed as numbers and frequencies for categorical variables, and as the mean standard deviation (sd) for continuous variables. for comparisons among groups, the chi - squared test was used for categorical variables, and the unpaired student 's t - test or the one - way analysis of variance (anova) was used for continuous variables. the observed genotype frequencies were compared with those expected for a population being in hardy weinberg equilibrium, using a chi - squared test with 1 degree of freedom. statistical significance is considered at p a, rs4244285), cyp2c19 3 (636 g > a, rs4986893), and cyp2c19 17 (806c > t, rs12248560) were identified by ligase detection reaction - real time polymerase chain reactions analysis system (perkin - elmer gene amp pcr systems 9600, perkin - elmer shanghai inc., shanghai, china), and genescan672 and genemapper (abi3730 dna analyzer, applied biosystems, california, usa) of the system were used to analyze the cyp2c19 alleles. duplicate samples and negative controls four milliliter of citrated whole blood and 4 ml of heparin - anticoagulated whole blood were drawn at least 3 days after the administration of the first clopidogrel maintenance dose. these samples were sent to the hemostasis laboratory and thromboelastography was performed within 3 h after blood collection according to the manufacture 's instruction (teg 5000, haemoscope corporation, heamoscope, illinois, usa) by trained technicians of the hospital. teg utilizes four channels to detect the effects of apt activity via the arachidonic acid and adp pathways. a teg pir of less than 30% after the maintenance dose of clopidogrel reached steady state was defined as htpr, while patients with nhtpr had pir of at least 30% according to the instruction and previous study. the patients were followed up at 1-, 3-month after the coronary angiography, respectively, and the clinical outcomes after the patients discharged were recorded. the primary endpoint was mace, which were a composite of cardiac death, nonfatal myocardial infarction (mi), and target lesion revascularization (tlr). cardiac death was defined as any mortality caused by cardiac diseases. a five - fold increase in creatine kinase and troponin levels, over the upper normal limit plus the occurrence of ischemic symptoms, tlr was defined as ischemia driven pci or coronary artery bypass graft on the target lesion. secondary endpoints included definite stent thrombosis, unstable angina (ua), nonfatal stroke, and major bleeding. definite stent thrombosis was defined according to the definition of the academic research consortium with angiographic evidence of thrombotic stent occlusion. ua was defined as the occurrence of ischemic symptoms in 1-month requiring a hospital stay but without an increase in creatine kinase and troponin levels. nonfatal stroke was defined as ischemic stroke caused by thrombosis or hypotension and hemorrhagic stroke because of intracranial or subarachnoid hemorrhage. major bleeding was defined as intracranial bleeding or clinically overt bleeding associated with a decrease in hemoglobin of 5 g / dl according to the thrombolysis in myocardial infarction criteria. the data were expressed as numbers and frequencies for categorical variables, and as the mean standard deviation (sd) for continuous variables. for comparisons among groups, the chi - squared test was used for categorical variables, and the unpaired student 's t - test or the one - way analysis of variance (anova) was used for continuous variables. the observed genotype frequencies were compared with those expected for a population being in hardy weinberg equilibrium, using a chi - squared test with 1 degree of freedom. statistical significance is considered at p 0.05). moreover, the administration of a -receptor antagonist, statins, ca antagonist, and acei / arb, proton pump inhibitors did not alter residual platelet reactivity (p > 0.05). demographic characteristics of the study population according to residual platelet reactivity htpr : high on - treatment platelet reactivity ; nhtpr : nonhigh on - treatment platelet reactivity ; bmi : body mass index ; dm : diabetes mellitus ; pci : percutaneous coronary intervention ; mi : myocardial infarction ; aceis : angiotensin - converting enzyme inhibitors ; arbs : angiotensin receptor blockers ; ccbs : calcium channel blockers ; ppis : proton pump inhibitor. genotype distribution and allele frequencies of the genetic variations studied are in table 2. among the 116 patients studied, 58 (50.00%) were carriers of at least one cyp2c19 2 loss of function (lof) allele and 12 (10.34%) were carriers of at least one cyp2c19 3 lof allele, for cyp2c19 17 gain of function (gof) allele, only one heterozygous carriers was detected. the distribution of all the cyp genetic variants did not deviate from hardy weinberg equilibrium. genotype distribution and allele frequency of investigated genetic variations on the basis of the distribution of genotypes, the patients were divided into extensive metabolizers (ems) without lof mutation allele (lof noncarriers), which is also the cyp2c19 1/1 (681gg/636gg) wild - type genotype, intermediate metabolizers (ims) carrying only one lof mutation allele (cyp2c19 2 or cyp2c19 3) with or without gof mutation allele, and poor metabolizers (pms) carrying two lof mutation alleles (cyp2c19 2 and cyp2c19 3), accounting for 41.38%, 56.90%, and 1.72% of all cases, respectively. in the study population, carriers of at least one lof allele (pms and ims) had higher residual platelet reactivity compared to noncarriers (ems), pms and ims had higher incidence of htpr than ems, (2 [100% ] vs. 9 [18.75% ], p = 0.000 and 27 [41.54% ] vs. 9 [18.75% ], p = 0.000, respectively) [figure 1a ]. and there are significant statistic difference between the incidence of htpr of pms and ims too, with a p = 0.000, pms had higher residual platelet reactivity than ims by teg detecting, which means the cyp2c19 polymorphism was a critical contributor of clopidogrel resistance. comparison of the high on - treatment platelet reactivity incidence (a) and the adenosine diphosphate - induced platelet inhibition rate (b) by thrombelastography detecting after clopidogrel among patients with acute coronary syndrome with different phenotype of cyp2c19 2, cyp2c19 3, and cyp2c19 17. htpr : high on - treatment platelet reactivity ; pm : poor metabolizers ; i m : intermediate metabolizers ; em : extensive metabolizers. furthermore, we evaluated the adp - channel platelet inhibition by teg (teg - pi) of each phenotype. it showed that the teg - pi of ems was significantly higher than that in pm and i m patients (12.25% 12.23% and 43.72% 28.30% vs. 60.39% 28.34%, respectively, p 0.05). moreover, the administration of a -receptor antagonist, statins, ca antagonist, and acei / arb, proton pump inhibitors did not alter residual platelet reactivity (p > 0.05). demographic characteristics of the study population according to residual platelet reactivity htpr : high on - treatment platelet reactivity ; nhtpr : nonhigh on - treatment platelet reactivity ; bmi : body mass index ; dm : diabetes mellitus ; pci : percutaneous coronary intervention ; mi : myocardial infarction ; aceis : angiotensin - converting enzyme inhibitors ; arbs : angiotensin receptor blockers ; ccbs : calcium channel blockers ; ppis : proton pump inhibitor. genotype distribution and allele frequencies of the genetic variations studied are in table 2. among the 116 patients studied, 58 (50.00%) were carriers of at least one cyp2c19 2 loss of function (lof) allele and 12 (10.34%) were carriers of at least one cyp2c19 3 lof allele, for cyp2c19 17 gain of function (gof) allele, only one heterozygous carriers was detected. the distribution of all the cyp genetic variants did not deviate from hardy weinberg equilibrium. genotype distribution and allele frequency of investigated genetic variations on the basis of the distribution of genotypes, the patients were divided into extensive metabolizers (ems) without lof mutation allele (lof noncarriers), which is also the cyp2c19 1/1 (681gg/636gg) wild - type genotype, intermediate metabolizers (ims) carrying only one lof mutation allele (cyp2c19 2 or cyp2c19 3) with or without gof mutation allele, and poor metabolizers (pms) carrying two lof mutation alleles (cyp2c19 2 and cyp2c19 3), accounting for 41.38%, 56.90%, and 1.72% of all cases, respectively. in the study population, carriers of at least one lof allele (pms and ims) had higher residual platelet reactivity compared to noncarriers (ems), pms and ims had higher incidence of htpr than ems, (2 [100% ] vs. 9 [18.75% ], p = 0.000 and 27 [41.54% ] vs. 9 [18.75% ], p = 0.000, respectively) [figure 1a ]. and there are significant statistic difference between the incidence of htpr of pms and ims too, with a p = 0.000, pms had higher residual platelet reactivity than ims by teg detecting, which means the cyp2c19 polymorphism was a critical contributor of clopidogrel resistance. comparison of the high on - treatment platelet reactivity incidence (a) and the adenosine diphosphate - induced platelet inhibition rate (b) by thrombelastography detecting after clopidogrel among patients with acute coronary syndrome with different phenotype of cyp2c19 2, cyp2c19 3, and cyp2c19 17. htpr : high on - treatment platelet reactivity ; pm : poor metabolizers ; i m : intermediate metabolizers ; em : extensive metabolizers. furthermore, we evaluated the adp - channel platelet inhibition by teg (teg - pi) of each phenotype. it showed that the teg - pi of ems was significantly higher than that in pm and i m patients (12.25% 12.23% and 43.72% 28.30% vs. 60.39% 28.34%, respectively, p ims > ems, p < 0.016) [figure 1a ]. ems teg - pir (60.39%) were higher than that of ims (43.72%) and pms (12.25%) significantly but there was no difference of teg - pir, between pms (12.25%) and ims (43.72%) [figure 1b ]. the htpr rate in this study was higher than the previous studies in chinese chd population (32.76% vs. 20.67%) which probably due to the difference of study population, different assays used for detecting platelet function may contribute to this observation, in additional to our small sample size. it is reported that the platelet function tests are highly test - specific, and even for the same testing method, the optimal cut - off value for the inferior response is also controversial. a standard definition for antiplatelet drug response has not been fully established. using teg to assess the residual platelet reactivity after clopidogrel administration is chosen due to its reproducibility. in addition, teg provides an overall assessment of ex vivo hemostatic function, thus the interaction of all the components of coagulation, including thrombin, platelets, fibrin, and clotting factors is considered in the test, which provides a graphical representation of the speed and strength of clot formation as well as clot stability. processing wise, it uses whole blood for the test, minimizing the lab workload. all these features provide an advantage of the teg method over other platelet function tests such as multiple platelet function analyzer and lta. in summary, teg provides specific and reliable results of the residual platelet activity after clopidogrel admission. the small sample size and short follow - up period are the limitations of this study. the duration of follow - up was 3-month and a follow - up of 12 or even 2436 months will provide more information for mace rate. moreover, it is noted that only part of the genetic polymorphisms that are considered in this study for clopidogrel resistance, the impact of other cyp p450 metabolism enzymes, such as cyp1a2, 2b6, and 2c9, and other allelic variants of 2c19 on platelet reactivity were excluded in this study. there is one patient with cyp2c19 17 mutation, thus the impact of cyp2c19 17 to clopidogrel response is inconclusive, this may be due to the infrequent cyp2c19 17 allele mutation observed low in the asian population. furthermore, other comorbidity, like diabetes and bmi as well as the interactions between clopidogrel and other drugs, such as statins, ca antagonist, and acei, and their effects on pi in different groups were not considered in this study. the small sample size and short follow - up period are the limitations of this study. the duration of follow - up was 3-month and a follow - up of 12 or even 2436 months will provide more information for mace rate. moreover, it is noted that only part of the genetic polymorphisms that are considered in this study for clopidogrel resistance, the impact of other cyp p450 metabolism enzymes, such as cyp1a2, 2b6, and 2c9, and other allelic variants of 2c19 on platelet reactivity were excluded in this study. there is one patient with cyp2c19 17 mutation, thus the impact of cyp2c19 17 to clopidogrel response is inconclusive, this may be due to the infrequent cyp2c19 17 allele mutation observed low in the asian population. furthermore, other comorbidity, like diabetes and bmi as well as the interactions between clopidogrel and other drugs, such as statins, ca antagonist, and acei, and their effects on pi in different groups were not considered in this study.	background : to investigate the contributions of cyp2c19 polymorphisms to the various clopidogrel responses tested by thrombelastography (teg) in chinese patients with the acute coronary syndrome (acs).methods : patients were screened prospectively with acs diagnose and were treated with clopidogrel and aspirin dual antiplatelet therapy. cyp2c19 loss of function (lof) and gain of function (gof) genotype, adenosine 5-diphosphate (adp)-channel platelet inhibition rate (pir) tested by teg and the occurrence of 3-month major adverse cardiovascular events and ischemic events were assessed in 116 patients.results:high on - treatment platelet reactivity (htpr) prevalence defined by pir < 30% by teg in adp - channel was 32.76% (38/116). with respect to the normal wild type, cyp2c19 2, and 3 lof alleles, and 17 gof alleles, patients were classified into three metabolism phenotypes : 41.38% were extensive metabolizers (ems), 56.90% were intermediate metabolizers (ims), and 1.72% were poor metabolizers (pms). of the enrolled patients, 31.47%, 5.17%, and 0.43%, respectively, were carriers of 2, 3, and 17 alleles. the htpr incidence differed significantly according to cyp2c19 genotypes, accounting for 18.75%, 41.54%, and 100.00% in ems, ims, and pms, respectively. eighteen (17.24%) ischemic events occurred during the 3-month follow - up, and there was a significant difference in ischemic events between htpr group and nonhigh on - treatment platelet reactivity group.conclusions:genetic cyp2c19 polymorphisms are relative to the inferior, the antiplatelet activity after clopidogrel admission and may increase the incidence of ischemic events in patients with acs.
type 2 diabetes (t2d) is one of the top five noncommunicable diseases globally, comprising a major, growing cause of morbidity and premature death. in 2012, the international diabetes federation (idf) estimated that 371 million people worldwide were living with diabetes, of which about half live in south asia, the western pacific, and eastern mediterranean regions. asia is now the epicenter of an escalating diabetes epidemic, chiefly due to population growth and ageing in india and china. projections suggest that by 2030, more than 60% of worldwide diabetes cases will come from asia [2, 3 ], with the vast majority of these being type 2 diabetes (t2d). t2d has an enormous economic, psychosocial, and physical impact on individuals, their families, and communities, both directly and indirectly. the direct economic burden of t2d includes both recorded expenditure by health services and unrecorded costs borne by patients and their families. indirect costs such as loss of productivity and disability are also substantial and may match or surpass direct costs. the proportion of worldwide disability - adjusted life years (dalys) due to t2d has soared in recent decades, rising from 43% in 1990 to 54% in 2010. temporary and permanent disability, excess morbidity, and premature death are the consequences of t2d vascular complications, including cardiovascular disease, retinopathy (blindness), nephropathy (kidney failure), and neuropathy (nerve problems) which can lead to amputation. intangible costs due to psychosocial effects on quality of life, diminished contribution to family tasks, and reduced income of care - giving family members are also likely substantial but difficult to assess. the enormous, growing global burden of t2d particularly in asia is now viewed as a crisis by the world health organisation (who) and the united nations (un). there is a major worldwide push to decrease the prevalence and impact of t2d by identifying risk factors, both genetic and nongenetic. explaining the distribution and variation of t2d susceptibility across asia will be vital for reducing the global burden of disease, due to the demographic, cultural, and genetic heterogeneity of asian populations, and t2d risk factor profiles between these populations [710 ]. the vast majority of t2d (about 80%) occurs in low- and middle - income countries (lmics), with india and china providing the largest absolute contributions. the prevalence of t2d is also rising most swiftly in lmics, particularly in asian countries experiencing rapid economic growth (figure 2). however, there are disparities in t2d prevalence among asian populations ; asians from the indian subcontinent (india, pakistan and bangladesh) have the highest prevalence (15.9% to 24.9%), with intermediate prevalence in malays (11.4% to 16.9%) and reduced prevalence in chinese (6.4% to 13.8%) [1113 ]. these risk profile differences may reflect population differences in t2d risk due to ethnicity - specific diet and lifestyle, body composition, genetic effects, or gene - environment interactions, as discussed further in the sections below. the pathogenesis of type 2 diabetes (t2d) involves deficient insulin secretion by pancreatic -cells, and diminished insulin effectiveness in target tissues (insulin resistance) t2d aetiology differs from that of type 1 diabetes (t1d), in which there is absolute insulin deficiency due to the destruction of insulin - producing -cells. impaired insulin secretion and insulin action led to an accumulation of glucose in the blood (hyperglycaemia), with adverse effects on health. clinical features of hyperglycaemia and t2d include excessive excretion of urine (polyuria), thirst (polydipsia), constant hunger, weight loss, vision change, and fatigue. these symptoms may occur suddenly but are often less marked, and t2d patients may be unaware of their illness for several years until further complications develop. glucose homeostasis depends upon a highly regulated feedback system comprising both insulin - secreting -cells and insulin - sensitive target tissues. the function of either component while accounting for the associated homeostatic response of the other can be evaluated using homeostasis model assessment (homa). studies assessing insulin resistance using homa (homa - ir) report continental differences in the relative contribution of insulin deficiency versus insulin resistance to t2d. compared to healthy european - ancestry participants matched for age and body mass index (bmi), asian indian individuals exhibit higher insulin resistance and a greater contribution of insulin resistance relative to insulin secretion to t2d pathogenesis. one study evaluating insulin response to a fixed glucose load also showed that japanese - americans displayed an insulin response more similar to native japanese than european - americans, in spite of sharing a highly westernized lifestyle with their european - american counterparts. there is also variation in the predisposition to insulin resistance between asian populations. for several decades, it has been recognised that the highest propensity is present in asian indians, in whom insulin resistance contributes substantially to t2d pathogenesis, potentially reflecting ancestry - related predisposition to abdominal obesity [21, 22 ]. a recent population based study of 4,136 chinese, malays, and asian indians living in singapore supported these findings, reporting substantially higher insulin resistance in asian indians, intermediate levels in malays, and the lowest levels in chinese (p < 0.001). differences between malays and chinese were removed after adjusting for body mass index (bmi) ; the remaining additional resistance in indians appeared to be mediated by a tendency to higher bmi and bmi - adjusted waist circumference, together with other unexplained factors. dickinson and colleagues studied postprandial hyperglycemia and insulin sensitivity after a 75 gram carbohydrate challenge in 60 lean, healthy individuals from five ethnic groups with similar age, bmi, waist circumference, and birth weight distributions. prior to carbohydrate consumption, fasting insulin was significantly higher in south indians and south east asians, compared to european caucasian, arabic, and chinese individuals (p < 0.001). following the challenge, hyperglycemia was significantly higher in south east asian and chinese participants compared with european caucasians, while indians and south east asians showed a 2 - 3-fold higher insulin response than europeans. a small singapore - based study of 30 individuals also showed significantly reduced insulin sensitivity in south indians compared with chinese or european individuals matched for age, bmi, and physical activity. impaired insulin secretion is associated with -cell dysfunction that results in a reduced insulin - secretion response to rises in blood glucose after eating. the insulin secretion response to various foods can be quantified using the insulin index ; more complex relationship between insulin secretion and insulin sensitivity can be measured using the disposition index (di), which is assessed by an intravenous glucose tolerance - test. a recent family - based study found that a high - fat, low - carbohydrate dietary pattern contributed to obesity, insulin resistance, and reduced -cell function. this finding might be explained by increased free fatty acids (ffas) reducing the expression of -cell specific transcription factors and impairing the -cells ' ability to respond to glucose with appropriate insulin secretion. similar to insulin resistance, insulin secretion also shows evidence of racial differences, being reduced in asians compared with europeans. the insulin index of asians is reduced almost 70% in the progression from impaired glucose tolerance (igt) to t2d, whereas in europeans the corresponding reduction is only 50% [29, 30 ]. a population based - cohort study of insulin resistance and -cell function during pregnancy also found a significantly lower -cell secretory response to pregnancy - induced insulin resistance in south asian and east asian women, compared to european participants with a similar level of insulin resistance. t2d complications can be life - threatening and include cardiovascular disease, nephropathy (kidney disease), retinopathy (blindness), and neuropathy (nerve impairment). observational studies in european american and african american population report that cardiovascular disease risk in individuals with t2d is more than double the rate in the general population and 50% of people with t2d die from cardiovascular disease, primarily heart disease and stroke. there is evidence for population differences in the rate of t2d complications, between asian populations and broader continental groups. a cross - sectional study of 5,707 chinese, indians, and malays showed that the population attributable risk of ischaemic heart disease related to t2d was the highest in indians (40.9%), intermediate in malays (27.9%), and the lowest in chinese (11%). a cohort study found that the progression of kidney dysfunction in t2d was faster in indo - asian (indian, pakistani, and bangladeshi) subjects - with an estimated 2 - 3-fold increase in the mean rate of rise of serum creatinine over a constant follow - up period compared to european - ancestry subjects. the prevalence of diabetic end stage renal disease (esrd) has also been reported as significantly higher in asian t2d subjects (52.6%) compared to caucasians (36.2%). another microvascular complication of t2d, diabetic retinopathy, represents about five percent of all cases of global blindness. visual impairment occurs as a result of long - term, accumulated damage to small blood vessels in the retina. a recent cross - sectional study conducted by the diabetic retinopathy in various ethnic groups in uk (drive uk) found that south asian t2d populations have significantly higher prevalence of diabetic retinopathy (42.3% versus 38%) and sight threatening diabetic retinopathy (10.3% versus 5.5%) compared to white europeans. combined with reduced blood flow, neuropathy (nerve damage) in the feet increases the risk of foot ulcers, infections, poor wound healing, and poor distal circulation, eventually increasing the risk of limb amputation. due to the elevated risk of these life - threatening complications, mortality risk among people with diabetes is at least double that of individuals without diabetes. a range of lifestyle and clinical factors contribute to risk of insulin resistance and t2d, including elevated body mass index (bmi), high waist - to - hip ratio (whr), physical inactivity, and diet (figure 1). according to the world health organisation (who), body mass index (bmi) is a simple index of weight - for - height that can be widely used to classify overweight and obesity in adults. it is defined as a person 's weight in kilograms divided by the square of their height in meters (kg / m). individuals with bmi greater than or equal to 30 kg / m are classified as obese for international standardised comparison. obesity elevates serum fatty acid concentrations, reducing glucose uptake and increasing fatty acid uptake by the liver, skeletal muscle, and pancreatic -cells. reduced glucose uptake elevates serum glucose, stimulating further insulin secretion ; it is the lack of response to this secreted insulin that induces insulin resistance. continually high insulin secretion in turn produces metabolic stress in pancreatic -cell mitochondria, inducing the release of reactive oxygen species that damage mitochondria. over time, mitochondria lose their ability to maintain cellular processes and -cells undergo apoptosis, irreversibly reducing insulin secretion potential. associations between bmi, percentage of body fat, and body fat distribution differ across populations, influencing the thresholds at which t2d risk increases. asian t2d patients have lower average bmi compared to european patients, which might reflect higher percentage body fat in asians (35% higher) than europeans for a given bmi [45, 46 ]. similarly, for a fixed body fat percentage, asians have a 3 to 4 unit lower bmi than europeans. the body fat percentage is also different between asian groups ; for fixed bmi, it tends to be the highest in indians, followed by malays and chinese. one study also showed that among asians, indians have the highest prevalence of obesity (35.8% (95% ci : 32.439.3)), followed by malays (32.0% (95% ci : 30.633.4)) and chinese (19.7 (95% ci : 17.921.6)). however, due to differences in body composition, recent studies have shown that waist circumference (wc) measurement or waist - to - hip ratio (whr) is a better predictor of t2d in asian populations than simple bmi or body fat percentage [48, 49 ], since these latter measures are insensitive to differences in body fat distribution. high waist - to - hip ratio (whr) and waist circumference (wc), or abdominal obesity, is a major cause of insulin resistance since subcutaneous abdominal adipocytes drain their lipolytic products (free fatty acids) directly into the portal vein. these free fatty acids are thought to decrease hepatic clearance of insulin and worsen systemic hyperinsulinemia, a precursor to t2d. additional factors such as reduced secretion of adiponectin by adipose tissue may also contribute to the insulin - resistant state in individuals with abdominal obesity. adiponectin is an adipose tissue - specific protein that controls a number of metabolic processes, including insulin sensitivity and fatty acid oxidation. the prevalence of abdominal obesity differs between ancestral groups and seems particularly marked in certain ethnic populations such as native americans, african - americans, asians, and pacific islanders [5456 ]. the multi - ethnicity study of atherosclerosis found that for a given waist circumference, chinese have the highest diabetes incidence, followed by hispanic, african, and european - ancestry individuals, a finding that may be explained by higher levels of visceral adipose tissue (vat) in chinese compared with europeans, at a fixed waist circumference. the same study also found that south asians have substantially higher visceral adipose tissue compared to europeans for given waist circumference. this might explain increased lipid and insulin levels observed in south asians compared with europeans at the same wc and/or whr. such differences are apparent not only between asian and other continental populations, but also among asian populations. among three major asian groups, the prevalence of abdominal obesity seems to be significantly higher among indians (61.8% (95% ci : 58.365.2)) compared with malays (45.3% (95% ci : 43.846.8) or chinese (40.4% (95% ci : 38.042.7)). the increasing global prevalence of t2d parallels escalating obesity rates resulting from reduced physical activity, increased intake of total calories, saturated fat (especially in fast food), and sugar - sweetened beverages (ssbs) in many societies. asian populations are undergoing a nutrition transition in conjunction with the increasing adoption of westernized lifestyles. in india and china, for example, caloric intake from animal fat has almost doubled in recent decades [60, 61 ]. high consumption of red and processed meat, ssbs, and refined grains with associated low consumption of cruciferous and yellow vegetables is strongly associated with increased in t2d. at the same time, physical activity has reduced in asian populations due to rapid urbanization and modernization [63, 64 ], further increasing t2d risk. metabolic features including elevated blood pressure, hyperglycaemia, and hyperlipidaemia increase t2d risk by several - fold. a recent multi - ethnic population - based survey indicated population differences in the prevalence of metabolic syndrome features, irrespective of t2d status. indians seem to have higher levels of triglycerides and hyperglycaemia and lower hdl cholesterol, compared with malay and chinese. these findings parallel those of a case - control study in which indians from uk and indians from india had higher total insulin and triglycerides and lower hdl cholesterol compared to european individuals, irrespective of shared environmental influences. other factors that have been associated with t2d risk include short sleep duration [67, 68 ], increasing age, which may reflect reduced exercise and muscle mass, history of gestational diabetes, polycystic ovary syndrome, severe mental illness, and having a family history of the disease. a recent randomized, crossover study found that sleep deprivation impairs peripheral metabolic pathways, thereby reducing insulin sensitivity. the loss of skeletal muscle mass with age, or sarcopenia, is also related to insulin resistance, with sarcopenia thought to cause insulin resistance and thereby increase risk of diabetes. in turn, finally, patients with severe mental illness such as schizophrenia or bipolar disorder have 3-fold higher risk of developing t2d compared to the general population ; this may result from underlying lifestyle factors, adverse effects of pharmacotherapy, and possible common genetic and/or environmentally linked pathophysiologic processes. in addition to conventional risk factors, family, twin and genetic studies show that t2d susceptibility has a substantial genetic component. full siblings of t2d probands have a 3060% increased risk of disease, compared with the general population [74, 75 ] and children with one affected parent have a 40% lifetime risk of developing t2d, which rises to almost 70% if both parents are affected. twin studies also show higher t2d concordance in monozygotic (6070%) compared with dizygotic twins (2030%) [7779 ]. the proportion of trait variance due to additive genetic effects is termed heritability and twin study heritability estimates are on the order of 3070% for t2d and about 60% for impaired glucose tolerance (igt) [80, 81 ]. twin studies also demonstrate a substantial genetic component for quantitative phenotypes related to glucose homeostasis, with heritability estimates of 7585% for in vivo insulin secretion, ~50% for peripheral insulin sensitivity, and ~50% for glucose metabolism. population differences in t2d pathophysiologic and risk factor profiles have been discussed in previous sections. it has been suggested that such differences may partly reflect population differences in the frequency of particular genetic risk factors and/or population - specific interactions between genetic and environmental factors. observed patterns of t2d inheritance, combined with the results of recent large - scale genetic studies, suggest that the genetic component of t2d is complex, involving multiple genetic variants of individually small effect (polygenic model). there have been three main approaches employed to identify genetic risk variants for such common complex disorders : linkage studies, candidate gene association studies (cgas), and, more recently, genome - wide association studies (gwas). familial linkage studies seek to identify broad genomic regions harboring disease risk variants by tracking disease and genetic marker segregation through multiple generations of families. familial linkage studies are challenging for disorders with advanced age at onset, as parents may no longer be alive. further challenges include difficulty in collecting accurate genealogical information and genetic (locus) heterogeneity, meaning that a particular risk locus contributes to disease in only a subset of families. more broadly, this approach is limited by low power for common variants of small effect and its inability to precisely localise underlying risk variants. earlier linkage studies found four (4) genetic loci linked with t2d ; capn10, enpp1, hnf4a, and acdc (adipoq). however, only the hnf4a locus has been confirmed by recent large - scale genome - wide association studies (gwas). hnf4a, together with the related locus, hnf1a and also gck also account for up to 80% of rare monogenic forms of diabetes. these diabetes cases present as familial, young onset, noninsulin dependent diabetes mellitus (maturity onset diabetes of the young or mody) and are inherited in a mendelian dominant pattern. unlike common polygenic forms of t2d, these monogenic forms require only one pathogenic genetic variant to produce disease. in contrast to linkage studies, the candidate gene approach searches for associations between common genetic variants and disease, restricting the search region to prespecified genes of interest. candidate genes are typically selected based on a priori knowledge or hypotheses reflecting the gene 's presumed biological role in disease. the most common study design is the case control study ; for a particular genetic variant, this involves comparing the frequency of genetic alleles between individuals with and without disease, aiming to identify alleles that are associated with disease status. although a mainstay of the initial era of disease gene mapping, the candidate gene approach has been limited by small sample sizes, restriction of hypotheses to known biology, and an inability to replicate many results. while candidate gene studies have reported numerous variants as beeing associated with t2d, just three loci, pparg, kcnj11, and tcf7l2, we note that the tcf7l2 association study was informed by prior genome - wide linkage study showing suggestive linkage between t2d and the 10q genomic region harboring tcf7l2. within the last five years, genome - wide association studies (gwas) have emerged as the method of choice for identifying common genetic variants associated with complex disease. gwas were facilitated by completion of the human genome project in 2003, the international hapmap project in 2005 that catalogued millions of common single nucleotide polymorphisms (snps), and the parallel development of high throughput genotyping arrays. single nucleotide polymorphisms (snps) are dna sequence variants in which a single nucleotide differs between individuals. snps have a low historical mutation rate, are amenable to high throughput genotyping, and are distributed abundantly across all 22 autosomes and the sex chromosomes. typically several million variants are screened genome - wide ; appropriate adjustment of the prespecified significance level is thus necessary to avoid an increase in false positive results. based on patterns of human genomic correlation, bonferroni correction for one million independent tests is the accepted approach, with variants required to reach a pointwise p value < 5 10 (or 0.05/1,000,000) to be declared very large sample sizes are necessary to identify associations of modest effect, which is often achieved via international collaboration and the formation of consortia. the existence of such collaborations also facilitates rapid replication of findings in independent samples, a requirement for publication. it has been eight years since the first notable gwas finding in 2005, identifying a common allele of large effect associated with age - related macular degeneration. the year 2007 was coined the year of gwas, due to the explosion of gwas publications in that year. from 2005 to september 2013, there have been more than 1,600 gwas published reports for a range of human diseases and traits, with an online catalogue established by the national human genome research institute at the us national institutes of health to collate major findings (http://www.genome.gov/gwastudies/). although complicated and costly, gwas have successfully identified thousands of genetic loci associated with common complex diseases under the common disease common variant (cdcv) hypothesis. the first t2d gwas was published in 2007, and as of september 2013, there were more than 40 publications on t2d and its complications listed in the online catalogue of published genome - wide association studies (http://www.genome.gov/gwastudies/). at the time of writing, the catalogue describes over 100 individual snps showing genome - wide significant association (p < 5 10) with t2d and related metabolic traits across diverse populations (table 1) and over 60 snps showing suggestive association (p < 1 10) (table 2). the first t2d gwas was conducted in european - ancestry participants 2007 by sladek and colleagues, using a discovery sample of 600 cases and 600 controls and a separate european replication sample of nearly 3,000 cases and 3,000 controls. this small study of early onset t2d reported t2d - associated variants in three novel susceptibility genes : tcf7l2 and hhex / ide which are associated with -cell function and slc30a8, encoding a zinc transporter highly expressed in pancreatic islets. several months later, four additional european studies [74, 98, 103, 104 ] confirmed association of variants at these loci and identified additional associated variants in igf2bp2, associated with -cell dysfunction, and cdkn2a / cdkn2b and cdkal1, which are both associated with -cell development [105, 106 ]. during this time, variants in the fto (fat mass and obesity associated) gene were also identified with important effects on obesity and hence, indirectly, t2d [107, 108 ]. interestingly, as the effect of fto variants on t2d is only via obesity, the fto locus was not identified in t2d gwas using cases and controls matched for bmi. two of these early publications also showcased the output of international consortia : the uk - based wellcome trust case control consortium (wtccc) and the usa - based diabetes genetics initiative (dgi), highlighting the benefits of large - scale collaboration in the gwas era. since these initial gwas had modest power to detect variants with modest effects on disease risk, follow - up studies employed meta - analysis to increase sample size and hence power to detect additional loci of similar or smaller effect. the first t2d gwas meta - analysis was published in 2008 and was also a european study, representing collaboration between three different consortia ; the wtccc, dgi, and the finland united states investigation of niddm genetics (fusion) which combined to form the diabetes genetics replication and meta - analysis (diagram) consortium. this study utilised an enlarged discovery sample of 4,549 cases and 5,579 controls with replication in further 24,194 cases and 55,598 controls, all of european - ancestry. this study identified associated variants at six additional novel loci : jazf1, cdc123, tspan8 and thada which are associated with -cell dysfunction [110, 111 ], adamts9 which is associated with insulin action, and notch2, associated with glucose - stimulated glucagon secretion by pancreatic islet cells. the first two large - scale t2d gwas conducted in asian populations were reported in 2008, each employing a multi - stage design in east asian groups. both studies reported association of variants in kcnq1, encoding the alpha subunit of a voltage - gated potassium channel expressed in the pancreas [113, 114 ]. these studies clearly demonstrated the utility of extending t2d gwas to non - european populations ; association of the kcnq1 variants with t2d was not detected in previous european gwas, due to markedly lower frequency of the risk allele in europeans (5% versus 40%), resulting in dramatically reduced power. a european meta - analysis subsequently confirmed association of the kcnq1 variants with t2d in europeans but at significance levels far below thresholds usually inspiring replication or follow - up studies (p ~ 0.02). a european study published in 2009 used multiple samples of french, danish, and finnish ancestry to identify association of variants in the insulin receptor substrate 1 gene (irs1), showing that the risk allele is also associated with insulin resistance and hyperinsulinaemia in large population - based cohorts. this contrasted with the apparent biology of previous associations, which principally related to impaired pancreatic -cell function. this first wave of t2d gwas was succeeded by a second wave beginning in 2010, in which existing and new datasets were combined into expanded meta - analyses. the most notable was a large european study reported by voight and colleagues, involving ~42,000 t2d cases and 100,000 controls split between discovery and replication stages and identifying twelve new associated loci. these included x - chromosomal association and an hnfa1a locus overlapping with the locus implicated in mendelian monogenic (single gene) forms of diabetes. other studies reported at this time included three east asian studies, one african american, and one european study, which together identified nine (9) additional loci [117121 ]. three of these genes have unknown function (rbms1, ptprd, and srr) [117, 118 ], while rps12, limk2, and auh are associated with diabetic nephropathy, c2cd4a is associated with -cell dysfunction [119, 122 ], spry2 is associated with obesity and insulin resistance [120, 123 ], and sash1 is associated with insulin growth factors. a subsequent 2011 meta - analysis included three southeast asian populations : chinese (3955 subjects), indian (2146 subjects), and malay (2034 subjects) and it further emphasized the value of surveying diverse ethnic groups. this study was the first to include individuals from the malay population, the largest group in southeast asia, with a population size of more than 300 million. this study alone contributed an additional 16 novel loci, in spite of its relatively modest sample size ; this partly reflected higher minor allele frequencies in southeast asian populations at some associated loci (e.g., rs3792615, number 18 in table 2). the first t2d gwas in south asian populations was also published in 2011, including individuals from india, pakistan, sri lanka and bangladesh. using a relatively modest sample size (5,561 cases and 14,458 controls in the discovery step) five additional novel t2d loci were discovered : hnf4a, involved in monogenic forms of diabetes and associated with -cell development ; grb14 which is associated with obesity and insulin resistance ; and another three loci with less clear functions ; ap3s2, st6gal1 and vps26a. another large meta - analysis in east asian groups were performed in 2012 and identified 10 further novel loci with mostly unknown function except for glis3, associated with -cell development. it is interesting that the maea variant discovered in this study is unique to east asian and african populations, being monomorphic in europeans and south asians (table 1 ; number 24). several other variants identified in this study have substantially higher risk allele frequency (raf) in east asians than europeans, for example, zfand3 (table 1 ; number 28 ; 34% versus 12%), fitm2-r3hdml (table 1 ; number 111 ; 41% versus 18%), and rps3p7-maf (table 2 ; number 56, 18% versus 1%), enhancing their detection in east asian samples of relatively modest size. reflecting the success of initial t2d gwas and the fast pace of technology, in 2012 voight and colleagues designed the metabochip, a custom genotyping array enriched for variants shown to be associated with cardiometabolic traits via gwas. these traits include t2d, coronary heart disease, myocardial infarction, body mass index, glucose and insulin level, lipid levels, and blood pressure. this new platform offers a powerful and cost - effective approach to both the discovery and follow - up of variants associated with these related traits, due to comprehensive coverage of previously associated loci (~120,000 snps). morris and colleagues used the metabochip to discover and characterize t2d - associated variants via meta - analysis of 34,840 cases and 114,981 controls of european descent, finding ten novel loci not reported in previous european studies, all of which reached genome - wide significance. another study using the metabochip combined newly available samples with samples from previous discovery meta - analyses, using genotype data for 66,000 follow - up snps. this study identified 41 novel glycaemic associations, 33 of which were also associated with t2d. this study implicated new loci in the aetiology of t2d and increased the overlap between loci associated with both glycaemia and t2d. these studies highlight the metabochip as a promising tool for identifying novel and robustly associated loci, facilitating future research into underlying biology. taken together, the results of t2d gwas signify tremendous progress in our quest to understand the genetic causes of t2d. genetic variants showing replicable association with t2d uniformly exert only a modest effect on disease risk, with per - allele odds ratios typically in the range of 1.11.3 (table 1). the combined effect of all variants reported to date explains only about 10% of observed familial clustering. while some appear to be associated with -cell function and insulin resistance, the biological role of many of them remains unknown. this suggests that the findings to date represent the first stage of a long journey to understanding t2d genetic risk. the distribution of odds ratios observed for validated t2d - associated snps suggests that numerous, associated loci exist with even smaller effects than those identified to date. one would not expect such loci to have reached genome - wide significance in previous gwas due to insufficient power. the existence of such additional small effect loci is consistent with the pattern of additional associated variants being discovered as sample sizes have increased ; it is also consistent with validated snp associations explaining only a small proportion of the t2d heritability estimated from twin studies, known as the missing heritability problem. two methods have recently been developed for assessing the contribution of common snps not reaching genome - wide significance to the heritability of a trait. both methods test the combined effects of thousands (or hundreds of thousands) of snps upon a trait of interest. a recent study by stahl and colleagues used polygenic analyses and linear mixed modelling to show that thousands of snps contribute to t2d risk, estimating that about 50% of observed variance in t2d risk could be attributed to the combined effects of all snps genome - wide. these investigators suggested that at least 70% of t2d heritability can be attributed to common snps represented on gwas arrays, with most having very small individual effects upon disease risk. the frequency of t2d risk alleles often varies between populations, producing population differences in the attributable risk due to a particular genetic risk factor or combination of risk factors. association of kcnq1 variants was found in east asian populations [113, 114 ] using a considerably smaller sample size than that required to detect the association in with european populations, reflecting higher allele frequency (33% in east asian versus 8% in europeans) and hence statistical power in asian groups. in addition, variants at the tcf7l2 locus showed the inverse ; a high risk allele frequency in europeans (30%) compared to a low frequency in east asians (3%) enhanced the detection in european studies. similarly, the syk variant demonstrates a raf of 26% in east asians and only 2% in europeans and is monomorphic in africans (table 2 ; number 36). further, a number of t2d risk variants are monomorphic (not variable) in some populations, preventing the detection of an association in these groups. the recently reported scgg variant is unique to indian punjabi sikh, being monomorphic in both european and african populations (table 1 ; number 92). other instances include the thada variant, which was discovered in european populations but is monomorphic in east asians (table 1 ; number 4), rnd3-fabp5p10 that was discovered in african americans but is monomorphic in europeans (table 1 ; number 6), and g6pc2, discovered in europeans but monomorphic in africans (table 1 ; number 10). for a set of snps showing association with t2d across multiple populations, table 3 shows risk allele frequencies and odds ratios for different populations in which associations have been reported. for these 14 snps, risk allele frequencies commonly differ across populations ; however, allelic effects upon disease seem markedly consistent in both direction and magnitude, given overlapping confidence intervals for allelic odds ratios. taken together, these results suggest that population differences can have important effects on power to detect common genetic associations for t2d in samples of diverse ancestry but may have less impact upon disease risk within individuals carrying the identified risk alleles. nevertheless, at the population level, the attributable risk of such genetic variants will increase with allele frequency, thus potentially influencing population disease burden. significantly, a recent study assessing thousands of genetic associations showed that t2d has the most extreme population differentiation of risk allele frequencies among a broad range of complex diseases. t2d risk allele frequencies demonstrated clear gradient matching paths of early human migration, suggesting potential differences in evolutionary adaptation to food availability, dietary patterns, or agricultural practices. this is consistent with thrifty genotype hypothesis [139, 140 ], which proposes that susceptibility to obesity and t2d in some populations reflects historical positive selection for genotypes promoting efficiency of metabolism, and energy and fat storage, thus providing an advantage in times of nutrient shortage. this might explain the extraordinarily high prevalence of diabetes now seen among certain populations [34, 142, 143 ], potentially reflecting historical feast and famine cycles, increasing the frequency of thrifty genotypes and genetic predisposition to obesity and diabetes. while being unproven, this may partly explain higher susceptibility to abdominal obesity at lower bmi and reduced muscle mass with increased insulin resistance in asian compared with caucasian populations. nevertheless, pronounced population differentiation of t2d risk allele frequencies provides a strong rationale for further comprehensive genetic studies of t2d in diverse populations, expanding on the comprehensive studies in european samples. to date, a range of non - european t2d gwas have been conducted, including studies in japanese [114, 119, 138, 144 ], chinese [117, 145, 146 ], african - american [121, 147, 148 ], southeast asian, hispanic, mexican - american, south asian, indo - european, and indian punjabi sikh. these studies have led to new discoveries, including novel loci and loci that seem specific to certain populations [119, 127, 151, 152 ]. while many loci appear to contribute broadly to t2d risk, some loci have currently been confirmed in european populations only, including wfs1, notch2, thada, adamts9, tspan8/lgr5, ins - igf2, adcy5, gck, mtrnr1b, hmga2, hnf1a, zbed3, klf14, zfand6, prc1, tles / chchd9, and rbms1 [109, 116, 153155 ]. other loci currently show association specifically in east asian populations, including ptprd, srr, cdc123/camk1d, psmd6, maea, zfand3, kcnk16, gcc1/pax4, glis3, and pepd [117, 119, 120, 127 ]. on the other hand, tmem163 and sgcg appear unique to south asian and indian punjabi sikh, respectively. some of these discoveries may reflect the impact of population allele frequency differences, as previously discussed. in such cases, larger studies may eventually show that some loci contribute to disease across a broader range of populations. seemingly population - specific genetic associations for t2d may also reflect differences in the patterns of genomic correlation, or linkage disequilibrium (ld), between associated marker loci and the underlying unobserved functional variants. populations with different demographic histories will often display different patterns of ld reflecting population differences in evolutionary recombination. older populations such as those in africa have lower ld and can be helpful for finely localizing a risk variant following an initial association finding. this is because the genomic distance between disease - associated markers and true risk variants is likely to be smaller in such populations. thus, the apparent population - specificity of some known t2d risk alleles may reflect population differences in risk allele frequencies or ld between tagging and causal variants, rather than actual population - specificity of the underlying functional risk loci. we note that population - specific estimates of disease variance explained by all known t2d loci although not widely reported do seem largely similar between european and asian populations. in their large 2012 study, morris and colleagues estimated that known common variants explain about 5.7% of t2d disease variance in europeans. in 2013, tabassum and colleagues estimated that known loci combined with one novel indian - specific locus explained 7.65% of t2d risk variance in south asian indians. the slightly higher estimate in indians may potentially be explained by the inclusion of additional variants discovered between publications of the two studies, together with the inclusion of the indian - specific locus discovered in the tabassum study. thus, available evidence thus does not strongly suggest that differences in the cumulative variance explained by known common t2d risk alleles can explain the markedly higher t2d prevalence observed in south asians. interestingly, however, very recent studies show that population differences in linkage disequilibrium (ld) and the presence of multiple independent variants within a locus can markedly influence estimates of variance explained by known risk variants [158, 159 ]. detailed fine mapping of t2d susceptibility loci in diverse populations, combined with the identification of underlying functional variants, may thus reveal population differences in the contribution of known loci to disease. future research may also show the extent to which population differences in t2d risk can be explained by rare alleles, gene - environment interactions, or epigenetic effects. in addition to the effects of specific genetic and environmental risk factors, gene - environment interactions are likely important mediators of population differences in t2d risk and contributors to the missing heritability problem. indeed, given the relative stability of dna sequence within populations over decades, recent increases in t2d prevalence must largely reflect environmental changes. accordingly, the single largest contributor to t2d risk is obesity, and the global t2d epidemic chronologically parallels the global obesity epidemic. a paucity of studies has examined gene - environment interactions in the context of t2d in general, in asian populations. a study by qi and colleagues found that a high genetic risk score formed from 10 t2d - associated snps was further increased by the presence of a westernized dietary pattern characterised by increased red and processed meat intake and reduced dietary fibre. the westernized diet was not associated with increased risk among those with a low genetic risk score. several studies have also found evidence for interactions between t2d - associated variants in tcf7l2 and the quality and quantity of ingested carbohydrates in the context of t2d risk [161163 ]. these studies support a possible contribution of gene - environment interactions to t2d risk, together with a potential model where interactions between recent lifestyle transitions and genetic risk factors may be contributing to the rapidly increasing prevalence of t2d in asian populations. however, these preliminary findings require validation. future analyses in well - designed, well - powered studies will help to clarify the role of gene - environment interactions in population differences in t2d risk. similar to the thrifty genotype the hypothesis relates to the metabolic consequences of fetal malnutrition, proposing that adaptation to a low - calorie intrauterine environment induces permanent changes in chromatin structure and gene expression that influence insulin secretion and resistance, promoting more efficient energy utilisation and thus fetal survival. according to the hypothesis, such epigenetic changes may predispose to insulin dysregulation, obesity, and t2d in later life. in support, epidemiologic studies have shown associations between small birth size, a marker for fetal malnutrition, and adult - onset t2d [165, 166 ]. a study by van hoek and colleagues in the dutch famine birth cohort detected an interaction between an igf2bp2 polymorphism and prenatal famine upon glucose level in the offspring. interactions between other t2d risk variant alleles and birthweight have also been associated with increased t2d risk [168, 169 ]. we have discussed differences in prevalence, risk factor profiles, and genetic risk allele frequencies between different asian countries and between asian and other continental populations. given these differences, continued t2d genetic studies in diverse populations are likely to contribute crucially to the broadening terrain of shared and unique population genetic effects for this disorder. future studies will ideally include large, population - specific characterisation of risk variants, studies of gene - environment interaction, and epigenetic studies. well - powered, well - designed studies performed in diverse asian populations should enhance the benefits of genetic discoveries and their ultimate clinical translation for these large susceptible groups.	the prevalence of type 2 diabetes is rising rapidly in both developed and developing countries. asia is developing as the epicentre of the escalating pandemic, reflecting rapid transitions in demography, migration, diet, and lifestyle patterns. the effective management of type 2 diabetes in asia may be complicated by differences in prevalence, risk factor profiles, genetic risk allele frequencies, and gene - environment interactions between different asian countries, and between asian and other continental populations. to reduce the worldwide burden of t2d, it will be important to understand the architecture of t2d susceptibility both within and between populations. this review will provide an overview of known genetic and nongenetic risk factors for t2d, placing the results from asian studies in the context of broader global research. given recent evidence from large - scale genetic studies of t2d, we place special emphasis on emerging knowledge about the genetic architecture of t2d and the potential contribution of genetic effects to population differences in risk.
in the pokhara valley, nepal, there are many unpublished cases of wasp poisoning which take a heavy death toll annually. wasp stings usually cause local allergic reactions but can sometimes lead to intravascular haemolysis, rhabdomyolysis, thrombocytopenia, acute tubular necrosis, acute hepatic injury (1) and even myocardial infarction (2) in addition to various respiratory and neurological (3) manifestations. death from wasp envenomation is a rare event and results from acute renal failure (arf) involving various mechanisms. although arf after wasp stings is typically caused by atn in the setting of haemolysis or rhabdomyolysis, in some patients, renal failure may result from a direct nephrotoxicity of wasp venom or acute interstitial nephritis from a hypersensitivity reaction. we here we report two cases of acute renal failure after wasp stings (vespa affinis). a 55-year - old farmer who had been collecting fodder from a jungle was admitted with dyspnea, hoarse voice and myalgia within 8 hours of being attacked by several wasps. the patient was given intravenous saline, oxygen, salbutamol (2-adrenergic receptor agonist), chlorpheniramine (antihistamine), cyproheptadine (antihistamine), prednisolone (corticosteroid) and ranitidine (histamine h2-receptor antagonist). he also received fluid, mannitol and furosemide. by the next morning he had haemoptysis and had produced 400 ml of dark urine. on examination, the patient was drowsy, pale, icteric and cyanosed and had approximately one hundred and fifty red and swollen sting marks all over the body. he died on the 8th day following admission despite aggressive therapy with medication, blood transfusions, assisted ventilation, and 16 cycles of dialysis. he presented with approximately twenty - five sting marks in exposed areas of face, throat, hands and legs [fig. 1 ]. the patient was treated in the primary health centre with rubbing of saliva and papaya slices over the sting marks and referred to teaching hospital almost 24 hours after being stung. on examination, the patient had a rapid pulse, unrecordable blood pressure, icterus, an urticarial rash in exposed parts of the face, legs and hands, facial puffiness, and a swollen left ankle and right knee joint. he was treated with oxygen, salbutamol, chlorpheniramine, cyproheptadine, prednisolone and ranitidine. his hepatic and renal function improved gradually with fluid challenge, furosemide, mannitol, bicarbonate infusion, dopamine, and 12 cycles of dialysis in 3 weeks time. direct toxicity is rare, but has been reported in cases when a very large amount of venom is injected. immediate hypersensitivity reactions, such as bronchospasm in the first case and an urticarial rash in the second, are known to occur. in our report, both reactions responded to steroids and antihistaminics. the second patient had swollen joints indicating serum sickness - like reaction in a sensitized individual. wasp venom contains toxic melittin, apamine, phospholipases a1, hyaluronidase, acid phosphatase, histamine, and degranulating peptide mastoparan (4). these components have direct and indirect cytotoxic (hepatic, renal and myocyte membrane), hemolytic, neurotoxic and vasoactive properties, which can cause intravascular haemolysis and rhabdomyolysis (5, 6). wasp venom can cause arf by several mechanisms, which include atn, acute interstitial nephritis, pigment nephropathy resulting from rhabdomyolysis (myoglobinuria) or intravascular haemolysis (haemoglobinuria) and hypotension caused by an anaphylactic reaction (7, 8). sakhuja had postulated that direct toxic injury could be one of the possible mechanisms of arf following wasp poisoning (9). many cases of rhabdomyolysis - associated arf have been published, but those due to wasp stings are rare. the wasp venom has deleterious effect on renal tubules and glomeruli (albuminuria, haematuria and arf), red blood cells (haemolysis, reticulocytosis, unconjugated hyperbilirubinaemia), muscles (rhabdomyolysis, elevated creatinine phosphokinase and lactate dehydrogenase, myoglobinuria) and liver (elevated transaminases, hypoalbuminaemia and prolonged prothrombin time) (10). kularatne had described similar multi - organ failure with high mortality following wasp poisoning owing to direct toxic effect (11). in the first case we presented, the patient had myalgia, indicating muscle injury as evidenced by elevated cpk, ldh and ast and myoglobinuria (728 ng / ml). (12) reported for the first time that acute tubulointerstitial nephritis could lead to arf in wasp sting cases. in the second case we present, the patient had eosinophiluria, indicating interstitial nephritis. he did not report taking any medication which might have had nephrotoxic side - effects, and no other causes of atn could be found. hence it can be hypothesized that the atn was caused by a hypersensitivity reaction to the wasp venom. direct toxicity is rare, but has been reported in cases when a very large amount of venom is injected. immediate hypersensitivity reactions, such as bronchospasm in the first case and an urticarial rash in the second, are known to occur. in our report, both reactions responded to steroids and antihistaminics. the second patient had swollen joints indicating serum sickness - like reaction in a sensitized individual. wasp venom contains toxic melittin, apamine, phospholipases a1, hyaluronidase, acid phosphatase, histamine, and degranulating peptide mastoparan (4). these components have direct and indirect cytotoxic (hepatic, renal and myocyte membrane), hemolytic, neurotoxic and vasoactive properties, which can cause intravascular haemolysis and rhabdomyolysis (5, 6). wasp venom can cause arf by several mechanisms, which include atn, acute interstitial nephritis, pigment nephropathy resulting from rhabdomyolysis (myoglobinuria) or intravascular haemolysis (haemoglobinuria) and hypotension caused by an anaphylactic reaction (7, 8). sakhuja had postulated that direct toxic injury could be one of the possible mechanisms of arf following wasp poisoning (9). many cases of rhabdomyolysis - associated arf have been published, but those due to wasp stings are rare. the wasp venom has deleterious effect on renal tubules and glomeruli (albuminuria, haematuria and arf), red blood cells (haemolysis, reticulocytosis, unconjugated hyperbilirubinaemia), muscles (rhabdomyolysis, elevated creatinine phosphokinase and lactate dehydrogenase, myoglobinuria) and liver (elevated transaminases, hypoalbuminaemia and prolonged prothrombin time) (10). kularatne had described similar multi - organ failure with high mortality following wasp poisoning owing to direct toxic effect (11). in the first case we presented, the patient had myalgia, indicating muscle injury as evidenced by elevated cpk, ldh and ast and myoglobinuria (728 ng / ml). (12) reported for the first time that acute tubulointerstitial nephritis could lead to arf in wasp sting cases. in the second case we present, the patient had eosinophiluria, indicating interstitial nephritis. he did not report taking any medication which might have had nephrotoxic side - effects, and no other causes of atn could be found. hence it can be hypothesized that the atn was caused by a hypersensitivity reaction to the wasp venom. wasp stings pose a great environmental hazard in nepal and early recognition of anaphylactic shock, hepatic or renal dysfunction, rhabdomyolysis or haemolysis and rapid transport to hospital are essential steps of management to avoid fatalities. arf due to toxic or pigment nephropathy and tubulointerstitial nephritis should be considered in any oliguric and azotemic patient following wasp attack.	acute renal failure is an unusual complication of wasp stings. we report two cases of renal failure after multiple wasp stings (vespa affinis). both patients had evidence of intravascular haemolysis, hepatic dysfunction, oligo - anuria and azotaemia and required dialysis. the first patient had severe hemolysis, rhabdomyolysis, pigment and venom nephropathy and died on the 8th day in hospital. the second patient, who recovered completely in 3 weeks time with steroid and antihistaminic therapy, had interstitial nephritis. although acute renal failure after wasp stings is typically caused by acute tubular necrosis (atn) in the setting of haemolysis or rhabdomyolysis, in some patients, acute renal failure may result from a direct nephrotoxic effect or acute interstitial nephritis from a hypersensitivity reaction.
the neuroendocrine tumors (nets) are a heterogeneous group of neoplasms that share some biological characteristics, that have been addressed as a common entity. in 62%82% of the cases, the nets are located in the digestive system, being designated as gastroenteropancreatic neuroendocrine tumors (gep - nets). the latest figures from the uk, sweden, and switzerland suggested that their incidence is 2 - 3/100000 with a higher prevalence in females. also data from the surveillance, epidemiology, and end results (seer) program database report an increased incidence between 1973 and 2004 of 1.09 to 5,25/100000 [3, 4 ]. nets are classically characterized by their ability to secrete hormones and/or vasoactive peptides, which results in many of their clinical manifestations, ganging up on specific hormonal syndromes. the most frequent syndrome is the carcinoid syndrome, which results from the secretion of serotonin and includes symptoms such as skin flushing, severe diarrhea, abdominal cramping, and electrolyte abnormalities [4, 5 ]. their clinical course is variable, having been considered traditionally with an indolent natural history [1, 4 ]. all of them have the potential of metastatic dissemination and, therefore, are currently recognized as malignant neoplasms. the prognosis and the approach of nets the tumors with grade 3, mitotic count exceeding 20/10 high - powered fields and/or with ki-67 proliferative index exceeding 20% represent aggressive malignant disease with rapid clinical course and a low survival. the liver is the most important place of metastatic disease. more than 75% of patients with nets of the small intestine and 30%85% of patients with pancreatic nets have liver metastases at the time of diagnosis or during the course of their disease. additionally 5%10% of patients with nets of unknown primary location have liver metastases at the time of the diagnosis [6, 8 ]. patients with liver metastases have a worsening of their morbidity and mortality, as compared to patients without these lesions. according to touzios. survival rate to 5 years of patients with untreated liver metastasis is about 13%54%, against 75%99% in patients without liver metastases [6, 9 ]. this revision aims to address the theme of gep - nets with metastatic liver, their clinical significance, prognosis, and its current therapeutic approach with a special focus on medical treatment. patients with nets often develop liver metastases (nlms) affecting significantly their morbidity and mortality. about 75%80% of patients present with hepatic metastasis at the time of diagnosis (synchronous), while 20%25% the presence of liver metastases is closely related to the appearance / aggravation of symptoms. the presence of hepatic metastases leads to less metabolization of these peptides leading to an increase of their circulating levels. sometimes, only then its distribution, in the way it modifies therapeutic management, reflects the aggressiveness of the tumor. type i metastases are lesions only confined to one hepatic lobe or limited to two adjacent segments, corresponding to about 20%25% of cases. type ii metastases include the presence of dominant injury with small satellites contralateral (10%15% of cases). type iii metastases correspond to diffuse multifocal, being the most prevalent being (60%70% cases). because of the possibility of surgical resection, type i is associated with a favorable outcome. type iii metastases have a worse prognosis regarding the greater liver involvement and the lack of surgical approach. in this group of patients however, the complete excision of secondary lesions is only an option for a small number of patients (10%20%). this differs from the normal vascularization system which is derived from the venous system port. the handling of hepatic artery prevents the supply of nutrients and oxygen to the tumor cells with its consequent destruction. for that reason, several techniques have been developed by exploiting duality in the liver vasculature in order to control the disease process. these modalities are include the ablative techniques, the hepatic artery embolization, chemoembolization, and radioembolization (selective internal radiation therapy). patients with predominant metastatic liver disease are those who benefit the most from these therapies. curative intention depends on the stage and presentation of the disease [13, 14 ]. even with hepatic involvement, surgical approach, when feasible, is the best treatment option, with proven benefits in terms of overall survival and quality of life improvement [1, 6, 12 ]. patients treated with surgical resection of the primary tumor and liver metastases had a survival at 5 years of more than 60%, reaching 80% in some studies, with minimum mortality (less than 5%) and admissible morbidity (less than 30%) [6, 12 ]. patients with liver metastasis that are not candidates to surgical treatment (80%90% of cases), still benefit from primary tumor resection. this approach can be combined with other liver - direct therapies increasing patients ' outcome [12, 13, 15 ]. in these cases, where primary tumor was resected therefore, the palliation of the symptoms is also a criterion for surgery [5, 6 ]. regarding the criteria for selecting patients with nlms, there is no consensus, since the several studies developed included a small number of patients and different surgical procedures. however, when the complete excision of a significant number of liver metastasis is possible, surgery must always be considered. in a recent retrospective study, 74 cases were analyzed and the 5-year survival rate was more than 60% in all patients who underwent surgical resection of liver metastases. in 65% of these patients, excision was incomplete, and there was no worsening of the prognosis [6, 12 ]. however, the identification of other predictive variables in patients ' selection for surgery was needed. therefore, clinical studies were conducted, and patients were selected based on tumor grade. the analyses of the results from these studies showed that tumors with high grade had a worse postsurgical outcome when compared to low grade tumors. high grade net tumors are good candidates to chemotherapy and do not seem to beneficiate from surgery. translating these results into clinical practice, in 2008, the european neuroendocrine tumor society (enets) issued general guidelines for surgical resection based on the three types of liver involvement. for patients with hepatic involvement of type i surgical resection is the first therapeutic option, while in patients with the involvement of type iii it is totally excluded. in order to give the best possible treatment to patients with net tumors this evaluation should take into account the following aspects : performance status, localization of the primary tumor, tumor grade, possibility of primary tumor resection, presence and localization of metastases, possibility of complete resection of metastases, presence of extrahepatic disease (extrahepatic disease should be excluded based on adequate image methods), and the presence or absence of carcinoid heart disease [6, 17 ]. in patients with carcinoid heart disease, symptoms control and clinical stabilization should be achieved before aggressive surgery is considered. also, in patients with carcinoid syndrome, even in those under treatment with somatostatin analogues, intra- and postoperative carcinoid crisis must be prevented, using these same somatostatin analogues. preoperative administration of octreotide (300 micrograms subcutaneously) can reduce the incidence of carcinoid crisis and is recommended for patients with a history of carcinoid syndrome who require surgical procedures. calcium and catecholamines may provoke the release of mediators from the tumor and worsen, rather than ameliorate, the syndrome. during a carcinoid crisis, the blood pressure should be supported by the infusion of plasma and octreotide (300 micrograms iv) given immediately. a continuous iv drip of octreotide may be needed. in patients with high risk of carcinoid crisis, an iv octreotide drip may be initiated preoperatively [1, 1823 ]. the presence of abdominal lymph node involvement, including local recurrence, is not an absolute contraindication for surgery, if the excision of lymph nodes and liver metastases and/or recurrent lesions could be done simultaneously or in planned staggered phases. extensive lymphadenectomy and a careful exploration of the entire abdominal cavity should be carried out during surgical procedure. the use of intraoperative echography seems to be of additional value as it allows to define the extent of known lesions and to detect small additional lesions. it is usually recommended in cases where more than 90% of the tumor can be excised or in young patients [6, 12, 20, 25, 26 ]. symptomatic patients also benefit from cytoreductive surgery, as it seems to improve the effectiveness of medical therapy. liver transplantation is a therapeutic approach with an intention to cure, to prolong survival or control of carcinoid symptoms. liver transplantation is only indicated in cases of metastatic disease if the primary tumor is an net. in 1998, lehnert analyzed a total of 103 net patients with liver transplant. the tumor histology and its primary location did not affect the survival in this study, unlike the extent of surgery and the patient 's age [12, 27 ]. however, in a more recent clinical trial, primary location in the duodenum or pancreas was associated with worse prognosis after transplant [6, 12, 28 ]. these findings were, however, not supported by other investigations [12, 29 ]. regarding biomarkers analyses and their correlation with transplant outcome, the authors found that patients with a low ki-67 and normal expression of e - cadherin had a more favorable prognosis. on the other hand, the analysis of the data from the united network for organ sharing between november 1988 and march 2011 showed that only 185 liver transplants were due to nlms. the 5-year overall survival was 57.8%, lower than patients transplanted for other reasons / with other pathologies [12, 14 ]. in patients with diffuse and unresectable liver disease, with uncontrolled symptoms (resistant to medical therapy support), the main criteria for selecting patients for liver transplantation are in these cases being not candidate for resection, diagnosis, and complete resection of the primary tumor at least one year before, no extrahepatic disease, stable disease at least for one year, and failure of nonoperative treatments. patients with net tumors can be selected to treatment with different types of ablative techniques such as cryoablation, alcohol ablation (these two are less used), and radiofrequency ablation (rfa). decreasing temperature lowers cell viability, and this depends both on the rate of colling and the spatial relationship between the ice formation and the cryoablation probe. the lower the temperature (which should be 50c) the highest the chance of achieving tissue necrosis. alcohol ablation also known as percutaneous alcohol injection (pai) has already been studied in different series [33, 34 ]. this technique could be of advantage in patients whose liver metastases are located next to large vessels or the bile duct, that could be damaged by the heat released during rfa. pai should be used not as monotherapy but with other ablative techniques in order to achieve better results. rfa consists in intense heat production from radiofrequency waves through alternating electric current and can be performed by percutaneous or laparoscopic approach [12, 13 ]. it is mainly used in patients with a small number of liver lesions that can not be resected or as an adjuvant to other therapies. the largest clinical study until involving patients with hepatic lesions treated with rfa was reported by the cleveland clinic. eighty - nine patients were followed and submitted to a total of 119 laparoscopic rfas. results showed that about 90% of the patients experienced immediate relief of symptoms after the procedure, with a mean progression - free survival of 1.3 years. from these 89 patients, 22% had recurrence, and from these 22% recurrences, 63% corresponded to new liver lesions and 59% to extrahepatic disease. an important limitation of the effectiveness of this technique is the size of the tumor. it seems to be difficult to totally eradicate lesions greater than 3 cm, and lesions greater than 5 cm are unsuitable for rfa, as demonstrated by mazzagalia. rfa is a procedure with low morbidity (5%12%) and low 30-day mortality (0%-1%) [12, 17 ]. the most frequent complications include hepatic abscesses, carcinoid crisis, biliopleural fistula, bile leakage, and pleural effusions. hepatic artery embolization (hae) and hepatic artery chemoembolization (hace) are two different technics that can also be used in the treatment of liver metastasis. hepatic arterial embolization is frequently applied as a palliative technique in patients with hepatic - predominant metastatic net who are not candidates for surgical resection. it is based on the principle that tumors in the liver derive most of their blood supply from the hepatic artery, whereas healthy hepatocytes derive most of their blood supply from the portal vein. using this technic, we can block blood supply with nutrients and oxygen to the tumor with consequent ischemia / necrosis. this technique has also proven to achieve the reduction of lesions size as well as symptoms relive [1, 12 ]. retrospective evaluation showed that a high rate and a prolonged disease regression were achieved with chemotherapy after hae. therefore, cytostatic agents were added to this technique resulting in hepatic artery chemoembolization (hace). hace provides not only the embolic blocking but also the supply of direct chemotherapy to these cells, increasing the action / concentration of these agents (more than 20 times of the systemic chemotherapy). its benefits have been documented by different authors, but there were no comparative studies between hae and hace demonstrating superiority of one of these techniques. both hae and hace are palliative techniques used in patients with unresectable lesions, with diffuse and progressive liver disease, with symptoms not controlled with medical treatment and without impaired liver function [13, 24 ]. the main benefit is symptomatic relief (70%90% of the patients) and tumor growth control (50% of the patients) [1, 24 ]. the mean duration of response may be short (up to allow collateral circulation to develop) ranging from 642 months in uncontrolled series of patients [5, 6 ]. these therapies have proved to be a valuable option, particularly in patients with more than 75% of hepatic involvement, carefully selected, without additional risk factors. sepsis, liver and renal failure, carcinoid crisis, necrotizing cholecystitis, and peptic ulcer bleeding have been reported in about 7.5 to 23.8% of patients. the most frequent is the postembolization syndrome (i.e., fever, abdominal pain, leukocytosis, increased transaminases, and bilirubin), which occurs in 90% of patients, in most cases self - limited (2472 hours of duration) [6, 17, 24 ]. in order to minimize the complications, it is recommended to split the area to be treated in small portions in each session (one lobe per session) [5, 17 ]. multiple sessions are usually required and must be carried out with 46 week interval [6, 13 ]. concomitant administration of antibiotics and somatostatin analogues, as well as aggressive hydration, to prevent liver abscesses, carcinoid crisis, and tumor lysis syndrome is further recommended [1, 24 ]. the use of somatostatin analogues during the procedure is of extreme importance in order to prevent carcinoid crisis. the most relevant contraindications are bleeding disorders, renal failure, and/or hepatic vein occlusion. external radiation therapy has limited value in nets, being only indicated in the presence of symptomatic brain and bone metastases. sirt is a novel technique based on small microspheres radioisotopes, including yttrium 90 (90y), which are introduced directly into the hepatic artery, leading to embolic blocking of tumor cells and also exposing them to radioactive agents. saxena. investigated the safety and efficacy of this treatment in patients with nlms. in this study, 34 patients had long - term responses with a mean overall survival of 29.4 months with radiological improvement in 50% of the patients. cromogranina a levels decreased to 50%, with a maintained response for approximately 30 months [12, 38 ]. kennedy. also showed that the major benefit seems to stabilize advanced disease allowing an increase in overall survival although still under investigation results have been promising. complications such as postembolization syndrome are common, with a small risk of radiation gastritis and ulceration. since nets are a rare pathology, large randomized studies in patients with nlms are difficult to perform. this limitation leads to an overall assessment of restricted systemic therapies that can not be translated in to a consensual treatment algorithm. however, the data obtained from a single center study showed that the aggressive medical treatment of nlms instead of surgical therapy increases the 3-year survival rate to 76.4% and the 5-year survival rate to 63.9%. therefore, conservative treatment, that is, the majority of nets is expressed on the surface of their cells somatostatin receptors (sstr 15). their activation inhibits the secretion of peptides and amines by tumor cells and also the effect of tumor growth factors inducing apoptosis [12, 24 ]. since natural somatostatin has a very short half life (2 - 3 min) analogues with longer half lives have been developed for clinical use. in 1980, bauer. synthesized the first analogue - octreotide. the main use of ssa is in symptomatic control. in an initial study, the subcutaneous administration of octreotide at the dose of 150 mg tid improved symptoms, especially carcinoid syndrome, in 88% of patients. however, the short action formulations continue to play an important role in stabilizing the disease, especially in the situations of carcinoid crisis and its prevention. in more recent years, there has been evidence that ssa can have antitumor activity and also be able to reduce tumor growth by direct action on somatostatin receptors. the first randomized clinical study that demonstrates the possible anti - tumor effects of long - acting octreotide in comparison to placebo was the promid study. this study showed a decreased risk of disease progression of 66% and arrested tumor growth in 69% for a median of 14.3 months. treatment with lar octreotide seems to be more effective in patients with low hepatic tumor burden and resected primary tumor [4, 41 ]. scintigraphy with somatostatin allows, beyond the level of self - uptake, to predict therapy 's patient response. there are other ssas in use such as lanreotide and others in the study as pasireotide (this one can interact with four receptors (ssts1, ssts2, ssts3, and ssts5) while octreotide and lanreotide only interacted with two type of receptors). preliminary data suggest that pasireotide could be useful in patients that do not respond to octreotide and could be able to control symptoms in 27% patients. however, nausea, vomiting, diarrhea, steatorrhea, cardiac abnormalities, arrhythmias, hypothyroidism, and hypoglycemia were reported in different percentages [42, 44, 45 ]. cholecystitis can occur in more than 50% of cases due to its action as an inhibitor of the contractility of the gallbladder. in patients with risk factors, interferon is an immunomodulator that inhibits the production of several growth factors and also has antiangiogenic properties. when used in net 's treatment these agents seem to have an additional activity which is the hyperregulation of somatostatin receptors. interferon may be used in functioning and nonfunctioning nets, alone or in combination with ssa in view of the lack of response under maximum dose of these agents. in some clinical studies, treatment with inf- showed symptomatic control in 30%70% of cases, with stable disease in more than 70% of patients [12, 47 ]. however, other studies have not demonstrated their effectiveness even in association with the ssa. a 37% increase in 5-year survival with inf- alone to 57% in combination with octreotide was observed in a single study. however, the results were not statistically significant due to the small number of patients. two other small randomized studies showed that the combined therapy increased toxicity without additional gain in survival [49, 50 ]. prrt is a therapeutic option with particular advantages in patients with a symptomatic disease and tumors with positive somatostatin receptors, not candidates for surgery. this technique uses a radioactive peptide bound to ssa, which after the interaction with the receptor is internalized, releasing specific and localized radioactivity, allowing a precise destruction of tumor cells [42, 51, 52 ]. it has little interference with the nontumor tissue, except with the kidney, bladder, and bone marrow. the most used are lutetium and y, which differ from each other in particles emission, particles energy, and tissue penetration [6, 12 ]. low uptake indicates 20% of the possibility of the effect on liver metastasis, and high uptake indicates the possibility of 60% effectiveness. nets sensitivity to cytotoxic therapy appears to have correlation with the primary location of the tumor and tumor grade. clinical studies show their use in nets in general, without specifications for metastatic liver disease. the great effectiveness of their use is in combination with other agents such as 5-fluorouracil and doxorubicin, but with results of mean response of 9.3 months [12, 13 ]. dacarbazine is another cytostatic agent with proof of effectiveness in pancreatic nets, with a response rate of 34% in phase ii study. clinical use of dacarbazine with stz has had limitations due to its high toxicity. recently, the use of alkylating agents such as temozolomide appears to be promising in this tumor type. in a phase ii study thalidomide and temozolamide demonstrated a response rate of 45%. in a retrospective study with temozolamide and capecitabine, the response rate was 70%, with a mean progression - free survival of 18 months and 2 years, overall survival of 92%. platinum based chemotherapy may be useful in patients with high grade undifferentiated tumors and with liver metastases. in these patients response rate was 42%80% using cisplatin and etoposide and 78% with combination of oxaliplatin [5760 ]. although chemotherapy could be used as rescue treatment, it is not considered the first line of the nonsurgical treatment. furthermore, the presence of liver metastases is associated with a poor response compared to nets without liver metastasis. conventional chemotherapeutic agents have limited efficacy in metastasized nets. despite positive results in some way to the little differentiated carcinomas and primary for the pancreas in relation to other locations, overall response rates are low and the impact on survival is small. the growing knowledge about the biology of these tumors, along with the ability to synthesize new drugs that interfere with therapeutic targets, has given the possibility to develop new target therapies that can change the outcome of these patients. nets are highly vascular tumors that express on their cell surface vascular endothelial growth factor (vegf) receptors. tumor progression seems to be associated with high levels of circulating vegf, making this possible therapeutic target, such as in other neoplasms. bevacizumab (a monoclonal antibody), sunitinib, sorafenib, and pazopanib (tyrosine kinase inhibitors) have been the most studied drugs in pancreatic nets. bevacizumab was introduced in a randomized clinical study with patients being treated with ssa and bevacizumab compared to the combined therapy with peg inf-. superiority was seen in bevacizumab treatment arm with 95% of patients with a progression - free survival of 18 months. based on positive results from phase ii studies, sunitinib was tested in a double - blind phase iii study in patients with well - differentiated and progressive pancreatic net compared with placebo. the primary endpoint (progression - free survival) was statistically higher among patients with sunitinib (11.4 months compared with 5.5 months in the placebo group). also positive was the objective response rate of 9.3%, with stabilization of the disease in 63% of patients. in this clinical trial, there were 95% of patients with metastases including hepatic metastases in the treatment arm and 94% in the placebo arm (figure 1). however, this study was stopped early because of the high number of adverse events and deaths in the placebo arm [1, 12 ]. the mtor pathway is of great importance since it interferes with the control of cell growth, apoptosis, and protein synthesis. the presence of mtor pathway abnormalities is acknowledged in nets, and these abnormalities are also believed to be in part responsible for the development of these tumors. two mtor inhibitors have been studied in pancreatic nets, temsirolimus, and everolimus. in a phase ii clinical study with everolimus has demonstrated a response rate of 9.6% and stabilization of the disease in 67.8% of patients. the subsequent phase iii study (radiant study) confirmed its activity in patients with well and moderately differentiated progressive pancreatic net with a progression - free survival of 11.1 months and 4.6 months in the placebo arm. in the everolimus arm, 94.8% of the patients had hepatic metastases versus 93.3% of patients in the everolimus plus lar arm. most of the adverse effects were grade 1 - 2 (stomatitis, diarrhea, rash, fatigue, and infections). the nets frequently metastasize to the liver, and the presence of liver metastases worsens the prognosis of patients, increasing morbidity and mortality. surgery resection remains the gold standard, especially in well - differentiated resectable lesions and also improves symptomatic control in selected cases. other liver - directed methods are also advantageous, especially in the presence of predominantly liver disease. in addition, they can be combined with each other and with surgery and systemic therapeutic, allowing a greater range of treatment and increased overall survival and progression - free survival. those modalities include the rfa, in situations of localized liver damage up to 3 cm of diameter, and hace, in the case of diffuse liver disease systemic therapy is important in controlling symptoms, particularly in carcinoid tumors, with the ssa. for those patients who are not candidates to surgery, ssa can also offer control of disease progression in low grade net. these agents remain the core of medical therapy for metastatic carcinoid tumors. in pnets and particularly in high grade nets with liver metastases, chemotherapy has a substantial role. more recently, the inhibitors of the mtor pathway and vegf pathway has been shown very promising in controlling disease progression in pnets. it is not known their role in high grade tumors and whenever necessary they can be used together with ssa. for instance, the prevention of tumor progression is one of the main investigating areas. in this area, the role of lar octreotide (in advanced tumors other than small intestine), lanreotide (investigation is already ongoing in nonfunctioning net), and pasireotide (som230 in nets from all sites of origin) is being evaluated. the use of everolimus and sunitinib in early stage tumors is another interesting issue, and its role in tumor progression prevention is still to be defined. physicians and net patients are also waiting for guidelines that acknowledge the best treatment approach for patients with grade 3 tumors from any origin and site. the combination of molecular target therapy and chemotherapy, namely, ssa plus chemotherapy, temozolamide and everolimus or temozolamide, and sunitinib in pnet patients should also be addressed in clinical trials.	neuroendocrine tumors (nets) comprise a heterogeneous group of tumors that form a distinct entity. approximately 7580% of patients present with liver metastases at the time of their diagnosis, and 20%25% will develop these lesions in the course of their disease. the presence of secondary deposits in the liver significantly increases the morbidity and mortality in these patients. the only potentially curative treatment is the surgical resection of the primary tumor and hepatic lesions. however, only 10% of patients presents under ideal conditions for that approach. several techniques aimed at localized liver lesions have been applied also with interesting results in terms of survival and symptom control. the same has been demonstrated with new systemic therapies (target therapies). however, these are still under study, in order to define their true role in the management of these patients. this paper intends to address, in a general way, the various treatment options in patients with liver metastases from neuroendocrine tumors.
roughly 2.7 billion people depend on the burning of biomass and other solid fuels in three stone fires (tsf) and other traditional cookstoves for their day - to - day cooking purposes. cookstoves have environmental and health impacts on an enormous scale, due in large part to emissions of products of incomplete combustion (pic) such as co, pm2.5, methane (ch4), and polycyclic aromatic hydrocarbons (pahs). black carbon (bc), commonly known as soot, is an aerosol component formed during combustion that is estimated to have the second highest global warming impact after co2. approximately 25% of global annual bc emissions and 6080% of africa s and asia s bc emissions that are not from open burning (e.g., wildfires) are from domestic solid fuel combustion. bc is coemitted with organic carbon (oc), a component which is often regarded to have a cooling impact on climate, although recent studies suggest that the oc fraction (generally called brown carbon or brc) that absorbs radiation at short wavelengths contributes significantly to warming. the net climate impacts of cooking - related aerosol emissions are uncertain, though likely warming. replacement of traditional cookstoves with alternative technologies thus has the potential to provide considerable climate and health benefits by reducing emissions and human exposures. efforts to reduce these impacts have spurred the development of a range of alternative cookstoves with varying configurations, levels of sophistication, and performance. models range from rudimentary low - cost cookstoves often built from local materials to mass produced state - of - the - art forced draft cookstoves (fdcs) which use electrically driven fans for improved combustion efficiency. using laboratory emission factors (ef ; g (kg wood)), grieshop. estimated that health (quantified as daily intake of pm2.5 for users) and climate impacts (quantified as global warming commitment or gwc) of various cookstove - fuel combinations can each span 2 orders of magnitude, with all biomass - burning cookstoves having greater impacts than modern fuel stoves such as lpg and kerosene. this variation across cookstoves types and the need to benchmark performance has led to the development of a tier framework, with emissions and other parameters quantified during standardized laboratory testing (e.g., the water boiling test ; wbt). while laboratory testing is required for benchmarking, field data indicate that laboratory tests typically greatly overestimate performance relative to in - home use. for example, field pm efs are 25 times higher than those measured during wbt tests. this is important because benefit estimates for alternatives rely on accurate estimates of real - world performance. fdcss have the potential to greatly reduce emissions via improved combustion efficiency ; their laboratory pm2.5 and elemental carbon (ec) efs are an order of magnitude lower than those for traditional stoves, putting fdcss in the highest tiers (3 or 4) for indoor pm emissions. however, in - field measurements of emissions from fdcs are limited, report real - time bc concentrations (not efs), and neglect other species (e.g., co2, oc). carbon finance has been held up for its potential to yield cobenefits by enabling access to improved cookstove technologies by poor households. while flagging carbon markets and evidence from early efforts call the near - term practicality of this into question, it remains an important possible source of finance. current carbon finance methodologies include greenhouse gases (ghgs) but fail to account for the climate impact of pics such as bc, co, oc, and non - methane hydrocarbons (nmhc), mainly because of the high uncertainty and variability in their emissions and the differing spatial and temporal scales of their impacts relative to ghgs. bc dominates cookstove pic climate impacts and has become a focus for mitigating near - term climate change. in response, the gold standard foundation recently developed a simplified methodology, which relies on either laboratory or (preferred) field - based emission measurements, with the latter collected using the kitchen testing protocol (kpt), to incentivize bc mitigation efforts. however, the gold standard does not require field emission measurements to be made (only measurements of fuel use reductions), and as noted above few cookstoves have actually been measured in the field. therefore, an important missing piece is a rigorous understanding of in situ emissions from cookstove technologies and the extent to which the emission reductions indicated by laboratory testing are achieved under real - world conditions. to address this gap, we conducted an evaluation of in - field emissions in malawi, africa focusing on two pre - existing cookstove programs. the specific objectives of this work were : 1) to measure fuel use and emission factors from in - home use of alternative and baseline household and institutional cooking technologies ; 2) compare emission factors and rates with existing measurements and emission tiers ; 3) analyze real - time optical properties (absorption and scattering) of aerosols during in - home use ; and 4) estimate and compare health and climate impacts / benefits suggested by lab and field - based measurements. malawi is a small, landlocked country in southeastern africa in which over 90% of the population uses biomass as their main source of domestic energy. it is one of the most densely populated countries in sub - saharan africa and among the poorest in the world, ranking 173 among 188 countries in human development index. high poverty rates, dependence on unsustainably harvested firewood, and a predominantly rural population means there is a great need for improvements in household energy systems and makes malawi an ideal location to study the impacts of alternative cookstove technologies. household emission measurements of uncontrolled in - home cookstove use (following the kpt protocol) took place during routine cooking activities in september - october 2015 (the hot and dry season). emission tests were completed in two communities on cookstoves at opposite ends of the technology spectrum discussed above. in one, the cooking and pneumonia study (caps ; www.capstudy.org), led by the liverpool school of tropical medicine (lstm), distributed limited quantities of two fdcs models (philips hd4012ls and ace-1 ; cost 90 usd) as part of pilot activities for this community - level randomized controlled trial of the effects of the philips hd4012ls cookstove on the incidence of pneumonia in children under the age of 5. fdcs use was not widespread in this community ; on the order of 1020 cookstoves had been distributed. in the other, the nongovernmental organization (ngo) concern universal (cu ; www.concern-universal.org) helped establish nearly universal distribution of the chitetezo mbaula (cm) cookstove, a low - cost (12 usd), locally produced, natural - draft clay cookstove, with the main objective of reducing fuel use by users and with funding from the sale of carbon credits. in both communities, traditional three stone fires or simple mud stoves cookstoves are shown in figure s1 in the supporting information (si). in - home cookstove use included cooking of traditional foods such as nsima (a corn - flour porridge, the staple food of malawi) or rice, preparing vegetables or meat, and heating water for bathing and took place inside, in semicovered verandas and outside. in addition to in - home testing, controlled cooking tests (cct) were conducted on several larger, wood - burning institutional cookstoves being piloted at an orphanage. institutional cookstoves are used where a large number of people are fed ; they are much larger than household cookstoves to accommodate a dedicated, large cooking pot (80100 l) that sits inside the stove body, enabling more efficient heat transfer. institutional cookstoves tested included a large institutional three stone fire (i - tsf), aleva (al), mayankho (ma), and the jumbozama (jz). the jumbozama is a scaled - up version of the zama zama rocket gasifier cookstove (rocket works, durban, south africa) built inside a masonry housing. figure s2 (si) shows the institutional cookstoves tested, and table s1 summarizes tests conducted during the campaign. in - home emission measurements were performed using the portable stove emissions measurement system (stems ; figure s3 in the si), which utilizes the sensor board from a portable emission measurement system (aprovecho research, cottage grove, or). the stems runs on a 12 v battery and measures real - time (2 s) concentrations of carbon dioxide (co2), carbon monoxide (co), temperature, relative humidity (rh), and particle light scattering (bsp ; also used as a proxy for real - time pm2.5 mass concentration) with a laser photometer (optical wavelength, = 635 nm). integrated filter samples were collected on two 47 mm diameter filter trains with equal flows for gravimetric and thermo - optical oc / ec analysis (see the si for details). one of the filter trains contained a quartz filter, and the other contained a teflon filter followed by a backup quartz filter downstream to correct for gas phase absorption artifacts. additional details on the stems sensors, filter analysis and associated uncertainties, and quality assurance are provided in section s1 in the si. real - time pm light absorption at = 880 nm was measured using an ae-51 microaeth (aethlabs) incorporated within stems. to avoid excessive filter loadings and frequent filter ticket changes in the field, an external flow meter (honeywell awm3150v) and vacuum source were used in place of the internal pump and flow rate set at 1025 cm min. the microaeth filter loading artifact was corrected via the algorithm described by park. a six - armed stainless - steel probe with sampling ports radially centered in equal areas was used to capture a representative sample of naturally diluted emissions approximately 11.5 m above the cookstove. from the probe, emissions passed through conductive sampling tubing to the stems via a 2.5 m cut - point cyclone (bgi inc.). wood fuel was set aside before the start of cooking and wood moisture and weight recorded as per the kpt protocol. wood moisture content was measured with an electronic moisture meter (lignomat mini - ligno s / dc). the fire was started using matches or hot charcoal left from a previous cooking session ; the latter practice was more common. one kg of wood can result in up to 161 g of char being formed. neglecting starting char likely biases high the estimates of wood consumed, whereas neglecting left - over char results in a low bias to wood consumed. to account for this, we assume a conservative 20% uncertainty in wood consumed. a brief, anonymous survey was conducted after testing to collect user feedback on performance and perception of alternative cookstoves. fuel based emissions factors were calculated using the carbon balance method, assuming that carbon comprises 50% of dry wood by weight and all gaseous carbon in the wood is emitted as co and co2. since the summed carbon mass obtained from background - corrected co and co2 concentrations serves as a tracer for the fuel consumed, the carbon balance does not require all emissions to be captured. other carbonaceous species (e.g., gaseous hydrocarbons) contribute a relatively small fraction (80% reductions for both. the jz cookstove used a slightly smaller, different cooking pot (80 l vs 100 l for other cookstoves), which may have contributed to mean pm ef values (e.g., due to a quicker warm - up phase) and cooking times that were 61% and 32% lower, respectively, than those for other cookstoves. further extensive comparisons of these data are complicated by the small sample size and lack of emission data in the literature. figure s6 in the si shows real - time (2 s and 1 min average) concentrations of co, co2, particle absorption, and scattering coefficients (bap ; bsp) for representative fdcs and traditional household stove tests. gravimetric pm2.5 concentrations correlated well (r = 0.87) with averaged bsp, suggesting that real - time scattering was a reasonable proxy for real - time pm2.5 mass concentrations under these test conditions (si figure s7). figure s6 also shows single scattering albedo (ssa ; fraction of scattering to total extinction ; (bsp/(bap+bsp), here at = 880 nm) and modified combustion efficiency (mce ; co2/(co+co2), where indicates background - corrected concentrations in ppm). a lower ssa signifies a greater contribution from absorption to total aerosol light extinction, while higher mces indicate more efficient combustion. all cooking events were characterized by a scattering spike at startup (evident in figure s6). observations of cooking activity showed that addition and adjustment of fuel typically resulted in spikes in bap for fdcs and in bsp for traditional stove tests. fdcs tests typically had a large scattering peak only at startup and overall particle extinction was dominated by absorption, while extinction from traditional stove tests was dominated by scattering. as a result, traditional stove tests had comparatively higher ssa (shown in figure 1h) and lower mce than fdcs tests. test - average ssa was highest for traditional stoves (0.36), followed by cm (0.28) and fdcs (0.25) ; particles from all alternative cookstoves are more absorbing than those from traditional stoves and thus have greater specific warming. our optical measurements are at = 880 nm, which influences the quantities (e.g., ssa) presented here, but likely not the relative trends discussed here and below. for example, while ssa for pure bc aerosol is typically 0.150.3 at = 530 nm, we observe periods with ssa ranging from 00.2 for fdcs, despite the fact that the particles are not pure bc. section s5 and figure s8 in the si present mie theory modeling indicating that both the longer wavelength and smaller particle diameter in fdcs emissions likely strongly reduces the scattering efficiency of these particles. the average mass scattering cross section (msc ; ratio of scattering to gravimetric pm2.5 concentration) over all tests was 0.87 0.31 m g, substantially lower than mscs of 3.64.3 m g (= 550 nm) and 2.2 0.6 m g (= 530 nm) reported for emissions from dry biomass burning and in - field cookstoves, respectively. lower msc values in our study are consistent with the wavelength dependence of scattering. averaged mass absorption cross - section (mac ; ratio of bap to ec concentration) for all tests was 13.2 4.8 m g, overlapping with the mac of 12.5 m g assumed in the microaethalometer. the data show a general trend of decreasing ssa with increased mce and ec / tc. si figure s9 shows the relationships between mce and ec / tc with ssa for in - home cookstove testing. greater specific absorption correlates somewhat with higher mce (r = 0.29) and more strongly with higher ec / tc (r = 0.57). these relations are consistent with greater bc production in more efficient, contained combustion where mce is highest, while more scattering oc is produced during less efficient (perhaps lower temperature) combustion. relationships between mce, ec / tc, and ssa have been proposed based on measurements of open biomass burning for a range of fuels. while the general trends we observe are consistent with the published parametrizations, a direct comparison is not possible due to differences in measurement wavelength. however, it is likely that the relationships would be different because combustion technology strongly influences aerosol properties. test - average quantities do not reflect the contribution of distinct combustion phases to total emissions. analysis of real - time data can give insight into the variation of parameters such as mce and particle properties throughout a burn. patterns of real - time emissions data (parted) analysis approach of chen. to evaluate quantities and optical characteristics of emissions based on real - time data. in this analysis, mce and ssa are calculated for each minute of data (termed a combustion event). a bivariate histogram of mce and ssa, weighted by instantaneous scattering emission factor (iefscat, particle light scattering normalized by mass of fuel consumed) and normalized by total scattering emissions, is then constructed. the resulting plot shows the fractional contribution of combustion at specific conditions (mce ; ssa) toward total scattering emissions. this weighting is chosen to represent the distribution of total particle emissions, as scattering shows a strong correlation (r = 0.87) with pm2.5 from gravimetric analysis (figure s5). figure 2 shows parted plots for traditional, cm, and fdcs (ace-1 and philips combined) emission tests, with each panel representing all test data from that cookstove type. all display a cluster at mce > 0.9 and ssa 0.9 and ssa < 0.4 for fdcs), whereas only 12% of scattering emissions took place under these conditions. the corresponding fractions (fuel consumption / scattering) in this mce - ssa range are 57%/15% and 58%/25% for the cm and traditional tests, respectively. this reinforces that the relative distribution of combustion conditions varies between stoves and has substantial impacts on particle emissions and properties. (though less so for traditional / cm tests) but emit a large fraction of scattering particles / particle mass. time series data (figure s6) show that all tests had sharp peaks in scattering emissions during cookstove startup, consistent with other field measurements. the parted plots also suggest that these startup emissions may make outsized contributions to overall pm emissions. we examine the contribution of startup emissions in si figure s11, which plots the running average of iefscat against normalized time for each test of a cookstove type. in all tests, the average peaks strongly during the startup phase, confirming the importance of start emissions. although the test averaged iefscat (at the right edge of the graph) is highest for traditional and lowest for fdcs tests, we see a much higher peak for gasifiers and in nearly all cases a monotonic decrease in iefscat during the test, reinforcing the dominance of startup emissions for these cookstoves. the outsized contribution of startup to pm emissions from fdcs has important implications for exposure as during startup the cook is assured to be in close proximity to the cookstove. bivariate histogram of mce and ssa weighted by particle emissions (parted plots) for a. traditional ; b. chititezo mbaula, and c. fdcs. the bottom axis delineates bins of single scattering albedo (ssa) at 880 nm, and the left axis shows modified combustion efficiency (mce) bins. each location on the plot represents an ssa and mce at which a combustion event (1 min) may occur ; the color scale indicates the percent contribution of emissions at that condition to the total scattering (pm mass) emitted. three of the traditional stove tests were excluded from this analysis due to lack of either co2 (1 test) or absorption data (2 tests). 100- and 20-year gwc (tons of co2-equivalent per year of cookstove use) were estimated based on measured pollutant emission factors (co2, co, oc, and ec) for household and institutional cookstoves following the approach of previous work, as briefly discussed in si section s6. gwcs associated with the use of a modern fuel, lpg, are also included as a benchmark (based on laboratory efs). the intergovernmental panel on climate change (ipcc) default value of 0.81 for the fraction of nonrenewable biomass (fnrb) in malawi was assumed for all calculations, though this is highly uncertain due to factors including spatial heterogeneity in fnrb and uncertainties in data on fuel demand and its dynamics. a fixed energy demand was assumed and annual fuel use for each cookstove estimated based on fuel use rate reductions observed in this study (figure s5) relative to the baseline (traditional cookstoves). gwc calculations used global warming potential (gwp) values recommended by the gold standard foundation and ipcc and account only for emissions during fuel combustion. gwc associated with upstream processes (e.g., fuel production and transport) related to cookstove fuel use have been found to be negligible for woodfuel and relatively small (1020% of combustion stage) for lpg, though they may be considerable for other fuels such as coal or charcoal. ch4 makes a substantial contribution to the gwc associated with cookstove use but was not measured here. to approximate ch4 gwc, ch4:co ratios from the literature were used to estimate ch4 efs ; ch4:co ratios of 0.05 for ace-1 and philips and 0.08 for the traditional, cm, and institutional cookstoves were used in calculations. gwc contributions from other hydrocarbons and n2o are small for biomass emissions and are neglected here. also not included in this accounting is brown carbon (brc), the component of oc that absorbs energy across visible and ultraviolet wavelengths. we did not measure absorption at multiple wavelengths in this work but expect that the brc absorption would make a relatively small contribution to short - wavelength absorption considering the very high ec : oc ratios (mean for all tests was 1) observed in emissions from all cookstoves. brc is expected to have little additional impact on climate forcing for emissions with ec : oc ratios above 0.1, though this merits further study. figure 3 shows the gwc values estimated across a 100-year horizon (figure s12 shows 20 year gwcs). as expected, traditional cookstoves show the highest gwc, followed by the cm, ace, and philips. across a 100 year horizon for household cookstoves, cm, ace, and philips show overall reductions of 13%, 23%, and 55%, respectively, from the household traditional (hh - trad) stoves. across household stove models, the highest relative contribution by species is from co2 (5169%) followed by ec (2638%) with other species contributing less than 10%. calculations using laboratory efs suggest much larger reductions (59% and 93% for improved and fdcs, respectively) relative to traditional stoves, highlighting again the implications of the lab - field discrepancy in emissions. due to high combustion efficiency for lpg, pics contribute minimally to its gwc, and the vast majority is contributed by co2. the gwc of lpg is a factor of 4 lower than the cleanest cookstove tested in the field (philips). among institutional cookstoves, only the jz yielded substantial fuel use and emission reductions during our limited testing, leading to an 50% reduction in gwc relative to baseline. across a 20-year horizon (figure s12) ec contributes the most to biomass cookstove gwc, contributing 5365% across all cookstoves. 100 year gwc values for one year of use of in - home cookstoves (l) and institutional cookstoves (r) from major short- and long - lived climate forcing species emitted by cookstoves. the ch4 component is estimated based on the ch4:co ratio from other studies. note that the daily energy use is different for household and institutional cookstoves. in - home cookstoves institutional cookstoves are assumed to be used for twice a day for 5 days a week for 40 weeks a year with an energy basis based on average fuel use measured in this study. figure 4 combines gwc estimates with those for human exposure to pm2.5 to examine the cookstoves measured in a the exposure estimation applies an individual intake fraction of 1300 ppm (1 ppm = 1 mg inhaled per kg emitted) to link emissions to human exposure. the figure shows estimated daily pm intake (horizontal axis) and gwc (vertical axis) of several cookstove technologies evaluated in that study, with added estimates made based on data from in - home testing during this study. it should be noted these calculations assume complete adoption of the cookstove in question, while field trials have shown that new technologies are rarely used exclusively and the exposure - response relationship for all - age mortality risk from ischemic heart disease (ihd) from burnett. is used to estimate adjusted relative risk of mortality due to ihd (dose response for chronic obstructive pulmonary disease mortality is similar), shown on the lower horizontal axis. this extrapolation of our field emissions data suggests that the philips cookstove reduces pm emissions and intake by approximately 75% relative to the traditional stove ; this is associated with a smaller reduction in estimated mortality relative risk (from 2.2 to 1.9) due to the nonlinear dose this figure dramatically demonstrates the implications of the performance decrement observed in field measurements. field - tested biomass stoves do not meet expectations based on laboratory tests in terms of emissions / exposure. for example, relative to estimates based on laboratory measurements, the field - measurement - based daily pm intake and gwc for the philips are a factor of 8.6 and 2.8 times higher, respectively. the philips and jz have the lowest estimated intake and gwc among in - home and institutional cookstoves, respectively, but are still associated with far greater impacts than the benchmark lpg cookstoves. for example, compared to the estimated impacts of lpg cookstove use, in - home use of a philips cookstove results in 4.9 times higher gwc and around 66 times higher daily exposure, corresponding to increase in adjusted relative risk for ihd mortality from 1.25 to 1.95. impact estimates for hh - trad, cm, and three of the institutional cookstoves (i - tsf, ma, and al) are within the bounds of impact estimates for laboratory - tested traditional stoves, whereas the best performing fdcs is in the range estimated based on laboratory performance of a basic improved biomass cookstove (w - im - u). field observations of emissions from a range of cookstoves used in interventions give important insights into the potential for these technologies to mitigate the health and climate impacts associated with traditional cookstoves. health and climate impacts of various cookstove - fuel combinations based on laboratory emission test data (shown with circles ; adapted from grieshop. with fuel renewability and energy demand values described in text) along with the estimates from this study are shown, marked with diamonds and squares with error bars for in - home and institutional cookstoves, respectively. abbreviations for laboratory - based calculations : w - tr - u : wood - burning traditional unvented cookstoves ; w - im - u : wood - burning improved unvented cookstoves ; w - gas - u : wood - burning gasifier unvented cookstoves ; w - fan - u : wood - burning fan unvented cookstove. household traditional ; cm : chitetezo mbaula ; philips : philips hd4012ls ; ace : ace-1 ; i - tsf : institutional three stone fire ; al : aleva ; ma : mayankho ; jz : jumbozama. our results suggest that both simple improved cookstoves and more advanced biomass cookstoves provide some benefits but fall short of those indicated by laboratory testing or that may be possible through the use of modern fuels / devices. impact estimates shown here (figure 4) are rough approximations but are consistent with field trials that have seen less than expected benefits from cookstove interventions. for example, the fact that no effect on childhood pneumonia incidence was observed during caps may be partly due to poorer - than - expected performance of these cookstoves under real - world conditions ; other factors such as continued use of traditional stoves and exposure to air pollution from other sources likely also contributed to this outcome. forthcoming findings from other intervention trials will report on the health effects associated with other fuel / cookstove technologies including lpg and give insights into whether or not they provide the benefits that might be predicted. a range of factors contribute to reduced performance observed in the field, all related to the difficulty of controlling combustion of heterogeneous fuels under widely ranging conditions. part of the performance decrement observed is due to cookstoves not being used in accordance with manufacturer recommendations. for example, wood pieces sticking out of the top of fdcs were commonly observed in the field (figure s1 in the si) and lead to suboptimal combustion of the volatiles emitted from pyrolysis of wood. however, this practice is unsurprising when one considers that processing larger logs and branches to the size recommended for the fdcs models considered here (1 5 cm) represents additional work for the household. this source of variability may be addressed via a cookstove that is highly robust to changing fuel type / configuration or a situation in which a homogenized fuel source (e.g., pellets) is provided or readily available. the former is only likely possible in a more advanced combustion device (e.g., the enclosed heating stoves, often with catalytic after treatment of exhaust used in developed countries) that are beyond the budget of the target population, while the latter requires a close look at the broader system, beyond the cookstove. these observations highlight the need to expand the view beyond clean cookstoves to clean and controlled cooking systems, which could provide considerable health and climate benefits and perform consistently and reproducibly under both laboratory and field conditions. one approach advocated is to focus efforts on a switch to modern appliances (e.g., electrical induction cookers and lpg) rather than promoting improved biomass cookstoves, though such technologies would be out of reach in the short- to medium - term for the poorest of the world s poor (e.g., rural malawians). improvements using biomass can likely be made by improving the cookstove / fuel system in tandem. our findings emphasize that laboratory protocols do not fully anticipate real - world emissions when fuel properties and cookstove operation are variable and highlight the need for testing approaches that more accurately represent real - world cookstove use. field evaluation of emissions performance early in product development would be one way to achieve this ; another way would be to develop testing protocols that simulate the range of ways a cookstove may be used, including low - efficiency emissions such as smoldering, often observed in field emissions. these data were collected during a dry season, and fuel moisture may also have important impacts on stove performance ; future field studies should assess stove performance across an annually representative period if possible.	emissions from traditional cooking practices in low- and middle - income countries have detrimental health and climate effects ; cleaner - burning cookstoves may provide co - benefits. here we assess this potential via in - home measurements of fuel - use and emissions and real - time optical properties of pollutants from traditional and alternative cookstoves in rural malawi. alternative cookstove models were distributed by existing initiatives and include a low - cost ceramic model, two forced - draft cookstoves (fdcs ; philips hd4012ls and ace-1), and three institutional cookstoves. among household cookstoves, emission factors (ef ; g (kg wood)1) were lowest for the philips, with statistically significant reductions relative to baseline of 45% and 47% for fine particulate matter (pm2.5) and carbon monoxide (co), respectively. the philips was the only cookstove tested that showed significant reductions in elemental carbon (ec) emission rate. estimated health and climate cobenefits of alternative cookstoves were smaller than predicted from laboratory tests due to the effects of real - world conditions including fuel variability and nonideal operation. for example, estimated daily pm intake and field - measurement - based global warming commitment (gwc) for the philips fdcs were a factor of 8.6 and 2.8 times higher, respectively, than those based on lab measurements. in - field measurements provide an assessment of alternative cookstoves under real - world conditions and as such likely provide more realistic estimates of their potential health and climate benefits than laboratory tests.
polymorphic eruption of pregnancy (pep), also known as pruritic urticarial papules and plaques of pregnancy (puppp) is a common benign dermatosis of pregnancy mainly affecting primigravidae and multiple pregnancies. pep usually evolves in the third trimester and resolves rapidly postpartum (table 1). table 1clinical features of two patients with postpartum polymorphic eruption of pregnancy.patientmaternal ageprimagravidadelivery gestational ageoutcomeonsetduration of polymorphic eruption of pregnancydistributionmorphologytreatmenta2112 days postpartumabdomen, thighsurticarial plaquesb26yescaesarean section....... healthy boy5 days postpartum3 weeksabdomen, thighsurticarial plaquestopical steroids, antihistamines we describe two cases of pep with typical clinical and histological features presenting in the postpartum period. only few cases of pep developing postpartum have been described in the literature. however, a rash appearing in the mother shortly after delivery still can be a specific dermatosis of pregnancy. a 21-year - old woman was referred to our department of dermatology complaining of an intense pruritic rash starting 12 days postpartum. the eruption initially developed in and around the abdominal striae distensae with a periumbilical sparing (figure 1). the urticarial plaques and erythematous papules spread to the buttocks, thighs and lower lumbar region. on the abdomen tiny vesicles were observed. the patient was successfully treated with prednisolone 20 mg daily for five days combined with high potency topical corticosteroids tapered over four weeks. a punch biopsy showed slight dermal edema and a predominantly perivascular infiltrate of lymphocytes and eosinophilic granulocytes (figure 2). figure 1erythematous papules and urticarial plaques with periumbilical sparing and accentuation in striae in a 21-year old woman with polymorphic eruption of pregnancy. erythematous papules and urticarial plaques with periumbilical sparing and accentuation in striae in a 21-year old woman with polymorphic eruption of pregnancy. a 26-year old primigravida woman was referred 5 days after giving birth with an itchy rash on the abdomen spreading to the proximal thighs. in the last week of her pregnancy, she had noticed some itchiness on her abdomen but no visible changes besides well - marked striae gravidarum. objectively erythematous urticarial plaques and confluent papules were seen with a periumbilical sparing. high potency topical corticosteroids were initiated in combination with oral antihistamines and the itchy eruption subsided in the following 3 weeks. a 4 mm punch biopsy from the skin of the buttock showed a slight dermal edema and perivascular lymphocytic infiltrates with scattered perivascular and interstitial eosinophils. in pregnancy, complex endocrinologic, immunologic, metabolic and vascular changes influence the skin in various ways. the multiple alterations of the skin during pregnancy can be classified as physiological skin changes, alterations in pre - existing skin diseases and specific dermatoses of pregnancy. the specific dermatoses of pregnancy represent a unique group of disease processes caused or exacerbated by the pregnancy state and include gestational pemphigoid (herpes gestationis), prurigo of pregnancy, intrahepatic cholestasis of pregnancy, impetigo herpetiformis and pep / puppp. pep is a common distinct clinical entity with an estimated incidence in a single pregnancy of one in 130300 pregnancies. the condition is more common in multiple pregnancies, where both earlier presentation and recurrence in second pregnancy are seen. it has been postulated that excessive abdominal distension and weight gain may act as a trigger for the skin changes due to connective tissue damage caused by overstretching. found a high frequency of atopy (55%) among their patients, especially in those with longer disease duration. pep usually evolves in the third trimester at the average gestational week of 35 and resolves rapidly postpartum and only exceptionally does it appear in the postpartum period. the lesions start in the abdominal striae in two thirds of the patients with a periumbilical sparing distinguishing pep from other common rashes of pregnancy. the rash consists of very itchy small erythematous papules in the stretch marks which can coalesce to form larger urticarial abdominal plaques often surrounded by blanched halos. occasionally eczematous, polycyclic and target lesions can be seen or vesicles (but never bullae) eventually in an acral dyshidrosiform pattern. over days, the rash can spread over the thighs, buttocks, breasts, and arms with infrequent facial, hand and foot lesions. in spite of the severe pruritus, the absence of excoriations on the skin is a striking feature in contrast to excoriations related to cholestasis of pregnancy. the condition is harmless to the mother but can be very annoying because of the severe itching. the diagnosis of pep can be made clinically in typical cases based on the appearance of the rash. there are no specific laboratory abnormalities and only nonspecific histopathology with a perivascular lymphohistiocytic infiltrate with some edema and eosinophils in the dermis. skin biopsies are only performed to rule out other differential diagnoses such as pemphigoid gestationis, atopic dermatitis, contact dermatitis, drug eruptions, viral eruptions and scabies. pep is a self - limiting disor - der with resolution shortly after parturition and the treatment is symptomatic. general measures, such as mild to potent topical steroids can be helpful in treating symptoms from the disease together with systemic antihistamines. furthermore application of emollients is basic in the treatment. in the most severe cases, as seen in one of the cases above, oral steroids may be necessary to control itching. if systemic corticosteroid treatment is necessary during pregnancy non - halogenated glucocorticosteroids (e.g. prednisolone) which are inactivated enzymatically in placenta should be administered as a short time therapy in a dosage of 0.52 mg / kg / day depending of the severity of symptoms. pep is a frequently occurring pruritic, self - limited inflammatory dermatosis, most often seen in primiparous women and in the last trimester of pregnancy. if a rash develops after delivery, specific dermatoses of pregnancy still remains a possible diagnosis.	polymorphic eruption of pregnancy (pep), also known as pruritic urticarial papules and plaques of pregnancy, is a common benign dermatosis of pregnancy mainly affecting primigravidae and multiple pregnancies. we report here two cases of pep with typical clinical and histological features presenting in the postpartum period.
a phobia is defined as an irrational fear that produces a conscious avoidance of the feared subject, activity or situation and the presence or anticipation of it elicits severe distress in an affected individual. there are various types of specific phobias including acrophobia, claustrophobia, zoophobia, etc., specific phobia of vomiting, also known as emetophobia, is relatively understudied with respect to its etiology, clinical features, and treatment. it is a specific phobia (other type) according to the current edition of the diagnostic and statistical manual of mental disorders. this specific phobia can include a fear of vomiting in public, a fear of seeing vomitus, a fear of watching the action of vomiting or fear of being nauseated. the anxiety and fear can go from mild feelings of apprehension to a full - blown panic attack. emetophobia is implicated in social, educational, and occupational impairment and it causes significant restrictions in leisure activities. there is no available data on the prevalence in the general population and little is known about the etiology. most studies describe predominance in females, early (childhood) onset and chronic course. the most important differential diagnoses are : panic disorder with agoraphobia, social phobia, anorexia nervosa and obsessive - compulsive disorder. an 8-year - old girl, studying in second grade was brought by parents with complaints of fear of vomiting and feeling nauseated since about seven and half months. around 20 days before the starting of symptoms, child had episode of acute and severe abdominal pain, high grade fever with 3 bouts of vomiting with nausea for a day. she was diagnosed as having acute appendicitis by a surgeon and was operated subsequently. within around 10 days after operation, patient started having fear of similar episode of vomiting with repeated remembrance of the episode. she started eating less, avoiding outside food which she used to ask for previously. she would worry about the pungent smell of vomitus in toilet and ask mother to clean it frequently. the fear increased slowly to the extent that, she started avoiding playing with other children in a fear that they will avoid her and tease her if she vomited in front of them. she avoided school for the same reason and thought that teachers will have bad impression about her if she vomits in the classroom. but symptoms went on increasing and she started refusing to use a lift, travel in bus, going to park, market places, etc., fear became generalized and patient started worrying that her parent may suffer vomiting, she would request parents not to go outside, not to travel in bus or use lifts. she had persistent fear that he may suffer vomiting as he eats outside food and there is no one to take care of him. she also developed reduced and non - refreshing sleep with constant thoughts / worries about vomiting at night with complaints of nausea and regurgitation after having her dinner. she refused separate interview of parents because of the fear that they will hide the illness from her. i feel nauseated even when i hear a word vomitus or vomiting or if i see anybody vomiting. besides this, there was no history of depressive or obsessive - compulsive features or eating disorder and no symptoms suggestive of other phobias. there was significant family history and both of her parents are suffering from anxiety disorder and taking regular treatment from a psychiatrist. patient described her mood as anxious with appropriate affect. in thought, there was preoccupation about the worries of having nausea and vomiting along with number of questions e.g., whether i will get better ; do i have some severe illness ; will your medicines have side effect of vomiting, etc., she was diagnosed as a case of specific phobia of vomiting i.e., emetophobia. as it was very difficult to involve the child in counseling or psychotherapy because of severe anxiety, she was prescribed tablet clobazam 5 mg in divided doses and cap fluoxetine 10 mg. she was admitted to a child unit to reassure her as she was not willing to take medicines due to fear of side effects. after a week, she and her parents perceived mild improvement in anxiety and she was somewhat comfortable. on further follow - up, child was taught relaxation and started on graded exposure therapy along with the medications. first, she was asked to read hand written article which contained the word vomitus / vomiting (multiple times) as many times as possible. after about a week, she was asked to witness the action of vomiting by parents which they were pretending, followed by behavior as if vomiting has caused no trouble and anyone can suffer it for a short duration. after around 15 days, she was advised to attend school with a facility to go to rest room whenever she has thoughts of vomiting followed by feeling of nausea. she used the rest room only for initial 2 days after which she was as regular to the classes as before the start of illness. she was asked to play with other children only for 15 minutes to begin with. when she did nt have even a single vomiting during this period, she started accepting the fact that it was an irrational fear and that she can achieve a mastery over it. slowly, she was exposed to the activities that can induce vomiting like smelling the toilet, spinning around, etc., she had nausea and she hesitated to do it initially, but with intermittent counseling and relaxation and frequently doing above activities, she could face the feeling of nausea with less fear than before. in view of severity of symptoms and family history of anxiety disorders in parents, this case clearly presents with a persistent fear of vomiting, which is called as emetophobia. our patient did not have symptoms suggestive of other types of phobia. according to some studies, the prevalence rate of fear of vomiting in the community sample is 8.8% (female : male ratio = 4:1). in this case, symptoms started with the remembrance of episode of acute appendicitis and research shows that people with emetophobia recall the memories of their own or others vomiting experiences from an earlier age and rate them as significantly distressing. the present case study also illustrates that exposure to distressing or otherwise aversive situations can lay down the groundwork for phobic conditions like emetophobia. this correlates well with the data of a survey among emetophobics showing about one - third of patients reporting past memories of actual vomiting, and more than half had observed someone else vomiting. the pattern of symptoms showed that severity increases from specific fear of vomiting to more generalized fear of suffering vomiting by family members over a period of 6 - 7 months. like any other phobias, emetophobia is also associated with the avoidance behavior and the child stopped going to school, playing, traveling in bus, etc., because of fear of vomiting in front of others. evidence suggests that fear of vomiting is a chronic and disabling condition that may cause significant impairment in daily functioning. there are no treatment protocols and randomized controlled trials for the treatment of emetophobia, and exposure - based therapies are the most commonly used approaches for vomit phobia as described in literature. other treatments that have been reported include the use of (combinations of) hypnotherapy, cognitive behavior therapy including stimulation of nausea or vomiting, the use of counter conditioning, exposure in vivo to cues of vomiting, re - scripting of past aversive experiences of vomiting, behavioral experiments, dropping of safety - seeking behaviors, and role play of vomiting using the smell of vomit. research shows that some psychotropic medications (such as benzodiazepines and antidepressants) do help in this phobia and some said gastrointestinal medications are also beneficial. the result of the present case study is in line with previous studies on phobias with people that have developed it following a disturbing event. more studies are needed for a better understanding of the epidemiology, clinical picture, etiology, and treatment of emetophobia. emetophobia is relatively neglected illness although it can cause as much distress as other major psychiatric disorders do and any patient presenting with these symptoms need to be evaluated in detail. fear of vomiting is not uncommon and can be disabling condition for a child leading to socio - occupational impairment. the present study has certain limitation that it was not entirely clear whether the improvement was caused by medications or psychotherapy.	emetophobia is an intense, irrational fear of vomiting including fear of feeling nausea, seeing or hearing another person vomit, or seeing vomitus itself. it may occur at any age and we need to understand its symptomatology. we report a case of emetophobic child whose fear of vomiting started after an attack of acute appendicitis. in the initial stage, fear was limited to vomiting, later it became generalized to a fear of seeing the vomitus, worries that parents may suffer vomiting, fear of vomiting in public places followed by avoiding social activities. patient improved on short course of anti - anxiety drugs and graded exposure therapy.
exercise is recognized as an important health - related behavior conducive to good mental and physical health and well - being. regular physical activity (pa) has many health benefits, including reduced risk of cardiovascular disease, ischemic stroke, noninsulin - dependent diabetes (type 2), colon cancers, osteoporosis, and depression. despite these benefits, a large proportion of the population in many countries fails to participate in sufficient pa to achieve these outcomes. pa among us adults is lower than of recommended level for health promotion and this issue determines as a target of public health intervention. today, many jobs have been created with advances in technology ; unfortunately, these jobs are associated with the inactivity or sedentary lifestyle. employees health will be affected by their depriving of the benefits of pa. in finland, more than one - third of the working population engages in pa less than recommended for health one study in female japanese showed a high percentage of japanese women are not regularly active. in iran, a study conducted by robabi. in bank employees (iranshahr) have shown that a high percentage of employees did not have enough pa. also in jalilian. study, on employees women in hamadan university of medical sciences, indicated that 65% of women employees did not have sufficient pa. based on our knowledge, there are no any available data regarding the pa levels and its relevant affecting factors in isfahan university of medical sciences employees. nowadays, health educators use health education models to explore health promotion behaviors and behavior change. the transtheoretical model (also called the stages of change model), developed by prochaska and diclemente in the late 1970s. the ttm model has four constructs ; stages of change, self - efficacy, decisional balance, and processes of change. the ttm consists of five stages of exercise behavior change : (1) precontemplation, (2) contemplation, (3) preparation, (4) action, and (5) maintenance. processes of change cognitive processes are consciousness raising, dramatic relief, self - reevaluation, environmental reevaluation, and self - liberation and behavioral processes are social liberation, counter - conditioning, stimulus control, reinforcement management, and helping relationships. another construct of the model is decisional balance that focuses on the importance of perceived positive (pros) and negative (cons) outcomes of a behavior change. self - efficacy is one 's perceived confidence in the ability to carry out a specific behavior successfully ; and thus have different levels of confidence in their ability to maintain exercise benefits and to overcome exercise barriers. study in japanese female employees and showed that 90% of subjects were in inactive stages (precontemplation to preparation). in iran also, study showed 45.7% of employees women placed in precontemplation, contemplation and preparation stage and a significant relationship was found between the benefits, barriers, self - efficacy and stages of change. study also stated that more than70% of the people in yazd were investigated in preaction stages. the aim of this study was to determine the pa among employee women in isfahan university of medical sciences based on the transtheoretical model. this cross - sectional study was conducted in 100 female employees of isfahan university of medical sciences in 2013. all participants were informed about the details of the study and were asked to read and sign a consent form. the following are criteria for inclusion of women to the study : ruled employment as employees for more than 6 months, the desire to participate in the study, and not being pregnant. data collected by self - administered questionnaire. the demographic information (age, weight, height, number of child, level of education) were obtained. for measuring exercising behaviors, weekly pa was used. in order to measure exercise behavior, the stage of exercise behavior change questionnaire developed by marcus and forsythused in this study. this structure was measured by the process of change questionnaire developed by nigg. this questionnaire contains 30 items that measure the 10 process of change (cognitive and behavioral process). in this questionnaire, participants were asked to determine the frequency of occurrence of each item on a five - point likert scale (ranging from 1, never to 5, repeatedly). the decision balance scale for exercise, developed by plotnikoff. with 10 items based on lickert scale was used to assess pros and cons participants were asked to determine, on a five - point likert scale (ranging from 1, not at all important to 5, extremely important). the perceived self - efficacy scale consisted of 6 items with four point scale ranging from 1 (can not do) to 4 (certainly can do). all the questionnaires were translated using forward translation, back - translation in farmanbar. finally, content validity of questionnaires was assessed by an expert panel (five expert in health education and pa). the reliability was determined by cronbach 's alpha, and the scores of alpha for the process of change, self - efficacy, and decisional balance were 0.94, 0.81, and 0.78 continuously. we used descriptive (mean and frequency) and analytical statistics (independent t - test and one - way anova test). anova test was used to compare the mean of processes of change construct and self - efficacy with stages structure. the results showed that the average age was 37.61 7.99, and there was no any significant relationship between age and level of pa. the relationship between physical activity and demographic information distribution of people in stages of change was as follows : 26% precontemplation, 22% contemplation, 20% preparation, 13% action, and 19% maintenance. there were significant differences between consciousness raising, dramatic relief, counter - conditioning, stimulus control, helping relationships, reinforcement management, and self - liberation with stages of change constructs [table 2 ]. the relation between stages of change and processes of change furthermore, anova test showed there was no significant difference between environmental reevaluation, self - reevaluation, and helping relationship with stages of change constructs [table 2 ]. the mean of self - efficacy in stages of change the mean of pros are, respectively, as follows : precontemplation 58.15 22.29, contemplation 70.73 22.81, preparation 73.00 19.59, action 75.69 16.93, maintenance 86.10 18.40. the statistical test showed there were significant differences between pros and stages of change (f = 5.304, p = 0.001). in relation to cons, anova do not show differences between cons and stages of change. while, the mean of cons was in precontemplation 82.00 11.66, contemplation 77.82 13.11, preparation 76.00 15.41, action 70.15 14.11, maintenance 75.58 17.17 (f = 1.645, p = 0.169). this study was conducted with the aim of determining the pa among women employees in isfahan university of medical sciences based on the transtheoretical model. in this study, 26% of women were in contemplation, 22% in contemplation, 20% in preparation, 13% in action, and 19% in maintenance. indeed, 68% of subjects were inactive, and 32% were active. it consist with this study, and this matter could be due to cultural similarities between people in two city (isfahan and yazd). there were significant differences between consciousness raising, dramatic relief, counter - conditioning, stimulus control, helping relationships, reinforcement management, and self - liberation with stages of change construct, also with progressing to maintenance, the mean of constructs is increasing. study, all structures of the model were significantly different in stages of change. in kirk. study, helping relationships, self - liberation, and consciousness raising increased from precontemplation to maintenance. it seems that special attention needed to environmental reevaluation, self - reevaluation, and helping relationship for women. based on the findings, the mean of self - efficacy is increasing from precontemplation to maintenance and the mean of action and maintenance stage is more than other stages. in marcus study, finding show that people who were physical active had higher self - efficacy than inactive people. other studies such as nishida. and gorely and bruce stated that those who have high self - efficacy for pa, they are more physically active. purath and miller also showed women have more self - efficacy with an increase in the stages of change. results indicated that in the movement from precontemplation to maintenance pros are increasing, and cons are decreasing. this study has been done among the women, so there was no chance for inter gender comparison of pa in staff and also this limitation led to the small sample size. long questionnaire and impossibility of assessing pa in different jobs were other limitations of this study. this study has been done among the women, so there was no chance for inter gender comparison of pa in staff and also this limitation led to the small sample size. long questionnaire and impossibility of assessing pa in different jobs were other limitations of this study. because of a significant relationship between cognitive and behavioral processes and pa in this group, designing and implementing an educational program based on the transtheoretical model may be useful in promoting pa of a female employee	introduction : today, many jobs are associated with the inactivity or sedentary lifestyle. employees health will be affected by their depriving of the benefits of physical activity (pa). therefore, the present study was undertaken to determine the pa among employee women in isfahan university of medical sciences based on the transtheoretical model.materials and methods : this is a cross - sectional study has been performed in isfahan university of medical sciences employee women (2013). a convenience sample of 100 women was selected. data were collected by validated and reliable questionnaire in three parts (demographics information, pa scale, and ttm constructs). data were analyzed by spss spss (version 16.0 ; spss, ibm, inc, chicago, il, usa) and descriptive and analytical statistics such as anova and independent t - test were used. a two - tailed p < 0.05 was considered statistically significant.results:the mean of pa was 21.17 27.30 min in a day. weekly heavy, moderate, and light exercise mean was 0.72 1.81, 0.89 1.87 and 0.57 1.57 days, respectively. in this study, 26% of women were in contemplation, 22% in contemplation, 20% in preparation, 13% in action, and 19% in the maintenance stage. furthermore, there were significant differences between consciousness raising, dramatic relief, counter - conditioning, stimulus control, helping relationships, reinforcement management, and self - liberation with stages of change constructs.conclusion:because of a significant relationship between cognitive and behavioral processes and pa in this group, designing and implementing an educational program based on the transtheoretical model may be useful in promoting pa of a female employee.
like many other noncommunicable diseases, hypertension is gradually assuming epidemic dimensions with the dawn of epidemiological transition. it is the most common cardiovascular disorder, affecting approximately one billion people globally and causing approximately 7.1 million deaths annually.1 hypertension is the most common cardiovascular disease in africans,2 and congestive cardiac failure is its commonest complication.3 other complications include stroke, renal failure, atherosclerotic cardiovascular disease, and death.4 according to the world health organization, cardiovascular diseases are the leading cause of mortality and morbidity in developed countries and are emerging as a prominent public health problem in developing countries.5 in nigeria, common cardiovascular diseases reported with a high incidence include heart failure, hypertension, and congenital heart disease, with hypertension being the commonest.6 the nigerian national noncommunicable disease survey in 1997 reported an adult hypertension prevalence of 11.4%, with a variation of 14.8% and 9.8% in urban and rural areas, respectively.7 the cutoff value of a blood pressure (bp) 160/95 mmhg for hypertension was used, so giving an underestimation of the prevalence of hypertension. this would have been higher if the new cutoff value of 140/90 mmhg was used. various community surveys in nigeria have shown an adult prevalence of 15%36.6%.811 oladapo recently reported a prevalence of 20.8% in the rural community of egbeda, southwest nigeria.12 the prevalence in semiurban ile - ife was 36.6%, while that in ipetumodu, osun state, nigeria, was 26.4% for mild hypertension (bp 140/90 mmhg) and 11.8% for moderate hypertension (bp 160/100 mmhg).12 the overall prevalence of hypertension ranges from 14.5% in nigeria, 16.9% in cameroon, 28.7% in accra,13 to 32.6% for blacks in the united states.14 cross - migration from rural to urban areas and vice versa, the asymptomatic nature of the disease, changing lifestyles, and the environment are contributing to the rising problem of hypertension. hypertension has been associated with various factors, including age, gender, family history, alcohol consumption, smoking, obesity, level of education, and occupation.15 hypertension is a silent killer and most patients are detected to have it incidentally when they are admitted to hospital for unrelated disease or subjected to pre - employment or preoperative medical checkups. many patients are also diagnosed when they seek medical advice because of target organ damage. target organ damage and associated clinical conditions are very common in newly diagnosed hypertensive patients in nigeria, with a recently reported prevalence of 66.7%.16 however, the majority of the rural population in nigeria is marginalized and has inadequate access to health care and quality education.17 a significant number of rural dwellers seek consultations with indigenous and private health care practitioners, where regular screening for hypertension is not practiced. clinic - based (opportunistic) screening of hypertension may not detect a large proportion of adults with hypertension. few studies have demonstrated the occurrence of hypertension and its predisposing risk factors in rural areas of nigeria. the objective of this study was to determine the prevalence of hypertension in two selected rural communities in southwestern nigeria. this was a community - based, descriptive, cross - sectional study carried out in two rural communities in osun state in southwestern nigeria. the two rural communities were alajue and ibokun, with a total population of 5000 people. factors predisposing to a western lifestyle appeared minimal in these communities, although they have roads making the communities accessible to town and cities. this study was carried out as a part of a community - based screening program to raise awareness of the 2011 world kidney day celebrations. entry commenced with holding a series of meetings with kings, traditional chiefs, politicians, heads of households, and other gatekeepers in the community. community town hall meetings were organized for each of the communities to mobilize and sensitize community members further. at these meetings, community members eligible for screening were encouraged to come out en masse, with the assurance that all those who came out and consented to the exercise would be screened. the two communities were mobilized and sensitized regarding the importance of screening for noncommunicable diseases, in particular, hypertension and diabetes. all consenting adults who participated in the screening exercise had their bp recorded, in addition to anthropometric measurements. an interviewer administered a pretested questionnaire to collect basic data pertaining to sociodemographic characteristics and risk factors for hypertension among the respondents. ethical approval to conduct the study was obtained from the ethical research committee of osun state university college of health sciences. a limitation of this study was its inability to study complications associated with hypertension in these rural communities, although this was actually beyond the scope of the present study. to standardize survey measurements and procedures, ten research assistants were trained using specially prepared survey manuals that conformed to recommended noncommunicable disease survey protocols.18 before the main study, a field test was carried out to fine - tune instruments and procedures. hypertension was defined as systolic bp 140 mmhg and/or diastolic bp 90 mmhg, based on the guidelines of the international society of hypertension / world health organization19 and the seventh joint national committee on hypertension,20 or being on regular antihypertensive drugs. overweight / obesity was defined as bmi 25 kg / m and 30 kg / m, respectively. a standardized calibrated mercury column type sphygmomanometer, stethoscope, common weighing machine, and measuring tape were used for physical examination. the subjects rested quietly for 10 minutes, and bp was measured in the sitting posture with an appropriate - sized cuff encircling the arm. with the left arm of the subject held at the level of the heart, the maximum inflation level was determined using a mercury sphygmomanometer, using a 15 cm stethoscope and a cuff of appropriate size (a cuff of larger width was used for large arms). bp was measured to the nearest 2 mmhg on two occasions at an interval of one minute. where the two readings differed by over 10 mmhg, a third reading was obtained, and the three measurements were averaged. the pressures at which sounds appeared and disappeared were taken as systolic bp and diastolic bp, respectively. body weight was measured (to the nearest 0.5 kg) with the subject standing motionless on the calibrated weighing scale, with feet about 15 cm apart and weight equally distributed on each leg. subjects were instructed to wear minimum outerwear (as culturally appropriate) and no footwear while the weight was being measured. height was measured (to the nearest 0.5 cm) using a calibrated tape and with the subject standing in an erect position against a vertical surface, and the head positioned so that the top of the external auditory meatus was level with the inferior margin of the bony orbit. bmi was calculated as weight in kilograms divided by height in meters squared. based on their bmi, individuals were classified into four groups, ie, thin (bmi 30.0), according to who classification. the statistical package for social sciences (spss) software version 17 (spss inc, chicago, il, usa) was used for data entry and analysis. validity of data was ensured by double entry and random checks for errors and outlier values. multivariate logistic regression analysis was done using systolic and diastolic bp as the dependent variable and risk factors identified to be significant in the bivariate analysis as independent variables. the level of significance was set at p values 0.05 for all inferential analysis of categorical variables. to standardize survey measurements and procedures, ten research assistants were trained using specially prepared survey manuals that conformed to recommended noncommunicable disease survey protocols.18 before the main study, a field test was carried out to fine - tune instruments and procedures. hypertension was defined as systolic bp 140 mmhg and/or diastolic bp 90 mmhg, based on the guidelines of the international society of hypertension / world health organization19 and the seventh joint national committee on hypertension,20 or being on regular antihypertensive drugs. overweight / obesity was defined as bmi 25 kg / m and 30 kg / m, respectively. a standardized calibrated mercury column type sphygmomanometer, stethoscope, common weighing machine, and measuring tape were used for physical examination. the subjects rested quietly for 10 minutes, and bp was measured in the sitting posture with an appropriate - sized cuff encircling the arm. with the left arm of the subject held at the level of the heart, the maximum inflation level was determined using a mercury sphygmomanometer, using a 15 cm stethoscope and a cuff of appropriate size (a cuff of larger width was used for large arms). bp was measured to the nearest 2 mmhg on two occasions at an interval of one minute. where the two readings differed by over 10 mmhg, a third reading was obtained, and the three measurements were averaged. the pressures at which sounds appeared and disappeared were taken as systolic bp and diastolic bp, respectively. body weight was measured (to the nearest 0.5 kg) with the subject standing motionless on the calibrated weighing scale, with feet about 15 cm apart and weight equally distributed on each leg. subjects were instructed to wear minimum outerwear (as culturally appropriate) and no footwear while the weight was being measured. height was measured (to the nearest 0.5 cm) using a calibrated tape and with the subject standing in an erect position against a vertical surface, and the head positioned so that the top of the external auditory meatus was level with the inferior margin of the bony orbit. based on their bmi, individuals were classified into four groups, ie, thin (bmi 30.0), according to who classification. the statistical package for social sciences (spss) software version 17 (spss inc, chicago, il, usa) was used for data entry and analysis. validity of data was ensured by double entry and random checks for errors and outlier values. multivariate logistic regression analysis was done using systolic and diastolic bp as the dependent variable and risk factors identified to be significant in the bivariate analysis as independent variables. the level of significance was set at p values 0.05 for all inferential analysis of categorical variables. table 1 shows that the age group 5069 years constitutes the highest proportion (n = 109, 42.0%) among respondents. one hundred (38.6%) were males, 111 (42.9%) were traders, and 84 (32.4%) were farmers by occupation. seventeen (6.6%) had isolated systolic hypertension, 11 (4.2%) had isolated diastolic hypertension, and 189 (73.0%) were normotensive. two hundred and thirty - six (91.1%) undertook daily exercises lasting at least 30 minutes. forty - eight (18.5%) had ever taken antihypertensive drugs, mostly the centrally acting agent, alpha methyldopa, and the diuretic combination of amiloride and hydrochlorothiazide. four (1.6%) claimed a family history of hypertension and 48 (18.5%) had ever taken antihypertensive drugs on a regular basis. the prevalence of overweight and obesity in the study population was as follows : 51 respondents (19.6%) had a bmi of 25.029.9 while 30 (11.5%) had a bmi of 30. figure 1 shows that more females (n = 19) were hypertensive as compared with males (n = 15). similarly, more females were overweight or obese (n = 35 and n = 19, respectively) than males (n = 16 and n = 11). a significant association existed between age older than 40 years and having hypertension (p 0.05). respondents with bmi 25 had a three times greater likelihood of developing hypertension compared with those having a bmi < 25 (odds ratio 2.9, 95% confidence interval 0.0070.056, p = 0.011). older age and increased bmi were significantly more common in hypertensive respondents than in their normotensive counterparts. this compares well with another study showing a mean age of about 42 years in rural communities in nigeria.21 just over 10% of the rural respondents we screened were hypertensive. this is consistent with the finding of a 10% prevalence in a typical community in rural india.22 however, the prevalence in our study is lower than that reported by other studies from nigeria (14.5%) and cameroon (16.9%,23 18.3%,24 and 19.04%).25 it is also much lower when compared with other studies reporting prevalences of 25.4%,21 28.3%,13 28.7%,26 30.5%,27 and 37.7%.28 however, our figure is slightly higher than that from a study conducted in sudan, which reported a prevalence of 7.5%.29 in this study, about 6.6% had isolated systolic and 4.2% diastolic hypertension, which is similar to another study reporting isolated systolic hypertension in 10.5% and isolated diastolic hypertension in 9.0% of subjects.30 a diverse pattern in the prevalence of hypertension can be said to exist in rural populations around the world. typical rural populations, being marginalized and vulnerable communities in nigeria, show a pattern of people migrating to urban areas or still living rurally but working in urban areas. in the process, some may be adopting the western lifestyle in the form of diet and physical activity related to urbanization and modernization, and western lifestyle has been identified as a risk factor for hypertension.31 like many other developing countries, disparity exists between rural and urban populations in terms of health facilities, education, and economic pursuits.15 for the same reason, about 10% of our rural population had taken antihypertensive drugs, mostly methyldopa and a combination of amiloride and hydrochlorothiazide, and were not on newer antihypertensive drugs for reasons of affordability and accessibility. further, the primary health care centers that serve this rural population only stock first - line antihypertensive drugs requiring minimal biochemical monitoring. a bp survey of 20 rural ghanaian villages in 1973 found a hypertension prevalence of 2%5%, and concluded that hypertension was not a significant health problem in rural ghanaians.32 thus, an increase in the prevalence of hypertension can be said to be occurring in rural populations undergoing modernization. this supports the fact that hypertension, which was considered to be nonexistent or extremely rare in most african societies, particularly in rural communities, is now emerging as a public health problem in sub - saharan africa,32,33 and its prevalence will increase even further unless broad and effective preventive measures are implemented.34 there is a need for primary prevention efforts targeted to all communities, both rural and urban. furthermore, the clinical significance of our findings is that, given that hypertension is now on the rise in rural areas of southwestern nigeria, and probably related to the recent rise in occurrence of modifiable risk factors of hypertension occasioned by adoption of lifestyle changes in these rural populations, it is imperative that health policy - makers give priority to improved allocation of health resources to rural health facilities for effective management of hypertension. in this study, this supports a study in which hypertension has been found to be associated with older age,29 and yet others finding an association in people older than 40 years.25,3538 it had been reported that the age - standardized prevalence of hypertension is significantly higher in urban than in rural areas for both men and women.39 in this study, hypertension was associated with being overweight and obese, and corroborates similar studies showing that bmi is strongly and independently associated with systolic and diastolic bp.4042 in fact, overweight and obesity are no longer rare in rural residents.13,43 the prevalence of hypertension is on the increase in rural areas, contrary to speculation, and is associated with certain sociodemographic factors and bmi. hence, there is a need for primary prevention efforts targeted at modifiable risk factors on a large scale.	backgroundthe purpose of this study was to determine the prevalence of hypertension in two rural communities of osun state, nigeria.methodsa consenting adult population of the alajue and obokun rural communities in southwestern nigeria that presented for the screening exercise participated in this community - based cross - sectional descriptive study. two hundred and fifty - nine respondents aged older than 18 years completed a standardized, pretested, structured questionnaire as part of activities celebrating world kidney day and world glaucoma day in 2011. anthropometric data and blood pressure were recorded, and the data were analyzed using the statistical package for social sciences version 17.resultsthe mean age of the respondents was 49.7 1.6 years, 100 (38.6%) were males, 84 (32.4%) were farmers, and 111 (42.9%) were traders. the prevalence of hypertension was 13.16% (present in 34 respondents). seventeen (6.6%) had isolated systolic hypertension, while 11 (4.2%) had isolated diastolic hypertension. two hundred and thirty - six (91.1%) undertook daily exercise lasting at least 30 minutes and 48 (18.5%) had ever taken antihypertensive drugs on a regular basis. four respondents (1.6%) claimed a family history of hypertension. the average body mass index (bmi) among respondents was 23.4 4.9 kg / m2, 51 (19.6%) had a bmi of 25.029.9, and 30 (11.5%) had a bmi 30. a significant association existed between age older than 40 years and having hypertension (p 0.05). rates of older age and high bmi were significantly higher among hypertensives than among normotensives. respondents with bmi 25 had at least a three times greater likelihood of developing hypertension than those with bmi < 25 (odds ratio 2.9, 95% confidence interval 0.0070.056, p = 0.011).conclusionthe prevalence of hypertension is high in this study population and we recommend scaling up primary prevention efforts to reduce this in nigerian communities.
cu / zn superoxide dismutase or sod1 is a soluble protein acting as a 32 kda homodimeric enzyme to convert naturally occurring, but harmful, superoxide radicals to molecular oxygen and hydrogen peroxide. sod1 is one of the three human superoxide dismutases identified and characterized in mammals : copper - zinc superoxide dismutase (cu / znsod), manganese superoxide dismutase (mnsod or sod2), and extracellular superoxide dismutase (ecsod or sod3). when sod1 was isolated for the first time, it was thought to be a copper storage protein ; the catalytic function of sod1 was discovered in 1969 by mccord and fridovich, and it was clear that sod1 acts as a scavenger of superoxide, through a two - step reaction involving reduction and reoxidation of the copper ion in its active site. primarily, this reaction occurs in the cytoplasm where sod1 is highly expressed. however, immunohistochemical analysis in rat hepatocytes identified sod1 in other different subcellular organelles, such as nucleus, lysosomes, and mitochondria. in the 1990s, the scientific community focused their studies on the genetic and biochemical characterization of sod1, demonstrating that sod1 plays an important role in diseases as heart failure, cancer, diabetes, down 's syndrome, and amyotrophic lateral sclerosis. in fact, in 1984, the first paper about down 's syndrome was published, and in 1993, the first sod1 gene mutations associated with als were described. in this paper, we focused on sod1 gene structure and organization, transcriptional and post - transcriptional gene regulation, and their contributions in the pathogenesis of als. the human sod1 gene (entrez gene i d 6647) is located on chromosome 21q22.11, and it codes for the monomeric sod1 polypeptide (153 amino acids, molecular weight 16 kda). more precisely, this gene is located from base pair 33,031,935 to base pair 33,041,241 with a genomic size of 9307 bp, according to ucsc genome browser (grch37/hg19 ; http://genome.ucsc.edu/). the coding region consists of five exons interrupted by four introns (figure 1). several polymorphisms have been identified in sod1 gene, mainly distributed in the regulatory regions, including promoter, utrs, and introns. with regards to sod1 mrna splicing, it has been demonstrated that the donor sequence at the first intron presents a t to c variant and, consequently, it deviates from the highly conserved 5 gt... ag 3 consensus sites. nevertheless, it has been demonstrated that this unconventional splice junction (5 gc... ag 3) is functional. the proximal promoter of human sod1 gene, involved in the basal transcription, has been well studied, and it contains not only the tata box, but also a ccaat box and a gc - rich region, which are recognized by ccaat / enhancer - binding proteins (c / ebps) and specificity protein 1 (sp1), respectively. other binding sites for egr1, ap1, ahr, nrf2, nf-b, and tr transcription factors have been also identified and verified by functional studies (figure 1). these transcription factors are involved in the regulation of sod1 inducible gene expression under specific extra- and intracellular conditions. different studies performed in rat cellular lines have identified several other regulatory sequences in the rat sod1 promoter [1518 ]. considering that the rat sod1 gene is very similar to that of human, especially with regard to the proximal part of the promoter region, further efforts will be necessary to identify possible other regulatory elements in the human promoter. moreover, in view of the fact that regulatory elements can be located up to several hundred kilobases away from the gene they control, experiments should be carried out to test whether the long - range control of transcription may represent a mechanism involved in human sod1 gene expression. with regard to the 5 untranslated region (5utr), sherman and colleagues demonstrated that sod1 mrna posseses various 5 termini, which are mapped by both primer extension and sl mapping. the authors showed that the vast majority of the mrna species has a 5 start site located 23 bp downstream the tata box (tataaa), while the other mrna molecules have 5 termini 30, 50, and 65 bp upstream from the major transcription start site. it would be interesting to perform in - depth investigations about the functional relevance of these multiple transcription start sites, mainly to determine the potential cell and tissue specificity of the different mrna species. furthermore, two sod1 mrnas of about 0.7 kb and 0.9 kb have been identified in a variety of cells, and it has been shown that they are transcribed from the same gene and differ in the length of their 3utrs caused by multiple polyadenylation sites. indeed, analysis of the dna sequence at the 3utr region of the sod1 gene revealed the presence of two groups of processing / polyadenylation signals ; the first one contains two signals (aataaa and attaaa). the former is fused to the terminal portion of the coding region and it is not utilized. on the contrary, the second site (attaaa), localized 76 bp downstream the stop codon, is the one involved in the production of the 0.7 kb mrna (figure 2). the second group includes three polyadenylation signals, located 200250 bp further downstream ; the middle one (aataaa) is involved in the formation of the 0.9 kb sod1 mrna (figure 2). both the mrna species are functional, since they can be translated in vitro to immunoprecipitable sod1 proteins [19, 20 ]. the longer transcript is approximately four times less abundant than the 0.7 kb mrna. even if sod1 has been often considered a housekeeping gene due to its high and ubiquitous expression, it is now clear that its induction is fine - tuned modulated by complex intracellular events which probably involve multiple positive and negative regulatory elements acting in concert. indeed, the diversity of sod1 inducers means that there are multiple cis - acting elements for this gene, and many studies have been performed to precisely identify the location and the functional relevance of both these dna sequences and the corresponding trans - acting factors. these proteins are a family of transcription factors, all containing a highly conserved, basic leucine zipper (bzip) domain at the c - terminus. it has been demonstrated that c / ebp - related factors are necessary for sod1 constitutive expression ; both c / ebp and c / ebp can interact with the caat box (located at position 64 to 55 from transcription start site), playing similar and nonmutually exclusive roles on sod1 basal transcription. this c / ebp consensus element partially overlaps the sp1/egr1 sequence, suggesting that an interlaced network among these transcription factors may act in the fine control of regulation of sod1 gene expression. furthermore, it has been recently demonstrated that also the transcription factor ccaat / enhancer binding protein delta (cebpd, c / ebp, nf - il6) is involved in the regulation of human sod1 transcription. specifically, cebpd enhances sod1 mrna expression in cisplatin - treated human urothelial carcinoma cell line (ntub1) via direct promoter transactivation. it binds gc - rich motifs (such as 5-g / t - gggcgg - g / a - g / a - c / t-3 or 5-g / t - g / a - ggcg - g / t - g / a - g / a - c / t-3) with high affinity and enhances transcription with one of the two glutamine - rich domains. egr1 is a nuclear phosphoprotein of 80 kda that functions as a regulator of transcription and belongs to the family of early response genes ; it is rapidly induced by mitogens to transduce the proliferative signal. it has also been demonstrated that cytokines and stress signals such as radiation, injury, and oxidative or mechanical stress can induce the expression of this transcription factor [2529 ]. in a paper published by minc and coworkers, it has been demonstrated that sod1 mrna level is rapidly increased after the treatment of hela cells with phorbol-12-myristate-13-acetate (pma), and the region between nucleotides 59 and 48 has been identified as the one responsible for pma - induced expression. this region presents noncanonical consensus recognition sequences for sp1 and egr-1, and it is bound by sp1 in a constitutive manner and by egr1 in response to pma exposure. ap-1 is a homo- or heterodimeric transcription factor made by proteins from jun, fos, and maf subfamilies. activating transcription factor (atf) the activity of ap-1 proteins can be regulated by a broad range of environmental cues, including growth factors, cytokines, and oxidative stress, which initiate a variety of intracellular pathways to transduce the information from the extracellular milieu to the nuclear compartment, thus leading to specific cellular responses. it has been demonstrated that ap1 represses sod1 transcription by sequestrating essential coactivators, such as sp1, rather than interacting directly with sod1 gene promoter. furthermore, in agreement with the previous results demonstrating ap1 transcriptional repression activity, it has been shown that neuronal nitric oxide synthase (nnos) over - expression causes the downregulation of sod1 in terms of mrna, protein, and activity levels and that this is caused by two events : the decreased binding of sp1 to sod1 promoter, caused by nnos interaction with sp1, and a concomitant increased binding activity of ap1 to the same site. ahr is a ligand - activated transcription factor belonging to the helix - loop - helix (bhlh) family. it is well known that, prior to ligand binding, ahr exists in a latent state in the cytosol associated with hsp90 and hsp90 accessory proteins [3234 ]. many synthetic halogenated and nonhalogenated aromatic hydrocarbons activate ahr signal pathway through a direct interaction : upon ligand binding, hsp90-bound ahr translocates into the nucleus where it exchanges hsp90 for another bhlh protein, known as hydrocarbon receptor nuclear translocator (arnt). this new heterodimeric complex binds to the xenobiotic responsive element (xre) that functions as a cis - acting enhancer in the promoter region of numerous phase i and ii drug - metabolizing enzyme genes [35, 36 ]. cho and coworkers observed an increased expression of sod1 mrna and protein after the exposure of human hepg2 and hela cells to one of the most toxic man - made hazard, the 2,3,7,8-tetrachlorodibenzo - p - dioxin (tcdd), an environmental contaminant belonging to the halogenated aromatic hydrocarbons class and interacting with ahr. the authors identified the presence of a xenobiotic responsive element in the 5flanking region of human sod1 gene (located between 255 and 238 from the transcription start site), which is responsible for the induction by tcdd. nrf2 is a capncollar (cnc) transcription factor that regulates the expression and the coordinated induction of a battery of defensive genes encoding phase ii detoxifying enzymes and antioxidant proteins. the activity of nrf2 is controlled by the cysteine - rich cytosolic inrf2 (inhibitor of nrf2), also known as keap1 (kelch - like ech - associated protein1). the activation of nrf2 pathway requires its cytosolic stabilization via oxidative modification of distinct keap1 cysteine residues, keap1 preoteasomal degradation, and/or phosphorylation of nrf2. the dissociation from keap1 is a prerequisite for nrf2 translocation to the nucleus. in this compartment, nrf2 heterodimerizes with a small maf protein and binds to the cis - acting antioxidant / electrophile responsive element (are / epre), activating the transcription of various cytoprotective genes involved in detoxification from xenobiotics, electrophile conjugation, ros scavenging, and regulation of intracellular redox homeostasis [3841 ]. an antioxidant responsive element (located between 356 and 330 from the transcription start site) has been identified in human sod1 gene promoter [42, 43 ]. park and colleagues demonstrated that sod1 gene transcription is induced in human hepg2 hepatoma cells after the treatment with the dioxin tcdd, which produces reactive oxygen species thus leading to the activation of nrf2 signalling. moreover, it has been shown that low - dose and nontoxic proteasome inhibition enhances mrna and protein expression of sod1 in different human endothelial and vascular smooth muscle cells through transcriptional induction mediated by nrf2. the term nf-b refers to a family of five structurally related transcription factors (p50, p52, rela / p65, c - rel, and relb), all containing the rel homology domain (rhd) within the n - terminus and acting as homo- and heterodimeric dna binding complexes. their functionality and nuclear localization are controlled by a family of inhibitor proteins, known as ikappabs (ibs). in nonstimulated cells, nf-b dimers are bound to inhibitory ib proteins and are thereby sequestered in the cytoplasm as inactive complexes. several studies showed that nf-b activity is induced in most cell types in response to a broad variety of stimuli, ranging from cytokines, radiation, and oxidative stress (such as exposure to h2o2), with major roles in coordinating innate and adaptive immunity, cell activation and proliferation, survival, development, and apoptosis [45, 46 ]. nf-b was one of the first transcription factors shown to be redox regulated [4750 ], and rojo and colleagues showed that cell treatment with h2o2 initiates the pi3k / akt cascades, which participates in nf-b activation and in subsequent sod1 transcriptional induction. indeed, the authors identified a p65-nf-b binding site in the human sod1 promoter (ggtaagtccc), and they demonstrated that akt - activated nf-b presents increased binding to this sequence, mediating the upregulation of sod1 expression. the thyroid hormone receptors are encoded by the tralpha and trbeta genes and are ligand - dependent transcription factors, since they bind both thyroid hormones (ths) and th - response elements (tres) that are located in the promoters of target genes. these proteins, which belong to the nuclear receptor superfamily, regulate development and a broad variety of critical cellular functions including growth, differentiation, basal metabolic rate, and metabolism of protein, fat, and carbohydrate. hormone - dependent repression requires binding to negative tres (ntres) ; in particular, the unoccupied receptor increases transcription on ntres, and ligand binding to tr reverses this induction. the responsive sites of these negatively regulated genes are generally localized to the proximal promoter region. in agreement with this observation, santos and co - workers have identified a thyroid hormone inhibitory element between 157 and + 17 of the human sod1 promoter and demonstrated that t3 exposure reverses the induction of sod1 transcription caused by the ros - producing paraquat and pma agents, through the direct tr / t3-dna interaction. on the contrary historically, efforts aimed at decoding the molecular mechanism of gene expression have been principally focused on transcriptional control. however, post - transcriptional regulation of mrnas is now considered as an important step in the flow of genetic information providing additional opportunities by which gene expression could be rapidly modulated. indeed, post - transcriptional events such as mrna processing and nuclear export, mrna stability, translational efficiency, and microrna - dependent modulation create a complex intracellular network contributing to determine the global levels of specific mrnas. most mrna regulatory elements are located within the 5 and 3utrs, where they function as platforms for the binding of numerous proteins and noncoding rnas. the 5utr is principally involved in controlling mrna translation, while the 3utr regulates multiple steps of mrna metabolism and stability. very few works on post - transcriptional regulation of sod1 have been published so far. as mentioned above, two species of sod1 mrna with different 3utr lengths kilk and colleagues performed functional study to investigate the effect of this regulatory region on sod1 protein expression, and they observed that cells transfected with a long cdna, containing the long 3utr, produce three times more sod1 protein than cells transfected with a cdna presenting a deletion of the last 185 bp from the 3utr. the authors hypothesized that the ability of the long mrna to produce more sod1 enzyme may depend on specific sequences located in the 3utr, and they identified the presence of various a / u - rich elements (ares) in this region (auuua, cuuua, auuug, guuuua, auuuu, and auuuc) (figure 2). ares were initially defined by the sequence auuua, although subsequent studies showed other sequences with u - stretches presenting the same properties as the canonical element. several are - binding proteins, which function as trans - acting factors, have been identified, and interaction of these proteins can correlate negatively or positively with the stability and the translatability of the target mrna. considering the biological relevance of these sequences, the identification of the proteins potentially interacting with sod1 mrna may shed new light on sod1 gene expression modulation. moreover, another important question is whether these sod1 mrna species, differing for their 3utr lengths, could be somehow linked to pathological conditions, since it is possible that specific cellular stresses may vary the relative proportions of such variants with relevant implications for cell phenotypes. in addition, also sod1 post - transcriptional regulation mediated by micrornas (mirnas) represents a field still almost unexplored. mirnas, which are small non - protein - coding rnas, function as key post - transcriptional regulators of gene expression by usually base pairing to the 3utr of the target mrnas to cause translational repression and mrna decay [59, 60 ]. recently, wang and co - workers showed, through computational and biological approaches, that sod1 is a target of mir-377 in human and mouse mesangial cells and mir-377 diminished sod1 protein levels. owing to the importance of mirnas in mrna metabolism control, future in - depth researches should be carried out in this field to unravel previously unrecognized complex regulatory and interactive pathways that may cooperate to modulate sod1 quantities and whose dysregulation may be relevant for als disease states. amyotrophic lateral sclerosis disease (als) is a multifactor and multigenic disorder with still unknown aetiology and pathogenesis. even if several new genes associated to als have been described, sod1 gene is considered the major gene involved in als pathogenesis. in 1991, siddique and collaborators identified a linkage of familial als to the sod1 locus on chromosome 21q22 and demonstrated genetic locus heterogeneity in fals studying 23 als families. in 1993, rosen and collaborators have reported tight genetic linkage between als and sod1 gene, establishing sod1 as the first causative gene for als (genetic nomenclature, als1). mutations in sod1 gene are responsible for 1223% of all fals cases [63, 64 ]. more than 150 sod1 mutations have been reported in 68 of the 153 codons, spread over all five exons (als online genetic database, alsod : http://alsod.iop.kcl.ac.uk/), most of them cause disease. although most mutations are missense, nonsense mutations and deletions have also been found [66, 67 ]. sod1 exonic mutations have occasionally been described in patients with apparently sporadic onset, and it has been estimated that 1% of sals could be due to sod1 mutations. correlation between mutations and phenotype has been investigated, because of a large variability in phenotype in term of disease progression, extramotor features, and age of onset but is generally difficult to predict on the basis of the sod1 mutations. even if sod1 gene has been considered fundamental in als, numerous studies have been done concerning protein expression and gene mutations, but mrna level studies are very rare. for the first time, in 1997, nishiyama studied sod1 expression in cervical and spinal cord of als patients using a quantitative in situ hybridization technique. there were no significant differences between the amounts of sod1 mrna level observed in patients with sporadic or familial disease, and normal control subjects. moreover, motoneurons in the normal spinal ventral horn and precentral motor cortex exhibited significantly higher levels of sod1 messenger rna than did other neurons. in recent years, expression studies often produced conflicting data about up- or down- regulation of als relevant genes, such as sod1 [7072 ]. the confounding data were probably due to differences in the central nervous system (cns) tissue areas analyzed and possibly differences in the analytical techniques used. the latest sod1 gene expression study demonstrated that sod1 mrna level is elevated in specific nervous areas typically affected by als disease (i.e., brain stem and spinal cord) and not in other brain areas not involved in the neurodegenerative process (i.e., cerebellum and cerebral cortex) in sals patients. moreover, increased sod1 mrna expression has been detected in peripheral system as lymphocytes from sals patients compared to healthy people. however, in the same work, western blotting analysis showed lower or similar expression of the protein both in lymphocytes and in nervous tissue - affected areas thus confirming a previous observation obtained in the peripheral cells. on the contrary, histopathological analysis of spinal cord tissue from sals patients evidenced an increased sod1 protein expression compared with controls. this result correlates with the evidence of higher mrna level and suggests the hypothesis that, in at least a subgroup of sals patients, misfolded and aggregated sod1 protein would precipitate in the insoluble fraction becoming undetectable after extraction with routinely used lysis buffers. actually, proteinaceous inclusion bodies that may contain sod1 have been already described in motor neurons of sals patients [7577 ], and the presence of aberrant sod1 species associated with human sporadic als has been extensively demonstrated [7880 ]. insoluble sod1-containing aggregates are also a characteristic of the familial als linked to sod1 mutation. in mutsod1 transgenic mouse, the detergent - insoluble accumulation of sod1 appears before or coincident with symptom onset, and in the human sod1-associated als, mutant sod1 aggregation is a pathological hallmark. considering the few literature data about sod1 gene regulation, it appears clear the importance of understanding how the expression of this gene can be controlled and modulated in normal and als pathological conditions. the molecular pathways regulating sod1 expression at the transcriptional level have been studied, and many cis - elements and the relative trans - acting protein factors have been identified. nevertheless, the details of most of these interactions and the in vivo consequences of their modulation for the most part are yet to be determined, mainly in relation to pathological states. moreover, also post - transcriptional mechanisms may exert important functions in determining the global levels of functional sod1. indeed, the formation of specific ribonucleoprotein complexes and rna - silencing events may provide additional mechanisms by which sod1 expression could be rapidly and precisely modulated. in particular, considering that micrornas are emerging as master regulators of gene expression due to their capability to finely tune gene dosage, investigation in this field should become a preferred topic for new researches. future gain of knowledge about these processes may help to discover previously unanticipated integrated networks, leading to new and exciting directions in the field of als medical research with promising prospects. indeed, the progress in understanding the mechanisms of transcriptional and post - transcriptional control could offer hope for the development of new - generation drugs or medical treatment strategies.	copper - zinc superoxide dismutase (sod1) is a detoxifying enzyme localized in the cytosol, nucleus, peroxisomes, and mitochondria. the discovery that mutations in sod1 gene cause a subset of familial amyotrophic lateral sclerosis (fals) has attracted great attention, and studies to date have been mainly focused on discovering mutations in the coding region and investigation at protein level. considering that changes in sod1 mrna levels have been associated with sporadic als (sals), a molecular understanding of the processes involved in the regulation of sod1 gene expression could not only unravel novel regulatory pathways that may govern cellular phenotypes and changes in diseases but also might reveal therapeutic targets and treatments. this review seeks to provide an overview of sod1 gene structure and of the processes through which sod1 transcription is controlled. furthermore, we emphasize the importance to focus future researches on investigating posttranscriptional mechanisms and their relevance to als.
evidence that patterns of human health and disease are predicated on socioeconomic and environmental conditions is compelling. as a population that has witnessed significant transitions over the past few decades, black south africans exemplify how overall health status is moulded by political and socioeconomic changes. not until the first democratic elections in 1994 did the enshrined structural inequities limiting their freedom of natural growth and potential to participate more equally in the economic and social life of south africa, give way to a freedom to choose where to live and work. with the end of the oppressive history of colonisation and apartheid, most black south africans have had to grapple with a new set of challenges affecting their health. contributing to a recent lancet review on health in south africa, coovadia. noted that the distinctive features of south africa 's history that account for the current health problems include racial and gender discrimination, income inequalities, migrant labour, the destruction of family life, and persistent violence spanning many centuries but consolidated by apartheid in the 20th century. indeed, even a cursory analysis of the country 's current health profile will not miss the simultaneous occurrence of epidemic infectious diseases, perinatal and maternal disorders, injuries, and chronic noncommunicable diseases (ncds). chronic ncds including hypertension is growing substantially as a cause of death and disability in all strata of the south african society, but most predominantly among poor people living in urban settings [4, 5 ]. this pattern mirrors the prevalence of risk factors for hypertension and underlying determinants including demographic and nutritional transitions arising from socioeconomic development and increasing globalisation.. analysed results of the south african demographic and health survey (sadhs) to identify population groups with a high prevalence and poor control of hypertension in the country. they showed that hypertension risk was lowest in rural blacks and significantly higher in obese black women than in women with a normal body mass index. high risk of hypertension was associated with education below tertiary level, older age groups, being overweight and obese, excess alcohol use, and a family history of stroke and hypertension. the growing trend has been exacerbated by the rapid increase in urbanization, notably the unprecedented levels of migration from century - old traditional lifestyles in rural areas to the large periurban settlements of cities. this migration contributes to the increasing inequities between the rich and poor in respect of hypertension risk ; people who live in poverty tend not to benefit from the higher living standards of urban life. urbanization has an effect on almost all aspects of the migrant 's lifestyle contributing to increasing risk of hypertension and other ncds. these include the influence on the migrant 's diets, level of physical activity, and use of alcohol and tobacco products. in many developing countries including south africa there has been a shift in dietary intake from a traditional diet high in fibre to a more western diet that is high in fat and refined carbohydrates. in addition, data have shown that south africans consume more salt than the recommended maximum of 6 g / day. regarding physical activity, urban dwellers more often make use of motorized transportation, accessible public transport, and thus less walking or less labour - intensive work, while having more sedentary leisure activities like watching television than rural dwellers. studies have shown that even moderate intensity activity is protective [911 ]. according to norman and colleagues, an estimated 3.3% of total deaths in south africa was due to physical inactivity. the contributing factors cited as driving the nutrition transition include the limited availability of affordable healthy food in poorer periurban areas, combined with the increased availability of fast foods that are high in fats and sugar. other contributing factors to physical inactivity are limited outdoor space, high rates of street violence, and cultural beliefs and practices. along with changes in dietary intake and physical inactivity, urban african women had the highest rate compared to other racial groups [14, 15 ]. some studies have reported that obesity among african women was associated with being affluent, attractive, and healthy [16, 17 ], and although they were aware of the consequences of being overweight, they perceived weight loss as a source of stigma and a sign of disease, particularly of hiv / aids [13, 18 ]. despite awareness of these different factors, the focus of hypertension prevention remains largely on advocating change in individual lifestyles. satcher has argued that hypertension control is an appropriate goal and certainly the elimination of disparities in hypertension control must take place in the context of physical and social environments and human behaviour. approaches that recognise the centrality of individual behaviour and community - based health promotion, and which exploit local initiative and opportunities, are required. khayelitsha (meaning new home) is a township for black africans located on the cape flats, approximately 35 km outside cape town. this is the fastest growing township in south africa, which mainly consists of informal settlements. however, a third of the houses are formal brick structures pointing to the relative affluence and better socioeconomic status of some parts of khayelitsha compared to others. it further suggests the increasing formalization of settlements within khayelitsha as more permanent residential structures now exist. the population of khayelitsha is continuously growing with newcomers arriving mainly from the adjacent eastern cape province. although health clinics serve the community, the staff members are not always able to meet the preventive care or needs of their chronic patients. therefore, the help of community health workers (chws) were introduced to assist with the current shortfalls in human resources. a nongovernmental organization invited us to collaborate with them by utilizing local chws to increase the community 's awareness about the risk factors for ncds. initially, we worked with chws, who were also at risk for ncds. a large percentage (97%) of them had a body mass index (bmi) > 30 jointly, a training programme was developed to equip chws with knowledge and skills to assist them to work with the community. community health workers were expected to develop and implement the interventions for primary prevention of ncds in the community for increasing awareness about risk factors and prevention strategies, upon completion of the training program. numerous activities for increasing awareness about risk factors for ncd were implemented by the chws and these included organising fun walks in the township, and staging a drama to disseminate messages about causes and prevention of hypertension and diabetes. community members in the township recognised and appreciated the activities of the chws and requested regular weekly meetings where they could meet and be educated about risk factors and preventive measures for hypertension and diabetes, which was seen to be affecting many people in this community. the health club was developed by the chws who had received training on the primary prevention of cardiovascular disease during october 2001 to october 2002. training of chws on facilitation of health clubs with a focus on exercise, diet, and anthropometry (blood pressure, weight, and height) was conducted over six months and commenced in 2005. on completion of the training, each chw recruited five people around their place of residence to join the health club. chws also chose the name of the health club masiphakame ngempilo yethu meaning let us stand up for our health. the process of the development of a health club has been described by puoane.. on the first day of the club meeting, only eight participants registered and all were females, subsequently the numbers increased weekly. by the end of three months, 76 participants had been recruited and every week new members joined the health club. eighty - two percent of the participants had a bmi > 25, falling in the overweight to grossly obese categories (figure 1). two years after the intervention, there was a reduction in the number of participants who were obese (i.e., bmi > 30 however, overweight (2529.9 kg / m) and obesity (30 kg / m) still remain a problem in this population. although there were 76 participants who initially joined the health club, only 22 attended the sessions regularly (table 1). anthropometric measurements including weight, height, and blood pressure were collected at baseline (table 2). nutrition education sessions were held once a month and cooking demonstrations were incorporated into the sessions. t - shirts with the name of the club were issued to all new members after recruitment. during the past 6 years, four fun walks, two diabetes workshops, and three community - based introductory sessions two additional clubs have been formed since inception and additional training was offered to the chws. during the health club meeting sessions, chws facilitated the physical activity sessions, followed by the cooking demonstration, tasting of food, and sharing of recipes to try them out at home. at each cooking demonstration, participants evaluated the meals for palatability and recipes were issued to all participants. health talks were given at every session and guided by the food - based dietary guidelines (fbdgs, box 1) which were developed by the south african department of health. this was done to ensure that the messages conveyed at the health clubs were in agreement with those of the department of health. once a month all club members joined a 25 km walk around the township. various topics were covered and these included the following : (1) basics on the aetiology of hypertension and diabetes, (2) signs and symptoms of these conditions, (3) prevention of the conditions, (4) importance of medication, and (5) the role of nutrition and physical activity in the prevention of hypertension and diabetes. in addition, the fbdgs were used to influence adoption of healthy living among participants. the implementation of a health club in a resource - poor setting had numerous challenges. data from health club records show that 76 participants were initially recruited, however, only 22 were regular attendees and two years later the number had increased to 30. this slow growth in numbers can be explained by the constant movement of people living in these settlements. informal settlements serve as temporal accommodation while people are waiting to be allocated to formal housing. implementation of such initiatives in mobile communities can be a challenge when conducting an evaluation. census data show that 51% of the population in khayelitsha is unemployed, with more males being unemployed as compared to females. furthermore, a majority of the health club members moved to the city in search of employment. therefore, the program ran during the day, thus limiting participation of the economically active individuals. furthermore, employed residents who may have had an interest in the health club could not participate. mbombo reported that although hypertension is a common condition among poor south africans, a large percentage is undiagnosed due to a lack of routine physical checkups ; people often seek medical aid only when they feel pain. poor health - seeking behaviour in this population explains the inability to attract healthy individuals to partake in interventions that focus on prevention of diseases. this paper demonstrates that education on lifestyle modification focusing on healthy eating and physical activity is possible even in resource - limited settings. however, lack of a supportive environment hampered the maintenance of healthy practices. for example, in a study to identify environmental risk factors for ncds conducted in khayelitsha, it was reported that generally there was a shortage of healthy, low - fat food and little fresh fruit and vegetables available. in addition, local shops sold cheap fatty foods, while street vendors often sold fatty meat and sausages. thereby confirming that to promote healthy living in a disabling environment complicates decisions on healthy choices. although the primary focus of the health club was physical activity and healthy eating, it yielded unexpected outcomes. as reported in the testimonies (box 2), participants saw the health club as a place of sharing problems with people who might be experiencing the same problems. studies show that individuals who face more stressful events are more likely to benefit from the buffering effects of social support. other studies suggest that social support may add more value to those who face increased stressors. the experience in implementing a health club in south africa lends learning for other developing countries in transition and witnessing rapid urbanization. a health club is sustainable and culturally appropriate when facilitated by community or local people who have an insight and a deeper understanding of the culture and environment of the people they serve. although a health club can be used as a platform to facilitate lifestyle changes, an enabling environment is necessary for the maintenance of healthy living. therefore, in order to adequately address the problem of hypertension and other ncds in disadvantaged communities, there is a need to focus on social determinants of health.	chronic noncommunicable diseases (ncds) are increasing substantially as a cause of death and disability in all strata of the south african society, particularly among the urbanised poor. hypertension is a risk factor for many of these diseases and becoming a burden in a growing population in a cape town township, khayelitsha. to alleviate healthcare demands at clinics in this area, a health club was initiated and community health workers (chws) were trained to empower community members about ncds and create public awareness. after training, a health club was initiated. three months after initiation of the health club, 76 participants had been recruited of whom 22 were regular attenders. new members joined the health club weekly. anthropometric and blood pressure measurements were taken, and various hypertension topics were covered at the club meetings which included healthy behaviours, such as the benefits of being physically active and eating healthy. nutrition education sessions based on the south african food - based dietary guidelines were also held. consequent to the initial group that was established, two more clubs were formed in the area. health clubs are sustainable and culturally appropriate when facilitated by local people who have an insight and deeper understanding of the culture and environment of the people they serve.
surface - dependent activation of factor xii and plasma prekallikrein is not believed to be a major component of the normal in vivo blood coagulation activation process. however, contact activation readily occurs in vitro, as a result of contact with surfaces such as glass, kaolin, and other materials and during procedures such as cardiopulmonary bypass [25 ]. under conditions such as these, factor xiia could convert factor xi to factor xia and result in unwanted thrombin generation. therefore, it remains critical to understand the mechanisms of contact activation, as this has important implications for the thrombogenic properties and biocompatibility of many materials. although collagens are naturally occurring molecules, if they are present in nonphysiological situations or at elevated concentrations, they might have unwanted properties, such as being thrombogenic. c1-inhibitor is a proteinase inhibitor in the serpin family which is an important physiological inhibitor of plasma kallikrein and factor xiia. previously we have shown that c1-inhibitor can bind to type iv collagen and that this can modulate the reaction with the complement proteinase c1s. as c1-inhibitor is the main inhibitor of plasma kallikrein, we considered it to be important to investigate the effect of type iv collagen on the inhibition of plasma kallikrein by c1-inhibitor. in this study we have investigated binding of c1-inhibitor to type iv collagen in more detail and studied the effects of type iv collagen on plasma kallikrein and factor xiia activities and on the inhibition of plasma kallikrein by c1-inhibitor. human plasma kallikrein, factor xiia, and c1s were from enzyme research labs (south bend, in). human types i, iv, and v collagen and mouse type iv collagen were from research diagnostics (flanders, nj, usa). the chromogenic substrates s-2302 (for kallikrein, factor xiia and -factor xiia) and spectrozyme c1-e (for c1s) were from diapharma (westchester, oh) and american diagnostica, (greenwich, ct), respectively. biotinylation of c1-inhibitor was carried out by incubating 0.5 ml of c1-inhibitor in pbs (1 mg / ml) and 0.04 ml of freshly prepared biotinylation reagent (2.2 mg nhs - lc - biotin (pierce, rockford, il) in 0.08 ml water) on ice for 2.5 h. thereafter, excess reagent was removed using a desalting pd10 column (pharmacia, piscataway, nj, usa), with protein detection at 280 nm. to measure the binding affinity, immulon 2 dividastrip 96-well plates were coated with 100 l mouse type iv collagen or bovine serum albumin at 10 g / ml in carbonate / bicarbonate buffer at ph 7.5, for 18 hrs at 4c. wells were blocked with 1% bovine serum albumin, 0.1% tween 20, in tris - buffered saline, followed by washing with 0.1% tween 20 in tris - buffered saline. biotinylated c1-inhibitor was added to the wells at 1.9125 ng / ml. binding was measured by subsequent incubation with 125-i streptavidin, washing and counting the individual wells in a gamma counter. affinity was determined by scatchard plot. to confirm that binding of c1-inhibitor to the type iv collagen was specific, tryptophan fluorescence was also used to analyze the interaction between c1-inhibitor and type iv collagen, using a photon technology international fluorimeter. excitation was at 280 nm, and emission was measured between 290 nm and 400 nm. samples contained 1 mg / ml c1-inhibitor, 6 g / ml type iv collagen, or a mixture of both, all in 3 ml of 50 mm tris - hcl, ph 7.4, 20 mm nacl. plasma kallikrein, factor xiia, and -factor xiia were assayed using s-2302, and c1s was assayed using spectrozyme c1-e, as described previously. to determine the effect of collagen on activity of the proteinases, kallikrein (30 nm), factor xiia (0.649 m), -factor xiia (0.185 m), and c1s (0.46 m) were incubated in 20 mm tris - hcl, ph 7.4, 150 mm nacl, 0.1% polyethylene glycol 8000 (with kallikrein, factor xiia, and -factor xiia) or 1 mg / ml bsa (with c1s), at 37c in the absence of collagen, or with collagen at 20, 40, 60, 80, or 100 g / ml. aliquots were removed at various times for assay of residual proteinase activity. the inhibition of plasma kallikrein by c1-inhibitor was measured under pseudo - first - order conditions using a discontinuous assay as described previously. kallikrein (30 nm) was incubated with a 10-fold excess of c1-inhibitor in 20 mm tris - hcl, ph 7.4, 150 mm nacl, 0.1% polyethylene glycol 8000 at 37c in the absence of collagen, or with collagen at 20, 40, 60, 80, or 100 g / ml. aliquots were removed at various times (from 30 seconds to 150 seconds) for assay of residual proteinase activity with s-2302. we have shown previously in a qualitative assay that c1-inhibitor will bind to type iv collagen. to determine the affinity for this interaction, the binding of biotinylated c1-inhibitor to immobilized type iv collagen was measured (figure 1). the affinity was determined to be 0.86 m from the scatchard plot of this data (figure 2). confirmation of the specificity of binding of labeled c1-inhibitor was shown by displacement of the labeled protein by unlabeled protein (figure 3). further evidence for the interaction is shown in figure 4, which shows the fluorescence emission spectra of c1-inhibitor alone, collagen alone, and a mixture of the two. the mixture of type iv collagen and c1-inhibitor showed less fluorescence than the c1-inhibitor alone, and less fluorescence than when the individual spectra of type iv collagen and c1-inhibitor were added together. although no red or blue shift occurred, this small quench is indicative of an interaction between the two proteins. in addition, the magnitude of quench was dependent on the amount of collagen used (not shown). however, as this quench was small, it could not be reliably used to quantify binding affinity. the effect of type iv collagen on the activity of kallikrein, factor xiia, or -factor xiia activity was determined. the proteinases were incubated with increasing amounts of collagen, and the residual activity was measured. figure 5 shows a time course of loss of enzyme activity in the presence of 20 or 100 g / ml of type iv collagen. there was a rapid loss of factor xiia activity, with 50% of the activity lost after 14.5 minutes in the presence of 20 g / ml of collagen and 50% of the activity lost after 3 minutes in the presence of 100 g / ml of collagen. this loss of activity is attributed to factor xiia adsorbing to the collagen. in the absence of collagen, only 10% of the activity was lost after 30 minutes of incubation. with -factor xiia, the loss of activity was less, consistent with -factor xiia not containing the surface binding heavy chain. in the case of kallikrein, 20 g / ml of collagen caused only a slight loss of activity even after 5 hours, whereas with 100 g / ml of collagen, there was an initial rapid decrease of activity to about 40%. a similar loss of kallikrein activity by adsorption to surfaces has been observed previously [16, 17 ], and so these results are entirely consistent with published data. as a control, the complement proteinase c1s showed no loss of activity even after 6 hours with 100 g / ml of type iv collagen indicating that the effects of collagen were specific for the different proteinases (data not shown). the rate constant for the inhibition of kallikrein by c1-inhibitor in the presence of type iv collagen was determined. as these assays were performed over a short time frame (under 3 minutes), there was no loss of kallikrein activity during this time (compared to the longer times involved for loss of kallikrein activity seen in figure 5). the second - order rate constant for inhibition decreased by 50% with 100 g / ml of collagen. in other studies we have shown that kallikrein inhibition by c1-inhibitor is also reduced when the inhibition reaction is carried out in type iv collagen - coated microtiter plates. in this study we provide evidence that c1-inhibitor can bind tightly to type iv collagen (figures 1, 2, 3, and 4). we also show that type iv collagen caused a concentration - and time - dependent loss of kallikrein and factor xiia activities (figure 5). however, by measuring the inhibition of kallikrein by c1-inhibitor at early time points, we show that type iv collagen can dramatically reduce the rate of inhibition. the most likely explanation for these data is that the binding of c1-inhibitor to collagen reduces the concentration of c1-inhibitor available to react with kallikrein and so the rate of inhibition drops. given that the plasma c1-inhibitor concentration is 2 m, this is an interaction which potentially could occur in vivo. however, whether this has any regulatory significance for the contact system in vivo remains to be determined, particularly as high concentrations of collagen were used. perhaps a more important consideration is that the effects of collagen on the activities of the proteins tested indicate that studies with biomaterials should consider such reactions as part of their screening for biocompatibility, especially as high local concentrations of collagen would likely be present in such materials. thus we would suggest that all materials that use collagen (especially type iv) should be evaluated not only for their thrombogenic potential, but also for more specific actions on the contact system proteinases and c1-inhibitor, such as have been carried out with other biomaterials [5, 6, 1921 ].	the contact system of coagulation can be activated when in contact with biomaterials. as collagen is being tested in novel biomaterials in this study, we have investigated how type iv collagen affects plasma kallikrein and c1-inhibitor. firstly, we showed c1-inhibitor binds to type iv collagen with a kd of 0.86 m. the effects of type iv collagen on plasma kallikrein, factor xiia, and -factor xiia activity and on c1-inhibitor function were determined. factor xiia rapidly lost activity in the presence of type iv collagen, whereas plasma kallikrein and -factor xiia were more stable. the rate of inhibition of plasma kallikrein by c1-inhibitor was decreased by type iv collagen in a dose - dependent manner. these studies could be relevant to the properties of biomaterials, which contain collagen, and should be considered in the testing for biocompatibility.
orally administered bisphosphonates have very critical absorption profiles that depend on the postdosing fasting period, which is usually recommended to be at least 30 min. in practice, however, not all patients adhere to this recommendation, thereby reducing the absorbed fraction of the drug and jeopardizing the outcome of long - term treatments [15 ]. the critical dependence of bisphosphonate action on the postdosing fasting period is related to the chemical and physical properties of these molecules and their mechanism of absorption. bisphosphonates are poorly soluble drugs that can not cross the intestinal barrier through cell membranes ; many molecules most probably remain trapped by calcium - containing proteins in the intercellular space. absorption begins when the irritant effect of the molecules causes local edema, transiently broadening the intercellular spaces and thus enabling a small portion of the soluble bisphosphonate molecules to reach the bloodstream. this intricate absorption mechanism is the reason for the low bioavailability, and local irritation accounts for the typical digestive discomfort frequently reported by patients taking oral bisphosphonates [711 ]. the convenience of triggering this irritant mechanism intermittently once weekly or once monthly rather than daily is the rationale for the dosing regimens currently in use. at the same time, however, the risk of diminished therapeutic response due to impaired absorption increases proportionally with the intermittent schedules of administration. consequently, ensuring that soluble bisphosphonate molecules are available at the site of absorption within 30 min after dosing is essential for therapeutic success in clinical practice. experimental pharmacokinetic studies show that absorption can occur in the stomach and also in the first portion of the small intestine [1315 ]. quick delivery of the drug to the intestine is therefore expected to be accompanied by fewer upper digestive tract symptoms. the oral solution affords soluble alendronate in a drinkable formulation of pharmaceutical quality, at a concentration below 1 % -which is not irritant- and circumvents certain problems described with the solid formulations, for instance : adherence of the tablet to the digestive mucosa ; the challenge to overcome potential motility obstacles such as hernia, spasm, the body position of the patient during transit ; a slow, variable rate of disintegration which causes precipitation or reflux of irritant particles, acidity [710 ]. the alendronate that is retained in the stomach during the postdosing fasting period stimulates absorption through the gastric walls, provided it is not exposed to interaction with food or with the high mineral contents of the water [1721 ]. the current intermittent schedule of alendronate administration makes the use of drinkable forms more comfortable for patients on long - term treatment for osteoporosis. in view of the above, we studied the transit in the upper digestive tract of alendronate given either as tablets or as drinkable solution in a group of healthy adults, with the aim to obtain quantitative data on the differences between the administrations of the two formulations that might impact its use in clinical practice. before this study a randomized, single - dose, fasting, two - way crossover study tested the pharmacokinetic bioequivalence of fast disintegrating alendronate tablets and a drinkable alendronate solution in subjects recruited at the mtz clinical research site in warsaw. the study was conducted according to the world medical association declaration of helsinki and current good clinical practice guidelines and state regulations of poland. the study protocol was approved by the bioethics committee of the warsaw regional chamber of physicians and dentists and by the european regulatory authority. the reference product was the 70 mg fosamax tablet manufactured by merck sharp & dohme limited, uk, and the test product was a 70 mg/100 ml drinkable alendronate solution with thickening agents and orange color and flavor (xeolas pharmaceuticals ltd. prospective participants were informed about the study and then asked to sign an informed consent form prepared according to mtz s standard operating procedures. those who consented were examined and eventually 108 healthy men were accepted into the study, of which 104 successfully completed all stages of the protocol. there were four withdrawals : one subject withdrew voluntarily, two subjects experienced adverse events (one had increased blood pressure during period 1 and another one reported an adverse event not related to the study products during the washout period), and another subject was tested positive for unauthorized drugs. main inclusion criteria were : male gender, age 1850 years, and body mass index between 19.0 and 26.0 kg / m. in addition to other routine criteria, subjects were required to be able to refrain from smoking 3 days before the beginning of the study until its completion. exclusion criteria : current or chronic health problems and/or drug therapy ; a history of allergy, hypocalcemia, digestive, hepatic or renal disorders ; a history of excessive alcohol consumption (> 30 g / day during the previous year) ; positive test results for anti - hiv, hbsag or anti - hcv. in addition subjects with abnormal blood pressure, pulse or laboratory test results were excluded from the study. participants were randomized according to a numerical table and were given either a tablet to be swallowed with 240 ml of water or a drinkable solution to be swallowed with 140 ml of water, to ensure equivalent hydration. no additional water intake was allowed from 1 h before to 1 h after dosing, but subjects were afterward administered 2,000 ml of fluids according to a standardized distribution and a 2,5003,000 kcal / day diet divided in three meals, given at 4, 9 and 12 h after dosing. after a 14 day washout period, study participants were asked to return to the research center to be given the other alendronate formulation. on each of the two dosing days, urine samples were collected at the following time points : 11.5 h before dosing, and then after dosing during the following time intervals : 0 to 0.25 ; 0.25 to 1 h ; 1 to 2 h ; 2 to 3 h ; 3 to 4 h ; 4 to 6 h ; 6 to 8 h ; 8 to 12 h ; 12 to 24 h and 24 to 36 h. each urine sample was collected into a container from which two 6 ml aliquots were drawn and placed into plastic tubes. samples were frozen (20 c) and were sent to bioclin research laboratories ltd. (ireland), where urinary alendronate concentrations were determined under blind conditions using a hplc fluorescence method (bioclin test method number pr120) which involves the isolation of alendronate from human urine by solid - phase extraction. alendronate was coprecipitated with calcium phosphate and then its primary amino group was derivatized with 2,3-naphtalene dicarboxyaldehyde (nda) and n - acetyl - d - penicillamine (nap) to form the fluorescent derivative. a shimadzu lc-10as (masons, dublin) system, a waters 717 plus autosampler (waters, dublin) and a waters 474 fluorescence detector were used for these measurements. intra - assay precision (% cv) was in the 3.958.05 % range and mean percentage accuracy in the 105.5112.5 % range. the precision at the lower limit of quantitation (10 ng / ml) was 5.43 % (defined by % cv), with a 100.1115.4 % accuracy. interassay precision ranged from 4.37 to 14.1 % and mean accuracy ranged from 105.9 to 112.8 %. the method was validated in the concentration range 101,000 ng / ml. the primary evaluation parameter was the total amount of drug excreted in the urine from the time of dosing until 36 h after dosing (total ae(036 h)) and the maximum urinary excretion rate (rmax). secondary parameters were the amount of drug excreted (ae) and the rate of urinary excretion (re) in each collection interval, and the time of maximum urinary excretion rate (tmax). the log - transformed total ae(036 h) and rmax were statistically compared by anova analysis of variance considering the effect of the treatments, sequence and period of study. the 90 % confidence interval was tested by lsmeans for ae(036 h) for the 80125 % range, and by hodges - lehmann nonparametric methods for rmax for the 75133 % range. winnonlin version 4.0.1. was used for the kinetic parameters and sas version 9.1 for the statistical calculations. in order to assess safety, adverse events observed during the study were reported (using ctcae v. 3.0 software) and clinical examinations, including the following measurements and procedures, were performed before and after each dosing period : pulse, blood pressure, body temperature, ecg, x - rays, and clinical laboratory determinations such as urine analysis and hematology, biochemistry, serology and controlled substance (alcohol, opioids, barbiturates, benzodiazepines, amphetamines, cocaine, cannabis and nicotine) tests. a randomized, single - dose, fasting, two - way crossover study tested the pharmacokinetic bioequivalence of fast disintegrating alendronate tablets and a drinkable alendronate solution in subjects recruited at the mtz clinical research site in warsaw. the study was conducted according to the world medical association declaration of helsinki and current good clinical practice guidelines and state regulations of poland. the study protocol was approved by the bioethics committee of the warsaw regional chamber of physicians and dentists and by the european regulatory authority. the reference product was the 70 mg fosamax tablet manufactured by merck sharp & dohme limited, uk, and the test product was a 70 mg/100 ml drinkable alendronate solution with thickening agents and orange color and flavor (xeolas pharmaceuticals ltd. prospective participants were informed about the study and then asked to sign an informed consent form prepared according to mtz s standard operating procedures. those who consented were examined and eventually 108 healthy men were accepted into the study, of which 104 successfully completed all stages of the protocol. there were four withdrawals : one subject withdrew voluntarily, two subjects experienced adverse events (one had increased blood pressure during period 1 and another one reported an adverse event not related to the study products during the washout period), and another subject was tested positive for unauthorized drugs. main inclusion criteria were : male gender, age 1850 years, and body mass index between 19.0 and 26.0 kg / m. in addition to other routine criteria, subjects were required to be able to refrain from smoking 3 days before the beginning of the study until its completion. exclusion criteria : current or chronic health problems and/or drug therapy ; a history of allergy, hypocalcemia, digestive, hepatic or renal disorders ; a history of excessive alcohol consumption (> 30 g / day during the previous year) ; positive test results for anti - hiv, hbsag or anti - hcv. in addition subjects with abnormal blood pressure, pulse or laboratory test results were excluded from the study. participants were randomized according to a numerical table and were given either a tablet to be swallowed with 240 ml of water or a drinkable solution to be swallowed with 140 ml of water, to ensure equivalent hydration. no additional water intake was allowed from 1 h before to 1 h after dosing, but subjects were afterward administered 2,000 ml of fluids according to a standardized distribution and a 2,5003,000 kcal / day diet divided in three meals, given at 4, 9 and 12 h after dosing. after a 14 day washout period, study participants were asked to return to the research center to be given the other alendronate formulation. on each of the two dosing days, urine samples were collected at the following time points : 11.5 h before dosing, and then after dosing during the following time intervals : 0 to 0.25 ; 0.25 to 1 h ; 1 to 2 h ; 2 to 3 h ; 3 to 4 h ; 4 to 6 h ; 6 to 8 h ; 8 to 12 h ; 12 to 24 h and 24 to 36 h. each urine sample was collected into a container from which two 6 ml aliquots were drawn and placed into plastic tubes. samples were frozen (20 c) and were sent to bioclin research laboratories ltd. (ireland), where urinary alendronate concentrations were determined under blind conditions using a hplc fluorescence method (bioclin test method number pr120) which involves the isolation of alendronate from human urine by solid - phase extraction. alendronate was coprecipitated with calcium phosphate and then its primary amino group was derivatized with 2,3-naphtalene dicarboxyaldehyde (nda) and n - acetyl - d - penicillamine (nap) to form the fluorescent derivative. a shimadzu lc-10as (masons, dublin) system, a waters 717 plus autosampler (waters, dublin) and a waters 474 fluorescence detector were used for these measurements. intra - assay precision (% cv) was in the 3.958.05 % range and mean percentage accuracy in the 105.5112.5 % range. the precision at the lower limit of quantitation (10 ng / ml) was 5.43 % (defined by % cv), with a 100.1115.4 % accuracy. interassay precision ranged from 4.37 to 14.1 % and mean accuracy ranged from 105.9 to 112.8 %. the method was validated in the concentration range 101,000 ng / ml. the primary evaluation parameter was the total amount of drug excreted in the urine from the time of dosing until 36 h after dosing (total ae(036 h)) and the maximum urinary excretion rate (rmax). secondary parameters were the amount of drug excreted (ae) and the rate of urinary excretion (re) in each collection interval, and the time of maximum urinary excretion rate (tmax). the log - transformed total ae(036 h) and rmax were statistically compared by anova analysis of variance considering the effect of the treatments, sequence and period of study. the 90 % confidence interval was tested by lsmeans for ae(036 h) for the 80125 % range, and by hodges - lehmann nonparametric methods for rmax for the 75133 % range. winnonlin version 4.0.1. was used for the kinetic parameters and sas version 9.1 for the statistical calculations. in order to assess safety, adverse events observed during the study were reported (using ctcae v. 3.0 software) and clinical examinations, including the following measurements and procedures, were performed before and after each dosing period : pulse, blood pressure, body temperature, ecg, x - rays, and clinical laboratory determinations such as urine analysis and hematology, biochemistry, serology and controlled substance (alcohol, opioids, barbiturates, benzodiazepines, amphetamines, cocaine, cannabis and nicotine) tests. this was a prospective, randomized, controlled trial comparing the upper digestive tract transit of two alendronate formulations, conducted according to good clinical practice guidelines. the study protocol was approved by the institutional ethics committee of the maimnides university and by the national administration of drugs, food and medical technology (anmat, appl. after a period of training in procedures such as subject positioning and x - ray follow - up of the formulations, the study was opened. twenty - four healthy adult volunteers, mean age 51.6 years (range 3968 years), were recruited and randomized according to an age - cohort table in order to obtain a balanced distribution of ages within the range. patients were excluded if : there was clinical evidence of any chronic disease ; they were hypersensitive to bisphosphonates ; they consumed aspirin, other nonsteroidal anti - inflammatory drugs and/or alcoholic beverages on a regular basis ; or they had experienced a bone fracture or taken part in any other study during the 60 days before this study. both tested formulations were the same as in the previous study. in order to facilitate the x - ray follow - up, the tablets were drilled with a fine drill and partly refilled with contrast substance, and 5 ml of the 100 ml drinkable solution were replaced by contrast media. the in vitro disintegration test of the original and the modified tablets did not show any significant differences in the dissolution time (data not shown). participants were given either the tablet or the drinkable solution standing or lying in prone position (fig. the formulations were administered under fasting conditions, which were maintained until the subjects had finished the three series of experiments.. 1body position of the participants during the upper digestive tract transit study. left standing position. right bed rest (prone) position body position of the participants during the upper digestive tract transit study. left standing position. right bed rest (prone) position all participants were trained to comply with the study procedures. from a few minutes before dosing until the experiment was completed, subjects were positioned in front of a remotely controlled x - ray system of 750 ma, 150 kv, with a high - resolution magnifier and digitizer (pinnacle dc 1000 mpeg 2, stored in a hppavilion dv1025ia personal computer), operated by a skilled radiologist. only subjects whose images were clear for the entire sequence were considered. for statistical purposes, a mouth to stomach time (mst) of 480 s was considered for tablet disintegration before reaching the stomach and a disintegration time (dt) of 20 min for nondisintegration. after the tablet with water and the drinkable solution had been completely swallowed, the arrival of the tail of the swallowed volume was considered, and the drinkable solution was followed until the head of the volume was clearly detected in the first portion of the duodenum (mouth to duodenum time, mdt). the tablets were followed until they lost their shape they typically formed a disintegration cloud and disappeared (i.e., dt), whether this occurred in the esophagus, the stomach or the small intestine. after the images had been digitalized, a pc time counter was used to estimate mst for both formulations, which were compared for the standing or upright and horizontal positions by analysis of the mean and the variance values by t test for independent groups. variance (s) is a measure of how robust the mean value is as a function of the distribution of the individual values considered. likewise, mdts for the drinkable solution and dts for the tablet formulation were compared for the two body positions of the subjects by t test for dependent samples. nonparametric spearman rank order correlation test, with pairwise deletion of cases was used to associate mst, mdt and dt with the age of the participants, and within treatments. once the experiments were over, the subjects remained in the unit under clinical supervision for 2 h and were then discharged and instructed to come back or call should they experience any adverse events or discomfort during the next week. during this period, patients were called back for a new visit if any anatomic or functional abnormalities were detected in the video deglutition images. the study protocol was successfully completed by 104 subjects, aged 26.7 7.9 years, with a body mass index of 23.2 2.1. rmax were found to be within the acceptable range for bioequivalence, respectively 81.8105.7 and 81.7106.2. intrasubject variation for ae(036 h) was 60.361.8 % for rmax ; the statistical power of the sample was 0.89 and 0.88, respectively, which is sufficient for establishing bioequivalence.table 1pharmacokinetic parameters from a bioequivalence study comparing single 70 mg dose taken either via a tablet or a drinkable solution (0.7 %) in 104 healthy young men after an overnight fast and a 4 h postdosing fasting periodformulationtabletdrinkable formulationbe (ic90)alendronate urinary excretion after a single 70 mg administrationae(036 h), g, mean (sd)167.3 (147.3)140.7 (109.9)ae(036 h), g, median (min max)124.2 (8.8818.8)121.9 (32.1919.9)ae(036 h), % cv88.178.181.8105.7rmax, g / ml, mean (sd)54.4 (50.3)47.3 (37.4)rmax, g / ml, median (min max)41.9 (4.6334.7)37.1 (5.03266.34)rmax, % cv92.579.181.7106.2tmax, h, mean (sd)1.58 (0.67)1.68 (0.72)tmax, h, median (min max)1.5 (0.133.5)1.5 (0.635.0)bioequivalence between both formulations is within the acceptable range pharmacokinetic parameters from a bioequivalence study comparing single 70 mg dose taken either via a tablet or a drinkable solution (0.7 %) in 104 healthy young men after an overnight fast and a 4 h postdosing fasting period bioequivalence between both formulations is within the acceptable range median ae(036 h) was very similar for both products ; 121.9 g for the drinkable solution and 124.4 g for the tablets ; mean values were higher, especially for the tablets, which also showed a greater sd. % cv was 78.1 % for the drinkable solution and 88.1 % for the tablets (ie, 12.8 % higher for the tablets). the distribution of the number of cases according to ae(036 h) showed that in the 112.5212.5 g range, which represents the magnitude of urinary alendronate excretion found with the highest frequency (63 of 104 for the drinkable solution and 52 of 104 for the tablets), there were 21.2 % more subjects treated with the drinkable solution than with the tablets (fig. 2). on the other hand, the distribution of cases treated with tablets tended to be higher in subjects with the lowest (212.5 g) levels of urinary recovery. above 412.5 g, 9 patients were treated with tablets and 3 with the drinkable solution. median rmax values were 37.1 g / ml for the drinkable solution and 41.1 g / ml for the tablets. variations in rmax show a similar pattern, with % cv being 16.9 % higher for the tablets ; this parameter, however, is less clinically relevant for this type of product.fig. 2individual curves of the cumulative amount of alendronate excretion in urine in 104 healthy young men after receiving, in fasting conditions (> 8 h before and 4 h after administration), a single 70 mg dose in a tablet + 240 ml of plain water (left), or a single 70 mg/100 ml drinkable solution + 140 ml of plain water (right) ; truncated at 9 h after dosing. from hours 936, the cumulative amount is minor ; the shape of the curves does not change individual curves of the cumulative amount of alendronate excretion in urine in 104 healthy young men after receiving, in fasting conditions (> 8 h before and 4 h after administration), a single 70 mg dose in a tablet + 240 ml of plain water (left), or a single 70 mg/100 ml drinkable solution + 140 ml of plain water (right) ; truncated at 9 h after dosing. from hours 936, the cumulative amount is minor ; the shape of the curves does not change mean urinary alendronate concentration did not differ significantly between the two formulations at any of the collection periods. for example, during the first hour urinary alendronate concentration was 109.0 302.5 g l after the solution (not significantly different). however, the variance was significant lower with the solution (f test, p 212.5 g) levels of urinary recovery. above 412.5 g, 9 patients were treated with tablets and 3 with the drinkable solution. median rmax values were 37.1 g / ml for the drinkable solution and 41.1 g / ml for the tablets. variations in rmax show a similar pattern, with % cv being 16.9 % higher for the tablets ; this parameter, however, is less clinically relevant for this type of product.fig. 2individual curves of the cumulative amount of alendronate excretion in urine in 104 healthy young men after receiving, in fasting conditions (> 8 h before and 4 h after administration), a single 70 mg dose in a tablet + 240 ml of plain water (left), or a single 70 mg/100 ml drinkable solution + 140 ml of plain water (right) ; truncated at 9 h after dosing. from hours 936, the cumulative amount is minor ; the shape of the curves does not change individual curves of the cumulative amount of alendronate excretion in urine in 104 healthy young men after receiving, in fasting conditions (> 8 h before and 4 h after administration), a single 70 mg dose in a tablet + 240 ml of plain water (left), or a single 70 mg/100 ml drinkable solution + 140 ml of plain water (right) ; truncated at 9 h after dosing. from hours 936, the cumulative amount is minor ; the shape of the curves does not change mean urinary alendronate concentration did not differ significantly between the two formulations at any of the collection periods. for example, during the first hour urinary alendronate concentration was 109.0 302.5 g l after the solution (not significantly different). however, the variance was significant lower with the solution (f test, p 999.92.0480.0na3.8 (17)3.513.60.817.0tablet, bed rest position73.1 (14)5.0>999.93.0480.0na3.4 (15)2.322.70.5>20drinkable solution, standing position6.8 (18)5.072.70.540.02.6 (18)2.07.40.210.0nadrinkable solution, bed rest position8.4 (16)5.059.33.030.02.0 (16)2.01.70.35.0nana not applicable, n number of subjects evaluatedtable 3statistical analysis of the mouth to stomach transit time (mst) as described in table 1administrationmst, stablet, bed rest positiondrinkable solution, standing positiondrinkable solution, bed rest positiontablet, standing position x0.410.370.43 md0.520.610.31 s0.090.0010.001tablet, bed rest position x0.110.14 md0.100.04 s0.0010.001drinkable solution, standing position x0.58 md0.004 s0.69significance probability was calculated using student s t test for independent groups for mean (x) and variance (s) and wilcoxon rank test for median (md) valuesbold values indicate significantly different transit time from mouth to stomach (mst), mouth to duodenum (mdt) and disintegration time (dt) for a tablet plus 250 ml of plain water and for 100 ml of a drinkable solution containing 70 mg of alendronate, assessed by x - ray video - deglutition analysis in groups of healthy adults na not applicable, n number of subjects evaluated statistical analysis of the mouth to stomach transit time (mst) as described in table 1 significance probability was calculated using student s t test for independent groups for mean (x) and variance (s) and wilcoxon rank test for median (md) values bold values indicate significantly different regarding the mdt, access to the duodenum was quick after swallowing 100 ml of alendronate solution but the difference in access to the duodenum between both positions was not significant (p < 0.75). surprisingly, however, variance was significantly smaller in the group examined in the bed rest position (p < 0.05), suggesting that the formulation could be a good option for bedridden patients. indeed, with the drinkable formulation, the alendronate was available in the intestine in <3 min on average, and no test showed results longer than 10 min. on the other hand, most of the alendronate tablets disintegrated in the stomach as expected (differences between body positions were not significant, p < 0.79 for the means and p < 0.32 for the variances), including two cases in which the tablet remained intact after dosing for 17 min (in the standing position) and for more than 20 min (in the bed rest position), and passed intact to the duodenum in the latter subject. in addition, the only significant influence of age was on the mdt within the group that was administered the drinkable solution in the bed rest position, with r = 0.53 (p < 0.04 ; n = 11), which indicates that transit might be somewhat slower in older patients. mean height of the subjects was 167.4 cm (range 152200 cm) and tended to correlate with the values of mst of the tablet taken in the bed rest position (r = 0.51 ; p < 0.07). the body mass index of the subjects did not correlate with any of the transit measurements taken. the mst correlation coefficient was high, r = 0.79 (p < 0.02 ; n = 8) when comparing the groups who had been administered the tablets and drinkable solution in the bed rest position, suggesting that the transit for both formulations is equally good when subjects have no apparent motility problems. for the mst the group taking the drinkable solution in the bed rest position also correlates moderately with the group taking it in the standing position r = 0.67 (p < 0.02 ; n = 12). finally, accessibility to the duodenum with the drinkable solution tends to correlate moderately r = 0.55 but not significantly in this study (p < 0.07 ; n = 12). we show here that in young men under strictly controlled conditions, the alendronate drinkable solution is bioequivalent to the reference tablets and therefore suitable for the long term treatment of osteoporosis. in young healthy subjects under optimal conditions (precise compliance with dosing instructions, quantity and quality of water consumed, no food intake, proper body position) the pharmacokinetics of the alendronate released from the tablet are equivalent to those of the liquid formulation. as this type of studies. however, we also showed that there are differences between the two preparations when given to older adults which may have important implications for the treatment of elderly patients in clinical practice. mean esophageal transit for the drinkable alendronate solution is around 78 s, with the mean small bolus transit velocity described in 6 s. with the drinkable solution, the alendronate was delivered in a completely soluble form to the first portion of the intestine in <3 min on average, in both the standing and the lying positions, and the longest time to reach the absorption site was < 10 min in all studied subjects, including those with hiatus herniae. hence, the recommended minimum 30 min postdosing fast is a prudent period to enable further absorption of alendronate at the most suitable site, i.e., the first portion of the intestine. conversely, although the disintegrates in < 4 min on average, it sometimes remains intact in the stomach and parts of the tablet can even remain in the esophagus. this potential greater variability in the availability of the tablets to the absorption site suggests that alendronate might be absorbed in places were the mucosa is very sensitive. the findings also suggest that the specific instructions for the administration of the alendronate tablets (remaining upright for 30 min after dosing, dosing with nlt 200 ml water) represent only a minimum requirement which is not fully effective in some patients. these considerations are not applicable to the liquid formulation, which is delivered quickly to the intestine irrespective of body position. because volunteers older than 70 years, who may have more difficulties in swallowing, were not allowed by the cme to participate in the study, we can not extrapolate the results to this age group. in addition, the alendronate tablet used in this study was the reference formulation with a fast disintegration time manufactured by merck sharp & dohme limited, and differences may be greater when generic tablets are used. although the tablets used here were slightly modified by the test methodology, the fact that some units dissolved early in the esophagus, while others appeared intact in the duodenum seems to indicate that the tablets were neither physically weakened nor strengthened to any considerable extent by the contrast media used in this study. moreover, the generic alendronate tablets available in the market, even when bioequivalent, have been questioned as a result of potential differences in their in vitro disintegration time, which can be as long as 13 min [2528 ]. such rate suggests that the slower dissolution gradient of the solid generics exposes the digestive walls to protracted high concentrations of alendronate during a critical period, probably affecting the local reactivity of the tissue and the tolerability, or favoring the undesired interactions of the drug with the intradigestive environment and/or contents. so we agree with the authors who claim that even with approved bioequivalence studies, generic tablets may perform differently in practice if their disintegration characteristics and quality are not adequately controlled. a recent article by kanis. report a dissimilar profile of adverse effects, compliance and cost / utility variables when switching to generic tablets of bisphosphonates. in fact, the comparison of formulations under experimental conditions such as bioequivalence studies may mask broader differences that can appear in practice. in the typical bioequivalence test, healthy young adults are recruited and properly trained to follow all dosing instructions, pay attention to the warnings and drink adequate volumes of water. usually, the postdosing fasting period is strictly controlled by the staff and can be as long as 4 h, during which the influence of delays in transit, disintegration or reflux on the results can be compensated by a certain amount of late absorption [3136 ]. in practice the present study shows that in older subjects, administering alendronate via drinkable solution induces fewer variations in the access to the intestine. in agreement with this view, a 6 month placebo - controlled clinical study involving 392 postmenopausal women treated with a different oral solution formulation (alendronate 70 mg/75 ml plus extra water) showed that the urinary ntx corrected for creatinine and serum bone- specific alkaline phosphatase reductions were (95 % confidence interval) 56.1 to 38.8 and 44.9 to 32.6, respectively, a range of variability that is quite narrow for a bisphosphonate and entirely within the effective levels. as expected, the tolerability of the oral solution in such sample was slightly lower than for the placebo, but the rate of upper digestive tract adverse events that were serious or led to discontinuation of treatment was similar. finally, the pharmaceutical drinkable solution of alendronate taken with no added water as in this study precludes interactions with minerals that can occur when patients use water, whether tap or bottled, to swallow (dissolve) alendronate tablets. the 100 ml volume of the test formulation was enough to enable the alendronate to be available in the intestine rapidly ; this agrees with physiological studies of water emptying from the stomach, according to which even smaller volumes can be emptied faster. moreover, the addition of synthetic viscosity agents to the drinkable solution formulation enables it to progress through the digestive system with some syrup - like properties, that is, to spread a little bit more slowly than water in the stomach and to remain for a longer time in the first portion of the intestine. this thinly viscous mass may make completely soluble alendronate molecules available for a longer time at the proper absorption site. furthermore, even in cases in which only partial volumes are delivered to the duodenum within the 30 min fasting period, such quantity is enough to allow the expected average absorption of around 1 % of the active principle. besides volume considerations, the calorie content of the alendronate solution is negligible, and it is probable that if administered after cooling in the refrigerator, transit may be even quicker, improving palatability at the same time [3941 ]. oral solution formulations do not enhance alendronate absorption, as proved by bioequivalence studies vs. the tablets, provided the comparison test has been conducted under adequate experimental conditions, yet the drinkable solution of alendronate is less affected by certain variables in practice, as shown in this study. the video deglutition radiological test used in this study can be considered in patients treated with alendronate who do not appear to respond or report digestive intolerance. transit problems with the tablet formulation can be asymptomatic and more frequent than one might expect. conveniently, as it is the transportation mode and not the molecule that is the cause of the unsatisfactory result, patients should remain under treatment with alendronate and change the vehicle of administration before switching to a different active compound which might be less effective, have uncertain safety parameters or be more expensive. in conclusion, the alendronate solution has rapid access to the absorption site and is less subject to transit problems than the tablet formulation. therefore, alendronate, the drug of choice for the treatment of osteoporosis in many countries, can be administered on weekly basis in an optimized manner even in bedridden patients.	the bioequivalence and upper digestive tract transit time of a drinkable solution of 70 mg/100 ml alendronate was compared to reference tablets. a randomized, single- dose, two - way crossover study of the rate of urinary recovery of alendronate during 36 h (ae(036 h)) by hplc, in 104 healthy young male volunteers, showed that ae(036 h) and the maximum excretion rate (rmax) were within the accepted range of bioequivalence 81.8105.7 and 81.7106.2, respectively. to characterize the oesophageal passage time of the two alendronate formulations, we performed a randomized, controlled study, in 24 healthy men and women (mean 52 years old), who took the formulations standing or lying down, by an x - ray video deglutition system. when taken in the standing position, both formulations had equal mean transit times from mouth to stomach and tablet disintegration but data dispersion was significantly smaller with the liquid form. when taken in lying position, drinkable alendronate had shorter and less variable median transit times compared to the tablets. these results show that the drinkable alendronate formulation is bioequivalent to the tablets and may be advantageous in patients in whom the transit or disintegration of the tablets is impaired.
according to who s data from 2004 cardiovascular disease is the cause of death of over 17 million people worldwide. most often cvd is manifested in the form of coronary heart disease (chd), myocardial infarction and stroke. the main cause of their development is atherosclerosis which is a chronic inflammatory disease of the arteries. atherosclerotic plaques formation and pathological remodeling of vascular walls consequently lead to impaired tissue perfusion and ischemia. many studies have shown that cholesterol is one of the key component of atherosclerotic plaques, therefore hyperlipidemia is considered as an essential risk factor for atherosclerosis [2, 3 ]. the basic laboratory exponent of cardiovascular risk is the elevated concentration of total cholesterol (tc) which mostly results from elevated ldl cholesterol (ldl - c) [4, 5 ]. modified ldl particles, especially their oxidized forms (ox - ldl) may be freely taken up by macrophages whose scavenger receptors combine with apolipoprotein b100 (apob). subsequently arising foam cells form the fatty infiltrates in the artery walls which are the starting point for plaques. ldl - c concentration reflects only the amount of cholesterol contained in ldl particles but does not provide information about their number and structure. in addition, ldl - c does not include the participation of other lipoprotein fractions (lp (a), vldl) that are essential in the development of atherosclerosis. ldl - c value is usually calculated with the friedewald formula whereas this method has some limitations, predominantly hypertriglyceridemia. it was shown that ldl - c is estimated with approximately 17% and 25% error at serum triglyceride concentration (tg) from 151 - 200 mg / dl and 201 - 300 mg / dl, respectively. therefore apob, the main protein of potentially atherogenic lipoproteins, seems to be a more reliable indicator. clinical studies proved that apob concentration highly correlates with the number of ldl particles, including small dense ldl (sd - ldl), having particularly strong atherogenic properties. elevated apob is considered one of the best prognostic factors of acute coronary events and deaths due to cvd [9, 10 ]. nevertheless, determination of apob requires the use of suitable methods and yet is not widely applied in routine laboratories. modern laboratory diagnosis of lipid disorders and cardiovascular risk should be based on the use of indicators which present full impact of all plasma lipid components involved in atherogenesis. non - hdl - c is the sum of cholesterol accumulated in all lipoproteins, except hdl, such as : chylomicrones, vldl and their remnants, idl, ldl and lp(a). the concentration of non - hdl - c is calculated using a simple equation : non - hdl - c (mg / dl) = tc hdl - c actually, little attention is being paid to the use of non - hdl - c but the latest guidelines for both european and american cardiological societies emphasize the importance of this parameter for assessing the risk of atherosclerosis and coronary heart disease. non - hdl - c concentration, as recommended by the ncep adult treatment panel iii, should be higher by about 30 mg / dl than ldl - c (table 1). non - hdl - c is considered as the second, after the ldl - c goal of cvd therapy in patients with hypertriglyceridemia and should be calculated routinely in the lipid profile. the usefulness of non - hdl - c in the prevention of cvd was confirmed in numerous clinical trials. liu. compared the diagnostic value of non - hdl - c as a prognostic factor of acute coronary events and myocardial infarction among healthy subjects and diabetics. it was found that increased level of non - hdl - c by 1 mg / dl increases the risk of death due to cardiovascular disease by 5% and seems to be a better predictive indicator than the traditional lipid risk factors. significantly higher concentrations of non - hdl - c and higher relative risk of coronary events among patients with diabetes were observed and the risk in particular grade levels of non - hdl - c was 1.5 to over 2.5 times higher in diabetics than in healthy subjects. ruminska. evaluated the usefulness of non - hdl - c in the lipid disorders in children and adolescents with simple obesity. patients with elevated non - hdl - c (> 123 mg /dl) had significantly higher values of waist circumference and serum tc, ldl - c, tg, tc : hdl - c, tg : hdl - c and lower hdl- c. the impact of elevated tg levels in the calculation of ldl - c with the friedewald formula suggests that non - hdl - c is beneficial in determining the risk of atherosclerosis and cvd in patients with hypertriglyceridemia. it might be an important estimate for diseases such as diabetes and obesity, in which excessive triglyceride values increase the concentration of sd - ldl and decrease hdl - c. numerous studies including health professionals follow - up study, safari and the copenhagen city heart study [16, 17, 18 ] indicated that non - hdl - c correlates better with apolipoprotein b100 than ldl - c and its diagnostic value as a risk factor is similar or as high as apob. the role of non - hdl - c in predicting and reducing cvd risk in patients treated pharmacologically due to dyslipidemia is noteworthy. in a meta - analysis of lipid - lowering therapies a 1:1 correlation between the 1% non - hdl - c lowering and chd risk reduction by lipid - modifying drugs was observed. thus, not only lowering ldl - c, but also non - hdl cholesterol is an important goal of prevention and treatment of cardiovascular diseases. currently available clinical data describe the relationship between the concentration of non - hdl - c and methods of imaging of atherosclerosis. the effect of serum lipids on the process of coronary arteries calcification (cac), regarded as an early marker of subclinical atherosclerosis was described in recent study by orakzai.. of all lipid parameters, only the non - hdl - c showed a significant association with the process of atherogenesis. the bogalusa heart study proved a relationship between the value of non - hdl - c in childhood and risk of cardiovascular disease in adulthood. a strong association between non - hdl - c, denoted at the age of 5 - 17 years and the intima - media thickness (imt) in carotid artery in adults was documented. kawamoto observed excessive imt value and chd risk with the increasing non - hdl - c values in patients over 65 years old. there is still much controversy about the use of non - hdl cholesterol in routine clinical / laboratory practice. despite the numerous advantages it is not possible to exclude a higher diagnostic value of apolipoprotein b100 and apob : apoai ratio in the primary cvd prevention [23, 24 ]. estimated non - hdl - c value, combined with apolipoproteins, hscrp and ldl particle number (ldl - p) assessment is suggested to be the most optimal solution. in summary, based on the available data, the use of a simple non - hdl - c calculation in a lipid profile testing, complemented by determination of the new risk factors, will allow a better assessment of the cvd risk.	cardiovascular disease (cvd), such as coronary heart disease (chd), is the most frequent cause of death worldwide, especially in developed countries. the latest recommendations of european and american cardiological associations emphasize the role of non - hdl cholesterol (non - hdl - c) in evaluating the risk of cvd. although this parameter has a lot of advantages, it is rarely used by general practitioners in lipid profile assessment.the aim of this article is to present the recent informations on the usage of non - hdl - c in the primary prevention of cardiovascular disease and to compare its diagnostic value to traditional and new cvd risk factors.
the alkylating agent ifosfamide is an anti - neoplastic used in both pediatric and adult age groups to treat various malignancies. although its potential for urologic toxicity is tempered by mesna (sodium 2-mercaptoethane sulphonate) pretreatment, glomerulopathy, tubulopathy, and hemorrhagic cystitis still occur as adverse events in a considerable number of patients (1,2). fanconi 's syndrome, a proximal tubulopathy marked by excessive urinary excretion of glucose, phosphate, bicarbonate, amino acid and protein, is likewise a noted complication, with rickets and growth retardation as long - term consequences. adults are rarely affected, but this syndrome is not infrequent in children as a clinical expression of ifosfamide - induced nephrotoxicity (3,4). several published reports have also linked a combination of ifosfamide and cisplatin to persistent renal function decline requiring hemodialysis (5). we report here a 67-year - old patient who received a regimen of ifosfamide, adriamycin and mesna for epithelioid sarcoma and soon thereafter developed a proximal renal tubulopathy, in conjunction with hemorrhagic cystitis. to our knowledge, this is the first documented instance where fanconi 's syndrome (nephrotoxicity) and hemorrhagic cystitis (urotoxicity) jointly resulted from one - time ifosfamide exposure, without contributory cisplatin. the patient, a 67-year - old male, presented at our clinic with dysuria and gross hematuria. he had a 6-month history of type 2 diabetes mellitus, treated with an oral hypoglycemic agent. more recently, a malignancy was discovered when he sought treatment (debridement) for an inflamed right fifth toe. following amputation of the right fourth and fifth toes and right inguinal lymphadenectomy, a histologic diagnosis of epithelioid sarcoma was rendered. following pretreatment with mesna (4,350 mg / m), he subsequently received one course of adriamycin plus ifosfamide (5 g / m for one day). laboratory values at baseline included a serum creatinine of 1.3 mg / dl and a glomerular filtration rate of 51.5 ml / min/1.73 m. his urine had tested negative for both protein and occult blood, with a specific gravity of 1.1. seven days after chemotherapy, the patient was admitted with a blood pressure of 130/80 mmhg, a pulse rate of 80/min, a respiratory rate of 20/min and a body temperature of 36.3. percussion at the costovertebral angle was pain - free, but palpation of the abdomen elicited mild tenderness. the complete blood count showed white blood cells (wbc) at 310/mm ; lymphocytes, 220/mm ; monocytes, 40/mm ; neutrophils, 40/mm ; eosinophils, 10/mm ; hemoglobin, 13.1 g / dl ; and hematocrit, 50,000/mm. a serum profile revealed a creatinine of 1.9 mg / dl ; sodium 129 meq / l ; potassium 2.4 meq / l ; inorganic phosphorus 1.1 mg / dl ; total magnesium 1.2 mg / dl ; and uric acid 0.7 mg / dl. urinalysis displayed 1 + protein, 3 + glucose, and red blood cells > 100/hpf. arterial blood registered a ph of 7.28 ; hco3 9.7 mmol / l ; and anion gap, 10.3 mmol / l (table 1). the size and shadow of both kidneys were normal by ultrasound, and a diffuse thickening of the bladder wall was noted (fig. 1). by the second hospital day, the patient was febrile (39). antibiotics and granulocyte - colony stimulating factor (g - csf) were initiated, and he was given supportive fluid therapy. bladder irrigation was also scheduled, but was discontinued after one attempt due to severe pain and discomfort. most of the patient 's symptoms subsided by day 6 of hospitalization, when he was afebrile with a normal neutrophil count, and the gross hematuria had resolved. his urine samples were microscopically devoid of organisms, and cultures of the urine and blood (each in triplicate) were consistently negative. without laboratory evidence of acute cystitis or pyelonephritis (i.e., negative urinary nitrite and normalized wbc / crp), urinalysis and a 24-hour urine test were unremarkable seven days postdischarge, but his serum creatinine had climbed to 1.5 mg / dl. a score of 8 by naranjo adverse drug reaction probability scale indicated a strong likelihood that the proximal renal tubular dysfunction experienced in this setting was linked to ifosfamide therapy. risk factors for the nephrotoxicity of ifosfamide include cumulative dosing, unilateral nephrectomy, and concomitant use of cisplatin (1 - 3). some studies, conducted with children or relative youths, have also proposed that the associated risk may be age - dependent (1). our patient, while diabetic, was treatment nave with respect to cancer therapy and was not otherwise at risk as currently defined. he nonetheless developed a proximal tubulopathy and hemorrhagic cystitis (gross hematuria and lower abdominal pain) seven days following a single course of ifosfamide - containing chemotherapy, but finally recovered after two weeks of conservative treatment. this certainly upholds the concept that ifosfamide may prove nephrotoxic in the absence of reported risk factors. the spectrum of ifosfamide nephrotoxicity is broad, ranging from asymptomatic tubulopathy to overtly impaired renal function or outright renal failure, and has been well documented (4). even after discontinuation of this drug, the tubulopathy may progress to end - stage disease, so that hemodialysis is sometimes required. our patient displayed features typical of fanconi 's syndrome ; hypophosphatemia, hypokalemia, hypouricemia, proteinuria and glucosuria. although beta-2 microglobin and amino acids were not monitored, the 24-hour urine results confirmed a proteinuria of 1.5 g / day and high phosphorus output, together suggesting proximal tubular damage. the mechanism of ifosfamide nephrotoxicity has been attributed to the metabolites 4-hydroxyl ifosfamide, chloracetaldehyde and acrolein. chloracetaldehyde, especially, suppresses activation of complex - i (nadh - ubiquinone oxidoreductase) in the mitochondrial respiratory chain, resulting in decreased intracellular glutathione and atp and inhibiting ca signaling, thus inducing cell death. the cellular impact of chloracetaldehyde, and hence the threat of nephrotoxicity, can not be averted with preventative use of mesna (5). hemorrhagic cystitis is a fairly regular accompanimont of ifosfamide, manifested by dysuria, suprapubic discomfort, and gross or microscopic hematuria. although this complication is often circumvented with mesna, it still occurs in a considerable number of patients (6). it augments the production of nitric oxide synthase in urothelium and generates various cytokines (tnf- and il-1) through intracellular transcription modulation to increase reactive oxygen species and nitric oxide levels. hemorrhagic cystitis thereby results, variably involving mucosal edema, hemorrhage, necrosis and ulceration. the sulfhydryl radical of mesna combats ifosfamide urotoxicity by combining with and eliminating acrolein (1). proper hydration, of course, is required ; but the fact that mesna prophylaxis does not uniformly prevent hemorrhagic cystitis in ifosfamide recipients implies that direct urothelial insult by acrolein may not be the sole pathogenic determinant. perhaps cytokines such as tnf- and il-1 and their roles in nitric oxide production provide an over - riding mechanism for the urotoxicity of this drug (7). however, at follow - up, his serum creatinine level had edged up to 1.5 mg / dl, and the nephropathy of diabetes was implicated. because his renal status may continue to deteriorate, he is monitored periodically, with strict attention to blood glucose control. it is consequently our view that diabetic nephropathy, in addition to age as a risk factor, may trigger adult susceptibility to ifosfamide nephrotoxicity.	the alkylating agent ifosfamide is an anti - neoplastic used to treat various pediatric and adult malignancies. its potential urologic toxicities include glomerulopathy, tubulopathy and hemorrhagic cystitis. this report describes a case of proximal renal tubular dysfunction and hemorrhagic cystitis in a 67-year - old male given ifosfamide for epitheloid sarcoma. he was also receiving an oral hypoglycemic agent for type 2 diabetes mellitus and had a baseline glomerular filtration rate of 51.5 ml / min/1.73 m2. despite mesna prophylaxis, the patient experienced dysuria and gross hematuria after a single course of ifosfamide plus adriamycin. the abrupt renal impairment and serum / urine electrolyte imbalances that ensued were consistent with fanconi 's syndrome. however, normal renal function and electrolyte status were restored within 14 days, simply through supportive measures. a score of 8 by naranjo adverse drug reaction probability scale indicated these complications were most likely treatment - related, although they developed without known predisposing factors. the currently undefined role of diabetic nephropathy in adult ifosfamide nephrotoxicity merits future investigation.
in the past two decades, the use of 2-[f]fluoro-2-deoxy - d - glucose (fdg) and positron emission tomography (pet) has achieved widespread acceptance as an effective tool for detecting cancers with high rates of glycolysis. it is generally accepted that a high rate of glucose metabolism (warburg effect) is associated with changes in tumor - driven alternative gene expression. however, despite the tremendous promise of fdg - pet for detecting and monitoring tumor metabolism, a significant portion of malignant tumors are not fdg - positive and can be missed in a fdg - pet scan. accordingly, there is a clear and urgent need to develop additional metabolic tracers, particularly for cancers with low fdg - uptake. recent reports suggest that metabolic reprogramming may cause some cancers to switch their energy source from glucose to glutamine. glutamine, which is found circulating in the blood and is also concentrated in the skeletal muscles (0.51 mmol / l), has various critical functions : as a substrate for dna and protein synthesis, a primary source of fuel for cells lining the inside of the small intestine and rapidly dividing immune cells, and as a regulator of acid base balance by producing ammonium in the kidneys. enhanced glutamine utilization in cancers due to changes in the expression of oncogenic signaling pathways can lead to glutaminolysis. in these cases, blocking glutamine synthetic pathways may lead to tumor cell death. glutamine imaging agents may be useful for testing the therapeutic efficacy of antitumor agents aimed at reducing glutamine metabolism in tumors. chemical structures of l-5-[c]glutamine, 1, (c]gln), [f](2s,4r)-4-fluoroglutamine, 2 ([f](2s,4r)-4-fgln), [f](2s,4r)-4-(3-fluoropropyl)glutamines, 3, ([f](2s,4r)-4-fpgln), and [f](2s,4s)-4-(3-fluoropropyl)glutamines, 4, ([f](2s,4s)-4-fpgln). in order to study glutamine metabolizing tumors, we previously prepared and tested l-5-[c]glutamine. in tumor cell uptake studies, the maximum uptake of l-5-[c]glutamine reached 17.9 and 22.5% uptake/100 g protein at 60 min in 9l and sf188 tumor cells, respectively. at 30 min after incubation, > 30% of the activity appeared to be incorporated into cellular proteins. dynamic small animal pet studies in rats bearing xenografts of 9l tumor and in transgenic mice bearing spontaneous mammary gland tumors showed prominent tumor uptake and retention. the results suggested that l-5-[c]glutamine would be useful for probing in vivo tumor metabolism in glutaminolytic tumors. because c has a half - life of 20 min, it would not be practical for most clinical settings. to make an imaging agent that would be better for clinical use, we created an alternative metabolic tracer labeled with f, which has a half - life of 110 min. in vitro studies with [f](2s,4r)-4-fluoroglutamine, 2 ([f](2s,4r)-4-fgln) showed that both 9l and sf188 tumor cells displayed a high rate of glutamine uptake (maximum uptake 16% dose/100 g protein), and the radioactivity trapped inside the cell was associated with the macromolecular fraction precipitated by trichloroacetic acid (tca). the cell uptake of [f](2s,4r)-4-fgln, 2, by sf188 cells is comparable to that of [h]l - glutamine but higher than that of fdg. biodistribution and pet imaging studies showed that [f](2s,4r)-4-fgln, 2, localized in tumors with a higher uptake than that of surrounding muscle and liver tissues, suggesting that [f](2s,4r)-4-fgln, 2, is selectively taken up and trapped by the tumor cells. one of the drawbacks of [f](2s,4r)-4-fgln, 2, (and its related optical isomers) is the radiolabeling reaction, which is relatively difficult and prone to formation of stereoisomers due a secondary fluorination reaction. to avoid this complication, we have designed and tested [f](2s,4r)-4-(3-fluoropropyl)glutamine, 3 ([f](2s,4r)-4-fpgln), and [f](2s,4s)-4-(3-fluoropropyl)glutamine, 4 ([f](2s,4s)-4-fpgln), as alternative probes for imaging glutamine metabolism (figure 1). this will make it easier for the sn2 fluorine substitution with a good leaving group (ots) used in the labeling reaction. to preserve the amide functional group at the c5 position, we have synthesized two types of precursors suitable for radiolabeling (fluoro for tosylate substitution reaction). reported herein is the preparation and in vitro and in vivo studies of these glutamine analogs. all reagents used were commercial products and were used without further purification unless otherwise indicated. boc - glu(obzl)oh (boc - l - glutamic acid 5-benzyl ester, 15) was purchased from sigma - aldrich. flash chromatography (fc) was performed using silica gel 60 (230400 mesh, sigma - aldrich). h nmr spectra were obtained at 200 mhz and c nmr spectra were recorded at 50 mhz (bruker dpx 200 spectrometer). chemical shifts are reported as values (parts per million) relative to remaining protons in deuterated solvent. coupling constants are reported in hertz. the multiplicity is defined by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), br (broad), or m (multiplet). high - resolution ms experiments were performed using an agilent technologies lc / msd tof mass spectrometer. compounds 58 were synthesized according to the procedures reported previously. to a solution of compound 8 (554 mg, 2 mmol) in 10 ml of dry dichloromethane (dcm) was added et3n (1.4 ml, 10 mmol), 4-(dimethylamino)pyridine (dmap, 24 mg, 0.2 mmol), and p - toluenesulfonyl chloride (tscl, 764 mg, 4 mmol) at 0 c. ice - cold water (15 ml) was poured into the reaction mixture and the mixture was extracted with dcm (15 3 ml), the combined organic layer was dried with magnesium sulfate (mgso4) and was purified with flash chromatography (fc, etoac / hexane = 2/8) to give 739.6 mg colorless oil 9 (yield : 86.1%). hnmr (200 mhz, cdcl3) : 1.391.54 (m, 18h), 2.082.21 (m, 2h), 2.47 (s, 3h), 4.064.09 (m, 2h), 4.134.16 (m, 1h), 5.035.10 (m, 1h), 7.37 (d, j = 7.8 hz, 2h), 7.81 (d, j = 8.2 hz, 2h). hrms was calcd for c20h32no7s (m + h) : 430.1899. found : 430.1910. sodium iodide (240 mg, 1.6 mmol) was added to a solution of compound 9 (343 mg, 0.8 mmol) in 10 ml of acetone (hplc grade) at rt. the mixture was stirred at 60 c for 3 h. the solvent was then removed and the residue was dissolved in 15 ml of dcm. the precipitated solid was filtered out and filtrate was concentrated. n, n - dimethylformamide (dmf, 7 ml) and potassium cyanide (78 mg, 1.2 mmol) were added to the residue. the reaction mixture was quenched with 25 ml of ethyl acetate and was washed with h2o (10 3 ml). the filtrate was concentrated, and the residue was purified by fc (etoac / hexane = 2/8) to give 196.2 mg colorless oil 10 (yield : 86.3%). hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.902.04 (m, 1h), 2.202.30 (m, 1h), 2.402.46 (m, 2h), 4.204.30 (m, 1h), 5.125.22 (m, 1h). hrms was calcd for c14h25n2o4 (m + h) : 302.2080. found : 302.2078. lihmds (lithium bis(trimethylsilyl)amide) (1.5 ml, 1 mol / l solution in thf) was added to a three - necked 250 ml flask. compound 10 (201 mg 0.67 mmol) in 3 ml of dry thf solution was added dropwise over 30 min. after being stirred at 78 c for 2 h, allyl bromide (0.24 ml, 2.8 mmol) was added dropwise over 15 min. the mixture was then stirred at 78 c for another 4 h. the reaction was quenched with 20 ml of ethyl acetate and 15 ml of hcl (2 m) and extracted with ethyl acetate (20 3 ml). the filtrate was concentrated, and the residue was purified by fc (etoac / hexane = 2/8) to give 60.9 mg 11a (yield : 28.0%) and 32.6 mg 11b (yield : 15.1%). 11a : hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.902.24 (m, 2h), 2.442.62 (m, 2h), 2.712.85 (m, 1h), 4.354.45 (m, 1h), 5.225.27 (m, 1h), 5.305.36 (m, 2h), 5.705.91 (m, 1h). hrms was calcd for c17h29n2o4 (m + h) : 325.2127. found : 325.2125. 11b : hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.701.90 (m, 1h), 2.052.25 (m, 1h), 2.32.55 (m, 2h), 2.712.85 (m, 1h) 4.354.45 (m, 1h), 5.225.36 (m, 3h), 5.705.91 (m, 1h). (91 mg, 0.28 mmol) in 7 ml of thf was added 9-borabicyclo[3.3.1 ] nonane (9-bbn, 2.22 ml, 0.5 m solution in thf) dropwise at 0 c. after being stirred at 0 c for 1 h, the reaction mixture was moved to rt and stirred for another 48 h. the mixture was then cooled with an ice - bath. (0.31 ml of 30 wt % solution in h2o) and naoh (0.4 ml, 1 m) were added dropwise. the mixture was stirred at rt for 30 min, diluted with 15 ml of h2o and extracted with ethyl acetate. the filtrate was concentrated, and the residue was purified by fc (etoac / hexane = 1/1) to give 30.7 mg colorless oil 12a (yield : 32.1%). hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.801.95 (m, 4h), 1.962.15 (m, 2h), 2.702.85 (m, 1h), 3.693.75(m, 2h), 4.204.35 (m, 1h), 5.205.35 (m, 1h). hrms was calcd for c17h30n2o5 (m + h) : 343.2233. found : 343.2258. compound 12b was prepared from 11b (194 mg, 0.6 mmol), 9-bbn (4.8 ml, 0.5 m solution in thf), h2o2 (0.65 ml of 30 wt % solution in h2o), and naoh (0.9 ml, 1 m), following the same procedure described for compound 12a. hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.701.85 (m, 4h), 1.862.01 (m, 1h), 2.092.30 (m, 1h), 2.702.85 (m, 1h), 3.653.75(m, 2h), 4.254.40 (m, 1h), 5.105.25 (m, 1h). hrms was calcd for c17h30n2o5 (m + h)+ : 343.2233. found : 343.2258. to a solution of compound 12a (137 mg, 0.4 mmol) in 7 ml of dcm was added et3n (0.3 ml, 2.1 mmol) and dmap (5 mg, 0.04 mmol) at c, followed by tscl (153 mg, 0.8 mmol). the resulting mixture was stirred at 0 c for 1 and 24 h at rt. the reaction was quenched with 15 ml of water and extracted with dcm (10 3 ml). the filtrate was concentrated, and the residue was purified by fc (etoac / hexane = 2/8) to give 180.2 mg of colorless oil 13a (yield : 90.7%). hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.701.90 (m, 4h), 1.912.02 (m, 2h), 2.45 (s, 3h), 2.602.70 (m, 1h), 3.954.15 (m, 2h), 4.164.25 (m, 1h), 5.105.19 (m, 1h), 7.37 (d, j = 8.0 hz, 2h), 7.80 (d, j = 8.2 hz, 2h). compound 13b was prepared from 12b (102 mg, 0.3 mmol), et3n (0.21 ml, 1.5 mmol), tscl (115 mg, 0.6 mmol) and dmap (4 mg, 0.03 mmol), following the same procedure described for compound 13a. hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.701.90 (m, 4h), 1.901.98 (m, 1h), 2.152.30 (m, 1h), 2.50 (s, 3h), 2.602.70 (m, 1h), 3.954.15 (m, 2h), 4.204.35 (m, 1h) ; 5.105.19 (m, 1h), 7.37 (d, j = 8.2 hz, 2h), 7.80 (d, j = 8.2 hz, 2h). mg, 0.363 mmol) in 3 ml of dcm and 3 ml of dmf was added et3n(hf)3 (0.021 ml) dropwise followed by 13a (35 mg, 0.072 mmol) in 3 ml of dcm. the reaction was quenched by the addition of ice - cold water (5 ml) and diluted with 50 ml of etoac, and then washed with h2o (15 ml 2) and brine (15 ml) and dried with mgso4. the filtrate was evaporated in vacuo and the residue was purified by fc (etoac / hexanes = 2/8) to give 22 mg of colorless oil 14a (yield : 88.8%). hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.701.90 (m, 4h), 1.912.10 (m, 2h), 2.122.26 (m, 1h), 2.662.80 (m, 1h), 4.204.32 (m, 1h), 4.51 (dt, j = 46.4 hz, j = 6.4 hz, 2h), 5.105.15 (m, 1h). hrms was calcd for c17h33fn3o4 (m + nh4) : 362.2455 compound 14b was prepared from 13b (80 mg, 0.16 mmol), tasf (220 mg, 0.80 mmol), and et3n(hf)3 (0.046 ml) following the same procedure described for compound 14a. hnmr (200 mhz, cdcl3) : 1.391.52 (m, 18h), 1.701.92 (m, 4h), 1.912.30 (m, 2h), 2.702.85 (m, 1h), 4.204.35 (m, 1h), 4.51(dt, j = 48.2 hz, j = 5.8 hz, 2h), 5.105.22 (m, 1h). compound 14a (37 mg, 0.11 mmol) in concentrated hcl (1.2 ml) was stirred at rt for 6 h. the ph was then adjusted to 78 with 5% ammonium solution. the neutralized solution was submitted to a small column of dowex 50wx8 - 200 (h form, 10 g). the fraction containing product was concentrated in vacuo and dried under high vacuum overnight to afford the crude product as a white solid. it was further purified by recrystallization from etoh / h2o to provide 5.9 mg of white solid 4 (yield : 26.0%). hnmr (200 mhz, d2o) : 1.491.56 (m, 3h), 1.571.80 (m, 1h), 1.801.95 (m, 2h), 2.512.59 (m, 1h), 3.453.52 (m, 1h), 4.51 (d.t, j = 46.4 hz, j = 6.4 hz, 2h) ; cnmr (200 mhz, d2o) : 179.8, 174.0, 84.82 (d, j = 157.5 hz), 53.0, 42.2, 33.4, 27.2 (d, j = 20 hz), 28.1. hrms was calcd for c8h16fn2o3 (m + nh4) : 207.1145. found : 207.1169. compound 3 was prepared from 14b (39 mg, 0.11 mmol) and concentrated hcl (1.2 ml) following the same procedure described for compound 4. compound 3 : 6.8 mg (yield : 30%). hnmr (200 mhz, d2o) : 1.491.70 (m, 4h), 1.701.90 (m, 1h), 2.012.15 (m, 1h), 2.402.50 (m, 1h), 3.423.56 (m, 1h), 4.41 (dt, j = 47.4 hz, j = 5.0 hz, 2h). cnmr (50 mhz, d2o) : 179.9, 174.0, 84.82 (d, j = 157.5 hz), 53.0, 42.1, 33.4, 27.4 (d, j = 30 hz), 27.4. hrms was calcd for c8h16fn2o3 (m + nh4) : 207.1145. found : 207.1162. to a solution of boc - glu(obzl)oh, 15, (3.37 g, 10 mmol) in 20 ml of dcm was added tert - butyl 2,2,2-trichloroacetimidate (3.9 g, 18 mmol) in 20 ml of cyclohexane dropwise. the reaction mixture was stirred at rt for 4 h. the solid was filtered off. the filtrate was evaporated in vacuo and the residue was purified by fc (etoac / hexanes = 2/8) to give 3 g of white solid 16 (yield : 76.3%). hnmr (200 mhz, cdcl3) : 1.441.46 (m, 18h), 1.901.97 (m, 1h), 2.132.20 (m, 1h), 2.402.50 (m, 2h), 4.204.23 (m, 1h), 5.06 (s, 1h), 5.13 (s, 2h), 7.36 (s, 5h). hrms was calcd for c21h32no6 (m + h) : 394.2230. found : 394.2241. lihmds solution (8.8 ml, 1 m in thf) was added to a three - necked 250 ml flask and cooled down to 78 c. compound 16 (1.57 g, 4 mmol) was dissolved in 6 ml of thf was then added dropwise over 30 min. the mixture was stirred at 78 c for another 2 h. allyl bromide (2.4 g, 20 mmol, 1.72 ml) was added dropwise. the mixture was then stirred at 78 c for another 4 h. the reaction was quenched with 20 ml of ethyl acetate and 15 ml of hcl (2 m), the mixture was extracted with ethyl acetate (3 25 ml). the filtrate was concentrated, and the residue was purified by fc (etoac / hexane = 2/8) to give 1 g of colorless oil 17 (yield : 58%). hnmr (200 mhz, cdcl3) : 1.451.47 (m, 18h), 1.942.01 (m, 2h), 2.372.44 (m, 2h), 2.592.69 (m, 1h), 4.184.30 (m, 1h), 5.025.04 (m, 1h), 5.075.22 (m, 4h), 5.615.82 (m, 1h), 7.38 (s, 5h). hrms was calcd for c24h36no6 (m + h) : 434.2543. found : 434.2507. to a solution of compound 17 (860 mg, 2 mmol) in 7 ml of thf was added 9-bbn (8 ml, 0.5 m solution in thf) dropwise at 0 c. after stirring at 0 c for 1 h, the reaction mixture was moved to rt and stirred for another 48 h. the mixture was then cooled in an ice - bath. (2.2 ml of 30 wt % solution in h2o) and naoh (3 ml, 1 m) were added dropwise. the mixture was stirred at rt for 30 min, diluted with of 15 ml h2o, and extracted with ethyl acetate. the filtrate was concentrated, and the residue was purified by fc (etoac / hexane = 1/1) to give 700 mg of colorless oil 18 (yield : 77.4%). hnmr (200 mhz, cdcl3) : 1.431.45 (m, 18h), 1.751.86 (m, 4h), 1.891.93 (m, 2h), 2.492.63 (m, 1h), 3.59 (t, j = 6.4 hz, 2h) 4.244.27 (m, 1h), 5.045.1 (m, 3h), 7.36 (s, 5h). (530 mg, 1.2 mmol) in 10 ml of dcm was added 3,4-dihydro-2h - pyran (225 l, 2.4 mmol) and pyridinium p - toluenesulfonate (30 mg, 0.12 mmol). the mixture was stirred at rt for 3 h. the solvent was evaporated in vacuo and the residue was purified by fc (etoac / hexanes = 3/7) to give 578 mg of colorless oil 19 (yield : 90%). hnmr (200 mhz, cdcl3) : 1.431.45 (m, 18h), 1.501.64 (m, 6h), 1.681.73 (m, 4h), 1.902.01 (m, 2h), 2.532.60 (m, 1h), 3.303.53 (m, 2h), 3.643.87 (m, 2h), 4.114.21 (m, 1h), 4.53 (s, 1h), 4.894.93 (m, 1h), 5.155.31 (m, 2h), 7.36 (s, 5h). a mixture of the ester 19 (750 mg, 1.4 mmol) and 10% pd / c (90 mg) in absolute etoh (15 ml) was stirred under hydrogen overnight. this mixture was then filtered and the filtrate was concentrated to give 620 mg of colorless oil 20 (yield : 99%). hnmr (200 mhz, cdcl3) : 1.451.47 (m, 18h), 1.501.70 (m, 6h), 1.751.80 (m, 2h), 1.822.00 (m, 4h), 2.512.58 (m, 1h), 3.413.51 (m, 2h), 3.803.95 (m, 2h), 4.224.29 (m, 1h), 4.59 (s, 1h), 4.055.01 (m, 1h). hrms was calcd for c22h40no8 (m + h) : 446.2754. found : 446.2740. to a solution of compound 20 (534 mg, 1.2 mmol) in 2 ml of dcm and 2 ml of dmf was added et3n (1.9 mmol, 0.26 ml), hobt (1.44 mmol, 297 mg), 2,4,6-trimethoxybenzylamine hydrochloride (370 mg, 1.58 mmol), and n, n - dicyclohexylcarbodiimide (276 mg, 1.8 mmol) at 0 c. the mixture was stirred at rt for 24 h. a total of 30 ml of etoac was added to the reaction mixture. the mixture was then washed with citric acid (10% in h2o, 5 ml), h2o (5 ml 2) as well as brine (5 ml), dried over na2so4, and filtered. pyridinium p - toluenesulfonate (50 mg, 0.2 mmol) was then added. after heating at 50 c for 4 h, the solvent was evaporated in vacuo and the residue was purified by fc (etoac / hexanes = 3/7) to give 203 mg of colorless oil 21 (yield : 30.8%). hnmr (200 mhz, cdcl3) : 1.441.45 (m, 18h), 1.461.70 (m, 4h), 1.811.86 (m, 2h), 2.142.21 (m, 2h), 3.543.60 (m, 2h), 3.82 (s, 9h), 4.124.18 (m, 1h), 4.284.37 (m, 1h), 4.534.62 (m, 1h), 5.045.09 (m, 1h), 5.95 (s, 1h), 6.13 (s, 1h). (200 mg, 0.37 mmol) in 10 ml of dcm was added 4-(dimethylamino)pyridine (4.55 mg, 0.037 mmol), et3n (147 mg, 1.11 mmol), and tscl (105 mg, 0.55 mmol) at 0 c. the reaction was then quenched with 15 ml of h2o and extracted with ethyl acetate (15 ml 3). the filtrate was concentrated, and the residue was purified by fc (etoac / hexane = 3/7) to give 209 mg of white solid 22 (yield : 81.7%). hnmr (200 mhz, cdcl3) : 1.441.45 (m, 18h), 1.551.62 (m, 4h), 1.761.89 (m, 2h), 1.932.07 (m, 1h), 2.47 (s, 3h), 3.823.84 (m, 9h), 3.944.04 (m, 2h), 4.044.10 (m, 1h), 4.284.37 (m, 1h), 4.564.66 (m, 1h), 4.954.99 (m, 1h), 5.895.94 (m, 1h), 6.14 (s, 1h) 7.35 (d, j = 8.0 hz, 2h), 7.76 (d, j = 8.4 hz, 2h). hrms was calcd for c34h51n2o11s (m + h) : 695.3214. found : 695.3099. compound 23 was prepared from 18 (0.370 g, 0.84 mmol), et3n (424 mg, 4.20 mmol), tscl (319 mg, 1.68 mmol), and dmap (10.2 mg, 0.084 mmol) in 10 ml of dcm, with the same procedure described for compound 22. hnmr (200 mhz, cdcl3) : 1.42(s, 9h), 1.45(s, 9h), 1.481.60 (m, 4h), 1.771.95 (m, 2h), 2.452.51 (m, 4h), 3.973.99 (m, 2h), 4.084.18 (m, 1h), 4.90 (d, 1 h, j = 9.2 hz), 5.10 (dd, 2h, j = 12.2 hz, j = 22 hz), 7.327.35 (m, 7h), 7.77 (d, 2h, j = 8.2 hz). hrms was calcd for c31h44no9s (m + h) : 606.2737. found : 606.2784. to a solution of tris(dimethylamino)sulfonium difluorotrimethylsilicate (1.31 g, 4.75 mmol) in 5 ml of thf and 5 ml of dmf was added et3n(hf)3 (0.273 ml) dropwise followed by 23 (0.700 g, 1.16 mmol) in 5 ml of thf. the reaction was quenched by the addition of ice - cold water (5 ml) and diluted with 100 ml of etoac, then washed with h2o (25 ml 2) and brine (25 ml), and dried with mgso4. the filtrate was evaporated in vacuo and the residue was purified by fc (etoac / hexanes = 2/8) to give 0.430 g of colorless oil 24 (yield : 82.0%). hnmr (200 mhz, cdcl3) : 1.43 (s, 9h), 1.45 (s, 9h), 1.591.72 (m, 4h), 1.922.00 (m, 2h), 2.512.61 (m, 1h), 4.154.30 (m, 2h), 4.514.53 (m, 1h), 4.93 (d, 1h, j = 8.4 hz), 5.13 (dd, 2h, j = 12.2 hz, j = 23 hz), 7.317.36 (m, 5h). hrms was calcd for c24h37fno6 (m + h) : 454.2605. found : 454.2667. a mixture of the ester 24 (0.430 g, 0.95 mmol) and 10% pd / c (0.100 g) in absolute etoh (10 ml) was stirred under hydrogen overnight. this mixture was then filtered, and the filtrate was concentrated under vacuum to give 0.345 g of colorless oil 25 (yield : 100%). hnmr (200 mhz, cdcl3) : 1.45(s, 9h), 1.47 (s, 9h), 1.611.83 (m, 4h), 1.912.03 (m, 2h), 2.432.62 (m, 1h), 4.174.39 (m, 2h), 4.514.67 (m, 1h), 5.06 (d, 1h, j = 9.2 hz). hrms was calcd for c17h31fno6 (m + h) : 364.2135. found : 364.2166. to a solution of 25 (0.345 g, 0.95 mmol), n-(3-(dimethylamino)propyl)-n - ethylcarbodiimide hydrochloride (0.258 g, 1.34 mmol) and 1-hydroxybenzotriazole hydrate (0.227 g, 1.34 mmol) was added triethylamine (0.485 g, 4.80 mmol) and 2,4,6-trimethoxybenzylamine hydrochloride (0.339 g, 1.45 mmol) at 0 c. after stirred overnight, the mixture was diluted with 150 ml to etoac and washed with h2o (30 ml 2) and brine (30 ml). the organic layer was dried by mgso4 and concentrated to give an oil that was purified by fc (etoac / hexane = 1/1) to give 0.350 g of colorless oil 26 (yield : 64.5%). hnmr (200 mhz, cdcl3) : 1.42 (s, 9h), 1.45 (s, 9h), 1.601.74 (m, 4h), 1.881.98 (m, 2h), 2.292.48 (m, 1h), 3.82 (s, 6h), 4.024.14 (m, 1h), 4.254.40 (m, 2h), 4.494.68 (m, 2h), 5.02 (br s, 1h), 6.20 (s, 2h), 6.34 (br s, 1h). hrms was calcd for c27h44fn2o8 (m + h) : 543.3082. found : 543.3048. [f]fluoride was purchased from iba molecular (somerset, nj) as an [o]enriched aqueous solution of [f]fluoride. solid - phase extraction (spe) cartridges such as sep - pak qma light and oasis hlb cartridges were purchased from waters (milford, ma). high - performance liquid chromatography (hplc) was performed on agilent 1100 or 1200 series system with different hplc columns. [h]gln was purchased from perkinelmer (waltham, ma) with > 97% radiochemical purity and 1.112.22 tbq / mmol specific activity. briefly, an activated seppak light qma carb was loaded with [f]fluoride (740 to 1480 mbq (20 to 40 mci)) and eluted with 1 ml of 18-crown-6/khco3 (160 mg of 18-crown-6 in 18.6 ml of acn/29 mg of khco3 in 3.4 ml of water). the solution was blown with argon until dry and dried twice azeotropically with 1 ml of acetonitrile at 80 c under a flow of argon. the dried [f]fluoride was cooled in an ice bath and 5 mg of tosylate precursor (o - tosylate, 13a and 13b, respectively) was dissolved in 0.5 ml of dmso and added to the dried [f]fluoride. the mixture was heated for 10 min at 110 c in an oil bath. the mixture was then cooled in an ice bath and added to 6 ml of water/1 ml of acetonitrile. the mixture was loaded onto an activated oasis hlb 3 cm cartridge, pushed through, and washed twice with 3 ml of water. a mixture of 400 l of tfa/100 of l concentrated h2so4 was added and heated for 10 min at 120 c in a capped 10 ml vial. water (1 ml) was slowly added and the mixture was neutralized by the slow addition of a saturated na2co3 solution under heavy shaking (1000 l) (ph 8). the mixture was put through an activated oasis hlb 3 cm, which was topped with 0.3 g of ag11-a8 resin. the radioactivity was eluted with phosphate buffered saline (ph 7.0) in fractions of 0.5 ml volume to yield the desired radioactive [f](2s,4r)-4-fpgln, 3, and [f](2s,4s)-4-fpgln, 4, respectively. gradient, 1 ml / min : 03 min 95% b, 311 min 95%5% b, 1119 min 5%95% b, 1921 min 95% b. retention times for both 3 and 4 are 2.5 min. system 2. mobile phase (isocratic) : 2 mm cuso4 solution, 1 ml / min, column temperature at 30 c. alternatively, tmobnh precursor, 22, was used under a similar labeling condition as described above (18-crown-6/khco3/acn/80 c/20 min). it was found that the tmobnh intermediate, 26, was formed with a lower yield of 6.6 1.6%, radiochemical purity 98% (n = 3). the f intermediate, [f]26, displayed the same profile on the hplc as that of the cold compound. deprotection was performed with 500 l of tfa at 40 c for 8 min. the residue was treated with 1 ml of phosphate buffered saline (pbs) and filtered through a 0.45 filter and washed with 0.1 ml of pbs (ph 7.0) to give a crude dose. the solution was passed through an activated cartridge (oasis hlb 3 cm). the solid - phase extraction was further rinsed with 0.3 ml of pbs (ph 7.0) to yield (2s,4s)[f]4-fpgln, 4. 9l cells (atcc, manassas, va) were cultured in dulbecco s modified eagle s medium (dmem, gibco brl, grand island, ny) supplemented with 10% fetal bovine serum (hyclone, logan, ut) and 1% 100 units / ml penicillin, 100 g / ml streptomycin. the cells were maintained in t-75 culture flasks under humidified incubator conditions (37 c, 5% co2) and were routinely passaged at confluence. tumor cells were plated in 12 well plates 24 h prior to studies. on the day of the experiment, the culture media was aspirated and the cells were washed three times with warm pbs (containing 0.90 mm of ca and 1.05 mm of mg). [f](2s,4r)-4-fpgln, 3 (370 kbq) and l-[3,4-h(n)]-glutamine ([h]gln) (37 kbq) were mixed in pbs (with ca and mg) solution and then added to each well. the cells were incubated at 37 c for 5, 30, 60, and 120 min. at the end of the incubation period, the pbs solution containing the ligands was aspirated and the cells were washed three times with 1 ml of ice cold pbs (without ca and mg). after washing with ice - cold pbs, 350 l of 0.1 n naoh was used to lyse the cells. the lysed cells were collected onto filter paper and counted together with samples of the incubation dose using a gamma counter (packard cobra). after 24 h, h activity was counted using a scintillation counter (beckman ls 6500). a total of 100 l of the cell lysate the data was normalized as percentage uptake of initial dose (i d) relative to 100 g of protein content (% id/100 g of protein). to test the in vivo cell incorporation, we used 9l cells. cells were plated (5 10 cells / well) on six - well plates in culture media 24 h prior to the experiment. on the day of experiment, the media was aspirated and the cells were washed three times with 4 ml of warm pbs (containing 0.90 mm of caand 1.05 mm of mg). to measure the extent of protein incorporation of [f](2s,4s)-4-fpgln, 4, protein bound activity in 9l cells was determined at 30 and 120 min after incubation. [f](2s,4s)-4-fpgln, 4 (370 kbq) and l-[3,4-h(n)]-glutamine ([h]gln, 37 kbq) in 2 ml of pbs were mixed in the incubation media. to identify the ligand s stability in supernatant after precipitation with trichloroacetic acid (tca), cells were grown in 10 cm dishes and incubated with 1.8 mbq [f](2s,4s)-4-fpgln, 4, only. at the end of incubation, the radioactive medium was removed, the cells were washed three times with ice cold pbs without ca and mg, treated with 0.25% trypsin, and resuspended in pbs. the samples were centrifuged (18 000 g, 3 min), the supernatant removed, and the cells were suspended in 200 l and 1% triton - x 100 (sigma, st. after vortexing, 800 l of ice cold 15% tca was added to the solution. after precipitating for 10 min, the cells were centrifuged again (18 000 g, 3 min) and washed twice with 15% ice cold tca. the radioactivity of both gamma- and beta - emitting isotopes was determined separately for the supernatant and pellet. protein incorporation was calculated as a percentage of acid precipitable activity. to characterize the transport of [f](2s,4s)-4-fpgln, 4, competitive inhibition studies various inhibitors were then added to the cells in concentrations ranging from 0.1 to 5 mm in pbs solution. selected inhibitors included synthetic amino acid transport inhibitors such as n - methyl--aminoisobutyric acid (meaib) for system a, and 2-amino - bicylo[2.2.1 ] heptane-2-carboxylic acid (bch) for system l. natural amino acids, such as l - serine and l - glutamine, were also used as inhibitors, although they are not specific for a particular amino acid transport system. the data was compared in reference to uptake of [f](2s,4s)-4-fpgln, 4, without any inhibitor in pbs solution at ph 7.4. studies of the in vivo distribution of [f](2s,4s)-4-fpgln, 4, were performed in fischer (f344) rats bearing 9l tumors as reported previously. 9l tumor cells (10) in pbs (0.2 ml) were injected subcutaneously into the lower right flank of the rat. the tumors took 1215 days to reach appropriate size (1 cm diameter). the rats were anesthetized with isoflurane (23%) and 0.2 ml of saline solution containing 25 ci of the ligand was injected intravenously. the rats were sacrificed at 30 and 60 min postinjection by cardiac excision while under isoflurane anesthesia. the organs of interest were removed, weighed, and the radioactivity was counted with a gamma counter (packard cobra). the percent dose per gram was calculated by a comparison of the tissue activity counts to counts of 1% of the initial dose. dynamic small animal pet (apet) imaging studies were conducted with [f](2s,4r)-4-fgln, 2, and [f](2s,4s)-4-fpgln, 4 similar to that reported previously. all scans were performed on a dedicated animal pet scanner (mosaic by phillips) that has a field of view of 11.5 cm. a total of 2237 mbq of activity was injected intravenously via the lateral tail vein. all animals were sedated with isoflurane anesthesia (23%, 1 l / min oxygen) and were then placed on a heating pad in order to maintain body temperature throughout the procedure. the animals were visually monitored for breathing and any other signs of distress throughout the entire imaging period. all scans were conducted over a period of 120 min (dynamic, 5 min / frame). in order to produce [f](2s,4r)-4-(3-fluoropropyl)glutamine, 3, ([f](2s,4r)-4-fpgln) and [f](2s,4s)-4-(3-fluoropropyl)glutamine, 4 ([f](2s,4s)-4-fpgln), we employed two different schemes (schemes 1 and 2) for preparation of nonradioactive cold compounds (3 and 4) and the cyanide and -ots precursors for radiolabeling. one approach was to prepare the corresponding protected 4-cyanide derivatives, which led to the formation of the desired final products. commercially purchased boc - asp(obzl)oh, 5, was treated with tert - butyl 2,2,2-trichloroacetimidate / bf3et2o at room temperature to give the t - buo- ester, 6 ; the bno- ester group was converted to the acid, 7, by pd / c catalyzed hydrogenation. the aspartic acid, 7, was carefully reduced with nabh4 in thf / water at 15 to 0 c to the corresponding alcohol, 8. the alcohol group was successfully converted to the cyanide, 10, through the -ots intermediate, 9. the cyanide derivative, 10, was treated with lihmds and allyl bromide at 78 c to give (2s,4s)-tert - butyl 2-(tert - butoxycarbonylamino)-4-cyanohept-6-enoate (11a) and (2s,4r)-tert - butyl 2-(tert - butoxycarbonylamino)-4-cyanohept-6-enoate (11b) (11a to 11b ratio of 2:1 in 28% and 15% isolated yields). a similar reaction was reported previously for preparation of allyl derivatives of aspartate using the dianionic allylation reactions of amino acid derivatives. the allyl derivatives, 11a and 11b, were separated and purified by flash chromatography. they were converted to the corresponding alcohols, 12a and 12b, and following the treatment with tosyl chloride to the o - tosylated 13a and 13b in good yields. the o - tosylated 13a and 13b were treated with tasf, et3n(hf)3, dcm, dmf, 50 c, overnight to give the desired 14a and 14b, in good yields. optimization of the fluorination reaction condition using tasf and et3n(hf)3, as the reagents was reported previously for the preparation of 4-fluoroglutamine. deprotection using hydrochloric acid at room temperature produced the final end products, [f](2s,4r)-4-(3-fluoropropyl)glutamine, 3, and [f](2s,4s)-4-(3-fluoropropyl)glutamine, 4. the o - tosylated 13a and 13b were also successfully used for the radiolabeling reaction (see discussion below). reagents and reaction conditions : (a) tert - butyl 2,2,2-trichloroacetimidate (tbta), bf3et2o, dcm, cyclohexane, rt, overnight ; (b) pd / c, h2, meoh, rt, overnight ; (c) ecf, nabh4, thf, h2o, 150 c, 4 h ; (d) tscl, et3n, dmap, dcm, rt, overnight ; (e) nai, kcn, dmf, rt, overnight ; (f) lihmds, allyl bromide, thf, 78 c, 4 h ; (g) 9-bbn, h2o2, naoh, 0 c - rt, 48 h ; (h) tasf, et3n(hf)3, dcm, dmf, 50 c, overnight ; (i) hcl, rt, 6 h. reagents and conditions : (a) tert - butyl 2,2,2-trichloroacetimidate (tbta), bf3et2o, dcm, cyclohexane, rt, overnight ; (b) lihmds, allyl bromide, thf, 78 c, 4 h ; (c) 9-bbn, h2o2, naoh, 0 c rt, 48 h ; (d) dhp, ppts, dcm, rt, 3 h ; (e) pd / c, h2, etoh, rt, 6 h ; (f) tmobnh2hcl, edcihcl, hobt, et3n, dcm, dmf, rt, 24 h ; (g) ppts, etoh, 50 c, 4 h ; (h) tscl, et3n, dmap, dcm, rt, overnight ; (i) tasf, et3n(hf)3, dcm, dmf, 50 c, overnight. the second method introduced n - tmob protected precursors (as a protecting group to preserve the amide) for radiolabeling and deprotection. previously, we have tested for the preparation of n - tmob protected precursors for making isomers of 4-fluoroglutamine (4-fgln). we wanted to extend the same method to the synthesis and labeling of [f](2s,4s)-4-(3-fluoropropyl)-glutamine, 4. to achieve this after treating with tert - butyl 2,2,2-trichloroacetimidate / bf3et2o at room temperature, it gave the t - bu ester, 16. using the same dianionic allylation reactions of amino acid derivatives, the reaction preferentially produced the protected (2s,4s)-4-allyl - glutamate (in 58% yield). it is interesting to note that the reaction led to the (2s,4s) isomer only. the allyl group was converted to alcohol, 18, by 9-bbn / h2o2/naoh in 0 c. the alcohol was protected by thp, and the o - benzyl ester was hydrolyzed and the acid, 20, was transformed to tmob - protected amide, 21. the alcohol, 21, was treated with tosyl chloride to the o - tosylated, 22, which is a suitable precursor for a radioactive f labeling reaction. in order to provide an authentic sample, a cold standard, for the first step of the radioactive f labeling reaction, we also prepared compound, 26. to further confirm the chemical structure, a slow evaporating recrystallization method provided excellent crystals of cold (2s,4s)-4-fpgln, 4 and the x - ray crystallographic analysis data added support to the structure assignment (figure 2). the optically pure (2s,4s)-4-fpgln, 4, has never been prepared and presented before. in the x - ray crystallographic structures of (2s,4r)4f - gln, 2, and (2s,4s)-4-fpgln, 4, the amino acid groups on the right side of the molecules were comparable, whereas the amide group on the left appeared to be varied and different from each other. the results reported in figure 2 firmly establish the configuration, which may facilitate future use of other 4-fluoroglutamine isomers for biological and medical applications. comparison of x - ray crystallography structures for (2s,4r)-4-fluoro - glutamine, (2s,4r)-4-fgln, 2, and (2s,4s)-4-(3-fluoropropyl)glutamine, (2s,4s)-4-fpgln, 4. (crystal structure, (2s,4s)-4-fpgln, 4, was submitted to the cambridge crystallographic data centre (ccdc 991692). radiolabeling of the desired [f](2s,4r)-4-fpgln, 3, and [f](2s,4s)-4-fpgln, 4, was achieved by methods in scheme 3. the preparation can be accomplished by using the o - tosylated cyanide derivatives, 13a or 13b, or the o - tosylated tmobnh- protected precursor, 22. the substitution of o - ts with [f]fluoride using o - tosylated cyanide derivatives, 13a or 13b, was performed with 18-crown-6/khco3 in dmso at 110 c for 10 min, followed by a solid - phase extraction (oasis hlb 3 cm cartridge). the intermediate was eluted from this cartridge and treated with h2so4/tfa at 120 c for 10 min. the crude labeled products were cooled to room temperature and neutralized with a saturated na2co3 solution. the mixture was passed through an oasis hlb 3 cm cartridge topped with ag11-a8 resin. the cartridge was eluted with phosphate buffered saline (ph 7.0) to give the desired radioactive [f](2s,4r)-4-fpgln, 3, and [f](2s,4s)-4-fpgln, 4, respectively (scheme 3). the purity was measured with reversed - phase hplc (radiochemical purity) and chiral hplc (optical purity). the decay - corrected radiochemical yield was 6.2 3.9%, radiochemical purity 91.5 1.5%, optical purity > 99%, n = 2 (for 3) and 25.2 2.3%, rcp 92.8 2.6%, optical purity > 99%, n = 5 (for 4). it is important to note that radiolabeling of these two seemingly close analogs showed very different yields. we noted the disparity in radiolabeling yields, but we do not have a simple explanation for this phenomenon. additional studies may be needed to investigate the optical preferences in the substitution of o - ts with [f]fluoride. to improve the radiolabeling reaction for the more promising [f](2s,4s)-4-fpgln, 4, we made the effort to use a different o - tosylated tmobnh- protected precursor, 22. a similar 4-o - tosylated tmobnh - protected precursor was successfully employed for substitution of o - ts with [f]fluoride using similar reaction conditions to give the desired [f](2s,4r)-4-fgln, 2, in good radiochemical yields (3040%). however, much to our surprise, the radiochemical yield for precursor, 22, gave a lower labeling yield. reagent and conditions : (a) 18-crown-6/khco3/f / dmso, 110 c, 10 min ; (b) 400 l of tfa/100 l of h2so4 concentrated/120 c, 10 min ; (c) 18-crown-6/khco3/f / acn, 80 c, 20 min ; (d) 500 l of tfa/40 c, 8 min. profiles a and b were obtained using a gradient system : gemini 3u c18 150 4.6 mm ; gradient, 1 ml / min ; solvent a : acn, solvent b : 0.1% aqueous formic acid : 03 min 95% b, 311 min 95%5% b, 1119 min 5%95% b, 1921 min 95% b. profiles c and d were obtained using a chiral column and isocratic system : chirex 3126 (d)-penicillamine 250 4.6 mm, 2 mm cuso4 solution, 1 ml / min, column temperature at 30 c. the radioactive peak displayed the same retention time as that of the cold standard under the same hplc conditions. in order to test the specificity of this radiotracer, in vitro cell uptake and inhibition studies were performed in 9l cells. both [f](2s,4r)-4-fpgln, 3, and [f](2s,4s)-4-fpgln, 4, displayed excellent uptake in the 9l tumor cells in vitro. at all time points studied (5 to 120 min) because of this observation, we only used the [f](2s,4s)-4-fpgln, 4, tracer in further investigations on inhibition of cell uptakes and for the in vivo biological studies. the tracer, [f](2s,4s)-4-fpgln, 4, was incubated at 37 c for 30 min with different amino acid transport inhibitors. the results in figure 5 suggested that the system a inhibitor, meaib (n - methyl--aminoisobutyric acid), had no inhibitory effect on the uptake, indicating that the system a amino acid transport was not involved in the uptake of this new tracer. system l inhibitor, bch (2-amino - bicylo[2.2.1 ] heptane-2-carboxylic acid), system asc inhibitor l - serine (l - ser) and system asc (slc1a5), n inhibitor, l - glutamine (l - gln), exhibited similar concentration dependent reduction of cell uptake, thus indicating potential involvement of system l, asc, and n in the uptake (figure 5). in vitro cell uptakes of [f](2s,4r)-4-fpgln, 3, and [f](2s,4s)-4-fpgln, 4. all radiotracers were evaluated in the 9l tumor cell line. in vitro cell uptake inhibition studies of [f](2s,4s)-4-fpgln, 4, conducted in 9l cells using inhibitors : system lat inhibitor, bch (2-amino - bicylo[2.2.1 ] heptane-2-carboxylic acid) ; system asc inhibitor, l - serine (l - ser), system a inhibitor, n - methyl--aminoisobutyric acid (meaib) ; and system n inhibitor, l - glutamine (l - gln). one of the important issues to consider when developing tracers to image glutamine metabolism in tumors is the protein incorporation of the tracer once inside the cells. after incubation of [f](2s,4s)-4-fpgln, 4, and [h]gln with 9l tumor cells, the cell lysates were treated with tca and the radioactivity in the precipitates and supernatant were counted. results showed that the majority of [h]gln activity was associated with the tca precipitates suggesting that most of the [h]gln (> 90%) was incorporated into macromolecules, whereas the glutamine analog, [f](2s,4s)-4-fpgln, 4, remained predominantly in the supernatant (no incorporation). incorporation of [f](2s,4s)-4-fpgln, 4, and [h]gln into protein in 9l tumor cells was investigated. the comparison of cellular uptake of [f](2s,4s)-4-fpgln, 4, and [h]gln was performed using dual - isotope experiments at 0, 5, 30, 60, and 120 min incubation time periods. cell lysates were treated with tca and the radioactivity (both f and h) associated with the tca precipitates was counted. on the basis of the protein incorporation data above (figure 6), [f](2s,4s)-4-fpgln, 4, behaved very differently from that of [h]gln. it is reasonable to conclude that the new probe, [f](2s,4s)-4-fpgln, 4, is not associated with intracellular macromolecules, and thus, it is less likely to measure the intracellular metabolism associated with glutamine metabolism. biodistribution studies of [f](2s,4s)-4-fpgln, 4, were conducted in f344 rats (125149 g, n = 4) bearing 9l tumors on their thigh. this is a well - established animal model that resembles typical human glioblastomas in clinical settings. rats were sacrificed at 30 and 60 min postinjection by cardiac excision while under isoflurane anesthesia. [f](2s,4s)-4-fpgln, 4, showed respectable uptake within the 9l tumors, displaying 0.83% dose / g uptake at 30 min post injection. tumor uptake and retention slowly washed out of the 9l tumor to 0.60% dose / g. at 30 min, tumor - to - background (tumor - to - muscle, tumor - to - blood, and tumor - to - brain) ratios of [f](2s,4s)-4-fpgln, 4, were 6.91, 1.45, and 5.53, respectively. high pancreatic uptake is consistent with the fact that amino acids are precursors for digestive enzymes actively produced in the pancreas. low bone (femur) uptake was observed at 30 min (0.53% dose / g) and it stayed at that value at 60 min post injection. representative pet images of 9l tumor bearing rats after intravenous injection of [f](2s,4s)-4-fpgln, 4, or [f](2s,4r)-4fgln, 2, into a f344 rat bearing a 9l tumor. the images of the transverse, coronal, and sagittal views are from a summed 2 h scan. arrows represent the location of tumors on the hind leg region of the f344 rat. time activity curves of tumor (target) and muscle (background) uptake in f344 rats bearing 9l tumors : (blue) [f](2s,4s)-4-fpgln, 4, and (red) [f](2s,4r)-4-fgln, 2. comparison of ratios of tumor / muscle at different time points (0 to 120 min) for [f](2s,4s)-4-fpgln, 4, and [f](2s,4r)-4-fgln, 2. preliminary pet imaging studies of [f](2s,4s)-4-fpgln, 4, in rats with 9l tumors showed that the probe was clearly taken up by the tumors (n = 3) (figure 7). to further investigate the tumor uptake, dynamic small animal pet studies using one rat bearing two 9l tumors were carried out on two different days with either [f](2s,4s)-4-fpgln, 4, or [f](2s,4r)-4-fgln, 2. the direct comparison study using the same animal can avoid some of the complications related to differences in tumor growth in different animals. [f](2s,4r)-4-fgln, 2, was recently reported as a tumor pet imaging agent for glutaminolysis. pet images of [f](2s,4s)-4-fpgln, 4, and [f](2s,4r)-4-fgln, 2, were selected for visualization (figure 7). as these images demonstrate, the 9l tumors could be visualized with either of the ligands. defluorination / bone uptake was more apparent in the images of [f](2s,4r)-4-fgln, 2, compared to those of [f](2s,4s)-4-fpgln, 4. to assess the in vivo kinetics, region - of - interest analysis was performed (using amide software to generate the time - activity curves ; figures 8 and 9). the kinetic curves confirmed that all the tracers exhibited higher tumor uptake compared to the muscle (background) regions. [f](2s,4s)-4-fpgln, 4, showed a higher tumor - to - muscle ratio than [f]4-fgln. both ligands had rapid tumor uptake and reached their maximum uptake within the first 20 min. tumor uptake for [f]4-fgln remained rather consistent over 2 h, whereas [f](2s,4s)-4-fpgln, 4, displayed a faster tumor washout rate. also noteworthy, ([f](2s,4s)-4-fpgln, 4, showed less defluorination / bone uptake in comparison to that of [f]4-fgln. results of the in vivo pet imaging studies using the 9l tumor model suggested that the [f](2s,4s)-4-fpgln, 4, localized in the 9l tumor as well, if not better, than [f](2s,4r)-4-fgln, 2. glutamine is found circulating in the blood as well as stored in skeletal muscles in high concentrations (0.51 mmol / l). glutamine plays various critical functions : as an energy source and a substrate for dna and protein synthesis, a primary source of fuel for cells lining the inside of the small intestine and rapidly dividing immune cells, and as a regulator of acid base balance by producing ammonium in the kidneys. in the brain, the glutamine glutamate shunt is a critical pathway to control the inhibitory and excitatory neuronal signals. there are three known glutamine transporters, slc1a5 (asct2, km 20 mm), lat1 (slc7a5, na independent), snat (na / neutral transporter). in the context of tumor growth, slc1a5 is the most important glutamine transporter responsible for rapidly growing tumors. in these tumor cells, the expression of slc1a5 is up - regulated. just as fdg - pet is useful for imaging tumors in which the glucose transporter is overexpressed the tracer reported in this project appeared to be more sensitive to the inhibition by lat inhibitor, bch, not glutamine (slc1a5). the relationship between amino acid transporter expression and tumor growth is a rapidly expanding research field. many amino acid derivatives have been reported for imaging tumor growth based on different amino acid transporters, most of which were not designed to measure glutamine metabolism specifically. recently, a detailed study of transport mechanisms of trans-1-amino-3-fluoro[1-c]cyclobutanecarboxylic acid (anti-[c]facbc) has been published. the corresponding [f]facbc is now being tested in humans as a potential prostate tumor imaging agent. it may be desirable to further evaluate the significance of the observation on different levels of inhibition by lat vs slc1a5 subtypes of amino acid transporters. [f](2s,4r)-4-fluoroglutamic acid, bay 858050 has been reported as a tumor imaging agent. in order to improve the in vivo stability and to reduce defluorination in vivo, (4s)-4-(3-[f]fluoropropyl)-l - glutamate (f - fspg, or bay 949392) was also prepared and tested. it was found that f - fspg, a glutamic acid containing a 3-fluoro - propyl substitution group at the c4 position, showed good tumor uptake and reduced in vivo defluorination. in vivo human studies suggest that f - fspg is a tracer useful for assessing system xc (anionic amino acid) transporter activity in tumors with pet. piramal biotechnology is now developing the f - fspg for imaging tumors in which xc transporters are prominently expressed and oxidative stress is up - regulated. the new (2s,4s)-4-fpgln, 4, reported in this paper, is a structural analog of glutamine containing a 3-fluoro - propyl substitution group at the c4 position. the mechanisms of uptake for [f](2s,4s)-4-fpgln, 4, are associated with three main amino acid transporters, slc1a5 (asct2), lat1 (slc7a5, na independent), and snat (na / neutral transporter). based on the inhibition studies, it appears that lat inhibition was the most prominent, suggesting that lat may be a preferred transporter for (2s,4s)-4-fpgln, 4. for [c]gln, 1, and (2s,4r)-4-fgln, 2, the most important amino acid transporter appeared to be slc1a5 (asct2). the in vitro incubation of [f](2s,4s)-4-fpgln, 4, with 9l tumor cells showed a high cell uptake reaching 6% uptake/100 g of protein at 120 min after incubation. under the same incubation conditions, the lysate of the 9l cells showed that a significant portion of the [f](2s,4s)-4-fpgln, 4, inside the cells remained intact as the original chemical species (> 90% showed no metabolic changes). the [h]-gln incubated simultaneously under the same conditions showed substantial incorporation into macromolecules (> 90% activity associated with the tca precipitated fraction). previously, using the same procedure [c]gln, 1, and [f](2s,4r)-4-fgln, 2, also displayed very similar incorporations into the macromolecular fraction as that observed for [h]-gln. the results suggest that [f](2s,4s)-4-fpgln, 4, may be more similar to the neutral lat preferred amino acid analogs, such as o-(2-[f]fluoroethyl)-l - tyrosine (fet) all of these amino acid probes are transported into the cancer cells, without being incorporated into intracellular macromolecules. there are important differences between these seemingly very close glutamine analogs, [f](2s,4r)-4-fgln, 2, and [f](2s,4s)-4-fpgln, 4. further exploration may be needed to clarify the similarities and differences between these probes. for the development of effective probes for studying glutamine metabolism, one should consider more than simple factors, such as tumor cell uptake and in vivo tumor signal localization. it may also be necessary to consider the subsequent intracellular metabolic processes, or the lack thereof. compared to the natural [c]gln, 1, fluorine substituted [f](2s,4r)-4-fgln, 2, or [f](2s,4s)-4-fpgln, 4, may always be suspected of having a modified intracellular metabolism. more studies are necessary to characterize these analogs for studying glutamine metabolism in tumors. the most important difference between [f](2s,4r)-4-fluoroglutamic acid (bay 858050) and [f](2s,4s)-4-(3-fluoropropyl)glutamic acid (f - fspg, or bay 949392) is that f - fspg, displays a slower defluorination rate in vivo. preliminary human studies have demonstrated that the bone marrow uptake in the vertebral region is relatively low. the same scenario may or may not apply to the second pair of glutamine probes (figure 10). we have noticed a reduced bone uptake and good tumor uptake in the rats receiving [f](2s,4s)-4-fpgln, 4, as visualized by pet or by a dissection method. results from both methods suggest a reduced bone uptake (of 4) in rats as compared to the uptake of [f](2s,4r)-4-fgln, 2. chemical structures of analogs of glutamine and glutamic acid derivatives : [f](2s,4r)-4-fluoroglutamine, (2s,4r)-4-fgln, 2, vs [f](2s,4r)-4-fluoroglutamic acid, bay 858050 ; [f](2s,4s)-4-(3-fluoropropyl)glutamine, (2s,4s)-4-fpgln, 4, vs [f](2s,4s)-4-(3-fluoropropyl)glutamic acid, f - fspg, or bay 949392. these two pairs of probes are structurally very similar, containing a c5 amide vs a c5 carboxylic acid group, but the mechanisms of uptake and retention are dramatically different. in summary, a new glutamine analog, [f](2s,4s)-4-fpgln, 4, has shown tumor specific uptake in vitro and in vivo. however, the tumor uptake and retention mechanisms may be significantly different from other glutamine probes, such as [c]gln, 1, and [f](2s,4r)-4-fgln, 2.	although the growth and proliferation of most tumors is fueled by glucose, some tumors are more likely to metabolize glutamine. in particular, tumor cells with the upregulated c - myc gene are generally reprogrammed to utilize glutamine. we have developed new 3-fluoropropyl analogs of glutamine, namely [18f](2s,4r)- and [18f](2s,4s)-4-(3-fluoropropyl)glutamine, 3 and 4, to be used as probes for studying glutamine metabolism in these tumor cells. optically pure isomers labeled with 18f and 19f (2s,4s) and (2s,4r)-4-(3-fluoropropyl)glutamine were synthesized via different routes and isolated in high radiochemical purity (95%). cell uptake studies of both isomers showed that they were taken up efficiently by 9l tumor cells with a steady increase over a time frame of 120 min. at 120 min, their uptake was approximately two times higher than that of l-[3h]glutamine ([3h]gln). these in vitro cell uptake studies suggested that the new probes are potential tumor imaging agents. yet, the lower chemical yield of the precursor for 3, as well as the low radiochemical yield for 3, limits the availability of [18f](2s,4r)-4-(3-fluoropropyl)glutamine, 3. we, therefore, focused on [18f](2s,4s)-4-(3-fluoropropyl)glutamine, 4. the in vitro cell uptake studies suggested that the new probe, [18f](2s,4s)-4-(3-fluoropropyl)glutamine, 4, is most sensitive to the lat transport system, followed by system n and asc transporters. a dual - isotope experiment using l-[3h]glutamine and the new probe showed that the uptake of [3h]gln into 9l cells was highly associated with macromolecules (> 90%), whereas the [18f](2s,4s)-4-(3-fluoropropyl)glutamine, 4, was not (< 10%). this suggests a different mechanism of retention. in vivo pet imaging studies demonstrated tumor - specific uptake in rats bearing 9l xenographs with an excellent tumor to muscle ratio (maximum of 8 at 40 min). [18f](2s,4s)-4-(3-fluoropropyl)glutamine, 4, may be useful for testing tumors that may metabolize glutamine related amino acids.
a 10-month - old female infant was referred to our urology department for unresolved febrile urinary tract infection (uti) despite parenteral antibiotic management. voiding cystourethrography (vcug) revealed bilateral vur (grade 2 in the right, grade 3 in the left), and a dmsa renal scan demonstrated a focal photon defect on the lateral side of the left kidney. following this episode, she had another febrile uti despite prophylactic oral antibiotics when she was 9 months old. the third febrile uti developed when she was 10 months old, and her fever continued although empirical parenteral antibiotics were given for several days. ultrasonography and vcug revealed a newly developed, large (2210 mm) right ureteral stone at the l4 - 5 level and consequent moderate hydronephroureterosis containing turbid urine (fig. she was referred to our urology department at that time for management of the complicated, large upper ureteral stone. a percutaneous nephrostomy tube was inserted into the dilated pelvocaliceal system of the right kidney for drainage of the turbid urine. after further parenteral antibiotic treatment for 1 week, ureteroscopic surgery for the upper ureteral stone was performed. deflux (oceana therapeutics, inc., edison, usa) injection for correction of the contralateral vur was performed at the same time. no abnormalities were visualized in the bladder, and the bilateral ureteral orifices were wide open. a 0.035 inch guide wire was placed to the level of the right renal pelvis under direct fluoroscopic and endoscopic guidance. a 6/7.5 fr self - dilating ureteroscope was advanced over the guide wire just distal to the ureteral stone, and then the working guide wire was removed. a coil guide wire was passed by the ureteral stone and coiled just proximal to the stone for the prevention of upward stone migration. the stone was yellowish and very large but was easily fragmented with an electro - medical systems lithoclast. the fragmented stones were extracted with a stone basket, and the remaining small fragments were flushed down with normal saline irrigation via the nephrostomy tube. after confirmation of complete stone removal from the right ureter, deflux injection was performed in the left ureter with the hydrodistention implantation technique. a postoperative kidney - ureter - bladder (kub) demonstrated that the stone was completely removed (fig. antegrade pyelography showed a passage disturbance in the right urinary tract with narrowing of the right distal ureter (fig. 2b), so the clamped nephrostomy tube was re - opened and the passage disturbance in the right ureter resolved spontaneously. ultrasonography performed 2 months after surgery showed no hydronephrosis on the right kidney (fig. because urolithiasis is a rare disease in childhood, the management of pediatric urolithiasis is based on treatment regimens originally developed for adults. however, several clinical situations in the management of pediatric urolithiasis differ from those of urolithiasis in adults. in children, there are no clinically insignificant residual stones, and general anesthesia is always required for every procedure. therefore, careful consideration of the characteristics of pediatric patients should be made when designing treatment strategies for pediatric urolithiasis. extracorporeal shockwave lithotripsy (eswl), ureteroscopic stone removal (urs), open surgery, or a combination of these modalities could have been applied in our case. eswl is currently considered a reasonable treatment option for pediatric urolithiasis and has a success rate of 75% to 90% [5 - 7 ]. however, the success rate with a single session of eswl is only 66%. eswl monotherapy for large stones often requires multiple sessions under general anesthesia. on the other hand, ureteroscopic recent studies report that stone clearance after a single ureteroscopic surgery was 100% for stone burdens 10 mm or less and 97% for burdens although the stone in our case had grown to a large size in only 8 months, there is no definite evidence that pediatric urinary stones grow faster than do stones in an adult. during urs in young children, our patient was only 10 months old, but was female and had a moderate grade of bilateral vesicoureteral reflux, which allowed for easier ureteroscopic access. second, the use of urs in this case allowed for simultaneous correction of the contralateral vur. this infant had a history of two previous febrile utis without any evidence of ureteral stones, and the dmsa renal scan performed during the acute infection period showed a photon defect at the lateral portion of the left kidney. the presence of the photon defect suggested that the left kidney was more vulnerable to infection. therefore, it was a reasonable choice to simultaneously correct the left vur in the patient. upon reviewing our case, we learned that the ureteral mucosa of young children is very delicate, so ureteroscopic management can induce transient urinary tract obstruction secondary to postoperative edema, which may be clinically significant. the postoperative urine passage disturbance developed abruptly at 2 days after surgery rather than during the immediate postoperative period, however, so we suggest that it was caused by tiny, remnant stone fragments combined with postoperative mucosal edema. if anti - reflux surgery for the ipsilateral ureter had been simultaneously performed, it might have aggravated the disturbance of urine flow and resulted in prolonged urinary tract obstruction. percutaneous nephrostomy was very useful in our case for the preoperative drainage of retained and turbid urine, for the intraoperative flushing of small stone fragments, and for the postoperative management of the passage disturbance of the urinary tract. however, in our opinion, routine percutaneous nephrostomy is not necessary before urs if the case is not complicated, regardless of stone size. urs is the first - line surgical treatment for a ureteral stone disease in adults without regard to stone location, but ureteroscopic manipulation in infants is limited because of the ureteroscopic access into the small ureter. in our opinion, if an infant has a moderate degree of vur on the same side as a ureteral stone, urs is a feasible and moreover a good surgical modality for the ureteral stone regardless of the location or size of the stone. in the case of a female infant, the minimal likelihood of urethral trauma by the ureteroscope is a further indication for choosing ureteroscopic surgery as the initial surgical treatment method.	pediatric urolithiasis is a relatively rare disease that can have lifelong consequences. the management of pediatric urolithiasis should be individualized with careful consideration of the patients ' small body sizes, delicate tissues, needs for general anesthesia with every procedure, and risks of long - term complications. miniaturization of urological instruments has made the treatment of distal ureteral stones by ureteroscopy in children more common, but there are few reports of the ureteroscopic removal of large upper ureteral stones in infants. we present a case of a 10-month - old female who simultaneously underwent ureteroscopic surgery and endoscopic deflux injection for treatment of a 2210 mm unilateral upper ureteral stone and bilateral vesicoureteral reflux. we also review the current treatment options for pediatric urolithiasis.
most cardiovascular trauma in military conflicts occurs from fragmentary missiles such as artillery, rockets, grenades and bombs. experimental studies of high - velocity wounds show that a temporary cavity can be created nearly 30 times as large as permanent residual tract. it is estimated that 80 - 90% of patients with gunshot wounds of heart do not reach hospital. the standard approach has been to repair the lacerations using mattress sutures while controlling hemorrhage with the finger on heart. emergency temporary control of hemorrhage from cardiac laceration can be achieved with the use of skin stapler. following stabilization of the patient, the staples can be removed after definitive suture repair is performed in operating room. hemostatic sealants such as floseal, etc, offer significant promise as additional tool in the surgical armamentarium when dealing with laceration to the heart. regardless the usual surgical approach to a major vascular injury was simple ligation of the vessel and amputation of limb. during civil war, a policy of mandatory surgical exploration of all potential vascular injuries lowered the amputation rate to 32% for popliteal injuries. a policy of performing arteriography on all suspected vascular injuries combined with evolving vascular repair techniques, during vietnam war, reduced the amputation rate of these injuries to 15%. with current diagnostic and vascular repair techniques, almost all of these injuries can be revascularized, although severely mangled limb may still need amputation. this retrospective study comprised of data acquired from jan 1996 to oct 2008. during this period a total of 386 patients of missile cardiovascular injuries patients who were pronounced dead on arrival to the hospital after missile injuries to precordium and patients with penetrating cardiac injuries or vascular injuries due to causes other than missile injuries were excluded from the study. the patients of cardiac injuries were categorized into three groups depending on physiological parameters. group i : (stable) mean blood pressure 622.3sd, alert, no respiratory distress.group ii : (shock) mean blood pressure of 484.4sd, conscious, mild respiratory difficulty.group iii : (agonal) no recordable pressure, semiconscious, gasping. group i : (stable) mean blood pressure 622.3sd, alert, no respiratory distress. group ii : (shock) mean blood pressure of 484.4sd, conscious, mild respiratory difficulty. patients having systolic blood pressure of less than 90 mmhg or mean blood pressure of less than 60 mmhg were categorised as group ii i.e., shock. all these patients were operated in the emergency operation theatre as emergency room thoracotomy is not available in our hospital. patients of vascular injuries were initially resuscitated in emergency reception and were categorised into two groups based on clinical examination : category 1 (hard signs) : include pain, pallor, pulselessness, parasthesias, pulsatile bleeding and a large or expanding hematoma. category 2 (soft signs) : these include a relatively diminished but palpable pulse, a non - expanding hematoma and a peripheral nerve injury. all patients received a third generation cephalosporin and an amino glycoside at induction of anesthesia. we used standard techniques for management of missile vascular injuries i.e., resuscitation with fluids and blood, properly defining the extent of vascular injury, proper control of bleeding using different types of vascular clamps, proper freshening and denuding the edges of injured vessel, tension free repair by using graft whenever needed, small lacerations treated by lateral repair only, bony injury (fractures) taken care of, in the same sitting, fasciotomy was performed as and when needed and systemic anticoagulation using heparin in perioperative period followed by aspirin or clopidogrel. different surgical procedures like direct end - to - end anastomosis, saphenous vein graft interposition and lateral repair were performed for revascularization. heparin was used in perioperative period and later on patients were shifted to oral clopedogrel and aspirin. all fractures were fixed before vascular repair. hospital stay was 12 - 15 days depending upon other comorbid states or other organ injuries. statistical analysis was done using fisher 's exact test with spss software (spss usa) with 95% confidence interval. twenty - six patients had missile cardiac injury and 360 patients had missile vascular injury. the patients of missile cardiac injury were received in the accident and emergency department between 24 minutes and 8.6 hours after the injury (mean 4.1 hours). there were seven (26.92%) patients in group i, eight (30.76%) in group ii and 11 patients in group iii. eighteen patients (69.23%) underwent left anterior thoracotomy, sternotomy was done in four (15.38%) and in four (15.38%) both thoracotomy and sternotomy was needed. sixteen patients had right ventricular injuries, five had left ventricular injuries, four had right atrial injuries and one had injury in left atrium. clinical status at admission had a bearing on the outcome and the results are demonstrated in the table 1. ten patients (38.46%) in our series had firearm injuries (gunshot injuries) and the remaining 16(61.53%) had pellet or splinter injuries to the heart due to the bomb blasts. five (50%) patients survived in the gunshot group while nine 56.25% survived from the splinter / pellet group. ten patients (38.46%) had associated other organ injury, some having injury at more than one site. the survival in patients with isolated cardiac injury was 61.11%, while it was only 37.50% in those with associated injuries. however, none of the four patients with multiple chamber injury in our series survived. one patient was re - explored for excessive bleeding and a missed right ventricular perforation was repaired. outcome depending upon clinical status complications in resuscitated patients the patients of missile vascular injuries were received in accident and emergency department between 30 minutes to 24 hours after the injury. three hundred and one patients were revascularized within 6 hours of injury (83.61%). popliteal artery was the most common vessel involved (42.50%) followed by brachial artery (23.60%). transection was the most common type of injury followed by laceration (43.88% vs. 40.27%). most of the patients were revascularized by using venous graft or end - to - end anastomosis (52.22% vs. 39.16%). amputation rate was not influenced by type of injury, type of repair or presence of associated venous injury. however, associated skeletal trauma increased the amputation rate [(p value = 0.036) for limb salvage (significant) and (p value=0.65) for death rate (not significant) using fisher 's exact test with spss software with 95% confidence interval ] [table 6 ]. another important factor influencing amputation rate was, delay in revascularization since time of injury. patients who were revascularised after 6 hours of injury had higher amputation rates [(p value=0.045) for limb salvage (significant) and p value=0.061 for death rate (not significant) using fisher 's exact test with spss software with 95% confidence interval ] [table 7 ]. nine patients had severe functional loss because of delayed revascularization after severe trauma to neurovascular bundle. distribution of artery involved distribution of associated venous injuries and their management (n=70) distribution of associated nerve injuries and their management (n=70) effect of associated fracture on outcome on limb salvage outcome as per timing of repair cardiothoracic injury causes 25% of deaths immediately following trauma, and the majority of these fatalities involve cardiac or great vessel injury. penetrating cardiac trauma represents an increasingly important form of trauma due to more use of firearm and bombs in civilian violence. the management of penetrating cardiac injury has evolved from conservative management during world war ii to aggressive early surgical intervention.[68 ] penetrating cardiac trauma presents with two clinical manifestations, exsanguinating hemorrhage or pericardial tamponade. pericardiocentesis in patients with acute tamponade is unreliable and was not used in our series. this is comparable with survival rates reported by others for gunshot and missile cardiac injuries. in our series, survival of patients with pellet splinter injuries to the heart variable mortality rates ranging 12 - 81% have been reported after gunshot wounds to heart by others. in 2006, a retrospective study of penetrating cardiac injury in south africa found 81% mortality among the 21 patients who sustained gunshot wounds to the heart. only one patient in our series who was stable on admission died while eight out of 11 who were admitted in an agonal state (group 111) died. this high mortality of 72.72% in these agonal patients could be due to the lack of facilities for emergency room thoracotomy in our hospital. in the present series, survival was better in patients with isolated cardiac injury as compared to those who had associated injuries (61.11 vs. 37.50%). however, previous studies have shown that risk of mortality was not higher in patients with associated injuries. however, the risk of death was not found to be different between patients sustaining single or multiple cardiac injuries in the series by degiannis. the most commonly involved chamber was found to be the right ventricle in 61.53% of the patients followed by left ventricle, right atrium and left atrium. this is easily explained by the fact that the right ventricle covers the greatest part of the anterior chest wall and represents 55% of the anterior cardiac surface. in a review of 1,802 cases of penetrating cardiac trauma from 20 reports, the right and left ventricles were injured 43% and 33% of the time, respectively. the frequency of involvement of the other chambers is directly proportional to the area of the chest wall they cover. the coronary arteries are reported to be involved in 3.1 - 4.4% of penetrating cardiac injuries.[1416 ] in the present series they were involved in three (11.53%) of the cases. owing to its anatomically anterior placement, the left anterior descending artery is the most frequently involved, being injured in 87.5% of cases of coronary artery lacerations. the right coronary is the second most commonly involved. in the present series, only two patients had main coronary artery laceration both involving the left anterior descending branch. teflon pledgets have been used to assist in anchoring the sutures and prevent sutures from causing further myocardial damage. murphy in 1896 did first successful vascular end - to - end anastomosis in a man. because of improvement in vascular repair techniques, accompanied by substantial progress in anesthesia, blood transfusion and use of antibiotics, improved outcome in vascular injuries was obtained in korean conflict as compared to world war ii. most recent studies have documented the continuous improvement in limb survival after vascular injuries, a reflection of primary repair or interposition graft. a cardinal operative principle in managing vascular trauma is to obtain proximal and distal control of injured vessel before entering surrounding hematoma. in extremities as in neck, control is achieved using standard extensile vascular exposure techniques. although popliteal vein was successfully ligated by many authors,[2326 ] but we advise repair of popliteal vein which enhances the success of arterial repair by relieving acute venous hypertension, compartment syndrome and edema. the significant factor associated with increased limb loss is the time lapse between injury and revascularization.[242733 ] this is because of progression of muscle ischemia and small vessel thrombosis that prevents successful outcome of repair. this have favored the prognosis in our series as well having reduced the amputation rate remarkably to 4.66%. in our study, wound infection was very high because of contamination and improper asepsis at the site of injury, at the time of mass casualties. clinical status at arrival, time interval till management, nature of injury and associated injuries will tell upon the mortality.	background : missile cardiovascular injuries have taken an epidemic proportion in kashmir valley since the eruption of militancy in 1990. present study was undertaken to analyse the pattern, presentation and management of missile cardiovascular injuries.patients and methods : three hundred and eighty - six patients with missile cardiovascular injuries since jan 1996 to oct 2008 were studied retrospectively. all patients of cardiovascular injuries due to causes other than missiles were excluded from the study.results:all patients of missile cardiac injuries were treated by primary cardiorrhaphy. right ventricle was the most commonly affected chamber. left anterior thoracotomy was most common approach used. most of the patients of missile vascular group were treated by reverse saphenous vein graft or end - to - end anastomosis. most common complication was wound infection (20.83%) followed by graft occlusion (1.94%) in missile vascular group. amputation rate was 4.66%. amputation rate was higher in patients with delay of > 6 hours and associated fractures.conclusion:missile cardiac injuries should be operated early without wasting time for investigations. clinical status at arrival, time interval till management, nature of injury and associated injuries, tell upon the mortality. missile vascular injury needs prompt resuscitation and revascularization at the earliest. time interval till revascularization and associated fractures has a bearing on mortality and morbidity.
previous studies have reported relationships between the resting position and the dynamic motion (dyskinesis) of the scapula and shoulder pain and dysfunction1,2,3,4,5. decreased activation of the lower trapezius (lt) muscle and the serratus anterior (sa) muscle together with simultaneous excessive activation of the upper trapezius (ut) muscle some previous studies have reported that scapular muscle exercises positively affect not only shoulder pain but also shoulder function7, 8. restoration of balanced scapular muscle activity is one of the aims of scapular muscle exercises, and of decreased activation of the lt and the sa and increased activation of the ut indicate the importance of the intramuscular balance ratio (ut / lt) and intermuscular balance ratio (ut / sa)6, 9,10,11,12. cools. concluded that four exercises were able to facilitate lt activity with minimal activation of the ut, from the point of view of the scapular muscle balance (fig. ; b : prone horizontal abduction (prohabd) ; c : forward flexion in the side - lying position (sideflex) ; d : side - lying external rotation (sideer) ; e : shoulder flexion with glenohumeral horizontal abduction load (flexband) ; f : shoulder flexion with glenohumeral horizontal adduction load (flexball))13. a previous study reported that the scapular muscle rehabilitation exercises performed by athletes with impingement syndrome improved their pain and function14. all of the exercises are carried out in side - lying or prone postures, even though upper extremity activities of daily living frequently occur in anti - gravity postures such as sitting or standing. to the best of our knowledge, few studies have examined scapular muscle exercises in sitting or standing postures which aim to elicit balanced scapular muscle activity. a high level of maximum voluntary isometric contraction in the lt muscle was reported for exercises including scapular retraction motion15. sa muscle activity has been induced not only with shoulder horizontal adduction16, but also with shoulder horizontal abduction in an anti - gravity posture17. taking these previous studies into account, we hypothesized that if glenohumeral flexion with horizontal abduction directional load or with horizontal adduction directional load, both of which induce scapular retractor activity, were peformed, balanced scapular intramuscular and also intermuscular activation would be induced in an anti - gravity posture. thus, the purpose of the current study was to compare the intramuscular and intermuscular scapular muscle ratios (ut / lt, ut / sa) among commonly used scapular muscle exercises carried out in the side - lying and prone postures and flexion with glenohumeral horizontal adduction load or horizontal abduction load in an anti - gravity posture, such as sitting or standing. ; b : prone horizontal abduction (prohabd) ; c : forward flexion in the side - lying position (sideflex) ; d : side - lying external rotation (sideer) ; e : shoulder flexion with glenohumeral horizontal abduction load (flexband) ; f : shoulder flexion with glenohumeral horizontal adduction load (flexball) sixteen healthy males volunteered for this study (mean age ; 21.9 years 0.83 years). exclusion criteria included shoulder pain and an orthopedic and/or a neurological history of the shoulder. all participants read and signed an informed consent form prior to their inclusion in this study. this study was approved by the institutional review board of the faculty of health sciences, hokkaido university (i d : 13 - 76). surface electromyographic (emg) activities of the ut, lt and sa muscle were measured. before placing the surface electrodes, the skin was cleaned with alcohol on a cotton swab in order to get a good electrode - skin contact. bipolar surface electrodes (blue sensor p-00-s, ambu, denmark) were placed over the ut and the lt muscles and the lower region of the sa muscle with a 2 cm inter - electrode distance. placement of the electrodes for the ut and the lt followed seniam18, and the electrode placement for the sa followed the descriptions of previous studies13, 16. all of the electrodes were connected to a myosystem 1,200 electromyographic receiver (noraxon usa inc., unit specifications include a differential input impedance of greater than 10 m, a gain of 1000, and a common mode rejection ratio of greater than 100 db at 60 hz baseline noise was filtered with a band - pass filter of 10500 hz. before performing the series of the scapular muscle activation tasks, the emg signal quality was verified for each muscle during the performance of the maximal voluntary isometric contraction (mvic) tests specified for each muscle of interest as described by seniam and previous studies13, 16. during the mvic tasks, subjects performed and held each posture for 5 seconds against manual resistance and each trial was repeated three times. after signal filtering with a sixth order butterworth 6 hz low - pass filter, emg value of the middle one - second window of the 5 seconds was averaged for each trial. each participant performed the four exercises, which were selected as exercises representative of those used for promoting balanced scapular muscle activity. they were prone extension (proext), prone horizontal abduction with external rotation (prohabd), forward flexion in the side - lying position (sideflex), and side - lying with external rotation (sideer) (fig. d). in addition, shoulder flexion with glenohumeral horizontal abduction load (flexband) and horizontal adduction load (flexball) in standing posture (fig. the order of the tasks was randomized to avoid the effects of muscle fatigue. as in the previous study by cools.13, all exercises, except for those performed in the side - lying posture, were performed bilaterally. before data collection, the participants performed the exercises without resistance to familiarize themselves with their execution. the subjects held 1 kg dumbbells in both hands, and completed 3 trials of each task. between trials, the participants had 30 seconds rest, and between exercises, they took a 1 minute rest to avoid muscle fatigue. during the exercises, when necessary, performance correction was verbally given to the subjects by the examiner (m. h). all raw emg signals were transferred to a windows computer through a usb analog / digital (a / d) converter at 1,000 hz and a 16 bit a / d board. they were full - wave rectified and filtered with a 6th order butterworth 6 hz low - pass filter. for each phase, the data were averaged across the middle 3 seconds of the 5 seconds. the mean emg data, expressed as a % mvic, were used to assess the activities of the ut, lt and sa muscle in each exercise. in order to assess intermuscle and intramuscle balance of the scapular muscle, the ut / lt ratio and the ut / sa ratio were calculated. one - way repeated analysis of variance (anova), and the tukey hsd test as a post hoc test, were conducted for the comparison of data across the exercises in each phase. the statistical analysis was performed using the ibm spss statistics 18 software program (ibm, chicago, il, usa). in addition, following the study of cools.13, the exercises were divided into 3 subgroups based on the ratios of 100 to 80% (moderate), 80 to 60% (good), and 0.05 among all exercises), whereas the flexball exercise demonstrated a significantly greater ut / lt ratio than those of the all other exercises in the concentric, isometric and eccentric phases (p<0.05, respectively). : significantly greater than the other exercises as the result of the anova, significant differences were found in the ut / sa ratio (table 2table 2. the ut / sa ratios of all the exercisesproextprohabdsideflexsideerflexloopflexballconcentric265.8313.1%714.3596.0%244.1644.9%431.9660.9%81.855.6%46.132.7%isometric380.8534.5%1,335.71,038.9%93.6103.1%358.7532.9%67.441.7%56.129.5%eccentric338.3436.4%1,100.2939.1%134.4121.3%372.6567.3%103.776.4%49.630.2% : significant differences between all exercises except the flexball exercise. : significant differences between all exercises. : significant difference from the prone horizontal abduction exercise) in each phase (p<0.001). the prohabd exercise demonstrated a significantly greater ut / sa ratio than the other exercises in each phase (p<0.001). the sideer exercise demonstrated a significantly greater ut / sa ratio than the other exercises in all phases (p<0.05), except the flexball exercise in the concentric phase. the ut / sa ratio of the flexball exercise was significantly smaller than those of the prohabd and the sideer exercises in the concentric phase (p<0.05). : significant differences between all exercises except the flexball exercise. : significant differences between all exercises. : significant difference from the prone horizontal abduction exercise according to the described above classification, the ut / sa ratio of the flexball exercise belonged to category one, whereas the ut / lt ratio of the flexband and the flexball exercises belonged to category four. the purpose of the present study was to compare the ut / lt ratio and the ut / sa ratio among commonly used scapular muscle exercises which are carried out in side - lying and prone postures and flexion with glenohumeral horizontal abduction or horizontal adduction load in an anti - gravity posture. to the best of our knowledge, this is the first study to have investigated a strategy that induces balanced scapular muscle activity in anti - gravity postures such as sitting and standing. in our study, when the intramuscular and intermuscular activity ratios of the exercises in an anti - gravity posture were significantly lower than or similar to the exercises in the side - lying and prone postures, we concluded that the exercises in the anti - gravity posture had induced balanced scapular muscle activity. our study revealed that the ut / lt ratio of the flexband exercise was not significantly different from those of the four exercises performed in the side - lying and prone postures in each phase. because horizontal abduction load elicits lower trapezius muscle activity in the flexband exercise, the flexband exercise induced balanced intra - scapular muscle activity. however, the flexball exercise elicited a significantly greater ut / lt ratio than the other exercises in each phase. based on these results, we recommend that the flexband exercise be used to enhance balanced muscle activity between the ut and lt muscles. a previous study by cools. reported that the proext, prohabd, sideflex, and sideer exercises were not suitable for inducing balanced ut / sa activity13. our present results indicate that the prohabd exercise elicited a significantly greater ut / sa ratio than the other exercises in all the phases, while the flexball exercise demonstrated a significantly smaller ut / sa ratio than the prohabd exercise in the concentric phase. however, under the classification criteria, the flexball exercise belonged to category one. therefore, we propose that the flexball exercise might induce balanced ut / sa activity in an anti - gravity posture. first, during the flexball exercise, the participants held a ball and dumbbells, and this might have changed muscle activity.	[purpose ] the purpose of this study was to compare the intramuscular balance ratios of the upper trapezius muscle (ut) and the lower trapezius muscle (lt), and the intermuscular balance ratios of the ut and the serratus anterior muscle (sa) among prone extension (proext), prone horizontal abduction with external rotation (prohabd), forward flexion in the side - lying position (sideflex), side - lying external rotation (sideer), shoulder flexion with glenohumeral horizontal abduction load (flexband), and shoulder flexion with glenohumeral horizontal adduction load (flexball) in the standing posture. [methods ] the electromyographic (emg) activities of the ut, lt and sa were measured during the tasks. the percentage of maximum voluntary isometric contraction (% mvic) was calculated for each muscle, and the ut / lt ratios and the ut / sa ratios were compared among the tasks. [results ] the ut / lt ratio with the flexband was not significantly different from those of the four exercises in the side - lying and prone postures. the ut / sa ratio with the flexball demonstrated appropriate balanced activity. [conclusion ] in an anti - gravity posture, we recommend the flexband and the flexball for inducing balanced ut / lt and ut / sa ratios, respectively.
treatment protocols for such complication are debatable, however, surgical intervene have been advocated. 14 years old male presented with pain and redness around the knee with no history of injury. among all skeletal tumors, osteochondroma constitutes about 10 - 15 % non - traumatic fracture of osteochondroma through the stalk is a known, but rare complication. a 14 years old male patient presented to our outpatient department with pain, redness, and swelling over distal and posterolateral aspect of left thigh with 4 days. on history, taking patient stated that he had a gradual progressive painless mild swelling over that area for 6 month. for 4 days patient increased pain, redness, along with increments in size of previous. he did not reveal episode of recent significant trauma. on physical examination, there was tender, palpable mild swelling over posteromedial aspect of distal thigh. on radiographic examination, it showed fracture through base of pedunculated osteochondroma, which was situated in distal left thigh posteromedially (fig-1). on meticulous questioning patient gave the history of mild intermittent pain over osteochondroma site after moderate to severe exertion. showing fracture from base of pedunculated osteochondroma for excision posterolatearl approach was used, vastus lateralis was reflected anteriorly and plane was developed between it and lateral intermuscular septum covering hastring muscles (internervous plane of femoral and sciatic nerve) to reach the base of growth. with the help of chisel, extra - periosteal excision was done (with cartilaginous cap) and raw surface filed (fig-2) and confirmation was done after histopathological examination. recuperating period was uneventful and patient resumed pre - disease activity status after two week. fracture, bursa formation, malignant transition and neurovascular compromise are mentioned complications of osteochondroma which entail it for being symptomatic [5, 6 ]. malignant transition in solitary lesion of osteochondroma is very less (1%) in comparison to autosomal dominant hereditary multiple exostoses, in which it may be up to 5% [1, 8, 9, 10 ]. usually pathognonomonic radiological features are sufficient for delineating it, and ct scan is further needed for diagnostic confirmation and pre - op management. it also measures the cartilaginous cap, which (1.5 cm) may be the herald of malignant changes [1, 2, 11 ]. biopsy usually not used, until other diagnostic tools fail, due to chance of spreading. commonest site of fracture of osteochondroma is around knee [1, 2, 12, 13 ]. only few cases of non - traumatic fracture of pedunculated osteochondroma has been reported [4, 7, 13, 16, 17 ]. growth of stalked osteochondroma leads to weakness at its stalk, so the violent muscle contraction or relative friction of muscles may cause the fracture and pain in the absence of history of trauma [14, 15 ]. our patient did not give the history of significant trauma so indirect muscle contraction was the probable cause of fracture. observation and conservative management has also been suggested for such a rare complication, but surgery gives quicker recovery and return to normalcy [2, 7 ]. in our case report, it was also true in which patient got full recovery in two week. in conclusion, it was an uncommon type of presentation of non - traumatic fracture of pedunculated osteochondroma. strong muscle contractions are postulated to cause this complication and surgical excision provides prompt relief.	introduction : non - traumatic fracture of pedunculated osteochondroma is a known, but rare complication. treatment protocols for such complication are debatable, however, surgical intervene have been advocated.case report:14 years old male presented with pain and redness around the knee with no history of injury. radiograph showed fracture of solitary osteochondroma of femur. excision was done through posterolateral approach and confirmed with histopathology. patient returned to activities at 2 weeks post surgeryconclusion : atraumatic fractures may occur due to strong muscular contractions in cases of pedunculated osteochondroma. surgical excision gives prompt relief from symtoms
injury to the central nervous system (cns) is devastating for the patient and in general is irreversible. strategies to repair the damaged cns are complex and challenging, and it is thought that the best approach is to use a combination of treatments including cell transplantation and pharmacological treatments. one of these includes the use of biodegradable bridges or scaffolds to bridge the lesion and encourage neurons and glial cells to cross into the noninjured tissue. however, to assess if these scaffold prototypes can promote the survival and differentiation of cns cells across a lesion is difficult as they would require in vivo models. therefore, the use of cell cultures that mimic the target cns tissue would benefit the development of potential scaffolds. the major strength of cell culture compared to in vivo work is their simplicity and accessibility. for example, cultures allow the study of many parameters over a relatively short period of time but in general can not replicate the complex architecture and local environment of endogenous tissue. however, with the advancement of three - dimensional (3d) culture systems intending to mimic tissue architecture and specific organs or tissues, e.g., bone, or even to mimic critical systems of an entire organism, e.g., human on a chip, the development and testing of increasingly complex cultures can mimic aspects of animal models and be used as a pre - test on potential scaffolds before use in vivo. previously, we have developed a method of fabricating tubular constructs (coined swiss - roll ; figure 1a) with potential applications in vascular and nerve tissue engineering. the swiss - rolls were made of a thin (1.5 cm) chambers. moreover, viable cells tended to concentrate at the open edges of the longer chambers, while being distributed uniformly in the shorter (1 cm) chambers. (a, b) representative images of mtt stained astrocytes at 21 div within mini - chambers of different lengths (13 cm), showing along the top the different positions of the 0.33 cm wide analysis windows (0 at the center, maximum ; four at the periphery) and below their corresponding graph symbols in b. (b) graph showing the density of mtt stained astrocytes within each analysis window along the mini - chambers. (c) representative images of mtt stained astrocytes at 21 div in 3 cm long mini - chambers with between 1 (i) and a maximum of 6 (vi) porous lids and a nonporous lid (c). (d) graph of mtt density correlates directly to that of viable cells within the mini - chambers shown in (c). all results shown (b, d) are the mean sd (n = 3). porous pcl lids were fabricated to simulate swiss - rolls with different thicknesses. mini - chambers (1 cm wide and 3.0 cm long) with 1 layer of flat pcl as a lid were used as the controls and stained with mtt. astrocytes (0.5 10 cells / ml) were seeded into these chambers, cultured for 3 weeks, and then stained with mtt. as shown in figure 2c, there were significantly more viable cells in the chamber with porous lid(s) than in the control chambers. however, much less viable cells were detected within the chambers with 6 layers of porous lids compared with the chambers with 13 layers of porous lids, suggesting that scaffold thickness will have a significant influence on cell survival within the swiss - rolls. these results demonstrated that open pores are an effective approach to circumvent the limitation of mass transfer. however, its efficiency was compromised if multiple layers were used in 3d scaffolds. thus, other approaches such as perfusion culture might be necessary to enable better mass transfer especially within thick or multilayered scaffolds. as demonstrated by the mini - chambers containing only cortical astrocytes, culture viability and cell density of the mixed myelinating cultures were affected by the length of the mini - chamber, with only mini - chambers of 0.5 cm length demonstrating viable cultures at 28 days (data not shown). in mini - chambers with a nonporous pcl lid, there was no cell survival whatsoever in chambers beyond 1 cm in length (figure 3a) and very poor viability (max 10% neurite density in any region) in mini - chambers of 1 cm in length. however, the inclusion of pores into the mini - chambers pcl lid enabled the survival of a viable myelinating culture in scaffolds of up to 2.5 cm in length (figure 3b). unlike that seen for single cell cultures in mini - chambers with nonporous lids, the neurite density was comparable throughout the length of the mini - chamber, with no preference for either edge (figure 3c). (a) representative images of a myelinating culture at different locations inside a 2.5 cm mini - chamber possessing a nonporous lid showing poor viability of cultures. (b) representative images of a myelinating culture inside a 2.5 cm mini - chamber possessing a porous lid at different locations. (c) graph showing mean neurite density (smi-31 immunoreactivity), an indicator of myelinating culture viability, in each location of the 2.5 cm long mini - chamber possessing a porous lid (gray bars) and nonporous lid (black bars). all images are at 28 d.i.v., and the graph is representative of this time point myelinating cultures were seeded into mini - chambers of 0.5 cm length, with porous or nonporous lids, and maintained for at least 28 days in vitro (d.i.v.). the inclusion of pores in the lids, although not essential for the survival of the myelinating cultures, with a comparable neurite density observed between the two conditions (figure 4c) demonstrated a significant enhancement of the levels of myelination (t test, n = 3, p < 0.05) compared with that of mini - chambers of possessing nonporous lids (figure 4d). in cultures expanded beyond 28 d.i.v. this difference was still detectable (figure 4a, b) (a, b) representative images of myelinating cultures within a 0.5 cm long mini - chamber at both 28 d.i.v. and 46 d.i.v. (a) with a nonporous lid and (b) with a porous lid. all images were labeled for smi-31 (red) and plp (green). graphs show neurite density (c) and percentage of myelination (d) for the myelinating cultures at 28 d.i.v. mini - chambers with grooved / pillared bases, flat pcl lids, and defined size (1 cm wide, 2.5 cm long, and 70 m high) were divided into 4 groups for the different angles. after seeding with fibroblasts / astrocytes (0.5 10 cells / ml), they were immediately placed in 6-well plates and rotated to different angles (0, 45, 90, and 180). after incubation at 37 c and 95% air/5% co2 for 1 h, the cells were fixed, stained with coomassie blue, and imaged to analyze cell distribution. as shown in figure 5a, when the chambers were placed horizontally (0 or 180), cells were always detected on the substrates that faced upward even though the gap between the two substrates was only about 75 m. when the chambers were rotated to 45, cells adhered all over the upward facing side, with slightly more cells toward the lower part of the mini - chamber. more cells were also observed on the sides of the pillars facing up, while microgrooves had no obvious influence on cell distributon (figure 5b). when chambers were rotated to 90, most of the cells could be found concentrated in the lower part of the mini - chamber, with very few cells in the middle part, and almost no cells could be observed in the upper part of the mini - chamber. more cells were again detected on the sides of the pillars that faced up, and microgrooves had no obvious influence on cell distributions. (a) coomassie blue stained cultures illustrating the influence of gravity on the distribution of fibroblasts within horizontally (0 or 180) placed mini - chambers, 1 h after seeding. (b) coomassie blue stained cultures illustrating the influence of gravity on the distribution of fibroblasts within tilted (45, 90) mini - chambers, 1 h after seeding. (c) the influence of fluid flow, microstructure, chamber orientation, and gravity on the distribution of cells within mini - chambers tilted at 45 or 90 over time. the black arrows indicate the direction of gravity (g), and the white arrow indicates the direction of fluid flow (f). the micrographs to the right in the same row were taken at different time points (0, 10, 20, and 40 min). the schematic diagram of the chamber cross - section to the left in each row illustrates the mini - chamber orientation, and the gray frame indicates at which layer the micrograph to the right in the same row was taken after the cells were introduced. scale bar : 100 m. to investigate how fluid flow influences cell distribution and how it interacts with gravitation, fibroblasts (2 10 cells / ml) were seeded into tube - connected chambers (1 cm wide and 2.5 cm long) on a tilted (45 or 90) time - lapse microscope and videoed. as shown in figure 5c, when the chambers were placed vertically (90) most of the cells were observed to quickly flow toward the lower part of the mini - chamber. within 20 min after cell seeding, very few cells remained attached to the pillars, and those which did only attached to the upper sides (with respect to flow and gravity). when the chambers were rotated to 45, some cells were still observed to flow downward, but most attached to the base within 10 min. compared with microgrooves, micropillars also influenced cell distribution as a substantial number of cells adhered to the upper - sides of the pillars. however, when the long axis of the micropillars was placed in line with respect to fluid flow and gravity, the pillars influence on cell distribution was reduced significantly. in this study, novel scaled down mini - chambers were developed to evaluate how cells interact with our previously fabricated tubular scaffold, with several important cell responses identified and tested. it is accepted that a major problem with 3d culture systems is the survival of cells within the culture itself, with oxygen and nutrient depletion leading to a proportional necrosis of the cells, based upon their distance from the edge of the 3d system. the mini - chambers, due to their configuration, also demonstrated this effect on cell survival in the absence of a porous lid, as demonstrated by the variable cell density in mini - chambers of different length, with the preference of cells to be located in areas proximal to the edges. this effect is even more extreme when a complex cns culture is added, with the entirety of cells in the culture consistently dying out in mini - chambers over 0.5 cm in length. this is most likely due to the limitations of mass transfer within the mini - chambers, which we investigated by observing trypan blue diffusion (data not shown), where the dye was present in the longer mini - chambers for up to a week after media change. this limitation could be overcome by the inclusion of pores within the mini - chamber lid with significantly greater cell survival in mini - chambers of all lengths compared with mini - chambers lacking a porous lid. again, when a complex cns culture was present, the relevance of the effects of the pores was even more apparent, with a consistent neurite density extending throughout the entire mini - chamber, a feature of importance for the tubular scaffold s primary purpose as a conduit for axonal growth through cns injury sites. the inclusion of pores also had an effect beyond simply enhancing the survival of the cells within the mini - chambers, with a significant enhancement of myelination within the myelinating cultures seeded into mini - chambers with porous lids. however, the neurite density between the two cultures was comparable, and cultures could be maintained in mini - chambers with both porous and nonporous lids for extended culture periods (46 d.i.v.) with no loss of neurite density. this suggests that the nutrient transfer permitted by the pores is important for critical processes undergone by the cells within the mini - chambers and, by extrapolation the tubular scaffold, by suggesting that different processes have different thresholds of nutrient requirements. the seeding experiments with the different angles of the mini - chambers suggested that the influence of gravity will play a critical role in the seeding of the cells into the tubular scaffold, which would possess a surface angled at every degree of rotation. the results illustrated the influences that chamber rotation, 3d configuration, micropillars, and gravity had on the distribution of the cells within mini - chambers or indeed swiss - rolls. all of these observations demonstrated that the distribution of cells freshly introduced into the tubular 3d scaffolds was influenced by various factors including gravity, scaffold configuration, larger it was almost impossible to achieve uniform cell distribution by simply loading the tubular construct with a cell suspension. as gravity, scaffold orientation, and configuration were demonstrated to be the main causes for heterogeneous cell distribution, approaches to circumvent their influences might be necessary to achieve uniform cell distribution. in summary, the mini - chambers demonstrated the capacity to support complex cultures of cells while isolating individual components of a complex 3d scaffold design in an environment where they are more accessible for analysis. as a simple 3d model, the mini - chambers displayed various technical and operational advantages : first of all, they are cost - effective. multiple chambers can be employed for intensive in parallel experiments, as they are cheap and easy to fabricate. mini - chambers with different sizes, multiple lids, and a variety of chemical, micro / nanotopographic features can be designed and fabricated enabling the simulation of more complex 3d scaffolds with different lengths, thicknesses, and complex internal features. cells can be easily seeded using a micropipet or injected using an attached tube into the chambers, which will be rotated to different angles within petri dishes or 6-well plates to simulate various configurations and orientations of a 3d scaffold during cell culture. because of the adaptation of two layers, various behaviors of living cells can be investigated in situ ; as the chambers can be integrated with light microscopy, it is possible to directly observe during culture. moreover these mini - chambers charged with complex neural cells represent a simplified version of the 3d environment, as well as the cells that would be involved in cns repair and encountered in vivo. thus, these devices allow the in - depth study of cellular interactions, drugs, and scaffolds in vitro and limit the necessity for in vivo studies by allowing prescreening of various potentially useful combinations of cells, drugs, and specific scaffold parameters. in addition, they allow the mimicking of effects of nutrient and oxygen deprivation on basic parameters such as cell adhesion, proliferation, and survival. with various scaffold modifications, the cells can have progressively less potential to spread and proliferate with increasing numbers of lids or toward the center of a closed lid chamber. deprivation of these factors also influences the more subtle parameters of axon extension, as well as oligodendrocyte differentiation and myelination. these chambers, therefore, could form a suitable in vitro test system for the influence of drugs targeted at repair under conditions where the access to nutrients is limited over prolonged periods of time (e.g., stroke). the most widely used in vitro model for stroke is oxygen nutrient (glucose) deprivation using a combination of low oxygen tension or an inhibitor of oxidative phosphorylation with desoxyglucose as a replacement for glucose. here, the physical barrier to nutrient and oxygen access, in combination with the volume restriction to a fluid layer of only 50 m in height, which although small compared to tissue culture dishes (mm) is much larger than the submicrometer sized free space between cells in the nervous tissue, is probably essential in contributing to this potential model. the space under the lid (50 m) closely resembles the average distance between capillaries in the cns (40130 m). considering the lactic acid production by, e.g. fibroblasts 40pmol / cell / h, and the volume under the lid over an area of about 14 cells (50 50 m), glucose (5 mm = 0.875 pmol) the ability of the devices to be connected up to, e.g., a syringe pump, would also allow experimenters to mimic the cellular environment in greater detail and investigate the relevance of fluid flow (which in the interstitium is ca. 0.6 m / h) to drug delivery and clearance, as well as to the study of reperfusion injury. the detailed control over the pore size and position would allow local access to nutrients and oxygen as well as the disposal of waste products to be tightly controlled. thus, these mini - chamber cell - charged systems represent a viable, novel, and scale down approach for the evaluation of complex 3d scaffolds, a microbioprocess strategy for tissue engineering, and offer opportunities to study drug testing and therapeutics in a range of models of cns injury and disease.	tubular scaffolds which incorporate a variety of micro- and nanotopographies have a wide application potential in tissue engineering especially for the repair of spinal cord injury (sci). we aim to produce metabolically active differentiated tissues within such tubes, as it is crucially important to evaluate the biological performance of the three - dimensional (3d) scaffold and optimize the bioprocesses for tissue culture. because of the complex 3d configuration and the presence of various topographies, it is rarely possible to observe and analyze cells within such scaffolds in situ. thus, we aim to develop scaled down mini - chambers as simplified in vitro simulation systems, to bridge the gap between two - dimensional (2d) cell cultures on structured substrates and three - dimensional (3d) tissue culture. the mini - chambers were manipulated to systematically simulate and evaluate the influences of gravity, topography, fluid flow, and scaffold dimension on three exemplary cell models that play a role in cns repair (i.e., cortical astrocytes, fibroblasts, and myelinating cultures) within a tubular scaffold created by rolling up a microstructured membrane. since we use cns myelinating cultures, we can confirm that the scaffold does not affect neural cell differentiation. it was found that heterogeneous cell distribution within the tubular constructs was caused by a combination of gravity, fluid flow, topography, and scaffold configuration, while cell survival was influenced by scaffold length, porosity, and thickness. this research demonstrates that the mini - chambers represent a viable, novel, scale down approach for the evaluation of complex 3d scaffolds as well as providing a microbioprocessing strategy for tissue engineering and the potential repair of sci.
a vast amount of literature is devoted to the investigation of the early stages of malignization, especially of the covering epithelium. the literature deals with the problems of viral and chemical carcinogenesis, and in recent decades the genetic and molecular disturbances in epithelial cells. we do not wish to diminish the importance of the above - mentioned factors in the etiology of cancer but, as pathologists, we have to acknowledge the existence of serious gaps in our understanding of the pathogenesis of malignant growth. in fact, one of the most reasonable questions that arises in the process of assessing different factors responsible for malignization of the cell is : how can we explain the existence of different rates of mutation penetrance, the capacity to eliminate oncogenic viruses from cells and other facts which favor the resistance of cells to carcinogenic stimuli ? the latter is probably the result of coaction of the adaptational mechanisms, both of intracellular structural and functional type, and external environmental ones. one of the most important environmental factors is vascular microcirculation under normal circumstances as a part of the protective mechanisms in the process of malignization, which unlike the phenomenon of angiogenesis in the stroma of tumors [1, 2 ] has not yet been investigated. the process of induction of angiogenesis in the stroma of tumors starts at relatively late stages of oncogenesis. information about the possible events which occur in the vascular network at earlier, precancerous stages is practically absent. nevertheless, several preliminary communications have been published in which the authors have persuasively demonstrated the occurrence of blood vessels in the covering of various types of epithelium in normal and different pathological entities, mainly of a non - neoplastic nature, although predominantly in animals. in many species of the animal world, for instance, the epidermis of amphibians contains many capillaries, especially in the foci rich with keratinocytes. many blood vessels are surrounded by connective tissue in the pseudo - multilayered ciliary epithelium of the oral mucosa of the cane toad. the vomeronasal neuroepithelium is also vascularized in rats, and intraepithelial blood vessels can be detected in the nasal mucosa of dogs. the same authors supposed that the penetration of blood vessels into rat retina induces polarization of the cells adjacent to the capillaries. it is of interest to note that the appearance of capillaries in the surface epithelium of several organs is found to be a routine event at specific stages of embryogenesis. observed intraepithelial capillaries in the neuroepithelium of human embryos at 1224 weeks of gestation, although they gradually disappear in later stages. as far as human pathology is concerned, the phenomenon of blood vessel penetration into the covering epithelium is well documented in the esophageal wall of patients with portal hypertension, in vulvar dermatosis, and in pharyngeal tonsils. in the pterygial epithelium, seifert and sekundo found capillaries with perivascular connective tissue in 11 out of 26 patients, and interpreted the findings as a reaction to hypoxia or deficiency of any other substances transported via the bloodstream. the authors hypothesized that the fibroblasts may contribute to the pathological dedifferentiation of the conjunctival epithelium. the interpretation of the nature of the vascular network in epithelium is not always easy. there are little data on the presence of lymphatic vessels at preinvasive stages of oncogenesis [1517 ]. some authors have documented the growth of lymph vessels in the conjunctiva at the preinvasive stage of melanoma. the study involved the investigation of histologic slides of 310 patients with tongue, cervical, laryngeal (all locations), esophageal (all cases : middle thoracic part), lung, penile, vulvar and vaginal squamous cell carcinoma and transitional cell carcinoma of the bladder (table 1). all patients received some type of adjuvant therapy ; the majority chemo- and radiotherapy with a mean interval of 23 months before surgery. eleven patients with penile carcinoma had phimosis : one of whom had undergone surgical removal 20 years earlier at the age of 21. many cervical carcinomas were diagnosed after repeated biopsies.table 1characteristics of the clinical materialno.sitenumber of casesgenderage (range)age (modal)mf1tongue352213258150692larynx21174418050593esophagus33276297550704penis26399050705cervix49277130496vagina340, 52, 647vulva46348260808lung53458419050699bladder4438641885069 characteristics of the clinical material in addition to the above - mentioned material, we also investigated the tissues of 50 embryos and fetuses of 840 weeks of gestation. all tumors except bladder three patients had foci of epidermization, 9 had poorly differentiated transitional cell carcinoma, and 10 had non - invasive carcinoma. as a control, we investigated 180 cases of corresponding healthy organs (20 cases each) removed during surgical interventions for non - neoplastic pathology, traumas. slides, 5 m thick, of formalin - fixed, paraffin - embedded material were used for the study. taking into consideration the possibility of artificial results obtained on the tangentially cut slides, very often we performed additional serial cuts. in some cases, we even dismounted the specimen, re - embedded it perpendicular to the initial position axis and prepared new set of slides. in addition to traditional staining methods (h&e, van - gieson), immunohistochemical stainings with cd31 (clone bc2, biocare, dilution 1:700), cd34 (clone qbnd/10, dako, diluted 1:700) factor viii (clone rb, biocare dilution 1:700), podoplanin (d2 - 40, dako, dilution 1:350) were performed for identification of the blood and lymphatic vessels. the study of the distribution of blood vessels in patients with squamous carcinoma as well as in healthy persons revealed that the normal peritumoral squamous epithelial covering is always vascularized. capillaries are encountered in the epithelium regardless of its thickness, and may be seen even in very thin linings (fig. a blood capillaries surrounded by stroma in the normal squamous epithelium of the patient with vulvar carcinoma. b successive stages of sequestration of the stroma with blood capillaries in patient with carcinoma of the tongue. original magnification 120. d absence of capillaries in the focus of high - grade dysplasia in patient with esophageal carcinoma. e contrast between vascularized normal squamous epithelium (on the left) and dysplastic epithelium devoid of blood vessels (on the right) in patient with cervical carcinoma. original magnification 160. g schematic representation of the previous (1 g) figure. a blood capillaries surrounded by stroma in the normal squamous epithelium of the patient with vulvar carcinoma. b successive stages of sequestration of the stroma with blood capillaries in patient with carcinoma of the tongue. original magnification 120. d absence of capillaries in the focus of high - grade dysplasia in patient with esophageal carcinoma. e contrast between vascularized normal squamous epithelium (on the left) and dysplastic epithelium devoid of blood vessels (on the right) in patient with cervical carcinoma. 160 throughout the epithelium, the vessels are randomly distributed at all levels, having both vertical and horizontal orientations. reconstruction of the vascularization processes of the squamous epithelial covering on consecutive and serial cuts shows that in fact the process is the changing of the relief of subjacent stroma. the latter penetrates in the form of excrescences and sprouts into the covering epithelium capturing the blood vessels (fig., there is sometimes an impression that the proliferating basal cells of the epithelium invaginate deeper, enfold the underlying stroma and incorporate blood capillaries. so what we perceive on the histological slides as intraepithelial blood vessels is a reflection of alterations on the epithelial all the above - mentioned describes the angiogenic patterns in the covering epithelium without visible signs of precancerous alterations. we found that as soon as dysplastic alterations start, the vascular network undergoes important changes. comparison of normal and dysplastic epithelial vascularization showed that the fully developed, high - grade dysplastic foci do not contain capillaries (fig. 1d). as a result of this lack of blood vessels, dysplastic foci contrast sharply with the neighboring normal epithelium (fig. the disappearance of blood vessels is a gradual process ; their amount decreases as the dysplasia progresses from low to high grades. foci represented by a slight degree of dysplasia still contain capillaries predominantly in the basal layers of the epithelium or even in the upper externally normal layers (fig. no blood vessels were observed in the foci of in situ carcinoma (fig. 1h). in those rare cases where the carcinoma in situ structures were vascularized, either they were indistinguishable from incipient invasive carcinoma, or the capillaries were localized to the uppermost layers of the epithelium and thus may be of a residual nature. all the above findings correspond to the peculiarities of the vascularization of the squamous epithelium. unexpected results were obtained from the analysis of the glandular (respiratory) and transitional types of epithelium. no capillaries were found in the normal respiratory lining epithelium of any cases of squamous cell lung carcinoma regardless of the thickness of the epithelium (fig. 2all slides except immunohistochemical reactions are stained with hematoxylin and eosin. a normal respiratory epithelium in the bronchial lining without blood vessels. b transition from normal respiratory epithelium to the metaplastic squamous epithelium in the bronchial lining. c penetration of capillaries in the metaplastic squamous epithelium in the bronchial wall of the same patient original magnification 200. g blood capillaries in the vagina of the fetus of 24 weeks of gestation. h eosinophilic membranous substance under the glandular lining of the future uterus in embryo of 23 weeks of gestation original magnification 100. the layer of the membranous substance is indicated by arrows all slides except immunohistochemical reactions are stained with hematoxylin and eosin. a normal respiratory epithelium in the bronchial lining without blood vessels. b transition from normal respiratory epithelium to the metaplastic squamous epithelium in the bronchial lining. c penetration of capillaries in the metaplastic squamous epithelium in the bronchial wall of the same patient original magnification 200. g blood capillaries in the vagina of the fetus of 24 weeks of gestation. h eosinophilic membranous substance under the glandular lining of the future uterus in embryo of 23 weeks of gestation original magnification 100. the layer of the membranous substance is indicated by arrows of special interest is the secondary metaplastic epidermization in the glandular epithelium. metaplastic foci showed different rates of vascularization, low in the lung, and in the larynx (fig. 2b, c) but in the penile glands in the surroundings of the cancer the foci of epidermization always had a rich vessel network and were clearly visible within the lining of the same gland. the pattern seen in transitional epithelium of the bladder and transitional cell carcinomas differed from that observed in the squamous and respiratory epithelium. very seldom a rich capillary network may be encountered in the basal layers, mainly in patients with some circulatory disturbances or inflammations. blood vessels do penetrate the transitional epithelial lining of the bladder as carcinogenesis starts, but as a rule the process runs parallel to the growth of exophytic tumors. blood vessels are one of the components of the mesenchymal stalks containing blood capillaries, perivascular stroma with stromal cells, etc. which make up the architectural body for the future exophytic papillary tumors (fig. as far as flat lesions of the bladder are concerned, foci of dysplasia, carcinoma in situ do not contain any capillaries at all (fig. the epithelial coverings of healthy persons in the control group showed the same characteristics as those of cancer patients : squamous epithelium is vascularized, glandular covering is avascular. no parallelism between the amount of blood vessels in the stroma and the corresponding epithelial covering, or emergence of blood capillaries in the metaplastic epidermoid foci was found. to discover any peculiarities of the epithelium vascularization at the frontier of the stroma, we paid attention to the fact that the respiratory epithelium, unlike squamous, is separated from the underlying tissues by broad amorphous eosinophilic material (fig. this material also disappears in the foci of epidermization of the respiratory epithelium in non - cancer patients. in embryos and fetuses, squamous epithelium almost always contains blood vessels, at least in the basal layer of the covering. the existence of blood capillaries may be detected very early at 12 weeks of gestation (in the vaginal wall, skin etc.) glandular epithelium does not contain blood vessels in the gestational period. as in the postnatal period, the embryonal glandular epithelium is linked to the underlining stroma by a thick eosinophilic material under the basal membrane (fig. this is clearly visible in the linings of the female genital tract, with a sharp divide between future utero - cervical, nonvascularized epithelium on underlying thick eosinophilic membranous structures, and the squamous future vaginal epithelium rich in blood capillaries without amorphous material. our results seem to a certain extent obvious, but their association with the processes of carcinogenesis did not attract much attention probably due to the assumption that some of these structures are rather artifacts, although nobody has tried to from whence comes the dynamic changes of blood vessel movement and regular character of these patterns ? coexistence of normal epithelial squamous covering containing blood capillaries and adjacent high - grade dysplasia without blood vessels in the same field of vision can not be the result of tangential cuts or artifacts. data on the antenatal development in human embryos and fetuses show that the penetration of blood vessels (of stromal excrescenses) into the epithelial covering often occurs in several organs, but towards the end of gestation this capacity is restricted, though it may recapitulate in the postnatal period. the appearance of blood capillaries in the developing antenatal organs can not be ascribed to methodological errors either. it is worth mentioning that the reduction of the epithelial vascularization at the end of the antenatal period coincides with the emergence of thick eosinophilic amorphous material under glandular epithelial coverings. it is well known that the respiratory epithelium is pseudo - multilayered, each cell attached to the basal membrane and in direct contact with stromal blood circulation (fig., squamous epithelium is a true multilayered structure, where only basal cells are in contact with the membrane and the cells of the higher layers receive blood nutrients through diffusion ; hence the necessity of developing excrescences, which facilitate the delivering of blood vessels to the superficially located cells of the epithelial covering (fig. reduction of the amount of blood vessels in the squamous epithelium most probably impairs normal metabolism and promotes dysplastic alterations. this may explain one of the most interesting findings the absence of blood vessels in the high - grade dysplastic epithelial layers (fig. 4a, b). in typical cases, this is well demonstrated at the border of normal and dysplastic epithelium. there are two possible explanations : dysplastic epithelium may suppress angiogenesis (stromal proliferation), or primarily the blood supply is reduced, lowering the proper level of nutrition thereby inducing or promoting dysplasia. the latter assumption seems more justified since it is favored by a certain parallelism in the gradual increase of dysplastic alterations and decrease in the rate of vessel density. besides, several well - documented investigations show that hypoxia is one of the most important environmental factors in the process of carcinogenesis. it favors activation of c - myc, vegf receptors and production of biologically active proteins (hif-1, glut-1 and ca9), which promote malignization [2022].fig. a, b in the pseudo - stratified respiratory covering, all cells are attached to the basal membrane and receive equal blood supply. c, d squamous epithelium is a true stratified structure ; only basal cells are attached to the membrane and thus have access to the stromal blood capillaries. the rest of the cells compensate for the lack of sufficient nutrients through the additional close contacts with the intraepithelial blood vessels which accompany the stromal excrescencesfig. 4normal squamous epithelium a is vascularized but the foci of fully developed dysplasia b do not contain blood capillaries at all. the foci of squamous metaplasia in the respiratory epithelium c need transformation of the blood supply network. the amount of blood capillaries invading metaplastic epithelium d, e may vary from case to case and result in different levels of blood flow (arrows blood vessels) schematic representation of the relationship between different types of epithelium and underlying stroma. a, b in the pseudo - stratified respiratory covering, all cells are attached to the basal membrane and receive equal blood supply. c, d squamous epithelium is a true stratified structure ; only basal cells are attached to the membrane and thus have access to the stromal blood capillaries. the rest of the cells compensate for the lack of sufficient nutrients through the additional close contacts with the intraepithelial blood vessels which accompany the stromal excrescences normal squamous epithelium a is vascularized but the foci of fully developed dysplasia b do not contain blood capillaries at all. the foci of squamous metaplasia in the respiratory epithelium c need transformation of the blood supply network. the amount of blood capillaries invading metaplastic epithelium d, e may vary from case to case and result in different levels of blood flow (arrows blood vessels) metaplasia and epidermization of the glandular epithelium change the pattern of blood supply in the metaplastic epithelium towards the reduction of normal metabolism since metaplastic foci generally have a sparse vascular network (fig.. transitional surface epithelium does not contain blood vessels, but as mentioned earlier, the penetration of blood vessels into this type of epithelium runs parallel with the building up of the papillary neoplasms (both benign and malignant) and is accompanied by a pronounced connective tissue component. this type of vascular architecture with marked perivascular stromal cuffs and high capillary density probably guarantees sufficient blood supply to maintain a normal metabolism in the cells of papillary tumors, whereas the metabolism is definitely dysregulated in flat lesions high - grade dysplasia and carcinoma in situ devoid of capillary network. these alternative vascularization patterns of early stage malignancies in the bladder mucosa may determine the morphogenesis of future urothelial tumors. we believe that the specific characteristics of the basal membrane may play an important role in the regulation of the capillary network. this assumption is supported by recent data from several authors who have demonstrated that the chemical composition of the basal membrane, of its various components especially proteins, favors the differentiation of the endothelium, converting the individual endothelial cells into tube - like structures. it seems logical that the different chemical composition of the basal membrane induces different morphogenetic events at the stromal epithelial junction. under normal physiological conditions, each type of epithelium has its own specific form of contact between the cells and subjacent stroma to maintain maximal access to nutrients for all the cellular population. in squamous epithelium, only basal cells are in direct contact with the stroma, the rest of the cells receive blood by the penetration of stromal excrescences upward into the covering. in respiratory epithelial coverings, each cell is in contact with the stroma, and thus has direct access to the blood supply. the boundary between squamous epithelium and stroma becomes flat, the number of stromal ingrowths (resp. blood vessels) is markedly reduced, and as a consequence the epithelial cells suffer ischemia. similarly, the foci of squamous metaplasia in respiratory epithelium lack an adequate capillary network and serve as a substrate of malignization in the initially well - vascularized glandular epithelium. looking to the future epithelial relationship in the process of malignization- (increasing dysplasia of the covering epithelium) and devise therapeutic approaches for the restoration of an adequate vascularization or nutrient supply. in the future, the above - mentioned data may be used in diagnostic processes as well the dynamics of the gradual disappearance of intraepithelial blood capillaries could serve as an indicator of the precise rate of dysplasia and may also serve as an objective differential diagnostic tool in excluding reactive polymorphism in epithelial coverings.	purposeto study the peculiarities of vascularization at the stromal epithelial interface in different types of epithelia and their alterations in precancerous lesions.materials and methodsperitumoral tissues of 310 patients, tissues of 180 healthy persons and of 50 human embryos and fetuses were used. traditional histological as well as immunohistochemical methods have been used.resultsthe study reveals that the occurrence of blood capillaries in surface squamous epithelium is an ordinary event, both in healthy persons and in peritumoral regions of the patients with squamous cell carcinoma. glandular epithelial coverings, as well as transitional epithelium, do not contain blood vessels. in squamous epithelium, only basal cells are in contact with the membrane and underlying stroma, the cells of the upper layer receiving nutrients through diffusion. thus, the cells of squamous epithelium are more vulnerable to blood deficiency, since for instance in the pseudo - multilayered respiratory epithelium each cell is attached directly to the basal membrane and has more ample access to the blood supply. metaplastic squamous epithelium has a markedly reduced vascularization and seems to be more sensitive to carcinogenic stimuli. high - grade dysplastic squamous epithelium and carcinoma in situ do not contain blood vessels.conclusionthe process of redistribution of vascular network occurring at the interface of epithelial stromal frontier plays an important role in maintaining the adequate metabolism of cells including those of epithelial covering. impairment of this mechanism most probably promotes precancerous alterations.
the primary objective of endodontic therapy is to render thorough debridement of the root canal throughout its length to ensure reduced postoperative symptoms, enhanced healing and increased longevity of the endodontically treated tooth. the root canal anatomy often presents obstructions in the coronal one - third, especially in the curved canals, which hampers the smooth access of endodontic instruments, medications, and irrigants to the apical one - third, thus leaving behind a significant residual bacterial load in the canal. use of cervical or coronal preflaring and the crown - down technique removes these obstructions to allow an unimpeded and straighter access of the instruments to the apical third of root canal. straight line access (sla) refers to a preparation path which results in an unobstructed or straight path from the occlusal to the apical end or the first curvature of the canal. this not only reduces unnecessary deflection of the file and allows better operator control over the instrument, but also improves tactile sense, increases the accuracy of working length (wl) determination and aids in adequately achieving the desired apical working width. one of the pioneer instruments used for achieving sla were the gates glidden (gg) drills. these are used sequentially in a circumferential manner, but do not completely remove the cervical interferences, especially in the cementoenamel junction. furthermore, they tend to cut more dentin in the furcal region (danger zone) thus compromising tooth integrity. today, most of the ni - ti rotary systems offer orifice openers and shapers for preflaring of the canals. quite popular among these, the auxiliary shaping ni - ti file protaper sx, offer advantages such as superelasticity, lesser chance of canal transportation and strip perforation when compared to the stainless steel gg drills. more recently, titanium nitride treated, stainless steel la axxess burs (sybronendo, orange, ca, usa), with safe ended tips have been introduced, which promise a safer and more effective preflaring compared to the conventional drills and files. the mesial root of the mandibular first molar often presents with a flat and ribbon - shaped canal anatomy with significant curvature in the mesiobuccal canals, thus making it difficult to negotiate the apical third area. the present study thus aims at cone - beam computed tomography (cbct) evaluation of the ability of gg drills, protaper sx, and la axxess burs to produce an sla in mesiobuccal canals of mandibular first molars. forty - five freshly extracted mandibular teeth with a canal curvature of 10 - 20 (schneider 's method) in the mesial roots and two separate foramina were collected and stored in 5.25% sodium hypochlorite for disinfection. exclusion criteria included teeth with incompletely formed apices, external resorption, or canal calcification. to determine the wl an iso size 10 file teeth were mounted on a clear acrylic block, and standard access cavities were prepared. a 0.5 mm depth groove was prepared on the mesiobuccal cusp tip of the teeth and gutta - percha was condensed in it so that it could serve as a radiopaque marker for a fixed reference point. the teeth were divided into three groups (n = 15) according to the system used for preflaring. group i : size 20/0.06 taper line angle stainless steel la axxess (sybronendo, orange, ca, usa) burs used for preflaring the cervical and middle third of the canal, 3 mm short of wlgroup ii : gg drills (dentsply, maillefer, switzerland) were used middle third preflaring for cervical and at a length till resistance was felt in middle third of canal in a sequence of size 4, 3 and 2 (iso sizes 110, 90, and 70) at a speed of 2500 rpm on a conventional micromotor. the canal was recapitulated with size 15 k file and irrigated with 5.25% naocl after each size changegroup iii : cervical preflaring with protaper sx (dentsply, maillefer, switzerland) 3 mm short of wl after determining canal patency with a size 15 file on an electric endomotor at 300 rpm and a torque setting of 0.2 n. group i : size 20/0.06 taper line angle stainless steel la axxess (sybronendo, orange, ca, usa) burs used for preflaring the cervical and middle third of the canal, 3 mm short of wl group ii : gg drills (dentsply, maillefer, switzerland) were used middle third preflaring for cervical and at a length till resistance was felt in middle third of canal in a sequence of size 4, 3 and 2 (iso sizes 110, 90, and 70) at a speed of 2500 rpm on a conventional micromotor. the canal was recapitulated with size 15 k file and irrigated with 5.25% naocl after each size change group iii : cervical preflaring with protaper sx (dentsply, maillefer, switzerland) 3 mm short of wl after determining canal patency with a size 15 file on an electric endomotor at 300 rpm and a torque setting of 0.2 n. cross - sectional sagittal cbct (planmeca promax 3d, usa inc., romexis software) images were taken with size 10 file inserted in mesiobuccal canal till wl, and these were termed as presla images. the images were saved for further comparison with images after coronal flaring (postsla images). after cervical and middle third preflaring, postsla images were taken with a file that was bound in the canal till full wl (tip visible at the apex). the presla and postsla changes in the file deflection were compared as presented in figure 1 and explained as follows. line c was drawn from radio opaque marker to the beginning of canal curvature where line a left the canal long axis. the point where the lines a and c meet was point b and the angle abc is the canal - file angle on the cbct image, line a represented the file long axis. line c was drawn from radio - opaque marker to the beginning of canal curvature. the point where the lines a and c meet was point b and the angle abc (canal - file angle) was noted for all teeth in the presla images. after preflaring, the angle abc was again measured. the pre- and post - readings were noted, and mean change was recorded [figures 24 ]. one - way anova was used to compare the pre and postsla mean change among the groups, followed by tukey 's post - hoc comparison test. pre and postinstrumentation cone beam computed tomography images of la axxess group pre and postinstrumentation cone beam computed tomography images of gates glidden group pre and postinstrumentation cone beam computed tomography images of protaper sx group there was no significant change (p = 0.06) in the angle in the presla images of la axxess group i (12.37 1.01), gg group ii (13.39 1.74), and protaper sx group iii (13.90 1.74). the mean decrease in the angle from presla to postsla was significant for all the three groups (p = 0.0001). however, the mean change was higher in group i (4.25 1.14), group ii (3.28 1.22), and group iii (2.89 1.53) [tables 1 and 2 ]. the mean percentage change was 34.7% in group i, 11.9% in group ii, and 10.5% in group iii. comparison of axis among the groups mean percentage change from prestraight line access to poststraight line access owing to the canal irregularities and curvature of the root canal, a significant area of the canal remains un - instrumented even after completion of cleaning and shaping. studies have shown that coronal and middle third preflaring gives a better chance for maximum instrumentation of the radicular dentin by increasing the contact between the instrument and dentin surface and also provide an sla to the apical region of the canal. however, this should not be achieved at the expense of tooth integrity, thus the instrument selection for this purpose should be done with conservation in mind. the present study intended to assess the canal preflaring efficacy of la axxess bur in comparison to protaper sx and gg drills, as the former claims to be more conservative owing to its safe ended tip design and titanium nitride coating. although studies have been done comparing the efficacy of la axxess bur, very few have utilised cbct imaging to compare the pre- and post - cervical flaring with these burs. the canal - file angle (angle abc) was made use of to study the ability of various instruments to achieve sla that is a reduction in the cervical curvature. as the instrument removes radicular dentin in the coronal third, the file tends to be placed straighter in the canal, and the canal - file angle decreases. according to the present study, la axxess burs caused a maximum reduction in the canal - file angle and thus maximum reduction of cervical curvature, (mean change 4.25 1.14, p = 0.0001) which was significantly higher than the protaper sx and gg drill groups. these instruments cut dentin at orifice level and dentin walls at pulp chamber at the same time, thus allowing more controlled apical cutting. the present results are in accordance to a study by duarte. in which la axxess bur was most effective in removing coronal dentin, however, the authors cautioned against the use of size 35/0.06 taper burs as they may cause strip perforation, especially in the mesial roots of mandibular first molars. the results of their study suggested that size 20/0.06 taper was safe for preflaring mesial canals of mandibular first molars. in a stereomicroscopic study by sharma., la axxess burs were found to be most effective in cervical preflaring among protaper race, hyflex, flexmaster, and gg drills and produced the least discrepancy between the anatomical diameter and first file to bind at the wl. gg drills ranked second in reducing the canal - file angle and creating a straighter canal (mean change 3.28 1.22) among the three systems. although gg drills are conventionally and one of the most commonly used instruments for cervical preflaring, these have an aggressive cutting action and do not have a taper and tend to cut more toward the furcal wall. it has been shown that even anticurvature filing motion does not reduce the risk of perforation by gg drills. however, studies have shown that if used with caution they can be an inexpensive tool for coronal preflaring. protaper sx produced least change in the canal curvature among the three groups (mean change 2.88 1.53). this may be due to the tendency of these instruments to remain centered in the canal and possess less aggressive radial lands. one of the limitations of these instruments is that they cut dentin uniformly toward outward, thus increasing risk for more dentinal loss at furcal level. cbct proved to be an effective tool in assessing the pre- and post - instrumentation changes in the canal curvature as it is more accurate, three - dimensional and provides better spatial resolution, especially in areas like mesial root of mandibular first molars (as in the present study) which have grooves in the mesial or distal aspect thus confounding the conventional two - dimensional radiographic images. the mean change in the canal file angle demonstrated a significant change in pre and postgroups of all the groups and proved to be a convenient way to analyze the adequacy of sla produced. in a similar study, using presla and postsla cbct images, farhad mollashahi. compared the sla produced and effect of preflaring on the fit of the initial apical file after preparing the canals with flexmaster intro file, pre - race, and gg drills. however, according to results of their study, gg drills were more effective than niti instruments in reducing the cervical curvature. within the limitations of the present study, it can be said that cervical preflaring helps to achieve an sla to the apical one - third of the root canal. the instrument selection for preflaring should be able to achieve this goal without causing much damage to the remaining tooth structure. la axxess bur, which has safe ended head design, was most effective in reducing the cervical curvature and thus producing a better sla compared to gg drills and protaper sx files, but more studies are required to evaluate its effect on remaining dentin thickness of the radicular dentin.	objective : this study aims at cone - beam computed tomography (cbct) evaluation of the ability of gates glidden (gg) drills, protaper sx, and la axxess burs to produce a straight line access (sla) in mesiobuccal canals of mandibular first molars.methodology:forty-five freshly extracted mandibular teeth with a canal curvature of 10 - 20 were taken for the study and divided into three groups according to the instruments used for cervical preflaring : group i (la axxess burs), group ii (gg drills), and group iii (protaper sx). pre- and post - instrumentation cbct images were evaluated for comparing the ability of gg drills, protaper sx and la axxess burs to produce an sla in mesiobuccal canals of mandibular first molars.results:there was no significant change (p = 0.06) in the angle in the presla images of la axxess group i (12.37 1.01), gg group ii (13.39 1.74), and protaper sx group iii (13.90 1.74). the mean decrease in the angle from presla to postsla was significant for all the three groups (p = 0.0001). however, the mean change was highest in group i (4.25 1.14), followed by group ii (3.28 1.22) and group iii (2.89 1.53).conclusion : la axxess burs were the most effective in reducing the coronal curvature and produced a straighter access to apical third compared to gg drills and protaper sx.
childhood obesity remains one of the most serious threats to the public 's health, with 1 in 3 children and adolescents overweight or obese (body mass index (bmi) 85th percentile). there is limited evidence for effective behavioral prevention interventions. to fill this gap, the institute of medicine, the strategic plan for nih obesity research, shaping america 's youth, and the white house task force on childhood obesity have called for community - engaged, family - centered approaches to pediatric obesity prevention. these approaches are thought to have the greatest potential for sustained efforts and effects in our obesogenic environment. in parallel, comparative effectiveness research is being discussed within the national health reform debate as a mechanism for improving healthcare quality and decreasing healthcare spending. clinical research typically examines the effectiveness of one prevention or treatment method at a time. comparative effectiveness research compares multiple methods to determine the effectiveness of an intervention relative to alternatives. identifying the most effective and efficient interventions has the potential to reduce unnecessary treatments, which should lower costs. it is estimated that 8.4 million children attend after - school programs (asp), and an additional 18.5 million would do so if a program was available. the purpose of this study was to evaluate the comparative effectiveness of a community - driven asp designed to combat physical inactivity versus a standard - of - care school - based asp available to working parents. the community asp was derived directly from local input and sustained through the collaboration and sharing of resources by the parks department and the public school system (davidson county, tn, usa). this community - engaged effort has the potential to serve as a new model for youth obesity prevention because (1) it systematically addresses the top four barriers, identified by shaping america 's youth, that prevent children from being active (lack of access to safe and appropriate places to be active, parental time constraints, cost of programs, and lack of parental motivation) and (2) it engages multiple sectors of society to support program attendance and sustainability. systematic reviews suggest that it is possible to improve activity levels, physical fitness, body composition, and blood lipids in the after - school setting and that limitations in study design, lack of statistical power, and problems with implementation have hindered the evaluation of most asps to date. to assess the comparative effectiveness of this community - driven asp as a pediatric obesity prevention intervention, we compared it to the routine aftercare available to working parents in the community and asked two research questions : (1) are children in the alternative asp more physically active than children in the standard asp ? (2) this study was guided by principles of community - based participatory research (cbpr). cbpr is an important research approach that equitably involves community members who are affected by the issue being studied in all phases of the research process. the community asp was developed by the parks department to address the community 's need for an affordable asp. families provided input about their needs and preferences (e.g., transportation from school to the community center, flexibility in pick - up times, homework time, reduction of screen time, and increased physical activity). the city 's public school system changed its policy around permissible bus stops allowing buses to deliver students to the community recreation center to support program attendance. the leadership and staff of the parks department were involved in all aspects of this research project : grant acquisition, study design, implementation, interpretation of results, and dissemination. the study design was an observational prospective cohort study and a natural experiment in nashville, tn, usa. the naturally occurring event, the parks department 's new asp, was the intervention, and children attending this community asp formed the intervention group (n = 54). comparison participants (n = 37) were recruited from an asp located in the same (low income) school district and operated by a national company that operates a high proportion of school - based asps in the city, making it de facto standard - of - care for the majority of the city 's school - aged children with working parents. children were eligible for the study if the following was true : (1) age 5 and < 13 years ; (2) attended one of the glencliff [neighborhood ] cluster of public elementary or middle schools ; (3) enrolled in the community or school - based asp. parents of eligible children underwent a 15-minute oral consent process before providing written consent for their child. children provided assent. the consent / assent process was conducted in the preferred language of english or spanish. the study was approved by the vanderbilt university institutional review board (# 090986). the two asps followed similar formats, and operated from 36 pm every day public schools were open. both asps included time for snack, homework, and play and did not focus on a single activity (e.g., tutoring, chess, and team sport). the community asp was set in a community recreation center and involved staff - led games. the school - based asp was set in a school cafeteria and involved opportunities for arts and crafts and playing on the playground. the main differences between the two asps were (1) format of active play time (adult - led versus unstructured) and (2) location (community recreation center versus public school). all measures were collected at the asps at three time points over approximately 12 weeks (february may 2010), with six weeks separating each wave of measurement. the measurement period was selected based on the cochrane review that states that obesity prevention interventions should last at least 12 weeks for behavior change to be observed. physical activity was assessed using actigraph gt1 m accelerometers (actigraph, pensacola, fl, usa) only during asp programming time. accelerometry is considered an objective measure of physical activity and has been used with children [15, 16 ], including latino and african - american children, with high reliability : r = 0.93. the actigraph is a small monitor that is worn on an elastic waist belt and measures the intensity of physical activity associated with locomotion. monitors were programmed to record in continuous 10-second epochs to capture the short, spurt - like activity characteristic of children. at each measurement period, the children wore monitors for five consecutive days, from the time they signed into the asp until they were picked up. measurement start and stop times were recorded by study staff at each site ; these were used as precise wearing cut - off points, eliminating the need for wearing / nonwearing time analysis. data were retained in analysis if the child wore the accelerometer a minimum of 3 days of the given measurement period [18, 19 ]. freedson 's age - dependent cut points were used to determine time spent in sedentary, light, moderate, and vigorous activity. trost 's validation study comparing various accelerometer cut points for predicting physical activity in children supports the application of freedson equations in field - based studies of school - aged children. in particular, trost found that, for classification of mvpa (moderate - vigorous physical activity), freedson cut points exhibited excellent classification accuracy. the analyses described below were also conducted using pate 's cut points and resulted in similar findings (not reported here). daily percentage of time spent in each level of physical activity (i.e., sedentary, light, moderate, and vigorous) was determined by dividing the minutes spent in each activity level by the sum of minutes the actigraph was worn in a day (i.e., time in attendance at the asp). thus, the continuous outcome measures were the proportion of time spent in lmvpa (light - moderate - vigorous physical activity) or mvpa (moderate - vigorous physical activity) out of total time in attendance, rather than the number of minutes the program was open, to allow for a meaningful comparison within individuals and across groups. daily percentages were averaged across days to create individual participants ' physical activity (pa) scores at each measurement period. body weight was measured after voiding while children wore light clothing without shoes. calibrated digital scales (detecto, webb city, mo, usa, model#758c) were accurate to the nearest 0.1 kg. body height without shoes was measured to the nearest 0.1 cm with the scale 's stadiometer. weight categories were defined by bmi percentile, according to centers for disease control growth charts : underweight : < 5th percentile ; healthy weight : 5th to < 85th percentile ; overweight : 85th to < 95th percentile ; obese : 95th percentile. body composition was measured by the rjl systems bia quantum ii (rjl systems, clinton, mi, usa) after voiding. standard procedures for whole body bioelectrical impedance measurement were used, along with the vendor - provided child - specific regression equation to estimate percent fat mass from total body water. children were asked to complete a 1/2 mile run as fast as possible on a running track. parents completed a survey asking about child 's date of birth (used to calculate age), gender, race / ethnicity, and name of school. because children were not randomly assigned, preexisting differences between groups were potential confounders. therefore, we compared children enrolled in the asps to test for differences on basic demographic and process variables, using bootstrap t - tests that controlled for the familywise false discovery rate. to assess change in pa over time, a conditional linear latent growth model was used with random intercepts and slopes that were free to covary and time varying error variances. this approach offers important advantages over older analysis of variance (anova) models, such as (a) better accuracy in assessing change over time, (b) graceful handling of missing values and unequal time intervals between waves and participants, and (c) repeated measurements that increase statistical power. the key result is a group by time interaction, which shows whether groups differ in their slopes / rates of change in pa. centering time zero at the first measurement let us answer two questions : (1) did the groups start out equally ? and (2) did their time slopes differ ? the analysis assumed data were missing at random and used full information maximum likelihood to maximize sample size by including all participants with at least one wave of data. using the ingredient method, we estimated the implementation costs, without estimating indirect costs or externalities associated with the programs, to indicate how much it would cost to replicate each asp. instead of accounting expenditures paid during the implementation, we valued resources using standard costs to society. all personnel time (including volunteer time) was valued by using the median earning per hour of a comparable worker published by the bureau of labor statistics 2010 ; thus, differences in human capital endowment did not affect our estimates of implementation costs. of the 91 children who attended the asps, baseline demographics were obtained from parents of 83 children. the analytic sample included the 82 children with pa data from at least one time point ; one child in the school - based asp did not provide at least 3 days of pa data in any measurement period and was not included in the analyses. of the 82 participants, 62 had data for all three time points, 16 had data for two time points, and 4 had data for only one time point. the baseline sample was 65% female and 7.9 years of age (sd = 1.7) on average ; 57% were healthy weight, 23% overweight, and 20% obese ; 40% were african - american, 40% white, and 20% latino. on average at baseline, children spent 77.4% (sd 10.3%) of the asp in lmvpa and 27.5% (sd 14.3%) in mvpa. at baseline, children in the two asps did not differ in gender, age, bmi, percent body fat, fitness (table 2), or physical activity level (table 3). however, children in the community asp were less likely to be white than children in the school - based asp (p =.027, table 2). at baseline, children spent approximately 30% of their asp time in mvpa (sd = 15.6). the linear latent growth model implied that, on average, children in the community asp became more active over time (average change between baseline - week 6 and week 6-week 12), compared to the children in the school - based asp (table 3). children in the school - based asp reduced their total physical activity (lmvpa) by an average of 3.4 percentage points over each measurement period (p =.002), for a total 6.8 percentage point decrease over the 12-week study period. in contrast, children in the community asp increased their total physical activity (lmvpa) by an average of 3.0 percentage points over each measurement period (p =.006), for a total 6 percentage point increase over the 12-week study period (figure 1). children in the school - based asp did not significantly change their mvpa on average (p =.12). however, children in the community asp increased their mvpa by an average of 2.8 percentage points over each measurement period (p =.006), for a total 5.6 percentage point increase over the 12-week study period (figure 2). taken together, the model - implied average difference between the two groups of children at week 12 was 15.4 percentage points in lmvpa (p <.001) and 14.7 percentage points in mvpa (p <.001), favoring the activity - enhanced community asp. however, as a more conservative indicator, the observed average difference between the two groups of children who had data at week 12 was 10.8 (p =.001) percentage points in lmvpa and 13 percentage points in mvpa (p <.001). the community asp served 54 children ; the school - based asp served 37 children. total implementation costs (valued in 2010 dollars) for the 12-week study period were $ 1,184 per child ($ 19.25 daily per child) for the community asp, compared to $ 1,087 per child ($ 17.67 daily per child) for the school - based asp (9% difference ; table 4). the facility cost represented 66% and 65% of the total implementation costs to run the community asp and school - based asp, respectively. the main source of cost differential between programs was the child to staff ratio (6 : 1 at the community asp, 12 : 1 at the school - based asp). to run the asps for 12 weeks, the personnel cost was $ 380 per child for the community asp compared to $ 314 per child for the school - based asp (21% difference). with more than 23 million parents of school - aged children employed full - time, this study demonstrated that, compared to a standard - of - care school - based asp, an asp set in a community recreation center with activities directed by recreation staff significantly increased total physical activity in a multiethnic sample of public school children by 6 percentage points over 12 weeks. most (5.6%) of this increase was in mvpa, which is the type of physical activity that has the greatest health benefits [3335 ]. the incremental cost of implementing the activity - enhancing asp compared to the traditional asp was $ 1.59 per day per child. the main source of cost differential between programs was their child to staff ratios. assuming the improvement in activity was solely due to the intervention ; these findings suggest that children attending traditional school - based asps, already costing an average of $ 17.67 per day, would need an additional daily investment of $ 1.59 per child over 12 weeks to increase their lmvpa by 15.4 percentage points or their mvpa by a model - implied 14.7 percentage points. the annual cost of childhood obesity - related health expenses in the us is $ 14.1 billion for outpatient care and $ 237.6 million for inpatient care which translates to about $ 5 in healthcare expenses per day per child, without including other relevant long - term costs related to school performance, labor market involvement, quality of life, welfare needs, and so forth. given this, providing structured pa programming by qualified staff in a community recreation center in the after - school hours could be a reasonable low - cost investment. asps have long played a critical role in supporting academic achievement, safety, discipline, and avoidance of risky behaviors. they could now be leveraged as part of a broader approach to address physical inactivity. community centers operated by local parks and recreation departments (20,000 nationally) provide an ideal venue for structured pa programming for children in large part because these centers, in conjunction with school transportation departments, can address community - based barriers to increasing children 's activity levels. it is noteworthy that the parks staff initiated and led this program on their own. their intimate knowledge of the community and the respect they commanded from both the children and adults in the community likely contributed to the program 's success. we speculate that the combination of the built environment that supported activity, low child to staff ratio, and intentional activity leadership resulted in the increased pa levels. the school - based asp could have let children play in the school 's gymnasium if there had been additional staff available to supervise. thus, we speculate that importing adult - led activities with lower child to staff ratios into school - based asps might be a cost - effective approach to increasing activity in that setting as well first, accelerometers do not adequately measure body movements of upper and lower extremities, but they are considered the gold standard for measuring pa under free - living conditions. this should not have biased our results since the limitation of accelerometry was the same across groups. second, our sample was small but having three waves of data increased statistical power and was sufficient for detecting a significant increase in pa under free - living conditions. third, despite efforts to select a comparable comparison group and measure potential confounders, we can not rule out all systematic differences between the two groups. we did rule out the most important possible confounds in the literature : body composition, fitness, age, and gender. it is possible that the difference in racial composition of the groups could explain baseline variance but is unlikely to explain change over time in activity levels. fourth, for the community asp, there were significant differences between the observed and model - implied averages at weeks 6 and 12. these discrepancies highlight the fact that the final specified model did not perfectly recreate the observed data. this could have been partially due to missing data for this group at either time point. the discrepancies could also have arisen because the community asp 's growth rate was not linear ; yet, a model with only three time points does not have the degrees of freedom to investigate more sophisticated growth parameter specifications (e.g., quadratic). nonetheless, applying latent growth models has provided further insight into how asps might impact children 's pa over time (e.g., what effect does asp type have on pa change over time ? what is the typical growth rate of pa for children over time ? do some programs increase the growth rate of certain types of pa (e.g., light, moderate, or vigorous) more than others ? an asp set in a community recreation center and led by recreation staff incorporating structured physical activity opportunities was associated with significant increases to physical activity during asp time in a multiethnic sample of public school children in 12 weeks, compared to a standard school - based asp. utilizing community recreation centers ' built environment and staff could be a promising low - cost proposition to improve health trajectories among school - aged children.	background. we conducted a comparative effectiveness analysis to evaluate the difference in the amount of physical activity children engaged in when enrolled in a physical activity - enhanced after - school program based in a community recreation center versus a standard school - based after - school program. methods. the study was a natural experiment with 54 elementary school children attending the community asp and 37 attending the school - based asp. accelerometry was used to measure physical activity. data were collected at baseline, 6 weeks, and 12 weeks, with 91% retention. results. at baseline, 43% of the multiethnic sample was overweight / obese, and the mean age was 7.9 years (sd = 1.7). linear latent growth models suggested that the average difference between the two groups of children at week 12 was 14.7 percentage points in moderate - vigorous physical activity (p <.001). cost analysis suggested that children attending traditional school - based asps at an average cost of $ 17.67 per day would need an additional daily investment of $ 1.59 per child for 12 weeks to increase their moderate - vigorous physical activity by a model - implied 14.7 percentage points. conclusions. a low - cost, alternative after - school program featuring adult - led physical activities in a community recreation center was associated with increased physical activity compared to standard - of - care school - based after - school program.
proteins are inherently flexible, displaying a broad range of dynamics over a hierarchy of time - scales from pico - seconds to seconds.(1) this molecular plasticity enables conformational changes in protein backbone and side chains that play critical roles in biomolecular function. nuclear magnetic resonance (nmr) spectroscopy has emerged as the method of choice for studying biomolecular structure and dynamics in solution. all experimentally measured nmr data are affected by motions occurring with characteristic exchange rates that are faster than the so - called chemical - shift range, giving rise to average peaks that represent a potentially complex dynamic average over relatively long time scales (up to the millisecond range for proteins in solution). in addition spin relaxation experiments reflect motions occurring on time scales faster than the molecular rotation diffusion coefficient c (520 ns), whereas relaxation dispersion can be used to identify sites of slower (sms) conformational exchange. although the importance of molecular flexibility is generally recognized, standard nmr - based structure determination protocols ignore the presence of protein dynamics, implying that, in common with x - ray crystallography, rapidly exchanging conformational equilibria are routinely represented in terms of a single static structure.(9) the specific averaging properties of different structurally dependent parameters are rarely incorporated into the structure determination procedure, such that the resulting set of coordinates represent a poorly defined average. the aim of this study is to actively use the rich dynamic information encoded in motionally averaged nmr parameters to develop a structural, dynamic and statistical mechanical molecular representation of the conformational behavior of proteins in solution. chemical shifts alone are not yet able to describe the dynamics giving rise to the average spectrum, and interproton cross relaxation rates, although rich in structural information, are dependent upon the time scales of the motional processes, and are therefore difficult to interpret quantitatively unless the time scales of the dynamics are known. however, other interactions such as scalar, and possibly more powerfully, residual dipolar couplings (rdcs), are exquisitely sensitive to conformational detail and therefore may hold the key to resolving this long - standing problem. over the past decade, rdcs have emerged as powerful tools for studying proteins in solution, providing simultaneous information about time- and ensemble averaged structural and dynamic processes occurring up to millisecond time - scales and thereby encoding key information for understanding biomolecular function. numerous approaches have been proposed to characterize protein backbone conformation from rdcs ; most notably the direct determination of dynamic amplitudes and anisotropies of bond vectors or structural motifs from multiple rdc measurements. molecular dynamics (md) simulation can also provide access to slower motions that can be compared to measured rdcs, however despite increasing computational power, trajectories are usually restricted to time - scales of hundreds of nanoseconds, and millisecond trajectories are still not viable. relatively long simulations (up to 1.2 s) have identified slow dynamic processes occurring on time - scales beyond the range probed by spin relaxation, which would affect the rdc data, but such long simulations provide only a single trajectory in phase space and do not avoid the problem of statistical mechanical sampling. a popular alternative to performing long simulations is to implement time- or ensemble - averaged restraints, thereby constraining a multiple copy molecular description to reproduce the conformationally averaged rdcs. although efficient for identifying conformational ensembles in agreement with experimental data, adding an arbitrary pseudopotential to a physical force field can perturb the simulated dynamics in a nonpredictable manner, making further analysis of the resulting trajectories uncertain. more importantly, the generation of an ensemble of structures that can reproduce the experimental data do not necessarily include the relative free energy weighting of each member of the ensemble. the potential energy surface of a protein may be rugged and highly structured, resulting in a broad distribution of populations in conformational space. to accurately reproduce rdcs or any other nmr observable, it would be necessary to include an accurate population analysis. in this paper, we present a novel approach aimed at providing a self - consistent structural dynamic representation of protein conformational sampling using a combination of state - of - the - art md simulation and a large set of experimental nmr data. in contrast to previously proposed techniques, we avoid using empirical energy terms to guide the conformational search, a procedure that has the potential to perturb both the nature and the time scale of simulated dynamics in a nonpredictable way. rather, we sample conformational space in an unrestrained way, such that boltzmann statistics can be respected. to sample conformational space efficiently, we use a recently proposed accelerated molecular dynamics (amd) approach, which biases the actual potential energy surface of the protein to enhance transition probabilities between low energy conformational substates. the appropriate level of acceleration, and therefore sampling of conformational space, is directly determined by matching the reproduction of millisecond - averaged experimentally measured dipolar and scalar couplings to those predicted from the different ensembles. the method is used to describe conformational dynamics occurring on time scales over many orders of magnitude in the prototypical protein system ubiquitin and validated against experimental data sensitive to the diverse time scales. figure 1 shows averaged order parameters for nh vectors obtained from md trajectories seeded from amd simulations using increasing levels of acceleration (see methods). a heterogeneous distribution of long time - scale dynamics is observed, with increasing amplitude motions occurring predominantly in loop regions (residues 811 and 4648, and to a lesser extent, residues 19, 20, 22, 36 and 6062). slower motions are generally seen in regions identified from previously applied long time - scale classical md simulations of ubiquitin. a single amd trajectory, using the equivalent number of steps to 8 ns of classical md, with relatively low acceleration parameters of (eboost vdih = 100 kcal mol, = 60 kcal mol) results in essentially identical conformational sampling to a 150 ns classical md trajectory (nh order parameters are compared in figure s1, supporting information). this demonstrates that the amd approach samples meaningful conformational space analogous to standard long mds. (a) order parameters are shown after performing a free energy weighting correction, and are averaged over the trajectories. from top to bottom, the boost energy is 0 (standard 5 ns md control set), 100, 150, 200, 250, 300, 350, 400, and 450 kcal / mol. the acceleration parameter,, was fixed at a value of 60 kcal / mol. (b) change in the trajectory - averaged cumulative r - values for rdcs as a function of the acceleration level. in all cases, the boost energy of 0 represents the control set of 5 ns standard md simulations starting from the x - ray crystal structure(47) using a different random seed generator. (c) change in the trajectory - averaged cumulative r - values for j - couplings as a function of the acceleration level. in all cases the acceleration parameter = 60 kcal / mol. to assess the most appropriate level of acceleration, we have analyzed the ability of ensembles derived at each value of (eboost vdih) to reproduce experimental rdcs and j - couplings (see methods). figure 1 shows the trajectory averaged cumulative r - factor (rcum) for rdcs and scalar j - couplings as a function of the acceleration level, and clearly identifies the optimum level at (eboost vdih) = 250 kcal / mol for an acceleration parameter = 60 kcal / mol. ensembles generated with less aggressive acceleration sample too little conformational space, while more aggressive acceleration samples too much conformational space to reproduce experimental data. at the optimal acceleration level, the trajectory - averaged nh rdc cumulative r - factor across all 23 alignment media is 2.496 (average 0.1085). figure 2 shows correlations between experimental and theoretical nh rdcs for four representative data sets with r - factors of 0.096, 0.098, 0.100, and 0.111. residue specific trajectory - averaged rcum values are compared to a control set calculated from standard 5 ns md simulations (figure 3). only residue 54 shows any increase in the rcum value. long time - scale dynamics are predominantly located in residues 811 and the residue specific rcum values for these residues show improvement compared to the control set. significant improvement is also observed in residues that show little or no long time - scale dynamics, (e.g., 15, 17, 34, 42, and 67). the improvement here arises from the more appropriate representation of the time- and ensemble- averaged alignment tensor. experimental vs theoretical rdcs for four representative alignment media out of the 23 alignment media. the trajectory averaged cumulative r - factors for the shown alignment media are respectively 0.096, 0.098, 0.100, and 0.111. the trajectory averaged cumulative r - factors across all alignment media varied from 0.090 to 0.129. the optimal extended conformational space molecular ensemble [(eboost vdih) = 250 kcal / mol ] is shown in red and compared to a cumulative r - factors for the extended conformational space molecular ensemble are in general lower than those for the control set, confirming the observation of a global improvement in the theoretical rdc data. the same level of acceleration also produces the best trajectory - averaged cumulative r - factors for the three scalar j - couplings (hh, hc and hc). in comparison to rdcs, scalar j - couplings are less sensitive to the inclusion of long time - scale dynamics, as seen by the relatively small improvement in the rcum values (figure 2b). this agrees with the previously described phenomenon whereby fitted karplus parameters can absorb a component of the motion. figure 4 shows the correlation between experimental and theoretical results for the three scalar j - couplings for the molecular ensemble associated with an acceleration level of eboost vdih = 250 kcal / mol. optimized karplus curves are compared for jnh - h obtained from a static structure (1d3z), standard 5 ns md simulations and the amd ensembles and quantum chemistry calculations performed using sum - overstates (sos) density functional theory (dft).(44) the curve for the optimal amd acceleration is almost identical to the dft - based karplus curve. a similar effect is observed for the other scalar j - couplings (data not shown). (a) experimental vs theoretical scalar j - couplings for the optimized extended conformational space molecular ensemble [(eboost vdih) = 250 kcal / mol ]. the three scalar j - couplings are jhn - h [black circles ], jhn - c [red circles ], and jnh - c [blue circles ]. red : optimal karplus curve for. black : optimal karplus curve for standard 5 ns md simulation. cyan : dft karplus curve for nme - ala - ace. the free energy weighted molecular ensembles at the rdc - optimum acceleration level provide a representation of the conformational space sampled on time - scales up to the milli - second. these molecular ensembles are composed of individual substates, each with a relative free energy weighting, and from which standard md simulations have been seeded to probe the fast motions occurring in the local conformational vicinity. figure 5 depicts the nh order parameters for the fast (ps - ns) dynamics and the effective order parameters probing dynamics on the millisecond time - scale calculated from the free energy weighted ensembles. the fast time - scale nh order parameters obtained by averaging the weighted order - parameters from each substate are in very good agreement with experimental spin relaxation data.(45) in agreement with earlier studies on gb3,(41) we observe an improvement in the agreement between experimental and predicted spin relaxation order parameters when averaging over the extended conformational space ensemble, compared to standard 5 ns md simulations (data not shown). figure 6 shows a representative bundle of structures for ubiquitin obtained from the molecular ensemble generated from the rdc - optimal acceleration level. the n spin relaxation experimental data are represented by the blue line.(45) the theoretical fast time - scale (psns) order parameters are shown as black circles and the slow time - scale (rdc - optimized) order parameters are shown in red. the error bars depict the variation in the magnitude of the order parameters for the different molecular ensembles generated from the 20 amd simulations at the same acceleration level (eboost vdih = 250 kcal / mol). the residues are color - coded according to the value of the rdc order parameters (blue : 1.0, red : 0.0). standard nmr structure refinement against experimental observables generates a time- and ensemble - averaged static conformational representation. in the case of ubiquitin, a high resolution static structure has been optimized (1d3z),(46) against extensive rdcs, noes, scalar couplings, and hydrogen bonding restraints. the average r - factor per alignment medium for this structure is 0.093 compared to 0.107 for the optimal amd ensemble. a direct comparison between unrestrained md and the nmr structure is complicated by the similarity of the different alignment tensors with those used to refine the structure of 1d3z. the average rdc r - factor for the optimal amd ensemble is better than the x - ray crystal structure for ubiquitin (1ubq)(47) (0.116). similarly, the amd approach provides a mean trajectory - averaged j - coupling r - factor (0.143) that reproduces the couplings better than 1ubq (0.153), and identically to 1d3z (0.143) despite the fact that these scalar j - couplings were used in the refinement of 1d3z. average backbone coordinates obtained over all free energy - weighted molecular ensembles generated at the rdc - optimal acceleration level are much closer (0.35) to the 1d3z structure than those obtained from a control set of 5 ns standard md simulations (0.55). thus although the rdc - optimal amd trajectories sample broader conformational space they are distributed about a mean conformation that resembles the time- and ensemble - averaged static structure. average structures obtained from the optimal molecular ensembles also exhibit negligible violations to the noe upper- and lower - bounds. although a quantitative analysis of cross - relaxation rates would require a more rigorous analysis,(48) this demonstrates that the rdc - optimal amd ensembles are in qualitative agreement with available structural data. we have also compared the results of the amd approach to a recent ensemble restrained molecular dynamics (eros)(33) description of ubiquitin using extensive noe data and, in this case, all of the 23 rdc data sets treated in our amd study. not surprisingly, as the rdcs were used to directly restrain the eros ensemble, an svd analysis of the nh rdcs using this ensemble gives a lower average r - factor (0.066). principal component projections of the conformational sampling (supporting information figure s2) reveal that the amd / svd approach described here samples essentially the same conformational space as the eros ensemble, without the need to employ ensemble - averaged restraints. this is remarkable considering that no structural restraints are applied, and the conformational space is only defined via global agreement with the entire data set. closer inspection reveals that the amd approach further refines the available conformational space sampling following free energy weighting, therefore providing a more realistic structural dynamic representation of the system. finally in the recent eros study, the order parameters derived from the 116 member ensemble were further scaled by a factor of 0.93, on the basis that, in the opinion of the authors, the ensemble may not include a sufficient representation of librational motions. in our case no an additional scaling is applied, so that the amd ensemble can be considered as a true molecular representation of the dynamic ensemble giving rise to the experimental data. we note that the extent and nature of the dynamics determined using the amd approach are quantitatively very similar to that determined using the recently developed three - dimensional gaussian axial fluctuation analysis of the experimental rdcs, where, in comparison to spin relaxation derived order parameters, ubiquitin was shown to be essentially rigid on ns - ms time scales, except for the 811 hairpin region and some additional surface loops.(22) a more detailed comparison of our results to those obtained from alternative representations, such as 1d3z and eros is provided in the supporting information. all nmr parameters are affected by motions occurring on time - scales that are faster than the so - called chemical - shift time - scale, resulting in resonance peaks that represent potentially complex dynamic averages over relatively long times (up to the millisecond range for proteins in solution). in this paper, we have combined a novel amd / svd approach with extensive experimental nmr data, to provide an accurate description of the structural dynamic behavior of the protein ubiquitin on time - scales ranging from the picosecond to the millisecond. the results of the amd simulations performed at different acceleration levels confirm that this method can efficiently and accurately sample extended conformational space explored by globular proteins. the svd analysis allows the model - free determination of the optimal rdc alignment tensor and the optimal j - coupling karplus parameters, for a given molecular ensemble, obviating the need for calibration against external references or rescaling of order parameters. the problems of statistical mechanical sampling associated with the incorporation of additional terms into a hybrid potential energy force field, and thereby perturbing the simulated dynamics, are avoided by using restraint - free trajectories seeded at different points of conformational space sampled by the accelerated md. the accuracy of the resulting rdcs and scalar couplings is however hardly compromised by this procedure, with a similar level of reproduction compared to state - of - the - art single - structure or restrained - ensemble approaches. importantly fast motional (psns) order parameters derived from experimental spin relaxation data are well reproduced by the population weighted average over md simulations performed within the different conformational substates. this important result nicely illustrates the potential, inherent to this approach, of resolving the time scales of different motions for comparison with appropriately sensitive experimental data. the optimal amd molecular ensemble is therefore in agreement with all available experimental data, giving excellent reproduction of rdcs and scalar j - couplings, experimentally determined noes, as well as n spin relaxation data. interestingly, the average backbone structure of the optimal molecular ensemble compares very closely with that of the experimentally refined 1d3z structure, indicating that although these ensembles sample more conformational space, they appear to be distributed about a mean that resembles the experimentally determined time- and ensemble - averaged structure. in all cases, the free energy weighted extended conformational space ensembles reproduce the experimental observables to a substantially greater degree of accuracy than a control set of 5 ns standard md simulations and provide better reproduction compared to the static x - ray crystal structure (1ubq). the ability to provide an explicit description of protein dynamics in terms of conformational substates and associated populations will undoubtedly improve our understanding of the molecular basis of their biological function, and simultaneously provide an essential basis for interpreting dynamically averaged nmr spectra of proteins. a full characterization of protein dynamics requires an integrated experimental and computational approach. in this study we have therefore used enhanced sampling from biased potential molecular dynamics simulation, combined with extensive experimental dipolar and scalar coupling data, to define a self - consistent representative molecular ensemble for solution state protein conformational dynamics. this approach presents a unified structural dynamic representation of the motional properties of proteins in solution that will provide the basis for furthering our understanding of molecular stability, folding, and function, while simultaneously proposing a new methodology for the interpretation of nmr data in terms of molecular ensembles that will be applicable to a wide range of experimental systems. the amd approach involves adding a continuous non - negative bias potential to the potential energy surface of the protein to raise and flatten the potential energy landscape, thereby enhancing the escape rate between low energy conformational substates. boost energy, eb, which is fixed above the minimum of the potential energy surface. at each step in the amd simulation, if the potential energy of the system lies below this boost energy, a continuous, non - negative bias is added to the actual potential. this results in a raising and flattening of the potential energy landscape, decreasing the magnitude of the energy barriers between low energy states, and therefore enhancing the escape rate from one low energy conformational state to another, while maintaining the essential details of the underlying potential energy surface. the extent to which the potential energy surface is modified depends on the difference between the boost energy and the actual potential. explicitly, the modified potential, v(r), is defined as : if the potential energy, v(r), is equal to or greater than the boost energy, and if the potential energy is less than the boost energy. the energy modification, or bias is given by : the extent of acceleration (i.e., how aggressively we enhance the conformational space sampling) is determined by the choice of the boost energy and the acceleration parameter,. conformational space sampling can be enhanced by either increasing the boost energy, or decreasing the acceleration parameter. in the present work, the extent of conformational space sampling was controlled by systematically increasing the boost energy using a fixed acceleration parameter. during the course of the simulation, if the potential energy is modified, the forces on the atoms are recalculated for the modified potential. the use of the bias potential defined above ensures that the derivative of the modified potential will not be discontinuous at points where v(r) = eb. a series of 20, 8 ns, accelerated molecular dynamics (amd) simulations of ubiquitin were performed at increasing levels of acceleration using the program amber8.(49) in each case was fixed at 60-kcal / mol and the boost energy for the eight acceleration levels was set at 100, 150, 200, 250, 300, 350, 400, and 450-kcal / mol above the dihedral angle energy (estimated from the average dihedral angle energy from the unbiased 5-ns md simulations). in all simulations, a time - step of 1 fs and periodic boundary conditions were used with a langevin thermostat and a berendsen weak - coupling pressure - stat. electrostatic interactions were treated using the particle mesh ewald(50) method with a direct space sum limit of 10. the recently developed ff99sb force field was used.(51) after reweighting the conformational space to obtain the correct canonical boltzmann distribution, a clustering protocol was implemented to identify low energy conformational substates. a series of short 3 ns standard md simulations were then seeded from the amd simulations, to sample the low energy substates. the initial 0.5 ns were discarded, and a mmpb / sa(52) analysis on the resulting md simulations was used to confirm the amd free energy weighting protocol. using these approximate free energies, a set of large (free energy weighted) structural ensembles resulting ensembles represent free energy weighted trajectories, sampling the conformational space explored by the amd trajectories at the relevant acceleration level. this method represents an efficient equivalent to performing numerous long time - scale md simulations. as a control, the next step is to identify which ensembles can best reproduce the experimental rdc data. at each increasing acceleration level, we have sampled an increasingly large amount of conformational space. ideally there should exist an optimum sampling on the time - scale relevant to the rdc data. however, for each molecular ensemble, the optimum alignment tensor for a given alignment medium must be calculated. this is achieved in a model free way using a singular value decomposition (svd) approach, and the analysis is performed for available nh rdcs in 23 different alignment media (see details below). using the optimized alignment tensors, theoretical rdcs were calculated for each molecular ensemble associated with a given acceleration level. as each molecular ensemble represents a single long time - scale trajectory, the theoretical rdcs for each ensemble associated with the same acceleration level were averaged and the agreement between experiment and theory was monitored using the trajectory averaged cumulative r - factor. the principal difficulty concerned with the direct calculation of rdcs and scalar j - couplings arises in the determination of parameters defining the strength of the interaction. in the case of rdcs, five unknown parameters are required to explicitly define the alignment tensor. as our simulations are performed in explicit solvent, in the absence of any alignment medium, it is not possible to define explicitly from the simulation alone the preferential alignment of the molecule in a given alignment medium. the issue is further complicated by the fact that the structure, dynamics and preferential alignment of the molecule are mutually dependent : the alignment tensor depends on the shape and anisotropy of the molecule, which is specifically related to the structure. dynamic motions on different time - scales result in small changes in the shape and anisotropy of the molecule, which, in turn result in small changes in the preferential alignment tensor for a given alignment medium. the approach taken in this work involves the use of an svd analysis to determine the optimal alignment tensor for each molecular ensemble directly from the experimental data. explicitly, the optimal alignment tensor and rdcs for each molecular ensemble were calculated in a reduced form as : where x, y, z are the cartesian components of the normalized bond vector of interest (in the case of nh rdcs, the nh bond vector), aij is a vector containing the five components necessary to completely define the 3 3 alignment tensor (bearing in mind that this tensor is symmetric and traceless) and dired is a vector containing the experimental rdcs for the particular alignment medium. the matrix on the left - hand side of the equation, which describes the bond vector fluctuations, has dimensions (n,5), where n is the number of rdcs in the given alignment medium. this matrix is formulated from the molecular ensemble where the brackets... represent ensemble averages. svd of the matrix of bond vector fluctuations liberates the optimal alignment tensor components, from which the theoretical rdcs can be calculated. the principle behind such an analysis is that there should exist some optimal ensemble which represents the conformational space sampled by the system over the time - scales to which rdcs are sensitive (i.e., up to 10-ms for nh rdcs). for this optimal molecular ensemble, and its optimal svd - calculated alignment tensor, the resulting theoretical rdcs will be in best agreement with the experimental observables. for molecular ensembles that sample too little or too much conformational space, the svd analysis will attempt to find the best possible alignment tensor for that particular ensemble, but the resulting rdcs will not be optimal. as mentioned above, we perform a series of amd simulations at increasing acceleration levels to obtain a set of free energy weighted molecular ensembles that systematically sample an increasing amount of conformational space. by using the svd analysis to obtain the optimal alignment tensor and hence the theoretical rdcs for each molecular ensemble, we can identify the most appropriate acceleration level (ie. the optimal conformational space sampling) to reproduce the experimental rdcs. a similar approach was also applied to calculate the backbone scalar j - couplings : we calculated three backbone scalar j - couplings, j(h, h), j(h, c), and j(h, c). the magnitude of all these j - couplings is strongly related to the backbone angle and can in general be described using the well - known karplus equation:(54) where a, b, and c are the karplus parameters, and is an offset angle, which typically has a value of 180 for j(h, c), 60 for j(h, h) and 60 for j(h, c). to calculate these scalar j - couplings, we used the svd analysis to obtain the optimal karplus parameters for each molecular ensemble : in each case, the analysis was initially performed using the typical offset angles defined above. the -offset values were then optimized by changing the -offset value in 1 steps and repeating the svd analysis until the best reproduction of the experimental scalar j - couplings was achieved.	an atomic resolution description of protein flexibility is essential for understanding the role that structural dynamics play in biological processes. despite the unique dependence of nuclear magnetic resonance (nmr) to motional averaging on different time scales, nmr - based protein structure determination often ignores the presence of dynamics, representing rapidly exchanging conformational equilibria in terms of a single static structure. in this study, we use the rich dynamic information encoded in experimental nmr parameters to develop a molecular and statistical mechanical characterization of the conformational behavior of proteins in solution. critically, and in contrast to previously proposed techniques, we do not use empirical energy terms to restrain a conformational search, a procedure that can strongly perturb simulated dynamics in a nonpredictable way. rather, we use accelerated molecular dynamic simulation to gradually increase the level of conformational sampling and to identify the appropriate level of sampling via direct comparison of unrestrained simulation with experimental data. this constraint - free approach thereby provides an atomic resolution free - energy weighted boltzmann description of protein dynamics occurring on time scales over many orders of magnitude in the protein ubiquitin.
excessive variations in blood pressure (bp) are guarded against by baroreflexes. impaired baroreflex mechanisms in the perioperative period can result in hemodynamic instability. anesthetistsare aware of life - threatening bradycardia and asystole that can occur because of the manipulation around the carotid sheath during a radical neck dissection (rnd). perhaps less well known is that an acute form of baroreflex failure can manifest as a hypertensive crisis in patients who have had surgery on the neck. we present two cases of severe postoperative hypertension developing in patients who underwent neck dissection. an 80-year - old, 48-kg, hypertensive man was scheduled for wide local excision of carcinoma lip and supra - omohyoid neck dissection. he was a smoker for 25 years and a known hypertensive (controlled with amlodepine and atenolol). lignocaine 75 mg iv was administered to attenuate sympatho - adrenergic response to laryngoscopy and tracheal intubation. were stable intraoperatively. wide local excision of carcinoma lower lip and supra - omohyoid neck dissection was performed. surgery lasted 3.5 h and blood loss was about 350 ml while urine output was > 0.5 ml / kg / h. at the end of surgery after tracheal extubation the patient was conscious and following verbal commands, hr was 62/min, bp 156/94 mmhg, and spo2 99% on 100% oxygen. ten minutes following tracheal extubation there was a progressive increase in bp up to 202/115 mmhg, with hr 65/min. morphine 1.5 mg and frusemide 5 mg were administered iv. as there was an inadequate decrease in bp (196/118 mmhg with hr 58/min), nitroglycerine (ntg) infusion arterial blood gas (abg) analysis at a fio2 of 1.0 revealed po2 65 mmhg, pco2 46 mmhg and ph 7.42. the patient was transferred to the intensive care unit (icu) for further management. the vital signs remained stable with hr 68/min, bp 130/ 86 mmhg, spo2 > 95% at fio2 0.4 and urine output > 0.5 ml / kg over 4 h. abg analysis (at fio2 0.4) revealed po284 mmhg, pco2 44 mmhg, and ph 7.43. the patient was observed overnight and transferred to the ward the following day and discharged home on the seventh postoperative day. a 68-year - old, 58 kg, normotensive male, a case of cervical lymph node metastasis with unknown primary, was scheduled for right sided rnd. surgery lasted 3.5 h and blood loss was about 250 ml with urine output > 0.5ml / kg / h. at the end of surgery the trachea was extubated as respiratory efforts were adequate and as the patient was awake and not tolerating the tracheal tube. fentanyl 50 mcg and midazolam 1 mg iv were given, but the bp increased to 246/114 mmhg with hr 96/min. a rising trend in bp was observed and ntg infusion was started at 0.5 mcg / kg / min and gradually increased to 1.5 mcg / kg / min. as the obstruction persisted, the trachea was reintubated after administering propofol 80 mg and succinylcholine 100 mg iv. tracheal intubation was not associated with a hypertensive response and surprisingly bp dropped to 90/54 mmhg with hr 69/min following propofol administration. the patient remained hemodynamically stable and was transferred to the icu for further management with elective ventilation. on arrival in the icu, hr was 70/min, bp 140/70 mmhg, and spo2 was 100% at fio2 0.4 on controlled ventilation. ntg infusion was restarted at 1 mcg / kg / min and increased to 2 mcg / kg / min. at 10 h postoperatively, bp had stabilized at 110/70 mmhg, hr 82/min, and spo2 100% at fio2 0.4, with ntg infusion at 0.5 mcg / kg / min. amlodepine 5 mg orally was given via a ryle 's tube. by 20 h postoperatively mechanical ventilation was gradually weaned to a t - piece and the trachea extubated 22 h postoperatively. the patient remained stable and was transferred to the ward on the third postoperative day and was discharged from the hospital on day 12, on amlodepine 5 mg once daily therapy. an 80-year - old, 48-kg, hypertensive man was scheduled for wide local excision of carcinoma lip and supra - omohyoid neck dissection. he was a smoker for 25 years and a known hypertensive (controlled with amlodepine and atenolol). lignocaine 75 mg iv was administered to attenuate sympatho - adrenergic response to laryngoscopy and tracheal intubation. were stable intraoperatively. wide local excision of carcinoma lower lip and supra - omohyoid neck dissection was performed. surgery lasted 3.5 h and blood loss was about 350 ml while urine output was > 0.5 ml / kg / h. at the end of surgery after tracheal extubation the patient was conscious and following verbal commands, hr was 62/min, bp 156/94 mmhg, and spo2 99% on 100% oxygen. ten minutes following tracheal extubation there was a progressive increase in bp up to 202/115 mmhg, with hr 65/min. morphine 1.5 mg and frusemide 5 mg were administered iv. as there was an inadequate decrease in bp (196/118 mmhg with hr 58/min), nitroglycerine (ntg) infusion arterial blood gas (abg) analysis at a fio2 of 1.0 revealed po2 65 mmhg, pco2 46 mmhg and ph 7.42. the patient was transferred to the intensive care unit (icu) for further management. the vital signs remained stable with hr 68/min, bp 130/ 86 mmhg, spo2 > 95% at fio2 0.4 and urine output > 0.5 ml / kg over 4 h. abg analysis (at fio2 0.4) revealed po284 mmhg, pco2 44 mmhg, and ph 7.43. the patient was observed overnight and transferred to the ward the following day and discharged home on the seventh postoperative day. a 68-year - old, 58 kg, normotensive male, a case of cervical lymph node metastasis with unknown primary, was scheduled for right sided rnd. surgery lasted 3.5 h and blood loss was about 250 ml with urine output > 0.5ml / kg / h. at the end of surgery the trachea was extubated as respiratory efforts were adequate and as the patient was awake and not tolerating the tracheal tube. fentanyl 50 mcg and midazolam 1 mg iv were given, but the bp increased to 246/114 mmhg with hr 96/min. a rising trend in bp was observed and ntg infusion was started at 0.5 mcg / kg / min and gradually increased to 1.5 mcg / kg / min. as the obstruction persisted, the trachea was reintubated after administering propofol 80 mg and succinylcholine 100 mg iv. tracheal intubation was not associated with a hypertensive response and surprisingly bp dropped to 90/54 mmhg with hr 69/min following propofol administration. the patient remained hemodynamically stable and was transferred to the icu for further management with elective ventilation. on arrival in the icu, hr was 70/min, bp 140/70 mmhg, and spo2 was 100% at fio2 0.4 on controlled ventilation. ntg infusion was restarted at 1 mcg / kg / min and increased to 2 mcg / kg / min. at 10 h postoperatively, bp had stabilized at 110/70 mmhg, hr 82/min, and spo2 100% at fio2 0.4, with ntg infusion at 0.5 mcg / kg / min. mechanical ventilation was gradually weaned to a t - piece and the trachea extubated 22 h postoperatively. the patient remained stable and was transferred to the ward on the third postoperative day and was discharged from the hospital on day 12, on amlodepine 5 mg once daily therapy. postoperative hypertension has been reported following carotid endarterectomy, rnd, and laryngectomy.[15 ] the reported incidence of hypertension after carotid endarterectomy is 19- 38% and after rnd it is 9.6 - 20.2%. carotid sinus denervation caused by mobilization of the carotid bifurcation during the operation has been implicated in its etiology. the carotid sinus reflex plays a central role in bp homeostasis [figure 1 ]. changes in stretch and transmural pressure are detected by baroreceptors in the heart, carotid sinus, aortic arch, and other large vessels. afferent impulses are transmitted by the carotid sinus, glossopharyngeal, and vagus nerves to the nuclei tractus solitarius and the para - median nucleus in the brain stem. efferent limbs are carried through sympathetic and vagus nerves to the heart and blood vessels, controlling hr and vasomotor tone.carotid sinus nerve injury during neck surgery leads to an interruption of signals from the carotid baroreceptors that causes stimulation of the vasomotor centre, resulting in hypertension and tachycardia. carotid sinus baroreceptor reflex pathway and physiology mcguirt and may found the incidence of hypertension following rnd to be 9.6% and that it usually occurred in the first two postoperative hours and lasted approximately 9 hours. we have earlier reported a case of severe postoperative hypertension following maxillectomy and rnd for squamous cell carcinoma of the maxilla. venkatesan. reported a hypertensive crisis following laryngectomy under general anesthesia attributed to baroreflex failure due to manipulation around the carotid sheath. acute baroreflex failure following bilateral carotid sinus denervation may produce severe labile hypertension, headache, diaphoresis and emotional instability. unilateral denervation of the carotid sinus results in temporary elevation of the bp and pulse, and no change in the postural vascular reflexes. reports vary from normalization of bp, to increased bp variability and sustained hypertension in individual patients. advanced age, hypertension, and preoperative radiotherapy have been considered important in the etiology of such hypertensive episodes. both patients described in the present report were elderly, case 1 had preoperative hypertension, but neither had received preoperative radiotherapy. bp increase in both the cases occurred after cessation of anesthetic agents (20 and 10 min in case 1 and case 2, respectively), when anesthesia - induced vasodilatation had abated. there was a drastic reduction in bp observed in case 2 following postoperative propofol administration in preparation for re - intubation. venkatesan. also found propofol to be effective in treating the hypertensive response when their patient did not respond to high doses of ntg. however, the pharmacological treatment of choice for bp surges in procedures in which baroreceptor dysfunction is anticipated is clonidine. clonidine acts centrally and peripherally to attenuate sympathetic activation and limit the extent to which pressor surges can occur. availability of alpha adrenergic agonists such as clonidine or dexmedetomedine in the operation theatre should be ensured during surgical procedures that can cause baroreflex failure in the postoperative period. inadequate analgesia and preoperative hypertension can both lead to a hypertensive response in the post operative period. analgesia was adequate in both cases as is evident from the stable intraoperative hemodynamic profile and lack of complaints of pain on awakening. additional sedation and analgesia were administered as a first measure in both patients to rule out the possibility of inadequate analgesia with no beneficial response. case 1 was an elderly hypertensive, well controlled on amlodepine and atenolol, had no episodes of intraoperative hypertension, and vital signs were within normal limits immediately following tracheal extubation. development of severe postoperative hypertension following rnd in both patients suggests baroreflex failure as the possible etiology. in conclusion, it should be suspected in elderly patients with otherwise unexplained labile hypertension in the postoperative period.	baroreflex failure results in wide excursions of blood pressure and heart rate. we report two cases that developed severe postoperative hypertension after radical neck dissection. carotid sinus denervation during neck dissection may be the cause of the reflex hypertension once general anesthesia - induced vasodilatation has ended.
management of distal ureter by en bloc resection during radical nephrectomy for upper urinary tract transitional cell carcinoma (tcc) is the standard for care. this holds true for large high - grade invasive tumours of the renal pelvis and the proximal ureter. removal of the entire ureter and bladder cuff with ureteric orifice is vital to prevent recurrence. various other minimally invasive techniques described are the pluck technique, ureteral intussusception, needloscopic technique, unroofing and electrocoagulation, laparoscopic nephrectomy with open ureterectomy and the latest being robotic nephroureterectomy. concerns remain as regards the various methods, oncologic effectiveness and the need for long - term catheter drainage. management of the lower ureter in such cases that include complete excision of the lower ureter with bladder cuff is one of the most challenging parts of surgery due to the anatomy and the need for in - depth operation of the pelvis. in this report, we describe our technique for the management of the lower ureter, which utilizes both endoscopic and laparoscopic approaches. the technique retrograde intra - vesical reconstructive surgery (rivrs), described in this article, notes the feasibility of intra - mural resection of the ureter and primary closure of the bladder with a combination of endoscopic resection and suturing. a total of three patients (two males and one female) underwent this procedure. all the patients had renal pelvic tcc and underwent laparoscopic radical nephrectomy ; the bladder cuff and the intra - mural ureter were managed with rivrs. in addition to standard laparoscopic instruments required for laparoscopic nephrectomy, 26-fr nephroscope / sheath (storz) and electrocautery hook (covidien) were used [figure 1 ]. the endosuturing was done with the help of sew - right sr5 endosuture needle (lsi solutions, victor, ny, usa) and its cartridge [figures 1 and 2 ]. the device is modified in length and diameter to enable its use through the working channel of a 24-fr nephroscope on a 26-fr sheath. (a) semi - oblique position for simultaneous laparoscopy and endoscopic procedure (b) sew - right sr5 endosuture (c) diagrammatic representation of needle passage at tissue end (d) perurethral nephroscopy endosuture needle and cartridge. (a) endosuture needle (b and c) deploying the cartridge (d) extra - corporeal knotting for loading the suture, the following steps were followed. each end of the suture used with the sew - right sr5 has a metal ferrule and comes pre - packed in a cartridge. the tip of the suturing device fits on a groove on the suture cartridge and the loop of the main body is threaded through the length of the shaft and exits through an opening on the handle [figure 1 ]. in addition, as shown in the figure, the suture is passed through the handle. step 1 : the patient is placed in a semi - oblique position, with straps over the thigh and chest as shown in figure 1. laparoscopic ports are placed, as in case of trans - peritoneal nephrectomy. following this, the point that specifically merits mention is once the uretero - gonadal packet is lifted, the ureter is immediately clipped to prevent possible inadvertent migration of malignant cells to the lower ureter. the position helps to simultaneously dissect the lower ureter and perform transurethral intra - mural dissection of the intra - mural ureter. the urethra is dilated and a 24-fr nephroscope on a 26-fr sheath is introduced per urethral. a 5-fr hook is introduced through the channel of nephroscope and ureteral orifice is circumferentially scored by electrocautery. although not done in the present patient cohort, this step can be alternatively done with a resectoscope sheath and a collings knife. the depth of incision is extended up to perivesical fat, which is laparoscopically monitored. pneumoperitoneum helps in reducing the theoretical chance of a bowel injury during intra - mural dissection and suturing. the laparoscopic surgeon simultaneously dissects the ureter up to the vesicoureteric junction (vuj), guided by the cystoscopic movement and light. the dissection of the intra - mural ureter transurethrally helps in securing adequate margin of the bladder cuff. in addition, it also guides the surgeon in performing the dissection of the lower ureter. step 3 : the next step includes suturing the bladder cuff with the sew - right device. the loaded sew - right sr5 is passed through the working channel of the 24-fr nephroscope. the initial suture approximates bladder incision in mid part, thereby dividing the suture line into two equal halves. additional sutures are placed on either side of the initial stitch to complete the procedure. typically three to five sutures are required, one to two on either side of initial stitch ; however, the number of stitches may vary. with the help of a needle selector, the first needle passes through the tissue and it automatically pulls the suture through the tissue, after which the second needle is passed and the procedure is repeated. the end result is suture - passed though both ends of the tissue, which is followed by an extra - corporeal rodders knot passed through the channel of nephroscope using a pusher [figure 3 ]. a 68-year - old male underwent left lap nephrectomy and endoscopic management of bladder cuff by rivrs the unique feature of our technique is the use of endoscopic method of suturing for closure of bladder by a special instrument designed for endosuturing, a suturing technique that has already been described for percutaneous endopyeloplasty [figure 4 ]. in addition to standard laparoscopic instruments required for laparoscopic nephrectomy, 26-fr nephroscope / sheath (storz) and electrocautery hook (covidien) were used [figure 1 ]. the endosuturing was done with the help of sew - right sr5 endosuture needle (lsi solutions, victor, ny, usa) and its cartridge [figures 1 and 2 ]. the device is modified in length and diameter to enable its use through the working channel of a 24-fr nephroscope on a 26-fr sheath. (a) semi - oblique position for simultaneous laparoscopy and endoscopic procedure (b) sew - right sr5 endosuture (c) diagrammatic representation of needle passage at tissue end (d) perurethral nephroscopy endosuture needle and cartridge. (a) endosuture needle (b and c) deploying the cartridge (d) extra - corporeal knotting for loading the suture, the following steps were followed. each end of the suture used with the sew - right sr5 has a metal ferrule and comes pre - packed in a cartridge. the tip of the suturing device fits on a groove on the suture cartridge and the loop of the main body is threaded through the length of the shaft and exits through an opening on the handle [figure 1 ]. in addition, as shown in the figure, the suture is passed through the handle. step 1 : the patient is placed in a semi - oblique position, with straps over the thigh and chest as shown in figure 1. laparoscopic ports are placed, as in case of trans - peritoneal nephrectomy. following this, the point that specifically merits mention is once the uretero - gonadal packet is lifted, the ureter is immediately clipped to prevent possible inadvertent migration of malignant cells to the lower ureter. the position helps to simultaneously dissect the lower ureter and perform transurethral intra - mural dissection of the intra - mural ureter. the urethra is dilated and a 24-fr nephroscope on a 26-fr sheath is introduced per urethral. a 5-fr hook is introduced through the channel of nephroscope and ureteral orifice is circumferentially scored by electrocautery. although not done in the present patient cohort, this step can be alternatively done with a resectoscope sheath and a collings knife. the depth of incision is extended up to perivesical fat, which is laparoscopically monitored. pneumoperitoneum helps in reducing the theoretical chance of a bowel injury during intra - mural dissection and suturing. the laparoscopic surgeon simultaneously dissects the ureter up to the vesicoureteric junction (vuj), guided by the cystoscopic movement and light. the dissection of the intra - mural ureter transurethrally helps in securing adequate margin of the bladder cuff. in addition, it also guides the surgeon in performing the dissection of the lower ureter. step 3 : the next step includes suturing the bladder cuff with the sew - right device. the loaded sew - right sr5 is passed through the working channel of the 24-fr nephroscope. the initial suture approximates bladder incision in mid part, thereby dividing the suture line into two equal halves. additional sutures are placed on either side of the initial stitch to complete the procedure. typically three to five sutures are required, one to two on either side of initial stitch ; however, the number of stitches may vary. with the help of a needle selector, the first needle passes through the tissue and it automatically pulls the suture through the tissue, after which the second needle is passed and the procedure is repeated. the end result is suture - passed though both ends of the tissue, which is followed by an extra - corporeal rodders knot passed through the channel of nephroscope using a pusher [figure 3 ]. a 68-year - old male underwent left lap nephrectomy and endoscopic management of bladder cuff by rivrs the unique feature of our technique is the use of endoscopic method of suturing for closure of bladder by a special instrument designed for endosuturing, a suturing technique that has already been described for percutaneous endopyeloplasty [figure 4 ]. mean age of the patients in our series was 58 years (range : 44 - 68 years), mean operative time for bladder cuff was 56 min (45 to 65 min) and mean time for catheter removal was 4 days (range : 3 - 5 days). average follow - up period was 9 months, with no recurrence in any case. conventional management of the distal ureter is done by the open method using extra - vesical method, intra - vesical method or a combination of both these methods. disadvantage of the intra - vesical method is the need for anterior cystotomy and prolonged catheterization period ranging 5 - 7 days. with extra - vesical dissection, cystotomy is avoided but there is a risk of incomplete dissection of the intra - mural portion of the ureter and accidental risk of contralateral ureter injury due to excessive traction. total laparoscopic techniques are associated with high risk of local recurrence, the most plausible reason being retained distal ureter in the bladder. with the intussusception technique, very high failure rate up to 18% have reported in cases where there was a disruption in the functioning of the ureter and associated with the need for additional surgical incision. few authors have reported high recurrence rate with endoscopic methods but these reports have not mentioned the location of the tumour, which might have resulted in potential bias in interpreting the results. others have reported that endoscopic methods are less invasive and equally effective with less morbidity than the open approaches. with the wide application of robotics in urology, the techniques of laparoscopic nephrectomy with robotic ureterectomy as well as total robotic nephroureterectomy have been described recently. the robotic - assisted surgery has the advantage of being able to emulate the open technique with good results, the disadvantages being the cost and the non - universal access to robot at all institutes. this report highlights the fact that management of the lower ureter in tcc pelvis can be done endoscopically without compromising the oncological principles. advantages of this minimally invasive technique include simultaneous laparoscopic endoscopic intervention, decreased chance of injury to the opposite ureter, controlled adequate tissue dissection, primary bladder closure, short catheterization period, early ambulation and decreased hospital stay. the disadvantages are the requirement of specially designed instrument(s) for intra - vesical suturing and the need for two trained surgeons and additional expertise required for endoscopic suturing. the described technique is not suitable for the management of lower ureteric tumours encroaching in the bladder, as the defect would be challenging to suture. we conclude that rivrs is a safe, efficient, minimally invasive and reproducible technique for lower ureter resection in tcc pelvis with primary bladder closure.	management of distal ureter by en block resection during radical nephrectomy for upper urinary tract transitional cell carcinoma (tcc) is considered as standard of care. in this report, we describe our technique for management of lower ureter which utilizes both the endoscopic and laparoscopic approach. the nephrectomy including the dissection of the lower ureter was completed laproscopically. the ureteral orifice was scored using a hook passed through a 24 fr nephroscope and secured.transurethral suturing of the defect with sewright sr5 device passed through the working channel of the 24 fr nephroscope was done. our report highlights the fact that management of lower ureter in tcc pelvis can be done endoscopically / laproscopically without compromising the oncological principles. our novel technique demonstrates feasibility of intra mural resection of the ureter and primary closure of the bladder endoscopically.
overaction of the inferior oblique muscle (ioom) is manifest by overelevation of the adducted eye. inferior oblique muscle overaction may be primary or it may be secondary to ipsilateral superior oblique palsy or contralateral superior rectus palsy. the primary and secondary overactions of the inferior oblique muscles have different clinical presentations (1, 2, 3, 4, 5). primary overaction of the inferior oblique muscle may have its onset equally in the two eyes or it may occur only in one eye. primary ioom is characterized by elevation of the eye in adduction, with smaller or minor vertical deviation in the primary position, with smaller or minor torticollis and negative bielschowsky test. the vertical deviation in the primary position in secondary overaction of the inferior oblique muscle usually ranges between 10 to 25 pd (prism diopters). secondary hyperfunction of oblique muscle is caused by ipsilateral or contralateral paresis of superior oblique muscle, when we, also, have the eye elevation in adduction, but vertical deviation is present in primary position too, with strong torticollis and positive bielschowsky test. overaction of the inferior oblique muscle or hypertropia can be isolated or combined with other types of deviations, according to parks, 72% of patients with congenital esotropia, 34% of patients with accommodative esotropia, and 32% of patients with intermittent exotropia (1, 2, 3, 4, 5, 6, 7). surgeries of weakening of iooa are done because of functional, but the aesthetic reasons too. the variety surgical procedure have been performed including : recession, hang - back recession, myotomy, myectomy, anterior transposition, nasal transposition, denervation and muscle fixation. myotomy is simplier version of myectomy, is not technically complicated and is easy to perform ; freed oblique muscle is cutted - down, and bonds are freed, after cauterization. recession is, according to many authors, the best method of inferior oblique muscle weakening and according to parksu dominant to all other methods. isolated muscle is recessed 2 mm laterally and 3 mm temporally or more towards lateral edge of insertion of inferior rectus muscle. by this method, recession can be adopted and dosed in accordance to the hyperfunction degree ; for 1 + overaction, the inferior oblique muscle is recessed 6 mm ; for 2 + overaction 10 mm and for 3 + overaction 14 mm, which is the maximum recession (2, 3, 4, 5, 6). but, the decision on choosing th surgical procedure is individual and is based on personal experience of surgant (3). indications for these surgeries, whether inferior oblique overaction is primary or secondary, are to treat hypertropia, diplopia, binocular vision compromise in the field of adduction, torticollis, and to improve cosmetic appearance. however, these surgeries may have effect not only on vertical deviation, but also on horizontal deviations in varying degrees (4). when the hyperfunction is unilateral, usually the only one muscle is weakened, and if the hyperfunction is bilateral, weakening procedure is done symmetrically. also, inferior oblique muscle weakening procedure, in case of existence of horizontal deviation, is done at the same time with correction of horizontal deviation (3, 4). during and after surgery, in spite of procedure the possible surgery and postoperative complications must be considered. one of them is intraoperative and postoperative bleeding and hematoma in orbit, adherent syndrome, postoperative and continued hyperfunction of iom, hypotropia, internal ophthalmoplegia etc (3, 4, 5, 6, 7). the study was conducted as a retrospective, clinical, controlled and descriptive study of a period of five years at the department of strabology and pediatric ophthalmology in eye clinic of the university clinic center sarajevo. we evaluated total number of 33 patients who had primary inferior oblique muscle overaction, isolated or combined with esotropia. was measured ; vertical deviation with the prism cover test or krimsky test, on primary position and 9 diagnostic gaze positions, hess - lancaster test for diplopia testing and binocularity which we tested with lang and titmus fly tests 3, 6 and 12 months after surgery. patients were divided into two groups : in one group, depending on vertical deviation, the weakening of inferior muscle overaction done by recession and in other group ; weakening of inferior muscle overaction myotomy was done. the date is shown in tables according to the number of cases, percentage, arithmetic mean, standard deviation, median, and range were given as descriptive statistics for quantitative data. for testing of predicted hypothesis and proving the goals, we used the hi - square test, student s test and pearson s or spearman s correlation coefficient test. results were accepted as statistically significant on significance level of p0,05 (figure 1). x2=0,337 ; p=0,419 analysis of average age during the surgery shows that the patients were operated in the average age of 10,67,5 (age 4 to 36), and that the patients in recession group were older with average age of 12,48,1 (age 5 to 36), comparing with patients in myotomy group with average age of 6,53,8 (age 4 to 17). statistical analysis shows the significant difference in average age during the surgery in accordance to type of surgery presented with significance level of p0,05. t=2,599 ; p=0,117 in total number of patients, postoperative angle of vertical deviation was in average 1,40,96 (range 0 - 4). the values in myotomy group were 1,50,8 (range 0 - 3) and 1,31,0 (range 0 - 4) with patients in recession group, but without statistically significant difference, in accordance to type of surgery p>0,05 (figure 5). t=2,599 ; p=0,117 binoculararity was at 65,2% in recession group and at 20% of the patients in myotomy group, which is statistically significant shown with significance level of p0,05 (8). he observed a persistent inferior oblique muscle overaction in 37% of patients and inferior oblique muscle underaction in 8% of patients after an inferior oblique muscle myectomy and 13% incidence of inferior oblique muscle adhesive syndrome when the myectomy was performed at the inferior oblique muscle insertion (2, 3, 4, 5, 7). after the surgery, the angle of vertical deviation decreased, and was in average 1,40,96 degrees. in myotomy group there was 1,50,8 (range 0 - 3), in recession group 1,31,0 (range 0 - 4), but without statistically significant difference according to type of surgical procedure p>0,05. cooper and sandall found that a measured recession will decrease the hyper deviation by 6.88 prism dioptry in primary position and by 12.3 prism diopters in the field of action of the overacting inferior oblique muscle. they analyzeded recession and myotomy group, bat no data about binocularity (9). shipman and burke had total of 23 patients, 12 myectomies and 11 recessions and follow - up period of 12 months. the average preoperative hyper deviation in contralateral gaze was 26.5 prism diopters in the myectomies and 20 prism diopters in the recessions. this was reduced at 12 months postoperatively to 1.75 prism diopters in the myectomies and to 3 prism diopters in the recessions. both procedures were largely self - grading, so that the larger the preoperative hyper deviation, the greater the effect of surgery (10). sekeroglu and coauthors did the qualitative analysis at 76 patients, 30 male and 36 female, average age 11(1 - 49). out of 76 patients, 54,5% had secondary hyperfunction of iom, and 45% primary hyperfunction of iom. they did not found any difference between different types of surgery in terms of horizontal deviations correcting effect. ehrt and others had retrospective studies of 234 patients in the age of 2 - 81, during the period of 9 years, where the quantitative analysis iom weakening effect by recession, and described reduction of vertical deviation from 25 degrees to 6. they propose this method as safe and suitable, but with smaller risk from consecutive hypotropia and limited elevation, which they get in one case (12). risovi and all in their study had 79, divided into two groups : first as a primary overaction iom, and other and dissociated vertical deviation. binoculararity was found at 67% patients from first group and 55,6 % patients from second group, but without big statistical significant difference between groups (13). the intention of the present study was not to evaluate effect of weakening of inferior oblique muscle on horizontal deviation, but to investigate it s effect to condition of binocular vision. when it comes to this, the better results were at recession as method of inferior oblique muscle weakening was used. recession is safe and effective procedure to eliminate inferior oblique overaction and can be dosed and adopt to overaction. but, when planning of surgery of inferior oblique muscle weakening, the knowledge and experience on it s influence to horizontal deviation and binocularity i needed, as well as dosing and possible operative and postoperative complications.	introduction : inferior oblique overaction (iooa) can be primary or secondary, isolated or combined to other types of horizontal deviation, mostly with esotropias. surgical weakening of iooa means several techniques like ; recession, myotomy, myectomy, anteroposition etc.goals:we analyzed the effect of inferior oblique muscle surgical weakening comparing two groups of patients with primary hypertropia.material and methods : in 5-years retrospective study, we observed 33 patients on which we did the surgical procedure of weakening inferior muscle overaction by two methods ; recession and myotomy.results:in total number of 33 patients, there were 57,6% male and 42,4% female patients with average age of 10,67,5 (in range of 436). there was 33,3% of isolated primary hypertropias, and 66,7% combined with esotropias. at 23 (69,9%) patients the recession surgical procedure was done, and with 10 (30,1%) myotomy. better effect and binocularity was in 65,2% of patients in recession group which was statistically significant with significance level of p<0,0, 2=5,705 ; p=0,021.conclusion : comparing of two surgical procedures of weakening inferior oblique muscles overaction, recession is better procedure than myotomy.
thiazide diuretics include all diuretics believed to have a primary action to inhibit nacl reabsorption in the distal convoluted tubule (table 1) and have been used in the management of hypertension for over 50 years1, 2). these agents decrease blood pressure (bp) when administered as monotherapy, enhance the efficacy of other antihypertensive agents, and reduce hypertension - related morbidity and mortality. however, the use of thiazide diuretics is often limited by concerns about some metabolic change, e.g., hyponatremia, hypokalemia and insulin resistance3). hyponatremia is an occasional but potentially fatal complication of diuretic therapy. the first detailed description of diuretic - induced hyponatremia was published over 35 years ago4). thiazides are a common cause of severe hyponatremia in hospitalized patients5) and are associated with high morbidity and mortality6, 7). virtually all cases of severe diuretic - induced hyponatremia have been due to a thiazide - type diuretic6 - 12). this observation is probably derived from the different nephronal sites of action of these two classes of diuretics. thiazide diuretics inhibit nacl reabsorption in the distal convoluted tubule, the main diluting site of the nephron. thus, thiazide diuretics interfere with maximum dilution of urine because sodium (na) excretion is increased along with diminished free - water excretion13, 14). on the other hand, loop diuretics inhibit nacl reabsorption in the thick ascending limb of the loop of henle. the reabsorption of nacl without water in the medullary thick ascending limb is normally the primary step in the generation of the hypertonicity in the medullary interstitium, and loop diuretics mainly impair urinary concentration and limit water retention and development of hyponatremia15). although thiazide diuretics are frequently recommended as the first - line anti - hypertensive according to the practice guidelines and results of the major clinical trials such as the antihypertensive and lipid - lowering treatment to prevent heart attack trial (allhat)16 - 18), the mechanism and optimal treatment of thiaizde - induced hyponatremia (tih) remain unclear. in this review, we will explore the pathogenesis, discuss the clinical features, and suggest an approach to the treatment of tih. electrolyte disturbance is common after administration of thiazide diuretics, and hyponatremia is a typical complication with an estimated incidence of 11% in one series of 114 geriatric patients19). in the systolic hypertension in the elderly program (shep), which focused on older patients, hyponatremia was observed in 4.1% of patients treated with chlorthalidone versus 1.3% in the control group20). thirty percent of patients were receiving 12.5 mg / day of chlorthalidone, and 60% were receiving 25 mg / day20). in another study on elderly patients, fifty three percent of patients were receiving a thiazide diuretic and 24% a loop diuretic21). in patients with tih, an average daily hydrochlorothiazide dose of 35 mg has been reported, with 44% of patients having received 50 mg22). on the other hand, in 10% of the cases with tih the dose was only 12.5 mg23). considering the high prevalence of hypertension in the elderly and the routine use of thiazide diuretics to treat it, tih is clearly a prevalent problem. the question arises as to whether a certain subgroup of patients is susceptible to this adverse effect of thiazides. the risk factors of tih are old age, women, reduced body mass7, 9, 13, 14, 22, 24), and concurrent use of other medications that impair water excretion13, 25). as shown in table 2 which was obtained by the recent nephrology consultation in our hospital, old women are clearly predisposed to tih. according to the experiment done by clark.13), healthy elderly subjects do not excrete free water as efficiently as younger ones do. in particular, elderly subjects with a previous history of tih have impaired urinary dilution capacity, and hence, increased susceptibility to tih. among the explanations, blunted prostaglandin synthesis is an attractive possibility25), and could result from aging or propensity among the elderly for polypharmacy, including non - steroidal anti - inflammatory agents. the reason why individuals with reduced body mass are more prone to complications from diuretics remains puzzling. plasma na level is determined by the ratio between the amount of exchangeable na and potassium (k) and the total body water26). thus, it is conceivable that na concentration might change to a greater degree in a subject with smaller body size, and hence less total body water. besides numerous case reports have indicated that hypertensive women are particularly at risk to develop hyponatremia7, 9, 13, 14, 22, 24). the reason that women are more susceptible to the effects of thiazide diuretics than men is unknown. women probably drink more than men to compensate for the diuretic and therefore develop more dilutional hyponatremia28), and hypertensive women may be more often treated with diuretics than hypertensive men29). moreover, women may be more symptomatic than men at comparable serum na levels and therefore may be diagnosed more often than men30). the clinical manifestations of tih are similar to those due to other causes of hyponatremia. one of the most remarkable features of tih is the rapidity with which it can occur. in susceptible individuals, the serum na may fall within hours of administration8), and severe hyponatremia can develop within less than 2 days7, 14). in most reported cases (50% to 90%) the duration of thiazide use was less than 2 weeks7, 8, 14, 22). however it can occur any time during thiazide therapy when sub - sequent contributory factors are complicated such as old age, increased water intake, low salt intake7, 9, 13, 14, 22, 24), and concurrent use of other medications that impair water excretion13, 25). mild hyponatremia, with serum na concentration in the range of 125 to 132 meq / l, is usually asymptomatic, although vague symptoms such as fatigue or nausea are possible3). more severe hyponatremia, as reported in patients who required hospitalization, can be asymptomatic or associated with symptoms including weakness, vomiting, nausea, confusion, dizziness, abdominal pain, and manifestations as serious as lethargy, seizures, and coma11, 22). the volume status of tih may be variable. while some patients are volume depleted on presentation, most appear to be euvolemic10, 14, 31, 32). reduced free - water clearance can explain why many patients with tih behave as if they are volume expanded ; the body weight may initially increase8). in patients with tih, blood urea nitrogen (bun) and plasma creatinine concentration are generally low - normal10, 33), and hypouricemia due to enhanced urinary uric acid excretion may be present32, 33). all of these findings are similar to those in the syndrome of inappropriate antidiuretic hormone secretion (siadh), which is also associated with initial volume expansion. the laboratory data from our tih patients are also compatible with those of siadh (table 2), similarly to reports from other studies8 - 11, 13). according to sonnenblick.32), elevated fractional uric acid clearance and low serum uric acid level were useful to determine that tih was more favored than furosemide - induced hyponatremia. although the traditional view is that diuretic - induced na or volume loss results in vasopressin - induced water retention, the following 3 main factors are implicated in tih : increased water intake, reduced free - water clearance and renal na and/or k loss (table 3). there is evidence that water intake is maintained or increased in many patients with tih6, 8, 27). friedman.8) showed that within 6 hours of ingesting a single hydrochlorothiazide - amiloride tablet, previously affected patients had a small rise in urine osmolality and a fall in serum na of 5.5 meq / l in association with a small gain in weight ; controls had only a slight fall in serum na, and their mean weight fell. although water intake was not measured, the authors suggested that thiazides might cause polydipsia, which, when combined with the renal effects, results in expansion of total body water and development of hyponatremia. stimulation of water intake would not only help defend the extracellular volume, but would also explain why thiazides are one of the most common causes of severe hyponatremia. increased water intake would not lead to hyponatremia unless impairment in water excretion exists. while thiazide diuretics do not inhibit concentrating ability as mentioned above, they inhibit electrolyte transport at the cortical diluting sites, thereby raising the minimum urinary osmolality34 - 36). diuretics may also reduce glomerular filtration rate and enhance reabsorption of na and water in the proximal nephron, diminishing fluid delivery to the distal diluting sites35). besides, thiazide - induced volume depletion probably contributes to the genesis of tih in some cases. hypovolemia may stimulate the release of vasopressin because non - osmotic vasopressin secretion is very common in normovolemic and hypovolemic types of hyponatremia31). a previous in vitro microperfusion study showed that hydrochlorothiazide enhanced water absorption in the inner medullary collecting duct from normal rats and brattleboro rats as well38). consistent with this, kim. reported that the paradoxical antidiuresis induced by hydrochlorothiazide in nephrogenic diabetes insipidus was associated with upregulation of aquaporin-2 in the collecting duct39). first, virtually all relevant studies have found that during the development of tih, na balance is negative4, 6, 10). argued that changes in external na and water balance did not completely account for the fall in serum na4). furthermore, chlorothiazide induced hyponatremia in their patients despite na supplementation and positive na balance. these observations led the authors to conclude that a shift of na into the intracellular space contributed to the pathogenesis of hyponatremia. second, once diuretics are withdrawn, urinary na excretion falls to very low levels4, 10). the great majority of their 25 patients was hypokalemic, and hyponatremia was corrected in 4 of them by k repletion despite continued diuretic use and na restriction. these investigators argued that k depletion predisposes the patients to hyponatremia because the serum na concentration is dependent upon the ratio of the sum of exchangeable na and k to total body water. they also speculated that k depletion might cause a shift of na into the intracellular space, thereby further compromising the extracellular volume and stimulating vasopressin release. na and/or k depletion alone would not be expected to cause marked hyponatremia because water excretion normally increases as the serum na concentration falls. however, because thiazides interfere with renal water excretion, they impair this normal osmoregulatory response. thus, as in most patients with hyponatremia, urine osmolality is inappropriately high relative to the osmolality of plasma and often exceeds it4, 6, 10, 31, 32). nonsteroidal anti - inflammatory drugs, through prostaglandin inhibition, decrease free - water clearance13, 25). drugs such as chlorpropamide or selective serotonin reuptake inhibitors may be associated with siadh3). the acute management of tih is more determined by the presence or absence of neurologic symptoms than by the na level per se. treatment consists of discontinuing thiazides, regular diet (usually supplemented with k) restricting water, administration of furosemide and either isotonic saline or, if the hyponatremia is severe or symptomatic, hypertonic saline. in patients with hypovolemic tih, in whom vasopressin secretion stimulated by hypovolemia contributes to the inability to excrete free water, normal saline infusion is appropriate because it will restore volume and suppress vasopressin release. in patients with severe manifestations such as seizures or coma, initiation of treatment with hypertonic saline to assure rapid onset of response is recommended3, 16). we treated our patients with tih using either isotonic or 3% hypertonic saline (table 4). the data suggest that the severity of hyponatremia might influence the choice between isotonic and hypertonic saline. in patients with euvolemia or hypervolemia - associated hyponatremia, who tend to have lower serum levels of uric acid, bun, and creatinine, normal saline will not restore free - water clearance or serum na concentration. in such patients, administration of furosemide in addition to the hypertonic saline can increase free - water clearance and thus hasten the increase of the serum na concentration while avoiding volume overload5). in asymptomatic or minimally symptomatic patients, stopping the offending diuretic and restricting water intake to < 1 l / day is usually all that is needed. normal saline is not needed unless correction of volume depletion is indicated. increasing oral salt intake in combination with fluid restriction can increase serum na concentration3). how can tih be prevented ? there are no guidelines regarding measures to prevent tih. the first step in prevention of tih is awareness that it can happen, particularly in the elderly, female, or small individuals. in susceptible individuals, the serum na may fall in hours of diuretic ingestion, and severe hyponatremia can develop within 2 days7, 14). therefore in susceptible patients, serum na should be measured within one day after beginning therapy. if the serum na level falls more than a few meq / l, the diuretic should be stopped. if there is little or no change in the serum na, it should be rechecked 1 - 2 days later to be sure the level is stable14). identifying patients with excessive fluid intake and counseling them to reduce their intake would be extremely helpful and cost - effective. when prescribing thiazides, low doses should be used3). a lower dose would increase the ability to excrete free - water, would reduce the hypovolemic stimulus for vasopressin secretion, and can correct borderline hyponatremia in many cases3). even patients who have done well on chronic thiazide therapy may develop severe hyponatremia if water intake increases. therefore, all patients using these drugs should be educated about the danger of drinking excessive water3, 14).	the importance of thiazide - induced hyponatremia (tih) is reemerging because thiazide diuretic prescription seems to be increasing after the guidelines recommending thiazides as first - line treatment of essential hypertension have been introduced. thiazide diuretics act by inhibiting reabsorption of na+ and cl- from the distal convoluted tubule by blocking the thiazide - sensitive na+/cl- cotransporter. thus, they inhibit electrolyte transport in the diluting segment and may impair urinary dilution in some vulnerable groups. risk factors predisposing to tih are old age, women, reduced body masses, and concurrent use of other medications that impair water excretion. while taking thiazides, the elderly may have a greater defect in water excretion after a water load compared with young subjects. hyponatremia is usually induced within 2 weeks of starting the thiazide diuretic, but it can occur any time during thiazide therapy when subsequent contributory factors are complicated, such as reduction of renal function with aging, ingestion of other drugs that affect free water clearance, or changes in water or sodium intake. while some patients are volume depleted on presentation, most appear euvolemic. notably serum levels of uric acid, creatinine and urea nitrogen are usually normal or low, suggestive of syndrome of inappropriate secretion of antidiuretic hormone. despite numerous studies, the pathophysiological mechanisms underlying tih are unclear. although the traditional view is that diuretic - induced sodium or volume loss results in vasopressin - induced water retention, the following 3 main factors are implicated in tih : stimulation of vasopressin secretion, reduced free - water clearance, and increased water intake. these factors will be discussed in this review.
the obesity rate in the united states has reached an epidemic level, with approximately one - third of the adult population classified as obese [body mass index (bmi) 30 ]. according to the national health nutrition examination surveys (nhanes), 64.1% of women were overweight or obese between the years 2007 and 2008 (2). differences in overweight and obesity prevalence exist among adults of various ethnic and racial backgrounds (2). in addition, certain sociodemographic factors such as income / socioeconomic status (ses) and education may contribute to overall weight status (3). overweight and obesity are concerning conditions because numerous studies have suggested a strong correlation between obesity and the onset of chronic diseases such as cardiovascular disease and type 2 diabetes (4). the centers for disease control and prevention estimate that 17% of youth aged 219 years are obese (5). research also suggests that parents are children 's immediate role models and can impact their children 's lifestyle, including food consumption and physical activity (6). according to the world health organization (who), obesity is a concerning global epidemic that is growing at an exponential rate. who states that the potential cause of obesity is consumption of foods that are high in energy density, are low in nutrient density, and lack vitamins, minerals, and macronutrients (7), combined with sedentary lives. obesity is caused by an energy imbalance resulting from excessive caloric intake and low energy expenditure, as discussed below. thus far, all research indicates that women who consume diets that are high in fat and low in fiber tend to be the most vulnerable to development of overweight or obese weight status. the overall recommendation for weight control is to consume diets that are rich in fiber, fruits, and vegetables and low in fat, especially saturated and trans fat, while engaging in regular physical activity (7). according to the 2008 physical activity guidelines for americans, adults between the ages of 18 and 64 years should get at least 150 min of moderate - intensity physical activity per week to maintain good health or 300 min of moderate - to - vigorous physical activity (mvpa) for weight loss (8). physical activity has been suggested to help lower hypertension, type 2 diabetes, heart disease, and metabolic syndrome. evidence also suggests that physical activity combined with a well - balanced diet is the most effective obesity preventative method (9, 10). studies have shown that parental modeling is an important factor in determining the level of physical activity and weight status in children (11). indeed, a greater level of mvpa is seen in children whose parents also have a higher level of mvpa (12). additional studies further suggest the influence of parenting style on children 's objectively measured physical activity (13). conversely, the presence of sedentary behaviors such as television time also correlates well between parents and their children (14). in line with these findings, hills. proposed that one productive approach to childhood obesity would be to support parents, teachers, and healthcare workers to encourage children to be physically active (15). since children have been shown to be influenced by parents modeling of physical activity and sedentary behavior and by encouragement of healthy eating in all socioeconomic conditions, it is important to identify the parental behaviors of children who are fit. we hypothesized that parents of children who exhibit healthy behaviors and lower bmis than the average us pediatric population are characterized by lower rates of obesity, higher physical activity, and lower energy intakes. to test this hypothesis, we determined the strongest predictors of weight status of female parents whose children are fit. more specifically, we determined (1) what parental behaviors are predicted to be associated with parents overweight and obesity, (2) any relationship between parents and children 's bmi, and (3) what is different about parents whose children exhibit healthy behavior. we studied mothers of fit children who are enrolled in a creative afterschool program to examine whether these mothers were behaviorally and anthropometrically different from the rest of the nation. the study reported here was part of a larger project investigating health behaviors and practices of adolescents (n=4,608) who participated in the destination imagination (di) global finals held in knoxville, tn, in may 2008 and 2009. the current study surveyed a total of 1,188 parents, of which 1,032 were female parents / guardians of this adolescent population. self - reported survey and anthropometric data were collected from a sample of parents who gave consent for their children to participate in the student study. parents received a letter and code number 1 week after their children competed in the di global finals. the letter thanked the parents for allowing their children to participate in the study and requested parents participation in a survey, either online or via mail. the letter contained the url for the online survey, informed consent for parents participation, and the code number, which linked parents data to their children 's data. we report here results obtained for the female parents, who were 87% of parents responding. surveys consisted of dietary, physical activity, and weight perception / management questions from the 2007 behavioral risk factor surveillance system (brfss) (16), along with (1) the adult version of the macarthur scale of subjective social status and (2) the rapid food screener (17) (conducted on a subset of the participants), which was developed at the university of california, berkeley, ca. specifically, the survey collected the following data : (1) how often respondents consumed fruit juice, fruit, green salad, potatoes (not including french fries, fried potatoes, or potato chips), carrots, and other vegetables ; (2) an inventory of foods present in the home (various categories of milk / dairy, cheese, salad dressing, cereal, bread / pasta / rice, baked goods, fresh vegetables, frozen vegetables, fresh fruit, meat / sausage, crackers / chips, and legumes) ; (3) grocery - shopping habits ; (4) frequency of eating away from home ; (5) amount and frequency of moderate or vigorous physical activity ; (6) standing in society in relation to money, education, and employment ; (7) standing in the community ; (8) assessment of weight (underweight / overweight, change in weight, and efforts to change weight) ; (9) relationship to di participant ; (10) age ; (11) race ; (12) marital status ; (13) education ; (14) income ; and (15) height and weight. data analyses included descriptive statistics, chi - squared test for cross - tabulation of weight status between parents and their children, correlations between bmi and the variables listed above, and linear regression for predictors of bmi. demographic information describing the parents is shown in table 1, which includes race / ethnicity, household income, marital status, and age. of respondents, 95.8% were white and 54.7% had annual incomes over $ 100,000, with 77.1% over $ 75,000. the overwhelming majority (92%) was married, and the mean age was 43.56 years. figure 1 shows the bmi categories for these female parents, the majority of whom were within the normal weight and with only 45.2% being overweight / obese. bmi distribution in each of the following four categories is indicated : underweight, normal weight, overweight, and obese. female parents demographic characteristics and descriptive statistics (n=1,032) table 2 shows a significant association between parent and child weight status. clearly, the weight category of children was significantly related to that of their parents [(9) = 83.82, p<0.001 ]. for parents who were underweight, none of their children were overweight or obese. for parents who were obese, however, 40% of their children were either overweight or obese. 2, our study showed that the percentage of calories from fat was significantly lower in women with normal weight status compared to women who were overweight or obese (p=0.001). this specific analysis was conducted only on a subset of participants (681 parents of children participating in the 2008 surreys). a validated rapid screener was used to assess fat intake in a subset of the participants (n=681) ; p<0.05. to determine the factors that influence bmi, we used linear regression with physical activity (total minutes of mvpa), income, social ladder, and weight perception as independent variables (see table 3). since bmi and minutes of physical activity were positively skewed, we took the natural logarithms of each. all factors were significant and explained 65% of bmi 's variance [f(3,953) = 352.52, p<0.001 ]. influence of bmi by physical activity, income, social ladder, education, and weight perception as independent variables dependent variable : bmi_ln. we also used pearson correlations to study the impact of each of the above variables one by one (table 4). as expected, log bmi correlated negatively with physical activity (0.25), education (0.16), and income (0.25). since a small value on the social scales indicated a high score, log bmi was positively correlated with social (0.27) and even community (0.18). self - perception of weight provided the highest correlation (0.79), indicating that the subjects had an accurate perception of their weight. given our large sample size, most of these correlations were significant (0.01 or 0.05, two - tailed), including for the community ladder, which did not add significantly to the overall linear regression (table 3). pearson correlations (two - tailed for various variables) correlation is significant at the 0.05 level (two - tailed). in this study, we tested the hypothesized that parents of children who exhibit healthy behaviors and lower bmis than the average us pediatric population are characterized by lower rates of obesity, higher physical activity, and lower energy intakes. of female parent participants, 53.5% were normal weight, 27.1% were overweight, and 18.1% were obese (fig. self - reported data from the 2007 brfss indicate that only 44.3% of female respondents were normal weight, 29.8% were overweight, and 25.6% were obese (16). thus, our sample seems to have lower rates of overweight and obesity than the national sample. among multiple variables assessed, the strongest predictors of bmi were income, fat intake, and time spent in moderate and vigorous physical activity. in a substudy of the bogalusa heart study that examined the relationship between diet, ses, and demographic factors, adults with higher incomes had healthier diets than adults in the study with lower incomes. these findings are important, as strong evidence exists suggesting that diet quality significantly impacts weight status (3). we believe that the differences in bmi found in our study are not related to these factors, however, as we compared a homogenous sample of participants with similar sex (female), income, education, and ses. those females who were the most physically active (mvpa) self - ranked highest in the macarthur scale of subjective social status. among multiple variables assessed, the strongest predictors of bmi were income, fat intake, and time spent in vigorous physical activity. interestingly, self - reported bmi correlated strongly with how these females described and perceived their weight, which is not usually the case according to the trend of underestimating weight and bmi by adults as documented by gorber. (18). the females who were within the normal weight category in our study had significantly lower percentage energy intake from fat compared to those who were overweight or obese. indeed, other studies, including one by miller., compared food consumption of lean adults to that of obese adults and found that leaner women consumed less energy from fat and more fiber compared to obese women. further, the study found that obese adults consumed more energy from sugar - added foods compared to their lean counterparts (19). nevertheless, fat intake in our study population was consistent with reported average fat intakes in the united states (20). similar findings were shown by a 12-year longitudinal study of diet macronutrient consumption and its association with overweight development of 737 non - overweight women. the study reported a 29% risk of developing overweight for the subjects consuming high fat diets, while women consuming more fruit, vegetables, and low - fat dairy and meat had overall lower risks, when compared to other dietary groups in this study (21, 22). thus far, research evidence supports weight and health benefits from consuming diets, which are rich in fiber, fruit, and vegetables and low in fat. this recommendation also is supported by major professional and scientific associations. in our study, the mean mvpa of female parents was 360 min / week. according to nhanes 20052006 data, only 55.8% of adult females in the united states meet the recommended physical activity guidelines (23). thus, the findings about the physical activity of the mothers surveyed in our study are consistent with their lower bmis. indeed, previous studies have shown that a higher fitness level is associated with leisure - time physical activity in young to middle - aged adults (24). overall, research strongly suggests that women following the physical activity recommendations had significantly lower bmis and waist - to - hip ratios and improved cardiorespiratory fitness (10). we have previously shown that children participating in a creative afterschool program were more physically active (compared to national averages in meeting recommendations) and exhibited overall lower bmi rates, lower screen times, and higher intake of fruits, vegetables, and milk (25). in the current study, we found that the female parents were highly physically active (based on recommended amounts of mvpa). although we previously reported that their children also were physically active, a direct cause - effect can not be established within this study. bmi of children and their parents were closely associated, as the prevalence of obesity and overweight in children was higher when parents also were overweight or obese (table 2), indicating close association between parent and children weight status ; this relationship could be due to both genetic and environmental influences. given the high physical activity of this parent population, however, it is possible that the family lifestyle influenced children 's weight status. these results also are consistent with previous reports showing associations between parents bmi and that of their children (26). of female parent participants, 53.5% were normal weight, 27.1% were overweight, and 18.1% were obese (fig. self - reported data from the 2007 brfss indicate that only 44.3% of female respondents were normal weight, 29.8% were overweight, and 25.6% were obese (16). thus, our sample seems to have lower rates of overweight and obesity than the national sample. among multiple variables assessed, the strongest predictors of bmi were income, fat intake, and time spent in moderate and vigorous physical activity. in a substudy of the bogalusa heart study that examined the relationship between diet, ses, and demographic factors, adults with higher incomes had healthier diets than adults in the study with lower incomes. these findings are important, as strong evidence exists suggesting that diet quality significantly impacts weight status (3). we believe that the differences in bmi found in our study are not related to these factors, however, as we compared a homogenous sample of participants with similar sex (female), income, education, and ses. those females who were the most physically active (mvpa) self - ranked highest in the macarthur scale of subjective social status. among multiple variables assessed, the strongest predictors of bmi were income, fat intake, and time spent in vigorous physical activity. interestingly, self - reported bmi correlated strongly with how these females described and perceived their weight, which is not usually the case according to the trend of underestimating weight and bmi by adults as documented by gorber. (18). the females who were within the normal weight category in our study had significantly lower percentage energy intake from fat compared to those who were overweight or obese. indeed, other studies, including one by miller., compared food consumption of lean adults to that of obese adults and found that leaner women consumed less energy from fat and more fiber compared to obese women. further, the study found that obese adults consumed more energy from sugar - added foods compared to their lean counterparts (19). nevertheless, fat intake in our study population was consistent with reported average fat intakes in the united states (20). similar findings were shown by a 12-year longitudinal study of diet macronutrient consumption and its association with overweight development of 737 non - overweight women. the study reported a 29% risk of developing overweight for the subjects consuming high fat diets, while women consuming more fruit, vegetables, and low - fat dairy and meat had overall lower risks, when compared to other dietary groups in this study (21, 22). thus far, research evidence supports weight and health benefits from consuming diets, which are rich in fiber, fruit, and vegetables and low in fat. in our study, the mean mvpa of female parents was 360 min / week. according to nhanes 20052006 data, only 55.8% of adult females in the united states meet the recommended physical activity guidelines (23). thus, the findings about the physical activity of the mothers surveyed in our study are consistent with their lower bmis. indeed, previous studies have shown that a higher fitness level is associated with leisure - time physical activity in young to middle - aged adults (24). overall, research strongly suggests that women following the physical activity recommendations had significantly lower bmis and waist - to - hip ratios and improved cardiorespiratory fitness (10). we have previously shown that children participating in a creative afterschool program were more physically active (compared to national averages in meeting recommendations) and exhibited overall lower bmi rates, lower screen times, and higher intake of fruits, vegetables, and milk (25). in the current study, we found that the female parents were highly physically active (based on recommended amounts of mvpa). although we previously reported that their children also were physically active, a direct cause - effect can not be established within this study. bmi of children and their parents were closely associated, as the prevalence of obesity and overweight in children was higher when parents also were overweight or obese (table 2), indicating close association between parent and children weight status ; this relationship could be due to both genetic and environmental influences. given the high physical activity of this parent population, however, it is possible that the family lifestyle influenced children 's weight status. these results also are consistent with previous reports showing associations between parents bmi and that of their children (26). only 45% of these female parents were overweight / obese, compared to a national average of 64.1% for females. most parents (76%) had completed a college degree and earned over $ 75,000 a year. parents who reported the lowest income were the most obese in this population. among multiple variables assessed, the strongest predictors of bmi were income, fat intake, and time spent in vigorous physical activity. those females who were the most physically active self - ranked highest in the macarthur scale of subjective social status. interestingly, self - reported bmi correlated strongly with how these females described and perceived their weight, which is not usually the case. these studies demonstrate that female parents of children participating in an afterschool creative program were physically active, which likely accounts for the parents lower overweight / obesity rates. we found that this group of mothers exhibited much healthier weight status and lifestyle behaviors compared to their counterparts in the general population. these findings further support the importance of parent modeling (6) in the management of overweight and obesity in children. race was not considered in our study, as the minority sample was small (4.2%). according to flegal., however, significant differences exist in women of various minority backgrounds, compared to the overall overweight and obesity prevalence among women in the united states (64.1% in 2008). in non - hispanic white women, obesity prevalence was 33%, whereas obesity was found to be at 49.6% among non - hispanic black women. further, the study found that, in comparison to non - hispanic white women, non - hispanic black and mexican - american women were more significantly more likely to be obese (odds ratio 2.26 and 2.51, respectively). approximately 7% of us women are classified to be at the grade 3 level of obesity (bmi 40), with this obesity level more commonly found among non - hispanic black women (14.2%) (2). our population was primarily caucasian, which is important to take into consideration as it is well established that differences in overweight and obesity prevalence exist among women of various ethnic and race backgrounds (2). given our population 's higher income and education status and low proportion of minorities, our findings can not be directly translated across all ethnic backgrounds and education / socioeconomic levels. fewer responses were obtained from male parents / guardians, so males were excluded in our methods. interestingly, unlike our observed low rates of overweight and obesity in the female parents, male parents exhibited comparable rates to the average bmis rates for the country. the number of fathers responding (n=156 for 2008 and 2009 surveys) may not be a statistically valid sample, so future studies should consider the difference between male and female parents of fit children, including whether fathers are education oriented. review of the literature strongly supports the role of parent / adult modeling and styles in influencing physical activity and dietary intakes. thus, more public health strategies are needed to tackle the obesity epidemic, especially in relation to parental influence on children 's health - related behavior. future studies also should consider (1) parents of child populations who are not involved in a program such as di and (2) fit children from diverse backgrounds. nmm, cc, bg, ef, ms, and nsk approved the research design and participated in data collection and analysis.	backgroundrecent findings from our research indicate that children participating in a creative afterschool program exhibit overall healthier lifestyle practices compared to the average us pediatric population. this observation led us to investigate the prevalence of overweight / obesity and lifestyle practices of their parents.objectiveto determine the strongest predictors of weight status for female parents whose children were participating in such creative afterschool program.designsurveyed subjects were parents of children who competed in the 2008 and 2009 destination imagination global finals in knoxville, tennessee. a total of 4,608 children participated in data collection, with parental consent. for the combined 2 years, 1,118 parents, 87% of whom were females (n=1,032) completed online questionnaires, which were based on the behavioral risk factor surveillance system and included self - reported height, weight, dietary intake, physical activity, and socioeconomic status. the majority of this population was white, and less than 5% were african american or hispanic.resultswe report here results obtained for the female parents. only 45.2% of these female parents were overweight / obese, compared to a national average of 64.1% reported by the national health nutrition examination surveys for 20072008. furthermore, this population was significantly more physically active compared to national average. most parents (76%) had completed a college degree and reported high incomes. parents with the lowest income were the most obese in this population. finally, we found a significant association between parent and child weight status.conclusionsthese studies demonstrate that female parents of children who have healthy lifestyles were physically active, which likely accounts for the parents lower overweight / obesity rates. in addition to physical activity, income and percentage of calories from fat were all predictors of weight status.

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