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Hiv / aids is a major cause of morbidity and mortality in zambia, where in 2009 the national hiv prevalence in the 1549 year age group was estimated at 13.5%, but effective treatment is complicated by a high degree of malnutrition in the hiv - infected population . A review of hiv - infected adults starting antiretroviral therapy (art) in lusaka, zambia, found 33% of patients were undernourished by world health organization criteria (e.g., a body mass index (bmi) <18.5 kg / m), and 9% were severely malnourished (bmi <16 kg / m). Mortality in the early art treatment period is higher among patients with low bmi, low cd4 + t - cell counts, low hemoglobin, low serum albumin and phosphate levels, advanced hiv / aids stage, and immune reconstitution inflammatory syndrome [35]. In zambia, our group recently reported a higher risk of early art mortality among individuals with moderately elevated triglyceride concentrations, a finding which may be related to the interaction of fatty acids and systemic inflammation . The essential fatty acids (n-3 and n-6 polyunsaturated fatty acids (pufa)) are nutrients of primary importance for health, and many research works in the last decades have shown the role of an adequate intake of n-3 and n-6 pufa in the prevention of several diseases, in particular of cardiovascular diseases [79]. Omega 3 fatty acids have been known to modulate biomarkers such as c - reactive protein (crp) and cd4 count which are important determinants in the progression of hiv disease and other inflammatory conditions [10, 11]. To our knowledge, apart from one small clinical trial that assessed the benefit of combining fenofibrate with n-3 fatty acids in improving hiv - related clinical outcomes, studies on effects of fatty acids on metabolic parameters associated with morbidity and mortality in hiv are lacking, especially in resource - limited settings . Reliable identification of individuals with key nutrient deficiencies that could be improved with nutritional support would inform the design of nutritional rehabilitation programs to reduce morbidity and mortality in hiv / aids patients . The zambian diet is mainly composed of cereals, predominantly maize, starchy roots, and, to a lesser extent, fruits and vegetables . Cereals provide almost two - thirds of the dietary energy supply . In urban areas of zambia food consumption patterns urbanization and globalization are responsible for changes in dietary patterns, as consumption is shifting from fresh and minimally processed traditional foods to imported processed foods acquired from supermarkets . Because fish intake in much of zambia is relatively low, we hypothesized that dietary intake of long - chain pufa may be inadequate in hiv - infected adults and could influence markers of cvd risk and hiv disease progression . In this study, we sought to determine whether 24-hr dietary recalls (dr) conducted in an urban art clinic setting in zambia could be used to effectively estimate pufa intake and whether pufa and saturated fatty acids (sfa) measured in plasma are associated with markers of cardiovascular disease risk . We enrolled hiv - infected adults (age 1660 years) eligible for art initiation into the diet, genetic polymorphisms in lipid - metabolizing enzyme genes, and antiretroviral therapy - related dyslipidemia (dgplead) study at chawama clinic, a government health centre in lusaka, zambia, between january and december 2007 . All participants had a bmi 16 kg / m and cd4 + lymphocyte count 50 cells/l, and were art eligible according to the zambia national hiv guidelines . The research protocol was approved by the vanderbilt university institutional review board in nashville tennessee, usa, and the university of zambia biomedical research ethics committee in lusaka, zambia . Data collection was conducted by a study nurse, a clinical officer, and a supervising physician (ckn). At the first encounter, medical history and physical examination with anthropometric measurements were performed . A fasting blood sample was drawn on the same day as the 24 hr dietary recalls (dr). Intake of fatty acids as well as that of total energy and other nutrients was assessed using a 24 hr dr that was administered by a study nurse or a clinical officer, each with training by a registered dietitian and aided by commercial food models . Foods and amounts consumed over the preceding 24 hours were recorded by the interviewer . Food composition and nutrient quantity were computed from a modified food composition database using nds - r software (nutrition data system for research software version 2006, developed by the nutrition coordinating center, university of minnesota, minneapolis, mn, usa (http://www.ncc.umn.edu/)). The nds - r nutrient database was supplemented with nutrient composition data for local staple foods already published by the zambian national food and nutrition commission (available from http://www.nfnc.org.zm/). Participants were asked to come to the clinic after an 8-hour fast before their blood draw . Plasma and serum specimens were collected from each participant and used to determine total cholesterol (tc), high density lipoprotein - cholesterol (hdl - c), low - density lipoprotein - cholesterol (ldl - c), triglycerides, insulin, glucose, creatinine, crp, and albumin . Methods for metabolic assays in dgplead have been described previously [6, 17]. Lipid profiles and glucose were measured using a roche cobas integra 400 + autoanalyzer (roche diagnostics, indianapolis, in, usa). Triglycerides, ldl - c, and tc concentrations were measured using an enzymatic colorimetric assay while hdl - c was measured using a homogeneous enzymatic colorimetric assay . Serum creatinine, crp, and albumin concentrations were determined on a roche modular p analyzer using bromocresol purple assay for albumin and immunoturbidimetric assay for crp (roche diagnostics, indianapolis, in, usa). The extraction and quantification of fatty acids were performed at the university of minnesota using a standard validated assay that has been described in detail [1820]. This assay identifies 29 individual fatty acids ranging from 12:0 through 24:1n9 . For the first objective, we focused on pairwise spearman rank correlations between diet and plasma pufa measurements . In the second objective, the dependent variables were markers of hiv / aids disease progression (i.e., bmi, cd4 + cell count, and serum albumin) and cardiovascular disease risk (i.e., crp, triglycerides, hdl - c, and ldl - c). In secondary analyses with plasma arachidonic acid as the main independent variable we investigated bmi, cd4 + cell count, plasma albumin, crp, triglycerides, hdl - c, ldl - c, and plasma albumin as dependent variables . Fatty acids in plasma were expressed as a percentage of the total fatty acids analyzed while dietary fatty acids were expressed as a percentage of total energy per day . To validate fatty acid intakes, we computed pairwise spearman correlation coefficients for each pufa estimated from 24 hr dr against a corresponding pufa measured in plasma . The exception was plasma -linolenic acid in plasma which was tested for correlation with total dietary linolenic acid since - and -linolenic acid could not be separated in our 24 hour dr . Next we determined whether plasma fatty acids are associated with crp and lipid profiles using multivariable linear regression models . For each of the dependent variables, that is, crp, triglycerides, hdl - c, and ldl - c we estimated associations with the exposure variables, namely, total plasma pufa and sfa adjusted for age, sex, bmi, plasma monounsaturated fatty acids (mufa), trans fatty acid, alcohol use, and smoking . Additional analyses were conducted to understand whether individual pufa was associated with markers of cvd risk and hiv disease progression (e.g., cd4 + counts and plasma albumin). In this analysis, spearman rank correlations between plasma aa and each of the markers of cvd risk and hiv disease progression (e.g., crp, lipids, cd4 + count, albumin, and bmi) were determined . We then distributed plasma aa concentrations into quartiles and used anova with robust variance estimator to determine whether markers of cvd risk and hiv / aids disease progression significantly varied by quartiles of aa before and after adjustment for age, sex, smoking, and alcohol consumption . Data were analyzed using sas version 9.4 (cary, nc) and stata version 12.1 (college station, tx). Most participants were women (54%) and relatively young, with a median age of 32 years for women and 35 years for men . Although the median cd4 + cell count was somewhat higher in women (143 cells/l) compared to men (129 cells/l), this difference did not reach statistical significance (p> 0.05). Concentrations of crp also tended to be higher in women than in men but the differences were not statistically significant (p> 0.05). The frequency of smoking was quite low in our study population with only one (0.9%) current smoker among women and 10 (11%) among men (p <0.05). The proportional distributions of fatty acids in the diet expressed as percent energy intake per day and in the plasma as percent of total fat are shown in figure 1 . The highest median percent energy was from linoleic acid (la) (11.5%) and the lowest was from epa (0.007%). The highest median percent of total fat in the plasma was from la (16.1%) and the lowest was from -linolenic acid (ala) (0.15%). The highest significant correlation was for epa (r = 0.36, p <0.01) and the second highest was for dha (r = 0.21, p = 0.005). The correlations for ala, dpa, la, and aa were not statistically significant . In multivariable linear regression analyses, total plasma pufa concentrations were not significantly associated with crp on a log scale (= 0.10; 95% ci: 0.22 to 0.02, p = 0.09) in analyses adjusting for age, sex, bmi, mufa, trans fatty acids, current smoking, and alcohol status (data not shown). A positive, though weak, association was observed between total plasma pufa and triglycerides on a log scale (= 0.03; 95% ci: 0.002 to 0.06, p = 0.04). No significant associations were observed between total plasma pufa and hdl - c or ldl - c . Plasma aa was inversely correlated with crp (spearman correlation coefficient, r = 0.19, p = 0.02) and triglycerides (r = 0.14, p = 0.05) and positively associated with cd4 + count (r = 0.16, p = 0.02), albumin (r = 0.44, p <0.001), hdl - c (r = 0.26, p = 0.01), and ldl - c (r = 0.29, p = 0.001). As shown in table 3, the associations between aa and crp, serum albumin, triglycerides, hdl - c, and ldl - c remained significant after adjustment for age, sex, smoking, and alcohol consumption in anova models . In multivariable regression, a positive association was also observed between total plasma sfa and crp (= 0.24; 95% ci: 0.08 to 0.40, p = 0.003) and between sfa and triglyceride concentrations (= 0.08; 95% ci: 0.03 to 0.12, p <0.01). We observed modest correlations between epa and dha estimated from 24 hr dr and corresponding measures in plasma but weak correlations for other pufas . We also observed null associations between total plasma pufa and markers of hiv / aids disease progression and cvd - risk, but significant beneficial associations were observed between plasma aa and these markers . Higher plasma aa concentrations were significantly associated with higher hdl - c, ldl - c, cd4 + cell counts, and serum albumin, a profile consistent with improved patient survival . The finding that ala provided the highest median percent energy intake for the n-3 dietary fatty acids (1.5% or 2.5 g / day) is consistent with the notion that ala is the major form of n-3 fatty acids obtained from dietary sources [22, 23]. The reported intake for ala in our study is slightly higher than the range reported among western adults (0.5 to 2 g / d), probably due to higher content of foods from plant sources in zambia compared to western countries . The highest median percent proportion of total fat in the plasma among the n-3 fatty acids was from dha (3.5%). The observation that la contributed the highest median dietary percent energy (11.5% or 19.7 g / day) is consistent with la's position as the major n-6 fatty acid obtained from the diet [22, 23]. As in the diet, dietary la undergoes a metabolic process which converts it to various n-6 fatty acids including aa . This may explain why even if our study reported very low median dietary intake of aa (0.04%) compared to la (11.5%), the plasma proportions were closer (aa 11.1%, la 16.1%). The sources of ala and n-6 pufa in the zambian diet are likely to include vegetable oils such as soybean and sunflower oil . . These food items are readily available and generally affordable in urban areas, which may explain the relatively high consumption levels reported in our study . Fish and other forms of seafood are much better sources of very long chain n-3 fatty acids such as epa and dha but are not readily available . In zambia, despite the availability of large bodies of fresh waters from rivers and lakes, the fish industry is not well developed; very little fish farming is practiced . Thus, consumption of long - chain fatty acids in zambia (0.08 g / day for epa, dpa, and dha) is lower than what is reported among adults in northern and eastern europe, north america, and australasia (mean intakes ~0.150.25 the correlation coefficients for epa and dha measured in 24 hr dr and plasma in our study were 0.36 and 0.21, respectively . These correlations were somewhat higher than those observed for epa (r = 0.21) and lower than that for dha (r ranges from 0.42 to 0.48), la (r = 0.25), and ala (r = 0.23) as measured from food frequency questionnaire and plasma studies [26, 27]. The correlations were also higher than those observed for epa and dha measured from a food frequency questionnaire and adipose tissue (r = 0.15 and 0.18, resp . ). Comparable correlations of 0.15 and 0.61 for epa and higher correlations of 0.47 and 0.57 for dha in men and women, respectively, have been reported in a previous study . In another diet - adipose tissue study, the finding from our study that total plasma pufa was not associated with lower crp and lipid profiles may be a result of opposing actions among fatty acids when combined as total pufa, in contrast with the roles they play individually . For example, supplementation with fish oil rich in epa and dha (n-3 pufas) has been reported to reduce inflammation in conditions such as colitis in both animal and clinical studies [30, 31]. In contrast, aa (an n-6 pufa) is known to exert both proinflammatory and anti - inflammatory effects through its metabolites [32, 33]. Our finding that aa was inversely associated with crp and triglycerides and positively associated with hdl - c, cd4 + cell count and plasma albumin suggests that individual fatty acids may influence clinical outcomes in hiv / aids patients . The observation that sfa was positively associated with crp (= 0.24, p = 0.003) is consistent with findings from a previous study in which, after adjusting for other covariates, sfa emerged as the single most important nutrient contributing to an increase in serum crp levels . The finding that sfa was positively associated with serum triglyceride concentration (= 0.08, p <0.01) supports evidence from previous studies that found sfa to be positively associated with coronary heart disease risk [35, 36]. In a clinical trial, daily supplementation with 1 g of n-3 fatty acids did not reduce the rate of cardiovascular events in patients at high risk for cardiovascular events mmol / l), more among patients receiving n-3 fatty acids than among those receiving placebo (p <0.001), without a significant effect on other lipids . Similarly, in our secondary analyses total n-3 fatty acids were not associated with reductions in crp or improved lipid profiles and this may justify the need to explore the role of individual fatty acids in improving the cvd risk profiles . The cross - sectional nature of our study limits causal inference between the exposure variables and the dependent variables of interest . The study could have been prone to interviewer bias arising from inconsistency in the way the dr was administered by different interviewers . The study could also have been prone to reporting bias which may have arisen from the participants being inclined to report healthier foods more than the less healthy foods . To mitigate these potential biases, the interviewers were specifically trained to elicit complete dietary histories from all participants lastly, the study population of adult hiv patients was recruited at a single health facility, which could limit generalizability to hiv - infected individuals in other settings . We also acknowledge that because the study was done among patients not yet on art, we do not know the associations between fatty acids and markers of hiv / aids disease progression or cvd risk among those already on art . Studies that assess effects of art before and during art are warranted so as to determine whether supplementation with fatty acids will modulate outcomes from art in resource - limited settings . The significant but generally low diet - plasma long - chain pufa correlations could suggest that a single self - reported 24 hr dr may be inadequate for assessing pufa intake in hiv / aids patients in zambia . The study also suggests that sfa, which were positively related to markers of cvd risk, could play a role in hiv - related cardiovascular disease . Our study has found no evidence that total pufa are inversely associated with cvd risk markers in hiv patients . However, there was evidence from secondary analyses that individual fatty acids, particularly aa, may play a role in improving cvd risk profiles and markers of hiv / aids disease progression.
Autoimmune polyglandular syndrome (aps) is a rare genetic disease that causes multiple endocrine disorders . The vague presentation of endocrine dysfunction can cause a delay in both the diagnosis and the treatment of life - threatening conditions . We, herein, present a case of aps in a 21-year - old man whose condition did not respond to initial treatment and in whom a definitive diagnosis was subsequently achieved by workup for hypocortisolism . A 21-year - old man with a history of hashimoto's thyroiditis with hypothyroidism presented to the emergency department for evaluation of an altered mental status . He was very lethargic, stared blankly, and was unable to respond appropriately to questions . He reported no fever, difficulty swallowing, shortness of breath, muscle or joint pain, headache, vomiting, or excessive thirst or urination . Physical examination revealed a temperature (t) 34.3c, heart rate (hr) 68/min, respiratory rate (rr) 16/min, and blood pressure (bp) 87/57 mmhg . He was very lethargic, confused, and able to answer only yes - or - no questions . The patient exhibited hyponatremia (128 meq / l), hypoglycemia (29 mg / dl), and a high thyroid - stimulating hormone (tsh) level (14.31 iu / ml). Test for other metabolic panel, complete blood count, triiodothyronine and thyroxine levels, head computed tomography, and urine toxicology screening results were all normal . He was diagnosed with severe hypothyroidism and received 2 l of fluid for resuscitation, 25 g of dextrose, and a single intravenous (iv) dose of 100 mcg levothyroxine at the emergency department . A random serum cortisol level was <1.0 g / dl . A 250 g adrenocorticotropic hormone (atch) stimulation test was <0.1 g / dl . He was diagnosed with primary adrenal insufficiency and received a treatment with iv hydrocortisone . His mental status, generalized fatigue, and metabolic parameters including glucose and sodium levels substantially improved . Aps type 2 (aps2) was suspected based on his history of hypothyroidism and primary adrenal insufficiency . The results of an additional liver function test, anti - gad65 antibody titer, testosterone level, and plasma renin activity were all within normal limits . The patient had returned to his normal level of activity by the time of the outpatient follow - up and reported feeling much better after treatment with hydrocortisone, fludrocortisone, and levothyroxine . The present case illustrates an example of aps2, the clinical presentation of which may overlap that of primary adrenal insufficiency and hypothyroidism due to autoimmune thyroiditis . The present patient exhibited lethargy, an altered mental status, hypothermia, hypotension, hypoglycemia, an elevated tsh level, and normal thyroxine levels . He was later found to have primary adrenal insufficiency and his clinical symptoms improved after receiving corticosteroid replacement therapy . Other autoimmune diseases, especially of the skin, stomach, and gonads, may occur with increased frequency in patients with aps2 . The greatest danger associated with aps2 is treatment of the presenting hypothyroid state without recognition of concomitant hypoadrenalism . Hypothyroidism reduces cortisol clearance, and the addition of thyroid hormone replacement increases cortisol clearance . Additionally, hypothyroidism reduces the metabolic rate, thereby, reducing the need for cortisol . The increased metabolic rate accompanying thyroxine replacement increases the requirement for cortisol . Furthermore, a high serum concentration of tsh in the absence of primary thyroid failure can be a feature of adrenal insufficiency . The development of an adrenal crisis due to underlying adrenal insufficiency has been reported after the administration of thyroxine replacement therapy to patients initially thought to have only hypothyroidism . The management of aps2 is based on individualized, lifelong replacement therapy for the affected endocrine organs plus monitoring for development of insufficiency in other organs or associated conditions . The use of medical alert sensors and other measures should be taken to ensure that adrenal function is considered during illness, surgery, or emergency situations . In summary, we have reported a case involving a patient with aps2 who presented with lethargy, hypotension, hypoglycemia, and an elevated tsh level . The vague presentation of this syndrome can cause missed or delayed treatment for adrenal insufficiency resulting in a life - threatening adrenal crisis.
Crestal incision placed about 1.5 mm palatal to the greatest bony height with an angulation of 45 with no . 15 bard parker blade [figure 1] a vertical releasing incision placed mesially and distally to the crestal incision without disturbing the adjacent teeth papilla and extending about 3 mm labially [figure 2] soft tissue reflected to expose the cover screw [figure 3] palatal extension of the incision followed closer to the adjacent teeth on both sides, and a release incision made around the palatal portion of the exposed cover screw [figure 4] then healing abutment is placed after thoroughly cleaning the soft tissue around internal hex of the implant [figure 5] soft tissue approximated with 40 suture material (vicryl)sutures removal has to be done after 10 daysafter 2 weeks, second stage prosthetic procedure should be carried out . Crestal incision placed about 1.5 mm palatal to the greatest bony height with an angulation of 45 with no . 15 bard parker blade [figure 1] a vertical releasing incision placed mesially and distally to the crestal incision without disturbing the adjacent teeth papilla and extending about 3 mm labially [figure 2] soft tissue reflected to expose the cover screw [figure 3] palatal extension of the incision followed closer to the adjacent teeth on both sides, and a release incision made around the palatal portion of the exposed cover screw [figure 4] then healing abutment is placed after thoroughly cleaning the soft tissue around internal hex of the implant [figure 5] soft tissue approximated with 40 suture material (vicryl) sutures removal has to be done after 10 days after 2 weeks, second stage prosthetic procedure should be carried out . Incision and flap outlined (after reflection) more importantly it plays an important and crucial role in an esthetic component of a natural dentition . Hence, it is very vital to maintain the integrity of the papilla during second stage implant surgery . Several methods with unique incisions and suturing designs have been proposed to regenerate interdental papilla around implants . Palacci suggested that a full thickness flap be raised from the buccal and palatal side of the implant on the ridge and rotated 90 to accommodate the interproximal space of the implant . Palatal sliding strip flap to form papilla between implants and natural dentition in the anterior maxilla . Nemcovsky et al . Suggested an u- shaped incision, with opening toward the buccal aspect of the implant site with divergent incisions . Tinti and benfenati reported a ramp mattress suture design, which pulled the buccal flap coronally, to obtain a papilla between two implants . Suggested a split finger surgical method in which three interlacing finger like incisions were made and each of the fingers was sutured over the desired interdental papillary position . Proposed a method for papilla regeneration wherein the healing abutment is buried beneath the gingival flap to produce a dead space with expectation of the space being filled by the soft tissue . Even though these procedures provided papillary height, the results were not predictable . Various factors were involved in the success of any papilla regeneration procedures, such as preservation of blood supply, prevention of scar formation, and more importantly ideal placement of the implant into the bone . This new and innovative surgical technique provided significantly excellent results compared to the conventional mid - crestal technique [figures 6 and 7]. In this case report, in order to overcome the limitations of above - mentioned techniques a novel window technique was followed to regenerate papilla around implants during second stage surgery prior to prosthetic restoration . Unlike most other procedures mentioned above, this technique avoided the labial papilla on the adjacent teeth and involved wholly the palatal papilla, which is considered to be the key element in the success of this technique . Moreover, there is no cross incisions on to the papilla, which may lead to, disturbed wound healing and scar formation . Above all avoiding more invasive surgery is considered one of the best methods of soft tissue regeneration, which is being incorporated in this technique . The major advantages of this technique, when compared with earlier ones, are minimal invasive incision, less postoperative discomfort, and excellent esthetic and emergence profile obtained . The aim of any soft tissue procedure should be to obtain a resilient and scar less soft tissue . This can be obtained with atraumatic tissue handling, minimal tension during approximation, adequate vascularity, adequate hydration, and expedient surgical procedures that do not compromise blood supply . This case report shows the excellent results following the above requisites to obtain esthetic soft tissue profile . The patients were followed for 2 years at regular intervals and still the esthetic results obtained during the surgery and after 2 years were the same [figure 8]. With above considerations with respect to follow - up and esthetic component, this technique provided the required and desired results . In order to obtain esthetic papillary height and tissue integrity around implants proper and meticulous implant positioning is needed . Being a long term follow - up report this technique can be considered as a simple, minimal invasive procedure, which can be followed during 2 stage surgery.
Until the 1980s, most of our knowledge about drugs, drug mechanisms and drug receptors could fit in a few encyclopedic books and a couple dozen schematic figures . However, with the recent explosion in biological and chemical knowledge, this is no longer the case . There is simply too much data (images, models, structures and sequences) from too many sources . The limited drug or drug receptor data that is electronically available is either inaccessible (except through expensive subscriptions), inadequate or widely scattered among many different public databases ., the wealth of electronic sequence / structure data that exists today has never been well linked to the enormous body of drug or chemical knowledge that has accumulated over the past half century . Recently informatics gap. The therapeutic target database or ttd is one such example (1). This very useful web - based resource contains linked lists of names for> 1100 small molecule drugs and drug targets (i.e. Proteins). In addition to the ttd, a number of more comprehensive small molecule databases have also emerged including kegg (2), chebi (3) and pubchem (). Each contains tens of thousands of chemical entries including hundreds of small molecule drugs . All three databases provide names, synonyms, images, structure files and hyperlinks to other databases . Furthermore, unfortunately, these databases were not specifically designed to be drug databases, and so they do not provide specific pharmaceutical information or links to specific drug targets (i.e. Sequences). Furthermore, because these databases were designed to be synoptic (containing <15 fields per compound entry) they do not provide a comprehensive molecular summary of any given drug or its corresponding protein target . More specialized drug databases such as pharmgkb (4) or on - line pharmaceutical encyclopedias such as rxlist (5) tend to offer much more detailed clinical information about many drugs (their pharmacology, metabolism and indications) but they were not designed to contain structural, chemical or physico - chemical information . Ideally, what is needed is something that combines the strengths of, say, pharmgkb, pubchem and swiss - prot to create a single, fully searchable in silico drug resource that links sequence, structure and mechanistic data about drug molecules (including biotech drugs) with sequence, structure and mechanistic data about their drug targets . Beyond its obvious educational value, this kind of database could potentially allow researchers to easily visualize and explore 3d drug interactions, compare drug similarities or perform in silico drug (or drug target) discovery . Here fundamentally, drugbank is a dual purpose bioinformatics cheminformatics database with a strong focus on quantitative, analytic or molecular - scale information about both drugs and drug targets . In many respects it combines the data - rich molecular biology content normally found in curated sequence databases such as swiss - prot and uniprot (6) with the equally rich data found in medicinal chemistry textbooks and chemical reference handbooks . By bringing these two disparate types of information together into one unified and freely available resource, we wanted to allow educators and researchers from diverse disciplines and backgrounds (academic, industrial, clinical, non - clinical) to conduct the type of in silico learning and discovery that is now routine in the world of genomics and proteomics . The diversity of data types and the required breadth of domain knowledge, combined with the fact that the data were mostly paper - bound made the assembly of drugbank both difficult and time - consuming . To compile, confirm and validate this comprehensive collection of data, more than a dozen textbooks, several hundred journal articles, nearly 30 different electronic databases, and at least 20 in - house or web - based programs were individually searched, accessed, compared, written or run over the course of four years . The team of drugbank archivists and annotators included two accredited pharmacists, a physician and three bioinformaticians with dual training in computing science and molecular biology / chemistry . Drugbank currently contains> 4100 drug entries, corresponding to> 12 000 different trade names and synonyms . These drug entries were chosen according to the following rules: the molecule must contain more than one type of atom, be non - redundant, have a known chemical structure and be identified as a drug or drug - like molecule by at least one reputable data source . To facilitate more targeted research and exploration, drugbank is divided into four major categories: (i) fda - approved small molecule drugs (> 700 entries), (ii) fda - approved biotech (protein / peptide) drugs (> 100 entries), (iii) nutraceuticals or micronutrients such as vitamins and metabolites (> 60 entries) and (iv) experimental drugs, including unapproved drugs, de - listed drugs, illicit drugs, enzyme inhibitors and potential toxins (3200 entries). These individual drug types are also bundled into two larger categories including all fda drugs (approved drugs) and all compounds (experimental + fda + nutraceuticals). In addition,> 14 000 protein (i.e. Drug target) sequences are linked to these drug entries . More complete information about the numbers of drugs, drug targets and non - redundant drug targets (including their sequences) is available in the drugbank the entire database, including text, sequence, structure and image data occupies nearly 16 gigabytes of data most of which can be freely downloaded . Drugbank is a fully searchable web - enabled resource with many built - in tools and features for viewing, sorting and extracting drug or drug target data . Detailed instructions on where to locate and how to use these browsing / search tools are provided on the drugbank homepage . As with any web - enabled database, drugbank supports standard text queries (through the text search box located on the home page). It also offers general database browsing using the browse and pharmabrowse buttons located at the top of each drugbank page . To facilitate general browsing, drugbank is divided into synoptic summary tables which, in turn, are linked to more detailed all of drugbank's summary tables can be rapidly browsed, sorted or reformatted (using up to six different criteria) in a manner similar to the way pubmed abstracts may be viewed . Clicking on the drugcard button found in the leftmost column of any given drugbank summary table opens a webpage describing the drug of interest in much greater detail . Each drugcard entry contains> 80 data fields with half of the information being devoted to drug / chemical data and the other half devoted to drug target or protein data (see table 1). In addition to providing comprehensive numeric, sequence and textual data, each drugcard also contains hyperlinks to other databases, abstracts, digital images and interactive applets for viewing molecular structures (figure 1). In addition to the general browsing features, drugbank also provides a more specialized pharmbrowse feature . This is designed for pharmacists, physicians and medicinal chemists who tend to think of drugs in clusters of indications or drug classes . This particular browsing tool provides navigation hyperlinks to> 70 drug classes, which in turn list the fda - approved drugs associated with the drugs . A key distinguishing feature of drugbank from other on - line drug resources is its extensive support for higher level database searching and selecting functions . In addition to the data viewing and sorting features already described, drugbank also offers a local blast (8) search that supports both single and multiple sequence queries, a boolean text search [using glimpse; (9)], a chemical structure search utility and a relational data extraction tool (10). These can all be accessed via the database navigation bar located at the top of every drugbank page . The blast search (seqsearch) is particularly useful as it can potentially allow users to quickly and simply identify drug leads from newly sequenced pathogens . Specifically, a new sequence, a group of sequences or even an entire proteome can be searched against drugbank's database of known drug target sequences by pasting the fasta formatted sequence (or sequences) into the seqsearch query box and pressing the submit button . A significant hit reveals, through the associated drugcard hyperlink, the name(s) or chemical structure(s) of potential drug leads that may act on that query protein (or proteome). Drugbank's structure similarity search tool (chemquery) can be used in a similar manner to its sequence search tools . Users may sketch (through acd's freely available chemical sketching applet) or paste a smiles string (11) of a possible lead compound into the chemquery window . Submitting the query launches a structure similarity search tool that looks for common substructures from the query compound that match drugbank's database of known drug or drug - like compounds . High scoring hits are presented in a tabular format with hyperlinks to the corresponding drugcards (which in turn links to the protein target). The chemquery tool allows users to quickly determine whether their compound of interest acts on the desired protein target . This kind of chemical structure search may also reveal whether the compound of interest may unexpectedly interact with unintended protein targets . In addition to these structure similarity searches, the chemquery utility also supports compound searches on the basis of chemical formula and molecular weight ranges . Drugbank's data extraction utility (data extractor) employs a simple relational database system that allows users to select one or more data fields and to search for ranges, occurrences or partial occurrences of words, strings or numbers . The data extractor uses clickable web forms so that users may intuitively construct sql - like queries . Using a few mouse clicks, it is relatively simple to construct very complex queries (find all drugs less than 600 daltons with logps less than 3.2 that are antihistamines) or to build a series of highly customized tables . The output from these queries is provided as an html format with hyperlinks to all associated drugcards . Every effort is made to ensure that drugbank is as complete, correct and current as possible . Each drugcard is entered or prepared by one member of the curation team and separately validated by second member of the curation team . Additional spot checks are routinely performed on each entry by senior members of the curation group, including a physician, an accredited pharmacist and two phd - level biochemists . Several software packages including text mining tools, chemical parameter calculators and protein annotation tools (10) have been modified or specifically developed to aid in drugbank's data entry and data validation . These tools collate and display text (and images) from multiple sources allowing the curators to compare, assess, enter and correct drug or drug target information . In addition to using a cvs (current versioning system), all changes and edits to the central database are monitored, dated and displayed on the drugbank a second text tracking system has been implemented to monitor the completeness (0100%) of each field (for all approved drugs) and to display up - to - date statistics on the number of drugs, drug targets and non - redundant sequences in various drug categories . To ensure drugbank is current, new drugs (approved and experimental) are identified using continuously running screen - scraping tools linked to the fda, the pdb and rxlist websites . Drug targets are identified and confirmed using multiple sources (pubmed, ttd, fda labels, rxlist, pharmgkb, textbooks) as are all drug structures (kegg, pubchem, images from fda labels). In summary, drugbank is a comprehensive, web - accessible database that brings together quantitative chemical, physical, pharmaceutical and biological data about thousands of well - studied drugs and drug targets . Drugbank is primarily focused on providing the kind of detailed molecular data needed to facilitate drug discovery and drug development . This includes physical property data, structure and image files, pharmacological and physiological data about thousands of drug products as well as extensive molecular biological information about their corresponding drug targets . Drugbank is unique, not only in the type of data it provides but also in the level of integration and depth of coverage it achieves . In addition to its extensive small molecule drug coverage, drugbank is certainly the only public database we are aware of that provides any significant information about the 110 + approved biotech drugs . Drugbank also supports an extensive array of visualizing, querying and search options including a structure similarity search tool and an easy - to - use relational data extraction system . It is hoped that drugbank will serve as a useful resource to not only members of the pharmaceutical research community but to educators, students, clinicians and the general public . A screenshot montage of the drugbank database showing several possible views of information describing the drug ramipril . Summary of the data fields or data types found in each drugcard a more complete listing is provided on the drugbank home page.
The characteristics of keratoconus (kc) have been well documented in numerous populations worldwide . Characteristics such as age, gender, visual acuity, severity, associated factors, and management have been reported in studies conducted in australia, england, india, iran, israel, jordan, malaysia, new zealand, scotland, singapore, turkey, and united states . To the best of our knowledge, there is no published study, from africa, of the characteristics of kc . The aim of the present study is to provide the first description of patients diagnosed with kc in an african community seen in a specialized contact lens clinic at a children's hospital in kenya . A retrospective chart review was performed on all the ophthalmic records of patients with kc evaluated at the muthaiga eye clinic at gertrude's children hospital in nairobi, kenya from january 2007 to october 2014 . All patients included in this had been initially diagnosed with kc by an ophthalmologist and referred either directly or through a third party to this clinic . Data were collected on gender, age at initial presentation, source of referral, main complaint at referral, severity of kc, best - corrected visual acuity (bcva), prior management, if any at first presentation, including optical correction, collagen cross - linking, and keratoplasty . During the study, this study was approved by the african medical research foundation, kenya and cardiff university, uk . Patients with systemic or ocular pathology (other than kc) were excluded, as well as those whose records were incomplete . Corneal curvature was measured with a keratometer (bausch and lomb, rochester, ny, usa). The severity was assessed based on the classification used in the collaborative longitudinal evaluation of kc study, which is: mild kc <45 d in both meridians, moderate kc 4552 d in one or both meridians, severe kc> 52 d in one or both meridians . Visual acuity was determined using a keeler (finesse) snellen chart at 3 m. snellen readings were converted to logmar . Smirnov test . If normality was satisfied, the t - test and chi - square test was used . In cases of nonnormal distribution, the mann all tests were two - tailed, and p <0.05 was considered statistically significant . The mean age at presentation was 20.97 11.13 years (n = 249 patients; age was not recorded in the charts of five patients). The age data were not normally distributed (z = 0.157, p <0.001) and slightly skewed towards the younger age (median 18.0). The ages ranged between 6 and 84 years although 75% of the patients were between the age of 6 years and 25 years . The percentage of male subjects was higher (59.8%, n = 152) than female subjects (40.2%, n = 102), (p = 0.002), resulting in a ratio of 3:2 . The mean age at initial presentation was 21.06 11.91 years (range, 684 years) for males and 20.84 9.93 years (range, 771 years) for females . The remaining patients were directed by a teacher (6.7%) or other sources, or very rarely an optometrist (0.4%), they had all been diagnosed initially by an ophthalmologist . About one - third of patients at first presentation had no refractive correction (33.9%). The remaining patients were managed with spectacles (28.7%), contact lenses (19.7%), or a combination of spectacles and contact lenses (17.7%). Of these patients, the most common patient complaints at initial presentation was blurred vision (50%) followed by poor visual acuity with spectacles (33.5%), contact lens intolerance (11.8%), unspecified (4.3%), and frequent change in refraction (0.4%). Corneal curvature data were available in only 306 eyes as data were missing in the remaining eyes or the keratometric data were removed for eyes that had undergone corneal transplantation . Based on the keratometry data available, 98.3% of the patients had bilateral kc of whom 6.2% had mild kc, 22.9% had moderate kc and 71% kc had severe kc . Mean binocular bcva (n = 248) was 0.24 0.23 (n = 248) and the median was 0.20, indicating a distribution skewed toward better visual acuity . Seventy - five percent of kc patients had binocular bcva of 0.4 logmar (6/15) or better . Bcva was 0.37 0.38 for right eyes (n = 234) and 0.36 0.36 (n = 233) for left eyes . The age at initial presentation and the corresponding bcva of the eyes of different severity are given in table 1 . The bcva data were not normally distributed (z = 0.197, p <0.005). Patients with severe kc presented at an earlier age and had poorer visual acuity than those with mild and moderate kc (p <0.005). Age at first presentation and best corrected visual acuity of eyes with keratoconus based on severity although 66.1% of the patients had refractive correction at presentation after assessment, 98% required some form of refractive correction, 2% required no refractive correction . Of those corrected, 34.6% were prescribed spectacles, 31.1% received gas permeable contact lenses and 32.3% were prescribed both spectacles and contact lenses and 16.5% of the patients were referred for keratoplasty . The reasons for referral were poor bcva with optical correction (55.2%), inadequate contact lens fitting (20.7%), hydrops (13.8%), and contact lens intolerance (10.3%). After keratoplasty, 92% required spectacles, 4% required contact lenses, and 4% required contact lenses and spectacles . In kenya, kc is occurring in people as young as 6 years of age ., a large proportion of patients in the present study were very young (75% were between the ages of 6 years and 25 years). Notably, however, the age at presentation does not usually represent the age of disease onset, which is usually younger as the development of the disease is generally insidious and asymptomatic, and the patients had already been diagnosed by an ophthalmologist before referral . Previous studies from india, iran, malaysia, israel, and jordan are similar to those obtained in the present study [table 2]. However, kc seems to occur at a somewhat older age in the us and among caucasians in the uk . Younger age at presentation means that the condition has an earlier onset and most likely faster progression in african, middle eastern and indian populations . The higher proportion of bilateral cases in this study (98.3%) substantiates faster progression of the disease developing in the less affected or sub - clinical fellow eye . Early occurrence of the disease suggests a more severe form of the disease, which progresses more rapidly to surgery . Contrary to this observation, a lack of correlation between severity and age of onset was reported in one study . Early onset may reflect the variability of the disease process and genetic differences between the various ethnic groups for which some evidence has been reported . The high proportion of bilateral cases in the current study concurs with other studies, where only 0.5% of patients had unilateral kc which was documented using the same procedure . In contrast, 14.2% of bilateral cases were reported in a study of 600 eyes conducted before the development of corneal topography . Mean age of keratoconus patients at initial presentation, diagnosis, or self - reported onset of the disease in various countries the preponderance of males with kc in the current study is consistent with the majority of studies published in the last 30 years . Papers published over 30 years ago report the opposite gender distribution . In a retrospective study from the netherlands using data from over 100,000 contact lens wearers from four university clinics and five contact lens centers between 1950 and 1986, the ratio of males - to - females remained <1.0 until the 1970s when the ratio significantly increased and reached 1.58 for patients diagnosed in 1985 and 1986 . A male majority may be explained by hormonal differences, and it has been noted that kc develops more rapidly in males than females which could account for the higher prevalence in some study samples . There could also be a gender bias in some societies where services are offered more so to males than females . The source of referral in kenya differs markedly from that in western countries . In kenya, the majority of referrals came from ophthalmologists similar to iran . In the uk and scotland, optometrists were the main source of referrals (72.2% and 79%, respectively). In a large study conducted at moorfields eye hospital in london however, in kenya patients are generally more likely to follow the advice of physicians such as ophthalmologists rather than nonphysician care providers . The most common complaint of the subjects was blurred vision followed by poor visual acuity with spectacles (83.5% total) which is consistent with the large number of cases of severe keratoconus (71%) who exhibited significantly poorer acuity than either patients with moderate or mild kc . This observation concurs with two other retrospective studies in the uk that reported symptoms of blurred vision of 78% and 93% . Other complaints were contact lens intolerance (11.8%) followed by unspecified reasons and frequent change in refraction . A high percentage of the patients in the current study had severe kc, which is not surprising as the study sample was from a hospital eye clinic to which patients are referred to by primary or secondary eye care practitioners and the age of presentation was younger . Similar results are reported from studies from india and jordan [table 3] from hospital contact lens clinics, except studies from the usa . In this study, 66.1% of the patients used some form of correction at presentation, 37.4% of whom had already been prescribed rigid contact lenses by a secondary eye care practitioner and 14.2% of the patients had already been treated by other specialists with either collagen cross - linking or keratoplasty before referral to this tertiary contact lens clinic . Management of the vast majority of patients (98%) was with some form of optical correction, which provided very good visual acuity and 72% of eyes had a bcva 0.3 logmar (6/12). Only rigid gas permeable contact lenses were used because the cost of hybrid lenses is too high for the patients presenting to this clinic and soft lenses were inappropriate for such irregular corneas . In the current study, this proportion of referral to corneal transplantation is well within the range of 2%29% reported in other studies (with one outlier) [table 4]. However, recent studies report lower percentages of referral to keratoplasty likely due to the well - documented success of corneal collagen cross - linking . For example, a study from malaysia reported 52% of patients were prescribed contact lenses, 24% had cross - linking, and only 2.5% had a corneal graft procedure . Severity of keratoconus reported in several studies from different countries percentage of keratoconus patients who have undergone unilateral or bilateral keratoplasty there are some limitations to this study . Although this was a retrospective study conducted over a few years, all assessments were performed by the same practitioner . In addition, this is a hospital - based study, and the patients may have a more severe form of the kc compared to cases presenting to a routine eye care practice . The percentage of patients prescribed contact lenses would most likely be higher if hybrid or other customized lenses could have been used; however, these lenses were cost - prohibitive for most patients in this study . This is the first study describing a cohort of kc patients from a tertiary hospital clinic in kenya, and to the best of our knowledge, in africa . The vast majority of patients were referred to this clinic by ophthalmologists which is similar to middle eastern countries . The age at referral tended was low with some cases as young as 6 years of age, which concurs with the findings in the middle east and india where there is a high prevalence of kc compared to europe and the us . The percentage of patients requiring corneal graft and the reasons for this procedure was similar to other studies.
Multicystic dysplastic kidney (mcdk), a variant of renal dysplasia, is one of the most frequently identified congenital urinary tract abnormalities (cakut). Its incidence varies, depending on the study and country, but ranges from 1 in 3640 to 1 in 4300 live births [1, 2]. Renal dysplasia is characterized by structural disorganization of the renal tissue, involving undifferentiated epithelium and primitive ducts surrounded by fibromuscular connective tissue . Although mcdk can be an isolated finding, it is often associated with other cakut . Additionally, this condition has also been described in association with other multisystemic disorders of known genetic etiologies . Most mcdks undergo involution within the first years of life; nevertheless, a continuous follow - up should be performed not only to identify other urologic abnormalities, but also because hypertension and abnormal renal function have been reported [57]. Cowden disease (cd, mim 158350) is an autosomal - dominant condition with variable expression that results most commonly (80%) from a mutation in the pten gene on chromosome 10q . It is characterized by multiple hamartomatous neoplasms of the skin and mucosa, gastrointestinal tract, bones, central nervous system, eyes and genitourinary tract . This disease is associated with the development of several types of malignancies during the adult life, making recognition of individuals with this condition important . We report a 7-year - old male patient with antenatal diagnosis of unilateral mcdk and recently identified cd an unknown association and discuss its approach as well as the obligatory multidisciplinary follow - up in the future . A 7-year - old male patient with no relevant family history had a prenatal diagnosis of probable unilateral mcdk and macrocephaly . Renal ultrasound examination at 30 weeks gestation showed a small right kidney (24 mm), globally hyperechogenic with multiple cysts of variable size . After birth, isolated macrocephaly (95th percentile) was confirmed without other abnormalities at physical examination . Renal ultrasound at 2 and 15 days of life confirmed a small right dysplastic kidney (35 mm) together with a normal, non - dilated and well - differentiated left kidney (52 mm). A voiding cystourethrography showed bilateral vesicoureteric reflux (right grade 4 and left grade 3). The rest of the neonatal period was uneventful . During infancy, the patient had normal weight and stature growth (50th percentile) but notable macrocephaly (> 97th percentile) with prominent forehead and small jaw . Psychomotor development was slightly delayed (walking at 20 months) and mild mental retardation was established . Both karyotype analysis and cerebral magnetic resonance imaging a renal ultrasound showed progressive right dysplastic kidney involution but still detectable by 7 years of age . Macrocephaly and psychomotor delay were suggestive of cd and prompted us to perform mutation screening of the pten gene . The emergence of a nodular lesion (6 mm) above the right eyebrow (histological analysis confirmed a leiomyoma) and a slightly squamous appearance of the lower lip were convincing additional evidence for cd . A single nucleotide substitution (c.210 - 14a> g) within intron 3 of the pten gene created a new acceptor - splicing site 13 nucleotides upstream of exon 4 . This frameshift variant creates a premature stop codon, predicted to result in the synthesis of a truncated pten protein, therefore probably not functional . This mutation occurred de novo since it was absent in both parents' dna extracted from blood leucocytes . Multicystic dysplastic kidney is characterized by the presence of multiple, non - communicating cysts of various sizes separated by dysplastic parenchyma and the absence of a normal pelvicaliceal system . Mutations in genes, such as eya1, six1, tcf2 and pax2, which are known to have important roles in ureteric bud development, have been identified in multiple syndromes with renal dysplasia, including mcdk . Indeed both branchio - oto - renal syndrome (mutations in eya1 or six1) and renal - coloboma syndrome (mutations in pax2 gene) result in dysplastic kidneys mcdk has also been reported in other syndromes, including alagille syndrome (mutations in the jag1 gene), beckwith wiedemann syndrome (imprinting disorder at 11p15.5), hypoparathyroidism - deafness - renal dysplasia syndrome (mutations in the gata3 transcription factor), maturity - onset diabetes of the young type 5 (mody5, mutations in the tcf2 gene), trisomy 18, vacterl association, waardenburg syndrome type 1 and williams syndrome [1, 4, 10]. To our knowledge, mcdk has not been reported in association with cd . Originally described in 1963 by lloyd and dennis, cd (or multiple hamartoma syndrome) was named after the family in which it was first reported . Pten (phosphatase and tensin homologue) hamartoma tumour syndrome refers to a broader category, which includes cd, bannayan rare cases of cd are due to a germline mutation in the bmpr1a (bone morphogenetic proteins) gene . The members of the bone morphogenetic protein family, namely bmp4 and bmp7 genes, have already been shown to encode important signalling molecules throughout kidney development in mice, and deletions / mutations in these genes have been associated with segmental multicystic dysplasia [12, 13]. The pten tumour - suppressor gene, located on chromosome 10q23.3, encodes a lipid and protein phosphatase, which exerts its lipid phosphatase activity through dephosphorylation of phosphoinositide-3-kinase (pi3k) products, which in turn decreases the activity of kinases downstream of pi3k such as akt and mtor and its protein phosphatase activity on proteins of the mitogen - activated protein kinase pathway . Thus, a loss or reduction in pten activity leads to increased phosphorylation of key cellular proteins involved in cell - cycle progression, metabolism, migration, survival and spreading [15, 16]. Cd is an inherited autosomal - dominant trait with incomplete and age - related penetrance and a wide range of expressivity . It is characterized by multiple hamartomas and neoplasms of ectodermic, endodermic and mesodermic origin affecting skin, oral mucosa, gastrointestinal tract, bones, central nervous system, eyes and genitourinary tract . This disease is associated with the development of several types of malignancies, especially breast and thyroid carcinoma . Morbidity and mortality from cd are primarily due to increased frequency of malignant tumours; benign tumours also cause significant morbidity [18, 19]. Cd is a rare condition with around 300 published cases internationally . Since the identification of the susceptibility pten gene, the estimated incidence of cd has increased from 1 in 1 000 000 to 1 in 200 000 individuals . This most likely remains an underestimation as cd is associated with a high degree of phenotypic variability and hallmark features are under - recognized within the medical community . The diagnosis of cd is typically considered between 13 and 64 years of age, with an average of 22 years; the pathognomonic criteria of cd (facial trichilemmomas, acral keratoses, papillomatous papules and mucosal lesions), unfortunately, are not specific enough . The national comprehensive cancer network 2008 has proposed operational criteria for the diagnosis of this condition (table 1). Table 1.international cowden consortium diagnostic criteriapathognomonic criteriamucocutaneous lesions: facial trichilemmomas and acral keratosespapillomatous lesionsmajor criteriabreast cancerthyroid carcinoma (especially follicular)macrocephalylhermitte duclos diseaseendometrial cancerminor criteriaother thyroid lesions (adenoma, multinodular goiter)mental retardation (intelligence quotient <75)gastrointestinal hamartomasfibrocystic disease of the breastlipomasfibromasgenitourinary tumours (uterine fibroids and renal cell carcinoma) or malformationsoperational diagnosis in an individualmucocutaneous lesions alone meet the criteria if (i) six or more facial papules are present, of which three or more must be trichilemmomas; (ii) cutaneous facial papules and oral mucosal papillomatosus are present; (iii) oral mucosal papillomatosus and acral keratoses are present or (iv) six or more palmoplantar keratoses are present.two major criteria, but one must include either macrocephaly or lhermitte duclos diseaseone major and three minor criteriafour minor criteriaoperational diagnosis in a family in which one individual is diagnostic for cdone pathognomonic criterionany single major criterion with or without minor criteriatwo minor criteriahistory of bannayan riley ruvalcaba syndrome international cowden consortium diagnostic criteria in our patient, supraorbital leiomyoma and squamous appearance of the lower lip were very suggestive of cd . He also had congenital macrocephaly (major criterion), genitourinary malformation and mental retardation (two minor criteria). Molecular analysis confirmed the presence of a mutation in the pten tumour - suppressor gene . The overall prognosis of patients with unilateral mcdk and normal contralateral kidney is usually considered to be excellent . Morbidity and mortality may result from other urological malformations, urinary tract infections, arterial hypertension and chronic kidney disease . Despite the dysplastic structure of the renal parenchyma, there is no epidemiological evidence for an increased risk of cancer in the standard forms of mcdk [6, 7]. However, concerns about such complications have resulted in historical nephrectomies [21, 22]. Nowadays, a more conservative approach is universally recommended . In our patient, the voiding cystourethrography showed bilateral vesicoureteric reflux, but no urinary tract infection has occurred, and both blood pressure and renal function were normal . However, the presence of such vesicoureteric reflux in a single kidney is a risk factor for further parenchymal scarring, so that periodic assessment of glomerular filtration rate, proteinuria and blood pressure should be emphasized . According to recent studies, the risk of malignant transformation of mckd to wilms' tumour and renal or transitional cell carcinoma appears to be minimal . To our knowledge, there is little, if any, consensus recommendation about the management of paediatric patients with cd, particularly with regard to the risk of tumours, although different supervision protocols are established for patients> 18 years [1820, 23]. In the patient presented here, the association of mcdk with cd raises the problem of an increased risk of renal malignancy, thus a periodic imaging follow - up must be recommended . In the case of any doubt from ultrasonography in addition, a periodic multidisciplinary surveillance should be maintained including psychomotor development, dermatology consultation, evaluation of thyroid morphology and function, assessment of any potential sign that could be related to gastrointestinal involvement as well as any sign potentially related to a growing intracerebral mass . Although signs or pathognomonic features of this syndrome are not often observed in early childhood, it is important to inform paediatricians about this disease, mainly with regard to the increased risk of cancers.
The anterior cruciate ligament (acl) is the most frequently injured knee ligament, and accounts for about 50% of all ligament injuries1 . Acl injury is a common cause of knee instability and alters the kinematics of the knee2 . Most are caused by non - contact injury mechanisms, such as landing from a jump, or rapid deceleration3 . An acl injury typically occurs during the landing portion of a jump in ordinary sports activities and has a greater incidence among women than among men4 . Patients with acl injury often have symptoms of knee instability, which may lead to functional change in and damage to other joint structures, and interfere with activities of daily living5 . Patients with acl injuries have difficulty recovering normal walking ability and full function of the lower limbs6 . Knee instability following acl injury results in various negative factors, which can be circumvented using acl reconstruction . Reconstructive techniques have been refined to achieve better stabilization of the knee and other joints in order to improve functional recovery7 . It has been suggested that the lack of full recovery of knee function after acl reconstruction is due to sensory and motor behavior deficits . Muscle strength, endurance, flexibility and balance training are used as therapeutic exercises in rehabilitation settings . Proprioceptive afferent neural input is also important in functional control during sports activities8 . In the clinic, patients are always asked to walk normally, but few exercise methods provide normal proprioceptive input during walking training . In recent years, robot - assisted therapy (rat) rehabilitation, which simulates normal walking, has also been used to help patients with spinal cord injury or stroke9 . The major difference between robot - assisted weight - supported treadmill training and existing supported ambulation training is the addition of a mechanical assistant in the rat, so that alternative steps with both feet can be achieved by patients10 . Moreover, simulation of a normal walking pattern facilitates input from the peripheral nerves . In this manner, the remaining central nervous system can be stimulated so that regeneration of the nerves involved in traumatic spinal cord injury can be completed . Recent studies have shown that this method is associated with significant improvements in reaction time and bowel function11, 12 . The aim of this study was to examine the long - term interventions effects of rat rehabilitation on functional activity levels after acl reconstruction . This study recruited 8 patients who underwent arthroscopic acl reconstruction in boai hospitals in beijing, china, between september and december 2015 . Patients performed rehabilitation exercises for at least 2 months at the department of physical therapy . The subjects were limited to patients who had undergone acl reconstruction with autografts, performed by the same surgeon, and showed no differences in anatomical stability based on radiographic and magnetic resonance imaging . The characteristics of the 8 subjects (6 males and 2 females) are shown in table 1table 1.subject characteristicsmean sd (n=8)age (yrs)35.2 3.2height (cm)173.4 8.6weight (kg)79.5 16.8 . The subjects participated in rat for one month . The purpose and content of this research were explained to the subjects, who gave their informed consent to participation in this study . The study was approved by the research ethics committee of china rehabilitation research center (irb no . Was carried out as traditional physiotherapy, which included the muscle strength training, joint mobilization, balance training, and endurance training . The walking training used a rehabilitation training robot (mbz - cpm1, manbuzhe [tian jin] rehabilitation equipment co., ltd ., china). In the treadmill and rat training, the initial training speed was 1.5 km / h, which was progressively increased to 1.8 km / h as quickly as possible while maintaining gait quality13 . The body weight system was initiated at 35%, and 70% guidance force was provided for the participants14 . The walking training lasted for 20 min a day, and was performed 5 times a week . The extension strength of the knee joint, and walking and balance abilities were measured before and after the one - month intervention . Walking ability was evaluated using the 10-meter walk test (10mwt) and the timed up - and - go (tug) test . Balance ability was assessed using the functional reach test (frt). For the assessment of knee extension strength, surface electromyography (semg) and the maximal extensor strength of the knee joint were measured with the subjects seated and the knee flexed at 45. in the semg evaluation of the vastus lateralis (vl) and vastus medialis (vm) muscles, maximum isometric contraction in the start position was maintained for 5 s, during which the maximum and average semg values of the muscle were measured by an semg system (telemyo 2400 t; noraxon, scottsdale, az, usa). The maximum and average extensor strengths of isokinetic movement of the knee joint were measured using an isokinetic dynamometer (prima plus, easytech, italy). The wilcoxon test was used to compare the 10 mwt, tug, frt, the maximum and average semg values of vl and vm, and the maximum and average extensor strengths of the knee joint isokinetic movement before and after the one - month intervention . All the subjects in this study received rat training 20 times over one month . The results of the 8 subjects are shown in table 2table 2.the long - term interventions effects of rat10mwt (s)tug test (s)frt (cm)maximum semg value of lateralis(v)average semg value of lateralis(v)maximum semg value of medialis (v)average semg value of medialis (v)maximum extensor strength (nm)average extensor strength (nm)before one month rat intervention8.8 1.1 * 10.0 0.819.3 5.1*1,872.2 966.0280.3 131.11,405 676.5189.5 60.3 41.0 22.3 * 37.0 21.7*after one month rat intervention7.3 0.3 9.5 1.626.5 4.8*1,915.7 827.4315.0 113.51,590 261.7230.3 67.2 52.3 24.1 * 47.7 22.5**p<0.05; * p<0.01 . 10mwt: 10-meter walk test; tug: timed up - and - go; frt: functional reach test; rat: robot - assisted therapy . 10mwt: 10-meter walk test; tug: timed up - and - go; frt: functional reach test; rat: robot - assisted therapy the average semg value of vm, frt, and the maximum and average extensor strengths of the knee joint isokinetic movement increased significantly . However, the changes the maximum and average semg values of vl, the maximum semg values of vm and tug were not significant . According to the results, the walking ability, balance ability and the extensor strength of the knee joint were improved by the rat treatment, and the average semg value of the vastus medialis muscle significantly increased . The reason for these results is that robotic walking training enabled the bilateral lower limbs to perform alternative and circulatory movements, which can effectively adjust lower limb movement after acl reconstruction . In this manner, walking ability can also be improved . There was no significant change in the semg value of the vastus lateralis, presumably because weakness of knee joint muscle is often observed in the vastus medialis, which is the first muscle to show weakness among the quadriceps muscles15 . Many studies have attempted to suggest the best angular velocity during isokinetic exercise to selectively strengthen the vastus medialis16 . However, compared to the vastus lateralis, the vastus medialis was clearly enhanced by rat training . These results suggest that walking ability and muscle strength can be improved by robotic walking training as a long - term intervention . Future studies with a control group and more subjects are needed to investigate the effects of different walking models using rat after long - term intervention for patients with acl reconstruction.
Inflammation plays a key role in innate immune responses during infections with invading pathogens such as bacteria, fungus, virus, and parasite . Inflammation can be protective by recruiting immune and inflammatory cells and eliminating pathogens; however, excessive inflammation with overexpression of inflammatory factors and cytokines commonly leads to kinds of autoimmune diseases and host tissue injury, such as inflammatory bowel disease (ibd), psoriasis, and sepsis [24]. Pattern - recognition receptors (prrs) are essential to regulate immune responses by targeting pathogenic microbes and presenting antigens to the adaptive immune system . Prrs can be divided into four categories: toll - like receptors (tlrs), nucleotide - binding domain leucine - rich repeats (nlrs), nucleotide - binding oligomerization domain (nod), and retinoic acid - inducible gene i - like receptors (rlrs). In the presence of microbial stimuli, host prrs such as tlrs promote nuclear factor - kappa b (nf-b) mediated proinflammatory cytokines expression such as interleukin- (il-) 1, il-6, tumor necrosis factor- (tnf-), and nitric oxide (no). Meanwhile, another group of prrs including nlr family pyrin domain - containing 3 (nlrp3) recruit the adaptor protein apoptosis - associated speck - like protein containing card (asc) and procaspase-1 . Formation of this complex leads to the activation of caspase-1 by triggering procaspase-1 self - cleavage and promotes the precursor forms of cytokines such as pro - il-1 and pro - il-18 into active forms that are secreted . Activation of macrophages triggered by prrs further induces the maturation of dendritic cells (dcs) by releasing proinflammatory cytokines and promotes the induction of adaptive immune responses . The nlrp3 inflammasome is mainly expressed in immune and inflammatory cells such as macrophages, monocytes, neutrophils, and dcs when challenged by microbial stimuli . It has been well - established that multiple pathways participate in inflammasome activation such as cellular and mitochondrial reactive oxygen species (ros), cathepsin b, and cytosolic protein kinase r (pkr) [1012]. Among all of these triggers, ros generation is reported to play an essential role in the activation of nlrp3 inflammasome when challenged by stimuli such as lipopolysaccharides (lps), adenosine triphosphate (atp), and urate crystals . More fundamentally, a more recent study demonstrated that ros derived by xo, the oxidized form of xanthine dehydrogenase, is the main source of nlrp3 activation in macrophages, leading to excessive il-1 and il-18 secretion . Inhibition of xo by febuxostat, well - documented xo inhibitor, could significantly decrease the ros generation and il-1 secretion in macrophages stimulated by lps, indicating the predominant role of xo in lps - induced il-1 mature and secretion [14, 15]. Hsya (2d and 3d structure in figure 1) is a water soluble monomer extracted from carthamus tinctorius l. (safflower), which has long been used for treatment of cardiovascular diseases in traditional chinese medicine . Recent researches showed that, besides the therapeutic effects upon cardiovascular system, hsya exhibits promising anti - inflammatory properties by suppressing innate immune tlr4-inducing pathway, bettering lps - induced inflammatory injury, scavenging excessive ros, and inhibiting proinflammatory cytokines generation [1719]. However, few studies have attempted to uncover the direct target of hsya and interpret the mechanisms of its anti - inflammatory properties . In this study, we tried to find the potential target of hsya via inverse prediction method and computation docking and further assessed the role of hsya in regulating nlrp3/caspase-1/il-1 pathway in macrophages . Hsya [> 98% high - performance liquid chromatography (hplc) purity] was purchased from tauto biotech (shanghai, china). Lps (escherichia coli o55:b5) was purchased from sigma - aldrich chemical (st . Louis, mo, usa). Fetal bovine serum (fbs), dulbecco's modified eagle medium (dmem), antibiotic - antimycotic, and trizol reagents bicinchoninic acid (bca) protein assay kit was purchased from pierce (rockford, il, usa). Enzyme - linked immunosorbent assay (elisa) kits for mouse il-1 and il-18 were purchased from cusabio (wuhan, china). Antibodies for mouse -actin, gapdh, pro - il-1, il-1, nlrp3, asc, procaspase-1, and caspase-1 were purchased from cell signaling technology (danvers, ma, usa). Antibody for xo was purchased from santa cruz (dallas, tx, usa). The goat anti - mouse antibody was purchased from li - cor odyssey (lincoln, ne, usa). Xo activity assay kit (c / f) was purchased from biovision (milpitas, ca, usa). Raw264.7 macrophage cell line was purchased from the american type culture collection (rockville, md, usa). Cells were cultured in dmem supplemented with 10% fbs and antibiotics (100 u / ml penicillin and 100 u / ml streptomycin) at 37c in a humidified incubator under 5% co2 . To detect the levels of cytokines (il-1 and il-18) released by raw264.7 macrophages in vitro, cells were preincubated in 24-well plates (4 10 cells / well) for 12 h and pretreated by hsya (25, 50, and 100 m) for 3 h. cells were then washed twice with pbs and challenged by lps (1 g / ml) for another 24 h at 37c under 5% co2 . The supernatants were collected and assayed immediately using sandwich technique . Cells (1 10) were preincubated at 37c under 5% co2 for 12 h and then treated by appropriate experimental reagents . Cells were then harvested on ice, washed three times with cold pbs, and suspended in 500 l lysis buffer supplemented with protease inhibitors . After incubation on ice for 30 min, cell extracts were centrifuged at 14,000 rpm for 15 min at 4c to isolate total cell proteins, which were quantified using a bca protein assay kit . Proteins were separated by sds - page and electrotransferred to nitrocellulose membranes (pierce, il, usa) before hybridization with the appropriate detection antibodies (xo, pro - il-1, il-1, nlrp3, asc, procaspase-1, and caspase-1). Gapdh was used to correct for differences in loading of the proteins and for normalization of the densitometric values of immunoblot signals obtained from three separate experiments using li - cor odyssey detecting system . Macrophages were preincubated in 6-well plates (1 10 cells / well) for 12 h and pretreated by hsya (25, 50, and 100 m) 3 h prior to lps (1 g / ml) stimulation . The concentration and integrity of total rna samples were evaluated by measurement of the a260/280 ratio . Quantitative polymerase chain reaction (pcr) analysis was performed using the dna engine mx3000p (agilent, santa clara, ca, usa) fluorescence detection system according to optimized pcr protocols . The following primers were used for amplification: -actin: f: 5-cccatctacgagggctatgc-3, r: 5-ggtgtaaaacgcagctcagta-3; xo, f: 5-cacgatgacgaggacaacgg-3, r: 5-taggctcaggcttgtttcgg-3. Ros production was detected by measuring intracellular ros formation using probe dcfh2da . Briefly, raw264.7 cells were pretreated with hsya (100 m) or positive control febuxostat (30 m) for 3 h and then stimulated with lps (1 g / ml) for 6 h to promote ros generation . Cells were washed twice with pbs and then incubated with probe dcfh2da (20 nm) for 15 min . Fluorescence staining was visualized using a fluorescence microscopy (olympus, ix71) and fluorescence assays were measured with a fluorescence microplate reader (tecan, sunrise) at excitation / emission 525/610 nm . Theoretically, xo oxidizes xanthine to hydrogen peroxide (h2o2) which reacts stoichiometrically with oxired probe to generate color at = 570 nm and fluorescence at ex / em = 535/587 nm . Since the color or fluorescence intensity is proportional to xo content, the xo activity can be accurately measured . To detect the direct inhibition of hsya on xo, we first added hsya at various concentrations (0, 10, 20, 40, 80, 160, 320, 640, and 1280 m) into active xo solution and incubated at 37c for 30 min . After that, this mixed solution was added into a reaction system that consisted of assay buffer, substrate mix, enzyme mix, and oxired probe and incubated for another 30 min at 37c . The ic50 of hsya on xo was calculated by spss probit regression . To detect the effect of hsya on xo activity in lps - stimulated raw264.7 macrophages, we treated cells with hsya at different concentrations (25, 50, and 100 m) for 3 h before lps challenge (1 g / ml, 12 h) clear xo extract was obtained by centrifuge (16,000 g, 10 min) and the activity of xo from cell extract was added into reaction system and measured as described above . Pharmmapper server is a web server for potential drug targets identification using pharmacophore mapping approach at http://59.78.96.61/pharmmapper/. Briefly, 2d mol2 file of hsya (pubchem cid: 6443665) was submitted to pharmmapper server . During the procedure, the maximum conformations were set up to 300, and the number of reserved matched targets was 300 . Other parameters were kept as default . The submission i d can be stored and used to check the prediction results . Further, to characterize the combination between hsya and xo, we used autodock and autogrid to calculate the binding affinity and binding sites . Briefly, the two - dimensional (2d) structure of hsya was drawn and converted to mol2 format by chemdraw . Then the mol2 file of hsya was optimized and saved as final coordinate pdb file for docking study . The 3d crystal structure of xo (entry code 1f04) was downloaded from the protein data bank (pdb) at http://www.rcsb.org/. The grid procedure was handled, by autogrid, in a grid box of 606060 with a 1.0 to enclose all the active sites and to allow for the flexible rotations of hsya . Then the data were presented as means standard error of the mean (sem) and differences between mean values of normally distributed data were assessed by the one - way analysis of variance (anova) followed by duncan's test for multiple comparisons . Via pharmacophore mapping approach, 300 potential candidates out of 7302 were listed and sorted according to the fit score (submission i d 151015052728). Based on the disease information and potential roles in inflammation and redox related signaling pathways, endothelial nitric oxide synthase (pdb i d: 1p6 m), nadph cytochrome p450 reductase (pdb i d: 1j9z), and xo (pdb i d: 1f04) were selected as potential targets of hsya . Based on the pharmacophore model, endothelial nitric oxide synthase had one hydrophobic, four donors, and three acceptors; nadph cytochrome p450 reductase had two hydrophobics, one positive, two negatives, one donor, eight acceptors, and one aromatic; xo had one negative, two donors, and five acceptors . However, since inhibition tests showed that hsya possessed no depression effects on activities of endothelial nitric oxide synthase and nadph cytochrome p450 reductase (data not shown), only the pharmacophore model of xo was exhibited in figure 2 . To confirm if there is a direct interaction between hsya and xo, we detected the inhibitory effect of hsya on xo activity both out of cells and within cells . Febuxostat was used as positive control . Because hsya liquor appears yellow, we employed fluorescence detection, other than od detection, to reduce error . Results showed that hsya exhibited remarkable inhibitory effect on xo activity with ic50 = 40.04 m out of cells (figure 3(a)). The ic50 of positive control febuxostat was 3.24 m (data not shown). In addition, as shown in figure 3(b), 50100 m hsya treatment started to exhibit suppression on the activity of xo in lps - stimulated macrophages (p <0.05). However, results demonstrated that xo treatment had no noticeable action on the protein and mrna expression of xo in macrophages (p> 0.05) (figure 4). Since we confirmed that hsya inhibits the activity of xo, we tried to further understand the interaction between hsya and xo by computation docking . From the molecular docking data, we concluded hsya directly implanted into the activity pocket of xo by hydrophobic characteristic and hydrogen bonds, which are essential to this interacting system with a binding energy of 5.77 kcal / m (table 1). In this hsya - xo complex, hsya was surrounded by leu 648, leu 712, his 875, leu 873, ser 876, glu 879, phe 649, and asn 650 in hydrophobic interactions (figures 5(a) and 5(b)). Moreover, glu 879, asn 650, and his 875 were observed to form hydrogen bonds with the hydroxyl groups of hsya (figure 5(c)). As a main source of ros, xo in macrophages if xo inhibition by hsya results in less ros production, we evaluated the ros level via fluorescence detection . As shown in figure 6, lps challenge markedly increased the amount of ros in macrophages (p <0.01); however, 100 m hsya pretreatment for 3 h significantly suppressed lps - induced ros generation (p <0.05), with no remarkable difference compared with 30 m positive control febuxostat (p> 0.05). Since latest research demonstrated that activation of nlrp3 inflammasome requires the ros generated by xo, we further detected the effect of hsya on activation of nlrp3 inflammasome . As shown in figure 7, 1 g / ml lps challenge significantly enhanced the expression of nlrp3, asc, and procaspase-1 proteins (p <0.01), indicating that nlrp3 inflammasome was activated by lps in macrophages . Hsya treatment from 50 to 100 m notably inhibited the expression of nlrp3 (p <0.05); however, no significant regulatory effects were observed on expression of asc and procaspase-1 (p> 0.05). Result showed that hsya treatment from 50100 m exhibited remarked inhibitory effect on the mature caspase-1 expression in macrophages (p <0.05). To further estimate the inhibition of hsya on nlrp3 inflammasome activation as shown in figure 8(a), hsya treatment at concentrations from 50 to 100 m notably decreased the protein expression of il-1 in a dose - dependent manner (p <0.05). Results based on western blot also displayed that 100 m hsya possessed an observable negative regulatory effect on pro - il-1 expression, but with no significant difference compared with lps group . Moreover, data in figure 8(b) showed that, as expected, hsya treatment at concentrations from 50 to 100 m markedly decreased the secretion level of il-1 and il-18 in supernatant (p <0.05). Xo is one of the major enzymatic sources of ros by utilizing molecular oxygen as a substrate to break down xanthine and hypoxanthine into o2 and hydrogen peroxide . It has been well - documented that the activity of xo can be enhanced by kinds of stimuli including lps and cytokines in macrophages, resulting in many pathologic conditions characterized by oxidative stress and inflammation [21, 22]. Inhibition of xo by pharmacological inhibitors such as febuxostat and allopurinol markedly decreases xo - induced excessive ros generation and betters the pathologic conditions, suggesting the essential role of xo in inflammation and providing potential strategy of therapies to cure inflammation diseases [14, 23, 24]. By employing pharmmapper, a server computationally predicting candidates of submitted small molecule, drug, and compounds using inverse docking technique, we found that endothelial nitric oxide synthase, nadph cytochrome p450 reductase, and xo may be potential disease - related targets of hsya . However, data from subsequent inhibitory tests showed that hsya only possessed promising regulatory effect on xo instead of endothelial nitric oxide synthase and nadph cytochrome p450 reductase . The pharmacophore outcome disposed to the molecule characteristics of hsya, which convincingly supports the inverse docking result provided by pharmmapper . To better understand the chemical - protein combination information of hsya and xo, we employed autodock and autogrid to investigate the interaction of hsya - xo complex . Results demonstrated that hsya embedded into the active region of xo via hydrophobic interaction, a critical binding force for complex stabilization, with many amino acids including leu 648, leu 712, his 875, leu 873, ser 876, glu 879, phe 649, and asn 650 . Besides, the hydrogen bond interactions between hsya and xo at residues such as glu 879, asn 650, and his 875 also contribute to the combination and endow the inhibitory effect of hsya on xo activity . Since we proved that hsya directly inhibits the activity of xo, we tried to further investigate whether hsya affects the expression of xo and leads to changes in downstream signaling pathways . However, results based on rt - pcr and western blot demonstrated that hsya possesses limited regulatory effect on lps - induced overexpression of xo at mrna and protein level, suggesting that hsya exhibits subsequent regulating properties through inhibiting the activity but not amount of xo in lps - challenged macrophages . Ros overproduction triggered by phagocytes is critical for the cellular signaling cascades in macrophages during immune activation [2729]. Since it has been well - documented that xo plays an important role in ros generation, we then detected the effect of hsya on lps - induced ros production in raw264.7 macrophages . Results indicated that 100 m hsya pretreatment notably suppressed lps - induced excessive ros generation, with no significant difference compared with 30 m xo inhibitor febuxostat . Knowing that hsya owns considerable antioxidant property [30, 31], our findings may partially uncover the mechanism of which hsya decreases ros generation . Since it has been proved that xo - induced ros generation is the dominant trigger of nlrp3 inflammasome, we further investigated the effect of hsya on nlrp3 inflammasome activation . Nlr family, well - established prr, is critical for promoting inflammation process in host innate immune response when challenged by infectious stimuli . Among these inflammasomes, nlrp3 has been characterized in most mammalian cells . Studies in macrophages and various animal models demonstrated that the activation of nlrp3 inflammasome can be observed in kinds of autoimmune and inflammatory diseases such as experimental autoimmune encephalomyelitis (eae), multiple sclerosis, inflammatory bowel disease including ulcerative colitis, and crohn's disease [35, 36]. Moreover, nlrp3 inflammasome activation may contribute to insulin resistance and type ii diabetes, uric acid accumulation, and gout . Mechanically, when stimulated by prrs, nlrp3 inflammasome activates and then promotes the recruitment of asc and the procaspase-1, resulting in the formation of caspase-1 p10 fragment which contributes to active caspase-1 enzyme that cleaves pro - il-1 into mature il-1 form . Results based on western blot illustrated that hsya treatment notably decreased the cleaved caspase-1 expression, indicating that hsya could suppress lps - induced activation of nlrp3 inflammasome . To better understand the regulatory effect of hsya on nlrp3 inflammasome, we detected the protein expression of nlrp3, asc, and procaspase-1 complex . Results revealed that hsya notably inhibited lps - induced nlrp3 expression; however, no noticeable suppressions were observed on the upregulated expression of asc and procaspase-1 . A more recent study in human and mice macrophages demonstrated that ros generation caused by xo, instead of nox, is the major source that promotes nlrp3 inflammasome activation . Taken together, we conferred that hsya treatment restrained the activation of nlrp3 inflammasome via directly binding to xo and inhibiting the ros overproduction . Il-1 and il-18 are two well - documented proinflammatory cytokines that play central role in inflammation and autoinflammatory disorders . It has been established that secretion of il-1 and il-18 needs the participation of nlrp3 inflammasome [39, 40]. Hence, we measured the protein expression of pro - il-1 and il-1 in macrophages and the secretion of il-1 and il-18 in the supernatant . As expected, hsya pretreatment notably decreased lps - induced cleaved il-1 expression, indicating that hsya inhibited the cleaving process of il-1 mainly by suppressing the nlrp3 inflammasome activation . Meanwhile, mild abatement of pro - il-1 expression was observed under hsya pretreatment . Considering the important role of ros generation in the activation of nf-b, a classical nuclear transcription factor that encodes kinds of proinflammatory cytokines including il-1, we speculated that ros scavenging by hsya partially depressed the activation of nf-b and led to lower pro - il-1 protein expression . However, the inhibition of nlrp3 eventually blocked the transformation process from pro - il-1 to cleaved il-1. Detection on secretion of il-1 and il-18 by elisa further proved that hsya inhibited nlrp3 inflammasome activation and finally suppressed its downstream cytokines secretion . But, since we did not find the suitable antibodies for pro - il-18 and il-18 detection, we could not prove whether the downregulation of il-18 was caused by blocking mature process . Taken together, we found that xo is a potential target of hsya using pharmmapper inverse docking and computer simulation . The inhibitory effect of hsya on xo activity contributes to the ros scavenging and nlrp3 inflammation suppression triggered by lps, leading to decreased il-1 and il-18 secretion in raw264.7 macrophages (figure 9).
Volumetric - modulated arc therapy (vmat) has been characterized as intensity - modulated radiotherapy (imrt) in a single gantry arc, in which gantry speed, dose rate and leaf speed of the multileaf collimator (mlc) are varied during gantry rotation . In contrast, 180 partial - arc vmat has been employed for lung cancer treatment to reduce doses in contralateral healthy organs . Dosimetric and delivery efficiency comparisons between 360 single - arc and 180200 partial - arc vmat for lung cancer have been made, reporting that partial - arc vmat significantly reduced mean dose to the contralateral lung with decreased delivery time . To our knowledge, however, no further comparisons of full - arc and half - arc vmat for tumors other than lung cancer have been reported . We speculated that a regular - shaped peripheral cancer such as a maxillary cancer may benefit from half - arc vmat due to its location . The purpose of this paper is comparison of dosimetric and delivery characterizations between full - arc and half - arc vmat for maxillary cancer . Delivery was characterized by recording gantry angle error and gantry acceleration during vmat delivery in a log file . The patients were helically scanned on an aquilion lb (toshiba, ootawara, japan) computer tomography unit with a gantry rotation time of 0.5 s and the images were reconstructed with a slice thickness of 2 mm . Gross tumor volume (gtv) was defined as a visible tumor at the head window settings . Clinical target volume (ctv) planning target volume (ptv) was further defined by adding an isotropic margin of 5 mm to the ctv to account for setup uncertainty and mechanical inaccuracy . The normal brain, brainstem, spinal cord, optic chiasm, ipsilateral and contralateral eyes, and ipsilateral and contralateral optic nerves were contoured as organs at risk (oars). The tumor sizes and positions for the six cases are shown in table 1 . Table 1.tumor sizes, positions and gantry angle ranges used for half - arc vmat deliveriescase 1case 2case 3case 4case 5case 6ptv (cm)280.1129.8347.9239.7302.8217.5ctv (cm)181.276245.1150.7199.3129.4gtv (cm)100.832.2142.280.5112.662.8lesion positionrlrlllgantry angle range ()2254530012022545315135315135315135r: right side, l: left side . Tumor sizes, positions and gantry angle ranges used for half - arc vmat deliveries r: right side, l: left side . Vmat plans were created using the monaco 3.0 (elekta, maryland heights, missouri, usa) treatment planning system (tps) with gantry rotation angles of 360 (full - arc vmat) and 180 (half - arc vmat). Gantry angle ranges used for the half - arc vmat deliveries are shown in table 1 . The collimator and couch angles were fixed at 0. a dose of 66 gy in 33 fractions was prescribed to 95% of the ptv, and maximum dose was restricted to 110% of the prescribed dose . Dose constraints for oars were given as follows: brainstem max dose 50 gy, spinal cord max dose 45 gy, optic chiasm mean dose, contralateral eye max dose and contralateral optic nerve mean dose 45 gy . Ipsilateral eye and optic nerve maximum dose were minimized while maintaining the target d95 prescription . The monaco tps provides monte carlo dose calculations with a grid size of 3 mm and variance of 3% . Dosimetric comparisons between the full- and half - arc vmat were performed with identical isocenter positions and dose constraints . For the ptv, a homogeneity index (hi) was calculated using the following formula: (1) where d2%, d98% and d50% are doses that cover 2%, 98% and 50% of the ptv, respectively . A conformity index (ci) was calculated using the following formula: (2) where vri was the volume of the reference isodose and tv was the target volume . Ci95%, ci80% and ci50% were calculated, for which the reference isodose percentages were 95%, 80% and 50%, respectively . With regard to oar, a 6-mv photon beam with an mlc leaf width of 10 mm was used for vmat delivery using the synergy linear accelerator (elekta, crawley, uk). Treatment plans were transferred from the tps to a desktop pro 7.01 linac controller via a mosaiq v1.6 (elekta, sunnyvale, california, usa) record and verify system . Total monitor units (mu), mean dose rate, delivery time, gantry angle error and gantry acceleration were evaluated using a log file that recorded cumulative mu as well as planned and actual gantry angles every 250 ms . The gantry angle error was calculated by subtracting the planned gantry angle from the actual gantry angle for each treatment time . Data were analyzed using the wilcoxon signed - rank test with statistical significance set at p <0.05 . Statistical analysis was performed with spss v.19.0 (ibm, chicago, il, usa). Figure 1 shows representative dose distributions and dose volume histograms of full - arc and half - arc vmat for the same patient . Compared with the full - arc vmat plan, the half - arc plan provided substantially lower doses to the normal brain and brainstem while maintaining a nearly identical dvh for the ptv . Figure 1.dose distributions calculated by (a) full - arc and (b) half - arc vmat planning for a patient with maxillary cancer; (c) comparison of dose volume histograms (dvhs) between full - arc (solid line) and half - arc (dashed line) vmat plans for the same patientthe ptv is shown as a translucent pink region . Compared with the full - arc plan, the half - arc plan provided substantially lower doses to the normal brain and brainstem while maintaining a nearly identical dvh for the ptv . Dose distributions calculated by (a) full - arc and (b) half - arc vmat planning for a patient with maxillary cancer; (c) comparison of dose volume histograms (dvhs) between full - arc (solid line) and half - arc (dashed line) vmat plans for the same patient table 2 compares the hi of the ptv, ci and mean doses to the oar between the full- and half - arc vmat plans, with data shown as group averages with ranges (n = 6). The hi, ci95%, ci80%, the mean doses to the spinal cord, optic chiasm, ipsilateral and contralateral eyes, and ipsilateral and contralateral optic nerves did not significantly differ between the full- and half - arc vmat plans (p> 0.05). In contrast, mean doses to the normal brain and brainstem in the half - arc vmat plans were on average 16% and 17% lower than those in the full - arc plans, with these differences being statistically significant (p <0.05). Table 2.dosimetric comparisons between full - arc and half - arc vmat plans(n = 6)full - archalf - arcp valueptvhi0.075 mean0.190.21 range0.140.320.150.31ci95%0.345 mean1.11.1 range0.91.21.01.2ci80%0.249 mean1.61.6 range1.41.81.41.9ci50%0.028 mean2.82.6 range2.53.42.33.2normal brain dmean (cgy)0.028 mean918.7768.3 range405.51623.2346.81335.0brainstem dmean (cgy)0.028 mean2344.71943.8 range1805.92655.61403.12273.2spinal cord dmean (cgy)0.080 mean1129.8890 range161.62341.9164.01709.5optic chiasm dmean (cgy)0.463 mean2226.22117.5 range568.73224.3615.43344.8ipsilateral eye dmean (cgy)0.280 mean3442.63254.3 range1487.45157.21338.44943.6ipsilateral optic nerve dmean (cgy)0.600 mean4256.54212.3 range2548.46088.72274.66134.2contralateral eye dmean (cgy)0.753 mean989.51006 range712.11512.8769.31542.0contralateral optic nerve dmean (cgy)0.345 mean2843.62724 range1572.73454.31721.23378.1the wilcoxon signed - rank test resulted in a statistically significant mean dose reduction to the normal brain and brainstem in the half - arc vmat plans (p <0.05). Dosimetric comparisons between full - arc and half - arc vmat plans the wilcoxon signed - rank test resulted in a statistically significant mean dose reduction to the normal brain and brainstem in the half - arc vmat plans (p <0.05). Figure 2 shows plots of gantry acceleration and gantry angle errors as a function of treatment time during full- and half - arc vmat deliveries for a patient study set . Full - arc vmat led to a larger gantry acceleration and larger gantry angle error than the half - arc vmat . In addition, gantry acceleration and gantry angle error appeared to show a considerable correlation . Figure 2.plots of gantry acceleration (black line) and gantry angle errors (gray line) as a function of treatment time for a patient study set, with (a) full - arc and (b) half - arc vmat . On average, the full - arc vmat led to larger gantry acceleration and a larger gantry angle error than the half - arc vmat . Plots of gantry acceleration (black line) and gantry angle errors (gray line) as a function of treatment time for a patient study set, with (a) full - arc and (b) half - arc vmat . On average, the full - arc vmat led to larger gantry acceleration and a larger gantry angle error than the half - arc vmat . Gantry acceleration as a function of arc angle divided by total mu during each of the full - arc and half - arc vmat deliveries . Gantry angle acceleration was linearly related to arc angle divided by total mu with an r value of 0.69 . Figure 3.relationship between full - arc (gray square) and half - arc (black diamond) vmat deliveries. (a) r.m.s . Gantry acceleration during each delivery (n = 6), and (b) r.m.s . Gantry acceleration as a function of arc angle divided by total mu for each delivery . Relationship between full - arc (gray square) and half - arc (black diamond) vmat deliveries . Table 3 compares total mu, mean dose rate, delivery time, r.m.s . Gantry acceleration and r.m.s . Gantry angle error between the half - arc and full - arc vmat plans . On average, the half - arc plans resulted in 11% less total mu, 20% shorter delivery time, 12% higher mean dose rate, 30% less r.m.s . Differences in all parameters were statistically significant according to the wilcoxon signed - rank test (p <0.05). Table 3.comparison of delivery parameters between full - arc and half - arc vmat plans(n = 6)full - archalf - arcp valuetotal mu0.028 mean506.2449.2 range430.0570.0378.0541.0mean dose rate (mu / min)0.046 mean135.6152.7 range116.7164.3135.0175.9delivery time (s)0.028 mean223.2178.3 range179.0264.0143.0210.0r.m.s . Gantry angle error ()0.028 mean0.280.21 range0.190.340.160.31group averages with ranges in parentheses are shown (n = 6). The wilcoxon signed - rank test resulted in statistically significant differences (p <0.05) for all parameters shown below . Comparison of delivery parameters between full - arc and half - arc vmat plans group averages with ranges in parentheses are shown (n = 6). The wilcoxon signed - rank test resulted in statistically significant differences (p <0.05) for all parameters shown below . In this study, we demonstrated that half - arc vmat plans provided significantly reduced doses to the normal brain and brainstem compared with full - arc vmat plans, while maintaining nearly identical dose homogeneity and conformity in the ptv . For other oars, no significant dvh differences were observed between the full - arc and half - arc vmat plans, possibly because these oars were located closer to the ptv . These results suggest that half - arc vmat planning provides a clinical advantage in maxillary cancer . In contrast, conventional 3d plans for a maxillary cancer resulted in much higher doses to oars, whereas four- and nine - field imrt plans provided target coverage and sparing ability similar to our half - arc plans . Our half - arc vmat plan may provide a better treatment option due to significantly decreased delivery time compared with the imrt and 3d plans . The dosimetric findings in the present study were consistent with those in a previous report in patients with lung cancer . We confirmed that the delivery time and total mu required for half - arc vmat delivery for maxillary cancer were less than those for full - arc delivery . The reduced mu may decrease the risk of secondary cancer . With regard to gantry acceleration and gantry angle error during vmat delivery, plots of r.m.s . Gantry acceleration during the half - arc and full - arc vmat deliveries showed an approximately linear correlation . The greater gantry angle acceleration during full - arc delivery may be due to increased arc angle per total mu . Values in half - arc deliveries were smaller than those in the full - arc deliveries . This finding might suggest that larger acceleration of the gantry and the accompanying greater inertia might result in a larger gantry angle error . We have demonstrated that half - arc vmat has clinical advantages over full - arc vmat for maxillary cancer . Advantages of the half - arc vmat planning include improved oar sparing with nearly identical hi and ci for the ptv . The advantages of the half - arc vmat delivery include reductions in total mu and delivery time, thereby leading to better patient comfort and treatment throughput.
Numerical investigations of cosmological spacetimes can be categorized into two broad classes of calculations, distinguished by their computational (or even philosophical) goals: 1) geometrical and mathematical principles of cosmological models, and 2) physical and astrophysical cosmology . In the former, the emphasis is on the geometric framework in which astrophysical processes occur, namely the cosmological expansion, shear, and singularities of the many models allowed by the theory of general relativity . In the latter, the emphasis is on the cosmological and astrophysical processes in the real or observable universe, and the quest to determine the model which best describes our universe . The former is pure in the sense that it concerns the fundamental nonlinear behavior of the einstein equations and the gravitational field . The latter is more complex, since it addresses the composition, organization and dynamics of the universe from the small scales (fundamental particles and elements) to the large (galaxies and clusters of galaxies). However, the distinction is not always so clear, and geometric effects in the spacetime curvature can have significant consequences for the evolution and observation of matter distributions . Any comprehensive model of cosmology must therefore include nonlinear interactions between different matter sources and spacetime curvature . A realistic model of the universe must also cover large dynamical spatial and temporal scales, extreme temperature and density distributions, and highly dynamic atomic and molecular matter compositions . In addition, due to all the varied physical processes of cosmological significance, one must draw from many disciplines of physics to model curvature anisotropies, gravitational waves, electromagnetic fields, nucleosynthesis, particle physics, hydrodynamic fluids, etc . These phenomena are described in terms of coupled nonlinear partial differential equations and must be solved numerically for general inhomogeneous spacetimes . The situation appears extremely complex, even with current technological and computational advances . As a result, the codes and numerical methods that have been developed to date are designed to investigate very specific problems with either idealized symmetries or simplifying assumptions regarding the metric behavior, the matter distribution / composition or the interactions among the matter types and spacetime curvature . It is the purpose of this article to review published numerical cosmological calculations addressing issues from the very early universe to the present; from the purely geometrical dynamics of the initial singularity to the large scale structure of the universe . There are three major sections: 2 where a brief overview is presented of various defining events ocurring throughout the history of our universe and in the context of the standard model; 3 where brief summaries of early universe and fully relativistic cosmological calculations are presented; and 4 which focuses on structure formation in the post - recombination epoch and on testing cosmological models against observations . With current observational constraints, the physical state of our universe, as understood in the context of the standard, or friedmann - robertson - lematre - walker (flrw) model, can be crudely extrapolated back to the planck epoch 10 seconds after the big bang, beyond which the classical theory of general relativity is invalid due to quantum corrections . At the earliest times, the universe was a plasma of relativistic particles consisting of quarks, leptons, gauge bosons, and higgs bosons represented by scalar fields with interaction and symmetry regulating potentials . It is believed that several spontaneous symmetry breaking (ssb) phase transitions transpired in the early universe as it expanded and cooled, including: the grand unification transition (gut) at 10 seconds after the big bang (here, the strong nuclear force split off from the weak and electromagnetic forces . This also marks an era of inflationary expansion and the origin of matter - antimatter asymmetry through baryon, charge conjugation, and charge + parity violating interactions and nonequilibrium effects . ); the electroweak (ew) ssb transition at 10 secs (when the weak nuclear force split from the electromagnetic force); and the chiral (qcd) symmetry breaking transition at 10 secs during which quarks condensed into hadrons . The most stable hadrons (baryons, or protons and neutrons comprised of three quarks) survived the subsequent period of baryon - antibaryon annihilations, which continued until the universe cooled to the point at which new baryon - antibaryon pairs could no longer be produced . A period of primordial nucleosynthesis followed from 10 to 10 secs during which light element abundances were synthesized to form 24% helium with trace amounts of deuterium, tritium, helium-3, and lithium . By 10 secs, the matter density became equal to the radiation density, identifying the start of the current matter - dominated era and the beginning of structure formation . Later, at 10 secs (3 10 years), the free ions and electrons combine to form atoms, decoupling the matter from the radiation field as the universe continued to expand and cool . This decoupling or post - recombination epoch marks the surface of last scattering and the boundary of the observable (via photons) universe . Assuming a hierarchical (cdm - like) structure formation scenario, the subsequent development of our universe is characterized by the growth of structures with increasing size . For example, the first stars are likely to form at t 10 years from molecular gas clouds when the jeans mass of the background baryonic fluid is approximately 10 m, as indicated in figure 1 . This epoch of pop iii star generation is followed by the formation of galaxies at t 10 years and then galaxy clusters . The solid and dotted lines potentially track the jeans mass of the average baryonic gas component from the recombination epoch at z 10 to the current time . A residual ionization fraction of nh+/nh 10 following recombination allows for compton interactions with photons to z 200, during which the jeans mass remains constant at 10 m. the jeans mass then decreases as the universe expands adiabatically until the first collapsed structures form sufficient amounts of hydrogen molecules to trigger a cooling instability and produce pop iii stars at z 20 . Star formation activity can then reheat the universe and raise the mean jeans mass to above 10 m. this reheating could affect the subsequent development of structures such as galaxies and the observed lyman - alpha clouds . The solid and dotted lines potentially track the jeans mass of the average baryonic gas component from the recombination epoch at z 10 to the current time . A residual ionization fraction of nh+/nh 10 following recombination allows for compton interactions with photons to z 200, during which the jeans mass remains constant at 10 m. the jeans mass then decreases as the universe expands adiabatically until the first collapsed structures form sufficient amounts of hydrogen molecules to trigger a cooling instability and produce pop iii stars at z 20 . Star formation activity can then reheat the universe and raise the mean jeans mass to above 10 m. this reheating could affect the subsequent development of structures such as galaxies and the observed lyman - alpha clouds . The isotropic and homogeneous flrw cosmological model has been so successful in describing the observable universe that it is commonly referred to as the standard model . Furthermore, and to its credit, the model is relatively simple; it allows for calculations and predictions to be made of the very early universe, including primordial nucleosynthesis at 10 seconds after the big bang, and even particle interactions approaching the planck scale at 10 seconds . At present, observational support for the standard model includes: the expansion of the universe as verified by the redshifts in galaxy spectra and quantified by measurements of the hubble constant h0 = 100h km sec mpc with hubble parameter 0.5 <h <1;the deceleration parameter observed in distant galaxy spectra (although uncertainties about galactic evolution, intrinsic luminosities, and standard candles prevent even a crude estimate);the large scale isotropy and homogeneity of the universe based on temperature anisotropy measurements of the microwave background radiation and peculiar velocity fields of galaxies (although the light distribution from bright galaxies seems more tenuous);the age of the universe which yields roughly consistent estimates between the look - back time to the big bang in the flrw model and observed data such as the oldest stars, radioactive elements, and cooling of white dwarf stars;the cosmic microwave background radiation suggests that the universe began from a hot big bang and the data is consistent with a black body at temperature 2.7 k;the abundance of light elements such as h, he, he and li, as predicted from the flrw model, are consistent with observations and provides a bound on the baryon density and baryon - to - photon ratio;the present mass density, as determined from measurements of luminous matter and galactic rotation curves, can be accounted for by the flrw model with a single density parameter to specify the metric topology;the distribution of galaxies and larger scale structures can be reproduced by numerical simulations in the context of inhomogeneous perturbations of the flrw models . The expansion of the universe as verified by the redshifts in galaxy spectra and quantified by measurements of the hubble constant h0 = 100h km sec mpc with hubble parameter 0.5 <h <1; the deceleration parameter observed in distant galaxy spectra (although uncertainties about galactic evolution, intrinsic luminosities, and standard candles prevent even a crude estimate); the large scale isotropy and homogeneity of the universe based on temperature anisotropy measurements of the microwave background radiation and peculiar velocity fields of galaxies (although the light distribution from bright galaxies seems more tenuous); the age of the universe which yields roughly consistent estimates between the look - back time to the big bang in the flrw model and observed data such as the oldest stars, radioactive elements, and cooling of white dwarf stars; the cosmic microwave background radiation suggests that the universe began from a hot big bang and the data is consistent with a black body at temperature 2.7 k; the abundance of light elements such as h, he, he and li, as predicted from the flrw model, are consistent with observations and provides a bound on the baryon density and baryon - to - photon ratio; the present mass density, as determined from measurements of luminous matter and galactic rotation curves, can be accounted for by the flrw model with a single density parameter to specify the metric topology; the distribution of galaxies and larger scale structures can be reproduced by numerical simulations in the context of inhomogeneous perturbations of the flrw models . Because of these successes, most work in the field of physical cosmology (see 4) has utilized the standard model as the background spacetime in which the large scale structure evolves, with the ambition to further constrain the cosmological parameters and structure formation scenarios through numerical simulations . The reader is referred to for a more in - depth review of the standard model . This section is organized to track the chronological events in the history of the early or relativistic universe; we focus mainly on four defining moments in our current understanding of the classical picture: 1) the big bang singularity and the dynamics of the very early universe; 2) inflation and its generic nature; 3) qcd phase transitions; and 4) primordial nucleosynthesis and the freeze - out of the light elements . The late or post - recombination epoch is reserved to a separate section 4 . Belinsky, lifshitz and khalatnikov (blk) [11, 12] and misner discovered that the einstein equations in the vacuum homogeneous bianchi type ix (or mixmaster) cosmology exhibit complex behavior and are sensitive to initial conditions as the big bang singularity is approached . In particular, the solutions near the singularity are described qualitatively by a discrete map [10, 11] representing different sequences of kasner spacetimes 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$d{s^2} = - d{t^2} + {t^{2{p_1}}}d{x^2} + {t^2}^{{p_2}}d{y^2} + {t^2}^{{p_3}}d{z^2},$$\end{document}ds2=dt2+t2p1dx2+t2p2dy2+t2p3dz2, with time changing exponents pi, but otherwise constrained by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${p_1} + {p_2} + {p_3} = p_1 ^ 2 + p_2 ^ 2 + p_3 ^ 2 = 1$\end{document}p1+p2+p3=p12+p22+p32=1 . Because this discrete mapping of kasner epochs is chaotic mixmaster behavior can be studied in the context of hamiltonian dynamics, with the hamiltonian 2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2{\mathcal h} = - p_\omega ^2 + p _ + ^2 + p _ - ^2 + {e^{4\omega}}(v - 1),$$\end{document}2=p2+p+2+p2+e4(v1), and a semi - bounded potential arising from the spatial scalar curvature (whose level curves are plotted in figure 2) 3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v = 1 + {1 \over 3}{e^{- 8{\beta _ +}}} + {2 \over 3}{e^{4{\beta _ +}}} \left [{\cosh (4\sqrt 3 {\beta _ -}) - 1} \right] - {4 \over 3}{e^{- 2{\beta _ +}}} \cosh (2\sqrt 3 {\beta _ -}),$$\end{document}v=1 + 13e8++23e4+[cosh(43)1]43e2+cosh(23), where e and are the scale factor and anisotropies, and p and p are the corresponding conjugate variables . Figure 2contour plot of the bianchi type ix potential v, where are the anisotropy canonical coordinates . Seven level surfaces are shown at equally spaced decades ranging from 10 to 10 . For large isocontours (v> 1), the potential is open and exhibits a strong triangular symmetry with three narrow channels extending to spatial infinity . For v contour plot of the bianchi type ix potential v, where are the anisotropy canonical coordinates . Seven level surfaces are shown at equally spaced decades ranging from 10 to 10 . For large isocontours (v> 1), the potential is open and exhibits a strong triangular symmetry with three narrow channels extending to spatial infinity . For v a solution of this hamiltonian system is an infinite sequence of kasner epochs with parameters that change when the phase space trajectories bounce off the potential walls, which become exponentially steep as the system evolves towards the singularity . Several numerical calculations of the dynamical equations arising from (2) and (3) have indicated that the liapunov exponents of the system vanish, in apparent contradiction with the discrete maps [17, 31]. However, it has since been shown that the usual definition of the liapunov exponents is ambiguous in this case as it is not invariant under time reparametrizations, and that with a different time variable one obtains positive exponents [13, 27]. Although blk conjectured that local mixmaster oscillations might be the generic behavior for singularities in more general classes of spacetimes, this conjecture has yet to be established . However, weaver et al . Have, through numerical investigations, established evidence that mixmaster dynamics can occur in (at least a restricted class of) inhomogeneous spacetimes which generalize the bianchi type vi0 with a magnetic field and two - torus symmetry . As noted in 3.1.1, an interesting and (as yet) unresolved issue is whether or not the generic cosmological singularity is locally of a mixmaster or blk type, with a complex oscillatory behavior as the singularity is approached . Most of the other bianchi models, including the kasner solutions (1), are characterized by either open or no potentials in the hamiltonian framework, and exhibit essentially monotonic or asymptotically velocity term dominated (avtd) behavior, the opposite dynamics to the complex blk oscillations . Considering inhomogeneous spacetimes, isenberg and moncrief proved that the singularity in the polarized gowdy model is avtd type . This has also been conjectured to be the case for more general gowdy models, and numerical simulations of one - dimensional plane symmetric gowdy spacetimes support the notion . Furthermore, a symplectic numerical method has been applied to investigate the avtd conjecture in even more general spacetimes, namely t r spacetimes with u(1) symmetry . Although there are concerns about the solutions due to difficulties in resolving the steep spatial gradients near the singularity, the numerical calculations find no evidence of blk oscillations . Berger attributes this to several possibilities: 1) the blk conjecture is false; 2) the simulations have not been run long enough; 3) mixmaster behavior is present but hidden in the variables; or 4) the initial data is insufficiently generic . In any case, the inflation paradigm is frequently invoked to explain the flatness (1) of the universe, attributing it to an era of exponential expansion at about 10 seconds after the big bang . The mechanism of inflation is generally taken to be an effective cosmological constant from the dominant stress - energy of the inflaton scalar field that regulates gut symmetry breaking, particle creation, and the reheating of the universe . In order to study whether inflation can occur for arbitrary anisotropic and inhomogeneous data, many numerical simulations have been carried out with different symmetries, matter types and perturbations . Kurki - suonio et al . Extended the planar cosmology code of centrella and wilson [22, 23] (see 3.5) to include a scalar field and simulate the onset of inflation in the early universe with an inhomogeneous higgs field and a perfect fluid with a radiation equation of state p = /3 . Their results suggest that whether inflation occurs or not can be sensitive to the shape of the potential . In particular, if the shape is flat enough and satisfies the slow - roll conditions (essentially upper bounds on v/ and v/ near the false vacuum 0), even large initial fluctuations of the higgs field do not prevent inflation . They considered two different forms of the potential: a coleman - weinberg type 4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v(\phi) = \lambda {\phi ^4}\left [{\ln \left({{{{\phi ^2}} \over {{\sigma ^2}}}} \right) - {1 \over 2}} \right] + {{\lambda {\sigma ^4}} \over 2}$$\end{document}v()=4[ln(22)12]+42 which is very flat close to the false vacuum and does inflate; and a rounder type 5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$v(\phi) = \lambda {({\phi ^2} - {\sigma ^2})^2}$$\end{document}v()=(22)2 which, for their parameter combinations, does not . Goldwirth and piran studied the onset of inflation with inhomogeneous initial conditions for closed, spherically symmetric spacetimes containing a massive scalar field and radiation field sources (described by a massless scalar field). In all the cases they considered, the radiation field damps quickly and only an inhomogeneous massive scalar field remains to inflate the universe . They find that small inhomogeneities tend to reduce the amount of inflation and large initial inhomogeneities can even suppress the onset of inflation . Suitable initial values over a domain of several horizon lengths in order for inflation to begin . Investigated the simplest bianchi model (type v) background that admits velocities or tilt in order to address the question of how the universe can choose a uniform reference frame at the exit from inflation, since the desitter metric does not have a preferred frame . However if inflation persists, the wave behavior eventually freezes in and all velocities go to zero at least as rapidly as tanh r, where is the relativistic tilt angle (a measure of velocity), and r is a typical scale associated with the radius of the universe . Their results indicate that the velocities eventually go to zero as inflation carries all spatial variations outside the horizon, and that the answer to the posed question is that memory is retained and the universe is never really desitter . In addition to the inflaton field, one can consider other sources of inhomogeneity, such as gravitational waves . Although linear waves in desitter space will decay exponentially and disappear, it is unclear what will happen if strong waves exist . Shinkai & maeda investigated the cosmic no - hair conjecture with gravitational waves and a cosmological constant in 1d plane symmetric vacuum spacetimes, setting up gaussian pulse wave data with amplitudes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$0.02\lambda \le \max (\sqrt i) \le 80\lambda$\end{document}0.02max(i)80 and widths 0.08lh l 2.5lh, where i is the invariant constructed from the 3-riemann tensor and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${l_h} = \sqrt {3/\lambda}$\end{document}lh=3/ is the horizon scale . They also considered colliding plane waves with amplitudes \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$40\lambda \le \max (\sqrt i) \le 125\lambda$\end{document}40max(i)125 and widths 0.08lh l 0.1lh . They find that for any large amplitude and/or small width with inhomogeneity in their parameter sets, the nonlinearity of gravity has little effect, and the spacetime always evolves into a desitter spacetime . The previous paragraphs discussed results from highly symmetric spacetimes, but the possibility of inflation remains to be established for more general inhomogeneous and nonperturbative data . To address this issue, kurki - suonio et al . Investigated fully three - dimensional inhomogeneous spacetimes with a chaotic inflationary potential v() = /4 . They considered basically two types of runs: small and large scale . In the small scale run, the grid length was initially set equal to the hubble length so the inhomogeneities are well inside the horizon and the dynamical time scale is shorter than the expansion or hubble time . As a result, the perturbations oscillate and damp while the field evolves and the spacetime inflates . In the large scale run, they maintain their shape without damping and, because larger values of lead to faster expansion, the inhomogeneity in the expansion becomes steeper in time, since the regions of large and high inflation stay correlated . The standard picture of cosmology assumes that a phase transition (associated with chiral symmetry breaking after the electroweak transition) occurred at approximately 10 seconds after the big bang to convert a plasma of free quarks and gluons into hadrons . Although this transition can be of significant cosmological importance, it is not known with certainty whether it is of first order or higher, and what the astrophysical consequences might be on the subsequent state of the universe . For example, the transition may give rise to significant baryon number inhomogeneities which can influence the outcome of primordial nucleosynthesis as evidenced in the distribution and averaged light element abundances . The qcd transition and baryon inhomogeneities may also play a significant and potentially observable role in the generation of primordial magnetic fields . Considered a first order phase transition and the nucleation of hadronic bubbles in a supercooled quark - gluon plasma, solving the relativistic lagrangian equations for disconnected and evaporating quark regions during the final stages of the phase transition . They numerically investigated a single isolated quark drop with an initial radius large enough so that surface effects can be neglected . The droplet evolves as a self - similar solution until it evaporates to a sufficiently small radius that surface effects break the similarity solution and increase the evaporation rate . Their simulations indicate that, in neglecting long - range energy and momentum transfer (by electromagnetically interacting particles) and assuming that the baryon number is transported with the hydrodynamical flux), the baryon number concentration is similar to what is predicted by chemical equilibrium calculations . Kurki - suonio and laine studied the growth of bubbles and the decay of droplets using a spherically symmetric code that accounts for a phenomenological model of the microscopic entropy generated at the phase transition surface . Incorporating the small scale effects of the finite wall width and surface tension, but neglecting entropy and baryon flow through the droplet wall, they also find that evaporating droplets do not leave behind a global rarefaction wave to dissipate any previously generated baryon number inhomogeneity . Observations of the light elements produced during big bang nucleosynthesis following the quark / hadron phase transition (roughly 10 10 seconds after the big bang) are in good agreement with the standard model of our universe (see 2.2). However, it is interesting to investigate other more general models to assert the role of shear and curvature on the nucleosynthesis process . Rothman and matzner considered primordial nucleosynthesis in anisotropic cosmologies, solving the strong reaction equations leading to he . They find that the concentration of he increases with increasing shear; this is due to time scale effects and the competition between dissipation and enhanced reaction rates from photon heating and neutrino blue shifts . Their results have been used to place a limit on anisotropy at the epoch of nucleosynthesis . Kurki - suonio and matzner extended this work to include 30 strong 2-particle reactions involving nuclei with mass numbers a 7, and to demonstrate the effects of anisotropy on the cosmologically significant isotopes h, he, he and li as a function of the baryon to photon ratio . They conclude that the effect of anisotropy on h and he is not significant, and the abundances of he and li increase with anisotropy in accord with . Furthermore, it is possible that neutron diffusion, the process whereby neutrons diffuse out from regions of very high baryon density just before nucleosynthesis, can affect the neutron to proton ratio in such a way as to enhance deuterium and reduce he compared to a homogeneous model . However, plane symmetric, general relativistic simulations with neutron diffusion show that the neutrons diffuse back into the high density regions once nucleosynthesis begins there thereby wiping out the effect . As a result, although inhomogeneities influence the element abundances, they do so at a much smaller degree then previously speculated . The numerical simulations also demonstrate that, because of the back diffusion, a cosmological model with a critical baryon density cannot be made consistent with helium and deuterium observations, even with substantial baryon inhomogeneities and the anticipated neutron diffusion effect . Gravitational waves are an inevitable product of the general einstein equations, and one can expect a wide spectrum of wave signals propagating throughout our universe due to shear anisotropies, primordial metric and matter fluctuations, collapsing matter structures, ringing black holes, and colliding neutron stars, for example . The discussion here is restricted to the pure vacuum field dynamics and the fundamental nonlinear behavior of gravitational waves in numerically generated cosmological spacetimes . Centrella and matzner [20, 21] studied a class of plane symmetric cosmologies representing gravitational inhomogeneities in the form of shocks or discontinuities separating two vacuum expanding kasner cosmologies (1). By a suitable choice of parameters, the constraint equations can be satisfied at the initial time with an euclidean 3-surface and an algebraic matching of parameters across the different kasner regions that gives rise to a discontinuous extrinsic curvature tensor . They performed both numerical calculations and analytical estimates using a green s function analysis to establish and verify (despite the numerical difficulties in evolving discontinuous data) certain aspects of the solutions, including gravitational wave interactions, the formation of tails, and the singularity behavior of colliding waves in expanding vacuum cosmologies . Shortly thereafter, centrella and wilson [22, 23] developed a more general plane symmetric code for cosmology, adding also hydrodynamic sources . In order to simplify the resulting differential equations, they adopted a diagonal 3-metric of the form 6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\gamma _ {ij}} = {a^2}(t, \, z)\,\,\left({\begin{array}{*{20}c} 1 & 0 & 0 \\ 0 & {{h^2}(t, \, z)} & 0 \\ 0 & 0 & 1 \\ \end{array}} \right),$$\end{document}ij = a2(t, z)(1000h2(t, z)0001), which is maintained in time with a proper choice of shift vector . The metric (6) allows an overall conformal factor a to simplify the initial value problem, and a dynamical transverse wave component in the variable h. the hydrodynamic equations are solved using artificial viscosity methods for shock capturing and barton s method for monotonic transport . The evolutions are fully constrained (solving both the momentum and hamiltonian constraints at each time step) and use the mean curvature slicing condition . [1, 3], implementing more robust numerical methods and an improved parametric treatment of the initial value problem . In applications of these codes, centrella investigated nonlinear gravity waves in minkowski space and compared the full numerical solutions against a first order perturbation solution to benchmark certain numerical issues such as numerical damping and dispersion . A second order perturbation analysis was also used to model the transition into the nonlinear regime . Considered small and large perturbations in the two degenerate kasner models: p1 = p3 = 0 or 2/3, and p2 = 1 or 1/3 respectively, where pi are parameters in the kasner metric (1). Carrying out a second order perturbation expansion and computing the newman - penrose (np) scalars, riemann invariants and bel - robinson vector, they demonstrated, for their particular class of spacetimes, that the nonlinear behavior is in the coulomb (or background) part represented by the leading order term in the np scalar and not in the gravitational wave component . For standing - wave perturbations, the dominant second order effects in their variables are an enhanced monotonic increase in the background expansion rate, and the generation of oscillatory behavior in the background spacetime with frequencies equal to the harmonics of the first order standing - wave solution . A unique approach to numerical cosmology (and numerical relativity in general) is the method of regge calculus in which spacetime is represented as a complex of 4-dimensional, geometrically flat simplices . The principles of einstein s theory are applied directly to the simplicial geometry to form the curvature, action and field equations, in contrast to the finite difference approach where the continuum field equations are differenced on a discrete mesh . A 3-dimensional code implementing regge calculus techniques was developed recently by gentle and miller and applied to the kasner cosmological model . They also describe a procedure to solve the constraint equations for time asymmetric initial data on two spacelike hypersurfaces constructed from tetrahedra, since full 4-dimensional regions or lattices are used . The new method is analogous to york s procedure where the conformal metric, trace of the extrinsic curvature, and momentum variables are all freely specifiable . Although additional work is needed to apply (and develop) regge calculus techniques to more general spacetimes, the early results of gentle and miller are promising . In particular, their evolutions have reproduced the continuum kasner solution, achieved second order convergence, and sustained numerical stability . The phrase physical cosmology is generally associated with the large (galaxy and cluster) scale structure of the post - recombination epoch where gravitational effects are modeled approximately by newtonian physics on an uniformly expanding, matter dominated flrw background . A discussion of the large scale structure is included in this review since any viable model of our universe which allows a regime where strongly general relativistic effects are important must match onto the weakly relativistic (or newtonian) regime . Also, since certain aspects of this regime are directly observable, one can hope to constrain or rule out various cosmological models and/or parameters, including the density (0), hubble (h0 = 100h km sec mpc), and cosmological () constants . Due to the vast body of literature on numerical simulations of the post - recombination epoch, it is possible to mention only a small fraction of all the published papers . Hence, the following summary is limited to cover just a few aspects of computational physical cosmology, and in particular those that can potentially be used to discriminate between cosmological model parameters, even within the realm of the standard model . The cosmic microwave background radiation (cmbr), which is a direct relic of the early universe, currently provides the deepest probe of cosmological structures and imposes severe constraints on the various proposed matter evolution scenarios and cosmological parameters . Although the cmbr is a unique and deep probe of both the thermal history of the early universe and the primordial perturbations in the matter distribution, the associated anisotropies are not exclusively primordial in nature . Important modifications to the cmbr spectrum can arise from large scale coherent structures, even well after the photons decouple from the matter at redshift z 10, due to the gravitational redshifting of the photons through the sachs - wolfe effect arising from potential gradients [49, 5] 7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\delta t} \over t} = {\phi _ e} - {\phi _ r} - \int\nolimits_r^e {{{2\overrightarrow l \cdot \nabla \phi} \over a}dt,}$$\end{document}tt=erre2ladt, where the integral is evaluated from the emission (e) to reception (r) points along the spatial photon paths \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\vec l,\phi$\end{document}l, is the gravitational potential, t / t defines the temperature fluctuations, and a(t) is the cosmological scale factor in the standard flrw metric . Also, if the intergalactic medium (igm) reionizes sometime after the decoupling, say from an early generation of stars, the increased rate of thomson scattering off the free electrons will erase sub - horizon scale temperature anisotropies, while creating secondary doppler shift anisotropies . To make meaningful comparisons between numerical models and observed data, all of these effects (and others, see for example 4.1.3) must be incorporated self - consistently into the numerical models and to high accuracy in order to resolve the weak signals . Many computational analyses based on linear perturbation theory have been carried out to estimate the temperature anisotropies in the sky (for example see and the references cited in). Although such linearized approaches yield reasonable results, they are not well - suited to discussing the expected imaging of the developing nonlinear structures in the microwave background . An alternative ray - tracing approach has been developed by anninos et al . To introduce and propagate individual photons through the evolving nonlinear matter structures . They solve the geodesic equations of motion and subject the photons to thomson scattering in a probabilistic way and at a rate determined by the local density of free electrons in the model . Since the temperature fluctuations remain small, the equations of motion for the photons are treated as in the linearized limit, and the anisotropies are computed according to 8\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\delta t} \over t} = {{\delta z} \over {1 + z}},$$\end{document}tt=z1+z, where 9\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1 + z = {{{{({k^\mu}{u_\mu})}_e}} \over {{{({k^\mu}{u_\mu})}_r}}},$$\end{document}1+z=(ku)e(ku)r, and the photon wave vector k and matter rest frame four - velocity u are evaluated at the emission (e) and reception (r) points . Applying their procedure to a hot dark matter (hdm) model of structure formation, anninos et al . Find the parameters for this model are severely constrained by cobe data such that 0h 1, where 0 and h are the density and hubble parameters . In models where the igm does not reionize, the probability of scattering after the photon - matter decoupling epoch is low, and the sachs - wolfe effect dominates the anisotropies at angular scales larger than a few degrees . However if reionization occurs, the scattering probability increases substantially and the matter structures, which develop large bulk motions relative to the comoving background, induce doppler shifts on the scattered cmbr photons and leave an imprint of the surface of last scattering . The induced fluctuations on subhorizon scales in reionization scenarios can be a significant fraction of the primordial anisotropies, as observed by tuluie et al . . They considered two possible scenarios of reionization: a model that suffers early and gradual (eg) reionization of the igm as caused by the photoionizing uv radiation emitted by decaying neutrinos, and the late and sudden (ls) scenario as might be applicable to the case of an early generation of star formation activity at high redshifts . Considering the hdm model with 0 = 1 and h = 0.55, which produces cmbr anisotropies above current cobe limits when no reionization is included (see 4.1.1), they find that the eg scenario effectively reduces the anisotropies to the levels observed by cobe and generates smaller doppler shift anisotropies than the ls model, as demonstrated in figure 3 . Figure 3the top two images represent temperature fluctuations (i.e., t / t) due to the sachs - wolfe effect and doppler shifts in a standard critically closed hdm model with no reionization and baryon fractions 0.02 (plate 1, 4 4, rms=2.8 10) and 0.2 (plate 2, 8 8, rms=3.4 10). The bottom two plates image fluctuations in an early and gradual reionization scenario of decaying neutrinos with baryon fraction 0.02 (plate 3, 4 4, rms=1.3 10; and plate 4, 8 8, rms = 1.4 10). The top two images represent temperature fluctuations (i.e., t / t) due to the sachs - wolfe effect and doppler shifts in a standard critically closed hdm model with no reionization and baryon fractions 0.02 (plate 1, 4 4, rms=2.8 10) and 0.2 (plate 2, 8 8, rms=3.4 10). The bottom two plates image fluctuations in an early and gradual reionization scenario of decaying neutrinos with baryon fraction 0.02 (plate 3, 4 4, rms=1.3 10; and plate 4, 8 8, rms = 1.4 10). The ls scenario of reionization is not able to reduce the anisotropy levels below the cobe limits, and can even give rise to greater doppler shifts than expected at decoupling . Additional sources of cmbr anisotropy can arise from the interactions of photons with dynamically evolving matter structures and nonstatic gravitational potentials . Considered the impact of nonlinear matter condensations on the cmbr in 0 1 cold dark matter (cdm) models, focusing on the relative importance of secondary temperature anisotropies due to three different effects: 1) time - dependent variations in the gravitational potential of nonlinear structures as a result of collapse or expansion; 2) proper motion of nonlinear structures such as clusters and superclusters across the sky; and 3) the decaying gravitational potential effect from the evolution of perturbations in open models . They applied the ray - tracing procedure of to explore the relative importance of these secondary anisotropies as a function of the density parameter 0 and the scale of matter distributions . They find that the secondary temperature anisotropies are dominated by the decaying potential effect at large scales, but that all three sources of anisotropy can produce signatures of order t / t 10 as shown in figure 4 . Figure 4the top two images represent the proper motion and rees - sciama effects in the cmbr for a critically closed cdm model (upper left), together with the corresponding column density of voids and clusters over the same region (upper right). The bottom two images show the secondary anisotropies dominated here by the decaying potential effect in an open cosmological model (bottom left), together with the corresponding gravitational potential over the same region (bottom right). The rms fluctuations in both cases are on the order of 5 10, though the open model carries a somewhat larger signature . The top two images represent the proper motion and rees - sciama effects in the cmbr for a critically closed cdm model (upper left), together with the corresponding column density of voids and clusters over the same region (upper right). The bottom two images show the secondary anisotropies dominated here by the decaying potential effect in an open cosmological model (bottom left), together with the corresponding gravitational potential over the same region (bottom right). The rms fluctuations in both cases are on the order of 5 10, though the open model carries a somewhat larger signature . In addition to the effects discussed in the previous paragraphs, many other sources of secondary anisotropies (such as gravitational lensing, the vishniac effect accounting for matter velocities and flows into local potential wells, and the sunyaev - zeldovich distortions from the compton scattering of cmb photons in the hot cluster medium) can also be significant . See reference for a more complete list and thorough discussion of the different sources of cmbr anisotropies . Observations of gravitational lenses provide measures of the abundance and strength of nonlinear potential fluctuations along the lines of sight to distant objects . Since these calculations are sensitive to the gravitational potential, they may be more reliable than galaxy and velocity field measurements as they are not subject to the same ambiguities associated with biasing effects . Also, since different cosmological models predict different mass distributions, especially at the higher redshifts, lensing calculations can potentially be used to confirm or discard competing cosmological models . As an alternative to solving the computationally demanding lens equations, cen et al developed a more efficient scheme to identify regions with surface densities capable of generating multiple images accurately for splittings larger than three arcseconds . They applied this technique to a standard cdm model with 0 = 1, and found that this model predicts more large angle splittings (> 8) than are known to exist in the observed universe . Their results suggest that the standard cdm model should be excluded as a viable model of our universe . A similar analysis for a flat low density cdm model with a cosmological constant \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$({\omega _ 0} = 0.3,\,\,\lambda/3h_0 ^ 2 = 0{. }7)$\end{document}(0=0.3,/3h02=0.7) suggests a lower and perhaps acceptable number of lensing events . However, an uncertainty in their studies is the nature of the lenses at and below the resolution of the numerical grid . They model the lensing structures as simplified singular isothermal spheres (sis) with no distinctive cores . Large angle splittings are generally attributed to larger structures such as clusters of galaxies, and there are indications that clusters have small but finite core radii rcore 20 30h kpc . Core radii of this size can have an important effect on the probability of multiple imaging . Flores and primack considered the effects of assuming two different kinds of splitting sources: isothermal spheres with small but finite core radii \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\rho \propto {({r^2} + r_{core}^2)^{- 1}}$\end{document}(r2+rcore2)1, and spheres with a hernquist density profile r(r + a), where rcore 20 30h kpc and a 300h kpc . They find that the computed frequency of large - angle splittings, when using the nonsingular profiles, can potentially decrease by more than an order of magnitude relative to the sis case and can bring the standard cdm model into better agreement with observations . They stress that lensing events are sensitive to both the cosmological model (essentially the number density of lenses) and to the inner lens structure, making it difficult to probe the models until the structure of the lenses, both observationally and numerically, is better known . In cdm cosmogonies, the fluctuation spectrum at small wavelengths has a logarithmic dependence at mass scales smaller than 10 solar masses, which indicates that all small scale fluctuations in this model collapse nearly simultaneously in time . Furthermore, the cooling in these fluctuations is dominated by the rotational / vibrational modes of hydrogen molecules that were able to form using the free electrons left over from recombination and the ones produced by strong shock waves as catalysts . The first structures to collapse may be capable of producing population iii stars and have a substantial influence on the subsequent thermal evolution of the intergalactic medium, as suggested by figure 1, due to the radiation emitted by the first generation stars as well as supernova driven winds . To know the subsequent fate of the universe and which structures will survive or be destroyed by the uv background, it is first necessary to know when the first stars formed . Ostriker and gnedin have carried out high resolution numerical simulations of the reheating and reionization of the universe due to star formation bursts triggered by molecular hydrogen cooling . Accounting for the chemistry of the primeval hydrogen / helium plasma, self - shielding of the gas, radiative cooling, and a phenomenological model of star formation, they find that two distinct star populations form: the first generation pop iii from h2 cooling prior to reheating at redshift z 14; and the second generation pop ii at z <10 when the virial temperature of the gas clumps reaches 10 k and hydrogen line cooling becomes efficient . Star formation slows in the intermittent epoch due to the depletion of h2 by photo - destruction and reheating . In addition, the objects which formed pop iii stars also initiate pop ii sequences when their virial temperatures reach 10 k through continued mass accretion . The lyman - alpha forest represents the optically thin (at the lyman edge) component of quasar absorption systems (qas), a collection of absorption features in quasar spectra extending back to high redshifts . Qas are effective probes of the matter distribution and the physical state of the universe at early epochs when structures such as galaxies are still forming and evolving . Although stringent observational constraints have been placed on competing cosmological models at large scales by the cobe satellite and over the smaller scales of our local universe by observations of galaxies and clusters, there remains sufficient flexibility in the cosmological parameters that no single model has been established conclusively . The relative lack of constraining observational data at the intermediate to high redshifts (0 <z <5), where differences between competing cosmological models are more pronounced, suggests that qas can potentially yield valuable and discriminating observational data . Several combined n - body and hydrodynamic numerical simulations of the lyman forest have been performed recently [26, 41, 60], and all have been able to fit the observations remarkably well, including the column density and doppler width distributions, the size of absorbers, and the line number evolution . Despite the fact that the cosmological models and parameters are different in each case, the simulations give similar results, provided that the proper ionization bias is used (bion (bh)/, where b is the baryonic density parameter, h is the hubble parameter and is the photoionization rate at the hydrogen lyman edge). A theoretical paradigm has thus emerged from these calculations in which lyman - alpha absorption lines originate from the relatively small scale structure in pregalactic or intergalactic gas through the bottom - up hierarchical formation picture in cdm - like universes . The absorption features originate in structures exhibiting a variety of morphologies commonly found in numerical simulations (see figure 5), including fluctuations in underdense regions, spheroidal minihalos, and filaments extending over scales of a few megaparsecs . Figure 5distribution of the gas density at redshift z = 3 from a numerical hydrodynamics simulation of the lyman - alpha forest with a cdm spectrum normalized to second year cobe observations, a hubble parameter of h = 0.5, a comoving box size of 9.6 mpc, and baryonic density of b = 0.06 composed of 76% hydrogen and 24% helium . The isosurfaces represent baryons at ten times the mean density and are color coded to the gas temperature (dark blue = 3 10 k, light blue = 3 10 k). The higher density contours trace out isolated spherical structures typically found at the intersections of the filaments . A single random slice through the cube is also shown, with the baryonic overdensity represented by a rainbow - like color map changing from black (minimum) to red (maximum). The he mass fraction is shown with a wire mesh in this same slice . To emphasize fine structure in the minivoids, the mass fraction in the overdense regions has been rescaled by the gas overdensity wherever it exceeds unity . Distribution of the gas density at redshift z = 3 from a numerical hydrodynamics simulation of the lyman - alpha forest with a cdm spectrum normalized to second year cobe observations, a hubble parameter of h = 0.5, a comoving box size of 9.6 mpc, and baryonic density of b = 0.06 composed of 76% hydrogen and 24% helium . The isosurfaces represent baryons at ten times the mean density and are color coded to the gas temperature (dark blue = 3 10 k, light blue = 3 10 k). The higher density contours trace out isolated spherical structures typically found at the intersections of the filaments . A single random slice through the cube is also shown, with the baryonic overdensity represented by a rainbow - like color map changing from black (minimum) to red (maximum). The he mass fraction is shown with a wire mesh in this same slice . To emphasize fine structure in the minivoids, the mass fraction in the overdense regions has been rescaled by the gas overdensity wherever it exceeds unity . Clusters of galaxies are the largest gravitationally bound systems known to be in quasi - equilibrium . This allows for reliable estimates to be made of their mass as well as their dynamical and thermal attributes . The richest clusters, arising from 3 density fluctuations, can be as massive as 10 10 solar masses, and the environment in these structures is composed of shock heated gas with temperatures of order 10 10 degrees kelvin which emits thermal bremsstrahlung and line radiation at x - ray energies . Also, because of their spatial size 1h mpc and separations of order 50h mpc, they provide a measure of nonlinearity on scales close to the perturbation normalization scale 8h mpc . Observations of the substructure, distribution, luminosity, and evolution of galaxy clusters are therefore likely to provide signatures of the underlying cosmology of our universe, and can be used as cosmological probes in the easily observable redshift range 0 z 1 . Thomas et al . Have investigated the internal structure of galaxy clusters formed in high resolution n - body simulations of four different cosmological models, including standard, open, and flat but low density universes . They find that the structure of relaxed clusters is similar in the critical and low density universes, although the critical density models contain relatively more disordered clusters due to the freeze - out of fluctuations in open universes at late times . The profiles of relaxed clusters are very similar in the different simulations, since most clusters are in a quasi - equilibrium state inside the virial radius and follow the universal density profile of navarro et al . . There does not appear to be a strong cosmological dependence in the profiles, as suggested by previous studies of clusters formed from pure power law initial density fluctuations . However, because more young and dynamically evolving clusters are found in critical density universes, thomas et al . Suggest that it may be possible to discriminate among the density parameters by looking for multiple cores in the substructure of the dynamic cluster population . They note that a statistical population of 20 clusters can distinguish between open and critically closed universes . The evolution of the number density of rich clusters of galaxies can be used to compute 0 and 8 (the power spectrum normalization on scales of 8h mpc) when numerical simulation results are combined with the constraint \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}${\sigma _ 8}\omega _ 0^{0.5} \approx 0.5$\end{document}800.50.5, derived from observed present - day abundances of rich clusters . Bahcall et al . Computed the evolution of the cluster mass function in five different cosmological model simulations and found that the number of high mass (comalike) clusters in flat, low 8 models (i.e., the standard cdm model with 8 0.5) decreases dramatically by a factor of approximately 10 from z = 0 to z 0.5 . For low 0, high 8 models, the data results in a much slower decrease in the number density of clusters over the same redshift interval . Comparing these results to observations of rich clusters in the real universe, which indicate only a slight evolution of cluster abundances to redshifts z 0.5 1, they conclude that critically closed standard cdm and mixed dark matter (mdm) models are not consistent with the observed data . The models which best fit the data are the open models with low bias (0 = 0.3 0.1 and 8 = 0.85 0.5), and flat low density models with a cosmological constant (0 = 0.34 0.13 and 0 + = 1). The evolution of the x - ray luminosity function, and the size and temperature distribution of rich clusters of galaxies are all potentially important discriminants of cosmological models . Investigated these properties in a high resolution numerical simulation of a standard cdm model normalized to cobe . Although the results are highly sensitive to grid resolution (see for a discussion of the effects from resolution constraints on the properties of rich clusters), their primary conclusion, that the standard cdm model predicts too many bright x - ray emitting clusters and too much integrated x - ray intensity, is robust, since an increase in resolution will only exaggerate these problems . Cosmological sheets, or pancakes, form as overdense regions collapse preferentially along one axis . Originally studied by zeldovich in the context of neutrino - dominated cosmologies, sheets are ubiquitous features in nonlinear structure formation simulations of cdm - like models with baryonic fluid, and manifest on a spectrum of length scales and formation epochs . Gas collapses gravitationally into flattened sheet structures, forming two plane parallel shock fronts that propagate in opposite directions, heating the infalling gas . Sheets are characterized by essentially five distinct components: the preshock inflow, the postshock heated gas, the strongly cooling / recombination front separating the hot gas from the cold, the cooled postshocked gas, and the unshocked adiabatically compressed gas at the center . Several numerical calculations [15, 51, 8] have been performed of these systems which include the baryonic fluid with hydrodynamical shock heating, ionization, recombination, dark matter, thermal conductivity, and radiative cooling (compton, bremstrahlung, and atomic line cooling), in both one and two spatial dimensions to ascert the significance of each physical process and to compute the fragmentation scale . In addition, it is well known that gas which cools to 1 ev through hydrogen line cooling will likely cool faster than it can recombine . This nonequilibrium cooling increases the number of electrons and ions (compared to the equilibrium case) which, in turn, increases the concentrations of h and h, the intermediaries that produce hydrogen molecules h2 . If large concentrations of molecules form, excitations of the vibrational / rotational modes of the molecules can efficiently cool the gas to well below 1 ev, the minimum temperature expected from atomic hydrogen line cooling . Because the gas cools isobarically, the reduction in temperature results in an even greater reduction in the jeans mass, and the bound objects which form from the fragmentation of h2 cooled cosmological sheets may be associated with massive stars or star clusters . Anninos and norman have carried out 1d and 2d high resolution numerical calculations to investigate the role of hydrogen molecules in the cooling instability and fragmentation of cosmological sheets, considering the collapse of perturbation wavelengths from 1 mpc to 10 mpc . They find that for the more energetic (long wavelength) cases, the mass fraction of hydrogen molecules reaches h2/nh 3 10, which cools the gas to 4 10 ev and results in a fragmentation scale of 9 10 m. this represents reductions of 50 and 10 in temperature and jeans mass respectively when compared, as in figure 6, to the equivalent case in which hydrogen molecules were neglected . Figure 6two different model simulations of cosmological sheets are presented: (a) a six species model including only atomic line cooling, and (b) a nine species model including also hydrogen molecules . The evolution sequences in the images show the baryonic overdensity and gas temperature at three redshifts following the initial collapse at z = 5 . In each figure, the vertical axis is 32 kpc long (parallel to the plane of collapse) and the horizontal axis extends to 4 mpc on a logarithmic scale to emphasize the central structures . Differences in the two cases are observed in the cold pancake layer and the cooling flows between the shock front and the cold central layer . When the central layer fragments, the thickness of the cold gas layer in the six (nine) species case grows to 3 (0.3) kpc and the surface density evolves with a dominant transverse mode corresponding to a scale of approximately 8(1) kpc . Assuming a symmetric distribution of matter along the second transverse direction, the fragment masses are approximately 10 (10) solar masses . Two different model simulations of cosmological sheets are presented: (a) a six species model including only atomic line cooling, and (b) a nine species model including also hydrogen molecules . The evolution sequences in the images show the baryonic overdensity and gas temperature at three redshifts following the initial collapse at z = 5 . In each figure, the vertical axis is 32 kpc long (parallel to the plane of collapse) and the horizontal axis extends to 4 mpc on a logarithmic scale to emphasize the central structures . Differences in the two cases are observed in the cold pancake layer and the cooling flows between the shock front and the cold central layer . When the central layer fragments, the thickness of the cold gas layer in the six (nine) species case grows to 3 (0.3) kpc and the surface density evolves with a dominant transverse mode corresponding to a scale of approximately 8(1) kpc . Assuming a symmetric distribution of matter along the second transverse direction, the fragment masses are approximately 10 (10) solar masses this review is intended to provide a flavor of the variety of numerical cosmological calculations that have been performed to investigate different phenomena throughout the history of our universe . The topics discussed range from the strong field dynamical behavior of spacetime geometry at early times near the big bang singularity and the epoch of inflation, to the late time evolution of large scale matter fluctuations and the formation of clusters of galaxies . Although many problems have been addressed, the complexity of the varied physical processes in the extreme time, spatial, and dynamical scales of our universe guarantees that many more interesting and unresolved issues remain . Indeed, even the background cosmological model which best describes our universe is essentially unknown . The topology of our universe, the generic singularity behavior, the fundamental nonlinear field and gravitational wave interactions, the existence and role of a cosmological constant, the correct structure formation scenario, and the origin and spectrum of primordial fluctuations, for example, are uncertain . However the field of numerical cosmology has matured, in the development of computational techniques, the modeling of microphysics, and in taking advantage of current computing technologies, to the point that it is now possible to perform reliable comparisons of numerical models with observed data.
An estimated 11.5 million incident ischaemic strokes (63% in low - income and middle - income countries) and 5.3 million incident hemorrhagic strokes (80% in low - income and middle - income countries) took place . Cv stroke is the leading cause of disability in adults and each year millions of stroke survivors have to adapt to a life with restrictions in activities of daily living as a consequence of cerebrovascular disease . Although cv stroke has an impact on the physical, social, cognitive, emotional or psychological functioning of individuals, research in this area has focused primarily on physical functioning, and psychiatric co - morbidity has been relatively neglected and under - recognized . In addition to neurological issues such as location of the stroke and the nature of the motor impairment, psychological factors are also important in determining quality of life (qol) and functional outcome in stroke survivors . Post - stroke depression impacts negatively on qol, functional recovery, cognitive function and rehabilitation of acute stroke patients . In addition, it increases mortality and is associated with a higher risk of recurrent stroke at 1 year . Apart from having an impact on the stroke survivors themselves, post - stroke depression also affects family . There is a paucity of literature from india about the prevalence of depression and its correlation with various factors in acute stroke patients . As there is uncertainty regarding the frequency and determinants of depression, this study was planned to assess the magnitude of depression in acute stroke patients admitted in a rural tertiary care hospital and to analyze the association of demographic variables (age, gender, socio - economic status and educational status), co - morbid conditions or risk factors and radiological imaging variables with depression . This study was conducted between april 1, 2013 and september 30, 2013 in the department of medicine, mahatma gandhi institute of medical sciences, sevagram which is a 750-bedded teaching rural tertiary care hospital located in a town in central india . The patients with the diagnosis cv stroke are admitted in the intensive care unit or general indoor ward of department of medicine . A cross - sectional design was used to assess the magnitude of depression through a questionnaire and to collect data related to demographic variables, co - morbid conditions, clinical and imaging variables . The study was approved by the institutional ethics committee of mahatma gandhi institute of medical sciences (irb00003623). We obtained a written informed consent from all study patients before enrolling in the study . (1) all consecutive patients admitted with a diagnosis of acute stroke (as defined by the world health organization criteria a rapidly developing clinical symptoms or signs of focal or global disturbance of cerebral function that last more than 24 hours, with no apparent non - vascular causes (primary and metastatic neoplasms, dural hematoma, head trauma, etc)) with a radiological (computed tomography (ct) scan or magnetic resonance imaging (mri) head) evidence of cv stroke . Acute stroke included acute ischemic stroke, intracerebral / intraventricular hemorrhage, and subarachnoid hemorrhage; (2) age of patient 18 years or older . (1) presence of communicative problems that preclude a psychiatric examination (e.g. Due to reduced level of consciousness, severe hearing or visual impairment, aphasia or severe dysarthria, severe cognitive dysfunction); (2) history of depression / psychiatric illness or on anti - depressant drugs; (3) illicit drug dependence; (4) patients who scored less than 10 on the glasgow coma scale (gcs); (5) history of chronic liver disease or liver enzymes (alt / ast) more than three times the upper limit of normal, end - stage renal disease, malignancy or thyroid disease; (6) refusal to consent . A research assistant collected the data of acute stroke patients including demographics (age, gender, per capita income (pci), education - status), and comorbid diseases or risk factors such as hypertension (history of hypertension or anti - hypertension drug), diabetes mellitus (dm) (history of diabetes or anti - hyperglycemic drug), history of previous cv stroke, history of ischemic heart disease (ihd), current smokers (persons who report smoking at least 100 cigarettes in their life and who currently smoke every day or on some days), and high alcohol consumption (5 standard drink / day). Socioeconomic status (ses) of the stroke patients was assessed by per capita monthly household income (pci) and was categorized into pci quartiles . Education status was categorized as illiterate, primary school, middle school and high school and above . Reports of laboratory investigations like serum creatinine, sodium, potassium, blood sugar, urea and liver function tests were collected from the patient's medical records . The radiological imaging data (type, location and size of the stroke lesion) was noted from the ct / mri head reports . The intensity of depression in patients with stroke was assessed by an another trained person who was blind of the diagnosis and investigations of the patient, through a face - to - face interview based questionnaire (see text s1), montgomery - asberg depression rating scale (madrs). The reliability and validity of madrs questionnaire for the assessment of depression have been established in the previous studies . Higher madrs score indicates more severe depression, and each item yields a score of 0 to 6 . Cutoff points for categorization of mild, moderate and severe depression were: 0 to 6 normal / no depression7 to 19 mild depression20 to 34 moderate depression>34 severe depression . 0 to 6 normal / no depression 7 to 19 mild depression 20 to 34 moderate depression> 34 severe depression . We used spss software (version 16.0) to analyze the characteristics of the study population . The demographic variables, comorbid diseases and radiological characteristics of the stroke patients with depression were compared with those without depression using the chi - square test or the fisher exact test as appropriate . This study was conducted between april 1, 2013 and september 30, 2013 in the department of medicine, mahatma gandhi institute of medical sciences, sevagram which is a 750-bedded teaching rural tertiary care hospital located in a town in central india . The patients with the diagnosis cv stroke are admitted in the intensive care unit or general indoor ward of department of medicine . A cross - sectional design was used to assess the magnitude of depression through a questionnaire and to collect data related to demographic variables, co - morbid conditions, clinical and imaging variables . The study was approved by the institutional ethics committee of mahatma gandhi institute of medical sciences (irb00003623). We obtained a written informed consent from all study patients before enrolling in the study . (1) all consecutive patients admitted with a diagnosis of acute stroke (as defined by the world health organization criteria a rapidly developing clinical symptoms or signs of focal or global disturbance of cerebral function that last more than 24 hours, with no apparent non - vascular causes (primary and metastatic neoplasms, dural hematoma, head trauma, etc)) with a radiological (computed tomography (ct) scan or magnetic resonance imaging (mri) head) evidence of cv stroke . Acute stroke included acute ischemic stroke, intracerebral / intraventricular hemorrhage, and subarachnoid hemorrhage; (2) age of patient 18 years or older . (1) presence of communicative problems that preclude a psychiatric examination (e.g. Due to reduced level of consciousness, severe hearing or visual impairment, aphasia or severe dysarthria, severe cognitive dysfunction); (2) history of depression / psychiatric illness or on anti - depressant drugs; (3) illicit drug dependence; (4) patients who scored less than 10 on the glasgow coma scale (gcs); (5) history of chronic liver disease or liver enzymes (alt / ast) more than three times the upper limit of normal, end - stage renal disease, malignancy or thyroid disease; (6) refusal to consent . (1) all consecutive patients admitted with a diagnosis of acute stroke (as defined by the world health organization criteria a rapidly developing clinical symptoms or signs of focal or global disturbance of cerebral function that last more than 24 hours, with no apparent non - vascular causes (primary and metastatic neoplasms, dural hematoma, head trauma, etc)) with a radiological (computed tomography (ct) scan or magnetic resonance imaging (mri) head) evidence of cv stroke . Acute stroke included acute ischemic stroke, intracerebral / intraventricular hemorrhage, and subarachnoid hemorrhage; (2) age of patient 18 years or older . (1) presence of communicative problems that preclude a psychiatric examination (e.g. Due to reduced level of consciousness, severe hearing or visual impairment, aphasia or severe dysarthria, severe cognitive dysfunction); (2) history of depression / psychiatric illness or on anti - depressant drugs; (3) illicit drug dependence; (4) patients who scored less than 10 on the glasgow coma scale (gcs); (5) history of chronic liver disease or liver enzymes (alt / ast) more than three times the upper limit of normal, end - stage renal disease, malignancy or thyroid disease; (6) refusal to consent . A research assistant collected the data of acute stroke patients including demographics (age, gender, per capita income (pci), education - status), and comorbid diseases or risk factors such as hypertension (history of hypertension or anti - hypertension drug), diabetes mellitus (dm) (history of diabetes or anti - hyperglycemic drug), history of previous cv stroke, history of ischemic heart disease (ihd), current smokers (persons who report smoking at least 100 cigarettes in their life and who currently smoke every day or on some days), and high alcohol consumption (5 standard drink / day). Socioeconomic status (ses) of the stroke patients was assessed by per capita monthly household income (pci) and was categorized into pci quartiles . Education status was categorized as illiterate, primary school, middle school and high school and above . Reports of laboratory investigations like serum creatinine, sodium, potassium, blood sugar, urea and liver function tests were collected from the patient's medical records . The radiological imaging data (type, location and size of the stroke lesion) was noted from the ct / mri head reports . The intensity of depression in patients with stroke was assessed by an another trained person who was blind of the diagnosis and investigations of the patient, through a face - to - face interview based questionnaire (see text s1), montgomery - asberg depression rating scale (madrs). The reliability and validity of madrs questionnaire for the assessment of depression have been established in the previous studies . Higher madrs score indicates more severe depression, and each item yields a score of 0 to 6 . Cutoff points for categorization of mild, moderate and severe depression were: 0 to 6 normal / no depression7 to 19 0 to 6 normal / no depression 7 to 19 mild depression 20 to 34 moderate depression> 34 severe depression . We used spss software (version 16.0) to analyze the characteristics of the study population . The demographic variables, comorbid diseases and radiological characteristics of the stroke patients with depression were compared with those without depression using the chi - square test or the fisher exact test as appropriate . The mean age was 59.13 11.66 years, ranging from 28 to 80 years . There were 21 (20%) stroke patients below the age of 50 years and 86 (80%) patients above the age of 50 years . Of the total 107 stroke patients, 60 (56%) were males and 47 (44%) were females [table 1]. There were 17 (16%) illiterates, 36 (34%) educated up to primary school, 26 (24%) educated up to middle school and 28 (26%) educated high school and above of the total 107 patients [table 3]. Gender distribution of stroke patients distribution of stroke patients by per capita income quartiles distribution of stroke patients by education status sixty - one (57%) of the 107 stroke patients had depression . Of the 107 stroke patients, 35 (33%) had mild depression, 22 (20%) had moderate depression and 4 (4%) had severe depression [table 4]. Ses (pci) was significantly associated with depression in our stroke patients (p <0.05) [table 5]. The association of age of the patient (<50 years vs> 50 years), gender, and education status with depression was not statistically significant (p> 0.05) [table 5]. Of the 107 patients, 69 (64%) had hypertension, 18 (17%) had dm, 16 (15%) had ihd and 18 (17%) had previous history of cv stroke . The association of co - morbid illnesses, i.e. Hypertension, dm, ihd, and previous history of stroke with depression was not statistically significant [table 6]. There were 22 (20%) current smokers and 14 (13%) alcohol drinkers of the total 107 stroke patients . The association of depression with either current smoking or alcohol drinking was not statistically significant [table 6]. Distribution of depression among stroke patients distribution of demographic variables among patients with or without depression distribution of comorbid diseases / risk factors among patients with or without depression in the radiological imaging (ct / mri head) of cv stroke patients, 61 (57%) patients had left - sided lesions, 42 (39%) patients had right - sided lesions and 4 (4%) had bilateral lesions . Ninety - three (87%) patients had cerebral infarcts and 14 (13%) had intracerebral hemorrhages (ich). Sixty - two (58%) of the 107 stroke patients had lacunar (lac) infarcts, 6 (6%) had total anterior circulation (tac) infarcts, 30 (28%) had partial anterior circulation (pac) infarcts and 9 (8%) had posterior circulation infarct (poc) infarcts . The association of left - sided lesions with depression was found to be statistically significant (p <0.05). Type of lesion (infarct / ich) and location of the lesion (lac / tac / pac / poc) were not associated with depression (p> 0.05) [table 7]. The overall magnitude of depression was 57% (61 of the 107 stroke patients had depression). Of the 107 stroke patients, 35 (33%) had mild depression, 22 (20%) had moderate depression and 4 (4%) had severe depression . In a study done by hsieh et al ., depressive symptoms and severe depression were found in 34.3% patients and 7.7% patients, respectively . Forty - four percent patients were found to have post - stroke depression (psd) in a study done by camoes barbosa et al . In a study done by raju et al . On 162 patients (interviewed after 1 month post stroke), 60 patients (37%) had depression . Of the 80 stroke patients, 53 (66%) were depressed, 41 (51%) were mildly depressed and 12 (15%) were moderately to severely depressed in a study done by glamcevski et al . In our study, the stroke patients were assessed for depression within a week of occurrence of stroke, i.e. During hospitalization . In most of the previous studies, depression was assessed in stroke patients from 1 month to 2 years after the occurrence of stroke . Acute stress exposure due to more functional dependency and physical disability during the acute phase of stroke might be responsible for high magnitude of depression in our stroke patients as compared to that in previous studies . In addition, use of different tools for the assessment of depression in different studies might lead to varied overall frequency of depression . However, the magnitude of moderate to severe depression (24%) in our study is comparable with previous studies . In our study, the association of female gender with depression was also observed in a study done by broomfield et al . Patient's ses was significantly associated with depression which is consistent with the previous studies . Worsening effects of financial strain, poverty, or in deprivation of personal resources may link ses with depression . The association of patient's education status with depression was not statistically significant in our study . In our study, co - morbid illnesses like hypertension, dm, ihd and previous history of stroke were not associated with depression . Observed that individuals with both stroke and depression were more likely to have history of hypertension, dm and heart disease compared with other groups . The association of depression with either current smoking or alcohol drinking was not statistically significant in our study . Stroke patients with left hemisphere lesions were significantly more depressed in comparison to patients with right hemisphere lesions . The similar finding was also observed in a meta - analysis done by narushima et al . Depression was significantly associated with left hemisphere lesions in previous studies done by robinson et al . And rajashekaran et al . The reason hypothesized by the authors that the behavioral and catecholaminergic response to injury on the left side of brain is different from the right side of brain . However, carson and colleagues in their systemic review did not find an association between depression and lesion side . Berg et al . And aben et al . In their respective studies also did not find any significant differences in depression prevalence between right- and left - hemispheric lesions . Type of lesion (infarct / ich) and location of the lesion (lac / tac / pac / poc) were not associated with depression in our study . Robinson et al . Reported an association of frontal lobe location with depression in stroke patients . Our patients represent typical rural indian patients from central india . By including every consecutive stroke inpatient we avoided the selection bias . We made a blind assessment (filling the madrs questionnaire and data collection) of the patients . If our sample size has been large with more number of patients with hypertension or dm or cardiovascular risk factors (smoking or alcohol drinking), we could get a significant association of these cardiovascular diseases or risk factors with depression . The patients with low gcs scores or aphasia which may be related to more severe strokes were excluded from the study . As our study is a hospital - based study, its results could not be generalized to the community or chronic cv stroke patients . Post - stroke depression was present in more than half of the acute stroke patients and was related to left - sided lesions and socioeconomic status . Moderate depression and severe depression were present in 20% and 4% of all the stroke patients, respectively . Early diagnosis and effective treatment of depression might improve functional recovery, cognitive performance, drug compliance, quality of life, and social and rehabilitation outcome of stroke patients . There is a need for further research to improve clinical practice in this area of stroke care.
The human immunodeficiency virus (hiv) pandemic has fuelled the tuberculosis (tb) epidemic by creating a population of immunosuppressed individuals that are highly susceptible to mycobacterium tuberculosis (mtb) infection . The last decade has seen an unprecedented increase in antimycobacterial drug resistance . Of the estimated 1.3 million deaths resulting from tb globally in 2012 appropriate treatment of patients with drug - resistant strains of mtb is of vital importance in limiting the transmission of the disease and reducing mortality rates.1 fluoroquinolones are potent antibiotics that have been used in clinical practice since the early 1980s.2 they display broad - spectrum antimicrobial activity, and have been used extensively in the treatment of bacterial infections of the respiratory, gastrointestinal, and urinary tracts, as well as in sexually transmitted diseases and chronic osteomyelitis.3 the fluoroquinolones have been advocated for the treatment of patients with multidrug - resistant (mdr) mtb, defined as resistance to at least isoniazid and rifampicin . Patients with extensively drug - resistant (xdr) mtb harbor mdr tb strains with additional resistance to fluoroquinolones and one of the second - line anti - mycobacterial injectable (kanamycin, amikacin, and capreomycin) agents . However, moxifloxacin (mxf), a new - generation fluoroquinolone, has been recommended by the world health organization (who) for the treatment of xdr . Studies that have explored the efficacy of mxf against xdr strains of mtb have concluded that the drug may be used in xdr cases provided that the infecting isolate has a minimum inhibitory concentration (mic) of <2 mg / l for mxf.1,4 mxf differs in structure when compared to ofloxacin (ofx) and ciprofloxacin (cpx). The structural difference, which includes a methoxy group in the c-8 position of mxf, results in increased bactericidal activity of the drug, lower mics, and a lower propensity for the development of resistance to the drug.5 although cross - resistance has been reported, it has been argued that the increased bactericidal activity of mxf and the lower mic allow for this drug to be effective against xdr isolates where cpx and ofx are ineffective.3,4 the fluoroquinolones inactivate the deoxyribonucleic acid (dna) gyrase enzyme, thereby preventing transcription during cell replication . Fluoroquinolone - resistant strains of mtb most frequently display mutations on codons 90, 91, and 94 of the gyra gene.69 additionally, double mutations in the gyra or concomitant gyra and gyrb mutations have been reported.6,8 the level of fluoroquinolone resistance is dependent on the mutation in the resistance - conferring gene and the fluoroquinolone tested.10 studies have demonstrated that mic levels of resistant isolates are higher for older - generation fluoroquinolones than for mxf.69 the use of mxf in xdr treatment regimens was introduced without prior testing for susceptibility against the circulating xdr isolates in kwazulu - natal (kzn) province . The aim of this study was to correlate the minimum inhibitory concentration (mic) levels of the fluoroquinolones with mutations in the gyra and gyrb genes in a subset of clinical isolates from the kzn province of south africa . The isolates used in this study were retrieved from the culture collection in the department of infection prevention and control, nelson r mandela school of medicine, school of laboratory medicine and medical science, college of health science, university of kwazulu - natal . We included the following phenotypes: ten fully drug - susceptible (ds), 20 mdr, and 30 xdr . The isolates were collected between 2005 and 2008 from patients in umzinyathi district, kzn, south africa . Ethical approval for the study (brec 247/09) was granted by the biomedical research ethics committee at the university of kwazulu - natal . Mic determination of the drugs was performed by means of the agar dilution method using middlebrook 7h10 (bd, franklin lakes, nj, usa) media supplemented with oleic acid albumin dextrose catalase (bd). The drug concentrations used ranged from 0.03 to 8 mg / l for cpx, ofx (sigma - aldrich, st louis, mo, usa), and mxf (bayer, leverkusen, germany). Following inoculation, mic values were recorded as the lowest concentration of the drug that resulted in complete inhibition of growth . The cutoff value for resistance 2 mg / l for cpx and ofx according to who recommendations11 and 0.5 mg / l for mxf, as described by angeby et al.12 all mic experiments were carried out in triplicate . Dna was extracted using cetyl - trimethyl - ammonium bromide - sodium chloride, as previously described.13 the quinolone resistance - determining region (qrdr) and flanking regions of the gyra and gyrb genes were amplified using primer pairs designed for this study: gyra forward (cgattgcaaacgaggaatag), gyra reverse (ggccagttttgtaggcatca), and gyrb forward (atcaacctgaccgacgagag), gyrb reverse (gccgagtcaccttctacgac).14 polymerase chain reaction (pcr) was performed using the expand high - fidelity pcr system (dntpack; hoffman - la roche, basel, switzerland). Cycling conditions were as follows: initial denaturation at 94c for 2 minutes; 40 cycles of denaturation at 94c for 45 seconds, annealing at 53c (gyra) or 56c (gyrb) for 45 seconds and extension at 72c for 45 seconds; and a final extension of 7 minutes at 72c . Sequencing pcr products were purified using the invitrogen purelink pcr purification kit (thermo fisher scientific, waltham, ma, usa). The sequencing reactions were performed using an abi prism bigdye terminator cycle - sequencing kit 3.1 (thermo fisher scientific) with the same forward primers as used for pcr amplification . Geneious version 5.5.7 sequence - analysis software was used to identify mutations in the final nucleotide sequences in comparison to the mtb h37rv reference strain.15 genotyping was performed by is6110 restriction fragment length polymorphism (rflp) analysis using the southern blot hybridization method, as previously described.16 the isolates used in this study were retrieved from the culture collection in the department of infection prevention and control, nelson r mandela school of medicine, school of laboratory medicine and medical science, college of health science, university of kwazulu - natal . We included the following phenotypes: ten fully drug - susceptible (ds), 20 mdr, and 30 xdr . The isolates were collected between 2005 and 2008 from patients in umzinyathi district, kzn, south africa . Ethical approval for the study (brec 247/09) was granted by the biomedical research ethics committee at the university of kwazulu - natal . Mic determination of the drugs was performed by means of the agar dilution method using middlebrook 7h10 (bd, franklin lakes, nj, usa) media supplemented with oleic acid albumin dextrose catalase (bd). The drug concentrations used ranged from 0.03 to 8 mg / l for cpx, ofx (sigma - aldrich, st louis, mo, usa), and mxf (bayer, leverkusen, germany). Following inoculation, plates were incubated in a co2 (5%)-enriched atmosphere at 37c for 21 days . Mic values were recorded as the lowest concentration of the drug that resulted in complete inhibition of growth . The cutoff value for resistance 2 mg / l for cpx and ofx according to who recommendations11 and 0.5 mg / l for mxf, as described by angeby et al.12 all mic experiments were carried out in triplicate . Dna was extracted using cetyl - trimethyl - ammonium bromide - sodium chloride, as previously described.13 the quinolone resistance - determining region (qrdr) and flanking regions of the gyra and gyrb genes were amplified using primer pairs designed for this study: gyra forward (cgattgcaaacgaggaatag), gyra reverse (ggccagttttgtaggcatca), and gyrb forward (atcaacctgaccgacgagag), gyrb reverse (gccgagtcaccttctacgac).14 polymerase chain reaction (pcr) was performed using the expand high - fidelity pcr system (dntpack; hoffman - la roche, basel, switzerland). Cycling conditions were as follows: initial denaturation at 94c for 2 minutes; 40 cycles of denaturation at 94c for 45 seconds, annealing at 53c (gyra) or 56c (gyrb) for 45 seconds and extension at 72c for 45 seconds; and a final extension of 7 minutes at 72c . Sequencing pcr products were purified using the invitrogen purelink pcr purification kit (thermo fisher scientific, waltham, ma, usa). The sequencing reactions were performed using an abi prism bigdye terminator cycle - sequencing kit 3.1 (thermo fisher scientific) with the same forward primers as used for pcr amplification . Geneious version 5.5.7 sequence - analysis software was used to identify mutations in the final nucleotide sequences in comparison to the mtb h37rv reference strain.15 genotyping was performed by is6110 restriction fragment length polymorphism (rflp) analysis using the southern blot hybridization method, as previously described.16 of the 60 isolates selected, four mdr isolates did not grow sufficiently on retrieval subculture and were excluded from further analysis . Mic and sequencing data were therefore obtained for ten ds, 16 mdr, and 30 xdr isolates . The ds and mdr isolates displayed mics for cpx, ofx, and mxf in the susceptible range . All 30 xdr isolates tested displayed mics for cpx, ofx, and mxf that were in the resistant range (table 1). Three mutations were observed in the nucleotide sequence of the gyra gene: e21q (gaacaa), s95 t (acgacc), and a90v (gcggtg). E21q and s95 t were present in all 56 isolates, regardless of mic values (table 1). The a90v mutation was present only in the 30 xdr isolates, correlating with an mic value of 8 mg / l for cpx and ofx . In the case of mxf, 23 of the 30 xdr isolates had an mic value of 2 mg / l, while seven had an mic value of 1 mg ten mdr isolates with an mic for mxf at the proposed breakpoint for resistance, ie, 0.5 mg / l, did not display the a90v mutation . Is6110 rflp analysis revealed that 35 of the 56 isolates belonged to the f15/lam4/kzn family of strains . Seventeen of the remaining isolates belonged to recognized strain families (f28, f11, and beijing) while four showed a unique rflp profile . Of the 60 isolates selected, four mdr isolates did not grow sufficiently on retrieval subculture and were excluded from further analysis . Mic and sequencing data were therefore obtained for ten ds, 16 mdr, and 30 xdr isolates . The ds and mdr isolates displayed mics for cpx, ofx, and mxf in the susceptible range . All 30 xdr isolates tested displayed mics for cpx, ofx, and mxf that were in the resistant range (table 1). Three mutations were observed in the nucleotide sequence of the gyra gene: e21q (gaacaa), s95 t (acgacc), and a90v (gcggtg). E21q and s95 t were present in all 56 isolates, regardless of mic values (table 1). The a90v mutation was present only in the 30 xdr isolates, correlating with an mic value of 8 mg / l for cpx and ofx . In the case of mxf, 23 of the 30 xdr isolates had an mic value of 2 mg / l, while seven had an mic value of 1 mg ten mdr isolates with an mic for mxf at the proposed breakpoint for resistance, ie, 0.5 mg / l, did not display the a90v mutation . Is6110 rflp analysis revealed that 35 of the 56 isolates belonged to the f15/lam4/kzn family of strains . Seventeen of the remaining isolates belonged to recognized strain families (f28, f11, and beijing) while four showed a unique rflp profile . We report on the correlation between mics of mxf and mutations in the gyra gene in a selection of clinical isolates in kzn, south africa . Fluoroquinolone resistance in mtb the qrdr of the gyra gene consists of a short region, coding for amino acids 74113 . In our study, we sequenced the qrdr of both the gyra and gyrb genes, as well as flanking regions . We found the c269 t mutation within the qrdr of the gyra gene, which corresponds with the amino acid change a90v, correlated with the high mics seen in the xdr mtb isolates that we studied . The a90v mutation in gyra has been described as one of the most frequent mutations associated with fluoroquinolone resistance.17 in our study, based on who - recommended breakpoints, the a90v mutation in xdr isolates was linked to resistance in all three fluoroquinolones tested.11 we did not find mutations in gyrb in any of the isolates tested . This supports previous observations that mutations within the gyrb gene are rare in mtb.8,17 maruri et al conducted a systematic review to evaluate gyrase mutations associated with fluoroquinolone resistance in mtb . The study reported on 534 fluoroquinolone - resistant mtb isolates, of which 17 (3%) harbored mutations within the qrdr of the gyrb gene . In addition, four different numbering systems were used to report on mutations in the gyrb gene, resulting in major discrepancies . The authors proposed a uniform numbering system in an attempt to improve the molecular detection in the gyrases.18 the significance of mutations within the qrdr of the gyrb gene cannot be ignored, and is thought to play an important role in resistance.9 who guidelines propose 0.5 mg / l as the breakpoint mic for susceptibility testing of mxf in the bactec 460 system (bd) and 0.25 mg / l in the bactec mgit 960 (bd) system.11 breakpoints for other test systems are not proposed . Angeby et al demonstrated comparable mic results for mxf on middlebook 7h10 agar and the bactec 460 system.12 with middlebrook 7h10 plates used for mic determination, all our xdr isolates had mics of> 0.5 mg / l . As per the who definition, these are classified as mxf - resistant, and this implies that mxf should not be recommended for treatment of cases harboring such isolates . However, there are reports of mxf efficacy in isolates with mxf mics <2 mg / l.3,4,6,10,19 poissy et al used the murine model to demonstrate that mxf is effective against ofx - resistant strains . They reported that mxf was most effective on mtb strains with mics 0.5 mg / l . Reduced mortality was observed in mice infected with strains, with mics 2 mg / l compared to untreated controls.4 fillion et al demonstrated similar findings using the murine model to determine the effect of a multidrug regimen containing mxf . The sterilizing activity of the multidrug regimen decreased in strains with increased mics to mxf . The impact of the sterilizing activity of the most effective second - line treatment regimen (ie, ethionamide, pyrazinamide, amikacin, and mxf) is dependent on the mic of mxf, and thus the mic of mxf has to be determined for all strains resistant to ofx . Mice infected with strains with mxf mics of 0.5 mg / l and 4 mg / l recorded relapse rates of 50% and 86%, respectively, compared to the wild type.10 sirgel et al proposed that mxf and ofx are possibly not equally affected by mutations associated with fluoroquinolone resistance.6 they concluded that the use of mxf for the treatment of infection with ofx - resistant strains is justified when combined with other drugs . They further suggested that the low recommended breakpoint of 0.5 mg / l determining mxf resistance may therefore give a false impression of clinical inactivity . Poissy et al and sirgel et al support who recommendations on the use of mxf for the treatment of xdr provided that the infecting isolate has an mxf mic of <2 mg / l.4,6 feasey et al reported on a case where high - dose mxf (600 mg / day) in combination with pza, cap, lin, pas, and amoxicillin clavulanic acid for 22 months successfully treated a case with an infecting isolate that was resistant to isoniazid, rifampicin, ethambutol, prothionamide, ofx, streptomycin, and mxf (mic of 2 mg / l). While high - dose mxf treatment increases the peak plasma (mxf), resulting in levels that remain constantly above the mic, it is difficult to assess the exact role of mxf in the successful management of this patient, since other drugs with known efficacy were also included in the treatment regimen.19 jacobson et al conducted a meta - analysis to assess treatment outcomes in patients with xdr . The report summarized 13 studies conducted mainly amongst hiv - uninfected people, who received a new - generation fluoroquinolone together with other anti - tb drugs . They concluded that the addition of mxf to xdr regimens may improve outcomes, but further evaluation in clinical trials is warranted.3 in kzn, the regimen used for xdr treatment is a combination of drugs with proven and putative efficacy . The national department of health in south africa recommends the use of mxf as part of xdr treatment in the presence of ofx resistance . Fluoroquinolones are added despite in vitro reports of resistance in the hope that there may be some residual activity . It is debatable whether the perceived benefit of using mxf under these circumstances outweighs the risks caused by side effects of this drug or the increased exertion of antibiotic pressure in the era of ever - increasing drug resistance.20 mendel and springsklee21 warned that the use of newer - generation fluoroquinolones in patients who display low - level resistance will be disastrous from a public health perspective . The use of mxf in such cases will result in the ready emergence of highly resistant strains unless drug concentrations are sustained above mutant - prevention concentrations at all infection sites . The latter is extremely difficult to achieve, and thus the use of mxf in patients with resistance to older - generation fluoroquinolones will only further drive resistance among xdr strains of mtb . Although all isolates used in this study were from different patients, the xdr isolates displayed a high degree of similarity and belonged to a single genotype, ie, f15/lam4/kzn . To date, all reports from kzn have attributed xdr to this strain . Ramtahal showed that the spread of xdr in kzn was clonal with the f15/lam4/kzn strain.22 clonal spread of this strain has been ongoing since at least 2005.23 during this period, further acquisition of resistance may have occurred . We therefore performed susceptibility testing and sequencing on 30 xdr isolates belonging to the only xdr strain family currently in kzn . Had mics of 1 mg / l, and 23 had mics of 2 mg / l . Basic microbiological principles regarding in vitro determination of mics allows for one twofold mic variation between tests . This implies that our xdr isolates may in their most susceptible form have mics between 0.5 and 1 mg / l and in their most resistant form from 2 to 4 mg / l.24 gandhi et al found that a large variety of strains were associated with ds, and this decreased as the degree of resistance increased . The low diversity of strains driving the mdr and xdr epidemics supports the theory of clonal expansion of drug - resistant phenotypes in kzn . This picture is different in other parts of south africa . In the eastern and western cape provinces, strains responsible for mdr and xdr in other provinces include the s, t1, and other families . Regardless of the strain family implicated in infection with xdr, the breakpoint for resistance to mxf remains the subject of debate . While there may be a role for mxf as part of individualized xdr treatment regimens, this cannot be advocated as part of empiric treatment protocols in the absence of mxf mic data of the circulating xdr strains in an area . In addition, validation from larger population - based studies using mxf in combination with various other antidrug regimes must be conducted.
Cryptosporidiosis is a parasitic disease that causes diarrhea lasting for about 1 - 2 weeks among the immunocompetent and becoming a more severe life - threatening illness among immunocompromised individuals . It constitutes 24% of human immunodeficiency virus (hiv) positive patients with diarrhea compared with 6.1% in the immunocompetent individuals . Association of cryptosporidiosis with malignancies is not as robust as acquired immunodeficiency syndrome (aids). Here, we report a case of cryptosporidiosis causing severe persistent diarrhea in a patient with multiple myeloma . The case we present here is the patient was a 64-year - old female who had been diagnosed to have multiple myeloma, stage iii b, type igg kappa 17 years ago in 1993 . She was being treated with oral lenalidomide (25 mg for 21 days) and dexamethasone (40 mg on days 1, 8 and 15) of 28 days cycle for the current progression of myeloma . The baseline serum m band prior to the start of this regimen was 3.4 g / dl . She presented on day 17 of the 2 cycle with the complaints of profuse watery diarrhea, 10 - 12 times / day for the previous 15 days . The diarrheal product was non - bloody, without any mucus and not foul smelling . This was associated with nausea, mild crampy abdominal pain and a sense of fecal urgency without any fever . There was anemia (hemoglobin of 9.7 g / dl) and leucopenia (white blood cells count 2800/mm) but the absolute neutrophil count (1700/mm) and platelets were normal . The liver and renal function tests were within normal limits but serum potassium was low (2.2 meq / l). The routine blood culture was sterile and the polymerase chain reaction test in peripheral blood for cytomegalovirus deoxyribonucleic acid was negative . Stool examination showed 2 - 3 polymorphonuclear leucocytes per high power field with mucus but no visible or occult blood . Colonoscopy was performed, which suggested colonic mucosal inflammation likely to be infective in origin [figure 1]. At this point, a diagnosis of cryptosporidial infection was suspected and the stool was submitted for examination with the modified kinyoun acid - fast stain . The patient was then started on oral nitazoxanide 500 mg 12 hourly for 10 days . The diarrheal frequency decreased after 5 days and it resolved completely by the 10 day of this drug . The patient was able to resume treatment with lenalidomide and dexamethasone later without any complications . Colonic mucosal inflammation on colonoscopy of the patient oocysts of cryptosporidium parvum in modified kinyoun acid - fast stain cryptosporidiosis is a protozoal intestinal infection caused by many species of the genus cryptosporidium that are either obligate human or human and bovine parasites . The first association with human diarrheal disease was reported in 1976, one case in an otherwise healthy child and one in an immunosuppressed adult . The infection is acquired by ingesting parasites in water or food contaminated by human or animal feces or by having contact with soil, a person, or an item that has been contaminated with the parasite . The diagnosis is established by demonstration of oocysts in the stool by the modified acid - fast kinyoun stain . Since the initial reports four clinical syndromes of cryptosporidial infection have been described in hiv positive patients: chronic diarrhea (affecting 36% of patients), cholera - like disease (33%), transient diarrhea (15%) and relapsing illness (15%). The severity of illness is related to the cd4 count in these patients . In immunocompetent patients, usually children <5 years of age the clinical presentation in our patient with severe diarrhea of 2 weeks duration that resulted in dehydration and dyselectrolytemia was more consistent with an underlying immunodeficiency which is expected in a patient with long standing multiply treated myeloma . Although uncommon, cryptosporidial diarrhea has been described in patients with malignancy . In one study, the incidence of cryptosporidiosis was 1.3% in the 560 cancer patients presenting with symptoms of diarrhea out of which 71% had hematolymphoid malignancy . In other studies it was found that the risk of severe disease is largely limited to children with acute leukemia and lymphoma . Interestingly, bone marrow transplantation does not seem to confer any additional risk over that of the underlying disease . Cryptosporidial diarrhea should be included in the differential diagnosis of any patient with persistent severe diarrhea, especially in patients with hematological malignancies . Diagnosis can be established by stool examination and administration of nitazoxanide is effective in controlling the infection.
Genomes vary from one another in multifarious ways, and the totality of this genetic variation underpins the heritability of human traits . Various recent studies show the landscape of genetic variation and allow estimation of the relative contributions of sequence (base substitutions) and structural variation (indels, insertions, or deletions; copy - number variations [cnvs], and inversions). Among dna sequence variations in the human genome, cnvs directly contribute to gene expression changes through gene dosage effects; the distribution of cnvs is now considered to be wider than previously thought [16]. Genome resequencing studies have shown that cnvs, involving the gain or loss of several hundreds of bases to several hundred kilobases (kb) of the genome, can be an important source of genetic variation among human populations of different ethnic groups as well as among individuals . Following the development of methodologies and the introduction of new research platforms [712], information regarding the nature and pattern of cnvs from representative populations has been accumulated . The functional impact of cnvs has been demonstrated across the full range of biology, from cellular phenotypes, such as gene expression, to all classes of human disease with an underlying genetic basis: sporadic, mendelian, complex, and infectious (reviewed in). Clinical geneticists need to discriminate pathogenic from benign cnvs in their patients and have made extensive use of data from cnv surveys of apparently healthy individuals . The mere presence or absence of a variant in such control data sets is only partially informative, as determination of the pathogenicity of inherited cnvs is at present limited by a lack of information on cnv frequency and combinations in apparently healthy individuals . Based on these considerations, examinations of a relatively large number of individuals from various specific ethnic groups have recently been conducted using different array platforms, such as bac arrays [6, 17, 18], oligo arrays [1921], and others . The results are not always consistent, and it is likely that different human populations bear different inherited cnvs . The numbers of japanese individuals examined to date are smaller than those in studies of other ethnicities . Polymorphic cnvs have received considerable attention since they might play an important role in the etiology of common diseases . We summarized the available data by focusing on highly polymorphic cnvs (in 5.0% of the individuals) in a previous paper . However, rare variations have recently received attention from scientists who espouse a hypothesis called common disease and rare variants, in contrast to the common disease - common variants hypothesis that underpins most genome - wide association studies . In this paper, we focus on cnvs observed at relatively low frequency (<5.0% of the individuals) in a population residing in hiroshima and nagasaki, japan, using cgh with bac clones as targets . The study was conducted in two stages: stage (1) 80 unrelated japanese individuals were examined by bac - acgh with an array having 2,241 bac clones; stage (2) 133 unrelated japanese individuals were examined by bac - acgh containing 2,622 bac clones . The majority of the clones used in stage 1 of this study were selected from the set of cytogenetically mapped p1 artificial chromosome (pac) clones and bacterial artificial chromosome (bac) clones reported by the bac resource consortium and obtained from either the children's hospital oakland research institute (oakland, ca, usa) or from invitrogen inc ., co. (carlsbad, ca, usa). In stage 2, together with the bac clones in stage 1, additional 381 bac clones were used, a majority of which were collaboratively obtained from dr . A total of 2,241 clones of chromosomal fragments from chromosome 1 to chromosome 22 were used in stage 1 and 2,622 clones in stage 2 . The clones were distributed every 1.2 mb across all human autosomes in stage 1 and every 1.1 mb in stage 2 . In addition to autosomal clones, four kinds of x - chromosomal clones were used as internal references . With respect to the examinations in stage 1, three sets of arrays the genomic dna samples used in this study were reported in a previous study [23, 27]. In brief, the dna used for the population studies and the family studies was extracted from lymphoblastoid cell lines obtained from the offspring of atomic - bomb survivors and their family members . Lymphoblastoid cell lines were derived from a cryopreserved archive of families consisting of father, mother, and offspring from hiroshima and nagasaki for whom permanent cell lines have been established by epstein - barr (eb) virus transformation . Since the offspring in some cases included siblings, we selected one representative offspring to create a group of unrelated individuals, thereby avoiding any duplication of cnvs from families containing two or more siblings, in accordance with the rules described in a previous report . In this study, 80 individuals (40 from each city) were included in examinations in stage 1 and 133 (84 from hiroshima, 49 from nagasaki) in stage 2 . The arrays were prepared as described in the previous paper . In brief, with respect to stage 1, cloned dna was digested by noti . On the other hand, in stage 2, cloned dna was digested with msei, and the fragmented dnas were amplified by ligation - mediated pcr carried out as described by snijders et al . . In both stages, the target dnas (0.5 g/l) were dissolved in 50% dimethylsuloxide and printed in triplicate onto the glass slides (matsunami glass co. ltd .) Using the affymetrix 417 arrayer (affymetrix). The screenings of both stages were conducted following the procedures described previously . In brief, for labeling dna, test and reference genomic dna (1.25 g each) was cut by bamhi and labeled by a random - priming method with cyanine-5- and cyanine-3-labeled dutp (cy5- and cy3-dutp; perkinelmer life sciences, wellesley, ma, usa). Prehybridization was conducted to block repetitive sequence binding of target dna on the arrays and to prevent nonspecific binding of probe dna to the targets . Following the initial incubation, the prehybridization solution was removed, and hybridization solution with cy - labeled dna (prepared as described above) was added . All of the procedures were conducted using the genetac hybridization station (genomic solutions inc . After washing procedures, fluorescent images of the hybridized arrays were obtained using a scanarray 5000 confocal laser scanner (perkinelmer life sciences)., fairfax, va, usa) in stage 1 and gene pix (axon instruments, sunnyvale, ca) in stage 2 were used to quantify the fluorescence of each spot on the array images . The population studies were conducted in accordance with accepted procedures as described in the previous paper . In brief, hybridization was performed as follows: mixtures of (a) cy5-labeled reference dna and cy3-test dna, and (b) inverse labeling with cy3-reference dna and cy5-test dna were applied to the slides . The two complementary hybridizations (i.e., (a) and (b)) were conducted for each individual . The variations in a given sample from a single individual that were identified consistently in both complementary hybridizations ((a) and (b)) were regarded as putative cnvs . Since only dna segments detected as putative cnvs in two individuals or more were regarded as true cnvs, further analyses were not conducted . On the contrary, when cnvs were detected in only one individual, all were confirmed with repeated examinations using arrays . The qpcr procedure was performed using sybr premix ex taq (takara - bio, ohtsu, japan) and the light cycler system (roche diagnostics japan, tokyo, japan), according to the procedures described in the previous paper . The results were analyzed with light cycler data analysis software using a second derivative maximum model . The relative copy number of each site was normalized using quantity of the amplified segment of a portion of a different chromosome (chromosome 16:7888854178888690; this position empirically showed the smallest deviation among many samples in our experiments) as a standard (relative copy number = 2) and was indicated as mean sd (n = 3). For the cnvs identified in only one offspring, further examinations were carried out for confirmation of inheritance . The family studies using qpcr for the cnvs were conducted using dnas from the mother, the father and sibling of offspring, if available . The main purpose of this paper is to report the data accumulated about cnvs found in <5.0% of the individuals . The results obtained from the population studies are shown in tables 1 and 2 . In table 1, the cnvs identified in two individuals or more are summarized . On the other hand, in table 2, a total of 126 cnvs were identified at 52 different bac regions in the genome . The cnvs observed at 27 of 52 bac regions were found in only one unrelated individual, and all of them were confirmed by repetitive analysis using the same array system as in the population studies . The family studies by qpcr were conducted for 23 of 27 cnvs, and the results revealed that those 23 cnvs were inherited from one of the parents . The inheritance of cnvs from the remaining two offsprings was not confirmed, since dna from the parents was not obtained . On the other hand, the cnvs on two bac clones (rp11 - 88b18, rp11 - 86b6) were identified in only one unrelated individual . In fact, however, they were observed in individuals who were not selected as the numbers of individuals who expressed the cnvs are described in the parentheses in table 2 . For that reason, we did not conduct family studies for those cnvs . As typical examples of family studies, the results of qpcr carried out for the cnvs observed in three bac - clone regions are described in figure 1 (rp11 - 81h5), figure 2 (rp11 - 90m13), and figure 3 (rp11 - 90o18 and rp11 - 90c3). For the first case, a single 180,264 bps deletion was observed in an offspring . The bac - clone (rp11 - 90m13) contains two genes, protein phosphatase 2, regulatory subunit b, alpha (ppp2r2a) and early b - cell factor 2 (ebf2). The deletion - type cnv was confirmed by qpcr, and the cnv was found to be inherited from the father . The deletion covered part of intron 7 of ppp2r2a and the promoter region of ebf2 . For the last case, as shown in figure 3, the cnv was covered by two bac clones (rp11 - 90o18 and rp11 - 90c3). This result was revealed by qpcr, and the cnv was found to be inherited from the father . The cnv contained the series of zinc finger protein families (znf 107, 138, 273, 117, 92). Many segmental duplications have already been summarized in public databases, such as the database of genomic variants (dgv database; http://projects.tcag.ca/variation/), ucsc human genome browser (ucsc database . When the cnvs reported in the databases or papers were too small to be detected by our bac - acgh methods, we assumed that they were different from our cnvs . The cnv data obtained in our studies, including an indication of the presence or absence of cnvs already reported in other databases, are summarized in tables 1 and 2 . Compared to caucasian populations, very little information about cnvs in japanese populations has been systematically screened . We compared our data with the data of japanese populations from the hapmap project as reported by redon et al ., in addition to the data by bac array, three groups, including redon et al ., reported the data of japanese populations based on examinations conducted using oligo - array methods, although each group used a different array platform [24, 25]. These results are also summarized in tables 1 and 2 . In the case where a multiple number of cnvs were identified in our study (table 1), moreover, regarding one individual, the cnvs identified in nine of 27 bac regions were reported in the data reported by the others, although we should emphasize again that the cnvs identified in our bac region are not exactly the same as those reported by the others . The majority of cnvs identified in our study tend not to be identified in the other reports . We summarized some of the genes and disease - related genes that overlap with the bac - clone region for our cnvs (table 3). In addition, mrnas also have been reported in the databases, but they are too numerous to describe here . All bac - clone regions contained at least one mrna, although the functions of a majority of the mrnas are not yet known (data not shown). We examined 213 unrelated japanese individuals using bac - acgh and found a total of 126 cnvs on 52 different bac regions in the genomes . The cnvs observed on 27 of the 52 bac - regions were found in only one unrelated individual . The family studies were conducted for 23 cnvs, and the results demonstrated that these were inherited from one of the parents . A fraction of the regions involved in the cnvs observed in our study (i.e., 34 of 126 or about 27%; tables 1 and 2) were not reported previously in other studies listed in the database . Thus, they are considered novel cnvs . In contrast, the data of highly polymorphic cnvs demonstrated that only 8% (55 of 680) had not been reported . This result showed that a significantly large number of novel cnvs have been identified among relatively less frequent cnvs (= 36.64, p = 5.10 10). This finding suggests that the structural rearrangements for creating less frequent cnvs are evolutionarily recent . We thus paid attention to differences in the number of cnvs identified in hiroshima compared to nagasaki . With respect to the cnvs observed multiple times, 12 of 25 were identified in one city . These cnvs appear to be so - called private polymorphisms occurring more recently than the others . On the other hand, quite a small number (21 of 126; about 17%) of the cnvs in this report were found on the bac clones that overlapped with segmental duplication (tables 1 and 2). A majority (about 63%) of highly polymorphic cnvs, however, were observed on the bac - clone overlapping with segmental duplication (see table 1 in). This result suggests that segmental duplication might play a significant role in the creation of highly polymorphic cnvs . These observations are supported by previous data from sharp et al ., who reported the sharing of cnvs among several populations, meaning that the specific genomic imbalances either predated the migration of modern humans from africa or arose independently in different populations . On the other hand, the data representing japanese populations are based on reports from three groups . We compared our cnv data to that of these other reports [4, 24, 25]. The cnvs found in 27 of 52 bac regions (about 52%) were not identified by the other groups . Speculation as to the reason for this includes the possibility that since the cnvs were private polymorphisms as mentioned above, they were not identified in the japanese populations used in the other works . In humans, it is estimated that around 23% of currently known cnvs reside in known or putative genes . Many cnvs that lie outside genic regions of the genome may still have a significant influence on gene expression by affecting gene regulatory elements . Another important area of exploration is linkage disequilibrium (ld), that is, nonrandom association between alleles at different loci, between cnvs and other genomic variants (especially snps), which may provide important insight into the genomic evolution (e.g., the recombination patterns) of cnvs [31, 32]. However, studies of ld between cnvs and snps are hindered by the fact that cnvs generally reside in genomic regions associated with segmental duplications and/or repeat - rich regions, which are difficult to sequence and use for detection of snps . Recent association studies have attempted to link cnvs with expression profiles, diseases, and known phenotypic differences . On the other hand, distribution of cnvs differs depending on genetic background, and population - specific cnvs might account for the divergence of some physiological traits and disease prevalence among populations . Like snps, cnvs also show variability among populations; therefore, comprehensive cnv information is needed when applying cnvs as genetic markers in disease or trait studies . For the reasons cited above, we made plans to accumulate japanese data in which we provide information about the relation between japanese cnv data and phenotypic differences correlated with reported diseases . In our previous paper, we summarized such data by focusing on highly polymorphic cnvs found in 5.0% of the individuals . More recent studies based on advanced molecular technologies, such as genome - wide association studies [33, 34] and next generation sequencing [35, 36], reported that many genes appear to play important roles in the etiology of common diseases . Depending on newly developed technologies, rare variations have recently received attention from scientists who espouse a hypothesis called common disease and rare variants . In this paper, we summarized cnvs that were identified in fewer than 10 individuals in our previous population study . We focused on genes reported in the dgv, ucsc, and ncbi databases (table 3), although many mrnas were also listed in them . Since these genes are good candidate markers for enabling us to examine the etiology of common diseases and phenotypical heterogeneities among individuals, our cnvs have the potential to become useful markers in future studies . In this study, 34 cnvs were new, indicating that cnv coverage of the human genome is still incomplete and that there is diversity between the japanese and other ethnic populations . Moreover, the cnvs found in about two - thirds of the bac regions examined in our study were not identified in the studies in which three groups examine japanese populations using different array platforms . The newly identified cnvs extend the coverage of cnvs in the human genome (also the general japanese population). Moreover, it is expected that the cnvs could now be taken into consideration when genetic studies, for example, cnv association studies, are conducted.
Rheumatoid arthritis (ra) is a systemic autoimmune disorder with progressive articular damage that may result in lifelong disability [1, 2]. It typically causes a symmetrical chronic arthritis that causes joint pain, swelling and in some cases a systemic illness [3, 4]. The central pathogenesis of rheumatoid arthritis (ra) is characterized by not only marked hyperplasia of the fibroblast - like synoviocytes (flss) in response to the production of autocrine, but also paracrine molecules produced by infiltrating mononuclear cells [3, 5]. The aberrant hyperplastic flss display properties of transformed cells and destroy periarticular bone and cartilage . Actually, flss actively participate in the invasive processes of ra . Since flss proliferate abnormally, they produce high levels of destructive enzymes and cytokines to decrease the susceptibility to spontaneous and induced apoptosis . Thus, it is important to elucidate the mechanisms of flss proliferation and apoptosis for the design of targeted drugs . Notably, because ra is a systemic autoimmune disease characterized by chronic inflammation of multiple joints and proinflammatory cytokines such as tumour necrosis factor (tnf)-, interleukin (il)-1, il-6, or il-17 may be reconsidered as the etiology for ra and further leads to the development of targeted therapies [3, 911]. Indeed, tnf- inhibitors, anti - tnf antibodies, a soluble tnf receptor fusion protein, and il-1 receptor antagonist have been attempted for the treatment of ra, but their side effects such as serious infections and inducible malignant tumors still remain not to be resolved . Moreover, about 25~30% of ra patients treated with tnf antagonists do not display any significant clinical improvement after initiation of therapy . Patients will display feature of secondary resistance to the delivered drug within the first 5 years of therapy . All these findings raise a question that it is still incompletely understood on the relationship between inflammation inhibitions and ra therapy . In mammalian cells, nuclear factor-b (nf-b) it includes subunits as nf-b1 (p50/p105), nf-b2 (p52/p100), rela (p65), c - rel, and relb and regulates immune and inflammatory responses, cellular proliferation, or cell death [12, 13]. Nf-b is constitutively activated in many autoimmune diseases, including ra [14, 15]. For example, flss release cytokines, chemokines, and growth factors to sustain inflammation and produce enzymes that degrade the organized extracellular matrix, destroying cartilage and bone [16, 17]. Moreover, nf-b activation facilitates synovial hyperplasia by promoting proliferation and inhibiting apoptosis of flss . Thus, nf-b seems to be a positive regulator of cell growth and inhibitor of cell apoptosis in flss . However, some studies have suggested that nf-b may also function in a proapoptotic fashion . For example, induction of apoptosis in human embryonic kidney cell line (293 cells) after serum withdrawal requires nf-b activation . Nf-b activation may promote apoptosis in neural cells and t cell hybridomas, and high levels of c - rel induce apoptosis in avian embryos and in bone marrow cells in vitro . Thus, the functions of nf-b in apoptosis may be distinct by different cell types . At least, all the paradox indicates that inflammations mediated by nf-b may not always be beneficial for cell survival . Ectopic elevations of inflammations may afford us new insights into the clinical ra therapy and explain why sometimes anti - inflammation drugs are not so efficacious . In this study, we proposed to determine whether enhanced inflammations may lead to cell apoptosis in flss . Thus, we screened and identified the inflammation alternations by 4-hydroxynonenal (4-hne) treatment in mh7a cells, a human rheumatoid arthritis synovial cell line . 4-hne is an aldehyde product of membrane lipid peroxidation, generated through peroxidation of omega 6-polyunsaturated fatty acids [24, 25]. Our results showed that, by 4-hne treatment, flss develop strong inflammation reactions, such as elevated il1-, il-6, and tnf- transcriptions and cox-2 expressions . Of note, 4-hne induces nf-b activation, indicated by rela (p65) nuclear translocations and subsequent cell apoptosis . The present findings may help to understand the molecular network of inflammations and apoptosis in flss and offer novel insights into the clinical improving of ra therapy . Dulbecco's modified essential medium (dmem), fetal bovine serum (fbs), and bay11 - 7082 (nf-b inhibitor) were purchased from gibco invitrogen (carlsbad, ca, usa). The millipore luminex kits for cell signaling multiplex assay were from millipore (billerica, ma, usa) (#48 - 611mag and 48 - 680mag). The hoechst kit for cell death detection was from beyotime biotechnology (haimen, jiangsu, china). The following antibodies, anti - cox-2, anti - lamin a / c, anti - beta actin, and anticleaved caspase 3 were from santa cruz biotechnology (santa cruz, ca, usa). The nf-b (p65) and gapdh antibodies were from cell signaling technology (danvers, ma, usa). A widely used mh7a human rheumatoid arthritis synovial cell was chosen as in vitro experiment system, which was obtained from shanghai institute of cell biology (introduced from american type culture collection). In our experiments, mh7a cells were plated in 6-well plates at 1.0 10 cells / ml . The cells were incubated in dulbecco's modified essential medium (dmem) containing 10% fetal bovine serum (fbs) plus antibiotics for 24 h in 5% co2 at 37c . For 4-hne and following bay11 - 7082 treatment, the final low and high concentrations (5 m and 50 m) of 4-hne and 10 m bay11 - 7082 were applied to these cells and then incubated from 0 to 12 h. no additives were used as internal controls . After culturing, the cells were harvested for subsequent examinations . To screen the targeted intracellular signaling of 4-hne on mh7a cells, cell signaling multiplex assay was carried out by millipore luminex kits . Briefly, the cell samples were loaded to 96 wells and coated with respective primary antibody mixtures . After washing, samples were detected in luminex multiplexing instruments (millipore, billerica, ma). Rna was subjected to reverse transcription with reverse transcriptase as the manufacturer's instructions (fermentas). Quantitative real - time pcr was performed using the bio - rad iq5 system, and the relative gene expression was normalized to internal control as beta actin . Primer sequences for sybr green probes of target genes are as follows, il-1: ctggtgtgtgacgttcccatta and ccgacagcacgaggcttt; il-6: ttccatccagttgccttcttg and ttggg agtggtatcctctgtga; tnf-: catcttctcaaaattcgagtgaca and tgg gagtagacaaggtacaaccc . Mh7a cell samples were lysed in lysis buffer, and insoluble material was removed by centrifugation at 12,000 g for 20 min at 4c . Final protein concentrations were determined using the bradford protein assay (bio - rad laboratories). Electrophoresis was performed using sds - page, and blots were transferred to nitrocellulose membranes . Resulting films were scanned, and optical densities were quantified using imagej . To detect the nf-b nuclear translocations, mh7a cells were harvested and isolated into cytosol and nuclear parts, according to instructions of nuclear / cytosol fractionation kit (biovision). As for the nf-b activity detections, plasmids encoding nf-b - responsive firefly luciferase and renilla luciferase, which is an internal control for normalizing transfection, were transfected to mh7a cells using lipofectamine 2000 . After transfection for 0, 3, or 12 h, cells were washed with phosphate - buffered saline, serum - starved in opti - mem overnight, and then assayed for luciferase activity using the dual - luciferase reporter assay kit (promega). For the preparation of cell death detection by hoechst staining, mh7a cells were plated with 1.0 10 cells / ml in 6-well plates . After pharmacological manipulations, cells were directly stained with hoechst kit from beyotime . The cell counting was carried out through the use of national institutes of health software imagej, which is available from http://rsbweb.nih.gov/. Data represent the mean and standard error of the mean (sem). Student's t - test was performed for all statistical significance analysis using graphpad prism software . To screen the effects of lipid peroxidation on synoviocytes, we examined the intracellular pathways by millipore luminex kits after 4-hne treatment . It is noted that, after 4-hne treatment, inflammation relative pathways, such as nf-b and stats, were significantly upregulated (figure 1), while the cell growth or survival relative pathways, such as insulin receptor (ir), akt, mtor, and gsk pathways, were downregulated (figure 1). These results are consistent with the previous findings, indicating that lipid peroxidation is able to induce cell stress to promote inflammations and inhibit cell growth . As for the ectopic upregulation of inflammations by 4-hne treatment, we determine to identify the subsequent inflammation alternations and cell viability in mh7a synoviocytes . To further confirm the inflammation inductions by 4-hne treatment, we next examined the expressions of canonical inflammation factors, including il1-, il-6, and tnf- . The results of quantitative real - time pcr showed that under low concentrations of 4-hne (5 m), the mrna levels of il1-, il-6, and tnf- were increased gradually with the increasing time up to 12 h, and the maximum folds increased by 6.79, 6.19, and 4.28 compared to the control, respectively (figures 2(a), 2(b), and 2(c)). Interestingly, when the concentration of 4-hne increased to 50 m, the mrna levels of il1-, il-6, and tnf- in rheumatoid arthritis synovial cells increased gradually with the increasing time up to 1 h and then decreased (figures 2(d), 2(e), and 2(f)). The shorter peak time of mrna increasing may indicate that inflammations are enhanced by high lipid peroxidation treatment . And the overwhelmed lipid peroxidation may terribly affect normal cell functions and lead to a subsequent reduction of inflammation gene transcriptions . Nevertheless, our results support the notion that lipid peroxidation may indeed induce inflammation reactions in synoviocytes . To further confirm the inflammation alternations by lipid peroxidation, we next examined the protein level of cox-2 in 4-hne - treated mh7a cells . Cox-2 is an inducible isoform of prostaglandin h synthase, which mediates prostaglandin synthesis during inflammations . We found that, under 5 m 4-hne conditions, cox-2 protein levels were increased gradually with the time up to 12 h, with the highest value being about 2.86-folds that of the control (figures 3(a) and 3(b)). This indicates that lipid peroxidations may induce cox-2 expressions in a time - dependent manner in mh7a synovial cells . When the 4-hne concentration came to 50 m, the protein levels of cox-2 were increased gradually with the increasing time up to 1 h and then decreased (figures 3(c) and 3(d)). These alternations are consistent with the changes of inflammation gene transcriptions, suggesting that lipid peroxidations could enhance inflammation reactions in rheumatoid arthritis synoviocytes . Inflammation is controlled by multiple signaling pathways in mammalian cells, and nf-b plays a pivotal role in the regulation of inflammatory responses . By millipore luminex kits, we have identified that nf-b pathway is induced by 4-hne . To confirm this finding, we further investigated the nf-b activations by 4-hne treatment . Upon activation of nf-b by stimulators, nf-b subunits (especially for p65) translocate into the nucleus to activate transcription of genes involved in cell inflammations or proliferations . Accordingly, we evaluated nf-b activity by detecting the nuclear localization of the p65 subunit . Subcellular fractionation of low 4-hne - treated mh7a cells showed that p65 localization was enhanced in the nucleus compared to controls (figure 4(a)). An nf-b - re - luc reporter assay also demonstrated that nf-b activity was 10.1-fold greater after 12 h 4-hne treatment compared with the level in controls (figure 4(b)). Under high 4-hne concentrations, nf-b translocation into nucleus was increased as early as 3 h but decreased at 12 h (figure 4(c)). The nf-b activity assay also exhibited that nf-b activity was increased in 3 h and decreased in 12 h, respectively . Thus, these results indicate that nf-b activity could be induced by lipid peroxidations, and overwhelmed oxidative stress may even impair the nf-b activation conversely . Lipid peroxidation has been proved to induce nf-b pathway and inflammatory responses in mh7a cells; we wonder how these inflammations affect cellular functions, especially on cell viability . To examine whether 4-hne treatment may induce the cell apoptosis in mh7a cells, we applied hoechst staining to mh7a cells treated by 4-hne . Quantitative results showed that 5 m 4-hne could induce dramatic cell apoptosis in mh7a cells, with nearly 87.8% cell apoptosis in the time of 12 hours (figures 5(a) and 5(b)). Comparably, high concentration 4-hne induces faster cell apoptosis than that in low 4-hne group (figures 5(a) and 5(c)). We also examined cleaved caspase 3, a well - known apoptotic marker to confirm the cell apoptosis by 4-hne . Results showed that the cleaved caspase 3 increased as 4-hne treatment in a time- and dose - dependent manner . Moreover, our findings also indicated that the cleaved caspase 3 increased and total caspase 3 decreased as 4-hne treatment in a time- and dose - dependent manner (figures 5(d) and 5(e)). It is noted that the protein level of cleaved caspase 3 seemed to decrease in 50 m 4-hne for 12 h, which could be explained as the solemn impairment of cell structures and functions for overwhelming 4-hne treatment . Based on the above results since nf-b is a master regulator of inflammatory responses, we would like to test whether inhibition of nf-b activation could reduce inflammations and subsequent cell apoptosis in mh7a cells . Firstly, we examined the inflammatory responses by bay11 - 7082 (nf-b inhibitor) treatment in mh7a cells under the condition of 4-hne . The mrna levels of il1-, a marker of inflammatory response, was lower in the presence of bay11 - 7082 compared to that of 4-hne alone group, either in low or high concentration of 4-hne group (figures 6(a) and 6(b)). Moreover, the induction of cox-2 was also reduced by bay11 - 7082 treatment (figures 6(c) and 6(d)). These findings indicate that the inhibition of nf-b signaling attenuates inflammatory responses in rheumatoid arthritis synoviocytes . Since inhibition of nf-b activation could reduce inflammations in mh7a cells, we further investigate whether the nf-b inhibitor could reduce 4-hne mediated apoptosis in mh7a cells . Quantitative results showed that in the presence of bay11 - 7082, the percentage of cell apoptosis reduced compared to the counterparts without bay11 - 7082 under 5 m while in 50 m 4-hne groups, the protective effect of bay11 - 7082 was weakened, especially in 6 and 12 h groups (figure 7(b)). This may be due to the solemn impairment of cell structures and functions by overwhelming lipid peroxidations . Nevertheless, these data at least verify that inhibition of nf-b and inflammations could partly reduce the 4-hne mediated cell apoptosis in rheumatoid arthritis synoviocytes . In the present study, we reveal a novel mechanism to clarify the role of inflammations on ra development . We demonstrate that products of lipid peroxidations, 4-hne, could induce synovial intrinsic inflammations by activating nf-b pathways and lead to cell apoptosis . Pharmacological inhibition of nf-b activation may reduce the 4-hne mediated inflammations and subsequent cell apoptosis (figure 8). Our work uncovers novel relationships between inflammations and ra development and offers new strategy to ra clinical therapy . Rheumatoid arthritis (ra) is a chronic inflammatory disease of synovium that can lead to severe joint damage . The central pathogenesis of ra is a proliferation of fibroblast - like synoviocytes (flss) in response to stimulators . During the process of flss proliferation, previous studies focus on drugs to inhibit inflammations, but they sometimes do not display significant clinical improvement after initiation of therapy, as well as bringing side effects such as serious infections and inducible malignant tumors . Now, our results could partly answer the question that why sometimes anti - inflammation drugs do not perform well in ra therapy . At least, the inhibitory effect of anti - inflammation drugs may partly block the inflammation - mediated cell apoptosis in rheumatoid arthritis synoviocytes . Therefore, appropriate drug compatibility should be important to balance the effect of inflammations on ra development and flss apoptosis . Since inflammation balance is important for the survival of flss, the precise regulation of synovial inflammations must be well achieved . It has been long appreciated that nf-b is a significant transcription factor that functions in immune and inflammatory responses . For example, nf-b plays a pivotal role in myocardial ischemia - reperfusion injury and induces many proinflammatory cytokines and chemokines . Moreover, nf-b is also thought to act as a redox sensitive transcription factor, that has been proposed to be the sensor for oxidative stress . Reactive oxygen species (ros) can activate nf-b directly, and ros is also involved in nf-b activation by other stimuli such as tnf . In the present study, we demonstrate that products of lipid peroxidation, 4-hne, may induce cell death in rheumatoid arthritis synovial cells by activating nf-b pathways . Our results confirmed that ros and its relative products, that is 4-hne, are enough to induce inflammation reactions via nf-b . Considering that ectopic activation of nf-b induces synovial apoptosis, it may afford us some new strategy to design antirheumatic drugs targeted at nf-b . Besides the ros itself, the products of lipid peroxidation may also cause the formation of reactive aldehydes and lead to inflammations . These lipid peroxidations have longer biological half - lives than free radicals and can diffuse from their site of formation to reach distant targets and cause cellular damage . 4-hne is an, -unsaturated hydroxyalkenal that is produced by lipid peroxidation in cells [32, 33]. It is found throughout animal tissue and in higher quantities during oxidative stress for the increase in the lipid peroxidation chain reaction and for the increase in stress events [34, 35]. However, the normal level of lipid peroxidation is limited to a low level, and even the modest upregulation could alter multiple intracellular pathways . Thus, we propose that lower levels of intracellular 4-hne are beneficial to cells, but higher levels can cause a toxic response in the cell and may lead to cell death . In this study, we applied different doses of 4-hne to treat mh7a synovial cells for different time points . Interestingly, we note that even a short overwhelmed treatment of 4-hne exhibits pretty effects on synovial apoptosis . Notably, the effect of low concentration 4-hne could be partly restored by nf-b inhibitors, while high concentration 4-hne could not . This suggests that at least the effects of lipid peroxidation to synovial apoptosis might be partly dependent on nf-b mediated inflammation reactions . In conclusion, the results of this study support the notion that lipid peroxidation - mediated inflammation promotes cell apoptosis through activation of nf-b pathway in rheumatoid arthritis synovial cells . Our results imply that blocking nf-b activation may be partly beneficial for human ra therapy.
Histologic confirmation of this lesion is difficult, although it is very important for decision of treatment method.1 various methods, including endoscopic ultrasound - guided fine needle aspiration (eus - fna),2,3 eus - guided trucut biopsy (eus - tcb), endoscopic submucosal - mucosal resection (esmr),4 and open surgery have been reported to be used for confirmation of the diagnosis.5 however, the best method for tissue acquisition has not been established yet . Here, we report a case with a 12 cm submucosal signet ring cell type adenocarcinoma, diagnosed by eus - tcb and immunochemical studies . A 57-year - old man presented with epigastric pain and a 6 kg weight loss over 3 months . Laboratory tests at admission showed white blood cell count of 13,050/mm (normal range, 4,800 to 10,800), hemoglobin 14.2 g / dl (13 to 18), carcinoebryonic antigen 1.5 ng / ml (0 to 5), and ca19 - 9 107.3 u / ml (0 to 36). The endoscopy showed an intraluminal protruding lesion covered with normal mucosa along the anterior wall of the lower body, antrum, and duodenal bulb (fig . A computed tomography of the abdomen showed about a 1012 cm exophytic mass at the antrum and lower body of the stomach, with invasion to the liver, pancreas, and transverse colon (fig . 2). Eus with a radial endoscope (ue 260; olympus, tokyo, japan) was performed subsequently to assess the gastric wall and evaluate the characteristics of the mass . The eus showed a heterogeneous echogenic texture with multiple hyperechoic deposits and anechoic necrotic zones inside the large tumor mass that was thought to have developed in the fourth hypoechoic layer (muscularis propria). However, the mucosal and submucosal layer were intact, and the extraluminal border could not be assessed due to the very large size of the mass (fig . 3). A non - operable malignant gastrointestinal stromal tumor (gist) was first suspected . Therefore, eus - tcb with a linear endoscope (uct240; olympus) was performed for rapid pathology to confirm the clinical suspicion with immunohistochemical staining and to save costs . The signet ring tumor cells were immunoreactive for cytokeratin (ck, 200) (fig . 4). Chemotherapy with ts-1 (ts - one; jeil pharmaceutical co., seoul, korea) plus iv cisplatin (cisplatin; ildong pharmaceutical co., seoul, korea) was started . Ts-1 was given orally at a dose of 40 mm / m twice daily for 2 weeks followed by a 1-week rest, and cisplatin was given intravenously on day 1 at a dose of 60 mg / m . During the admission, a percutaneous drainage (pcd) tube the general condition of the patient deteriorated rapidly after about 2 weeks from admission and the patient died 2 months after the diagnosis . This case report of a patient with gastric primary signet - ring cell carcinoma with features of gist was confirmed by eus - tcb . Gastric cancers have various endoscopic findings, and histology is used to confirm the diagnosis.6 however, it is often difficult to confirm the histologic diagnosis despite taking a biopsy specimen, because the tumor surface of gastric cancers mimicking a submucosal tumor (gcsmt) is covered by normal mucosa.1 gcsmts are very rare; they account for 0.1% to 0.63% of all resected gastric cancers.7 moreover, the histological confirmation of a signet ring cell type is uncommon . Eus alone can provide useful information on gcsmts;8 however, it is difficult to determine the histological nature of the lesions from the eus image alone.9 the methods used to overcome this problem include eus - fna,2,3 eus - tcb, esmr,4 laparoscopic excision biopsy,10 and open surgery.5 however, the best method for confirmation of the diagnosis has not been established yet . For a definite diagnosis of a submucosal tumor, tissue acquisition, and pathology confirmation recently, mekky et al.11 reported that the results of eus - fna in 141 patients with gastric smts were diagnostic, suspicious, and nondiagnostic in 43.3%, 39%, and 17.7% of cases, respectively, with an overall accuracy of 95.6% and the accuracy of differentiating potentially malignant lesions of 94.2% . Sftoiu et al.12 reported that the yield of adequate tissue sampling was similar for eus - fna and eus - tcb (96.4% vs. 89.3%, p = not significant). However, the accuracy for obtaining a specific diagnosis was significangly lower for the eus - fna compared to the eus - tcb (5.3% and 68.4%, p<0.005). Cantor et al.13 suggested that the esmr has a significantly higher diagnostic yield than jumbo forceps biopsy with the use of the bite - on - bite technique for the evaluation of subepithelial lesions limited to the submucosa . The diasgnositc yield of the jumbo forceps biopsy was four out of 23 cases (17%) compared to 20 out of 23 cases (87%) of the esmr (p<0.001). The esmr, however, accompanies major complications such as bleeding (0% to 24%) and perforation (0% to 5%). Therfore, the eus - tcb was performed in this case for rapid pathology in order to confirm the clinical suspicion with immunohistochemical staining, with a relatively lower complication rate . In conclusion, this is the first report of the eus - tcb used as a diagnostic tool in a case of gastric primary signet - ring cell carcinoma with features of gist . Eus - tcb has low complication rate and enables immunohistochemical staining unlike other methods such as eus - fna, esmr, or open surgery, which is why it should be considered when results of a mucosal biopsy are not diagnostic . The possibility of signet ring cell carcinoma, which has a poor prognosis, should always be considered even in cases with gcsmt developing in the fourth gastric layer.
Philadelphia positive acute lymphoblastic leukemia (ph+ all) is a high - risk, aggressive form of acute leukemia, affecting primarily adults and the elderly . The hallmark of this disease is the presence in all leukemia cells of a reciprocal translocation termed t(9; 22)(q34; q11) resulting in a chimeric bcr - abl (e1a2 breakpoint) fusion gene that encodes a 190 kd protein (p190) with constitutively active tyrosine kinase activity that can alter multiple signaling pathways, contributing to tumor growth and proliferation . Before the advent of tyrosine kinase inhibitors (tkis), the outcome of ph+ all patients not eligible for allogeneic stem cell transplant (allo - sct) was characterized by an extremely poor prognosis, a weak response to most chemotherapy combinations, short remission durations, and poor survival rates . The introduction of imatinib, a selective inhibitor of the abl tyrosine kinase, has revolutionized the treatment and the outcome of this subset of patients . However, a substantial proportion of imatinib - treated ph+ all patients develop resistance to imatinib . Second - generation tkis have demonstrated promising efficacy in the treatment of imatinib - resistant ph+ all patients, but despite these results, the relapse rate of ph+ all patients remains very high with an overall survival still unsatisfactory . The persistence of a measurable residual disease at molecular level appears to be the key issue for treatment failure [35]. The development of alternative strategies that could selectively target ph+ all cells and synergistically work in combination with tki may have a crucial impact on disease control and ultimately patients' survival . On this matter, a p190-specific active immune approach like a vaccine could meet these requirements . Due to bcr - abl fusion, the corresponding p190 joint region contains an amino acid sequence unique to the oncoprotein in addition to a novel amino acid, not belonging to either bcr or abl sequences, created at the exact fusion point . Thus, from an immunologic point of view, peptides derived from p190-breakpoint area are leukemia - specific antigens that may be employed as therapeutic vaccine with the purpose to induce a t cell response toward p190 + leukemia cells . Recently natural bcr - abl breakpoint - specific cytotoxic t lymphocytes (ctls) were found in the bone marrow of ph+ all patients treated with imatinib correlating with a better response to this tki . These findings suggest a potential activity of the immune system against this lethal disease and the crucial role of p190 itself as target . In the present work we searched for p190-derived breakpoint peptides previously, we have developed a p210-breakpoint derived penta - peptide vaccine for controlling minimal residual disease in chronic myeloid leukemia (cml) patients treated with imatinib . In this setting, we found that the best antileukemia immune response was mediated by cd4 + t cells specific for an hla class ii p210-breakpoint peptide - specific cd4 + t cells isolated from vaccinated patients were found to be either perforin+ or cd25+/foxp3 +: in both cases they exerted direct cytotoxic activity against a cml cell line . Based on these premises, in our vaccine strategy for ph+ all, we focused our efforts in the search for p190 breakpoint peptides as strong inducers of a peptide - specific cd4 + t cell response . Our results show a promising p190-derived breakpoint peptide suitable for a peptide vaccine therapeutic approach in these patients . To pursue our vaccine strategy for ph+ all we investigated the fusion region of p190 in search of novel 25-mer p190 breakpoint peptides with strong hla class ii binding prediction and thus potentially able to induce a strong cd4 + t cell stimulation . The length of 25 amino acids has been chosen as maximum length that should contain all possible hla class ii molecules binding epitopes, usually from 13 to 23 amino acids long, always including the breakpoint and the new amino acid produced at the fusion point . We analysed all 25 possible 25-mer long peptides that include the fusion point (table 1). We employed syfpeithi database for mch ligands and peptides motifs and bimas database which allow to estimate the ligation strength to a defined hla type for a sequence of amino acids . The level of binding was compared with our binding prediction mean database for hla class ii bcr - abl - derived peptides already employed in cml patients . Promising peptides will be synthesized on f - moc solid phase synthesis and purified by hplc for in vitro use . In order to evaluate the capability of p190-derived peptides to induce a peptide - specific cd4 + t cell in vitro cd4 + t cells freshly isolated from pbmc were cultured for 21 days in 5% ab human serum media while undergoing to 3 rounds of stimulation with autologous cd14 + cells previously isolated and frozen . Cd4 + t cells were seeded in 24-well plates at 1 10 cell / ml together with cd14 + cells at 0.25 10 cell / ml (4: 1 ratio) and maintained in a humidified incubator with 5% co2 and 95% air at 37c for 7 days . Each test peptide was added at 20 g / ml and il-15 was added at final concentration of 10 ng / ml . After this time cd4 + t cells underwent the second round of stimulation: the cells were collected, counted, and replaced in the same conditions with fresh cd14 + cells, peptides, and il-15 for other 7 days . The peptide - specific cd4 + t cell proliferation was measured by standard thymidine incorporation assay and expressed by a stimulation index (si = cpm cd4 + t cells plus test peptides / cd4 + t cell alone or cd4 + t cells plus control peptides). Briefly, cd4 + t cells were collected and incubated at 2 10 cells per well for 96 hours and maintained for 4 days in a humidified incubator with 5% co2 and 95% air at 37c with 20 g / ml peptide under the following experimental conditions: (1) no peptide, (2) each test peptide alone, and (3) wt1-derived 25 amino acid peptide alone as negative control . The level of p190-peptide specific t cell proliferation was compared with our stimulation index mean database for hla class ii bcr - abl - derived peptides already employed in cml patients . The latter allowed us to identify peptides with good probability to be sufficiently immunogenic and suitable for vaccination studies in vivo . Among all 25 possible peptide in the fusion region, we identified three promising 25-mer long p190 fusion peptides: pdngegafhgdaealqrpvasdfep (p190 - 13), dgegafhgdaealqrpvasdfepqg (p190 - 15), and egafhgdaealqrpvasdfepqgls (p190 - 17) with strong hla binding properties for hla - drb1 * 0101, hla - drb1 * 0401, hla - drb1 * 1101, and hla - drb1 * 0301 (dr17). Subsequently p190 - 13, p190 - 15, and p190 - 17 have then been synthesized and purified for in vitro t cell stimulation testing . In vitro p190-derived peptide cd4 + t cell stimulation was first performed in 5 normal subjects . In each healthy donor p190 - 13, p190 - 15, and p190 - 17 peptides were tested in separated experiments . Only p190 - 13 peptide was able to induce in all 5 subjects, regardless of their hla - dr phenotype, a peptide - specific cd4 + t cell proliferation as measured by standard thymidine assay, with an si ranging from 2.0 to 2.7 . All patients had previously received a standard induction treatment followed by tkis therapy and were at least in hematologic remission at the time of in vitro cd4 + t cell experiment . Three of six were receiving dasatinib at 100 mg / day and 3/6 were receiving imatinib at 400 mg / day (see table 2). Again p190 - 13 peptide induced peptide - specific cd4 + t cell proliferation in 4/6 ph+ all patients with an si value of 2.2, 2.0, 2.2, and 2.1, respectively (table 2). The ph+ all is a high - risk acute leukemia with poor prognosis, in which the specific t(9; 22)(q34; q11) translocation results in a chimeric bcr - abl (e1a2 breakpoint) and in a 190 kd protein (p190) with constitutive tyrosine kinase activity . Despite the promising results of tkis, the relapse rate of ph+ all patients remains very high with an overall survival still unsatisfactory . In ph+ all, leukemic cells could be targeted also by means of an active specific immune response raised against bcr - abl - derived p190 fusion protein that is indeed a leukemia - specific intracellular antigen . Thus vaccinations with p190-derived peptides could induce in ph+ all patients peptide - specific t cell response that could control or even eradicate minimal residual disease thus reducing the probability of relapse in these patients . In an antitumor vaccine strategy the choice of the appropriate peptide is crucial and we decided to focus our search on longer, hla class ii binder p190-breakpoint peptides, with the intent mainly to prompt a peptide - specific and possibly leukemia - specific cd4 + t cell response . The present study identified p190 - 13, a novel p190-derived 25-mer peptide that met these requirements . In fact p190 - 13 was able to easily induce, in vitro, a peptide - specific cd4 + t cell response in all healthy donors and in 4/6 ph+ all patients tested . Of note, this peptide includes in its sequence all the epitopes for which p190-specific ctls were naturally found in all patients and thus p190 - 13 could as well mediate a peptide - specific ctls response in this setting of patients . Regarding the choice of a breakpoint peptide for developing a peptide vaccine strategy in ph+ all we are aware that all has a much more instability than cml and thus p190 has more probability to undergo mutations . Even if the latter is true, it has to be underlined that p190 mutations do not usually appear in the breakpoint region which sequence is very stable through all the phases of the disease . The little number of patients studied and the variability of age, chemotherapy induction treatment, and type of tki do not allow us to speculate further about the quality and intensity of the in vitro peptide - specific immune response observed . However, it is encouraging that 4/6 ph+ all patients were able to mount an in vitro cd4 + t cell response against p190 - 13 while being treated with imatinib but also with second generation tkis, dasatinib . The magnitude of p190-derived peptide - specific t cell response, as measured by the stimulation index, was the same in patients and healthy donors and was in the range of what we previously found in cml patients, confirming that all patients treated with tkis maintain a functional and reactive immune system even against a poorly immunogenic self oncoprotein like p190 . For all these reasons our peptide could be a good candidate for developing an immune target vaccine strategy possibly synergizing with tkis for remission maintenance and reducing the probability of relapse in ph+ all patients . These results support the rationale to offer to ph+ all elderly patients or adult patients not eligible for intensive chemotherapy and allo - sct, a line treatment without chemotherapy, by using a tki as debulking induction treatment and subsequently by adding a potentially synergic leukemia - specific immune approach such as a p190-derived peptide vaccine . In conclusion novel identified p190 - 13 25-mer peptide is able to induce in vitro a peptide - specific cd4 + t cell response in ph+ all patients . Thus there appears a good candidate for developing an immune target vaccine strategy possibly synergizing with tkis for remission maintenance . A p190 - 13 peptide vaccine trial in ph+ all patients in complete hematologic remission during tkis is currently in planning.
The human skin forms a barrier to the external environment that is constantly exposed to colonizing microbiota, invasive pathogens, and allergens (belkaid and segre, 2014, pasparakis et al ., these encounters drive differentiation and colonization of tissue - resident memory t (trm) cells (gaide et al ., 2015, 2012), originally characterized in barrier tissues such as skin, gut, lung, and the female genital tract (gaide et al ., 2015,, 2014, watanabe et al ., 2015). Of the 20 billion t cells contained in human skin (clark et al ., 2006), 50%70% express the trm cell markers cd103 (-subunit of the e7 integrin receptor) and cd69 (watanabe et al ., 2015), both implicated in lodging trm cells in peripheral tissues . Cd103 binds e - cadherin, which is highly expressed on epithelia, whereas cd69 antagonizes sphingosine 1-phosphate receptor 1 (s1pr1)-mediated egress from tissues (mackay et al ., 2015, trm cells provide strong defense against recurrent infections (ariotti et al ., 2014, 2014). While such local immune responses contribute to immunity (gebhardt et al ., 2009, 2012), aberrant activation might cause disease (hondowicz et al ., 2016, jabri and abadie, 2015, park and kupper, 2015). In the skin, the patchy appearance of several t cell - mediated diseases, such as psoriasis and vitiligo (boehncke and schn, 2015, ezzedine et al ., 2015), suggests that tissue - resident rather than circulating cells drive immunopathology (cheuk et al ., 2014, demarcated, inflamed and hyperproliferative plaques are maintained by interleukin-23 (il-23) and il-17 in psoriasis (hueber et al ., 2010, leonardi et al ., 2008), whereas vitiligo presents with persistent depigmentations attributed to local interferon- (ifn-) production and t cell - mediated killing of melanocytes (harris et al . Thus, if trm cells drive these diseases, then vitiligo and psoriasis would be expected to involve the contribution of functionally different subsets . Trm cell subsets with distinct cytokine profiles have been reported in skin (naik et al ., 2015, sanchez rodriguez et al ., 2014, watanabe et al ., 2015), possibly reflecting a diversity of immune functionalities and responses to pathogenic and commensal microbiota nonetheless, phenotypic markers of trm cell subsets with specialized effector functions have not been established . In addition to cd103 and cd69, murine trm cells that create local immunity to recurrent viral infection homogenously express cd49a (gebhardt et al ., 2009, ray et al ., 2004, cd49a constitutes the -subunit of the 11 integrin receptor, also known as very late antigen 1 (vla-1), and binds collagen iv enriched in the basement membrane separating epidermis and dermis . As only 15% of human skin - derived t cells express cd49a (purwar et al ., 2011), cd49a expression might distinguish trm cell subsets with distinct effector functions and putative roles in immunopathology . Here, we determined the anatomical localization, transcriptional profiles, and functional properties of trm cell subsets in human skin with respect to cd49a, cd69, and cd103 . In healthy individuals, cd8cd103cd49a trm cells were present in both the dermis and epidermis, whereas cd8cd103cd49a trm cells specifically localized to the epidermis . Epidermal cd8cd103cd49a trm cells were the predominant il-17-producers, whereas cd8cd103cd49a trm cells excelled at ifn- production and rapidly gained a cytotoxic capacity following il-15 stimulation . This functional dichotomy was evident in the comparison of distinct immune - mediated skin diseases, with skin biopsies from vitiligo patients showing a predominance of cytotoxic cd8cd103cd49a trm cells while skin biopsies from psoriasis patients featured the accumulation of the il-17 producing cd8cd103cd49a counterparts . Together, our results reveal a functional specialization of distinct skin trm cell subsets in health and disease . To gain insights into the diversity among trm cells with respect to spatial niches, we determined the relative expression of tissue - residency markers cd69, cd103, and cd49a on cd4 and cd8 t cells from healthy human skin (figure s1a). Compared to the dermis, an increased proportion of epidermal cd4 and cd8 t cells co - expressed cd69 and cd103 (figures 1a and 1b) as previously reported (watanabe et al ., 2015). In these healthy skin samples, cd49a expression was restricted to cd8cd69cd103 t cells that were located in the epidermis (figures 1a and 1c). Considerable variation in the frequencies of cd8cd69cd103cd49a t cells was apparent among (figure 1c) and within different donors (figures s1b and s1c), indicating high inter- and intra - individual variation of trm cell compositions within the epidermal compartment . Skin cd4 trm cells were predominately cd62lcd45racd28, whereas cd8 trm cells were cd62l but heterogeneous with respect to expression of cd27, cd28, and cd45ra (figures s1d and s1e). To delineate the microanatomical localization of skin cd8 t cell subsets, we performed confocal imaging of healthy skin . Regardless of cd49a expression, epidermal cd8 t cells were juxtaposed to the collagen iv - rich basement membrane, embedded among basal keratinocytes (figures 1d and 1e). Perivascular dermal cd8 t cells were predominately detected in papillary dermis well separated from epidermis (figure 1d). In total, 24% (6/33) of epidermal and 6% (3/53) of dermal cd8 t cells expressed cd49a in direct contact with the basement membrane (figure 1e; data not shown). Bulk t cells in full thickness human skin display a diverse t cell receptor (tcr) repertoire that is in equilibrium with that of circulating t cells (clark et al ., 2006, to determine whether epidermal cd8cd103cd49a trm cells shared a clonal origin with circulating effector memory cd8 t cells or skin t cell subsets, cd8 t cell subsets from five healthy donors were sorted (figures s1f and s1 g) and analyzed by targeted, deep, high - throughput genomic dna sequencing of the tcr complementarity - determining region 3 (cdr3). A single v family dominated the epidermal cd8cd103cd49a trm cell population in three donors, with cdr3 diversity more restricted in cd8cd103cd49a versus cd8cd103cd49a trm cells (figures 1f and 1 g). Although only a small proportion of the detected clonotypes overlapped between epidermal cd8cd103cd49a and cd8cd103cd49a trm cells (figure 1h), a few dominant clones were shared and comprised the majority of total reads in two donors (figures 1h and 1i). Furthermore, whereas the most prevalent cd8cd103cd49a trm clonotypes showed some overlap with other epidermal and dermal cd8 t cell subsets (figure 1j), v family distribution differed in epidermal and dermal cd8cd103cd49a trm cells (figure 1f). The ten largest cd8cd103cd49a trm cell clones were highly enriched in the epidermis (figure 1j). Although the mechanical procedure of dermal and epidermal preparations might result in some degree of cellular cross - contamination, these results together demonstrate the epidermal localization of expanded, oligoclonal cd8cd103cd49a trm cells in human skin . To further interrogate the functional properties of epidermal cd8cd103cd49a and cd8cd103cd49a trm cell populations, we determined their transcriptional profiles, along with those of dermal cd8cd103cd49a and cd8cd103cd49a t cells as well as peripheral blood cd8cd62lcd45ra t cell populations expressing or lacking the cutaneous leukocyte antigen (cla). Cells were sorted from healthy donors (figures s1f and s1 g) and analyzed by rna - sequencing (ramskld et al ., 2012). Principal component analysis on all sorted t cells subsets from six donors distinguished circulating and skin t cell populations (figure 2a). Among skin t cells, principal component analysis discriminated epidermal cd8cd103cd49a and cd8cd103cd49a trm cells from dermal populations (figure 2b). Focusing on the epidermal trm cells, 92 genes were differentially expressed between cd8cd103cd49a and cd8cd103cd49a trm cells (figure 2c, tables s1 and s2). Gene enrichment analysis (david) indicated acquisition of cytotoxicity - related function as the most compelling difference between the populations (figure 2d). Specifically, expression of prf1, gzmb, gzmh, gzmk, gnly, and nkg7 transcripts, all encoding cytotoxic granule components, were elevated in epidermal cd8cd103cd49a trm cells (figure 2e), indicating that this subset might mediate cellular cytotoxicity . Moreover, genes mediating response to viruses, lymphocyte activation, and chemotaxis were also upregulated in cd8cd103cd49a trm cells (figure 2d). Conversely, gene enrichment analyses did not identify any significantly downregulated gene programs (figure 2d). Nonetheless, transcripts of genes associated with il-17 production, such as il17f, rorc, il23r, and ccr6, were significantly decreased in cd8cd103cd49a relative to cd8cd103cd49a trm cells, whereas transcripts for ifn- were elevated (figures 2d - e). Transcription of itga1, encoding cd49a, was not significantly different following correction for multiple comparisons (uncorrected p = 0.0007, adjusted p = 0.12). Quantitative pcr (qpcr) analysis demonstrated significantly higher itga1 transcription in cd8cd103cd49a versus cd8cd103cd49a trm cell subsets (figure 2f; p = 0.002). Moreover, regulation of gzmb, ifng, and ccr6 was confirmed by qpcr, whereas no difference in itgae, encoding cd103, was found (figure 2f). Further validating transcriptional data, cxcr3 expression was higher on cd8cd103cd49a trm cells, whereas il-23r and ccr6 were preferentially expressed by cd8cd103cd49a trm cells (figure 2 g). As such, transcriptional profiles as well as surface receptor expression suggested functional differences between cd8cd103cd49a and cd8cd103cd49a trm cells . Despite enrichment of transcripts for cytotoxic granule constituents in epidermal cd8cd103cd49a trm cells, perforin, and granzyme b protein expression was virtually undetectable when examined ex vivo (figures 3a and s2a and s2b). We reasoned that in situ inflammatory stimuli might be required to elicit expression of cytotoxic granule constituents in trm cell subsets, as recently proposed for il-15 (jabri and abadie, 2015). Following incubation of epidermal cell suspensions with a variety of proinflammatory cytokines relevant to skin inflammation, intracellular protein expression of perforin and granzyme b was detected in cd8cd103cd49a trm cells following il-2 and il-15 stimulation (figures 3a3c). Granzyme b expression was upregulated as early as 1 hr after il-15 stimulation, whereas perforin expression was detected at later time points (figure 3d). Antibody - mediated blockade of mhc class i did not diminish il-15-mediated granzyme b upregulation (data not shown) and other inflammatory cytokines, including il-1, il-6, il-7, il-12, il-23, and ifn-, did not elicit expression of cytotoxic granule constituents (figure 3 g). In an inflammatory milieu, several cytokines might interact for induction of cytotoxic granule constituent expression . Accordingly, il-15-dependent expression of perforin and granzyme b was augmented by il-6, but not other cytokine combinations tested (figures s2c s2e). Differential expression of il-2 and il-15 receptor components could have explained the preferential responses of epidermal cd8cd103cd49a trm cells . However, the il-2 receptor and chains were expressed in both epidermal subsets (figures s2f and s2 g). Thus, epidermal cd8cd103cd49a trm cells appeared quiescent in healthy skin, but il-2 from activated immune cells or il-15 from keratinocytes (loser et al ., 2004) besides the skin, trm cells are widely distributed in other barrier tissues . To determine whether cd49a expression more broadly defined trm cells with a cytotoxic potential, we examined two mucosal barriers, gut and cervix (figure s3a). Cd8cd103cd69cd49a trm cells were abundant in these tissues, with an overall higher frequency of cd49a t cells (figure 4a). Unlike skin, substantial proportions of cd8cd49a t cells lacking cd69 or cd103 expression were present in both gut and cervix and a population of cd4cd103cd69cd49a trm cells existed in cervical epithelium (figure 4a). With respect to expression of cytotoxic granule constituents, gut, and particularly cervical trm cells displayed higher basal expression of perforin and granzyme b relative to skin (figures 3a3c, 4b and s3b). Perforin and granzyme b were more highly expressed in cd8cd103cd69cd49a compared to cd8cd103cd69cd49a gut and cervical trm cells, being further augmented by il-15 stimulation (figure 4b). These results indicate that cd49a expression defines trm cell subsets with cytotoxic potential in a variety of epithelial tissues . Having found that cd49a generally identifies trm cells with cytotoxic potential and having elucidated inflammatory signals for their priming, we set out to determine their cytotoxic capacity . Sorted t cell subsets from blood and skin collected from healthy donors (figure s4) were incubated with cr - labeled target cells in 4 hr redirected antibody - dependent cellular cytotoxicity assays . The cytotoxic activity of epidermal cd8cd103cd49a and cd8cd103cd49a trm cell subsets was compared to that of peripheral blood cd8cd57 t cells, a highly cytotoxic effector memory subset (chiang et al ., 2013), as well as cd8cd57 t cells displayed strong cytotoxic activity, whereas skin - derived cd8cd103cd49a trm cells mediated very poor activity following anti - cd3 antibody stimulation (figure 5a). However, following 48 hr of il-15 pre - stimulation, epidermal cd8cd103cd49a trm cells manifested cytotoxic activity comparable to that of peripheral blood cd8cd57 t cells, whereas cd8cd103cd49a trm cells and cd8cd57 t cells remained poor cytotoxic effectors (figure 5a). Stimulation of epidermal t cells with anti - cd3 antibodies for 24 hr induced similar levels of cd8cd103cd49a trm cell granzyme b and perforin expression as il-15, whereas these stimulations in combination displayed an additive effect (figure 5b). In summary, skin - derived cd8cd103cd49a trm cells lack perforin expression, with short - term tcr stimulation alone being insufficient to induce cytotoxicity . T cell - derived cytokines are crucial for defense against invading pathogens at barrier sites . Generally, ifn- contributes to immunity toward intracellular infections while il-17 provides anti - fungal defense and both of these cytokines initiate inflammatory keratinocyte responses . Prompted by differences in transcription of ifng as well as rorc, a key transcriptional regulator of il-17 production, we determined whether cd49a expression also defined epidermal skin trm cell subsets in regards to cytokine production . Corroborating transcriptional profiles, cd8cd103cd49a trm cells produced il-17 while cd8cd103cd49a trm cells excelled in ifn- production upon stimulation with phorbol 12-myristate 13-acetate and ionomycin (figures 6a6c). Tnf and il-2 were abundantly produced by dermal and epidermal trm cell subsets (figures 6b and 6c). Tcr engagement using anti - cd3 antibodies also preferentially induced ifn- by epidermal cd8cd103cd49a trm cells (figure 6d). Moreover, il-15 stimulation potentiated tcr - dependent expression of il-17 and ifn- by epidermal cd8cd103cd49a and ifn- by cd8cd103cd49a trm cells, respectively (figure 6d), substantiating effectual chain receptor signaling in both subsets . Coating of wells with collagen iv in addition to anti - cd3 antibodies specifically augmented production of ifn- by epidermal cd8cd103cd49a trm cells (figure 6e), indicating vla-1-mediated regulation of cytokine production . Unconventional mucosal - associated invariant t (mait) cells represent a fraction of peripheral blood il-17-producing t cells (dusseaux et al ., 2011). Although up to 30% of skin cd8 t cells expressed cd161, cd8tcr - v7.2cd161 mait cells comprised less than 25% of dermal or epidermal skin cd8 trm cell populations (figures s5a and s5b) and made up less than 25% of the il-17-producing cd8cd103 t cells thus, cd49a expression delineated a dichotomy in trm cell cytokine production, augmented by il-15, with cd8cd103cd49a and cd8cd103cd49a trm cells preferentially producing il-17 and ifn-, respectively . A population of epithelial trm cells that upon controlled activation perform cytotoxic killing and ifn- production would be perfectly placed to safeguard against infections and malignant transformation in situ . Hypothetically, such specialized trm cells might also participate in focal immune pathology in vitiligo (figure s6a), a disease where cytotoxic t cells have been implicated in eradication of melanocytes . The proportion of epidermal cd8cd103cd49a trm cells was increased in lesional vitiligo but not in psoriasis (figures 7a and 7b). Cd49a was expressed on almost half of dermal cd8cd103 trm cells in lesional vitiligo (figures 7a and 7b), adjacent to collagen iv - expressing vessels (figure s6b), while healthy skin, non - lesional vitiligo and psoriasis was devoid of this population (figures 7a and 7b, s6c and s6d). In contrast, cd4cd103 t cells in lesional vitiligo did not express cd49a (figure s6e). In vitiligo, a substantial proportion of lesional cd8cd103cd49a trm cells recognized melanocyte - derived antigens (figures s6f and s6 g). Perforin and granzyme b - expressing trm cells were detected in vitiligo, but not in healthy skin or psoriasis (figures 7c and 7d). Moreover, cd8cd103cd49a trm cells co - expressed perforin and granzyme b in lesional vitiligo ex vivo (figures 7e and 7f). In line with increased cd49a frequencies, ifn- producing trm cells were enriched in vitiligo lesions (figure 7 g). Conversely, il-17-producing trm cells were enriched in psoriasis plaques (figure 7 g). As in skin from healthy volunteers, cd8cd103cd49a trm cells thus preferentially expressed ifn- and cd8cd103cd49a trm cells il-17 in both vitiligo and psoriasis (figures 7h7j). Similar to healthy skin and in agreement with a previous study (teunissen et al ., 2014), less than 5% of skin il-17 producing cells were cd161v7.2 mait cells (figures s6h s6j). Trm cells with the capacity to co - express ifn- and il-17 were present in psoriasis plaques (figure 7h and 7j), indicating a degree of functional plasticity in the context of chronic inflammation . However, constrained plasticity of healthy skin - derived trm cells was indicated, as sorted cd8cd103cd49a trm cells from healthy skin failed to produce il-17 following 72 hr of stimulation with il-1, il-6, and il-23 (figures s6l and s6 m). In resolved psoriasis, cd8 trm cells poised for il-17 production accumulate (cheuk et al ., 2014), and il-17 expression thus, functional dichotomies associated with cd49a expression were generally preserved in disease - associated trm cells arguing that cd49a marks subsets of trm cells with imprinted effector profiles and distinct functions in inflammatory skin diseases . At epithelial boundaries, trm cells differentiate and form a first line of adaptive defense against multiple pathogens, tailored to effectively control recurrent infections . Consequently, it would be expected that distinct subsets of trm cells respond to activation with heterogeneous cytokine responses (naik et al ., 2015, sanchez rodriguez et al ., 2014, schlapbach et al ., 2014, watanabe et al ., 2015) despite their protective role in recurrent viral infections, direct proof of trm cell - mediated cytotoxicity is lacking (mueller and mackay, 2016). In addition to anti - microbial defense, pathogenic trm cells are implicated in several inflammatory diseases (clark, 2015). Here, we identify cd49a expression as a marker delineating a subpopulation of cd8 trm cells in human skin that specifically localize to the basal layer of epidermis, preferentially produce ifn-, and display high cytotoxic capacity upon stimulation . We also find a high proportion of cd49a trm cells poised for cellular cytotoxicity in other epithelial tissues . Lastly, we determined that this functional dichotomy among trm cell subsets was preserved in the inflammatory skin diseases psoriasis or vitiligo . In healthy skin, we found cd49a trm cells confined to basal epidermis in contact with collagen iv expressed in the basement membrane . Mouse epidermis displays analogous spatial restrictions to the number of trm cells during homeostasis (zaid et al ., 2014). Interactions with collagen iv might anchor cd49a trm cells in the epidermis . In murine models, protective cd8cd49a trm cells develop in barrier tissues (gebhardt et al ., 2009, ray et al ., 2004, zhang and bevan, 2013) following viral infections . In line with our study of human skin, granzyme b - expressing trm cells are retained in mouse epithelium following epidermotropic viral infections (gebhardt et al . Epithelial tissues represent first sites of viral entry and with high cellular turnover of keratinocytes, offer an attractive niche for viral propagation and shedding . Thus, by expressing cd49a, trm cells seem well - positioned for immunosurveillance of infected epithelial cells via production of ifn- as well as cellular cytotoxicity . Collagen iv - mediated engagement of cd49a enhanced ifn- production by cd8cd103cd49a trm cells, possibly through stabilizing ifng transcripts (wang et al ., similar to mouse cd8 trm cells in the context of viral infection (mackay et al ., 2013,, 2013), the transcriptional profiles of human cd8cd103cd49a trm cells suggested that anti - viral defense and target cell killing represent key functions of this subset . Such cells might also contribute to local surveillance and defense against malignancy (jameson et al . Freshly isolated human skin cd8cd103cd49a trm cells displayed a transcriptional profile indicative of cytotoxic function, but did not express key mediators of cellular cytotoxicity . Rather, their cytotoxic capacity was primed through il-2 and il-15-mediated induction of perforin and granzyme b expression ., 2013) and entry into the epidermis (adachi et al ., 2015). Here we find that il-15 additionally acts both to potentiate cytokine responses and as a key mediator in cytotoxic licensing of cd8cd103cd49a trm cell . Our results suggest an important role for bystander keratinocytes or t cells in activation of trm cell - mediated effector functions within the skin . These observations validate the hypothesis that il-15 might generally act as a central danger signal regulating trm cell responses (jabri and abadie, 2015) and extend knowledge by providing a cellular marker of specific trm cell subsets capable of mediating cellular cytotoxicity . In the gut, il-15 is implicated in driving cytotoxic lymphocyte responses that turn pathological in celiac disease (meresse et al ., 2004). In our analyses, cd49a was abundantly expressed on cd8 trm cells in gut and cervix, representing mucosal barrier tissues . In these tissues, a greater proportion of trm cells expressed cd49a than in skin and displayed a more activated phenotype, with constitutive expression of perforin and granzyme b. it is possible that mucosa is a milieu more commonly challenged by pathogens, requiring activated, cytotoxic cd49a trm cells that provide continuous surveillance . Nonetheless, il-15-mediated potentiation of cd49a trm cells might represent a mechanism to safeguard tissues against immunopathology . Revealing functional specialization among epidermal trm cells with respect to cd49a expression, cd8cd103cd49a trm cells preferentially produced il-17, a cytokine required for control of bacterial and fungal infections . Epidermal cd8cd103cd49a trm cells excelled in il-17 production relative to dermal counterparts as well as dermal cd8cd103 t cells . This observation, combined with different gene - expression profiles, indicates distinct subsets of cd8cd103cd49a trm in dermis versus epidermis . To explore potential origins of epidermal cd8cd103cd49a trm cells, we sorted dermal cd8cd103 trm cells and stimulated them with il-15 and tgf-, cytokines implicated in trm cell differentiation (mackay et al ., 2013, watanabe et al ., 2015). A fraction of dermal cd8cd103 trm cells upregulated both cd103 and cd49a following stimulation (s.c . And l.e ., unpublished observations), indicating that inflammatory responses might differentiate dermal t cells to functionally distinct trm cells embedded in epidermis . Animal models provide further opportunities to dissect the molecular requirements for induction of cd49a and determine the role of cd49a in ensuring effective trm cell - mediated immunity . The patchy appearance and fixed recurrence of several inflammatory skin diseases implies the activity of trm cells . In vitiligo, there, they are well - positioned to eradicate repopulating melanocytes . In contrast, in active psoriasis, with up to 100-fold more trm cells within the hypertrophic epidermis compared to healthy skin (cheuk et al ., 2014), il-17-producing trm cells were profoundly expanded . Moreover, il-17 or ifn- production by distinct trm cells subsets was generally maintained even in the context of the vigorous tissue inflammation . Co - expression of il-17 and ifn- was detected in some cd8cd103cd49a and cd8cd103cd49a trm cells . However, we could not induce il-17 production by cd8cd103cd49a trm cells sorted from healthy skin following il-23, il-6, and il-1 stimulation . Thus, we speculate that during differentiation trm cells require tcr engagement in addition to il-17 polarizing inflammatory cues to display the level of functional plasticity we observe in psoriasis . In resolved psoriasis where numbers of trm cells are similar to healthy skin, we previously described enrichment of cd8cd103ccr6il-23r trm cells poised to il-17 production (cheuk et al ., 2014). In contrast, ifn--producing cd8cd103cd49a trm cells were enriched in vitiligo, further supporting the notion of a dichotomy of trm cell subsets according to cd49a expression even in the setting of skin inflammation and immunopathology (figure s6o). Given the role of il-2 and il-15 in potentiating both il-17 and cytotoxic effector functions in trm cells, blockade of the downstream jak / stat pathway represents a promising therapeutic target for skin diseases putatively caused by aberrant trm cell activation . Interestingly, a case report suggests clinical efficacy of toficitinib, a small molecule inhibitor of jak1 and jak3, on vitiligo (craiglow and king, 2015). Systemic administration of il-15r blocking antibodies prevented hair loss in a mouse model of alopecia areata (xing et al ., 2014), a disease characterized by aberrant killing of hair follicles . Most patients present with limited burden of disease that cause considerable suffering, yet do not require systemic treatment . Future development of topical treatments aiming at controlling pathogenic trm cells in situ would offer an attractive therapeutic strategy, with our data highlighting chain signaling an attractive target . In summary, our data reveal that human skin contains different subset of epidermal trm cells poised toward cytotoxicity and ifn- or il-17 production, respectively . Inflammatory cytokines unleashed tcr engagement - dependent cellular cytotoxicity by cd49a trm cells from healthy skin, whereas activated, perforin- and granzyme - expressing cd49a trm cells accumulated in vitiligo lesions . Further insights into the dynamics of the composition, retention, and activation of expanded, functionally specialized trm cell populations might contribute to improved management of both infections and chronic inflammatory skin diseases . Human peripheral blood and healthy skin samples from trunk were obtained from reconstructive skin surgery at advita clinic, stockholm, sweden, and karolinska university hospital, solna . Cervical tissue samples were obtained from non - malignant and non - inflammatory surgical specimens, st . Gut biopsies were obtained from the ileum of healthy patients with hereditary predisposition for colorectal cancer (lynch syndrome) at karolinska university hospital, huddinge . Patients with non - segmental vitiligo, or plaque psoriasis were collected at the swedish psoriasis association clinic or the dermatology clinic at karolinska university hospital, solna (table s3). Resolved psoriasis was collected as previously described (cheuk et al ., 2014). Lesional vitiligo biopsies were sampled from within 1 cm to and non - lesional at least 10 cm away from lesional borders . Ethical permits 2012/50 - 31/2, 2015/0041 - 31, 2015/933 - 32, 2012/1900 - 31/1, 2013/1800 - 32, 2015/1078 - 32, regional ethical committee of stockholm . 810 m thick sections were stained as previously described (cheuk et al ., 2014) with primary antibodies to collagen iv (clone col-94, abcam), melana (clone ep1422y, abcam), keratin 5/6 (cloned516, dako), cd8 (ab4055, abcam), cd49a (clone 550594, bd bioscience), and cd3 (clone cd3 - 12, abcam). Images were acquired by zeiss lsm700 and lsm800 (zeiss) and analyzed with fiji - imagej . Whole - skin punch biopsies and cervix samples were incubated in 5u dispase (life technologies) overnight at 4c followed by manual separation of epidermis and cervical epithelium from dermis or cervical submuocsa respectively followed by 90 min incubation in collagenase iii (3 mg / ml; worthington) with dnase (5 g / ml; roche) in rpmi 1640 . Dermis and submucosa were further processed by medicon tissue disruptor (bd biosciences) as previously described (cheuk et al ., 2014). Ileum biopsies were digested in collagenase ii (0.25 mg / ml; sigma - aldrich) with dnase (0.2 mg / ml; roche) in imdm (life technologies) for 3045 min . Complete rpmi medium was added and the cell suspension were subsequently passed through a 40 m (gut) / 70 m (skin or cervix) cell strainer (bd bioscience). Peripheral blood mononuclear cells (pbmcs) p815 cells were purchased from atcc and maintained in complete medium (rpmi 1640 supplemented with 10% fetal bovine serum [fbs], l - glutamine; all hyclone). Recombinant il-15, il-1, il-2, il-6, il-7, il-23, il-12 and ifn- (all r&d systems) were stored at 80c . Human collagen iv, phorbol 12-myristate 13-acetate (pma), and ionomycin were purchased from sigma . Sorting was performed within 2 - 4 hr using moflo xdp (beckman coulter) cell sorter or bd faczjazz (bd). Details on flow cytometry and sorting experiments, as well as functional evaluations are provided in the supplemental experimental procedures . Dna was extracted (puregene, qiagen) and the tcr- cdr3 regions were sequenced and mapped (immunoseq, adaptive biotech). Rna was purified from cell - qiazol lysate (mirneasy, qiagen) and quality was checked (bioanalyzer rna 6000, agilent) before library construction (smart - seq, clontech) and sequencing (illumina hiseqtm 2000). The cytotoxicity assay was performed and analyzed as described previously (chiang et al ., 2013). In brief, sorted and rested cell populations were used as effector cells against cr - labeled p815 target cells supplemented with 0.5 g / ml of anti - cd3 antibody (clone s4.1, invitrogen) with effector - to - target ratios between 10 and 0.3 . Cr release in supernatant was measured on a -counter (wizard, perkinelmer) and specific lysis was calculated as previously described (chiang et al ., 2013). Statistical analysis and graphical illustration of numerical data was performed by using either prism (v6, graphpad), jmp 13 or rstudio ., a.i ., and l.e . Provided reagents and clinical material: m.e ., l.e . Supervised ex vivo preparation and analysis of human skin cells, y.t.b.
Gossypiboma refers to a mass resulting from retained cotton, gauze, or sponge accidentally left within the body after surgery . As indicated in its name, gossypiboma is an iatrogenic outcome reported on rare occasions in various parts of the body with diverse clinical findings, often mistaken radiologically as a tumor or abscess . Now, we report a literature review and our experience of a case with incidentally found mass, who was first judged as a gastrointestinal stromal tumor (gist) of the gastric fundus by abdominal computed tomography (ct) and endoscopic ultrasonography (eus), but was later confirmed as gossypiboma resulting from retained surgical sponge . A 78-year - old female visited the hospital for sudden onset epigastric pain which lasted for one day . She received abdominal us at a private clinic indicating multiple gallbladder (gb) stones and common bile duct (cbd) stones and was transferred to the emergency room of this hospital . She had a history of partial gastrectomy 30 years ago in another hospital for a reason she cannot remember precisely . The physical examination revealed blood pressure 110/70 mm hg, pulse rate 76/min, respiration rate 20/min, and body temperature 36.6 without unusual finding except for the epigastric tenderness . Peripheral blood test showed hemoglobin 9.2 g / dl, white blood cell (wbc) 9,600/mm, and platelet 428,000/mm . Iu / l, alt 95 iu / l, total bilirubin 1.5 mg / dl, alkaline phosphatase 404 iu / l, -gtp 58 iu / l, amylase 66 u / l, and lipase 26 u / l . The abdominal ct performed at the emergency room indicated mild dilatation of intrahepatic bile duct and cbd with multiple gb stones, but the finding of cbd stone was not definite . In addition, a 5.5-cm sized well - demarcated round mass with irregular calcification density was found adjacent to the gastric fundus (fig . 1). Esophagogastroduodenoscopy (egd) was performed to search the cause of the abdominal pain, where 5-cm sized bulging area covered with normal mucosa was found at the gastric fundus but not any other lesions to cause the abdominal pain (fig . Cbd stone was not detectable on eus performed to exclude the presence of cbd stone . Eus also showed an exophytic hypoechoic mass which was connected with the fourth layer of the gastric wall at the area where subepithelial tumor was suspected . It appeared, from the operative field, that the mass was a foreign body covered by the inflammatory membrane rather than a gist of the gastric fundus . The mass was tightly adhered to the gastric fundus, making it difficult to dissect from the gastric wall . The resected mass was 6.54 cm in size and when the mass was cut in half, a surgical sponge was visible with the naked eyes (fig . The covering material was confirmed as inflammatory pseudomembrane accompanying fibrosis, abnormal calcification, hemorrhage, and necrosis . Gossypiboma, also called' textiloma,' is a compound of 2 words " gosysspium " meaning' cotton' in latin and " boma " meaning' place of concealment' in swahili, referring to a mass resulted from the retained cotton or gauze after the operation.1 the occasion of retaining surgical instruments inside the human body is rare but there have been always the possibility from the beginning of the history of surgery . Every surgery with incisional wound has the potential to leave a foreign body inside the human body, such as gauze, clamp, retractor, or electrode . Surgical sponge, among others, is most frequently left in the human body after surgery, as much as to be named separately as gossypiboma or textiloma . Gossypiboma within the abdominal cavity in most patients are asymptomatic and often leads to a medical - malpractice claim only by incidental discovery several years later, further complicating the recognition of precise incidence . The incidence of gossypiboma in south korea is unknown but reported about 1/1,000 - 10,000 surgeries worldwide.2 gossypiboma complication might occur in every form of surgery, ranging from general surgery (52%) to gynecology (22%), to urology and vascular surgery (10%), and to ophthalmology and spinal surgery (6%).2 a case - control study by gawande et al.3 found emergency operation, unexpected change in operation, and high body mass index of the patient were the risk factors of leaving a foreign material inside the human body after the operation . Cotton surgical sponge does not cause any specific biochemical reaction except for the adhesion or granuloma formation inside the body.4 the clinical manifestation of gossypiboma depends on the type of reaction, the location of gauze or sponge, and the degree of chronicity, but could be separated into 2 groups.5 the first group is the exudative inflammatory reaction which could be detected early after the surgery by symptoms caused by the abscess formation, or could be detected later with intestinal obstruction, erosion into the adjacent organs, and fistula formation . The retained gauze inside the abdominal or pelvic cavity could erode into the gastrointestinal tract causing extracorporeal extrusion on rare occasions . The second group is the aseptic fibrinous reaction where the encapsulated sponge does not induce symptoms and would not be detected for a long time . Our patient belongs to the latter, since she did not have any symptom for as long as 30 years, and the gauze was encapsulated and adhered to the gastric fundus . There are various ct findings but a cystic lesion with marked rim enhancement is most common.6 an investigator termed the calcium deposition around the rim of the retained surgical sponge appearing in the form of calcified reticulate rind as " calcified reticulate rind sign " and reported it as the characteristic ct finding of gossypiboma . Hypoechoic mass with strong posterior acoustic shadowing due to hyperechoic capsule suggests gossypiboma on the us.6 in this patient the mass showed irregular calcification at the center area of the mass . Every gauzes recently used for surgery contains radiopaque markers for easier detection by postoperative radiography . Gauzes in which these markers are not contained or degraded, or calcified may not reveal the characteristic radiopaque line on the radiography.7 there is no specific eus finding for gossypiboma . Eus could be performed for detailed observation when a gastrointestinal subepithelial lesion is suspected . In the patient of this study, it was considered that the 5 cm sized protruded area covered by normal mucosa on egd was the same mass discovered on ct appearing to originate from the stomach . Eus was performed for more detailed observation, revealing a hypoechoic mass originating from the fourth gastric wall layer . It is presumed that the inflammatory mass encapsulating the gauze was adhered to the serosal layer due to the fibrosis, and appeared as hypoechoic on eus . Subepithelial lesion on eus is diagnosed putatively based on the layer of origination and echogenic findings of the mass . When the pathologic diagnosis and eus impression of subepithelial lesions was compared in the prior studies, the diagnostic accuracy of eus was estimated about 78%.8 the factors contributing to the incorrect eus interpretation include inflammatory change, fibrosis, or tumor microinvasion around the tumor.9 the typical eus finding of gist is hypoechoic tumor originating from the fourth (proper muscle) layer, occasionally in multilobulated or pedunculated forms . Findings of necrosis, calcification, or ulceration of gist suggests the possibility of malignant transformation.10 our case did not show spongiform air bubbles with marked rim enhancement and cystic lesion on ct as typically found in gossypiboma patients . But the round well - demarcated mass originated from the stomach accompanying radiopaque material was noted and mistaken as a mass with irregular calcified density . This lesion was also found on eus as an exophytic hypoechoic mass connected with the fourth layer of the gastric wall and internal irregular hyperechoic spots with posterior acoustic shadowing . But the resected lesion was found to be a mass resulted from the retained surgical sponge.
Oncocytes are defined as modified epithelial cells with characteristic finely granular eosinophilic cytoplasm, which are abundant in mitochondria [1, 2, 3, 4]. Tumors composed of oncocytes have been described in the major and minor salivary glands, the thyroid, parathyroid, pituitary, and adrenal glands as well as in the kidneys, testes, pharynx, larynx, trachea, pancreas, respiratory tract, skin, and other organs [1, 5]. In the vast majority of cases, oncocytic lesions of the ocular adnexa occur in the caruncle; other locations rarely include the conjunctiva, plica semilunaris, lacrimal sac, eyelid, and lacrimal gland [1, 4, 6]. Oncocytic lesions occur more commonly in elderly patients, usually in their seventh decade of life, with a 2:1 female: male ratio, especially for those occurring in the caruncle [1, 5, 6]. The most common presenting symptom is a mass or lump, although patients are usually asymptomatic [4, 5]. Oncocytic lesions of the eye are generally benign, and complete surgical excision is the preferred treatment since it has often proved to be curative . A 69-year - old woman was referred to the ocular oncology service for the evaluation of a pigmented mass in the caruncular region of the right eye . The patient was regularly followed for chronic blepharoconjunctivitis in both eyes and dry eye syndrome, treated with lubricant eye drops . The patient took daily medications for hypertension and was suspected to have rosacea . Aside from this, her personal medical history and family history were unremarkable . The patient complained of a tan - colored lesion on her right caruncle, but denied symptoms of bleeding, discharge, an increase in size, or a color change of the lesion . On ocular examination, visual acuity was 20/20 and intraocular pressure was 16 mm hg in both eyes . Slit lamp biomicroscopy revealed a well - circumscribed, partially pigmented lesion in the caruncle of the right eye . No involvement of the lacrimal system was noted, and the dilated fundus examination of both eyes was unremarkable . The patient was diagnosed with conjunctival nevus of the right caruncle . Due to the lesion's unfavorable location, histopathological examination disclosed a benign, cystic, tubular, well - circumscribed lesion composed of tall, columnar, granular eosinophilic cells (fig . 1b). A thin fibrous tissue capsule penetrating the tumor from the periphery was also present . The pigmented macroscopic appearance of the lesion was thought to be due to concretions within the tumor (fig . 1b). A diagnosis of oncocytoma of the caruncle was made an oncocytoma is a rare benign tumor that usually arises in the glandular epithelia of tissues throughout the body . Among cutaneous sites, oncocytomas have also been known as oxyphil adenomas, papillary cystadenomas, adenolymphoma - like tumors, and warthin's tumor . The lesion is usually composed of large oval cells with eosinophilic granular cytoplasm filled with malformed mitochondria, most likely representing an age - associated metaplastic and neoplastic transformation of the glandular epithelium . Clinically, caruncular oncocyto - mas occur more frequently in female elderly patients [1, 4, 6] and usually present as a mass or lump that rarely shows a tendency to grow . They are commonly red - blue or orange tan in color, sometimes with a lobulated, fleshy, or cystic appearance . Histopathologically, oncocytomas have been attributed with four main architectural patterns: cystic - micropapillary, confluent - glandular, solid - organized, and solid - disorganized . Benign lesions are commonly described as having a cystic - micropapillary pattern, while potentially aggressive growth is associated with solid patterns . Reported atypical features of oncocytomas include the presence of pigmentation, feeder vessels, keratinization, a pedunculated growth pattern, and superficial lobulations . Furthermore, it has been reported that oncocytomas that are not dark blue in color are more likely to be misdiagnosed preoperatively . Surgical treatment outcomes are excellent for benign oncocytic neoplasms, with no recurrence found after surgical excision [1, 4]. Several entities must be taken into consideration in the differential diagnosis of carun - cular lesions . Nevi (40.7%), squamous papillomas (8.5%), and oncocytomas (5.1%) are the most common lesions at this body site . More specifically, oncocytomas are clinically often mistaken for hemangiomas, nevi, and cysts . The case we reported showed several atypical characteristics: the lesion was partially pigmented and lobulated and its macroscopic appearance resembled that of a conjunctival nevus . Conjunctival nevi tend to remain stationary throughout life, with less than 1% risk for transformation into malignant melanoma . Nevertheless, malignant melanoma of the conjunctiva is a potentially life - threatening tumor . Tumors found in unfavorable locations such as the palpebral conjunctiva, fornices, plica, caruncle, and lid margins have been associated with a higher rate of metastatic death . Given this knowledge, nevertheless, complete surgical excision had already been performed, and no further treatment was required . At follow - up after 12 months, the patient remained asymptomatic, with no signs of recurrence of the oncocytoma . In conclusion, although nevi are the most common lesions present in the caruncle, onco - cytoma should be considered in the differential diagnosis of a pigmented caruncular mass, especially in elderly female patients.
Alginic acid sodium salt (al) (viscosity: 20.0 - 40.0 cp in 1% water, mw: 7334), diclofenac sodium salt and cellulose acetate dialysis tube (mw: 7014) was procure from sigma louis, usa . Calcium chloride, hydrochloric acid, potassium chloride, potassium dihydrogen orthophosphate, sodium chloride and sodium hydroxide were purchased from s. d. fine chemicals, mumbai, india . Mmt rich bentonite clay was collected from akli mines, barmer district, rajasthan, india . De - ionized water was obtained from milli - q gradient a10 water purification system . To obtain mmt in na - form, the raw bentonite (300 g) was dispersed in 3 l solution of 0.1 m nacl and stirred for 12 h. finally, the slurry was centrifuged and washed with de - ionized water until the supernatant is free from chloride ion as tested by agno3 solution . The raw bentonite was purified by sedimentation technique as described earlier . According to the stoke's law of sedimentation, the purified clay was obtained by dispersing 150 g of raw bentonite in 10 l of de - ionized water and clay particles of <2 m size were collected from 15 cm height after 10 h at 30. the desired particles thus obtained was dried at 90 - 100, ground and sieved through 200 mesh sieve (astm). The cation exchange capacity (cec) of clay was measured by the standard ammonium acetate method at ph 7 and was found to be 91 meq/100 g of mmt on dry basis (dried at 110). The suitable amount of al dissolved in 25 ml of milli - q water with stirred till complete homogeneous solution was obtained . The desired quantity of ds was added into al and solution was kept at 35 into shaking water bath (100 rpm) for 3 h. to this mmt (200 mesh size) powder was added and the emulsion was stirred overnight at 40. this emulsion was dropped using a peristaltic pump (master flex l / s 7518 - 00, cole - parmer, usa) with helps of a 20-guage hypodermic needle fitted with a rubber tubing (falling distance 2 cm, pumping rate 1.5 ml / min) into 200 ml of 100 mm calcium chloride solution, gently agitated with a magnetic stirrer . The formed beads were allowed to stand in solution for 20 min for curing, then collected by filtration and were washed thrice with milli - q water . The mean diameter of the beads was determined by measuring 50 beads, using micrometer screw (mitutoyo, japan). The formulation code, content for the formulation, encapsulation efficiency and bead diameter are given in table 1 . Formulation codes and different characteristic of ds - mmt - al composites the ds content in beads was determined by a digestion method . The ds - loaded beads (900 mg) were smashed and incubated in 50 ml of phosphate buffer (ph 7.4). The resulting suspension was stirred to promote swelling and break - up of the cross linked structure to facilitate liberation of ds from ds - mmt - al composite into buffer media . The ds was quantified by centrifuging the suspension at 10,000 rpm for 30 min (kubota-6500, kubota corporation, japan) and ds was determined from the clear supernatant by uv - spectrophotometer at max = 276 nm . These studies were performed in triplicate for each sample and the average values were used in data analysis . The ds encapsulation efficiency, ee (%) was determined by using equation; ee = (aq / tq)100, where aq is the actual quantity of ds present in ds - mmt - al composite beads and tq is 100% theoretical quantity of ds present in ds - mmt - al composite beads . The intercalation of ds and al into interlayer of mmt was examined by powder x - ray diffraction (p - xrd) analysis . P - xrd was carried out on a phillips powder diffractometer x pert mpd using pw3123/00 curved cu - filtered cu - ka (=1.54056) radiation with slow scan of 0.3 deg / s in 2 range of 2 - 10. fourier transform infrared spectra (ft - ir) were measured with perkin - elmer, gx - ftir as kbr pellet over the wavelength range 4000400 cm . Thermogravimetric analysis was carried out within 30 - 800 at the rate 10/min in the nitrogen flow of 60 ml / min using mettler - toledo, tga / sdta 851e . Uv / vis absorbance of ds solutions was measured at a characteristic max = 276 nm by uv / vis spectrophotometer (cary 500, varian) equipped with a quartz cell having a path length of 1 cm . The surface morphology of composite was observed under scanning electron microscope (sem), leo-1430vp, uk . Buffer solution of ph 7.4 (simulated intestinal fluid) was prepared by mixing 500 ml of 0.1 m kh2po4 and 391 ml of 0.1 m naoh . In vitro release studies were performed using usp six stage dissolution rate test apparatus (thermonik, campbell electronics, mumbai, india) by using dialysis bag technique . The dialysis sac was equilibrated with the dissolution medium for few hours prior to experiments . Three hundred milligrams of ds loaded composite beads suspended in 5 ml of dissolution medium was taken in a dialysis bag and sealed at both ends . Dialysis bag was dipped into 500 ml of dissolution medium, stirred at 100 rpm at 371. at time intervals of 30 minutes, 5 ml of the dissolution medium was taken and the ds concentration was determined by uv / vis spectrophotometer at max = 276 nm . To obtain mmt in na - form, mmt slurry was treated with nacl solution . The raw bentonite (300 g) was dispersed in 3 l solution of 0.1 m nacl and stirred for 12 h. finally, the slurry was centrifuged and washed with de - ionized water until the supernatant is free from chloride ion as tested by agno3 solution . The raw bentonite was purified by sedimentation technique as described earlier . According to the stoke's law of sedimentation, the purified clay was obtained by dispersing 150 g of raw bentonite in 10 l of de - ionized water and clay particles of <2 m size were collected from 15 cm height after 10 h at 30. the desired particles thus obtained was dried at 90 - 100, ground and sieved through 200 mesh sieve (astm). The cation exchange capacity (cec) of clay was measured by the standard ammonium acetate method at ph 7 and was found to be 91 meq/100 g of mmt on dry basis (dried at 110). The suitable amount of al dissolved in 25 ml of milli - q water with stirred till complete homogeneous solution was obtained . The desired quantity of ds was added into al and solution was kept at 35 into shaking water bath (100 rpm) for 3 h. to this mmt (200 mesh size) powder was added and the emulsion was stirred overnight at 40. this emulsion was dropped using a peristaltic pump (master flex l / s 7518 - 00, cole - parmer, usa) with helps of a 20-guage hypodermic needle fitted with a rubber tubing (falling distance 2 cm, pumping rate 1.5 ml / min) into 200 ml of 100 mm calcium chloride solution, gently agitated with a magnetic stirrer . The formed beads were allowed to stand in solution for 20 min for curing, then collected by filtration and were washed thrice with milli - q water . The mean diameter of the beads was determined by measuring 50 beads, using micrometer screw (mitutoyo, japan). The formulation code, content for the formulation, encapsulation efficiency and bead diameter are given in table 1 . The ds content in beads was determined by a digestion method . The ds - loaded beads (900 mg) the resulting suspension was stirred to promote swelling and break - up of the cross linked structure to facilitate liberation of ds from ds - mmt - al composite into buffer media . The ds was quantified by centrifuging the suspension at 10,000 rpm for 30 min (kubota-6500, kubota corporation, japan) and ds was determined from the clear supernatant by uv - spectrophotometer at max = 276 nm . These studies were performed in triplicate for each sample and the average values were used in data analysis . The ds encapsulation efficiency, ee (%) was determined by using equation; ee = (aq / tq)100, where aq is the actual quantity of ds present in ds - mmt - al composite beads and tq is 100% theoretical quantity of ds present in ds - mmt - al composite beads . The intercalation of ds and al into interlayer of mmt was examined by powder x - ray diffraction (p - xrd) analysis . P - xrd was carried out on a phillips powder diffractometer x pert mpd using pw3123/00 curved cu - filtered cu - ka (=1.54056) radiation with slow scan of 0.3 deg / s in 2 range of 2 - 10. fourier transform infrared spectra (ft - ir) were measured with perkin - elmer, gx - ftir as kbr pellet over the wavelength range 4000400 cm . Thermogravimetric analysis was carried out within 30 - 800 at the rate 10/min in the nitrogen flow of 60 ml / min using mettler - toledo, tga / sdta 851e . Uv / vis absorbance of ds solutions was measured at a characteristic max = 276 nm by uv / vis spectrophotometer (cary 500, varian) equipped with a quartz cell having a path length of 1 cm . The surface morphology of composite was observed under scanning electron microscope (sem), leo-1430vp, uk . Buffer solution of ph 7.4 (simulated intestinal fluid) was prepared by mixing 500 ml of 0.1 m kh2po4 and 391 ml of 0.1 m naoh . In vitro release studies were performed using usp six stage dissolution rate test apparatus (thermonik, campbell electronics, mumbai, india) by using dialysis bag technique . The dialysis sac was equilibrated with the dissolution medium for few hours prior to experiments . Three hundred milligrams of ds loaded composite beads suspended in 5 ml of dissolution medium was taken in a dialysis bag and sealed at both ends . Dialysis bag was dipped into 500 ml of dissolution medium, stirred at 100 rpm at 371. at time intervals of 30 minutes, 5 ml of the dissolution medium was taken and the ds concentration was determined by uv / vis spectrophotometer at max = 276 nm . Basal spacing of mmt and ds - mmt - al was found to be 1.18 nm (2=7.4) and 1.5 nm (2=5.9), respectively (fig . The peak shifting from higher diffraction angle to lower diffraction angle can be attributed to the increase in the basal spacing during the formation of clay - alginate hybrid, indicating successful intercalation of ds and al into the interlayer of mmt . P - xrd pattern p - xrd pattern of mmt and ds - mmt - al ft - ir spectra of ds, mmt, al and ds - mmt - al composite beads are shown in fig . The virgin alginate showed asymmetric and symmetric stretching vibrations at 1617 and 1417 cm due to carboxyl anions, and at 1030 cm due to cyclic ether bridge . Mmt shows a broad band centered near 3400 cm due to -oh stretching band for interlayer adsorbed water . The bands at 3620 and 3698 cm are due to -oh band stretch for al oh and si oh . The shoulders and broadness of the structural oh band are mainly due to contributions from several structural oh groups present in mmt . The overlaid absorption peaks in the region of 1640 cm is attributed to oh bending mode of adsorbed water . (out - of - plane) and the peak at 1035 cm is attributed to si - o stretching (in - plane) vibration for mmt . The ir peaks at 915, 875 and 836 cm are attributed to alaloh, alfeoh and almgoh bending vibrations, respectively . Ft - ir spectra of ds showed n - h, c = o, c - cl stretching vibration at 3398, 1675 and 1335 cm, respectively . In the ftir spectra of ds - al beads, the asymmetrical band of carboxylate ion was shifted from 1617 cm to 1635 cm whereas carboxyl group of ds was shifted from 1575 cm to 1454 cm . The hydroxyl band of sodium al was shifted from 3493 cm to 3438 cm due the interaction between ds and al . Ft - ir spectra ft - ir spectra of (a) ds (b) al (c) ds - al (d) mmt (e) ds - mmt - al composites and (f) mmt - al pongjanyakul and puttipipatkhachorn explained the shift in the ir band of coo while al interacted with mmt . Incorporation of mmt into al caused a shift to a higher wave number and decreased the intensity of coo stretching peaks of al . The oh stretching peak of the silanol group (sioh) at 3698 cm disappeared in the spectra of mmt - al composites, and the oh stretching peak of al shifted to a higher wave number . This may be due to the intermolecular hydrogen bonding between silanol groups on the surface of mmt and the hydroxyl or carboxyl groups of al . With these results, the bond formation of al and mmt in the dry state intermolecular hydrogen bonding and electrostatic forces between al and mmt were confirmed by ftir studies . The possible structural model of the mmt - al composites the al molecules possessed a negative charge due to ionization of the carboxyl groups and could form intermolecular hydrogen bonding (fig . It can be seen that al and mmt formed electrostatic and intermolecular hydrogen bonds, which brought about numerous points of contact to create a three dimensional network . Al chains could also serve as a bridge between neighboring silicate layers when a higher content of mmt was incorporated . Possible structure model possible structure model of (a) al gel (b) mmt dispersion and (c) mmt - al composites the thermogravimetric analysis (fig . 4) of mmt shows two distinct steps, the first one is due to the loss of free water at <150 and the second weight loss at 550700 due to the loss of structural oh group from mmt . Al, mmt - al and the degradation of ds from ds - mmt - al was observed over a range of 200 - 500 while al moiety was degraded 550700. the degradation of alginate in mmt - al was not prominent, may be due to the reinforcement of cross linked ca - alginate by mmt, and therein increases the thermal stability of mmt - al composite . Thermogravometric analysis thermogravometric analysis of (a) mmt (b) mmt - al (c) ds - mmtal and (d) al the surface and cross - sectional morphology of ds - al beads and ds - mmt - al composite beads are depicted in sem images (fig . Ds - al beads displayed an unstable structure while ds - mmt - al composite beads are stable . Ds - mmt - al composite beads demonstrated a more rigid and flat surface as compared to ds - al beads, whose surface was convoluted . The rigidity imparted in ds - mmt - al composite beads may be due to the reinforcement effect of mmt in the al matrix . The sem image of ds - al and ds - mmt - al composite beads revealed their porous structure (fig 5 b d). Sem images sem images of (a) ds - al, scale: 100 m and (b) cross - section of ds - al, scale: 30 m; (c) ds - mmt - al composite bead, scale 100 m and (d) cross - section of ds - mmt - al composite bead, scale 30 m in vitro release profiles of ds from ds - alginate (formulation ads - al) and ds - mmt - alginate composite beads (ds - mmt - al - i to iv) in simulated gastric fluid and simulated intestinal fluid was done at 371. only 1 - 2% ds release was observer in simulated gastric fluid (ph 1.2) after 8 h. the release profile in simulated intestinal fluid showed (fig . 6) 9.8% and 17.7% ds released from ds - al composite in 1 h, and 4 h, respectively, thereafter the release of ds remained constant up to 8 h. while, in the case of ds - mmt - al - i to ds - mmt - al - iv formulation, the average release of ds was ~63% within 5 - 8 h depending upon the content of mmt in different formulations . The study reveals that the plateau for the release of ds was reached more rapidly in ds - al as compared to the ds - mmt - al - i to ds - mmt - al - iv . There was an increase in the release rate of ds from ds - mmt - al composite beads with an increase in the mmt loading . It may be due to the weak interaction between the ds and mmt as both ds and mmt are negatively charged . The ds encapsulation efficiency was lower in the case of virgin al beads as compare to ds - mmt - al composite beads . The ds encapsulation efficiency increases with the increase in the mmt content in ds - mmt - al composite beads (table 1). A possible explanation to this behavior is the presence of more hydroxyl groups on the surface of mmt facilitating hydrogen bonding with ds . A release of ds from ds - al and ds - mmt - al beads in 8 h was found to be 17.7% (ds - al) and 48.80%, 55.52%, 59.41% and 62.49% from ds - mmt - al - i to ds - mmt - al - iv, respectively . In vitrorelease profile in vitrorelease of ds from (a) ds - al (b) ds - mmt - al - i (c) dsmmt - al - ii (d) ds - mmt - al - iii (e) ds - mmt - al - iv composite beads ds - mmt - al composite beads were successfully synthesized by gelation methodology . The amount of ds released from ds - al and ds - mmt - al composite bead is 17.7% and 62.49%, respectively in simulated intestinal fluid . Mmt content in ds - mmt - al composite beads regulate the release rate of ds . This approach can be effectively use for the oral administration of ds to enhance its therapeutic efficacy.
There will be 60.5 million people with open angle glaucoma (oag) and angle closure glaucoma (acg) in 2010; the number will be increasing to 79.6 million by 2020 . An automatic and economic system is highly desirable for glaucoma screening for a wider range of people . The best approach to do screening is based on 2d color fundus image as it is cheap and easy to manipulate . The optic nerve head assessment based on the 2d color fundus image is the best way for glaucoma screening at the moment . But automatic glaucoma screening is based on the risk factors from the optic nerve head, so an accurate segmentation of optic nerve head is the basic and most important process in glaucoma screening . In retinal images, the optic disc generally appears as bright, yellowish, circular or slightly oval - shaped object as shown in figure 1 . Optic disc segmentation can be generally grouped into three categories based on the methods for extracting the optic disc boundary: template matching or hough transform methods, deformable models like snake, level sets, supervised classification or unsupervised classification . Template matching or hough transform methods, deformable models like snake, level sets, supervised classification or unsupervised classification . Shape - based template matching [3, 4] is a very easy approach to segment the disc as the optic disc is approximately circular or elliptical . But this method is not very accurate as the shape of the optic disc is not a perfect circle or ellipse due to some pathological changes and this method often fails as there is ppa near the optic disc . Use binary robust independent elementary features (brief) and a rotation invariant brief (obrief) features to find approximated boundary of the optic disc . Then a pixel classification method followed by circular template matching to segment the optic disc . Active contour model is one of the most promising approaches for od segmentation as it is better in capturing irregularity disc region . Gvf - snake algorithm is proposed by osareh et al . To extract the optic disc boundary . In order to remove the vessel occlusion during the deformable model, the blood vessel was first removed by morphology in the preprocessing step . Walter et al . Also use morphological filtering techniques to remove the blood vessels and they detect the optic disc boundary by means of watershed transformation . Li and chutatape used principal component analysis (pca) to locate the disc first and applied a modified active shape model (asm) for optic disc identification . By means of shape model, this approach can handle vessel occlusion and fuzzy edges . Use global elliptic model to estimate the optic disc location and radius; then they apply a local deformable model with variable edge - strength dependent stiffness . This algorithm often fails where there are large numbers of white lesions, ppa, or strongly visible choroid vessel . In order to solve the vessel occlusion and fuzzy contour shapes, xu et al . Propose a knowledge - based clustering and smoothing update deforming model after each deformation, the contour points are classified into two clusters by knowledge - based unsupervised learning . Then the contour is updated using both global and local information, so this method can achieve balance on contour stability and accuracy . They showed their proposed method achieves better success rate (94%) when comparing to gvf - snake (12%) and modified asm (82%) approach . However, they also show that this method fails where there is obvious white ppa region near the disc . Propose an automatic approach for od segmentation using a multiresolution sliding band filter (sbf) and the od boundary is regularized using a smoothing algorithm . This approach gets an average overlapping area of 83%, 89%, and 85% in three datasets . More recently, region - based active contour model is presented in; this method can detect the fuzzy edge and is not sensitive to initial contour . However, the chan - vese model cannot handle inhomogeneous image which may lead to erroneous segmentations . Propose a new segmentation method based on localized cv models using local image information from three - dimensional feature spaces . This is a novel method by using texture feature to segment the optic disc, and they show a very nice result in their paper . But the local chan - vese active contour model can lead to oversegmentation as the texture of the retinal image is inhomogeneous . The selection of local neighbor radius parameter which defines the local image domain around a point of interest is very important . If the radius is too small this model is just like gvf model, while a large radius will decrease the sensitivity to small gradients and make this model be similar to the traditional chan - vese model and ppa region will be misclassified to optic disc region . Another way to segment the optic disc the difficulty of this approach is the shape of the classification result is often unsmooth . Moreover, it is hard to find a good texture feature to distinguish the ppa and optic disc . And this approach also needs to be trained before being applied to classification and it is very time consuming to segment the optic disc by svm when exploiting lots of features for learning . The difficulty in optic disc segmentation is caused by ppa as shown in figure 1 . The traditional methods often mistake ppa region as a part of optic disc . In order to solve the problem this method is based on a balloon snake model, unlike other active contour models, such as snake or level set, which use gradient information to make the contour stop at the edge of disc . Thus, those methods require the initial contour to be near the true optic disc boundary . In this paper, we apply a fuzzy c means method to exclude those noises and keep the edge texture near the boundary . Then we choose the image texture rather than gradient to do segmentation, and the initial contour of the snake is a small circle in the disc region to avoid ppa region . Comparison with results from same database by other methods can also be found in this section . The optic disc segmentation methodology of this paper can be divided into 3 main steps: (a) optic disc location, (b) vessel removal, and (c) optic nerve head segmentation . We use the template matching to find the estimated disc region and then apply a circle hough transform to correct the center and find an estimated disc radius . Since the appearance of the od may vary significantly due to retinal pathologies, but the optic disc is the brightest feature of the normal fundus and shape of the optic disc is approximately vertically slightly oval (elliptical), a lot of templates have been proposed in to find the optic disc . In this paper, we adopt the template from lowell et al . As shown in figure 2 . In our dataset then the pearson correlation coefficient is used to measure the correlation between the intensity channel from hsi color space subimage and the template in general:(1)cij=x, yfx, yfmtxi, yjtmx, yfx, yfm2x, ytxi, yjtm2,where tm is the mean intensity values of the template, which need to be calculated only once . The method is robust to small exudate and can fast locate the optic disc . In the next step a circle hough transform hough transform can be used to find the circular shape with fixed radius in the edge image of the fundus . An estimated disc center which is detected in the template matching step is selected as the region of interesting center . Canny kernel edge detection with a threshold value of 0.4 is applied to the roi to remove weak boundaries and noises . Finally the hough transform is applied to the edge pixels in the edge map to accumulate evidence of circles with fixed radius r in the image . In this paper, the radius is ranging from 140 to 230 with a step size of 15 . The circle with the highest magnitude of evidence is chosen as the optic disc . Large blood vessels extending from optic disc may influence the precise edge of the optic disc . We also try to use morphological closing operation and mean pixels replacement method . In the morphological closing approach, a disc with radius of 15 is chosen as the structuring element, while in the mean pixels replacement approach the vessel mask pixel value is replaced by the mean value of the neighbor region . We apply above three vessel removal methods and the results are shown in figure 4 . The advantage of morphological closing operation is that you do not need to segment the vessel first but it destroys the image texture and makes optic disc boundary become blurred . After the vessel removal processing, there is little information about vessel left . In order to improve the segmentation result of the optic disc, we need to find those features which can distinguish the optic disc region and boundary . Moreover, in consideration of the ppa region which surrounds the optic disc, we need to find those features that can also have different values between the optic disc and ppa region . Schmid proposed texture feature filter banks which are rotationally invariant and are obtained by convolution with isotropic gabor - like filters . These filters combine different frequencies and scales together as follows:(2)fx, y,, = f0,+cosx2+y2ex2+y2/22,where is the number of cycles of the harmonic function within the gaussian envelope of the filters . Several filters are generated by taking different banks parameters (,) pairs . They are taken by ranges (2,1), (4,1), (4,2), (6,1), (6,2), (6,3), (8,1), (8,2), (8,3), (10,1), (10,2), and (10,3), 12 filters in all . The responses of the image from schmid filter bank are shown in figure 5(b). The rfs consists of 38 filters and is very similar to the leung - malik filter bank . It consists of first and second derivatives of gaussians at 6 orientations and 3 scales, 36 filters in all, and the last two filters are a simple gaussian and a laplacian of gaussian filter . To achieve rotational invariance, the authors get the maximum response filter bank from rfs by recording only the maximum filter response across all orientations for the two anisotropic filters . By measuring only the maximum response across orientations, the author reduces the number of responses results from 38 to 8 (3 scales for 2 filters, plus 2 isotropic). The results of applying mr8 filter to the image are shown in figure 6 . Cohen added a pressure term to make the model behave like an inflatable balloon or bubble that is trapped by strong edges but expands through edges that are weak relative to the pressure and smoothing forces . The energy functional of traditional snake is changed and a pressure force is added to the formulation:(3)eballoon=2us2dstension+22us22dsstiffness2usu dspressure+piudspotential, where the contour u depends on two parameters as below:(4)us, t = xs, t, ys, t.the third energy part in this equation is an isotropic pressure potential that controls the evolution of the area enclosed by the model . The energy of pressure is measured by size of the triangles region as in figure 7 . We can get the total energy change of balloon snake as below:(5)e=pu2us2+4us4+usu ds . At the limit of infinitesimal steps, the continuous descent equation is(6)ut=2us2tension force4us4stiffness forceuspressure forcepuimage force . Balloon snake is difficult to use when the edges of images are weak as the image force of balloon snake is still gradient . After lots of experiments we adopt the 3rd texture response from maximum response filter bank; see figure 8 . The third texture response is good for segmentation as the boundary information is clearer than gradient and the noise texture caused by optic cup and vessel is less notable . The energy equation of image force is as below:(7)eimagex, y=mrfilterix, y, where mrfilter is the maximum response filter banks and means convolution . Although this texture information is better than the image gradient for edge detection, there still remain some weak boundaries problems as shown in figure 9(b). We adopt the contrast - limited adaptive histogram equalization (clahe) to enhance the edge features . The clahe operation is directly applied to the image texture rather than the raw roi image, as the clahe is good at enhancing small objects such as edges, and the image texture is something like small objects . However, the noise texture which is formed by cup boundary is also clearer as shown in figure 9(d). This will reduce the segmentation result, as the initial contour is inside the optic disc and the balloon snake may stop at the false edges formed by cup boundary or the vessel in the nasal side . (4) noise textures remove . In order to remove all noises except the disc boundary, we need to exclude those noise features and retain the texture features near the boundary . In this paper, we proposed a clustering method to find the estimated edge region from the texture image by fuzzy c means approach . According to estimated radius from the circle hough transform, a larger estimated radius is selected to make sure the disc region is included in the new roi . In this paper, we add the estimated radius by 50 . We give up the image intensity space as there is serious inhomogeneous intensity in fundus image which will make wrong classification . They are the 1st response from the schmid filter bank and 5th and 7th response from maximum response (mr) filter bank . So every element point has three features; then they are classified into two clusters by fuzzy c means . After the classifications, the region is divided into two groups as shown in figure 10(b). The proposed knowledge - based clustering is based on the assumption that the background region is larger than estimated edge region . The max region is selected as the estimated background region, but there are still some holes in the background due to the cup boundary or the faked vessel edge . In order to remove the noise inside the background region, an open operation is applied to the background region as shown in figure 10(c); this step will link the edge region; then we choose the opposite region, the estimated edge region, and select the max connect region as the estimated edge region . After this operation, small faked edge region is excluded from the edge region as shown in figure 10(d). After we get the edges mask, we need to remove those noises features in the optic disc . Based on the background mask from the preview step, we cannot simply assign 0 or 255 to the background region as it will produce a local maximum or minimum between background region and other regions which makes the snake contour stop . In order to make the contour smooth cross the background region, an inpainting approach is employed to remove the texture noises . So in this step an inpainting operation is employed to the image again to remove the vessel edge and background region which is detected in the previous step . The final result of texture enhancement and texture noises removal is shown in figure 11 . (e) the deformable balloon contour . In this paper, a circle with half of the estimated radius is adopted to make sure that the initial contour is inside the optic disc . Moreover, if there is a ppa region near the disc, this contour will stop near the disc boundaries as long as there exists difference between the ppa and disc region . The database used in this paper is from high - resolution fundus (hrf) image database . The database contains 15 images of healthy patients, 15 images of patients with diabetic retinopathy, and 15 images of glaucomatous patients at the moment . There are often some large exudates or bleeding in diabetic retinopathy images which make it difficult to locate the disc . In this paper, we choose 15 images of healthy patients and 15 images of glaucomatous patients from this database . The manual optic disc mask which is used as golden truth was collected by one ophthalmology expert from he eye hospital, shenyang . In this paper, we adopt the dice coefficient to quantify the performance of the optic disc segmentation; the dice coefficient is defined as follows:(8)dc=2area(ab)areaa+area(b). A dc value of 1 indicates a perfect segmentation result with the ground truth, and a small dc value indicates a bad segmentation . The chan - vese (cv) and an improved approach, local gaussian distribution model (lgd), are state - of - the - art level set segmentation methods and they are employed to segment the optic disc . Although both of them are not sensitive to the initial contour, we make the initial contour be a circle with radius smaller than the estimated disc radius from the circle hough transform . However, we find that the cv and lgd are not suitable to do disc segmentation as shown in figure 13 . In cv model, the image intensity is used as energy to find an edge which makes the sum of inner and external energy be the minimum . This method often fails in optic disc segmentation because of inhomogeneity as shown in figure 13(b). This image has intensity inhomogeneity where the left part is brighter than the right part . So the cv model makes some bright region located in the left optic disc as a part of the optic disc . While the lgd model is designed for handling image inhomogeneity problem, this method efficiently utilizes local image information rather than the global information such as cv model . We can see from figure 13(c) that there are lots of small dark or bright regions near the optic disc which is caused by the light illumination or reflex . After we apply lgd to the roi image, we find that it has the problem of oversegmentation as the energy minimization is achieved by an interleaved level set evolution and estimation of local intensity means and variances in an iterative process . So we give up these two methods and regard the edge based active contour model . We compare our proposed method with traditional snake and another texture cluster methods which is fuzzy c means cluster method . And in the texture cluster segmentation method, we adopt the 5th response from mr8 and 1st response from schmid filter bank of the red channel values of image, 2 features for the fcm input . Then we apply morphological operation to the larger region, and an ellipse fit is applied to get this region boundary . As shown in figure 14, we can see from the image that the snake model often fails to detect the true edge of disc because of the fuzzy edge and ppa region . The results often include the ppa region as a part of disc . To sum up, our proposed segmentation method is more robust to the snake, as you can see from figure 15 . In the box - and - whisker figure, the proposed balloon snake is better than other two approaches, and most of segmentation dice coefficient is 94% as shown in table 1 . Although the mean dice coefficient of fcm texture cluster approach is almost the same as snake model, most of coefficient is distributed more than 90% . We utilize the matlab (mathworks, inc ., natick, ma) and mathematica (wolfram research, champaign, il) to implement our method mentioned in this paper . It costs about 2 minutes to process one image on pc (i5 intel cpu and 4 g ram). In this paper, we focus on automatic segmentation of the optic disc based on 2d fundus image . Template matching and circle hough transform are used to locate the optic disc and find an estimated circle radius . In the vessel erasing step, we use inpainting approach to remove vessel which is better than the closing operation and mean neighbor replacement . Our proposed optic disc segmentation method is based on image texture and active contour model . We use the balloon snake model which makes the contour expand like balloon from the inner optic disc . This method can avoid the ppa region outside the disc boundaries and is less sensitive to the initial contour, as the initial contour of normal snake model and gvf model are recommended near the true disc boundaries . The main problem of this balloon model is the texture noise which is caused by faked vessel edges, and sometime the cup is obvious in the red channel which will make this contour mistake the cup boundary as the disc edge . So we proposed a cluster based approach to remove those texture noises in the disc . The results from our proposed approach are better than the snake model and fuzzy c means cluster approach . The optic nerve head segmentation results show that the proposed method is able to achieve better segmentation accuracy for optic disc comparing to other existing methods . The works of this paper as a whole can be used in retina image processing system, which is highly desirable for large - scale screening programs . However, our proposed method is sensitive to optic disc location approach and vessel segmentation results; in the future we need to adopt a better optic disc location method which can handle large exudate in the diabetic retinopathy images and make our method more robust to handle inaccurate vessel segmentation results.
Tehran with approximately twelve million citizens is the largest city of iran, with most of its population being immigrants . This study was conducted from february 2010 to september 2011.the participants were defendants with a judicial order of evaluation for mental status and criminal responsibility . These participants were evaluated by two forensic psychiatrists with a doctoral - level education in psychiatry and an experience of twelve - month training in forensics while not being aware of the purpose of the study, using american psychiatric association's diagnostic and statistical manual 4th edition text revised (dsm - iv - tr) criteria for mental disorders . Malingerers were diagnosed according to misattribution of symptoms, unusual hallucinations, distortion of notifications, fictions of complaints, and exaggeration of their symptoms . Subjects excluded from our study were those without definite diagnosis of at least one forensic psychiatrist, subjects with positive screening tests of alcohol and drug at the time of arrest, subjects with a history of previous imprisonment, illicit drug abuse, determined mental, neurological or psychiatric problems and those who received psychoactive medications . Finally, forty five malingerers were included and assessed in another interview by the head of forensic psychiatry center . Because of the legal order to evaluate mental status of the subjects and intentional faking symptoms to deceit forensic psychiatrists, informed consent was not obtained from the fakers taking part in our study . Ethical approvals for this study were obtained from tehran university of medical sciences research ethics committee prior to the launch of the study . Demographic variables were obtained from family reports (if accessible) and court reports about educational level, ancestry, marital status, and employment . Symptoms were documented using an absolute list of 31 symptom presentation variables after a research pre - testing . The captions of manifestation used to construct the areas were (1) mood and affect, (2) thought content, (3) thought process, (4) perception, (5) cognitive function, and (6) behavioral disorder symptoms (14).motivations of malingering were assessed according to the court order; and financial gain or relief from responsibility in a felony background was concluded.the categories of charged with felonies versus not charged with misdemeanors were analyzed in 22 contingency tables with fisher's exact test due to small numbers of the felonies . The level of statistical significance was set at p <0.05 . Tehran with approximately twelve million citizens is the largest city of iran, with most of its population being immigrants . This study was conducted from february 2010 to september 2011.the participants were defendants with a judicial order of evaluation for mental status and criminal responsibility . These participants were evaluated by two forensic psychiatrists with a doctoral - level education in psychiatry and an experience of twelve - month training in forensics while not being aware of the purpose of the study, using american psychiatric association's diagnostic and statistical manual 4th edition text revised (dsm - iv - tr) criteria for mental disorders . Malingerers were diagnosed according to misattribution of symptoms, unusual hallucinations, distortion of notifications, fictions of complaints, and exaggeration of their symptoms . Subjects excluded from our study were those without definite diagnosis of at least one forensic psychiatrist, subjects with positive screening tests of alcohol and drug at the time of arrest, subjects with a history of previous imprisonment, illicit drug abuse, determined mental, neurological or psychiatric problems and those who received psychoactive medications . Finally, forty five malingerers were included and assessed in another interview by the head of forensic psychiatry center . Because of the legal order to evaluate mental status of the subjects and intentional faking symptoms to deceit forensic psychiatrists, informed consent was not obtained from the fakers taking part in our study . Ethical approvals for this study were obtained from tehran university of medical sciences research ethics committee prior to the launch of the study . Demographic variables were obtained from family reports (if accessible) and court reports about educational level, ancestry, marital status, and employment . Symptoms were documented using an absolute list of 31 symptom presentation variables after a research pre - testing . The captions of manifestation used to construct the areas were (1) mood and affect, (2) thought content, (3) thought process, (4) perception, (5) cognitive function, and (6) behavioral disorder symptoms (14).motivations of malingering were assessed according to the court order; and financial gain or relief from responsibility in a felony background was concluded.the categories of charged with felonies versus not charged with misdemeanors were analyzed in 22 contingency tables with fisher's exact test due to small numbers of the felonies . Characteristics of the defendants and statistical results comprising the two groups are demonstrated in table 1 . No significant difference was observed between groups in sex, provenance, occupation and marital status, but the misdemeanor group was fairly less educated and contained proportionally more married urban subjects . The mean age of the 45 defendants was 40.31 15.12 years with the range of 19 to 76 years . Participants charged with criminal accusation had significantly lower mean age than other non - criminal defendants . There were 44 immigrants (28.9% rural, 68.9% urban), and only one participant was citizen of tehran . Twenty four had nine years of education or less, three had bachelor's degree or higher . Sixteen were single, of whom only one was divorced; and 20 were unemployed . Misdemeanors were comprised of one domestic disturbance, three illicit drug trafficking, four robberies, ten claims on insurance policies based on a traumatic event (especially a minor head trauma), and 20 fraud and felonies including three sexual assaults and four violent offences and murders . Background information of felony and misdemeanor groups symptoms were assessed in six significant domains (14). Follow - up studies incorporated fisher exact tests for each dependent variable reported in table 2 . A statistically significant increase in memory function problems was demonstrated in the misdemeanor group regardless of time period . N value and percentages don't equal total of columns since some subjects had fake multiple symptoms . Differences among the other 25 (including seven subgroups) and the six captions of variables were not statistically significant . Table 3 demonstrates the concordant occurrence of feigned symptoms according to the denunciatory group of malingerers . Concordant occurrence of the caption variables with regard to concordant occurrence of two symptom domains (14), statistically significant associations were observed between the thought content and perceptual symptoms for felonies . Misdemeanor cases had significant correlations between moods & affect and thought process symptoms, mood & affect and behavioral symptoms as well as cognitive function and behavioral symptoms . Regarding the other symptoms in the present study, we analyzed the characteristics of the most relevant symptoms of malingering among the iranian population . In iran, no traditional use of tests as a part of a standard forensic psychiatric examination has been established; furthermore, the validity and reliability of such tests are not studied for iranian population; thus, there has been no indication of applying such tests . However, longitudinal assessments made by forensic psychiatry experts with appropriate information for determining the diagnosis can be applicable in most cases (15). In this study, there were 45 participants with the diagnosis of malingering confirmed by two separate psychiatric interviews from february 2010 to september 2011 . The mean age of participants was 40.31 15.12 years which is higher than reported by american studies (1618). In our study, we had no restrictions on major offenses, as investigation of more than a half of all cases was performed on fraud or plaintiffs who claimed psychotic problems to terminate financial contracts . The male to female ratio in our study was 41:4, which reflects male predominance as female malingering of psychotic symptom is less common in our study group compared to other studies (17, 18). This could be explained by sample selection from inmates of province county jails in american studies compared with some subjects in our study who were referred for outpatient evaluation . On the other hand, none of the women in our study were involved in complex felonies or criminal offences; and social rejection of psychotic patients, especially in iranian women, probably leads to avoid resorting simulations of mental disorders even in prisoners . A relatively low education level of 7.25 years was observed; namely, in the seven criminal cases the education level was 10.8 years and in 38 misdemeanors, it was 6.6 years . In american malingerers who were criminal defendants referred for evaluation of competency to stand trial or criminal responsibility (12), the mean level of education was 10.4 years which is similar to the seven criminal cases in our study . However, in another study carried out on adult inmates from maximum and minimum security jails in a rural southeastern county of the united states, malingerers had lower level of education compared to the genuine patients (18). The employed to unemployed ratio in our study was 25:20 . Considering that the unemployment rate in iran is about 11 percent, we could conclude that more unemployed individuals may be involved in criminal acts or attempt to commit fraud, and due to lack of adequate intelligence and genius, they often simulate psychotic symptoms poorly . The majority of faked psychiatric symptoms are more frequent in urban population, with a rural to urban ratio of 13:32 . This could be explained by less instability of social status in rural cases after immigration, probably because they are willing to do even hard jobs and live with fewer benefits . In comparison, in an american multi - center study examining base rates of malingering, differences were not significant in several locations (13). Typically, the clinical symptoms and signs obtained in the psychiatric interview should be congruent and lead to a significant diagnostic approach . For example, perceptual symptoms such as auditory hallucinations and some delusional thoughts usually indicate schizophrenic disorders . Furthermore, mood symptoms, behavior disturbances and psychomotor activities are the most frequent symptoms in mood disorders . Mood is a subjective symptom, but behavior disturbances or psychomotor activities are detected by psychiatrists during a psychiatric interview . A person with delusional disorder usually does not have a history of prominent auditory hallucination, and also, severe bizarre behaviors combined with depressed mood without other psychotic symptoms are rare, but faked symptoms do not usually introduce a known psychiatric diagnosis . For instance, a person may claim that he / she suffers from auditory hallucination but no abnormal behaviour may be observed . Some malingerers show mood disorder symptoms but cannot introduce congruent behaviour within psychiatric examination . The most common feigned psychotic symptoms in our participants were behavioral (77.8% of cases who charged with felonies or misdemeanors), mood & affect (68.9%), and cognitive function symptoms (60%). In another study focused on comparison of forensic and non - forensic malingerers, the most common symptoms feigned by forensic malingerers were medical syndromes or cognitive impairments (11), while the most common deceitful symptoms in criminal defendants referred for pretrial and outpatient evaluation of competency to stand trial or criminal responsibility were perceptual and auditory hallucination, followed by behavioral symptoms in cornel and hawk study (1989). As the role of a previous consort with other vicious people or prisoners and learning psychotic symptoms from video films is more prominent in foreign studies, defendants may be familiar with the symptoms of mental disorders (2, 19). However, in our study we excluded subjects with history of imprisonment, illicit drug abuse, determined mental, neurological or psychiatric problems to make a relatively pure sample of participants without psychotic symptoms . In our study, participants often used simple methods such as refusing to answer questions during interviews, having unusual or bizzare behavior, or claiming depression or memory loss . In the two of seven criminal cases, defendants claimed obsession and, at the same time, hallucination and pseudo hallucination . From 38 misdemeanors, 39.5% feigned mood & affect beside thought process disorder symptoms; 55.2% feigned cognitive function and mimic behavioral disorder symptoms; and 65.8% simulated mood & affect and behavioral disorder symptoms concurrently . The main restriction of this study was unavailability of standard tests to use in a control group . Therefore, the conclusions may not apply to other situations such as a forensic hospital, where the array of patients may be different . In general, it seems that differences among presenting symptoms among different offenses may not be useful in detecting malingering . However, unusual dual symptom imitation may be useful, particularly when standard tests are not performed.
Studies have reported the prevalence of psychological distress and psychiatric comorbidity in patients with epilepsy [14]. Seizure activity, poor seizure control, and kindling - like phenomena may be causes of depression . Sometimes depression may be a side effect of anticonvulsant medication [5, 6]. Psychosocial factors predisposing to depression in people with epilepsy include adjustment difficulties, limitations and restrictions in social settings which the disorder imposes, as well as the unpredictable nature of the seizures and the associated feelings of helplessness and loss of control over one's life . The unpredictability limits mobility, hinders the work and education, and may lead towards the psychological disorders . These issues gain more importance when there is limited knowledge about the disorder especially in the case of developing countries with low level of education and poor health care system . Three quarters of the 50 million people with epilepsy live in developing countries and 94% of them are untreated . This large percentage raises many questions related to the treatment facilities as well as the societal attitude towards the disorder . Several studies from developing countries have reported more negative attitudes and stigma about epilepsy as compared to the developed countries [810]. Overall prevalence of epilepsy is estimated to be 9.99 per 1000 population with the highest rate in young adulthood . Here the burden of epilepsy is twice as high in rural areas (14.8/1000) as compared to urban areas (7.4/1000). This high rate of pwe in rural areas increases the need to investigate more about the disorder with a focus on cultural variation and psychosocial variables to help in the treatment and management . Factors associated with depression include lack of occupation, the presence of an underlying disabling condition (with treatment), and the severity of epilepsy [1417]. In one study by the authors of [18, 19] it was found that women with epilepsy of childbearing age are at high risk of depression . Sometimes depression may be a side effect of anticonvulsant medication or from combinations [5, 6]. A number of researches have reported the comorbidity of anxiety along with depression in patients with epilepsy [2022]. The most common emotional responses of people with epilepsy include fear of the unexpected seizure, humiliation after a seizure, particularly if incontinence occurs, and feelings of alienation at work and social situations . These emotional responses are not limited to cognitive impairments, but also affect the social domain of functioning . Since the person who has seizures has no control over other people's reactions during a seizure, therefore they prefer solitary activities or reduce their social contacts . It has been reported that people with epilepsy have feelings of low life satisfaction in the areas of employment, peace of mind, and social relationships . In one study it was found that the family dysfunction contributes to psychiatric, emotional, and behavioral problems, to be worse in those with epilepsy than in the control group of patients with other chronic diseases . A research conducted by aziz, akthar, and hasan from pakistan reported that pwe face difficulty performing activities of daily living and find it hard to make decisions about whether to marry or to have children . It is therefore important to consider the above - mentioned clinical (seizure frequency) and demographic variables (gender, education, occupation) in assessment of psychological and social well - being in epilepsy . The existing literature suggests that in pakistan studies on epilepsy are mainly limited to distribution, prevalence, etiology, and biological aspect of the disease . Therefore it is necessary to assess the psychological, social, and behavioral aspects of epilepsy so the improvement can be made in the management plan keeping in consideration the cultural aspects . Based on the above - mentioned literature the following hypotheses have been formulated . There exists a significant relationship between seizure frequency and psychological distress . In this study, a total 50 individuals (31 males and 19 females) with epilepsy were selected as participants . Patients with a confirmed diagnosis of epilepsy were included with the help of psychiatrists or neurophysicians . Patients who had a diagnosis of epilepsy for more than 1 year were included in the sampling frame . Participants with tonic - clonic seizures and partial complex seizure were selected because these types are common among adults with epilepsy in pakistan . Patients were excluded from the sampling frame if they had serious physical or mental limitations that did not allow them to complete the questionnaire . In addition, patients with psychiatric or neurological disorders that would impair judgment or impact quality of life beyond the effects caused by epilepsy, including mental retardation, stroke, head injury, brain tumor, and cerebral palsy, were excluded from the sampling frame . Participants were divided into three categories: mild, moderate, and severe which is based on the number of seizures over the past 6 months . Individuals who had only one or no seizure in the past 6 months were categorized as being in the mild seizure state . Patients with one seizure in the past 3 months were included in the moderate state . Patients who had one or more seizures during the past month were categorized as being in the severe or uncontrolled state . The demographic information included name (optional), age, gender, education, occupation, area of residence, and monthly income (personal or family). The epilepsy - related variables included current medications and number of seizures during the last 6 months . General health questionnarie-28 (ghq-28) developed by goldberg and hillier in 1979 is wildly used instrument in detecting the psychological distress among clinical and nonclinical population . In pakistan this questionnaire has been translated in urdu and this urdu version has found to be valid and reliable . There are four dimensions in assessing the psychological distress . These are each item has four response choices ranging from the scoring of ghq-28 followed the test manual recommendations (0, 0, 1, 1) using the binary scale method . A cut - point of 5 is used to detect psychological distress . Higher score on ghq-28 subscales the scale is a valid and reliable instrument; in the present study the alpha reliability coefficient was computed to be 0.80 . The internal consistency reliability levels for the subscales is reported to be 0.74 (somatic complaints), 0.75 (anxiety / insomnia), 0.72 (social function), and 0.76 (depression) in this research . The data was collected from outdoor patients department at neurology and psychiatry departments of three government hospitals from rawalpindi and islamabad . Further questions related to day - time activity, seizure effects, medication effect, and treatment options tried before were also catered . After the data collection results were analyzed by using statistical product for social sciences (spss v.17). Descriptive (mean, standard deviation, cross - tabulation) and inferential statistics (independent sample t - test, multiple regression analysis) were computed and will be discussed in section 3 . The sample comprises of 50 adults including 31 men (62%) and 19 women (38%). Majority of the participant had education up to matriculation (n = 23, 46%). Thirty - eight percent of patients were in mild seizure category, 26% fall in moderate seizure category, and 36% had severe seizures . Furthermore no significant differences are present in seizure frequency, mean age, marital status and education . So epilepsy patients did not show the symptoms of anxiety like insomnia, sleep disturbances, and irritability as reported by the participants . There is need to assess anxiety particularly related with epilepsy, its characteristics and medication side effects . In regression seizure frequency is found to be a significant predictor of psychological distress (b = 1.20, p = .001) accounting for 42% variance in psychological distress (table 4). The results further indicate that education is also found to be predictor for psychological distress (b = 1.19, p = .01). Gender, occupation, and age are found to be non - significant in predicting psychological distress among patients with epilepsy . In order to identify gender differences a further analysis comprising cross - tabulation was also computed to see gender differences (see table 7), and results show that 68% female and 71% males with epilepsy experience psychological distress . Furthermore a descriptive analysis of mean scores was also computed to see the gender differences in psychological distress and the subscales of ghq-28 . As the table shows females have higher mean score on the total as well as all subscales except somatic complaints . The highest difference is reported on social dysfunction subscale which indicates high level of psychological distress in females as compared to males . The demographic profile of the participants has revealed that most of them were experiencing severe or uncontrolled epilepsy (n = 18, 36%). It has also been identified that none of female participant was employed (table 2). This condition is cultural specific because in pakistan it is preferable for women to stay in their homes, so they are totally depending on their families for financial support and long - term treatment of the disorder . It is also evident from the results of table 6 where female as compared to males had high mean scores in social dysfunction . The social dysfunction in people with epilepsy has increased the risk of accidents and unexpected sudden death especially in the communities where a great deal of social activities takes place around the fire . It is further reported that pwe face difficulty in performing activities of daily living and find it hard to make decisions about whether to marry or to have children . This social dysfunction is further linked to psychological morbidity, that is, presence of psychological disorders . Factors associated with depression include lack of occupation, antiepileptic medications, and the severity of epilepsy . Psychosocial factors predisposing to depression in pwe include adjustment difficulties, the limitations and restrictions which the disorder imposes, as well as the unpredictable nature of the seizures and the associated feelings of helplessness and loss of control over one's life . The present research has also investigated the relationship between these clinical and demographic variables with psychological distress including depression and anxiety (tables 3 and 4) which has supported the first hypothesis . In the present study participants have reported that they experience severe headache, drowsiness and lethargy for one to two days after a seizure . Society's lack of understanding of epilepsy is a psychosocial burden that is strongly felt, and many people with epilepsy try to keep their condition a secret . Feelings of anger, frustration, embarrassment, and vulnerability may develop as a result of society's attitude towards the illness . Other demographic variables leading to psychological distress include lack of education, unemployment, and age . The present study has also included regression analysis for these variables and the results indicate that education has inverse relation with psychological distress (table 4). Patients with epilepsy in pakistan do not appear to be highly stigmatized, but their education and grades are affected by the disorder . They have difficulty performing activities of daily living and find it hard to make decisions about whether to marry or to have children . Previous researches have reported that employment is a significant predictor of psychological distress [24, 29]. Table 1 also shows that even though 46% of the participants have education up to matriculation, however the unemployment rate is high (42%). There are number of factors related to this high unemployment rate . In one research by chung et al . 31% of respondents from the community believed that pwe should not be employed in jobs as other persons are . Depression is also affected by a number of demographic variables . In one study by beghi et al . It was found that women with epilepsy of childbearing age are at high risk of depression . The similar finding has been generated in the present research in which 13 out of 19 females with epilepsy have reported to suffer from psychological distress . It was also found that none of these women was employed (a cultural specific phenomenon) with a slightly high number of unmarried females (74%) as compared to males (71%). These gender differences reveal that women carry with themselves not only a diagnosis of epilepsy but relatively low position in the society especially with reference to decision making in major life events . In pakistan it is also evident from the present research where 42% unemployed participants are solely dependent on their families for maintenance and treatment expenditures . It has also been reported in one community - based research in karachi, pakistan that pwe do not appear to be highly stigmatized . The results further indicate that seizure frequency is the most significant predictor in epilepsy (table 4). Furthermore it is also reported that quality of life can be improved by controlling seizure frequency [15, 32]. It has also been reported that treating comorbid depression in epilepsy increases psychological well - being . Therefore it is important to assess psychological distress in epilepsy and to incorporate this component in treatment plan . On the basis of present study findings it can be concluded that seizure frequency is a significant predictor of psychological distress . Increase in seizure can also increase psychological problems such as depression and anxiety . Furthermore the social dysfunction and somatic complaints are also reported . It can be better understood in realm of physical as well as in psychological, social, and behavioral domains . This finding may help in management and rehabilitation of patients with epilepsy in developing countries . The finding can be used by health care providers and policy makers to propose interventions such as improved access to mental health care, job training, and self - management programs to improve health outcomes in people with epilepsy especially by using the knowledge of indigenous practices and social structure . So by understanding the relationship between clinical and psychosocial variables in epilepsy a good management plan can be devised for the patients with epilepsy with a focus on social and gender differences . The present research can also help to increase the awareness and to lower the stigmatization related to epilepsy . This is a small - scale research focusing on four major domains of psychological well - being . It is therefore suggested to conduct research on larger sample for result generalization and to cooperate other factors such as age at onset of illness, treatment mode, and perceived social support . The issues highlighted by this research are the gender differences especially in terms of work status and difference in depression . An in - depth analysis is required to see the economic hardships faced by the patient and its correlate with their social status . Moreover this research will be beneficial for health professionals to consider the societal attitude with reference to stigmatization, as this present research has included only those patients coming to hospitals not the traditional healers.
The incidence of colorectal cancer is one of the highest malignancies . In netherlands alone, colorectal cancer is diagnosed in 12,000 patients annually and it is the second most frequent cause of death due to malignancies . In an effort to improve the standard of care for these patients, new techniques have been introduced over the years, such as laparoscopy and tme surgery [3, 4]. Recently, much attention has been given to patient volume of both the hospital and the individual surgeon . Publications have shown that a high - volume surgeon operating in a high - volume hospital leads to an improved short - term outcome such as a lower number of adverse events, shorter hospital - stay, lower postoperative mortality, and cost reduction . Furthermore, a number of studies have reported increased long - term survival when patients are treated in high - volume centers . However, the relationship between operative volume of the surgeon and long - term outcome remains unclear . We therefore conducted the current study to evaluate survival rates of patients with colorectal cancer following a procedure performed by high - volume surgeons compared to low - volume surgeons . The rijnland hospital is a teaching hospital in leiderdorp, netherlands, serving approximately 200,000 people . For the current study a retrospective analysis was conducted from our prospectively collected database including all colorectal cancer patients who underwent surgery in our hospital between 2004 and 2011 . Eight hundred and twenty - four patients underwent a colorectal procedure between 2004 and 2011 . For our study we used the same inclusion criteria as the national web - based registry for the surgical treatment of colorectal cancer in netherlands: the dutch surgical colorectal audit (dsca). Patients were excluded in case the procedure was performed for metastatic disease following previous surgery (n = 13), in case the primary tumor could not be resected (n = 9), or when the pathology report showed a different type of tumor than an adenocarcinoma (n = 28). After applying these exclusion criteria our study population of 774 patients consisted of a homogenous cohort . In order to qualify as a high - volume surgeon a cut - off point of 25 colorectal resections per year, averaged over the study period, was chosen based on recent studies [6, 913]. Taking this criterion into account for our analysis, 13 low - volume surgeons operated on 453 patients and four high - volume surgeons operated on 321 patients . Perioperatively, all patients received equal care using the colorectal enhanced recovery after surgery (eras) protocol [14, 15]. The data collected in our database were the patient characteristics, including the american society of anesthesiology- (asa-) classification; the intraoperative data (high - volume surgeon versus low - volume surgeon); and the postoperative data, including the tnm - stage, resection margins, length of hospital - stay, and adverse events . In case of an adverse event, the type (surgical or nonsurgical) and the severity were recorded according to netherlands' society of surgery standard [18, 19]. Follow - up took place in our hospital according to netherlands' society of surgery protocol . This protocol dictates that patients are seen in the hospital for follow - up by an attending surgeon every 4 months for the first 2 years and every 6 months for the years after, with a minimum of 5-year follow - up . In the current study patients were followed up for a minimum of 3 years . At each visit, an ultrasound and cea levels were performed . Also the iknl (integral cancer centre netherlands) was consulted in case a patient deceased, which provided us with the date and cause of death . For some patients the follow - up did not take place in our hospital, mostly due to relocation of the patient . In those cases we consulted the general practitioner and the hospital where the follow - up was taking place for survival data . It was possible to evaluate the 5-year disease - free survival (dfs) of 761 patients (13 patients lost to follow - up: 8 low - volume and 5 high - volume) and overall survival (os) of 772 patients (3 low - volume patients were lost to follow - up). For assistance with the statistical analysis, the department of statistics in our hospital and the leiden university medical center were consulted . Comparisons were made between the high - volume and the low - volume group for all variables: perioperative characteristics, disease - free survival, and overall survival . The -test and the independent sample t - test were used to determine the association between perioperative characteristics and volume (high - volume versus low - volume). Multivariate poisson regression survival models were used to determine the effect of volume on dfs and os . Variables in the univariate analysis that showed a significant association were then introduced into a cox regression multivariate model . Preoperative clinicopathological characteristics of the 453 low - volume and the 321 high - volume patients are shown in table 1 . The groups were comparable except the fact that a greater number of the low - volume patients had a higher asa - classification (p <0.001) and laparoscopic surgery was more frequently performed in the high - volume group compared to the low - volume group, 78% (n = 249) versus 59% (n = 266), respectively (p <0.001). The type of resection also showed a difference (p <0.001), largely caused by a higher number of abdominoperineal resections (apr) in the high - volume group . The significantly larger number of patients who received chemoradiotherapy as neoadjuvant regimen in this group can be explained by the higher number of rectal cancer cases (p <0.001). The intraoperative data are listed in table 2 . In the high - volume group, significantly less blood loss was observed compared to the low - volume group, 308 ml versus 547 ml, respectively (p <0.001). Also the conversion rate in case of laparoscopic surgery was significantly lower in the high - volume group (18% versus 27%, p = 0.01). The postoperative characteristics are listed in table 3 . A significantly more advanced tumor (t) stage (p = 0.03) and metastatic (m) stage (p <a larger median number of lymph nodes were harvested in the high - volume group, 15.3 versus 13.5 (p <0.001). In the high - volume group the median postoperative hospital - stay was lower compared to the low - volume group: 10 versus 13 days, respectively (p <0.001). No difference was seen between nodal (n) stages or resection margins and the number and neither did the severity of both surgical and nonsurgical adverse events show a difference in both groups . The 5-year dfs in the high - volume group was 66% compared to 48% in the low - volume group (p <0.001, figure 1). We performed a univariate analysis to estimate the effect of all variables on the dfs . The high - volume group showed a significantly increased dfs (hazard ratio (hr) 0566; 95% ci 0.440.74; p <0.001). The other pre-, intra-, and postoperative variables that showed a statistical significance for dfs in the univariate analysis are listed in the left half of table 4 . We then incorporated the statistically significant variables of the univariate analysis into a cox multivariate regression model to determine which variables remained as prognostic factors for dfs . Surgeons' volume showed to be an independent prognostic factor for dfs in favor of the high - volume surgeon (hr 0.739; 95% ci 0.560.99; p = 0.04). Other independent prognostic factors for a longer dfs were lower patient's age (p <0.001), lower asa - classification (p = 0.05), and a lower t (p = 0.04), n (p <0.001), and m (p <0.001) stage . We also analyzed if the time periods (20042007 versus 20082011) had an influence on dfs; however, neither in the univariate or in the multivariate analysis did this show significance . The patients in the high - volume group showed a 5-year os of 75% as compared to 54% for the low - volume group (p <0.001, figure 2). Similarly to what is described above, we performed a univariate analysis for os . The high - volume surgeon was significantly associated with an increased os (hr 0.495; 95%ci 0.350.69; p <0.001). The other pre-, intra-, and postoperative variables that showed a statistical significance for os in the univariate analysis are listed in the left half of table 5 . After incorporating the statistically significant variables of the univariate analysis into a cox multivariate regression model to determine which variables remained as prognostic factors for os, the high - volume surgeon did not remain significant (hr 0.731; 95% ci 0.711.68; p = 0.09). We also analyzed if the time periods (20042007 versus 20082011) had an influence on os; however, neither in the univariate or in the multivariate analysis did this show significance . The factors that did prove to be an independent prognostic factor were advanced age (p <0.001), higher asa - classification (p = 0.01), and higher n (p <0.001) and m (p <0.001) stage which showed to have an independent negative influence on os . Laparoscopic surgery appeared to be a positive independent prognostic factor for os (p <0.001). In the current analysis we found that a high - volume surgeon is an independent prognostic factor for increased dfs for colorectal cancer surgery when compared to a low - volume surgeon . However, high - volume surgery did not remain as an independent prognostic factor for os in the multivariate analysis . Although increased dfs is an important outcome in research, ultimately a longer os is what is most desirable in medicine and what is important to the patient . Possibly in a larger cohort of patients we may show an increased os in the future since os did show to be significantly increased in the high - volume surgery patients in the univariate analysis . Previous studies have been performed to investigate possible variables of short - term and long - term outcomes following colorectal resection for malignancies . These outcomes depend on numerous patient-, surgeon-, and hospital - related variables [5, 9, 2227]. While the patient - related variables are difficult, if not impossible to adjust, efforts aimed at improving the perioperative care have been shown to have positive impact on the postoperative outcome . Some of these efforts include the administration of preoperative antibiotics, maintaining normothermia during surgery, and implementing an eras protocol [15, 16, 2830]. Another approach that has been shown to be effective is implementing high - volume surgery of colorectal procedures . Recent studies have shown an improved short - term outcome, when a high - volume surgeon performed the procedure [9, 23, 24, 31]. Studies reporting long - term effects for high - volume colorectal surgery, however, have shown less unanimous results [12, 26, 27, 32, 33]. Although high - volume surgery showed a significant relationship towards an increased os in the univariate analysis, it did not remain as an independent prognostic factor for os in the multivariate analysis . When looking at the difference in os between the high - volume group and the low - volume group (figure 2) in the univariate analysis, it seems likely that, with either a larger patient population or an increased median follow - up time, this observed difference could also become statistically significant in the multivariate analysis . This would of course be an important outcome for our patients, as increased os is even more relevant than an increased dfs . Our findings are in agreement with the outcomes of studies of low - volume surgical procedures, such as esophageal and pancreatic cancer surgery, in which it has been shown that the surgeon's caseload is an important predictor for outcome [34, 35]. Also support for our assumption of an increased os can be found in the article by rogers jr . Et al . Who showed that, in a group of 26,644 patients with a median follow - up of 6 years, those who were operated upon by a high - volume surgeon had an increased os following colorectal cancer . The same is seen in the cochrane analysis in which an improved survival is reported for both the high - volume surgeon and the high - volume hospital . However, not all studies reporting survival after high - volume surgery for colorectal cancer are in agreement . [27, 32, 33, 36, 37], showing that more research in this field is required before any definite statements can be made regarding the volume an individual surgeon should perform . Recently published literature has increasingly published a similar observation showing an improved os following laparoscopic colorectal surgery when compared to open colorectal surgery [3841]. It has been suggested that this improved os observed in the recent years is mostly caused by the increased experience with the procedure together with technical and procedural advances . Although this cannot be attributed to the implementation of high - volume surgeons alone, the fact that dfs remained as an independent prognostic factor in the multivariate analysis shows that high - volume surgery is attributed to improved survival . In a previous report, renzulli et al . Observed a similar increase in dfs for high - volume surgery . However, in the multivariate analysis this did not remain significant showing that the difference in dfs seen between both groups cannot be explained by the timing of the surgery and the on - call surgeon . Apart from the increased dfs in the multivariate and the increased os in the univariate analysis, a number of perioperative variables also showed statistical significance in favor of the high - volume surgeon . In case a high - volume surgeon performed a laparoscopic procedure, a significantly lower number of conversions were observed . Furthermore, intraoperative blood loss was significantly less in the high - volume group and a greater number of lymph nodes were harvested leading to a more accurate staging . A decrease in postoperative adverse events has been reported in case the operation was performed by a high - volume surgeon [9, 11]. Possibly, underreporting of adverse events may have taken place in our study: the number of days a patient was admitted to the hospital following surgery by a high - volume surgeon was significantly lower, suggesting a quicker and uncomplicated recovery . Therefore, in our hospital, high - volume surgery does not only improve the dfs and possibly os but also improve short - term outcome the low - volume group consisted of more patients with a higher asa - classification and a higher tnm - stage [16, 17]. The early drop of dfs seen in the low - volume patients that is demonstrated in the kaplan - meier curve is most likely due to the more advanced disease in this group (figure 1). For this reason a multivariate analysis was conducted to correct for these differences in patient population . Even after this correction high - volume surgery remained as an independent factor for dfs . Rectal cancer resections were performed more frequently by the high - volume surgeons, which could have caused fewer postoperative complications due to increased experience . On the other hand, one could have expected more complications following the neoadjuvant radiotherapy and chemotherapy in this group, but this was not the case in the statistical analyses . In conclusion, the current study shows that in our hospital high - volume surgery is an independent prognostic factor for increased dfs following surgery for colorectal cancer . Although high - volume surgery also significantly improved os in the univariate analysis, it did not remain statistically significant in the multivariate analysis . It is possible that, with either more patients included or a longer follow - up time, this observed difference will also become statistically significant for os . To our opinion, introducing high - volume surgeons will provide better perioperative care for patients suffering from colorectal cancer resulting in both improved short - term and long - term results.
Bruxism, also known as tooth grinding, is a distinct type of oral habit possessing rhythmic activity of the orofacial muscles causing a vigorous contact between the surfaces of the teeth . Although, there is no unanimous agreement on the definition of bruxism, some scholars defined it as a repetitive jaw - muscle activity characterized by clenching or grinding of the teeth and/or by bracing or thrusting of the mandible . Bruxers are usually divided into the day time bruxers (awake bruxers) and the night time bruxers (sleep bruxers). Awake bruxism is a day time parafunctional activity of the muscles of mastication and it is reported that usually the clenching of the teeth and grinding is loud enough for the partners to notice . The dental health care practitioners are particularly concerned with bruxism because of the detrimental effect it has on the oral hard and soft tissues . Though there have been quite a few studies conducted on bruxism which portray its link with various factors, the confirmed cause of bruxism has not been determined till date . In the early years, it was thought to occur due to occlusal interferences or problematic anatomy of a person; however, recent studies have shown specific factors to be linked to bruxism . In one study conducted among the study sample of a developed nation, bruxism was reported to be associated with psychological stress, smoking, genetic factors, emotional conditions, and mental instability . Some researchers have also shown its association with other parasomnias such as hallucinations and other violent injurious behaviors . The modifiable risk factors reported to be closely linked to bruxism are coffee, tobacco, and alcohol . All of these are known to have a high impact on the central nervous system of an individual . Studies have reported a high use of some of these modifiable risk factors such as tobacco and coffee by people of all age groups in the jazan region of saudi arabia . Apart from these risk factors, the local population in this region of saudi arabia is also seen to be indulged in the habit of khat chewing on a regular basis . Studies report that khat (catha edulis) use is highly prevalent among the people living in the eastern africa and southwestern arabian peninsula . The fresh leaves of this plant are reported to have a similar effect on the central nervous system to the use of amphetamine . The world drug report in 2001 has presented saudi arabia among the top 5 countries which have the population of khat chewers, and in this country most of the khat chewers are reported to be from the jazan region . One study reported that the urban part of this region had more khat users when compared to the rural population . The prevalence rate of khat users in the schools is seen to be 37.7% among the boys and 3.7% among the girls . It is also reported that the khat use increases with age and number of years of study . Thus, there is a lack of data on bruxism and its associated factors in the developing nations and at present there is no study which has evaluated the association of khat use with bruxism among any study population . The current study aims at assessing the prevalence of bruxism among the jazan university students and checking its association with their khat chewing habit . The current study was approved by the ethics committee at faculty of dentistry, jazan university, saudi arabia . This study was conducted in the jazan university campus which is located at the southern tip of saudi arabia bordering yemen . The current study is designed as a cross - sectional descriptive type using cluster random sampling technique without the intention of determining the cause and effect relationship . In the first stage of the study, the colleges under jazan university were divided into various clusters and 4 colleges were selected randomly . The medical and dental wings were excluded as the students may have prior knowledge on bruxism which can lead to a bias . In the second stage, the participants who had no history of cervical or facial injury and who are not undergoing orthodontic treatment were included in the study . The questionnaire originally prepared in english was subjected to translation and reverse translation in the local language by bilingual dentists, who were fluent in both english and arabic . Initially, a convenience sample size of 20 was randomly selected and the questionnaire was subjected to validity and reliability tests . To check the reliability of the questionnaire, a test / retest procedure and a measure of internal consistency using cronbach - alpha coefficient were calculated and it was found to be consistent as the minimum value of 0.68 was obtained . Apart from taking the sociodemographic details, the questionnaire was mainly divided into two sections . The first section consisted of questions pertaining to the list of signs and symptoms leading to the diagnosis of bruxism . It also had a direct question (have you ever been aware of clenching or grinding your teeth during wakefulness in the past 6 months? Yes / no) leading to the classification of the participants as a bruxer or a nonbruxer (awake bruxism). A similar type of questionnaire was previously used by winocur and his colleagues . The second part of the questionnaire consisted of the assessment of potential risk factors like coffee, khat, and tobacco use (both smoked and smokeless). Stress was also assessed using an inventory which included symptoms like depression, strong heartbeat, dry mouth, angry burst, inability to concentrate, weakness, fatigability, muscle stiffness in the morning, lock jaws, tight jaws, pain in the jaws, insomnia, headache, and excessive sweating . The values were indicated as percentages or mean sd (standard deviation) wherever necessary . The chi - square test at 5% significance was used for assessing the association between variables . Logistic regression with 95th percentile a total of 500 questionnaires were distributed and a high response rate of 95% (n = 476) was observed which can be attributed to the method of data collection . The session of questionnaire distribution and collection was on the same day during the break time of the respondents . Most of the male subjects fit the inclusion / exclusion criteria whereas few of their female counterparts had to be excluded as they had either prosthesis in their oral cavity or they were undergoing orthodontic treatment . Most of the subjects (77%) had the age ranging from 21 to 25 years (table 1). Subjective analysis revealed that 85% of university students (63% males and 22% females) had at least one episode of bruxism during the past six months . The university students between the ages of 21 and 25 were seen to be more affected with bruxism when compared to other age groups (table 2). Also, the same age group (2125 years) was seen to be more indulged in the use of khat, coffee, and tobacco when compared to the other age groups . Khat (36.9% to 14.9%), tobacco (31.7% to 16.6%), and coffee (60.1% to 24.8%) use was seen to be more in males when compared to females (tables 1 and 2). The stress assessed in the study population was significantly on a higher side, with 87% of males and 89% of females reporting stress in their life (inclusive of relationships and academics). About 47.2% of bruxers have reported pain in comparison with the nonbruxers (table 4). In the analysis of multiple logistic regression, a strong association of stress (p = 0.00; or = 5.902, 95% ci 2.61413.325) and the khat use (p = 0.05; or = 1.629, 95% ci 0.3607.368) with bruxism was seen among the study sample (table 3). Interestingly, it is observed that people who have the habit of chewing khat have nearly 3.56 times (95% ci; 2.6211.22) less chance of developing pain when compared to the nonusers (table 4). Thus, out of the variables assessed, only khat use and presence of stress were associated with the bruxism . Association of bruxism with khat among the university students was assessed for the first time in any study population . Bruxism is not a normal habit and in certain circumstances like when there is an increase in the frequency of episodes it may turn into a phenomenon with pathological consequences . It is also reported that bruxism may dramatically alter results and the duration of the delicate and careful treatments performed by the dentists . Hence, the current study is conducted in order to add to the literature on the field of bruxism . The study revealed that 85% of the university students had experienced at least one episode of bruxism during the past six months and this finding was high in comparison to another study conducted by lavigne et al . . The current study also portrays a strong association between bruxism and self - reported stress among the study population . Some studies in accordance with the current study confirmed the association [1820] while other studies failed to confirm such correlation [6, 21]. In couple of other studies performed, the investigators showed that the high prevalence of bruxism among the police officers and the air force pilots was due to its strong association of the stress involved in their jobs [22, 23]. Thus, the high prevalence of bruxism among the jazan university students can also be attributed to the high level of stress reported . Khat (catha edulis) use was assessed for the first time which showed a strong association with bruxism . There is no previous literature available till date to check such a relation, but there are some studies which were conducted to report the prevalence of khat among different age groups . One such study was done among secondary school children and the prevalence results obtained were a little less when compared to its use among the university students examined in the current study . Khat use is considered as a public health problem in saudi arabia and it is shown to affect the academic performance in children and is also seen to be associated with psychosis in young adults [25, 26]. The significant relationship of khat use with bruxism suggests that more research work should be conducted, especially focusing on the cause and effect relationship . A very interesting statistical finding in the current study was that the subjects who were in the habit of khat chewing were 3.56 times less likely of experiencing pain due to bruxism in comparison to the nonusers . This can be attributed to the cathinone content of the catha edulis (khat), which has a similar composition and effect to amphetamine . However, further investigations should be performed to confirm the association of khat in providing pain relief . Unlike other studies which demonstrated the association of tobacco and coffee use with bruxism out of the examined factors, only the stress attributed to the daily life and the khat chewing habit among the university students were seen to be significantly associated . In the limitations of the study, it could be said that the assessment of bruxism through the questionnaire may not be accurate and has a chance of recall bias . The gold standard of evaluations is expensive and may not have been feasible with the current budget of the study . The current method of assessment is widely acceptable by various researchers, both to diagnose and to check the association of potential risk factors with awake bruxism . To conclude, it is observed that bruxism is highly prevalent among the jazan university students . High amount of stress and khat use can be considered as important risk indicators for awake bruxism . Further studies are required in this field in order to portray the cause and effect association . Immediate public health measures should be taken to prevent the use of khat (catha edulis) among the university students.
A small subset of patients with chronic total occlusion of the middle cerebral artery (mca) are refractory to medical treatment due to inadequate collateral circulation.1 surgical revascularization with extracranial to intracranial bypass has been the mainstay of treatment but its efficacy remains controversial.2 development of new endovascular devices and improved operator experience have rendered angioplasty and stenting a potential treatment option for such patients . In these case reports, we illustrate the feasibility of elective (at least 1 month after the ischemic event) endovascular recanalization for symptomatic mca occlusion by angioplasty and stenting, and the long term outcome . A 43-year - old male smoker with hypertension and hyperlipidemia presented with a 2-month history of recurrent transient ischemic attacks (tia) of left - sided weakness . Angiogram demonstrated total occlusion of the right mca m1 segment with thrombolysis in cerebral ischemia (tici) grade 0 (figure 1a). Pial collaterals extending from the right anterior cerebral artery (aca) (figure 1a) and posterior cerebral artery to the right mca territory were evident . The risks and benefits of conservative medical treatment, extracranial to intracranial bypass and endovascular therapy were discussed with the patient and his families . In light of his recurrent symptoms despite medical therapy with antiplatelet agents, endovascular recanalization of the right mca was planned . Under general anesthesia, the lesion was traversed with a coaxial assembly of agility soft microguidewire (cordis endovascular, miami lakes, florida, usa) and prowler 14 microcatheter (cordis endovascular, miami lakes, florida, usa) (figure 1b). The microguidewire was then withdrawn, the patency of the lumen distal to the lesion confirmed through the microcatheter (figure 1c) and a transcend microguidewire (boston scientific, fremont, california, usa) was exchanged . The diameter of the proximal segment of the right mca adjacent to the lesion was approximately 2.5 mm and lesion length was 6 mm by visual estimation, compared with the 6 french guide catheter (boston scientific, natick, massachusetts, usa). After withdrawing the microcatheter, a 2.58 mm apollo balloon expandable stent (microport medical, shanghai, china) was advanced over the microguidewire and deployed at the lesion . Subsequent angiogram demonstrated resolution of the occlusion and good antegrade perfusion to the right mca territory (tici grade 3) (figure 1d). Postoperatively, blood pressure was maintained at 100120/6080 mm hg to minimize the risk of reperfusion injury . The patient displayed no periprocedural neurological complications verified independently by a neurologist . At his 29-month follow - up, the patient remained asymptomatic on treatment with antiplatelet agent and risk factor control . (a) angiogram demonstrated total occlusion of the right middle cerebral artery (mca) m1 segment (arrowhead), and pial collaterals extending from the right anterior cerebral artery territory (arrows). (b) the lesion was traversed with an agility soft microguidewire (arrowhead). (c) angiogram through the microcatheter confirming that the tip of the microcatheter was in the distal mca . (d) post stenting angiogram showed resolution of the occlusion and good antegrade perfusion to the right mca territory . A 66-year - old male smoker with diabetes mellitus and hypertension presented with a 2-month history of tia of left - sided weakness, similar to case no 1 . Angiogram demonstrated total occlusion of the right mca m1 segment with tici grade 0, and 70% stenosis of the right aca ostium (figure 2a). Pial collaterals extending from the right aca (figure 2b) and posterior cerebral artery contributed to the supply of the right mca territory . Under general anesthesia, the lesion was traversed with an assembly of agility soft microguidewire and prowler 14 microcatheter, which was then exchanged with a transcend microguidewire . The diameter of the c7 segment of the right internal carotid artery adjacent to the lesion was visually estimated to be 3 mm and lesion length 8 mm . The occlusion was predilated with a maverick 29 mm balloon angioplasty (boston scientific, natick, massachusetts, usa) (figure 2c), followed by deployment of a 2.59 mm wingspan stent (boston scientific, natick, massachusetts, usa) covering the entire lesion site (figure 2d). Subsequent angiogram demonstrated complete recanalization of the right mca with good antegrade perfusion to the right mca territory (tici grade 3) (figure 2e). Twelve months later, there was recurrence of tia of left - sided weakness while on medical treatment . An 80% in - stent restenosis (figure 2f) demonstrated on angiogram was successfully treated with maverick 29 mm balloon angioplasty without complications (figure 2g, h). At his next 12-month follow - up, (a) angiogram demonstrated total occlusion of the right middle cerebral artery (mca) m1 segment (arrowhead) and 70% stenosis of the right anterior cerebral artery (aca) a1 segment (arrow). (e) post stenting angiogram showed good antegrade flow to the right mca territory . (f) twelve - month follow - up angiogram demonstrated 80% in - stent restenosis . (g) microguidewire was placed in the right aca to avoid the snow ploughing effect during right mca angioplasty occluding the right aca ostium . A 43-year - old male smoker with hypertension and hyperlipidemia presented with a 2-month history of recurrent transient ischemic attacks (tia) of left - sided weakness . Angiogram demonstrated total occlusion of the right mca m1 segment with thrombolysis in cerebral ischemia (tici) grade 0 (figure 1a). Pial collaterals extending from the right anterior cerebral artery (aca) (figure 1a) and posterior cerebral artery to the right mca territory were evident . The risks and benefits of conservative medical treatment, extracranial to intracranial bypass and endovascular therapy were discussed with the patient and his families . In light of his recurrent symptoms despite medical therapy with antiplatelet agents, endovascular recanalization of the right mca was planned . Under general anesthesia, the lesion was traversed with a coaxial assembly of agility soft microguidewire (cordis endovascular, miami lakes, florida, usa) and prowler 14 microcatheter (cordis endovascular, miami lakes, florida, usa) (figure 1b). The microguidewire was then withdrawn, the patency of the lumen distal to the lesion confirmed through the microcatheter (figure 1c) and a transcend microguidewire (boston scientific, fremont, california, usa) was exchanged . The diameter of the proximal segment of the right mca adjacent to the lesion was approximately 2.5 mm and lesion length was 6 mm by visual estimation, compared with the 6 french guide catheter (boston scientific, natick, massachusetts, usa). After withdrawing the microcatheter, a 2.58 mm apollo balloon expandable stent (microport medical, shanghai, china) was advanced over the microguidewire and deployed at the lesion . Subsequent angiogram demonstrated resolution of the occlusion and good antegrade perfusion to the right mca territory (tici grade 3) (figure 1d). Postoperatively, blood pressure was maintained at 100120/6080 mm hg to minimize the risk of reperfusion injury . The patient displayed no periprocedural neurological complications verified independently by a neurologist . At his 29-month follow - up, the patient remained asymptomatic on treatment with antiplatelet agent and risk factor control . (a) angiogram demonstrated total occlusion of the right middle cerebral artery (mca) m1 segment (arrowhead), and pial collaterals extending from the right anterior cerebral artery territory (arrows). (b) the lesion was traversed with an agility soft microguidewire (arrowhead). (c) angiogram through the microcatheter confirming that the tip of the microcatheter was in the distal mca . (d) post stenting angiogram showed resolution of the occlusion and good antegrade perfusion to the right mca territory . A 66-year - old male smoker with diabetes mellitus and hypertension presented with a 2-month history of tia of left - sided weakness, similar to case no 1 . Angiogram demonstrated total occlusion of the right mca m1 segment with tici grade 0, and 70% stenosis of the right aca ostium (figure 2a). Pial collaterals extending from the right aca (figure 2b) and posterior cerebral artery contributed to the supply of the right mca territory . Under general anesthesia, the lesion was traversed with an assembly of agility soft microguidewire and prowler 14 microcatheter, which was then exchanged with a transcend microguidewire . The diameter of the c7 segment of the right internal carotid artery adjacent to the lesion was visually estimated to be 3 mm and lesion length 8 mm . The occlusion was predilated with a maverick 29 mm balloon angioplasty (boston scientific, natick, massachusetts, usa) (figure 2c), followed by deployment of a 2.59 mm wingspan stent (boston scientific, natick, massachusetts, usa) covering the entire lesion site (figure 2d). Subsequent angiogram demonstrated complete recanalization of the right mca with good antegrade perfusion to the right mca territory (tici grade 3) (figure 2e). Twelve months later, there was recurrence of tia of left - sided weakness while on medical treatment . An 80% in - stent restenosis (figure 2f) demonstrated on angiogram was successfully treated with maverick 29 mm balloon angioplasty without complications (figure 2g, h). At his next 12-month follow - up, the patient remained asymptomatic on medical treatment . (a) angiogram demonstrated total occlusion of the right middle cerebral artery (mca) m1 segment (arrowhead) and 70% stenosis of the right anterior cerebral artery (aca) a1 segment (arrow). (e) post stenting angiogram showed good antegrade flow to the right mca territory . (f) twelve - month follow - up angiogram demonstrated 80% in - stent restenosis . (g) microguidewire was placed in the right aca to avoid the snow ploughing effect during right mca angioplasty occluding the right aca ostium . Chronic mca occlusion as a cause of hemodynamic ischemic stroke is not a prominent clinical issue worldwide.1 while acute occlusion of the mca is commonly caused by an embolic thrombus,3 4 atherosclerotic occlusion of the intracranial arteries is more prevalent in asians . Patients with chronic proximal mca occlusion may have minor or no stroke because of well developed collaterals . Conversely, the prognosis for those with hemodynamic impairment is poorer.1 chronic occlusive lesion does not seem to represent a significant embolic source but threatens the patient with hemodynamic ischemia and infarct . In addition, patients with mca occlusion may have disabling cognitive impairment, especially for those with bilateral diseases.5 for our two patients, the etiology of their recurrent tias was likely to be hemodynamic impairment from total mca occlusion, arising from atherosclerotic stenosis and superimposed thrombosis based on angiographic findings . The optimal management of them was controversial.2 direct stenting of mca occlusion has been used for acute stroke but it is rarely performed electively in the chronic phase.6 our cases showed direct stenting without intra - arterial thrombolysis for symptomatic chronic mca occlusion is feasible . From our experience, the gateway balloon catheter and the wingspan stent are more suited for patients with tortuous vascular access, especially at c4 segment, as these afford better flexibility in traversing the curvature . The apollo stent is more rigid compared with the gateway wingspan system but it is preferred for patients with a smoother access path as delivery of the balloon expandable stent does not require exchanging and less procedural time was needed . Our immediate technical success rate was 100% (2/2), better than the 33% (1/3) reported in a previous study which used angioplasty alone.7 although no periprocedural complications occurred in our patients, vessel perforation from microguidewire manipulation or balloon angioplasty was a potentially serious complication . Great care must be taken in monitoring the movement of the microguidewire tip to ensure its position within the true lumen . In the event of dissection, stenting may help to minimize the intimal flaps and hence is superior to angioplasty alone . Hyperperfusion hemorrhage into the recanalized brain parenchyma is another major potential complication, and tight control of blood pressure is crucial postoperatively . During follow - up, one of our two patients the long term outcome of recanalization of chronic total occlusion of the mca remains uncertain . Direct stenting of chronic total occlusion of the mca is technically feasible but not without the potential for major complications . Further studies are needed to determine its efficacy in carefully selected patients with medically refractory cerebral ischemia due to mca occlusion.
The increase in respiratory diseases arising from allergies in industrialised countries in recent years is considered to be linked to changes in certain environmental factors . One such factor relates to the higher levels of atmospheric pollution and the greater presence and distribution of allergenic taxa . Climate change has brought about large increases in the concentrations of some airborne allergens, with the resulting higher incidence and/or severity of allergic illnesses (gilmour et al ., 2006). Significant increases in allergic responses are often reported, even in individuals who have never had symptoms previously, and this leads to the belief that these allergic responses might be caused by new allergens that are released by plants used for ornamental purposes or reforestation, or by invasive plants . In europe the increases in pollinosis appear to be caused by pollen from birch, hornbeam and cypress trees, and above all, from ragweed, rather than by the classic allergenic pollens, such as grasses, pellitory, olive and mugwort (corsico et al ., 2000; asero, 2004; ridolo et al ., 2006). Ambrosia artemisiifolia (short or common ragweed) is an annual, anemophilous, and extremely allergenic weed that can produce enormous amounts of pollen . As these pollen grains are small (1822 m), they can often be transported over long - distances (mandrioli et al ., 1998). Ragweed started its expansion in europe in the last decades of the 20th century, arriving from hungary, the country where it was most abundant . Ragweed is now also found in italy (damato et al ., 1998), and the two regions that have been most affected are lombardia and friuli venezia - giulia, which are located in the north - northeast of italy (mandrioli et al ., 1998; asero, 2002). Ragweed plants have been detected only sporadically in central - southern italy (pignatti, 1997). However, transboundary transport of ragweed pollen to central italy has been demonstrated, which could have a clinical effect on atopic patients (corsico et al . Ragweed pollen concentrations have been reported to have increased over the last decade (oswalt and marshall, 2008), mainly because ragweed is invasive in europe and is an opportunistic and pioneer plant, invading field crops and open disturbed habitats or roadsides (bassett and crompton, 1975). However, the increase in allergic disease might be attributed not only to greater concentrations of ragweed pollen in the atmosphere, but also to modifications to the allergenicity this pollen can promote . Although the role of atmospheric pollutants on the allergic sensitivity of airways is not yet completely clear, there is evidence to suggest that urbanisation increases allergic sensitization due to its high levels of exposure to ozone (o3), nitric oxides (nox), sulphur oxides (sox) and particulate matter (pm10) (damato, 2002). In addition to affecting the airways of allergic individuals, air pollutants can have direct effects on the aeroallergens in the atmosphere, which can result in changes in the antigenic characteristics of pollen . Air pollutants, and especially o3 and respirable pm10, can induce proinflammatory responses in the lung (bernstein et al ., 2004), and can have immunological adjuvant effects on ige synthesis, as has been found with polyaromatic hydrocarbons in the particles of diesel exhaust (nel et al ., 1998). Ozone is the main component in the so - called summer smog comprised of photochemical oxidants and appears to account for up to 90% of the total oxidant levels in cities that have a mild sunny climate (butkovic et al ., ozone is generated at ground level by photochemical reactions that involve ultraviolet radiation of atmospheric mixtures of no2 and hydrocarbons that can derive from vehicle emissions . These o3 trends depend not only on the substrate supply (no2 emitted by cars), but also on the sunny weather, because of the transformation of no2 into o3 during a photochemical smog . Current safety standards for o3 levels are exceeded frequently in most mediterranean countries . Indeed, the 8 h average levels of o3 for the period from 2000 to 2004 in different sites in italy showed that the background o3 pollution exceeded the european standards (paoletti et al ., 2007) that were fixed by the european union at 0.060 ppm (directive 2002/3/ec, 2004). Its toxicity is due to the generation of reactive oxygen species (ros), such as the superoxide anion radical (o2), hydrogen peroxide (h2o2), the hydroxyl radical (oh), and singlet oxygen (o2) (mudd, 1997). The o3 effect on pollen grains has been considered in particular in studies of plant germination in vitro . However, opposing effects have been reported: feder (1968, 1981) observed a reduction in nicotiana tabacum pollen tube elongation, and similar results were reported for corn pollen by mumford et al . (1983) reported that the ability of the pollen of pinus strobus to germinate was not significantly reduced by o3 fumigation . It has also been reported that o3 can influence allergen release from pollens grains, and masuch et al . (1997) found that o3 increases the content of group 5 allergenic proteins of lolium perenne . When the pollen comes into contact with the airway mucosa, it releases not only allergenic proteins, but also lipid immuno - modulators, such as pollen - associated lipid mediators (traidl - hoffmann et al ., 2002), and nicotinamide adenine dinucleotide phosphate (nad[p]h) oxidases (boldogh et al ., 2005). These latter are enzymes that have an oxidase activity that can produce the o2 ion, which is converted into h2o2 through the action of the enzyme superoxide dismutase (sod). Therefore the nad(p)h oxidases have fundamental roles in inflammatory processes, as they can increase the levels of ros in the epithelium of the respiratory apparatus, and promote the flow of neutrophils towards the respiratory apparatus (boldogh et al ., 2005). On the basis of the concomitance of episodes of high o3 levels (gabusi and volta, 2005) and the high concentrations of ragweed pollen in the atmosphere during the summer months (mandrioli et al ., 1998), the aim of the present study was to determine whether o3 affects mature ragweed pollen grains after their dispersal . In particular, we determine first whether o3 can increase the allergy potency of ragweed pollen by stimulating allergen and nad(p)h oxidase release, and secondly whether o3 can induce physiological alterations that affect pollen viability, which is the ability of the pollen to complete post - pollination events and to achieve fertilisation . For this purpose, under laboratory conditions, we exposed ragweed pollen to a high o3 concentration (100 nl l) during the day . This concentration corresponds to peak o3 levels measured during the summer in central italy (http//www.arpa.umbria.it). We specifically evaluated: (i) pollen ros and no content, as no is considered to be a mediator of inflammatory responses (moilanen and vapaatal, 1995); (ii) activity of the nad(p)h oxidase, which can generate ros; (iii) expression of the major ragweed pollen allergens; and (iv) pollen viability . Ragweed (ambrosia artemisiifolia) pollen was purchased from greer laboratories (lenoir, nc, usa). The pollen was aliquoted into 2 ml, sterile microcentrifuge tubes, and stored dry at 4 c until use . The o3 treatment was performed by exposure of 0.5 g pollen in petri dishes to 100 nl l o3 for 5 h (08:00 h to 13:00 h) per day for 7 consecutive days in a plexiglass chamber (0.32 m) under light with a photosynthetic photon fluence of 120 mol m s, as previously described (pasqualini et al ., 2009). A non - fumigated (o3) pollen sample was maintained in a filtered - air plexiglass chamber under the same experimental conditions . After each daily o3 fumigation, the pollen samples were all left in a growth chamber under controlled conditions (14 h photoperiod, photosynthetic photon fluence rate of 120 mol m s, day / night air temperature 25 c/20 c, and relative humidity 60%75%) until the next treatment . After the 7 days of this o3 fumigation, the pollen was either immediately analysed for viability and ros, h2o2 and no content, or frozen under liquid n2 and stored at 80 c for protein quantification, nad(p)h oxidase activity assay, and rna analysis . Louis, mo, usa), as reported by heslop - harrison and heslop - harrison (1970). Pollen grains (2 mg) were hydrated for 30 min in 2 then 20 l of these suspensions were placed on a microscope slide and stained with 4 m fda . The total number of pollen grains was visually counted using bright - field microscopy, while the fluorescent pollen grains in the same field of view were counted using a uv epifluorescence microscope (dmlb; leica, leica microsystems, wetzlar, germany) with a 450 nm excitation filter and a 535 nm emission filter . Pollen viability was determined as the percentage of fluorescing pollens relative to the total pollen grains . For each sample (control and o3 treated), ten slides were prepared, and for each slide, at least 100 pollen grains were counted . (2009) with slight modifications using the fluorescent no indicator dye 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (daf - fm diacetate; molecular probes, invitrogen, carlsbad, ca, usa) (kojima et al ., 1998). The pollen (1 mg) was first incubated for 30 min at 4 c in 1 ml mes kcl buffer containing 8% sucrose, 10 mm mes, 5 mm kcl, 50 mm cacl2 (ph 6.8), in the absence and presence of 200 m of the no - scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cptio; sigma daf - fm diacetate (10 m) was then added, and the samples were incubated in the dark for 20 min, to allow the dye to enter the pollen . The pollen suspensions were then centrifuged at 6500 g for 1 min, and the resulting supernatant was discarded . Fresh mes kcl buffer was then added to the pellets, which were resuspended and left at room temperature for 20 min . Finally, 50 l pollen suspension were placed onto a glass slide and covered with a glass coverslip before being examined with a uv epifluorescence microscope with a 450 nm excitation filter and a 535 nm emission filter . Intracellular no was determined as the percentage of fluorescing pollens relative to the total pollen grains . For each sample (control and o3 treated), six slides were prepared, and for each slide, at least 100 pollen grains were counted . Ros detection was performed using the fluorescent ros indicator dye 2,7-dichlorodihydrofluorescein diacetate (dcfh2-da; molecular probes) (setsukinai et al ., 2003). Pollen grains (2 mg aliquots) were hydrated at 4 c in 2 ml 100 mm phosphate - buffered saline (pbs) for 30 min, and then transferred to 2 ml microcentrifuge tubes and centrifuged at 2000 g for 30 s. after centrifugation, the supernatant was discarded and fresh pbs containing 2.5 m dcfh2-da was added to the pellet . After an incubation in the dark at 25 c for 15 min, it was centrifuged at 2000 g for 30 s. after centrifugation, the supernatant was discarded and the pellet was immediately resuspended in fresh pbs, gently shaken, and centrifuged as before . Finally, the pellet was resuspended in pbs, and 50 l of the pollen suspension was placed onto glass slides, covered with glass coverslips, and examined using a uv epifluorescence microscope with a 450 nm excitation filter and a 535 nm emission filter . Content of ros was determined as the percentage of fluorescing pollens relative to the total pollen grains . For each sample (control and o3 treated), six slides were prepared, and for each slide, at least 100 pollen grains were counted . Pollen grains (100 mg) were hydrated in 1 ml pbs for 25 min at room temperature, with gentle shaking . The pollen suspensions were then centrifuged at 14000 g for 10 min, and the supernatants were recovered (s25 supernatants). The pollen pellets were resuspended with 1 ml pbs, shaken for 25 min and then centrifuged again at 14000 g for 10 min, to obtain the second supernatants (s50 supernatants). This procedure was repeated once again to obtain the third supernatants (s75 supernatants). The pellets were then resuspended in 0.5 ml pbs, sonicated, centrifuged at 14000 g for 10 min, and the supernatants were recovered (sson supernatants). The soluble protein concentrations in the supernatants were measured using the dye - binding method of bradford (1976), with bovine serum albumin as the standard . The nad(p)h oxidase activity was measured in the samples of these four supernatants obtained as above (s25, s50, s75 and sson) using the nitroblue tetrazolium (nbt) assay (bacsi et al ., this activity was only detectable in the s25 supernatants . Here, 25 g protein was used for each assay and mixed with 2 mm nbt without or with 100 m reduced nicotinamide adenine dinucleotide (nadh), or 100 m reduced nad phosphate (nadph), and without or with the nad(p)h oxidase inhibitor diphenyleneiodonium (dpi, 100 m) or the o2-scavenging enzyme sod (100 u ml). Nbt was completely removed by repeated washing with fresh pbs, and the formazan precipitate was dissolved in 100% methanol ., 100 mg pollen grain aliquots were hydrated for 30 min at 4 c in 1 ml sterile distilled water; they were then centrifuged at 14000 g for 10 min . The supernatants were recovered and immediately used for h2o2 quantification using the xylenol orange method (jiang et al ., 1990), based on the peroxidase - mediated oxidation of fe followed by the reaction of fe with xylenol orange o - cresolsulfonephtalein 3-3-bis[methylimino] diacetic acid, sodium salt . H2o2 was determined by adding 250 l of distilled h2o to 250 l of supernatant and 500 l of assay reagent (500 m ammonium ferrous sulphate, 50 mm h2so4, 200 m xylenol orange, and 200 mm sorbitol). Absorbance of the fe - xylenol orange complex (a560) was detected after 45 min . The specificity for h2o2 was tested by eliminating h2o2 in the reaction mixture with catalase . Standard h2o2 curves were obtained by adding increasing amounts of h2o2 from 0 to 100 nmoles . Pollen grains (130 mg) were incubated in 1 ml sterile distilled water and shaken gently for 30 min at 4 c . The pollen suspensions were then centrifuged at 14000 g for 10 min, and the soluble protein was measured as reported above . Crude ragweed pollen extract (10 g total protein) and 6 g natural amb a 1 (namb a 1), kindly provided by prof . T. p. king (rockefeller university, ny, usa), were separated by denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds - page) and visualised by staining with coomassie brilliant blue r-250 (laemmli, 1970). For immunoblotting analysis, the protein was electroblotted onto nitrocellulose membranes (protan, schleicher & schuell, dassel, germany) and then incubated with mouse monoclonal anti - amb a 1 antibodies for 2 h. these mouse monoclonal anti - amb a 1 antibodies (clone e10) were obtained by immunisation of mice with the purified recombinant amb a 1 protein (ramb a 1), as described by wopfner et al . After washing, the membranes were incubated with alkaline - phosphatase - conjugated anti - mouse igg (1/1000; sigma labelling was detected using the nbt / bromo - chloro - indolyl phosphate substrate (sigma aldrich). For quantification, membranes were scanned and band intensities were determined with the image analysis software imagej (http://rsb.info.nih.gov/ij). The expression of the amb a 1.1, amb a 1.4, amb a 2, profilin 1 and profilin 2 allergens and of the house - keeping 18s rrna were analysed by reverse transcription - polymerase chain reaction (rt - pcr). Total rna was extracted from the control and o3-treated pollen grains using nucleospin rna plant (macherey nagel, dren, germany), with pollen samples (30 mg) ground in a mortar under liquid nitrogen, and processed according to the manufacturer instructions . Both the quality and concentration of the total rna were assessed by non - denaturing agarose gel electrophoresis and by measuring a260/a280 . The rna was immediately processed for rt - pcr or stored at 80 c . A given amount of total rna (1 g) was reverse transcribed, as described in pasqualini et al . Images of the rt - pcr ethidium bromide - stained agarose gels were acquired with a digital kodak camera (eastman kodak company, rochester, ny, usa) and quantification of the bands was performed with kodak 1d image analysis software . The o3-fumigation experiment was replicated 3 times, and for each experiment, three replicates were used . One or two - way analysis of variance (anova) was applied to the data reported in the figures, as detailed in the figure legends . The means were compared using duncan s multiple range tests, and the values followed by different letters are significantly different at p 0.01 . The data concerning the ragweed pollen viability after 7 days of o3 fumigation are shown in fig . The mean pollen viability in the non - fumigated pollen (control) was 55.9%, while fumigation with o3 significantly decreased this pollen viability (39%; p <0.01). In non - fumigated ragweed pollen, we measure a rather low viability; nevertheless, that the pollen used for the experiments is a commercial pollen must be taken into account, as it was purchased from a company and kept at + 4 c for more than 4 months before we carried out the viability assays . Many reports have shown loss of viability of pollen and loss of membrane integrity during pollen storage (jain and shivanna, 1989). However, although the use of a commercial pollen did not allow us to work with pollen that had a high viability, it provided good homogeneity of the samples and the possibility for other groups to reproduce these experiments . In addition, the objective difficulty of the need to collect a sufficient amount of pollen for the analyses needs to be considered, particularly as ragweed grows only sporadically in central italy . As the toxicity of o3 is due to its ability to generate potentially lethal ros, we evaluated the release of h2o2 from pollen grains after this o3 fumigation as a possible cause of an oxidative burst in the pollen . The release of h2o2 from pollen grains was evaluated by analysing the liquid medium in which the pollen had been incubated for 30 min . About 0.9 mole h2o2 g fw were released from the non - fumigated pollen grains after their hydration, with similar levels detected in the o3-fumigated pollen, showing that o3 fumigation did not significantly influenced the pollen h2o2 content (fig . Using fluorescent microscopy and the ros - sensitive fluorescent probe dcfh2da, we were able to qualitatively detect ros inside the pollen . Indeed, this probe lacks specificity among the ros and reacts only slowly with h2o2 or o2, but very fast with the oh radical and peroxynitrite [onoo (setsukinai et al ., 2003)]; it thus follows that this dcf fluorescence is an assay of generalised oxidative stress, rather than of any particular ros . Our results show that the 7-days of o3 fumigation induced only a small, and non - significant, increase in ros inside the pollen grains (fig . The nad(p)h oxidase in these ragweed pollen grains following the o3 fumigation was determined by nbt reduction to formazan, with this activity completely released after 25 min of hydration (s25 supernatant) (fig . For the supernatants obtained from the longer hydrations (s50 and s75) and from the sonication of the pollen pellets (sson), there was no measurable nad(p)h oxidase activity . Without nadph or nadh, nbt reduction by the pollen grains of ragweed was just detectable, with no significant difference seen between the non - fumigated and o3-fumigated pollen grains (fig . On addition of nadh or nadph, the activities measured from the o3-treated pollen increased by 41% and 21%, respectively, with respect to the non - fumigated pollen (fig . This reduction of nbt by the ragweed pollen extract was almost completely blocked by the addition of sod (absorbance reduction, 82% 5%) and of the nad(p)h oxidase inhibitor dpi (absorbance reduction, 85% 3%), which suggests that nad(p)h oxidase is the major source of these ros . Our data show here that high levels of constitutive no were detected in the ragweed pollen grains, and that the o3 fumigation induced a small, but significant, increase in no after 30 min of hydration (fig . 2 a, b; + 10%, p <0.01). To ensure that this fluorescence was no specific, the no - scavenger cptio was added to the probe . In this case, there was significant quenching of the green fluorescence, which indicates that the daf - fm probe was specific for no detection (fig . There was a large release of soluble proteins after the first 25 min of pollen hydration (81%), with 14.85 mg protein g fw and 14.71 mg protein g fw detected in the s25 samples of the non - fumigated and o3-fumigated ragweed pollen, respectively (fig . Less protein was released from the pollen after 50 min of hydration (s50; 19%). In the supernatants obtained from the further hydration (s75) and from the sonication of the pellets (sson) the o3 treatment did not induce significant changes in protein release from the ragweed pollen . Sds - page of the pollen extracts revealed similar profiles in both control and o3-fumigated pollen grains (fig . The major ragweed allergens ranged from 8 kda to 43 kda and the migration band visible at about 38 kda was identified as amb a 1 . Indeed, purified namb a 1 migrated at 38 kda, and also shows specific degradation into two fragments, of 26 kda (namb a 1) and 12 kda (namb a 1) (fig . The crude protein released after 30 min hydration of the pollen was analysed by immunoblotting and probed with a monoclonal antibody against amb a 1 (fig . Here, it has been detected a 38 kda protein band that corresponded to amb a 1, the major antigenic component of ragweed pollen, with no quantitative difference between non - fumigated and fumigated pollen grains . Altogether, these data indicate that this 7-day o3 fumigation did not induce changes in the released protein pattern, nor in the content of the major allergen amb a 1 . The expression of the major ragweed allergens, amb a 1, amb a 2, profilin 1 and profilin 2 was analysed by rt - pcr using the primer pairs given in table 1 . Fig . 4 shows that all of these allergens examined are expressed in these ragweed pollen grains, and that the o3 fumigation did not influence the transcript levels, substantially reflecting the unchanged amounts of the amb a 1 protein detected in the western blotting (fig . In this study, we have sought to simulate the effects of acute o3 exposure on ragweed pollen after its dispersion in the atmosphere . For this purpose, under laboratory conditions, the ragweed pollen was exposed to 5 h of 100 nl l o3 for 7 days, with some of the biochemical and molecular features of the pollen grains then analysed . However, it should also be considered that o3 can influence the pollen not only directly after its dispersion, but also indirectly by affecting the plant growth and the pollen maturation, since ragweed typically grows during the summer when the highest o3 peaks occur . Indeed, it has been reported that in l. perenne plants exposed to chronic o3 concentrations, the percentage of underdeveloped pollen was significantly increased compared to plants grown in filtered air (schoene et al ., 2004). This effect was said to arise from an inhibition of pollen starch accumulation, which was responsible for this disturbance of the pollen development (schoene et al ., 2004). Also, many reports have shown that ros and no accumulate in the reproductive tissues, such as the stigma, anthers and pollen, where they have different biological roles, depending on the stage and tissue (mcinnis et al ., 2006; hiscock et al ., 2007; however, whether o3 exposure of the plants during their growth can affect the ros and no contents of pollen grains is not at present known . A reduction in pollen viability by o3 has also been reported in other studies and in other species (feder, 1968, 1981; mumford et al ., 1972). The fluorochromatic reaction used to test pollen viability provides an indirect measurement of esterase activity and the intactness of the plasma membrane (heslop - harrison and heslop - harrison, 1970). As such, the reduction in pollen viability induced by this o3 fumigation is indicative of damage to the pollen membrane system . Furthermore, the exposure of pollen to a high o3 concentration after its release from the plant can reduce its viability, resulting in decreased fertility, and consequently, in a reduction in seed production . As irreversible damage to cell membranes can be caused by an o3-induced oxidative burst, we evaluated the ros production in pollen grains . The results show the presence of constitutive ros accumulation in the hydrated ragweed pollen, which has been suggested to promote pollen germination and/or pollen - tube growth to the stigma (potock et al ., 2007). Nad(p)h oxidase is the major source of ros in pollen grains (bacsi et al ., 2006), which produces the o2 ion; this is converted into h2o2 through sod activity or by spontaneous dismutation . The superoxide ion can also act as a reducing agent for transition metals such as fe and cu, which leads to h2o2-dependent formation of oh, an extremely aggressive ros . Hydroxyl radical accumulation in pollen grains can cleave polymers, including cell - wall polysaccharides (fry, 1998), which promotes the cell - wall loosening that is necessary to allow for the rapid cell elongation of growing pollen tubes (eckardt, 2005). In fumigated pollens, both the h2o2 released after hydration and ros localized in pollen grains by the fluorescent probe remained unchanged, indicating that 7 days of o3 fumigation did not induce oxidative stress in the pollen, at least when it was measured at the end of the o3 exposure . Thus the damage to the membrane system that is seen here as decreased pollen viability might be due to other factors in addition to ros . As reported above we found that the nad(p)h oxidase was rapidly released (in less than 30 min) after pollen grain hydration, as indicated by bacsi et al . (2006), who showed that subpollen particles containing the amb a 1 allergen and nad(p)h oxidase activity are released from ragweed pollen grains after 35 min of hydration . When ragweed pollen grains were exposed to 7-day o3 fumigation, we saw significant induction of nad(p)h oxidase enzymatic activity . Thus, as previously observed in leaves of o3-treated plants (pellinen et al ., 1999; ranieri et al ., there is evidence that ros have prominent roles in the pathogenesis of allergic diseases (bowler and crapo, 2002). (2005) indicated that oxidative stress generated by nad(p)h oxidase in ragweed pollen grains can augment the immediate - type hypersensitivity reactions and pollen - antigen - driven allergic conjunctivitis . Our data here indicate that when ragweed pollen grains are in the presence of o3, there is an induction of nad(p)h oxidase activity which is released into the hydration medium, and may, under natural conditions, be released into the airway mucosa when pollen grains are inhaled . No is a key signalling molecule that controls a variety of physiological responses in plant and animal systems, and it has also been demonstrated to be a signal in plant defence responses against pathogens (delledonne et al ., 1998) and o3 (ederli et al ., 2006; meier et al ., recently, it was shown that no is involved in plant reproductive processes, as it can modulate the growth of the pollen tube and the orientation of the pollen tube towards the stigma (prado et al ., 2004, 2008). It has been suggested that in interacting with h2o2 of the stigmatic papille, no can regulate the processes of recognition between pollen and stigma (mcinnis et al ., 2006). The presence of no has been reported in hydrating pollen grains of various angiosperms (bright et al ., 2009); in that study, no and nitrites were shown to be released after hydration of the pollen, with greater levels of release seen for allergenic species compared to non - allergenic pollens in the limited number of taxa examined . Ozone induces a weak, but significant increase in the no fluorescence signal, suggesting that the no acts as a signal molecule in response to stress also in the pollen . As no can produce onoo when in the presence of the o2 ion (wendehenne et al ., 2001), we hypothesise that the fluorescent signal detected in the pollen grains loaded with dcfh2da was attributable to the oh radical, which was potentially derived through the fenton reaction from nad(p)h - oxidase - generated o2, and to the onoo . The major allergenic proteins released from ragweed pollen ranged from 8 kda to 43 kda in all of the extracts examined, in agreement with results previously reported (weber and nelson, 1985). After o3 fumigation, the protein release by hydrated pollen grains was rapid, with similar protein pattern profiles in both control and o3-fumigated pollen . The major antigenic component of ragweed pollen, amb a 1, was identified as a band visible at about 38 kda, and western blotting analysis revealed that its content did not change after o3 exposure . We also examined the expression profiles of the major ragweed pollen allergens: amb a 1 and amb a 2, which are proteins belonging to the pectate lyase family (rafnar et al ., 1991), and profilin 1 and profilin 2, which are proteins involved in signal transduction from the outer cell membrane to the inside of the cell, and in the regulation of actin polymerisation (goldschmidt - clermont et al ., 1990). The profilins are defined as panallergens, as they are widespread among the pollens of plants that are both closely and distantly related botanically (van ree et al ., our data show that o3 exposure did not affect the expression of the major ragweed pollen allergens . Earlier studies concerning the effects of gaseous pollutants on the content of allergenic proteins provided no conclusive results . Recently, it has been reported that pollen extracts from o3-fumigated plants showed increased allergen content and higher ige reactivity (masuch et al . 2010). On the other hand, exposure of timothy grass pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts (rogerieux et al ., 2007). (2010) used a proteomic approach to show that the major allergens of birch (bet v 1, 2, 3 and 4) were not expressed differentially in their samples from rural and urban areas, but that the extracts collected in urban areas had higher chemotactic activities on human neutrophils . They then suggested that the greater allergenicity of pollen collected from polluted areas was determined by more than just the allergen content (bryce et al ., 2010). Our data support the concept that when ragweed pollen is exposed to o3 during its time in the atmosphere, it can suffer damage that is seen as decreased pollen viability, and it can become more allergenic through stimulation of inflammatory ros - generating nad(p)h oxidase, which would be released when pollen comes into contact with the cells of the respiratory apparatus.
Antibodies used were specific for atpase subunit and (invitrogen molecular probes), monoclonal and polyclonal acetyllysine (cell signaling technology), sirt3 (as described3), etf and lcad (generously provided by jerry vockley, university of pittsburgh). Oxidation of [1-c] palmitic acid by tissue homogenate was adapted from a previously established method26 . Briefly, tissue was homogenized in sucrose / tris / edta buffer, and was incubated for 3060 min in the reaction mixture (ph 8.0), containing [1-c] palmitic acid, and measured for acid - soluble metabolites (asm) and trapped co2 . Enzymatic activity for lcad was measured using the anaerobic electron transfer flavoprotein (etf) fluorescence reduction assay27 using 2, 6-dimethyheptanoyl - coa as a substrate in recombinant lcad expressed and purified from hek293 t cells with wild - type or catalytically inactive sirt3, or in e. coli in the absence (control) or presence of nicotinamide (nam, 50 mm)28.
Sub - saharan africa (ssa) has been under the scourge of communicable diseases (cds) for a long period of time, and sudden deaths (sds) are still responsible for most of the morbidity and mortality in the region . Cds continue to be a burden although the non - cds (ncds) are now coming up as an equally burdensome disease, thus leading to a double burden on the health care services and economy of this resource - poor region.1 the world health organization (who) defines sd as death occurring within 24 hours of an abrupt change in one s previous clinical status.2 sd refers to nonviolent, nontraumatic deaths, but studies have shown that psychologically and physically traumatic events can precipitate sd.3,4 ncds have been incriminated as the principal culprits in sd, but some deaths that occur out of hospital or within 24 hours of admission in our hospitals are caused by cds, and as such, these cds are pooled into the category of sds . The most common cause of death worldwide now is cardiovascular disease;5 this is similar for sd.6 before 1900, the world was in the first stage of epidemiologic transition, which is the stage of pestilence and famine, with infectious diseases and malnutrition emerging as the predominant causes of death; improvement in public health and nutrition has led to the emergence of the second stage of the epidemiologic transition known as the stage of receding pandemics, with concomitant decline in death rates due to malnutrition and infections.5 the second stage of epidemiologic transition includes hypertension, coronary heart disease, stroke (predominantly hemorrhagic), amongst others.5 a study in saudi arabia reported that the cause of sd in 45 (20.2%) out of 223 cases was due to infectious diseases.7 cds on their own usually present with signs and symptoms, which often continue to worsen over days if left untreated, are poorly treated, or if the correct diagnosis is missed altogether, before finally leading to death . In view of the fact that cds are easily amenable to treatment once sufferers present early, prompt diagnosis is made and appropriate treatment is instituted, patients with cd should therefore not present with sd, we therefore consider it important to conduct this retrospective study to find out the proportion of such cases that are presenting as sd . We are of the opinion that a study of this nature will help in mounting a public health campaign against the actions and inactions of our people that are making diseases, which can be easily cured with prompt and adequate antimicrobial treatment, the cause of sd in the 21st century . We conducted a retrospective study of sd caused by cds in adult patients aged 18 years and older, carried out at the ladoke akintola university of technology teaching hospital (lautech), osogbo, osun state, southwest nigeria . We retrieved the case notes of all the cases coded as sudden unexpected death from nonviolent, nontraumatic causes that occurred from january 2003 to december 2011 . These hospital records were comprised of cases indicating that there was unexpected death before arrival in the hospital and death within 24 hours of admission to the hospital . We then sorted out the subset of cases where the postmortem diagnosis was classified as cd . Further information was subsequently obtained from the patients case notes and autopsy reports pertaining to demographic data, symptoms on presentation, investigations done, and clinical diagnosis . Data entry was inputted into the computer using the statistical package for social sciences version 16 (ibm corporation, armonk, ny, usa) for statistical analysis . There were 48 cases of sd in the period spanning january 2003 and december 2011, inclusive . A subset of 17 (35.5%) of the sd cases were caused by cds, with ncds accounting for 29 (60.4%) of patients, while immunosuppressive disease and chronic undernutrition accounted for two (4.2%) patients . These aforementioned 17 cases were analyzed to get more information on the cd - related sd . The overall adult mortality of medical patients in the hospital during this period of study was 25.5% (718 out of a total admission of 2,821 patients). The total admission included all cases of death before arrival at the hospital that had autopsy done on them . The cd - related sd accounted for 2.4% (17/718) of all adult medical mortality and 0.6% (17/2,821) of all adult medical admissions . Table 1 shows the sociodemographic characteristics of the patients with cd as the cause of their sd . The mean age of the patients was 37.6 11.6 years, age range of 2562 years, mode of 25 years, and a median of 34.0 years . Table 2 shows the details of patients with a postmortem diagnosis of cd as the cause of their sd, while table 3 shows the postmortem diagnosis according to age group . However, there were two patients who did not fit into the group of cd - related sd that are worthy of mentioning . One was a 32-year - old female trader with severe immunosuppressive disease, and the other was a 60-year - old male security guard with chronic under - nutrition, who also had features of poor personal hygiene and neglect, but no evidence of natural disease or marks of violence . There were no symptoms recorded for 14 (82.4%) of the patients who were brought in dead . The rest (17.7%) consisted of the two patients with pulmonary tuberculosis (ptb) and severe hemoptysis who died of hemorrhagic shock . In addition, a 62-year - old female patient with lobar pneumonia, septicemia, and a huge obstructed incisional hernia presented with a 5 day history of cough and catarrh . She also had clinical evidence of a chest infection with septic shock; she died within a few minutes of admission . In the 19th century, the world witnessed the first stage of an epidemiologic transition known as the stage of pestilence and famine, with infectious diseases and malnutrition emerging as the most common causes of death;5 however, improvement in public health and nutrition has led to the emergence of the second stage of the epidemiologic transition known as the stage of receding pandemics, with accompanying decline in death rates due to malnutrition and infections.5 the second stage of the epidemiologic transition includes hypertension, coronary heart disease, and stroke (predominantly hemorrhagic), amongst others.5 ssa (nigeria inclusive) is not left out of this epidemiologic transition, but it is also still battling with cds due to poverty, ignorance, misplaced priorities, and widespread corruption . This has paved the way for cd - related sd to still be rearing its ugly head in our communities . In this study, cd - related sd accounted for 2.4% of all adult medical deaths and 0.6% of all adult medical admissions . This study further showed that 35.5% of sd was caused by cds, and typhoid sepsis accounted for 47.1% of sd as shown in table 2, which was the most common amongst the cds . The proportion of sd caused by cd (35.5%) in this study is higher than the 20.2% reported for infections in the saudi arabia study, which also reported lobar pneumonia separately as being responsible for 13.0% of sds, hence resulting in a total of 33.3% sds, which supports our finding from this study.7 lobar pneumonia alone accounted for 17.7% of the cd - related sd, which is also close to the percentage quoted in the saudi arabia study.7 all of the patients who had acute bacterial infection had septic shock and died on account of multiple organ failure . It is our view that a delay in seeking a prompt diagnosis and appropriate treatment, on the part of the patients, led to the so - called sd . Similarly, ptb was also responsible for 17.7% of the cd - related sd with two of the infected patients dying of hemorrhagic shock following massive hemoptysis, and the third patient died of respiratory failure . The case of a 60-year - old immigrant worker in new york who died suddenly is a good illustration of this . Investigation into the cause of his death revealed that he had been coughing for months before his death, and that ptb was the cause of his death; in addition, he had infected a good number of his coworkers.8 in another study of sd in suspicious circumstances in two young subjects, the cause of death at postmortem was found to be tuberculosis.9 ptb is a chronic infectious disease that would be present for 1 month or longer . Ptb is one of the most common causes of massive hemoptysis; a study that reviewed 123 cases of massive hemoptysis confirmed that 38.2% of patients had ptb, closely followed by 30.1% with bronchiectasis.10 massive hemoptysis is unpredictable, and eight apparently clinically stable patients died suddenly while awaiting surgery or endoscopy in the same review.10 the human immunodeficiency virus (hiv) epidemic has greatly increased the number of cases of active tuberculosis per capita in ssa to the extent that the region now has the highest rate.11 the patients with ptb in this study were not tested for hiv at autopsy . The mean age of the patients in this study was 37.6 11.6 years, with a mode of 25 years, median of 34 years, and an age range of 2562 years . The saudi arabia study showed that the greatest incidence of sd occurred at the extremes of life (<12 months and> 60 years).7 our study did not show this because our focus was on adults, so the pediatric age group was excluded from our study . We also did not notice this high incidence in the elderly adults, as the young adults and middle - aged subjects (aged 2059 years) constituted 94.2% (table 1) of those who had sd secondary to cds . The most affected age group was the 2029-year - old age group at 41.2%, followed by the age group of 4049-year - olds at a rate of 29.4% . Patients with an age <40 years accounted for 53.0%, while those in <50 years accounted for 82.4% of the cd - related sd . A study of sd in cape town, south africa showed that infections were responsible for most of the deaths (26.1%) in the youngest age group (1829 years).12 in this age group, 13.6% of patients died of pneumonia, while 12.5% of them died of meningitis; ptb was common in all the age groups.12 in a similar study in india, sd in the young showed that infections accounted for 54.6% of deaths.13 there was male preponderance, with a male - to - female ratio of 1.8:1 . This is in keeping with the study in india, which also showed a male preponderance, with sd mainly occurring in the age group of 3035-year - olds.13 this could be because the males are the breadwinners and they could not afford to stay away from work despite ill health; hence, they kept working while ill . They are also likely to eat at work (outside their homes) most times, thereby contracting food- and water - borne infections easily . It could also be because they considered going to queue in the hospital for treatment as a waste of time . All the occupational groups were affected, but the unskilled group took the lead in sds, with 35.3% of the deaths occurring among this group . The low- and middle - income countries still regard infectious diseases as a major challenge due to poverty, inadequate health and sanitation infrastructure, as well as due to political upheaval.14 the unskilled occupational group are also mostly the low - income earners, and usually have a low level of education, and as such they are more vulnerable to all the problems bedeviling the low - income countries like nigeria (where they live); hence, this high incidence of sd from cds among this group was recorded in our study . As mentioned earlier, typhoid sepsis accounted for 42.1% of all cds, and it also has a high incidence rate in all the age groups, except for the 60-year - old age group, as shown in table 3 . Typhoid sepsis is a water / foodborne disease that is endemic in developing countries like nigeria where potable water is not readily available to a majority of the people; this is coupled with poor sanitation . Typhoid and paratyphoid fever constitute a major public health burden worldwide, especially in the developing nations.15,16 the global estimate of typhoid fever episodes in 2010 alone was 13.5 million, although data for this study were unavailable for seven regions including central and west ssa.16 the diagnosis of typhoid fever in ssa is difficult to make in the midst of numerous causes of febrile illnesses such as malaria.17 a nigerian study on the rate of malaria coinfection with typhoid fever reported 0.5% coinfection rate of typhoid with malaria when diagnosis was done by blood culture, as opposed to 10.1% by a diagnosis made by the widal test.18 in a recent study in tanzania,19 a group of researchers found that the case definition of the who was better than the widal test in diagnosing typhoid fever . The case definition of the who states the following.20 a confirmed case of typhoid fever (fever of 38c and>) of at least 3 days duration with s. typhi confirmed positive culture from any of the following samples: blood, bone marrow or bowel fluid . A probable case of typhoid on the other hand has fever with character and duration as stated for confirmed case without isolation of s.typhi but only has positive sero - diagnosis or antigen detection test . Chronic carrier state also occurs after an acute typhoid fever episode, with the patient excreting s.typhi in stools or urine (or repeated positive bile or duodenal string cultures) for more than 1 year, however some patients may be excreting s.typhi without a history of acute typhoid fever . The widal test, on the other hand, is the oldest of the agglutination tests, having been introduced in 1896 by widal.21 it utilizes bacterial suspensions of salmonella typhi and s. paratyphi a and b that have been treated to retain only the somatic lipopolysacchride o antigen and the flagellar h antigen.21,22 there is an agglutination reaction between these antigens and the corresponding antibodies in the serum of patients suspected of having typhoid fever.22 the immunoglobulin m somatic o antibody, representing the initial serologic response of acute typhoid fever, is the first to appear, followed by the more slowly appearing but persisting immunoglobulin g flagella h antibody.23 two types of agglutination techniques are available: the slide test and the tube test.22 the slide test is a rapid screening procedure, and once the initial screening is positive, it is then done in serial dilutions of the patient s serum to determine the strength of the antibody.22 the result is scored 04, with 0 indicating no agglutination, 1 indicating 25% agglutination, 2 indicating 50% agglutination, 3 indicating 75% agglutination, or 4 indicating 100% agglutination.22 the endpoint of serum activity or titer is the smallest amount of serum that shows a 2 agglutination.22 the strongly reactive tests can be easily seen, while a very good light source is needed to see the weak reactions.22 the tube test is much more technical and macroscopic,24,25 and requires 37c incubation of the suspended antigen antibody for up to 20 hours in a water bath.22 results are also scored, as earlier mentioned, so are also the serial dilutions for the antibody strength.22 the tube agglutination test confirms the results and clarifies erratic or equivocal agglutination reactions of the more rapid slide agglutination test.22 the widal test is a cheap and rapid alternative to the expensive and cumbersome blood or bone marrow culture, which may need 7 days to isolate and identify the s. typhi; hence, it has continued relevance in patients with febrile illness in the developing world, such as in africa.21 the widal test, however, has several limitations, including nonspecificity,26 in that there are cross - reactions with other non - salmonella organisms such as malaria, dengue, miliary tuberculosis, endocarditis, brucellosis, and chronic liver disease, among others.22 low sensitivity is another limitation,27 but the widal test done on convalescent - phase serum has been reported to give more reliable results with higher specificity and sensitivity.27 other studies have shown that a positive widal test during the acute and convalescent period of typhoid fever (in sera taken 710 days apart) with a fourfold rise in antibody titer gives an index of suspicion.28 to further compound the raging controversies on the widal test, a study on four different brands of widal reagents showed a lot of discrepancies; also, a fourfold rise in antibody titer was not achieved with any of the brands.29 this study also found that for typhoid fever diagnosis, the use of two brands of widal reagents in sequence could be of value, and so a single - serum sample could also be useful in a typhoid diagnosis based on an anti-o titer.29 there is also a general consensus that the test is still valuable for the diagnosis of typhoid fever if a baseline antibody titer of the healthy afebrile individuals in the population being studied is known.26 in such a study done with 200 healthy afebrile blood donors in six centers spread across southwest nigeria, the baseline antibody titer was found to be <1:160,26 while that of turkey was found to be <1:200.27 the slide agglutination test is the prevalent method for performing the widal test in zaria and other parts of nigeria.30 there are conflicting reports in the literature as to whether blood culture or bone marrow culture is the gold standard for diagnosing the disease correctly;19,31,32 it is, however, important to note that the developing nations lack the facilities to conduct these tests in most of their health care facilities . Hence, clinical acumen is relied upon most times for the diagnosis to be made before either complications or death occurs . This lack of appropriate diagnostic tools is a major problem militating against the correct management of this infection.32 the patients also do not report their symptoms early, usually reporting when complications set in, thus leading to high morbidity and mortality . A mortality rate of 22.2% was reported amongst patients treated for typhoid in a tertiary health facility in southwest nigeria the same area as that of this current study.33 furthermore, intestinal perforation was found to be the most common complication, followed by hemorrhage with septic shock.33 another identified problem in the sub - saharan region is the emergence and spread of strains of s. typhi that are resistant to chloramphenicol, other readily available antibiotics, and recently to the quinolones.32 these antibiotics are also used for the treatment of other infections, thereby posing serious challenges to our health care systems, placing more financial burden on patients and government, and increasing the risk of complications and death.32 other cd - related sd reported in this study, as shown in tables 2 and 3, were lobar pneumonia, which accounted for 17.7%, and sepsis in many of the patients . One of the patients had hemorrhagic shock and metastatic abscesses, while another also had cerebral abscesses with raised intracranial pressure . The challenges of prompt diagnosis and treatment are late presentation of the patients in the hospital, lack of appropriate diagnostic facilities and inability of patients to afford the prescribed drugs . We are of the opinion that the patient with sepsis, hemorrhagic shock, and metastatic abscesses must have had disseminated intravascular coagulopathy as a complication of the sepsis, and this must have led to hemorrhagic shock and subsequently to death . The metastatic abscesses might also have been caused by staphylococcus aureus, or any of the other organisms that are notorious for this . A japanese study of eleven autopsy cases of fulminant bacterial infection in adults showed that all the patients had sudden onset of hypotension and multiple organ failure culminating in unexpected death.34 blood culture in all of them confirmed bacteremia with isolation of s. pneumoniae (group a, hemolytic streptrococcus) in four patients, s. pyogenes in another four patients, s. dysgalactiae subgroup equisimilis in one patient, and vibrio vulnificus in another patient.34 the same study further showed underlying chronic diseases in 82% of patients.32 circulatory collapse caused by sepsis, severe pulmonary congestion, and hemorrhage accounted for death in ten (90.9%) of the eleven patients.34 it is also important to note that symptoms for some days preceded sd in these patients, and the authors noted that the status of the host immune system and the virulence of the bacterium determined the onset of the fulminant bacteria infection.34 there are so many factors that can be responsible for suppression of an individual s immune system in a developing nation like nigeria, and they include chronic malnutrition, indiscriminate use of unprescribed drugs, herbal concoctions, alcoholism, substance abuse, other competing infections, hiv or acquired immunodeficiency syndrome (aids), and ncds such as diabetes mellitus, cancers, and so on . We are of the opinion that most of the patients in our current study must have had some form of fulminant bacterial infection with attendant circulatory collapse or their infections progressed to septic shock and death from multiple organ failure . There were two patients who had unusual autopsy diagnosis as the cause of their sd . These included a 32-year - old female with severe immunosuppressive disease, which we opined was likely to have been caused by aids, but no test was done to confirm this at autopsy . The current prevalence of hiv / aids in nigeria is 3.6%.35 mortality has continued to increase because people do not seek voluntary hiv counseling and testing (hct), as well as treatment when needed, despite the availability of free hct and treatment . Lautech is one of the recognized centers for this free hct and hiv / aids treatment, but this patient never showed up for any such assistance in her lifetime . The other patient was a 60-year - old male security guard with chronic under - nutrition and features of poor personal hygiene and neglect who must have had his problem for an appreciable duration, but since there was no relative to look after his welfare, he died and was also erroneously treated as a case of sd . This current study shows that the improvement in our public health and nutrition in nigeria is still inadequate, and as such, chronic under - nutrition and cds are not just causing death but they are also causing sd . The treatment of some of these cds responsible for sd in this study might have been covered by antimicrobials on the essential drug list in most of our primary and secondary health care facilities, where some form of free health care is being offered to citizens; in addition, there are health care services being provided by international organizations as a form of aid to developing nations like nigeria . The majority of the funding for the treatment of hiv / aids in nigeria comes from development partners, and the main donors are the president s emergency plan for aids relief (pepfar), the world bank, and the global fund.36 nigeria is the largest oil producer in africa and the 12th largest in the world,37 and yet is ranked by the united nations development program human poverty index as 156th out of 187,38 thus making it extremely difficult for the nation to cope with its numerous health challenges, amongst other challenges . The developed world has moved away from the burden of cds since the 19th century, but in nigeria, we are still battling with cds and malnutrition in the 21st century to the extent that these factors are even responsible for an appreciable percentage (35.5%) of sds in our communities . We must, therefore, launch a spirited public health effort to prevent both cds and ncds . Advocacy, health education, provision of potable water, continuous improvement in sanitation and personal hygiene, and enforcement of existing laws that ban over - the - counter sales of drugs without prescription are needed in order to prevent the emergence of antimicrobial resistance and other drug - related complications . It is also important for our government to devote greater percentages of its annual budgets to health, and tackle the issue of corruption in order to harness our resources to combat the various scourges on our health, education, and other essential areas.
Thirty years have passed after discovering human immunodeficiency virus (hiv), the etiological agent of the acquired immunodeficiency syndrome (aids) [14]. Two types of hiv are known: the most common hiv-1, which is responsible to the worldwide aids epidemic, and the immunologically distinct hiv-2, which is much less common and less virulent [6, 7] but produces clinical findings similar to hiv-1 . The hiv-1 type itself includes four groups m (main), o (outlier), n (non - m, non - o), and p [912], which have different geographic distributions but all produce similar clinical symptoms . The m group further splits into 9 subtypes (a through j) [1315], as well as at least 58 circulating recombinant forms (crfs, http://www.hiv.lanl.gov/content/sequence/hiv/crfs/crfs.html, last accessed 06 may 2013) and multiple unique recombinant forms (urfs). The vast majority of reports on drug resistance deal with hiv-1 subtype b infections in developed countries, and this is largely due to historical delays in access to antiretroviral therapy on a worldwide basis . Advances in antiretroviral therapy have revolutionized hiv management and the control of the spread of regional epidemics [1618]. Currently, a combination of several antiretroviral agents, termed highly active anti - retroviral therapy (haart), has been highly effective in reducing the number of hiv particles in the blood stream (as measured by a blood test called viral load) and delaying disease progression . Clinical trials and observational studies have shown profound reductions in morbidity and mortality in patients infected with hiv as a result of combination antiretroviral therapy [16, 1927]. Of relevance, advances in hiv treatment have had a positive impact on all the affected demographic and behavioral risk groups, with an expected longevity for hiv - infected patients that is now 73 years . Moreover, it should be considered that, thanks to the recent expansion in the number of antiretrovirals and antiretroviral classes, virological suppression has become achievable in most patients for whom numerous prior antiretroviral regimens had failed . In addition, antiretroviral therapy results in efficacious treatment of hiv-1, regardless of the viral subtype . However, despite advances in antiretroviral therapy, some treatments still fail . A major cause of treatment failure is the development of drug resistance both in hiv-1 b and non - b subtypes [2834]. The extreme variability and the high evolution rate of hiv-1 favour the development of antiretroviral resistance . Indeed, hiv-1 infection is characterized by a high degree of genetic variability within infected persons . This is explained by the fact that the virus population present at a certain time point within an infected person consists of a complex mixture of heterogeneous strains, termed the subsequent overgrowth or dominance of a certain viral strain over another is largely determined by its relative adaptation to a given intrahost environment, a factor particularly relevant to the emergence of drug resistant variants . Indeed, the intrapatient virus population is a highly dynamic system, characterized by a high turnover rate and a high mutation rate [37, 38]. These evolutionary dynamics are the basis for a diversified population that can quickly generate drug - resistant variants in response to the therapy [3942]. Escape mutants that have a selective advantage under therapy become dominant in the population and lead to an increasing virus production and eventually to therapy failure . The shifted population may be hit with a new drug combination, but finding such a potent regimen after treatment failure is challenging, since many accumulated mutations confer drug resistance not only to the administered drugs but also to structurally and functionally similar compounds [41, 43]. Identifying and understanding hiv-1 drug resistance therefore helps clinicians to avoid minimally active antiretrovirals in favor of newer drugs that are fully or nearly fully active [4446]. For this reason, resistance testing has become an important diagnostic tool in the management of hiv infections [4752]. With the aid of hiv resistance tests pharmacoeconomic studies have shown that these tests are also cost effective [53, 54]. For several years, national and international hiv treatment guidelines have been recommending the use of resistance testing both in drug - naive and drug - treated patients [4852]. Antiretroviral drug design, resistance research, and interpretation systems have been largely based on hiv-1 subtype b because of its predominance in the wealthy countries in which antiretroviral drugs were first introduced, as well as the availability of assays for drug resistance in such locations . However, it should be noted that hiv-1 b subtype represents only about 10% [5557] of the overall subtypes in the world . Several studies showed that non - b subtype hiv-1 infections have been rapidly increasing over time in previously subtype b homogeneous areas such as europe and north america [5873]. For example, in france, switzerland, and italy, it is estimated that non - b infections constitute roughly 15%35% of hiv-1 infections, with an increasing prevalence and complexity of intersubtype recombinants over the last years [59, 64, 66, 7377]. Although non - b infections are infrequent in north america, a study in new york identified non - b infections in a few us citizens who never travelled abroad, suggesting that transmission of non - b subtype occurs in the united states independently of travel history . Moreover, new hiv-1 strains are constantly emerging [78, 79]. Due to the spread of hiv-1 non - b subtypes and the introduction of antiretroviral drugs in resource - limited settings (known for the largest assortment of non - b subtypes), further knowledge concerning the responsiveness to antiretroviral therapy and hiv-1 drug resistance in non - b subtypes is required . In this regard, the development of specific mutations varies in different subtypes, and this can be explained mainly by the intrinsic properties of the virus and not only by a different pressure of antiretroviral drugs, as suggested in a recent study . To date, the improvement of the genotypic resistance test in terms of sensitivity, cost effectiveness and detection of drug resistance in non - b subtypes is a major topic . In order to achieve this goal, the introduction of new affordable assays is crucial . Group m subtype independent genotyping assays for detection of hiv-1 drug resistance were recently developed . These assays could represent an alternative to commercial assays for hiv-1 drug resistance genotyping in routine diagnostics and for surveillance and monitoring of drug resistance in resource limited settings [8082]. Several discrepancies are still present in the interpretation of resistance patterns present in hiv-1 non - b subtypes by using different interpretation algorithms . These discrepancies illustrate how hard it may be to reach an unambiguous conclusion, despite the efforts made over the last decade to interpret drug resistance in hiv-1 non - b subtypes [8387]. The increasing prevalence of hiv-1 non - b subtypes could have implications not only for the development of resistance but also for diagnosis, vaccine design, and the clinical management of hiv infection [65, 90, 91]. Moreover, patients infected by certain hiv-1 non - b subtypes present accelerated disease progression [9297] and higher cognitive impairment . In the light of the above mentioned, the aim of this review is to provide a complete picture about hiv-1 genetic diversity and its implications . Mutational escape results in a remarkable degree of viral diversity within hiv-1 and in its adaptation to both immune activity and antiretroviral therapy . However, not all escape mutations are advantageous to the virus since some of them can severely affect viral fitness [99, 100]. The extensive genetic diversity of hiv-1 is due to its high replication rate, the error - prone reverse transcriptase, and recombination events that may occur during virus replication [101, 102]. The molecular basis of hiv-1 variability is a highly error - prone reverse transcriptase enzyme . The activity of this enzyme, essential for viral replication, is specifically required for the conversion of single - stranded genomic rna into double - stranded viral dna, which is later integrated into the host genomic dna . For this reason, hiv-1 reverse transcriptase inhibitors are powerful inhibitors of hiv-1 replication and represent an important class of antiretroviral agents . Hiv-1 reverse transcriptase is a multifunctional enzyme that possesses rna - dependent and dna - dependent dna polymerase activities as well as an rnase h activity that specifically degrades the rna strand of rna / dna hybrids . As an intrinsic property, and in contrast to other dna polymerases, hiv-1 reverse transcriptase lacks a proofreading function . This error - prone nature of reverse transcriptase, together with the high rate of virus production sustained by hiv-1 infection in vivo, strongly contributes to the continuous generation of new viral variants [13, 106, 107]. The rate of nucleotide substitutions introduced by reverse transcriptase is approximately 10 per nucleotide per cycle of replication, which is equal to one nucleotide substitution per genome during a single replication cycle . Hiv-1 has a rapid turnover, and it is estimated that approximately 10 virions per day are generated in an infected individual . The composite lifespan of plasma virus and virus - producing cells is very short with a half - life of approximately two days, and an almost complete replacement of wild - type strains by drug resistant virus occurs in plasma within 24 weeks . During antiretroviral treatment, rapid viral turnover in combination with a high mutation rate is a primary factor behind the emergence of hiv variants with antiretroviral drug resistance . Each retroviral particle contains two copies of single - stranded rna, and template switches occur frequently during reverse transcription, thus generating mutations and recombination by intramolecular and intermolecular jumps . Recombination may link drug resistant mutations in hiv-1, leading to increased resistance to a particular drug, or the generation of multidrug resistant variants . In addition, recombination may lead to the acquisition of mutations that compensate for a loss in viral fitness or replicative capacity due to previous acquisition of resistance mutations . Since recombination can create a multiple drug resistant virus out of two single drug resistant strains, it is generally believed that the capacity of the virus to recombine facilitates the evolution of drug resistance [9, 110, 113115]. This rapid evolution of drug resistance in hiv remains a major obstacle for hiv therapy . Recombination is a strategy for viral rejuvenation, and it is likely that recombination between hiv strains may lead to the evolution of fitter forms and viral strains acquiring drug resistance to all major classes of hiv-1 inhibitors . A different scenario could be that a fitter virus can be generated by recombining parts of two parental genomes with lesser fitness, or alternatively a less fit virus can be generated by breaking up favourable combinations of mutations in the parental genomes . This interaction between recombination, mutations, and viral fitness is highly intricate, but nonetheless, recombination and its mechanisms, especially at the level of diverse subtypes, warrant further investigation . The potential for genetic differences among subtypes to yield different patterns of resistance - conferring mutations is supported by natural variation among hiv subtypes in genetic content (40% variation in the env gene, and 810% variation in the pol / gag genes). This issue acquires special relevance in view of the fact that the hiv pol gene is the major target for all major classes of anti - hiv drugs and most hiv strains show hotspots for recombination in gag - pol and env regions . On the basis of genetic homology, hiv-1 has been classified into four groups: m (main), o (outlier), n (non - m, non - o), and the recently identified p [912]. The m group further splits into 9 subtypes (a through j) [1315], as well as at least 58 circulating recombinant forms (crfs, http://www.hiv.lanl.gov/content/sequence/hiv/crfs/crfs.html, last accessed 06 may 2013) and multiple unique recombinant forms (urfs). In general genetic variation is of 2535% and 1520% between and within subtypes, respectively . For example, subtypes a and f have been subdivided into five (a1, a2, a3, a4, a5) and two subtypes (f1, f2), respectively . Crfs are intersubtype recombinant hiv-1 genomes that have infected three or more subjects who are not epidemiologically related, so they can be assumed to make an epidemiologically relevant contribution to the hiv-1 m group epidemic . The crfs are labelled with numbers rather than letters, and numbered in the order they were first adequately described as the first time . Urf variants are widely distributed worldwide, with recombination breakpoints different from those found in crfs . Very few isolates of hiv-1 n group have been identified and sequenced from humans, while the diversity of sequences within the o group is nearly as great as the diversity of sequences in the m group . Today, the hiv-1 m group, the cause of the worldwide pandemic, has a near global distribution, whereas the n and o groups are restricted to individuals of west african origin . The m and n hiv-1 groups have a common ancestor, one that is most closely related to the siv strain found in chimpanzees (sivcpz; pan troglodytes troglodytes) [117, 118] that live mainly in gabon, cameroon, and the republic of the congo . This hiv-1 progenitor probably was passed from chimpanzees to human hunters through blood borne transmission [119, 120]. Recent evidence suggests that the hiv-1 o and p groups may have originated in wild gorillas, in which the closest relatives of these two groups have been identified [10, 121]. Phylogenetic analysis of hiv-1 and related viruses from nonhuman primates suggests that three independent transmission events, which happened in early 20th century, spawned the hiv-1 m, n, and o groups [119, 120]. In particular, the earliest direct evidence of hiv infection in humans was found retrospectively in a serum sample and in a lymph node biopsy specimen stored in 1959 and 1960, respectively, in kinshasa in the democratic republic of the congo [122, 123]. Moreover, they have been instrumental in the validation and extrapolations of molecular clock computer programs used to estimate the time to the most recent common ancestors (tmrcas) and evolutionary rates of various hiv lineages [123, 124]. The hiv-1 m group appears to be the oldest hiv lineage in humans, with an estimated time to the tmrca in the first two or three decades of the 1900s [123, 124]. In particular, the tmrca shared between the m group and sivcpz is estimated at 1853 (17991904). Cross - species transfer is therefore inferred to have taken place sometime between 1853 and the early 1900s, although there is some uncertainty, given the size of the confidence intervals . Hiv-1 m group subtypes can be considered as a result of the high error rate of reverse transcriptase enzyme during virus replication and selective pressure exerted by the immune system . Since their introduction into humans, hiv-1 m group subtypes have expanded rapidly in west and central africa and established multiple epidemics around the world . Regional expansion of hiv-1 has come in waves with the rapid emergence of specific subtypes due in part to specific modes and routes of transmission . For example, intravenous drug use in southeast asia in the mid-1980s and in eastern europe and russia during the early 1990s led to the rapid spread of crf01_ae and of subtype a, respectively [126, 127]. A similar expansion of subtype b hiv-1 transmission occurred among men who have sex with men in north america and europe in the early 1980s however, hiv-1 subtype c (the most dominant subtype in the world responsible for more than 50% of overall infections) appears to have slowly emerged throughout the world over the past 10 to 15 years as a consequence of multiple introductions [126, 128]. In recent years a substantial increase of recombinant forms has been observed as a consequence of the increased genetic complexity of the global epidemic [57, 59, 64, 66, 7377, 129131]. However, the prevalence of recombinant forms (estimated to be around the 20%) is still underestimated . Indeed, genetic complexity is not always detected, and this is mainly due to the subtyping of only one genetic region and not of the full genome . Consequently, specimens previously considered pure variants may be classified as recombinants when additional viral genes are analyzed . The drugs currently approved for clinical use by the us food and drug administration (fda) and used to treat hiv infection belong to 6 distinct classes (http://www.fda.gov/forconsumers/byaudience/forpatientadvocates/hivandaidsactivities/ucm118915.htm, last accessed 06 may 2013): (1) seven nucleoside and one nucleotide reverse transcriptase inhibitors (nrtis); (2) five nonnucleoside reverse transcriptase inhibitors (nnrtis); (3) nine protease inhibitors (pis); (4) one fusion inhibitor (fi); (5) one cc chemokine receptor 5 (ccr5) antagonist; (6) two integrase inhibitors (inis). Each of these drug classes act at different steps in the hiv replication cycle (figure 1). The nnrtis and nrtis block the reverse transcription of the viral rna genome in cdna, which is catalysed by the reverse transcriptase . The inis block the integration of viral genome into the dna of the host cell . Although it seems that combination antiretroviral regimens are effective against all hiv-1 group m subtypes, there is emerging evidence of subtype differences in drug resistance relevant to antiretroviral strategies in different parts of the world . For this purpose, extensive sampling of hiv genetic diversity and ad hoc analyses are required to tailoring of antiretroviral therapies in different parts of the world . Hiv-1 non - b subtypes present clade - specific substitutions in positions related to drug resistance that could accelerate the emergence of drug - resistant viruses, change or induce alternative pathways of resistance, influence viral replicative capacity in vitro, impair the interpretation of genotypic resistance algorithms [70, 77, 134137], and affect drug - binding affinity [138, 139]. Specific mutation development varies with different subtypes because of the intrinsic properties of the virus as well as the different pressures of antiretroviral drugs . In particular, three factors are involved in this phenomenon.intersubtype differences in codon usage: subtype differences in nucleotide and mutational motifs, which are defined as the number of transitions or transversions needed to develop resistance to different classes of antiretroviral drugs, may affect the genetic barrier for resistance, as shown in the development of some mutations in different hiv-1 proteins, such as (i) mutations associated with resistance to nnrtis at the codon 106 of the reverse transcriptase in c and b subtypes (see section 4.2.2) [140, 141]; (ii) mutations at the protease codons 74 in c subtypes, 82 in g subtypes, and 89 in several non - b subtypes such as c and crf02_ag (see section 4.2.3) [87, 142145]; (iii) mutations in the integrase gene at codons 140, 148, 151, 157, and 160 (see section 4.2.4) [146, 147].intersubtype amino acid differences involved in minor structural changes in the targets of therapy: in this situation, different mutations emerge under the same drug pressure (e.g., the protease mutation d30n; see section 4.2.3).intersubtype differences in sequence motifs favoring nucleotide substitutions involved in drug resistance (e.g., the reverse transcriptase mutation k65r; see section 4.2.1). Intersubtype differences in codon usage: subtype differences in nucleotide and mutational motifs, which are defined as the number of transitions or transversions needed to develop resistance to different classes of antiretroviral drugs, may affect the genetic barrier for resistance, as shown in the development of some mutations in different hiv-1 proteins, such as (i) mutations associated with resistance to nnrtis at the codon 106 of the reverse transcriptase in c and b subtypes (see section 4.2.2) [140, 141]; (ii) mutations at the protease codons 74 in c subtypes, 82 in g subtypes, and 89 in several non - b subtypes such as c and crf02_ag (see section 4.2.3) [87, 142145]; (iii) mutations in the integrase gene at codons 140, 148, 151, 157, and 160 (see section 4.2.4) [146, 147]. Intersubtype amino acid differences involved in minor structural changes in the targets of therapy: in this situation, different mutations emerge under the same drug pressure (e.g., the protease mutation d30n; see section 4.2.3). Intersubtype differences in sequence motifs favoring nucleotide substitutions involved in drug resistance (e.g., the reverse transcriptase mutation k65r; see section 4.2.1). The following subsections describe the impact of hiv-1 subtypes on the different classes of antiretrovirals so far available . Table 1 provides an overview of the resistance associated mutations which are related to subtype diversity . The impact of subtype in terms of emergence of resistance in the nrti antiretroviral class is mainly due to the more rapid selection of primary resistance mutations in hiv-1 subtype c infected patients than in those infected by a subtype b virus . Some of the differences in rates of acquisition of the mutation k65r or thymidine analogue mutations (tams) are doubtless due to treatment regimens and disease stage, as well as access to viral load testing in many developing countries . However, one study suggests that increased rates of k65r acquisition in subtype c may be due to the nature of the subtype c rna template . The lysine to arginine mutation at position 65 (k65r) is a major mutation that confers broad high - level resistance to most nrtis, except zidovudine . The nucleotide sequence at codons 64 - 65 - 66 (containing a homopolymeric stretch of adenine bases) differs between subtypes b and subtype c viruses . This leads to reverse transcriptase pausing during the synthesis of double - stranded dna from the single - stranded dna intermediate template, a process that is template - specific but independent of the reverse transcriptase enzyme [14, 15, 148]. Subsequent misalignment of the subtype c template and primer leads to the aag to agg change responsible for the k65r mutation (figure 2). On the contrary, subtype b templates are prone to frequent pausing at codon 67, facilitating the generation of mutation d67n and tams instead of k65r (figure 2, table 1) [148, 150, 151]. A recent study showed a very high rate (> 65%) of k65r for patients infected by subtype c who failed tenofovir - based first - line regimens in south africa . Furthermore, a rate of 7 to 15% of k65r and/or k70e mutations was observed in subtype c endemic area in patients failing regimens including stavudine, didanosine, or zidovudine . Studies from israel reported a high frequency of k65r with treatment failure in subtype c viruses from ethiopian immigrants, while in indian patients a rate of k65r around 1012% at failure of first line combination therapy including stavudine, lamivudine, and nevirapine was observed . By using ultrasensitive pyrosequencing, variants carrying k65r mutation showed a higher frequency in subtype c infected patients than those infected by subtype b (1.04% versus 0.25%). In addition to the recruitment for monitoring k65r prevalence in patients with subtype c hiv infection, also the tam pathway (67n/70r/215y) deserves further attention . Indeed, in patients infected by subtype c from botswana, india, south africa, and malawi tams were also observed at treatment failure with zidovudine and didanosine [153, 155, 157, 158]. Larger numbers of patients and followups will be required to determine whether any consistent effect of the emergence of k65r in subtype c is clinically relevant . The emergence of mutations associated with resistance to nnrtis occurs after a single dose of nevirapine (sdnvp) [159161], a procedure recommended for the prevention of mother - to - child hiv transmission (pmtct) by initial guidelines from the world health organization (who) [162, 163]. Replacement of sdnvp may be necessary to reduce infant hiv infection risk and drug resistance development . Indeed, high frequency of drug resistance was observed in 69% of pregnant women infected with subtype c, 36% of those with subtype d, 19% of those with subtype a, and 21% of those with the crf02_ag, despite the absence of resistance before the administration of antiretroviral therapy [163167]. Moreover, by using ultrasensitive sequencing techniques, several studies revealed that in patients infected by subtype c a higher prevalence of nevirapine - resistance mutations (k103n and y181c) was found, reaching 70% to 87%, as compared with 42% of patients with subtype a with resistant viruses [168, 169]. These findings underscore the role of viral subtype in facilitating the development of drug resistance, which is exacerbated by the fact that resistance to nnrtis can also be transmitted through breast feeding . Indeed, the v106 m mutation is commonly selected in subtype c viruses (in approximately 30% of patients) after exposure to nevirapine or efavirenz, whereas a v106a substitution is only rarely selected (in approximately 5% of patients) by these two nnrtis in other subtypes (table 1). This peculiarity of subtype c results from the fact that v106 is encoded by gta in subtype b viruses and gtg in subtype c viruses [141, 171]. A single g - to - a transition at the first position of codon 106 in subtype c viruses results in v106 m, which confers high - level resistance to efavirenz and nevirapine . In contrast, in subtype b viruses, v106 m requires two nucleotide substitutions (gta - atg) and therefore occurs infrequently [140, 141]. K103n and y181c mutations remain important in both subtypes b and c, with k103n occurring in 40% of subtype b and 29% of subtype c viruses and y181c occurring in 23% of subtype b and 12% of subtype c viruses . In a recent italian study, the prevalence of mutation k103n in nnrti - treated patients was lower in subtype c infected patients compared with patients infected by b, f, or crf02_ag subtypes (table 1). Conversely, the mutations y181c and y188l were found with higher frequency in patients infected with subtype c compared to patients infected with subtype b . Another substitution that is seen more frequently among patients with a subtype c virus is g190a, which is also a result of naturally occurring g190a reverse transcriptase polymorphisms at residue 98 are common in subtype g, but they are also associated with nnrti resistance in subtype b (table 1) [15, 173]. The second - generation nnrti etravirine has demonstrated good efficacy across subtypes . A recent study highlighted the fact that hiv-1 genetic variant does not significantly influence the genotypic prediction of etravirine susceptibility in infected individuals failing efavirenz containing regimens . However, among the etravirine resistance associated mutations, some polymorphic substitutions resulted in drug - naive individuals infected with non - b subtypes, especially crf02_ag . Noteworthy, in a recent study it was found that the mutation e138k was the first mutation to emerge in either subtype b, c, or crf02_ag clinical isolates under etravirine pressure (table 1). Another study confirmed this finding for subtype c, while a preferential selection of y181c for the subtype b virus was observed (table 1). A novel mutation in the c - terminal domain of reverse transcriptase (n348i) has recently been reported to reduce susceptibility to etravirine in subtypes a, b, and c (table 1). In particular, in a south - african clinical trial, the n348i mutation was observed in 24% of subtype c infected patients failing a first - line nnrti regimen (most commonly with nevirapine). This mutation is not included in standard mutation lists or algorithms, but more data are urgently required to determine its clinical relevance . To date, the efficacy and the resistance patterns of the novel second - generation nnrti rilpivirine seem to be similar across the different subtypes [181, 182]. A study focused on hiv-1 subtype crf01_ae showed that the presence of mutations associated with resistance to rilpivirine are uncommon in patients infected by this subtype and failing a first - line nnrti - containing regimen . However, cross - resistance between rilpivirine and etravirine was high . Moreover, approximately 60% of patients had a high level of etravirine resistance, thus suggesting that the role of etravirine in hiv-1 subtype crf01_ae infected patients in second - line therapy is limited in late nnrti failure settings . Nnrti resistance associated mutations such as y181i, y188l, g190a, k101a, v106i, and v179i are natural polymorphisms in hiv-2, thus hiv-2 proves to be naturally resistant to all nnrtis . Nonpolymorphic substitutions in the protease gene have a greater impact on baseline susceptibility to pis than polymorphic mutations . However, intersubtype amino acid differences can create subtle structural differences in the targets of therapy . For example, subtype b infected patients receiving nelfinavir are more likely to develop d30n than those with viruses belonging to subtypes c, f, g and crf01_ae, which are more likely to develop l90 m or n88s (table 1) [186, 187]. More specifically, by comparing b and g subtypes, even in cases where the first mutation is l90 m in both these subtypes, indeed, in subtype b, l63p is the second mutation and occurs in almost all cases, suggesting that the progression of resistance is dependent on the emergence of this mutation, followed by the selection of v77i and other mutations . Differently, in subtype g, in almost 100% of cases, l89i follows the emergence of l90 m, suggesting a role in subtype g similar to that of l63p in subtype b. in this regard, findings indicate that the association of l89i with l90 m may further increase the pi resistance of subtype g viruses when compared with l90 m alone . Although subtype c isolates from ethiopian immigrants to israel favoured the l90 m pathway, patients with subtype c viruses in botswana did have d30n, suggesting that subtype c viruses from ethiopia and southern africa might be different [186, 189]. The basis for the higher preponderance of d30n in subtype b than in other subtypes may be related to the flexibility of the protease flap region and destabilization of the pi complex in subtype b, whereas an accessory n83 t mutation may be needed to rescue fitness and confer resistance in subtype c [142, 190]. Recent evidence has suggested that the polymorphism at codon 36 in the protease gene (m36 in subtype b and i36 in most other non - b subtypes) affects both the patterns of resistance that emerge under drug pressure and viral replication capacity (table 1). Similarly, the m89 polymorphism in subtypes a, c, and crf01_ae (l89 in subtype b) preferentially leads to the emergence under drug pressure of the m89 t mutation, which confers high - level resistance to nelfinavir, atazanavir, and lopinavir . There is also in vitro evidence that crf2_ag viruses with the 17e/64 m polymorphisms demonstrate hypersusceptibility to certain pis (nelfinavir, atazanavir, and indinavir). Differences in polymorphisms in the protease gene have been reported among several non - b subtypes; these include the following protease residues: 10, 20, and 63 in subtype a; 20, 53, 63, 74, and 82 in subtype c; 13 and 20 in subtype d; 10, 14, 20, and 77 in subtype f; 20, 67, 73, 82, and 88 in subtype g; 20, 63, 82, and 89 in the crf01_ae; 20 and 89 in the crf02_ag (table 1). In particular, in patients treated with pis, l89v was predominantly found in crf02_ag, while the tipranavir resistance mutation t74p was predominantly found in the c subtype in comparison to b subtype . The mutation v82 m was mainly found in subtype g while the mutations v82a / f / s were mainly present in other subtypes; the mutation n88s was found in subtypes c and crf02_ag, while n88d was present in subtype b . In southern brazil, scientists reported a lower relative frequency of primary resistance to pis in subtype c rather than in subtype b . Despite clear evidence for preferential emergence, diminished susceptibilities among wild - type isolates have been found for crf02_ag recombinant viruses in three different studies of nelfinavir and atazanavir [138, 173, 194]. A study suggested that distortions in the k26 pocket of the a / g protease may be responsible for a lower binding energy of nelfinavir and lower susceptibility of a / g viruses . The protease and gag genes coevolve as a functional unit when hiv is subjected to drug pressure from pis . Mutations in gag can act as compensatory substitutions that can increase both rates and levels of resistance to pis, as well as viral replication capacity . No genotypic system for the determination of drug resistance to pis currently monitors gag, despite the fact that relevant mutations in gag can increase resistance by a factor of 2 to 2.5, depending on the subtype . It is likely that different subtypes could develop compensatory gag mutations at different rates; this might ultimately justify the genotyping of gag in resistance testing . The primary mutations in the integrase gene associated with ini resistance are e92q, y143r / c, q148k / r / h, and n155h . The residues associated with primary resistance seem to be highly conserved across subtypes, but polymorphisms at the sites of secondary resistance mutations are more common in non - b subtypes [147, 198200]. Signature subtype differences in integrase at codons 140, 148, 151, 157, and 160 among hiv subtypes may affect the genetic barrier for resistance . These variations predict higher genetic barriers to the development of g140s and g140c mutations in subtypes c, crf02_ag, and a / crf01_ae, as well as higher genetic barriers to v151i in subtypes crf02_ag and crf01_ae [146, 147]. Regarding the impact of subtype on primary resistance development, it was recently observed that the subtype b integrase enzyme with the n155h mutation (e92q) exhibited increased resistance to raltegravir compared to the subtype c enzyme (table 1). In addition a new potential resistance pathway for raltegravir might include the mutation g118r, a substitution associated with lower susceptibility to raltegravir in crf_02ag subtypes . Notably, polymorphisms such as t97a, v151i, and g163r, already known as raltegravir secondary resistance mutations, showed a considerable prevalence (9%) in several recent studies also analyzing non - b subtype infected patients [203, 204]. The additional ini - resistance associated mutations k156n and s230n mutations were more frequent in b subtype while v72i, l74i, t125a, v201i, and t206s were more frequent in certain non - b subtypes [200, 205]. No differences in phenotypic raltegravir resistance were found in several non - b isolates tested . Dolutegravir is a second - generation ini which recently entered the battery of antiretrovirals against hiv-1 infection . It showed a higher genetic barrier to resistance and retains activity against viruses with resistance to raltegravir or elvitegravir . Interestingly, among the mutations associated with in vitro resistance to dolutegravir, the mutations l101i and t124a were polymorphic and significantly more prevalent in patients with non - b subtypes . In addition, mutation r263k seems to be preferentially selected in subtype b under dolutegravir pressure (table 1). Among the eis today available in clinics, both enfuvirtide (targeting gp41 hiv-1 protein) and maraviroc (targeting cellular ccr5 receptors) have shown antiviral activity against hiv-1 b and non - b subtypes [207, 208]. However, the high level of diversity (20 to 40%) in the env region predicts that this class of drugs will probably have higher potential for natural and emergent drug resistance . Clinical data have shown that the fi enfuvirtide is active against non - b subtypes owing to a highly conserved binding domain, although hiv-2 and hiv-1 class o viruses have natural resistance against this drug [209, 210]. So far, some studies suggest that there are significant differences in baseline susceptibility to hiv eis among the predominant hiv-1 subtypes [211, 212]. For instance, it was found that in drug - naive patients many more mutations associated with resistance to eis occur in subtype c compared with subtype b strains (table 1). Due to the worldwide spread of non - b viruses and the introduction of antiretroviral drugs in developing countries (known for the largest assortment of non - b subtypes), further knowledge concerning responsiveness hiv-1 drug resistance in non - b subtypes is required . Moreover, it should be taken into account that the increasing prevalence of hiv-1 non - b subtypes could have several implications not only for the development of resistance but also for other topics such as the response to antiretroviral therapy, disease progression, and transmission . There is a solid body of evidence indicating that the type and degree of hiv-1 resistance to nrtis, nnrtis, and pis vary between different subtypes [136, 186, 213, 214]. The development of nelfinavir resistance in subtypes b and g represents a classic example of this phenomenon . A different level of resistance has been observed among different subtypes . Indeed, the recombinant form crf02_ag is more susceptible to nelfinavir and ritonavir than subtypes c and f; subtype g is more sensitive to tipranavir and lopinavir than other subtypes, and the subtype c has accelerated risk in developing resistance to tenofovir [189, 215, 216]. An explanation for the extreme variability of hiv-1 subtypes in the response to antiretrovirals can be given by the presence of some polymorphisms that can influence both the emergence of drug - resistance mutations and the response to drugs . For example, polymorphisms at residues 20 and 36 of hiv-1 protease decrease the genetic barrier to tipranavir resistance in subtypes a, c, f, and g, while nucleotide heterogeneity at 64 and 65 positions in the reverse transcriptase accelerates development of k65r in subtype c [150, 189]. In some cases, drug exposure may lead to amplification of such polymorphisms as a98g / s in reverse transcriptase and m36i, k20i, and l89 m in protease, leading to a potential for resistance . It is commonly accepted that, as a result of a high degree of polymorphism in the protease gene, drug - naive patients infected with non - b subtypes present a certain degree of resistance to the pis . However, the data which support this assumption are largely controversial, as highlighted by two studies that showed conflicting results . Indeed, in a study analysing the hiv-1 phenotype from 42 patients (19, subtype g; seven, subtype c; six, subtype f; 10, crf02_ag) by the antivirogram assay (virco bvba), no differences in baseline susceptibility to any pi were found, with the exception of an unexpected hypersusceptibility to nelfinavir with crf02_ag . On the contrary, a second study, investigating drug susceptibility of 39 isolates from treatment - naive ghanaian patients, mostly infected with crf02_ag, showed reduced susceptibility to several pis, especially nelfinavir . This reduced in vitro susceptibility is not in agreement with the available clinical data, which show no difference in time to undetectable viral load among different subtypes [217219]. Continuous research on the role of polymorphisms in the development of drug resistance is therefore necessary . Studies are needed to assess genotypes both before and after therapy in the context of possible associations between polymorphisms and drug resistance . This area of research could include polymorphism variability in variants of the same subtype in different geographic regions . This information might improve the efficacy of certain drug combinations over others in the context of second- or third - line therapeutic strategies . Polymorphisms should improve current algorithms for interpretation of genotyping results in a subtype - independent way . As access to antiretroviral therapy in resource limited settings increases, where non - b subtypes are predominant, it remains imperative to establish appropriate treatment strategies for long - term clinical benefit that limits the emergence of drug resistance . The use of nontoxic, effective antiretroviral drugs should yield excellent clinical responsiveness, regardless of the viral subtype . Subtype differences, suboptimal therapies, and deficiencies in health care delivery systems can create conditions for accelerated development of resistance . Urgent recruitment for low - cost viral - load monitoring is needed to prevent and detect drug resistance, as well as to avoid unnecessary treatment switches [220, 221]. Unfortunately, pooling of resistance data often masks the role of regional differences in viral subtypes and antiretroviral therapies in the development of drug resistance . In resource - poor settings, such studies have used different nrti backbones (e.g., combinations of zidovudine and didanosine, zidovudine and lamivudine, or stavudine and lamivudine). In parts of africa, treatment failure has been reported in as many as 40% of patients after 2 years . Of note, resistance rates in india to two drug classes were observed in more than 80% of patients who failed their first - line regimen using combinations of nrtis and nnrtis . Larger longitudinal studies are necessary to determine the response to first - line combinations of antiretroviral drugs . The availability of genotypic resistance testing needs to be expanded to include all countries in which antiretroviral drugs are used . Cross - resistance among drugs is important, especially in settings where treatment options may be limited . Relatively few in vitro comparative data are available for pis in non - b subtypes, even if such data may be crucial for an understanding of cross - resistance to this class of antiretrovirals [191, 224]. Such data are important, since pis are often the only available option for drug sequencing in resource - limited settings after the failure of first- or second - line treatment . This means that resistance to pis usually requires the presence of large numbers of resistance mutations . For this reason, there is an advantage of using pis to avoid early resistance development . Therefore, differences among subtypes with regard to drug resistance are likely to be more important for nrtis, nnrtis, and inis than for pis . Our knowledge of the impact of hiv-1 subtypes on responses to antiretroviral therapy is very limited . Most studies have been small or poorly designed, and only a few have had an evaluation period longer than 48 weeks . An important limitation of these studies was to compare subtype b with non - b subtypes grouped together, a necessary oversimplification when dealing with small numbers of patients infected with non - b subtypes [217219, 225227]. A study by the paediatric european network for treatment of aids (penta 5 trial) investigated the response to therapy of 113 children infected with 7 different subtypes (a, 15%; b, 41%; c, 16%; d, 9%; f, 5%; g, 2%; h, 1%) and the two crfs crf01_ae (5%) and crf02_ag (7%). However, these results should be used with caution because the study did not have the statistical power to rule out minor differences between subtypes . Moreover, only four drugs were used (zidovudine, lamivudine, abacavir, and nelfinavir). Although polymorphisms at resistance - associated positions were highly prevalent in non - b subtypes, no negative impact of the mutations in virological responses was found; therefore, the authors concluded that polymorphisms at resistance - associated positions in subtype b could not necessarily be interpreted as conferring resistance in non - b subtypes . Retrospectively analysed the virological response in 362 patients: 265 europeans infected with subtype b and 97 africans infected with non - b subtypes . Subtype was presumed from ethnic and epidemiological data, with confirmation further extrapolated from genotyping the samples from 60% of the africans and 30% of the european patients . There was a significant imbalance between the two groups in several important parameters, including gender, transmission - risk groups, cd4 cell counts, and antiretroviral regimens used . Results showed no difference in time to undetectable viral load or recovery of cd4 count; however, a significant difference was observed in viral load over time, with a continuous increase in the african group after 9 months, suggesting a higher rate of therapy failure . Based on indirect evidence (i.e., the number of resistance mutations at time of failure), the authors concluded that the difference in virological response was mainly due to poorer adherence in the african group rather than the infecting viral subtype . As the study was not controlled for adherence, its conclusions remain controversial . A study which analysed retrospectively b and non - b subtypes (mainly focused on pure subtype a or recombinants with a subtype a protease) showed no differences in the time to undetectable viral load or in the viral load measured over time . However, the gain in cd4 count was significantly lower in the non - b group, which showed an increase of 161 cells / mm, compared with 236 cells / mm in the subtype b group . These results were consistent with those of camacho, who compared retrospectively the pathways to nelfinavir resistance in 101 patients (46 subtype b, 55 subtype g) failing a first - line regimen including this pi . At the time of failure, the only parameter significantly different between the two groups was the cd4 count (423 cells / mm in the subtype b group and 360 cells / mm in the subtype g group; p <0.001). Assessed virologic and immunologic responses to starting haart in a large cohort of 2116 patients, with the specific objective of comparing outcomes in patients with subtype b infection and those with subtype c, subtype a, crf02_ag, and subtype d infection, the predominant non - b subtypes circulating in the united kingdom . Overall, 1906 (90%) patients achieved viral load undetectability within 12 months after they started haart, of whom 335 (18%) subsequently experienced virologic rebound . In adjusted analyses, viral load suppression occurred more rapidly in patients infected with subtype c (hazard ratio (95% confidence interval, ci) 1.16 [1.011.33], p = 0.04) and subtype a (1.35 [1.041.74], p = 0.02) relative to subtype b infection . The virologic rebound occurred marginally faster in patients with subtype c infection (1.40 [1.001.95], p = 0.05), but the hazard of virologic rebound was similar with other subtypes . Although patients infected by subtype b viruses showed higher baseline cd4 cell counts and maintained the advantage throughout therapy, cd4 cell count recovery occurred at similar rates with all subtypes . The effect of pretreatment hiv-1 drug resistance on the response to first - line combination antiretroviral therapy in sub - saharan africa has recently been evaluated in a large prospective cohort . Pretreatment drug resistance results were available for 2579 (94%) of 2733 participants . Among them, 123 (5%) had pretreatment drug resistance to at least one prescribed drug, and 52 (2%) had pretreatment drug resistance and received fully active antiretroviral therapy . More than 50% of participants for whom it was possible to determine the subtype were infected by hiv-1 subtype c (1405 patients, 54%), while 638 (25%) harboured subtype a, 296 (11%) d, 117 (5%) a / g recombinant, 68 (3%) g, 48 (2%) other recombinants, seven (<1%) other subtypes, and five (<1%) b. compared with participants without pretreatment drug resistance, the odds ratio (or) for virological failure (or (95% ci): 2.13 [1.443.14], p <0.0001) and acquired drug resistance (2.30 [1.553.40]; p <0.0001) was increased in participants with pretreatment drug resistance to at least one prescribed drug but not in those with pretreatment drug resistance and fully active antiretroviral therapy . Cd4 cell count increased less in participants with pretreatment drug resistance than in those without (35 cells per l difference after 12 months; 95% ci 1358; p = 0.002). Further ad hoc studies are needed to better evaluate the therapeutic implications of specific non - b subtype differences in terms of long - term efficacy of different antiviral regimens . Several studies on disease progression showed that, among non - b subtypes, subtypes c and d were found to be more aggressive, followed by g, ae, ag, and a, the least aggressive of all hiv-1 subtypes [9297]. In this regard, it should be noted that because the different hiv-1 subtypes are not uniformly dispersed, comparisons of virulence and transmissibility are hampered by potential confounders, such as ethnic, socioeconomic, and other epidemiological factors . The report by kiwanuka et al . Concerning this topic provided a good opportunity to compare rates of disease progression associated with these different subtypes within a similar population, thanks to the cocirculation of hiv-1 subtypes a and d, and several intersubtype recombinants in the rakai district of uganda . In particular, kiwanuka et al . Compared rates of progression among hiv-1 seroconverters who were followed as part of the rakai health sciences program . Subjects identified during 19972002 were followed through 2004, when antiretroviral therapy became available in rakai district . The primary end point was the time to achievement of a cd4 cell count of 250 cells / mm or death due to aids . Progression to a cd4 cell count of 250 cells / mm was significantly less common among subjects infected with hiv-1 subtype a (20%), compared with subjects infected with subtype d (40%), recombinant forms (40%), or multiple subtypes (53%) (p = 0.03). Death from aids was also less common among subjects infected with hiv-1 subtype a. these differences were reflected in the longer time to aids onset for subjects infected with hiv-1 subtype a (8.05 years) compared with those infected with non - a subtypes (d = 6.49 years; recombinant forms = 5.57 years; multiple subtypes = 5.80 years). By multivariable models (adjusting for viral load, age, and sex), subjects infected with non - a subtypes more likely progressed to aids compared with those infected with hiv-1 subtype a. similarly, subjects infected with non - a subtypes had an approximately 68-fold greater risk of death from aids . Viral factors that could explain some of the hiv-1 pathogenicity are higher ex vivo replicative capacity, higher genetic diversity, and cxcr4 coreceptor usage [231233]. In this regard, a less common emergence of cxcr4-using (x4) variants was found in hiv-1 subtype a infection compared with hiv-1 subtype d infection, thus explaining the apparent lower virulence of hiv-1 a [234, 235]. A european study showed how subtype d has a fourfold higher rate of cd4 count decline, in the absence of antiretroviral therapy, compared with other subtypes in the study (a, b, c, and crf02_ag, which had similar rates of cd4 loss), even when adjusted for baseline cd4 count . Subtype d was also associated with higher rates of dementia in individuals with advanced immunosuppression if compared with subtype a . A study by kiwanuka et al . Showed how in uganda subtype a viruses have a significantly higher rate of heterosexual transmission than subtype d viruses . The faster disease progression and the lower rate of heterosexual transmission of subtype d compared with subtype a can explain the changes in the proportions of subtypes a and d observed over time in uganda and kenya [238, 239]. In particular, a significant decrease in the prevalence of subtype d with a concurrent increase of subtype a was observed . Despite the importance of viral characteristics in determining the rate of hiv-1 disease progression, recent findings from genomic studies show that host genetic factors also play a crucial role . The genetic determinants that influence susceptibility to hiv-1 and limit aids vary in different populations and among individuals . Meta - analyses of large cohort studies have identified several genetic variants that regulate hiv cell entry (particularly chemokine coreceptors and their ligands with copy number variations), acquired and innate immunity (major histocompatibility complex (mhc), killer immunoglobulin - like receptors (kirs), and cytokines), and others (trim5- and apobec3 g) that influence the outcome of hiv infection [240242]. Of the various genes that contribute toward host genetic propensity, mhc turns out to be the major contributor because it is responsible both for restriction of cytotoxic t lymphocyte (ctl) epitopes and for the emergence of ctl escape mutants . Leukocyte antigen (hla) alleles have been shown to be associated with the rate of disease progression in africans and caucasians [243245]. The interplay of viral, immune, and host genetic factors in the control of hiv-1 replication has been recently evaluated in hiv controllers [246, 247]. Similar efforts should be undertaken in diverse human populations infected with a variety of hiv-1 subtypes in order to understand fully the complex interplay of virus and host in aids pathogenesis . Hiv diversity plays a central role in the hiv pandemic; for this reason, it is today imperative that global molecular epidemiology surveillance is continued and improved using rigorous sampling strategies . The improved knowledge of the significance of non - b subtypes for resistance evolution and interpretation, in response to antiretroviral therapy, disease progression and vaccine design are becoming mandatory today not only because antiretroviral therapy is being introduced in countries where non - b subtypes are driving the epidemic but also because the number of infections by these variants is increasing sharply in previously subtype b homogeneous areas such as europe and north america [5769, 7173, 248]. It is reassuring that current antiretroviral strategies appear to be effective against a broad spectrum of hiv subtypes . However, further ad hoc studies in larger and homogenous populations infected by different specific non - b subtypes are required to better evaluate their therapeutic implications in terms of long - term efficacy of different antiviral regimens . Further elucidation of viral polymorphisms and properties associated with transmission, viral load set point, and disease progression may lead to new approaches to disease prevention and treatment . Efforts should be undertaken in diverse human populations infected with a variety of hiv-1 subtypes in order to fully understand the complex interplay of hiv and host in aids pathogenesis.
Transurethral resection of the prostate (turp) is still viewed as the benchmark for surgical therapies for benign prostatic enlargement (bpe). Even though the mortality and short - term morbidity rates following turp in contemporary series are much lesser than in the older series (0.1% vs. 0.2% mortality rate, 11.1% vs. 18% morbidity rate), they still remain an area of concern to the urologist . The learning curve for turp, as most urologists would agree, ranges from 50 - 100 cases and this level of experience is often not reached by the end of the residency training period because of the dramatic decrease in the overall turps being performed annually . These, among other factors have driven the search for a safer, easier to perform alternative that is as efficacious as turp for the surgical management of bpe . Among the emerging surgical therapies for bpe, it is selectively absorbed by hemoglobin within prostatic tissue, thus permitting photoselective vaporization of prostate (pvp). Ktp - pvp is considered to be easier to learn and perform than turp and competence occurs following 5 - 20 procedures . Even though ktp - pvp is being done now for well over a decade, data comparing ktp - pvp with standard we performed a prospective, randomized study to examine the efficacy and safety profile of ktp - pvp when compared to standard turp . The study protocol and all procedures were approved by the institutional ethics committee . Between february 2009 and august 2009, consecutive patients attending the urology opd with lower urinary tract symptoms (luts) secondary to benign prostatic enlargement (bpe) who satisfied the eligibility criteria (inclusion / exclusion criteria) and who were planned for surgery according to the international bph guidelines of the american urology association were included in this prospective, randomized study . Inclusion criteria: a] age> 50 years, b] ipss>7, c] prostate volume (trus):> 20 and <80 cc, d] q max <15 ml / sec . E] patient presenting with an indwelling foley's catheter where indication for catheterization was chronic retention(pvru> 300). G] patients receiving alpha blocker/5 alpha reductase inhibitor drugs / herbal medications believed to be active in prostate . Initial evaluation included a detailed clinical history including the international prostate symptom score (ipss), quality of life (qol) score and international index of erectile function (iief5) score, physical examination including digital rectal and focused neurological examination, urinalysis, serum prostate specific antigen (psa) measurement, prostate volume estimation by transrectal ultrasound (trus), post - void residual urine (pvru) measurement by abdominal ultrasound and qmax (maximum flow rate) measurement on uroflowmetry (ufr). {pvru and ufr measurement were not done in patients who were on catheter consequent to acute retention of urine . Such patients were eligible if all other criteria were met with}. If the digital rectal examination was abnormal and/or the psa was> 4 ng / ml, a 12-core trus - guided prostatic biopsy was performed preoperatively to rule out prostate cancer . All procedures were performed by one of the three consultant urologists in our department, each of whom were skilled in turp but had performed <5 ktp - pvp prior to this study . All three surgeons performed nearly equal number of ktp - pvps and turps . For pvp, a continuous flow 23f laserscope was used . The lens employed was a 30-degree lens and the irrigant used was 0.9% sodium chloride . The fiber was a 600 micron, 70 degree side firing laser fiber emitting green light at 532 nm . At first the median lobe (when present) was lased and thereafter the lateral lobes were lased in a symmetrical manner . Tissue was vaporized down to the prostatic capsule until an unobstructed view of the trigone and a turp like cavity was obtained . Vaporization was achieved by moving the laser fiber slowly and constantly in a paint brush fashion taking care to keep the fiber in near contact with the prostatic tissue . If any bleeding vessels were encountered during vaporization, coagulation was accomplished by defocusing the laser fiber (increasing working distance to 3 - 4 mm) or by reducing the power setting to 30w from 80w . A standard tungsten cutting wire loop at a setting of 160 w cutting and 80 w coagulation was used . The resection was carried down to the surgical capsule from bladder neck up to the verumontanum . Since all procedures (in both groups) were done under spinal anesthesia, in all patients, an indwelling 22f three - way foley's catheter was inserted into the bladder . Irrigation with 0.9% normal saline was started postoperatively if deemed necessary until the urine was sufficiently clear . As an institutional policy, catheter is removed 24 h after the irrigation is stopped and the urine is sufficiently clear . For the purpose of this study, catheter removal was decided by a urologist who was unaware of the procedure that the patient had undergone . Patients who failed trial without catheter were recatheterized and a voiding trial was given again after one week . All patients received an intravenous antibiotic at induction and an oral antibiotic was continued till five days post catheter removal . Intraoperative and postoperative parameters recorded included the operative time (measured as equivalent to the time the resectoscope / laserscope was inside the urethra), amount of irrigation fluid used intraoperatively, whether postoperative irrigation was instituted, amount of irrigation fluid used in the postoperative period, duration of postoperative irrigation, duration of catheterization and postoperative hemoglobin concentration . All patients were followed up in the urology outpatient department at 1, 3, 6 and 12 months . At each follow - up visit, ipss, qol, iief 5, qmax, pvru, residual prostate volume (assessed by transrectal ultrasound) and complications, if any were recorded . Neither the patient nor the outcome assessor was blinded to the procedure the patient had undergone . Primary outcome measures for group analysis (pvp vs. turp) included: subjective (ipss, qol, iief5) parametersobjective (prostate volume, pvru and qmax) parameters . Subjective (ipss, qol, iief5) parameters objective (prostate volume, pvru and qmax) parameters . Secondary outcome measures for group analysis included: perioperative parameters: operative time, amount of irrigation fluid used intraoperatively, whether postoperative irrigation was instituted, amount of irrigation fluid used in the postoperative period, duration of postoperative irrigation, duration of catheterization (defined as time to initial removal of the catheter), postoperative hemoglobin concentration (sample taken on the morning after surgery).complications, if any . Perioperative parameters: operative time, amount of irrigation fluid used intraoperatively, whether postoperative irrigation was instituted, amount of irrigation fluid used in the postoperative period, duration of postoperative irrigation, duration of catheterization (defined as time to initial removal of the catheter), postoperative hemoglobin concentration (sample taken on the morning after surgery). Observations were recorded and arranged on a microsoft excel spreadsheet (microsoft, seattle, wa usa) and analyzed by spss version 12.0 (spss inc ., chicago, il), software package . The calculated sample size was 130 patients (65 per arm) with a power of 80% . The two - tailed student t - test was used as a statistical tool to see the significance level between the groups . Categorical data in perioperative or complications outcome were analyzed by the nonparametric mann - whitney all procedures were performed by one of the three consultant urologists in our department, each of whom were skilled in turp but had performed <5 ktp - pvp prior to this study . The lens employed was a 30-degree lens and the irrigant used was 0.9% sodium chloride . The fiber was a 600 micron, 70 degree side firing laser fiber emitting green light at 532 nm . At first the median lobe (when present) was lased and thereafter the lateral lobes were lased in a symmetrical manner . Tissue was vaporized down to the prostatic capsule until an unobstructed view of the trigone and a turp like cavity was obtained . Vaporization was achieved by moving the laser fiber slowly and constantly in a paint brush fashion taking care to keep the fiber in near contact with the prostatic tissue . If any bleeding vessels were encountered during vaporization, coagulation was accomplished by defocusing the laser fiber (increasing working distance to 3 - 4 mm) or by reducing the power setting to 30w from 80w . A standard tungsten cutting wire loop at a setting of 160 w cutting and 80 w coagulation was used . The resection was carried down to the surgical capsule from bladder neck up to the verumontanum . Since all procedures (in both groups) were done under spinal anesthesia, in all patients, an indwelling 22f three - way foley's catheter was inserted into the bladder . Irrigation with 0.9% normal saline was started postoperatively if deemed necessary until the urine was sufficiently clear . As an institutional policy, catheter is removed 24 h after the irrigation is stopped and the urine is sufficiently clear . For the purpose of this study, catheter removal was decided by a urologist who was unaware of the procedure that the patient had undergone . Patients who failed trial without catheter were recatheterized and a voiding trial was given again after one week . All patients received an intravenous antibiotic at induction and an oral antibiotic was continued till five days post catheter removal . Intraoperative and postoperative parameters recorded included the operative time (measured as equivalent to the time the resectoscope / laserscope was inside the urethra), amount of irrigation fluid used intraoperatively, whether postoperative irrigation was instituted, amount of irrigation fluid used in the postoperative period, duration of postoperative irrigation, duration of catheterization and postoperative hemoglobin concentration . All patients were followed up in the urology outpatient department at 1, 3, 6 and 12 months . At each follow - up visit, ipss, qol, iief 5, qmax, pvru, residual prostate volume (assessed by transrectal ultrasound) and complications, if any were recorded . Neither the patient nor the outcome assessor primary outcome measures for group analysis (pvp vs. turp) included: subjective (ipss, qol, iief5) parametersobjective (prostate volume, pvru and qmax) parameters . Subjective (ipss, qol, iief5) parameters objective (prostate volume, pvru and qmax) parameters . Secondary outcome measures for group analysis included: perioperative parameters: operative time, amount of irrigation fluid used intraoperatively, whether postoperative irrigation was instituted, amount of irrigation fluid used in the postoperative period, duration of postoperative irrigation, duration of catheterization (defined as time to initial removal of the catheter), postoperative hemoglobin concentration (sample taken on the morning after surgery).complications, if any . Perioperative parameters: operative time, amount of irrigation fluid used intraoperatively, whether postoperative irrigation was instituted, amount of irrigation fluid used in the postoperative period, duration of postoperative irrigation, duration of catheterization (defined as time to initial removal of the catheter), postoperative hemoglobin concentration (sample taken on the morning after surgery). Observations were recorded and arranged on a microsoft excel spreadsheet (microsoft, seattle, wa usa) and analyzed by spss version 12.0 (spss inc ., chicago, il), software package . The calculated sample size was 130 patients (65 per arm) with a power of 80% . The two - tailed student t - test was used as a statistical tool to see the significance level between the groups . Categorical data in perioperative or complications outcome were analyzed by the nonparametric mann - whitney of 164 patients screened, 128 were found eligible and were randomized, 64 each to group a and b respectively, of which four and seven patients in group a and b respectively were subsequently excluded leaving 60 and 57 patients in group a and b respectively available for analysis . Follow - up data at 1, 3, 6 and 12 months was available for 60, 60, 58, 52 and 57, 55, 50, 50 patients in group a and b respectively [figure 1]. Allocation and dispersion of patients the baseline characteristics of the two groups including mean age, ipss score, qol score, iief-5 score, prostate volume, serum psa, qmax, pvru, and preoperative hemoglobin were similar with no significant differences noted [table 1]. Baseline characteristics follow - up data is summarized in table 2 . In both groups, there was significant improvement in the ipss score, qol score, prostate volume, qmax and pvru, as compared to the baseline at each of the follow - up visits with the most dramatic improvement being seen at the first month follow - up . Iief-5 scores did not show any significant improvement in either group at any of the follow - up visits . Overall ipss score decreased by 70.17% and 71.26%, qol score decreased by 61.71% and 62.15%, prostate volume decreased by 41.32% and 46.96%, qmax increased by 171.52% and 192.89%, pvru decreased by 83.53% and 85.76% at 12 months in group a and b, respectively . Between the two groups, there was no significant difference in the ipss score, qol score, iief-5 score, prostate volume, qmax and pvru at each of the follow - up visits except for qol score at one month which was significantly better in group b. data pertaining to the perioperative period is summarized in table 3 . Operative time was significantly longer in group a when compared to group b. the need, amount and duration of postoperative irrigation along with duration of postoperative catheterization were all significantly lesser in group a as compared to group b. the postoperative hemoglobin percentage was significantly higher in group a as compared to group b. the complications in each of the two groups are summarized in table 4 . Although the overall complication rate did not differ significantly between the two groups, the rate of clot retention and that of blood transfusion was significantly higher in group b when compared to group a. dysuria in the early postoperative period was significantly more common in group a as compared to group b [15 in group a vs. 5 in group b]. Dysuria resolved by the end of one month in all except one patient of group a in whom it took three months to resolve . As per the claviendindo classification of surgical complications, no significant differences between the two groups were noted insofar as the different grades of complications were concerned . Amongst the three operating surgeons, the green light laser system, initially introduced as part of a hybrid technique with nd: yag laser, evolved to stand alone green light laser with gradually increasing power as studies proved efficacy and safety . The first clinical experience with pure ktp - pvp was reported by malek in 1998 who used a 60w laser . Various authors with follow - up ranging from one to five years have reported favorably on the efficacy and safety profile of ktp - pvp. [815] further, favorable outcomes have been reported for ktp - pvp even in high - risk patients and in those with larger glands. [1619] insofar as data comparing ktp - pvp with turp is concerned, the literature is sparse . The first prospective though non - randomized comparison between ktp - pvp and turp with a six - month follow - up was reported by bachmann et al . They found that though the overall perioperative complication rates between the two groups were similar, patients undergoing pvp had significantly lesser drop in hemoglobin percentage and serum sodium postoperatively . As far as improvement in ipss, qol, qmax and pvru was concerned, no significant difference was noted between the two groups at six months . Intermediate term results with two - year follow - up of the above mentioned prospective non - randomized study were published in 2008 . The authors reported that the rates of intraoperative bleeding, blood transfusion, capsular perforation and postoperative clot retention were significantly lower in patients in the pvp group whereas the incidence of urethral and bladder neck strictures did not differ significantly between the two groups . Catheterization time and hospital stay were significantly lesser in the pvp group for patients in the <70 years and 70 - 80 years subgroups . Though improvement in qmax and decrease in prostate volume and psa were significantly higher in the turp group, improvement in ipss and pvru did not differ significantly between the two groups . The authors concluded that ktp - pvp was more favorable in terms of perioperative safety and had comparable functional outcomes . A single prospective randomized comparative study between ktp - pvp (80w) and turp has been published so far . The authors, bouchier - hayes et al ., having earlier published interim results in 2006, recently published their final data . Of 119 patients randomized, 10 refused surgery leaving 50 and 59 patients who underwent turp and ktp - pvp respectively . Of these 39 and 46 patients in the turp and ktp - pvp groups respectively, were available for analysis at 12 months . Both groups showed significant improvements in ipss, qol, bother score, qmax and pvru at 12 months when compared to the baseline with no significant difference noted between them . Patients undergoing ktp - pvp had significantly shorter catheterization time (13 vs. 44.2 h), shorter length of inpatient stay (1.09 vs. 3.6 days) and lesser blood loss . Our study has shown that at one - year follow - up, efficacy of ktp - pvp is comparable to turp with no significant differences noted . As far as safety is concerned, ktp - pvp was associated with significantly less blood loss, clot retention and transfusion rates, even though the overall complication rate did not differ significantly when compared to the turp group . Similar overall complication rates in this study must be interpreted in the context that while the operators were skilled in turp prior to the start of this study, the learning curve of the operators for ktp - pvp is part of this study . A case in point is the rate of transient dysuria in group a. in our study, 25% of ktp - pvp patients complained of transient dysuria in the early postoperative period, a much higher rate than that reported in other ktp - pvp series . Dysuria is primarily caused by coagulation rather than vaporization of the tissue and its severity correlates with the volume of coagulated tissue . . A possible explanation of the higher rate of dysuria in our study could be the limited experience of the operators with pvp to begin with, since in the first 10 cases of each operator, 3, 4 and 4 patients respectively developed transient dysuria . Subsequently, in the remaining 30 patients who underwent pvp, only 4 developed transient dysuria . One drawback of this study is that since patients with prostate volumes> 80 cc were excluded, the results of this study cannot be extrapolated to bpe patients with larger prostates . Further, though the efficacy of ktp - pvp is evident up to the one - year follow - up, no comment can be made on long - term durability . Ktp - pvp is an equally efficacious alternative to turp in the management of luts due to bpe with durable results at one - year follow - up . It has the added benefits of significantly lesser perioperative blood loss and transfusion requirements along with a shorter catheterization time . More long term studies are needed to clearly define the place of ktp - pvp in the management of patients with bpe.
Understanding the causes of human disease is one of the most fundamental goals of modern medicine . Identifying the factors contributing to these differences, and elucidating their interactions as they contribute to aspects of disease phenotype, is a precursor to improved prevention, detection and treatment of disease . Much of the understanding of human disease derives from the study of those diseases that segregate in families in a mendelian fashion, where the causative variants and the genes in which they reside have been identified through classical family linkage approaches and through studies in large pedigrees and in isolated populations based on founder effects . The vast majority of common diseases exhibit a more complex mode of inheritance, however, aggregating in families but rarely exhibiting mendelian inheritance . Examples of diseases of this type include diabetes, obesity, schizophrenia and asthma . Understanding of these' complex' diseases is improving, although still limited, but it is clear that genetic variation plays an important role in susceptibility to disease, for example in autoimmune and infectious diseases . Most complex disease is thought to be caused by the combined effect of genetic variants at a few loci or multiple loci, each with only modest functional effects on susceptibility . Mapping the genomic regions contributing to disease creates new directions for disease research and is an important step towards improving human health . Approaches to identifying the genes involved in complex disease can be generally grouped into two categories: candidate gene studies and linkage / association studies . Candidate gene studies use knowledge about the biology of a disease, and about genes in physiologically or biochemically relevant pathways, and attempt to correlate genetic variation at these' candidate genes' with disease phenotype . Unfortunately, for most diseases, this type of information is not available or complete enough to prove widely useful, and including them in some of the analyses is more likely to increase the noise than it is to reduce the search space for the disease . Genome - wide linkage studies and association analyses serve as alternative approaches to surveying the contribution to disease of genetic variants located anywhere in the genome . The genome - wide aspect means that these studies do not require any a priori hypothesis that a particular region is involved, although predictions about the potential effect of specific variants (eg non - synonymous single nucleotide polymorphisms [snps]) can be incorporated in the models . In this respect, these approaches are unbiased . Family - based linkage studies entail identifying genetic variants in families that co - segregate with disease more often than would be expected by chance . In general, linkage studies have achieved limited success in identifying genomic regions involved in complex disease, in part because they are underpowered to detect moderate genetic effects . Furthermore, because identification of a region or regions associated with the disease or trait requires identifying those alleles that segregate with the disease in families, which in turn depends on recombination within the families, it can be difficult to narrow a region exhibiting significant linkage . An alternative methodology is to perform association analysis, which looks for correlation of genetic variants with aspects of phenotype, but does not require a pedigree structure for the individuals . Association analyses are more powerful for the detection of common disease alleles with small to modest effects, and increasingly are being used successfully in studies to identify genes contributing to disease . Although many phenotypic differences among individuals are attributable to variants in coding dna, variants in noncoding dna can have profound effects on phenotypes, including disease phenotypes . For example, regulatory variants affecting transcription initiation, splicing, rna stability and translational efficiency are known to play roles in conditions including autoimmune disease (ctla4), malaria (darc), various cancers (smyd3) and other examples (shown in table 1). In studies of such diseases, gene expression may serve as an intermediate phenotype between disease phenotype and genotype . Gene expression, or mrna levels, can be modulated by variants in coding or non - coding dna (eg transcription factors or binding sites within promoters). Whole - genome association studies of gene expression (expression quantitative trait loci [eqtl] mapping) may generate hypotheses for disease susceptibility by identifying those regions of the genome with functional effects on gene expression . These might then serve as candidate regions for evaluating for association with disease phenotypes, as is discussed below . An efficient approach to the study of human disease benefits from the use of shared resources . For example, in order to perform genome - wide linkage or association analyses, suitable dna markers are required . The human genome is estimated to harbour more than 10 million snps, present at> 1 per cent frequency, and these snps are located throughout the genome in regions of coding and non - coding dna . Publicly available databases of snp alleles, assays and genotypes are accessible online (eg dbsnp and hapmap). High - throughput genotyping platforms and reductions in genotyping costs now make whole - genome genotyping feasible for large numbers of samples . Gene expression can also be quantified in a high - throughput manner using commercially available microarrays, permitting the detection of small differences in expression levels among samples . The establishment of cell lines creates resources that can be used by multiple research groups from around the world to survey various cellular phenotypes . With respect to the study of gene expression, it is desirable to establish cell lines from different tissues because gene expression is highly dependent on developmental and cellular context and, indeed, some diseases manifest their phenotypes only in specific tissues . In addition, the cell perturbations that accompany the establishment of cell lines suggest the study of gene expression in primary tissues, although, clearly, the choice of sample depends on the purpose, stage and feasibility of a study, the sample size required and its availability . Despite some shortcomings of cell lines as perfect proxies for the complete set of human tissues, data can be collected on a large scale with respect to sample size and reproducibility and can provide candidates for further study in other samples . Currently, there are relatively few data on gene expression across the diversity of healthy human tissues or across multiple individuals from different populations . These data from healthy individuals will provide important information on the range of naturally occurring gene expression variation and will serve as a baseline against which to compare disease - associated molecular phenotypes . Although genome - wide association studies are thought to have more power than family - based linkage studies, they present strong challenges in the form of statistical interpretation . For example, a simple genome - wide association study may test hundreds of thousands of snps for association to a phenotype (or, more typically, multiple phenotypes), and more complicated models allowing for snp - snp interactions vastly increase the already large number of statistical tests . With such a large number of tests, the significance threshold must be adjusted to control for the number of false - positive associations . Although procedures for multiple test correction exist--for example, bonferroni correction, false discovery rate and permutations of phenotypes relative to genotypes--it remains unclear which is the best method to apply in this context . It is also not trivial to infer the biological significance of an association from statistical significance, because allele frequencies, variance of the phenotype, density of markers and linkage disequilibrium (ld) can have a tremendous impact on the statistical significance inferred . Human genetic variation is structured into haplotypes, such that alleles at nearby loci often show strong statistical association with one another . Because of this association, known as ld, a large region may contain multiple snps exhibiting a significant association with a given phenotype . Although this structure of human genetic variation facilitates association mapping, it can complicate subsequent fine - scale mapping to narrow the associated region and locate the causal variant, as discussed below . Another concern in association studies is the potential for false associations caused by population stratification, so care must be taken to reduce these effects through appropriate experimental design and data analysis . The interrogation of gene expression to facilitate the design and interpretation of disease association studies can be a powerful tool for the identification of biologically functional variants and the interpretation of biological effects . By introducing a quantifiable and easily measurable biological outcome, it is possible to assess the relevance of statistical significance and eliminate some of the issues raised above . The use of gene expression variation in the context of disease mapping can be viewed in two ways (figure 1). On the one hand, hypotheses can be generated by discovering functional variation using eqtl mapping and subsequently testing those functional variants in large case - control samples . On the other hand, following a disease association study that has identified several signals located within regions of non - coding dna, eqtl mapping can be used to interpret and dissect the functional effect of the candidate disease variants . Flow charts demonstrating the use of genome - wide gene expression studies in relation to disease studies . . Expression quantitative trait loci (eqtl) mapping studies identify variable regions of the genome with functional effects on gene expression . (b) supporting disease association studies . Disease mapping studies often identify non - coding regions of the genome exhibiting significant association with disease . Eqtl studies can provide a link to an associated gene by providing annotation of the function of that non - coding region . Regions with functional effects on gene expression can be localised through the use of association mapping . Gene expression, or mrna level, is a quantitative phenotype that can be assayed in multiple individuals . When the same individuals are surveyed for genetic variation at marker loci, for example snps, association analysis tests whether variation at each snp can explain the observed phenotypic variation . The rationale behind this analysis is that markers themselves are either the causal variant or are highly correlated (in ld) with the causal variant . Association mapping of gene expression variation has been successful in many species, including human [42 - 45], yeast [46 - 48], mouse [49 - 52], rat, fish and maize . Together, these studies provide several striking observations related to the nature of functional variation influencing gene expression . First, variation in gene expression levels among individuals is common--and much of that phenotypic variation has a genetic basis . Much of the association signal is located cis- to the gene of interest, although trans - acting variants have also been observed . Hotspots of gene regulation (ie regions of the genome influencing expression of several genes) have been observed in some, but not all, studies . There are several ways in which the study of the regulation of gene expression can enhance disease studies as well as narrow the choice of candidate regions for disease association studies . Where information exists about the contribution of particular genes to a disease phenotype or susceptibility, understanding the regulatory control of those genes may assist in elucidating the complete set of effects . In addition, understanding the regulation of categories of genes, or genes of a particular pathway, may provide targets for further follow - up in disease studies . It may prove more time - efficient and cost - effective to have a list of many potential functional variants located throughout the genome, however, and test them against a large number of diseases . Whole - genome eqtl studies can provide a list of regions of the genome with functional effects on the expression of known genes (figure 1a). Snps located within these regions can then serve as candidates for disease association studies, much in the same way that non - synonymous snps are often considered because of their potential functional effect . There are several advantages to this type of targeted approach over a whole - genome scan . First, because the number of snps to be genotyped in each individual is reduced, many more individuals can be surveyed in a disease study without vastly increasing costs . The reduction in the number of markers tested can eliminate some of the problems of multiple test correction, more sensible thresholds can be used and smaller effect variants can be detected . Secondly, any significant associations detected between snp and disease phenotype provide both a mechanism (gene regulation) and the identity of the affected gene . Finally, the fact that potential causal regulatory variants were initially discovered in healthy individuals and subsequently have been associated with disease means that such variants are common and are likely to contribute significantly to the disease risk of the population . The methodology above carries the risk of focusing only on certain types of genomic variants, while it is known that much of genome function is still missing . A way to circumvent this problem is to enhance disease studies by incorporating the data on functional regulatory regions while using commercially available whole - genome snp genotyping chips in disease studies, in order to perform the association analysis using bayesian methods that assign different prior probabilities to snps on the array . Under such a scenario, snps located in regions with known functional effects on expression of specific genes--as identified through eqtl studies--would be assigned a higher prior probability of being associated with a phenotype . In addition, one might assign a higher prior probability to snps in known promoters, enhancers or transcription factors . Thus, one could focus on the effects of candidate variants without missing other important signals . Another substantial advance of knowing regulatory variants before performing a genome - wide association study is that one can correlate phenotypes and regulatory networks and utilise such information in the statistical modelling of the disease . Regions with functional effects on gene expression can be localised through the use of association mapping . Gene expression, or mrna level, is a quantitative phenotype that can be assayed in multiple individuals . When the same individuals are surveyed for genetic variation at marker loci, for example snps, association analysis tests whether variation at each snp can explain the observed phenotypic variation . The rationale behind this analysis is that markers themselves are either the causal variant or are highly correlated (in ld) with the causal variant . Association mapping of gene expression variation has been successful in many species, including human [42 - 45], yeast [46 - 48], mouse [49 - 52], rat, fish and maize . Together, these studies provide several striking observations related to the nature of functional variation influencing gene expression . First, variation in gene expression levels among individuals is common--and much of that phenotypic variation has a genetic basis . Much of the association signal is located cis- to the gene of interest, although trans - acting variants have also been observed . Hotspots of gene regulation (ie regions of the genome influencing expression of several genes) have been observed in some, but not all, studies . There are several ways in which the study of the regulation of gene expression can enhance disease studies as well as narrow the choice of candidate regions for disease association studies . Where information exists about the contribution of particular genes to a disease phenotype or susceptibility, understanding the regulatory control of those genes may assist in elucidating the complete set of effects . In addition, understanding the regulation of categories of genes, or genes of a particular pathway, may provide targets for further follow - up in disease studies . It may prove more time - efficient and cost - effective to have a list of many potential functional variants located throughout the genome, however, and test them against a large number of diseases . Whole - genome eqtl studies can provide a list of regions of the genome with functional effects on the expression of known genes (figure 1a). Snps located within these regions can then serve as candidates for disease association studies, much in the same way that non - synonymous snps are often considered because of their potential functional effect . There are several advantages to this type of targeted approach over a whole - genome scan . First, because the number of snps to be genotyped in each individual is reduced, many more individuals can be surveyed in a disease study without vastly increasing costs . The reduction in the number of markers tested can eliminate some of the problems of multiple test correction, more sensible thresholds can be used and smaller effect variants can be detected . Secondly, any significant associations detected between snp and disease phenotype provide both a mechanism (gene regulation) and the identity of the affected gene . Finally, the fact that potential causal regulatory variants were initially discovered in healthy individuals and subsequently have been associated with disease means that such variants are common and are likely to contribute significantly to the disease risk of the population . The methodology above carries the risk of focusing only on certain types of genomic variants, while it is known that much of genome function is still missing . A way to circumvent this problem is to enhance disease studies by incorporating the data on functional regulatory regions while using commercially available whole - genome snp genotyping chips in disease studies, in order to perform the association analysis using bayesian methods that assign different prior probabilities to snps on the array . Under such a scenario, snps located in regions with known functional effects on expression of specific genes--as identified through eqtl studies--would be assigned a higher prior probability of being associated with a phenotype . In addition, one might assign a higher prior probability to snps in known promoters, enhancers or transcription factors . Thus, one could focus on the effects of candidate variants without missing other important signals . Another substantial advance of knowing regulatory variants before performing a genome - wide association study is that one can correlate phenotypes and regulatory networks and utilise such information in the statistical modelling of the disease . Genome - wide association studies are now increasing in frequency, and although it would have been preferable to have identified all functional regulatory variants in advance, investigators will be faced with the challenge of interpreting some of the strongest association signals . Many of the association studies have a multi - phase design, wherein a fraction of the snps with the top statistical significance in the first phase are genotyped in a subsequent phase in a new set of individuals . The statistical exercise must eventually give way to biological interpretation, however, and the identification of the causal variant will be necessary . Although most of the confirmed disease - causing variants are located in coding regions, this observation is due to an ascertainment bias in the ability to predict the potential functional consequences of nucleotide variation . As the human genome is composed of only ~3 -5 per cent coding dna, and studies increasingly attribute function to non - coding dna, it might be expected that much of the disease - causing variation will be non - coding and that many of the significant peaks in an association analysis will fall in regions devoid of genes . Disease association studies often identify non - coding regions of the genome exhibiting significant association with disease . The exploration of those non - coding regions will benefit from the survey of gene expression variation and how it relates to genetic variation (eqtl mapping). For any disease - associated non - coding region (eg from a case - control study), it is possible to test whether the disease - associated snps and haplotypes are also associated with gene expression variation of nearby genes (as identified from eqtl studies; figure 2). This enables conclusions to be drawn about the nature of the function of the causal variant . For instance, if the same haplotype that appears to increase the disease risk also appears to be associated with high expression of a nearby gene, it is possible to start making some connections between the biology of the affected gene and the disease itself . Moreover, one can hypothesise (and hopefully test) how levels of expression of a gene might affect disease risk . This simple connection between the two types of study could provide not only the identity of the gene that is linked to the disease, but also the consequence of genome variation that linked the gene with the disease . It may also provide some clues about other candidates (upstream transcriptional regulators, interacting proteins etc). The three panels show the signal of association (as - log p - value of individual single nucleotide polymorphism (snp) - phenotype associations) of genomic regions with disease, gene expression of gene a and gene expression of gene b. from this plot, it is suggested that genetic variation that increases disease risk is also associated with gene expression variation of gene a (assuming that the associated snps and haplotypes are the same). This probably indicates that the disease risk is a regulatory effect and that the amount of transcript (or protein) of gene a is critical for the development of the disease . Several studies illustrate the utility and validity of using gene expression variation for disease fine mapping . Two of these studies have focused on identifying functional nucleotide variation by focusing primarily on the regions surrounding each of a set of genes (cis-), but also considering other regions located trans- to the genes . These studies showed that a large fraction of genes (10 - 20 per cent) have significant variation that affect their gene expression in cis, and in some cases in trans . The regulatory variants that affect gene expression variation can be mapped with the same resolution as disease variants in genome - wide association studies because, in both cases, the resolution depends on the ld structure of the human populations . These studies, which allow the identification of regulatory haplotypes, need to be verified before functional experiments can be performed . The most appropriate way to perform a first - pass verification is to test whether allelic imbalance in expression is correlated with heterozygosity in the same snps as those that showed genotypic association with gene expression . Even with this information, and the fact that the effect of a causal variant may be known to have an effect on gene regulation, it is still a long way from being able to identify the exact dna variant that causes the regulatory effect and subsequent increased disease risk . For example, although the genome - wide distribution of ld is quite variable, average ld in the human genome extends over large regions, which makes it challenging to fine map a causal variant in many regions . In the best case scenario, associated snps would be identified in a region of very low ld, thus reducing the number of potential causal variants to test subsequently . More often, an associated region of approximately 10 - 20 kilobases will be identified . Although fine mapping in a population with reduced ld (eg africans) might assist in identifying shorter associated regions, it is at this stage where extensive amounts of information about genome function are crucial . The diversity of methodologies for large - scale interrogation of the human genome for function is increasing; the resulting information will be very important for prioritising which of those associated dna segments to focus on first . Many studies to determine functionality within the human genome sequence are now in progress using high - throughput, genome - wide methodologies . The encyclopedia of dna elements (encode) project is the best example of this type of study . The aim of this project is to attribute a functional identity to each nucleotide of the human genome . In its pilot phase, 1 per cent of the human genome (44 genomic regions) has been studied extensively for function, interspecies conservation and population genetic variation . The comprehensive analysis of these 44 regions will provide important clues for the pattern and structure of genome function and will allow predictions for the nature of variations behind complex disease and phenotypic variation . This, and other ongoing studies, will offer a first - pass annotation of functional elements in the human genome and will provide the framework for detailed characterisation of functional variation . If an established and confirmed association of a region with disease and gene expression variation exists, and there is light annotation of the associated region for coding and non - coding elements, it is possible to apply brute force approaches to identify the specific dna changes that are causal . A recommended strategy is to perform extensive resequencing of potentially functional segments of the region in high and low expressing individuals . The number of individuals required to be assayed depends on the magnitude of the functional effect and the predicted within - population frequency of the causal variant . This can be assisted by initial power calculations that allow prediction of what is likely to be identified, given the study design . The optimal approach seems to be to sample sequences from individuals at each of the two ends of the phenotypic (expression) distribution, and then proceed inwards . As soon as a set of genomic segments have been resequenced, one should look for variants that appear to have equal or better correlation with the phenotype than that observed in the initial association study . This can be determined by genotyping all of the potentially functional variants (identified in the resequencing approach in a subset of the individuals) in the original complete sample . Depending on the strength of these correlations, and where the highly correlated variants are located, the appropriate approach should be adopted for direct functional testing of causal haplotypes . Such approaches can include reporter constructs, binding assays, rna stability assays and chromatin modification assays using all of the alternative haplotypes . In this paper, some issues have been discussed that arise from the incorporation of gene expression variation data in disease studies . The overall message is that gene expression can greatly assist the discovery of disease variants, as well as the interpretation of the biological effects of causal variants . Further exploration of gene expression variation in more samples and more cell types will greatly enhance both our understanding of phenotypic variation in humans and also the nature of regulatory variation and its impact on complex disease.
Francisella tularensis is a gram - negative facultative intracellular bacterium and the causative agent of tularemia, a systemic infection of many mammals including humans . Left untreated, the virulent type a subspecies of f. tularensis routinely caused lethal infection in people particularly after aerosol exposure to the pathogen; as few as 10 virulent type a bacilli can initiate severe disease . Consequently, f. tularensis is considered a category a biological warfare agent . Despite its clinical and biosecurity importance, the molecular basis for the immunopathogenesis of f. tularensis infection, particularly when initiated through the respiratory tract, remains largely unknown . Lymphotoxin- (lt) is a member of the tumor necrosis factor (tnf) superfamily of cytokines and has two distinct roles: as a membrane - bound heterotrimer in combination with lt, it binds the lt receptor and is critical in the development and maintenance of organized secondary lymphoid organs, and as a soluble homotrimer, it signals through the tnf receptor pathway and leads to activation of various inflammatory cytokines and chemokines [3, 4]. Indeed, lts (lt and lt), together with tnf and light (lt - related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells), form an integrated signaling network which is important for the regulation of both innate and adaptive immune responses . In this regard, lt has been implicated in the host defense against several different bacterial, viral, and parasitic pathogens (reviewed in). For instance, several studies have shown that after infection with mycobacterium tuberculosis, mice genetically defective in lt (lt/ mice) harbour increased bacterial burdens and exhibited a shorter median time to death when compared to lt+/+ mice [57]. Similarly, mice deficient in lt, lt, and the lt receptor (ltr) are more susceptible to listeria monocytogenes infection than wild - type mice [6, 7]. Given that lt is important in the control of these intracellular bacterial pathogens, in the present study we sought to determine whether it also plays a role in host defense against low - dose aerosol infection with a virulent type a strain of f. tularensis . Eight- to twelve - week old, age - matched b6.129s2-lt/j (lt/), and wild - type c57bl/6j (lt+/+) mice were used in this study . The foundation breeding pairs of lt/ mice were purchased from jackson laboratories (bar harbor, me, usa). Mice were bred and housed under specific - pathogen - free conditions in a federally licensed animal biosafety level-3 facility and given free access to sterile water and certified mouse chow . The animals were maintained and used in accordance with the recommendations of the canadian council on animal care guide to the care and use of experimental animals . Stocks of type a f. tularensis strain fsc33/snmf, originally isolated from a squirrel in georgia, usa . For low - dose aerosol exposure, thawed f. tularensis stocks were diluted in mueller hinton broth containing 20% (v / v) glycerol to maintain infectivity at the high - relative humidity employed . Aerosols of f. tularensis strains were generated with a lovelace nebulizer operating at a pressure of 40 p.s.i . To produce particles in the 46 m range required for inhalation and retention in the alveoli . Mice were exposed to these aerosols for 7 minutes (inhaled dose of ~10 organisms) using a customized commercial nose only exposure apparatus (in - tox products, albuquerque, nm) resulting in the implantation of 1020 organisms into the lungs . Groups of five mice of each strain were sacrificed at 2 and 4 days after inoculation (dpi) by co2 asphyxiation . The phenotype of the lt/ mice was confirmed by visual inspection at necropsy to confirm the absence of peripheral lymph nodes . The lungs and spleens were removed aseptically, cut into small pieces, and then homogenized using an aerosol - proof homogenizer for quantitative bacteriology or fixed immediately by immersion in 10% neutral buffered formalin for histopathology . For quantitative bacteriology, ten - fold serial dilutions of the tissue homogenates were plated on cysteine heart agar supplemented with 1% (w / v) hemoglobin and sulfamethoxazole and trimethoprim . The tissues were processed by standard paraffin embedding methods (department of pathology and laboratory medicine, university of ottawa, ottawa, ontario). Sections were cut 4 m thick, stained with haematoxylin - eosin (he) and examined by light microscopy . In some experiments, the lungs were lavaged with five 1.0-ml aliquots of pbs supplemented with 3 mm edta, and the total lavage cell numbers were counted on a haemocytometer, and differential cell counts were carried out on cytospin preparations stained with hema3 stain set (fisher scientific, middletown, va, usa). The bronchoalveolar lavage (bal) fluid was centrifuged at 3000 xg for 7 minutes, supernatants were removed, sterile - filtered, and stored at 80c . Serum and bal levels of cytokines and chemokines were determined using beadlyte mouse 21-plex cytokine detection system (upstate, temecuta, calif, usa) on a luminex 100is system (luminex, austin, tex, usa). Undiluted bal and 1: 2 diluted serum samples (50 l) were analyzed as specified by the manufacturer (http://www.millipore.com/userguides/tech1/ proto_mpxmcyto-70k). The analysis was done in duplicate, and the cytokine / chemokine concentrations were calculated against the standards using beadview software (ver 1.03, upstate). All data are presented as mean standard deviation (sd) for each group . Differences between groups were analyzed by two - way anova followed by the bonferroni multiple comparison test (graphpad prism 4.0, graphpad software, inc . Initially, a total of 22 lt/ and 14 lt+/+ mice were challenged by low - dose aerosol with virulent type a f. tularensis and their survival monitored . With the exception of two lt/ mice and one lt+/+ mouse, all mice succumbed to infection between day 4 and 7 with a median time to death of 5 days (range 46 days for lt/ mice and 57 days for lt+/+ mice, p>.05 by kaplan - meier survival analysis) (figure 1), indicating that lt/ mice are no more susceptible to low - dose aerosol challenge with this strain of the pathogen than control lt+/+ mice . It has been previously reported that increased susceptibility of certain immunocompromised mice to intradermal infection with the live vaccine strain (lvs) of f. tularensis and oral type a f. tularensis infection is only apparent when using a very high inoculum . To examine the possible effect of inoculum size on the need for lt expression to control respiratory infection with type a f. tularensis, groups of lt/ and lt+/+ mice were intranasally challenged with 10, 100, and 1000 cfu type a f. tularensis and their survival monitored . This study revealed that the ld100 of type a f. tularensis for lt/ and lt+/+ mice was comparable in that all mice died of the infection by dpi 5 (data not shown). These results indicate that lt does not appear to play a significant role in determining the clinical outcome of respiratory infection with various doses of type a f. tularensis in mice . Since type a strains of f. tularensis are extremely virulent for mice even at the minimum challenge dose, it remained possible that subtle effects of lt expression were overlooked by the above relatively crude survival experiments . Therefore, we next examined whether lt contributes to the control of f. tularensis replication and systemic dissemination by comparing the bacterial burdens in the lungs and spleens of lt/ and lt+/+ mice at dpi 2 and 4 following aerosol challenge (figure 2). There was no difference in the bacterial burdens in the lungs, the primary site of infection, between lt/ and lt+/+ mice at dpi 2 . However, the bacterial burdens in the spleens of lt/ mice were about 1 to 1.5 log, but not statistically significant, lower than those in lt+/+ mice at this time point . By dpi 4, lt/ mice had approximately 10-fold more bacteria in their lungs than did lt+/+ mice (p <.01), and the bacterial burdens in the spleens of lt/ mice were also higher, although not statistically significant, than those in lt+/+ mice . Hence, these data imply that although lt is not sufficient to control virulent f. tularensis infection, it may play a minor role in the initial dissemination of f. tularensis from the lung to spleen and subsequent multiplication of the pathogen in the lungs and spleen . Histopathologically, both lt/ and lt+/+ mice showed moderate inflammatory infiltrations in the livers and spleens and mild, focal bronchopneumonia at dpi 2, and by dpi 4 moderately severe necrotic hepatitis, lymphoid follicle destruction in the spleen, and bronchopneumonia . However, as would be expected from the quantitative bacteriology and survival data, no overt differences in tissue histopathology or blood clinical chemistry (data not shown) were observed between lt/ and lt+/+ mice following aerosol exposure to type a f. tularensis . Early pulmonary recruitment of inflammatory cells and local and systemic production of proinflammatory cytokines are considered important characteristics of innate host responses against respiratory infections including f. tularensis . Previous studies have shown that respiratory infection of mice with type a and attenuated live vaccine strain of f. tularensis upregulates a number of proinflammatory cytokines, which play important roles in host defense against f. tularensis infection [1315]. Therefore, we determined total and differential leukocyte counts in the bal fluid to identify the inflammatory cell influx into the lungs on dpi 2 and 4 . As can be seen in table 1, low - dose aerosol infection of mice with type a f. tularensis induced no significant difference in either the total cell number or the composition of cell populations (macrophage, neutrophil, and lymphocyte) in the lavage fluids of lt+/+ and lt / mice with the exception of a small but not significant increase in lymphocytes in lt/ mice on both dpi 2 and 4 . This is likely due to a higher baseline number of lymphocytes in the lungs of lt/ mice rather than a result of f. tularensis infection . To assess whether lt deficiency alters f. tularensis - induced cytokine responses following aerosol challenge with the pathogen, levels of a panel of 21 cytokines and chemokines, including ifn-, il-6, kc, and mcp-1, in the bal and the sera of lt/ and lt+/+ mice killed at dpi 2 and 4 were measured overall, there was little change in the levels of the majority of assayed cytokines in either the bal or the sera at dpi 2 or 4 in either mouse strain (data not shown). However, f. tularensis infection resulted in a substantial increase of mcp-1 and a moderate increase of kc in bal fluid at dpi 2 (figure 3(b)) and a substantial increase of ifn-, il-6, kc, and mcp-1 in both bal and sera at dpi 4 (figures 3(a) and 3(b)), but again no differences were observed between the two mouse strains with the exception of il-6, which was significantly higher in bal fluid of lt/ mice than that of lt+/+ mice (figure 3(b), p <.05). Recent studies have established that, in addition to its role in the organogenesis of secondary lymphoid organs, lt plays an important role in host defenses against microbial infections (reviewed in). However, the role of lt in host defenses against infection appears to be complex and varies from pathogen to pathogen . In this study, we utilized lt/ mice and performed some preliminary studies to examine the potential role of lt in the host resistance to respiratory infection with virulent type a f. tularensis . Our results showed that lt/ mice had lower bacterial burdens in their spleens on dpi 2 and higher bacterial burdens in their lungs on dpi 4 when compared to lt+/+ mice but showed no overt differences in clinical outcome, tissue damage, or host immune responses to the infection . Although our data suggest that lt exerts some subtle influence over the course of aerosol - initiated tularemia, its mechanism of action remains unknown . The possible reasons are as follows: (1) lt- is not crucial in host defense against this pathogen; (2) the role of lt- can be compensated by other cytokines / chemokines in this infection model; and (3) the pathogen is too virulent and even immunocompetent hosts have little resistance to the infection . In this regard, we have previously shown that a number of immunodeficient mice show similar clinical outcome to the immunocompetent mice . The lower bacterial burdens in the spleen of lt/ mice at dpi 2 could be explained by a delay / reduction in the dissemination of bacteria from the lung since lt/ mice lack tracheobronchial lymph nodes which normally are the major draining lymph nodes for the lung . Once f. tularensis reached the spleens, however, bacteria quickly multiplied and by dpi 4, bacterial burdens were no longer significantly different in the spleens of lt/ and lt+/+ mice . In fact, lt/ mice actually seem to have slightly higher burdens in their spleens at this time point despite starting on dpi 2 with substantially lower bacterial burdens (see figure 2). Also by this time, lt/ mice harbored significantly more bacteria in their lungs than lt+/+ mice (p <.01), suggesting that lt may play a role in host defense against f. tularensis infection which is distinct from its role in lymphoid organogenesis . Alternatively, the lack of draining lymph nodes in lt/ mice may simply cause a delay in antigen presentation leading to a delayed or otherwise impaired antibacterial host response . In summary, following a low - dose aerosol infection with the highly virulent type a strain of f. tularensis, lt/ mice consistently harbored approximately 10-fold fewer bacteria in their spleens at dpi 2- and 10-fold more bacteria in their lungs at dpi 4 than lt+/+ mice . However, the mortality and median time to death were indistinguishable between the two mouse strains . In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in bal fluid and histopathological analysis, were generally similar between lt/ and lt+/+ mice . These data suggest that although lt does not contribute significantly to the resistance and host responses of mice to airborne type a f. tularensis infection; it does play a subtle role in the multiplication / dissemination of f. tularensis.
In india, the regulation of genetically engineered (ge) organisms is prescribed in rules notified by ministry of environment and forests (now the ministry of environment, forests and climate change; moef&cc), government of india on december 5, 1989 under the environment (protection) act 1986 . These rules, commonly referred to as rules 1989, cover the manufacture, import, use, research and release of ge organisms and derived products in india . (rules for the manufacture, 1989) the key regulatory bodies responsible for implementation of the rules 1989 are the genetic engineering appraisal committee (geac) in moef&cc, and the review committee on genetic manipulation (rcgm) in the department of biotechnology (dbt). Regulatory systems cannot remain static; technological innovation is a dynamic driver of how biotechnology is being applied to develop new products and processes internationally . Since these are regulated activities in india, it is important for geac and rcgm to remain apprised of these changes so that risk assessment guidance, supporting resources, and outreach and training of stakeholders can be appropriately modified . In order to proactively identify emerging issues that may impact the risk assessment and risk management functions of the indian biosafety regulatory system, moef&cc conducted a survey to understand the nature and diversity of genetically engineered crops (also referred to as transgenic crops herein) that may move to product commercialization within the next 10 y. this is the first comprehensive overview of the r&d pipeline for ge crops in india . An electronic questionnaire was prepared to identify what ge crops and traits are currently under development or are anticipated to be developed within the next 10 y in india (20142024). It was distributed to 1050 individual scientists from 320 public and private sector organizations that are involved in plant breeding r&d in india based on known research activities e.g., through engagement with institutional biosafety committees . The questionnaire was comprised of 2 categories of questions: (1) general questions related to information about the respondent's institution and r&d activities; and (2) specific questions to provide information about specific transgenic plants currently under development (see box 1). A total of 243 responses to the questionnaire were received by the closing date of may 1, 2014 which was a response rate of 23% . Respondents to the product pipeline questionnaire identified over 85 different plant species currently being used in experimental work, including plants used for food, livestock feed, fiber, fuel, and dietary or medicinal purposes . The ten most prevalent crops are presented in fig . 1, and a comprehensive list of all of the crop species identified by respondents is in table 1 . Trait variation was also found to be extensive, ranging from resistance traits for biotic stressors, to abiotic stress tolerances (e.g., drought, salt, heavy metals, etc .) Figure 2.aggregated responses for traits that are being studied in r&d programs (total number of responses received for each trait). Table 1.a list of all crops / plants in the r&d pipeline as identified by respondents to the questionnaire (the number in parentheses indicates the number of respondents who listed the crop / plant)abelmoschus esculentus (12)malus domestica (2)allium cepa (4)mangifera indica (1)amorphophaullus sp . (1)manihot esculenta (3)arabidopsis thaliana (10)medicago sativa (1)arachis hypogaea (6)melia sp . (15)bambusoideae (2)nicotiana tabacum (22)brassica caranata (1)ocimum sanctum (1)brassica juncea (8)oryza sativa (73)brassica napus (1)panicum sp . Italica (2)phillanthus (1)brassica rapa (3)picorhhiza kurroa (3)brassica sp . (6)piper nigrum (3)cajanus cajan (13)pisum sativum (2)camellia sinensis (5)populus sp . (1)carica papaya (4)punica granatum (1)carthamus tinctorius (2)ricinus communis (5)casuarina sp . (2)sesamum indicum (2)centaurea depressa (1)setaria italica (1)cicer arietinum (14)solanum lycopersicum (51)citrullus lanatus (6)solanum melongena (28)cocos nucifera (2)solanum tuberosum (7)coffea sp . (7)sorghum bicolor (8)cuminum cyminum (1)spinacia oleracea (1)curcuma longa (1)stevia rebaudiana (1)dendrobian sp . (1)switennia mehagony (1)elettaria cardamomum (1)tectona grandis (1)eleusine coracana (4)theobroma cacao (2)eucalyptus sp . (2)triticosecale (1)glycine max (5)triticum sp . (5)vigna radiata (4)hevea brasiliensis (3)vigna unguiculata (2)ipomoea batatas (2)withania somnifera (2)jatropha sp . (2)zingiber officinale (1) the most prevalent crops in r&d programs by organizational category . Aggregated responses for traits that are being studied in r&d programs (total number of responses received for each trait). Ms / fr = male - sterility, fertility restoration pollination control system . A list of all crops / plants in the r&d pipeline as identified by respondents to the questionnaire (the number in parentheses indicates the number of respondents who listed the crop / plant) figs . 1 and 2 and table 1 demonstrate that indian plant biotechnology r&d is being used to develop plant products that are relevant to indian agriculture today, but it is also forward looking . For example, there is significant research on traits that are relevant to mitigating the impacts of climate change on agriculture, which will be important to ensuring that agricultural productivity is maintained and ultimately improved . (swaminathan and kesavan, 2012) productivity constraints in crops that are particularly relevant to smallholder farmers (e.g., pulses, millets) are also receiving significant attention, with important implications for improved food and nutrition security . (erskine et al ., 2011, national academy of agricultural sciences, 2013) the majority of r&d projects reported in the questionnaire are early phase . For example, 80% of the respondents indicated that their projects were limited to basic research, transformation and regeneration, or early phase event selection in contained (i.e., laboratory, growth chamber or greenhouse) conditions, and only 20% had progressed to event selection in confined field trials . Plant transformation is a commonly used and very important research tool, and it is likely that many of the r&d projects identified by respondents are experimental system or proof - of - concept projects . This is important information as it emphasizes that the biosafety regulatory system must be responsive to all types of agricultural biotechnology research, be it for knowledge generation or product development . Differentiating basic r&d projects from those that are intended for advancement through to commercial product release will help in informing prospective biosafety risk assessment and regulation needs, and so should be determined through a separate, systematic study . Public sector plant biotechnology r&d in india is very strong, with 73% of all respondents self - identifying from public sector research institutions, state agricultural or other universities . Of the 57 respondents who identified as private sector, 78% were from indian companies indicating that the domestic private sector is strongly represented in plant biotechnology r&d . The majority of all 127 respondents who anticipate eventual commercialization expect india to be the first adopter of the ge crops they are developing . This is a strong indication that the ge plants under development by indian scientists are intended for the indian market, and that the r&d investments made in india should result in benefits for indian farmers and consumers . Eighty - eight respondents to q21 indicated that they anticipate submitting applications for commercial release (i.e., cultivation, and use in food and/or feed) of their ge crops, with 8% and 6% of these identifying 2015 and 2016 as target years for approval . Anticipated products for commercialization from the private sector are limited to brinjal, cotton and maize, whereas applications from the public sector encompass a much broader range of crops (fig . Figure 3.applications for commercial approvals that respondents anticipate will be submitted to indian regulatory authorities by the end of 2016 . Applications for commercial approvals that respondents anticipate will be submitted to indian regulatory authorities by the end of 2016 . The results of the questionnaire also demonstrated a significant gap between respondents' expectations for product delivery to farmers (i.e., commercial release of ge crops) and the reality of what is actually required to achieve this, including the time it takes to address biosafety regulatory requirements . This means that many respondents are either unaware of what is required by regulators to demonstrate that a novel ge plant is as safe as its conventional counterpart, or have unrealistic expectations about how promptly products can advance from contained research, through biosafety research level i (brli) and biosafety research level ii (brlii) field trials (dbt and moef&cc, 2008), to approvals for cultivation and consumption / use . This means that, at a minimum, additional education and outreach is needed at the institutional level to improve understanding about the staged pathway to product deployment . Product developers must have a realistic understanding of both the time and financial implications of meeting regulatory requirements in order to make informed decisions about whether project objectives can be achieved . Internationally there is considerable experience as regards assessing the environmental and food safety of ge plants expressing insect resistance and/or herbicide tolerance traits, including published risk assessments conducted by competent authorities in many countries . While fig . 2 makes clear that these traits are already common in indian r&d programs, the indian regulatory system must also prepare for a potential scenario of new plants expressing new traits with which there is relatively less experience in india and elsewhere . This will be greatly facilitated if the development of tools and guidance needed to address these future challenges is initiated in the near term . Are comparative exercises requiring specific kinds of information about the non - transformed host plant and its derived food and feed products . Biology and crop composition documents such as those published by the organization for economic cooperation and development (oecd, 2006) are very useful resources for this purpose . The government of india has published biology documents that complement the oecd documents by providing information that is specific to the indian context for cotton, (moef&cc and dbt, 2011) maize, (moef&cc and dbt, 2011) okra, (moef&cc and dbt, 2011) and rice, (moef&cc and dbt, 2011) and moef&cc is currently developing the same for chickpea, pigeon pea, sorghum, papaya, mustard, tomato, rubber and potato . The intent of these crop - specific biology documents is to describe information that is directly relevant to environmental risk assessment in a format that is accessible to risk assessors and regulators . The document is an overview of pertinent biological information on the untransformed (i.e., conventional or non - transgenic) species that can be used as a reference to help define the baseline and scope for the conventional comparator against which transformed organisms will be compared in the risk assessment . As seen in table 1, there are many other crop species that could potentially be submitted for regulatory permits and approvals in india, and so additional biology documents will need to be prepared after a careful assessment that determines which of these new plant species are intended for eventual brli and brlii confined field trials and commercialization (as some are being studied for basic research under contained conditions only). For less familiar crop species, it is likely that gaps in information needed for risk assessment (and hence the preparation of biology documents) may have to be addressed through field research . For example, understanding the reproductive biology of the non - transgenic plant species is essential to determine appropriate isolation practices so that confined brli and brlii field trials of the experimental, transgenic plants can be effectively managed . Similarly, it will be necessary to identify important food and feed nutrients, as well as anti - nutrients, toxins or allergens that naturally occur in those plant species that will be used for food and/or feed so that any changes in these that lie outside of the normal range of variation can be identified and evaluated as part of the safety assessment process . Risk assessors may wish to consider if there are any new, plausible risk hypotheses (garcia - alonso and raybould, 2014, wolt et al ., 2010) to be addressed when assessing the potential environmental impacts of transgenic plants expressing abiotic stress tolerance traits (see fig . 2). If there are, then how these should be addressed must be clearly described in an appropriate guidance document . As seen in fig . 2, drought tolerance is the second most popular trait in the r&d pipeline, and so product developers need to know what additional kinds of studies (if any) will be necessary to meet their regulatory obligations . In regards to the safety assessment of foods derived from plants with altered nutritional or metabolic profiles (see fig . 2), the government of india's guidelines for the safety assessment of foods derived from genetically engineered plants was updated in 2012 to include a framework to address the safety assessment of foods derived from transgenic plants modified for nutritional or health benefits . (icmr, 2008) this update made sure that the indian guidance was consistent with the codex alimentarius commission's guideline for the conduct of food safety assessment of foods derived from recombinant - dna plants cac / gl 452003 . (cac, 2008) ensuring that risk assessors have the technical capacity to apply this framework is important as a significant number of r&d projects were identified that relate to nutritional or metabolic changes (see fig . Table 1 also lists a number of medicinal plant species which raises interesting questions relative to existing regulations for traditional medicines, and how ge versions of these plants should be managed from risk assessment and regulatory oversight perspectives . This survey, while being the first comprehensive overview of the r&d pipeline for ge crops in india, is preliminary only and the results indicate that more detailed follow - up is required . For example, it would be very instructive to follow - up with public sector institutions to get a much clearer idea as to how many of the research projects being undertaken are aspirational versus realistic in terms of future commercial release . This is needed to ensure institutional preparedness for compliance with the regulatory system, as well as for post - approval considerations such as stewardship, market access and trade implications . Please choose the item that best describes your organization: research institution; university / state agricultural university; private sector; non - governmental organization.2 . If you answered research institution in question #1, please indicate if you are under any one of the following agencies: council of scientific and industrial research; indian council of agricultural research; indian council of medical research; department of biotechnology; department of science and technology; ministry of human resource development.3 . If you answered private sector in question #1, please indicate if you are under one of the following companies: indian company; multinational company.4 . Indicate how many years your organization has been involved in: plant breeding research; germplasm enhancement; variety / hybrid development; biotechnology r&d.5 . Please indicate the crop species that are currently part of biotechnology r&d programs at your organization (respondents were asked to provide information for up to 5 crop species).7 . Do you anticipate that a new crop species (other than those listed in response to q6) will be used in biotechnology r&d programs at your organization within the next 3 years? If yes, " please indicate which species and traits are likely to be examined.8 . Are there transgenic plant materials from discontinued research programs currently stored at your institution?product - specific questions:1 . For each of the crop species identified in q6 please indicate which trait or traits are being studied (select as many as apply): insect resistance, fungal resistance, viral resistance, bacterial resistance, nematode tolerance, herbicide tolerance, drought tolerance, salt tolerance, other abiotic stress tolerance, nitrogen use efficiency, hybrid production system, micronutrient enhancement, macronutrient enhancement, modified fatty acid, modified amino acid, modified color, delayed ripening, other3 . Which stage of biosafety research level i trials when do you anticipate submitting this product for commercial approval?6 . Has this product been approved in any other countries? Gegenetically engineeredgeacgenetic engineering appraisal committeemoef&ccthe ministry of environment, forests and climate change, government of indiar&dresearch and developmentrcgmreview committee on genetic manipulation genetically engineered genetic engineering appraisal committee the ministry of environment, forests and climate change, government of india research and development review committee on genetic manipulation this work was conducted under the auspices of the phase ii capacity building project for implementation of the cartagena protocol on biosafety with funding from the united nations environment program.
Ventricular septal defect (vsd) is one of the most common congenital cardiac defects . Traditional open - heart repair has been established as the gold standard for closure of the perimembranous vsd . Due to the close proximity to the conduction system, complete atrioventricular block (avb) occurs in 03% of vsd patients and is a serious complication that can occur both during and after operation . On the other hand, the most significant risk factor for avb is down syndrome, and 2.7% of these patients with avb require permanent pacemaker implantation . But there are some problems, such as abrasion of leads due to the friction between leads, between the lead and generator, or between the lead and first rib or clavicle . A 6-year - old girl with down syndrome, mild mental retardation, congenital heart disease (patent ductus arteriosus (pda) and vsd) was referred to our outpatient pacemaker clinic (clinical course is summarized in table 1table 1clinical course of the casedateageheightweightbmievents1996.02.20birth1996.07.055 monthspda ligation1996.10.117 monthsoccurrence of avb during vsd patch closureindwelling epicardial ventricular lead1996.11.219 monthspacemaker implantation2000.11.174 yearsepicardial atrial lead implantation . Mode change: vvi to dddbattery exchange2002.03.166 yearsreferred to our pacemaker clinic2003.06.117 years116 cm18 kg13.38battery exchange2005.12.139 years133 cm26 kg14.70battery exchange2007.01.2410 years140 cm29.5 kg15.05intravenous pacemaker implantation (medtronic e2dr21 enpulse 2 dr)2013.08.0517 years140 cm40 kg20.41generator exchange (medtronic advisa)). She had suffered from congestive heart failure and been treated with a mechanical ventilator for approximately 6 months just after birth . Patch closure for vsd was performed when she was 7 months old . Due to a complete surgical avb, a vvi pacemaker with a permanent epicardial pacing lead was implanted on the right ventricle . When she was 4 years old, an atrial lead was implanted through a mini - thoracotomy on the anterior surface of her right atrium, and the pacing mode was switched from vvi to ddd . When she was 7 years old, we exchanged her generator again because of battery depletion . Due to the pacing threshold of the ventricular lead not being good when she was 10 years old, we implanted a permanent pacemaker transvenously under general anesthesia (medtronic e2dr21 enpulse 2 dr). The chest x - ray photos taken before and after implantation are shown in figure 1figure 1chest x - ray just before transvenous pacemaker implantation at the age of 10 (2006.12.22). And figure 2figure 2chest x - ray just after transvenous pacemaker implantation (2007.1.25). Note that both new leads have loops long enough in her right atrium to keep up with her growth .. since she has a persistent left superior vena cava variation, we performed cutdown of the right cephalic vein, fixed screw - in leads to her right ventricular apex and right atria, and implanted a generator under her right subpectoral muscle . She was discharged from the hospital on day 1 after the surgery and showed a satisfactory outcome thereafter . This was the youngest case to receive a transvenous permanent pacemaker in akita prefecture . Chest x - ray just before transvenous pacemaker implantation at the age of 10 (2006.12.22). Note that both new leads have loops long enough in her right atrium to keep up with her growth . The approach to permanent pacemaker implantation in young patients is determined by many factors such as age, structural congenital heart disease, venous access to the heart, venous thrombosis, and pacing - induced dyssynchronous cardiomyopathy . A previous paper showed that the probability of continued epicardial pacing in children increased to 76% at 10 years after implantation . Bipolar steroid - eluting leads and an automatic output adjusting system significantly increased pacing system longevity . A study showed pediatric pacing patients with epicardial lead systems have a high incidence of lead failures, and transvenous lead systems were recommended when anatomy permits . In our case, we were able to operate when her body size was large enough . Transvenous permanent pacemaker implantation has been demonstrated to be a safe procedure with fewer complications and a lower ventricular threshold than the epicardial route in children from 0.09 to 12 years of age (median, 2.3 years). In another report, transvenous permanent pacemaker implantation was proved to be a safe and effective method in children (mean age 9.2 4.7 years). Although, lead or generator exchange is inevitable, the long - term outcome is favorable . Their skin and subcutaneous tissue are fragile, and a generator is quite large for their small body size . As shown in figure 3figure 3enhanced computed tomography showed that the right subclavian vein and cephalic vein were large enough (2006 . The right external and internal jugular veins are identical ., our patient had a large enough right subclavian vein and cephalic vein . Her right jugular vein, which is an alternative access vein in the case of any trouble with the cephalic vein, was large enough, although it was not used . Enhanced computed tomography showed that the right subclavian vein and cephalic vein were large enough (2006 . Puncture into intrathoracic vessels can cause many complications such as vessel rupture, brachial plexus injury, pneumothorax, or hemothorax . Recently, the axillary vein puncture method has been demonstrated to be less invasive, more cosmetic, and without the complications encountered with the intrathoracic method . Cutdown of the cephalic vein can prevent such complications and enables perfect hemostasis at the access point of the vein . As it is less stressful to leads anatomically, lead fracture was significantly decreased . But since screw - in leads were used extensively, dislodgement of leads decreased significantly . However, heart perforation by screw - in leads should be noted . In our past 380 cases with these techniques, 1 patient had late tamponade due to perforation of the right atrium . In the present case, 3.5 cm of excess lead was formed into a loop in the right atrium and ventricle in order to prevent lead dislodgement due to growth of her body . The ventricular lead was anchored carefully so that it could not reach the orifice of ivc . The patient recovered well after 1 week of bed rest without any surgical intervention . A study reported an improved cosmetic result, less abrasion, less infection, no neurovascular and muscular damage, no generator damage by the ribs, no serious hematomas, and no chronic pain . So the subpectoral pocket is recommended as the preferred site for implantation of transvenous pacemakers in pediatric patients . We chose the smallest generator (e2dr21 enpulse 2 dr) as the replacement because we planned to change the generator when her growth stops at 17 or 18 years old . The small generator contributed to prevention of skin ulcer on her generator pocket and no restriction of movement of her shoulder joint . In fact, we exchanged her generator to a larger one (medtronic advisa) when she was 17 years old . The chest x - ray photos taken before and after exchanging are shown in figure 4figure 4chest x - ray taken 7 years after transvenous pacemaker implantation when she was 17 years old (2013.6.20). The lengths of both leads are long enough for fit with her grown up body . And figure 5figure 5chest x - ray taken after her generator was exchanged (2013.8.5).. chest x - ray taken 7 years after transvenous pacemaker implantation when she was 17 years old (2013.6.20). The lengths of both leads are long enough for fit with her grown up body . Our case indicated that transvenous permanent pacemaker implantation is safe and useful in young patients . When the patient s anatomy permits, transvenous lead systems should be considered as one of the treatment options in young patients.
The general secretory (sec) pathway, conserved throughout bacteria, is the canonical translocation pathway and is responsible for translocating the vast majority of secreted proteins across the cytoplasmic membrane . Like other general translocation pathways, sec requires the synthesis of its cargo as preproteins with n - terminal signal peptides, which target them to the sec machinery and from which the mature protein must be released after translocation (1, 2). The enzyme responsible for the liberation of most mature proteins translocated by sec is type i signal peptidase (spase) (35). Accordingly, spase has been demonstrated to be essential in both gram - positive and gram - negative bacteria . Staphylococcus aureus is a striking example of the importance of spase, as it mediates the secretion of a diverse range of virulence factors, including proteins required for adhesion and colonization, evasion of the host immune response, scavenging of nutrients and minerals from the environment, and dissemination (6). The arylomycin family of natural products are potent inhibitors of spase (710). As part of an effort to develop the arylomycins as therapeutics, we and others have been exploring the optimization of their spectrum of activity (1113). An arylomycin with particularly promising activity against s. aureus is arylomycin m131 (fig . We have also developed the arylomycins as chemical biology probes to assess secretion in different bacteria, including staphylococcus epidermidis (14) and s. aureus (6). As part of these efforts, we recently demonstrated that s. aureus responds to arylomycin - mediated spase inhibition by increasing expression of the four adjacent genes, sa0337 to sa0340, and that arylomycin resistance is conferred by loss - of - function mutations in sa0337 (15). (b) genomic organization of the ayrrabc operon (ayrr, sa0337; ayra, sa0338; ayrb, sa0339; ayrc, sa0340) and the region immediately upstream that includes sa0336 . Below is the sequence of the putative promoter region upstream of ayrr ending with its gtg translational start site . A 22-nt palindromic repeat (arrows) the positions of the putative 35 and 10 regions and ribosome binding site (rbs) are indicated in the coding strand . The transcriptional start site is marked as + 1, corresponding to 18 nt upstream from the translational start site . (c) rt - pcr analysis of the ayrrabc operon and divergent genes (sa0334 to sa0336). Gyrb was used as an external control, and gene expression was normalized using gmk . (d) gel shifts performed with 30 ng of each dna probe and an increasing concentration of ayrr as indicated at the top . The region of dna corresponding to each gel shift probe is shown above the gel data, with the ayrr binding site shown in red . Filled triangles denote free dna probe, and open triangles denote the indicated dna - ayrr complexes . All dna probes were amplified from wild - type n315 genomic dna with the exception of irpal, which was created by annealing synthetic oligonucleotides . (e) luminescence from the ayrr promoter (expressed from parc1) in wild - type n315 and n315sa0336-ayrrabc (labeled ayrr), expressed as counts per second per unit of optical density at 590 nm (cps / od590). (f) luminescence from n315 harboring parc1 in the presence and absence of arylomycin m131 (0.5 mic). Here, we demonstrate that the genes sa0337 to sa0340 constitute an operon and that sa0337 is a transcriptional repressor that controls its expression . Remarkably, derepression of these genes bypasses the need for spase, rendering spase nonessential and suggesting that the function of the operon is to mediate an alternate process by which translocated proteins are released from the cytoplasmic membrane . We have thus named the repressor gene ayrr and the downstream genes ayrabc for their role in arylomycin resistance . Sequence analysis identifies ayra as a 6-transmembrane - domain protein of unknown function (duf3169/pf11368) and ayrb and ayrc as two domains of an abc transporter, and we demonstrate that all three are required to bypass spase . Finally, we demonstrate that ayrabc is able to mediate secretion of the proteins normally processed by spase, although in some cases with reduced efficiency, but it does so either with the signal peptide still attached or after cleavage at a site within the signal peptide . The stop and start codons of ayrr and ayra overlap, as do those of ayrb and ayrc, while the stop and start codons of ayra and ayrb are separated by only 24 bp (fig . Using reverse transcription - pcr (rt - pcr), we examined transcription of ayrrabc in the arylomycin - resistant strain arc0001, which harbors a nonsense mutation in ayrr (15), and its parental wild - type strain n315 . As expected, no differences in transcript levels were observed for control genes (sa0334 to sa0336 and gyrb), but transcript levels of ayrr, ayra, ayrb, and ayrc in arc0001 were each increased ~8-fold compared to those in the wild type (fig . Moreover, analysis of our previously published rna - seq data (15) reveals reads that when overlapped align continuously across the entire ayrrabc locus, including the intergenic sequences (see fig . Collectively, these results suggest that ayrrabc is an operon and demonstrate that the mutation in ayrr that renders s. aureus resistant to spase inhibition results in operon derepression . Ayrr encodes a helix - turn - helix motif protein annotated as an xre family transcriptional regulator with homology to the phage cro repressor . Sequence analysis revealed an almost perfect 22-nucleotide (nt) palindrome, tttgacaaatatagttgtcaaa, upstream of ayrr which overlaps the 35 promoter element (fig . Such palindromes commonly compose the binding site of transcriptional regulators (16), and cro regulates its own transcription by binding such a palindrome (17, 18). To determine if ayrr binds its upstream palindrome, a gel shift assay was employed using various dna fragments (fig . The data clearly demonstrate that ayrr selectively binds the palindrome . To test the functional significance of this binding, we constructed the transcriptional reporter plasmid parc1, which harbors the intergenic region between sa0336 and ayrr upstream of the genes encoding luciferase (luxcdabe) (19). Wild - type n315 and a strain lacking the entire region from sa0336 to ayrc (n315sa0336-ayrrabc) were then transformed with either the empty luxcdabe vector or parc1, and luminescence was assayed in actively growing cultures (fig . Luminescence was significantly lower for n315 harboring parc1, consistent with repression by the genomically encoded ayrr protein . As an additional control, the ability of spase inhibition to release repression was tested by conducting the same experiment in the presence of a subinhibitory concentration of arylomycin m131 (0.5 mic). Two hours after arylomycin addition, the luminescence signal observed with wild - type n315 cells harboring parc1 was 7-fold higher than that observed in the absence of arylomycin (fig . These results confirm that ayrr acts as a repressor of ayrrabc and that spase inhibition induces derepression . Ayra encodes a putative membrane protein, predicted to possess a duf3169 domain of unknown function . Proteins in the duf3169 family are found in both staphylococcus and streptococcus species, with one homolog present per genome in sequenced strains, and while they share only ~30% sequence identity, six predicted transmembrane domains and a d - e(a / g)e motif located in the loop connecting the fourth and fifth predicted transmembrane segments are highly conserved . Interestingly, each ayra homolog identified appears to be immediately downstream of a gene that is homologous to ayrr . The downstream genes ayrb and ayrc are predicted to encode the two domains of a type 2 family abc transporter, with ayrc encoding the transporter domain and ayrb encoding the atp - binding cassette domain (conserved domain cd03230). Although ayra could be deleted in an otherwise wild - type strain (n315), we were unable to delete ayra in arc0001 . Thus, to test whether ayra is required to tolerate spase inhibition, we attempted to evolve arylomycin resistance in n315ayra . As a control, we constructed and analyzed a strain lacking sa0336, which is predicted to encode a hypothetical protein and which is not part of the ayrrabc operon . High - level arylomycin m131 resistance (> 16 g / ml) was evolved in n315sa0336 with frequencies indistinguishable from the wild - type parental strain (2.0 10) (15) (see table s2 in the supplemental material). In addition, sequencing revealed that 8 out of 8 of the resistant strains examined contained mutations in ayrr (see table s3 in the supplemental material), similar to the behavior observed with wild - type cells (15). In contrast, n315ayra developed high - level resistance with a significantly reduced frequency (2.9 10) (see table s2), and for the single resistant clone isolated, sequencing revealed a wild - type ayrr . Deletion of ayrbc in arc0001 (arc0001ayrbc) resulted in full resensitization to arylomycin m131, demonstrating that the abc transporter is required to tolerate spase inhibition (see table s2 in the supplemental material). Moreover, deletion of ayrbc in n315 (n315ayrbc) results in the same reduced frequency of resistance to arylomycin m131 that was observed with n315ayra (see table s2), and again, sequencing revealed wild - type ayrr in the single resistant mutant isolated . Collectively, these results demonstrate that each member of the ayrrabc operon is required to tolerate spase inhibition . Because derepression of ayrrabc in s. aureus confers high - level resistance to spase inhibition, we speculated that it may render spase nonessential . Using the same chromosomal integration approach, we attempted to delete the spase gene spsb in arc0001 and its parental wild - type strain, n315 . Not surprisingly, attempts to delete spsb in the wild - type strain resulted in the recovery of the wild - type sequence . In addition to confirming the absence of spsb at its chromosomal locus via sequencing, we demonstrated that spsb was not present elsewhere in the genome of arc0001spsb via pcr and rt - pcr of genomic dna and rna, respectively, using primers internal to the spsb gene (see table s4 in the supplemental material). Deletion of spsb was also verified phenotypically via an examination of the susceptibility of n315 and arc0001spsb to gentamicin and the -lactam antibiotics cefoxitin, oxacillin, and penicillin g. previously, we and others demonstrated that the activity of these antibiotics is uniquely synergistic with that of the arylomycins (13, 20). As controls, we also examined susceptibilities to cccp, vancomycin, daptomycin, erythromycin, trimethoprim, and tetracycline . For each control antibiotic tested, the observed susceptibilities of n315 were virtually identical to those observed for n315 and arc0001 (15). However, the susceptibility of arc0001spsb to gentamicin and each -lactam was 8- to 64-fold greater than that of n315 or arc0001 (see table s5 in the supplemental material), consistent with the absence of spase activity . We next evaluated protein secretion in n315, arc0001, and arc0001spsb using one - dimensional (1d) sds - page (fig . 2). Identical patterns of secretion were observed for the wild type and arc0001, which suggests that arc0001 still employs spase even though the ayrrabc operon is derepressed . In contrast, a small but clearly significant alteration in the pattern of secretion was apparent for arc0001spsb . To confirm that this altered pattern of secretion was the result of the loss of spase activity, secretion was examined in the presence of arylomycin m131 (4 mic). As expected, wild - type cells showed drastically reduced levels of secretion in the presence of the arylomycin . However, treatment of arc0001 cells with the arylomycin did not reduce secretion and resulted in a secretion pattern indistinguishable from that of arc0001spsb, clearly revealing that the altered pattern does indeed result from the absence of spase activity . Moreover, the pattern and quantity of secreted proteins observed with arc0001spsb were unchanged by the addition of the arylomycin, confirming that secretion did not depend on spase function . The extracellular proteomes of n315, arc0001, and arc0001spsb in the absence and presence of arylomycin m131 . To minimize cell lysis, strains were grown until wild - type n315 reached an od590 of 4.0, corresponding to late exponential growth . Strains were then balanced to an od590 of 4.0, pelleted by centrifugation, and washed twice with medium before resuspension in fresh medium . Strains were then grown in the presence or absence of 4 mic of arylomycin m131 (n315) for 20 min . Proteins in the supernatant were precipitated overnight with 10% (wt / vol) tca, washed with acetone, resuspended in 1 loading dye, and visualized by sds - page . Arrows denote bands where significant differences are observed in the extracellular proteomes of wild - type n315 and arc0001spsb . The secreted proteomes of arc0001spsb and n315 were next compared in more detail by isolating proteins from the medium, digesting with trypsin, using reductive dimethylation (redime) to label the wild - type fragments with heavy isotopes and label the arc0001spsb fragments with light isotopes, and then analyzing the combined samples via multidimensional mass spectrometry (mudpit) (21, 22). In total, tryptic peptides corresponding to 38 proteins that are encoded with signal peptides were identified (table 1), which is consistent with previous studies of the s. aureus secretome (6). The quantity of secreted proteins was then determined from the ratio of parent ion peak areas, which demonstrated that while tryptic peptides for the same proteins were detected from each strain, several were detected at different levels (table 1). Sixteen of the 38 secreted proteins were detected in both strains at similar levels (within 2-fold), while 20 were detected at 2- to 8-fold - reduced levels (for example, the proteases staphylokinase and sspp, the complement inhibitor sak, and a hypothetical surface protein sa2285). Two proteins (sa0663 and sdre) were detected at 6- to 8-fold - elevated levels in the spsb strain, while prsa was detected at a 16-fold - elevated level . Prsa is a peptidyl - prolyl isomerase involved in protein folding of secreted proteins in the extracellular environment (23), and we previously demonstrated that it is upregulated at the transcriptional level by spase inhibition . Comparison of the secreted proteomes of wild - type n315 and arc0001spsb reported as the average for all detected tryptic peptides for a given protein . Data are averages and standard errors of the means for three independent biological samples . Underlined amino acids correspond to tryptic peptides detected in arc0001spsb which contain part of the spase signal sequence . As expected for secretion mediated by spase, none of the tryptic peptides isolated from the wild - type sample contained any part of the signal peptide; however, peptide fragments for eight of the proteins were found with n termini that precisely matched the predicted spase cleavage site (see fig . In contrast, with arc0001spsb, tryptic peptides containing portions of the n - terminal leader peptides of 12 of the 38 proteins were detected (table 1; also, see fig . These data suggest that ayrabc mediates secretion of the same proteins as does spase but either via cleavage at a more n - terminal site or perhaps by extricating the entire intact preprotein from the cytoplasmic membrane . Spase functions at the terminal step of the general secretory pathway by releasing translocated proteins from the cytoplasmic membrane at a defined cleavage site . We have provided evidence of an inducible alternative terminal step in s. aureus encoded by the ayrrabc operon, which under normal conditions is repressed by ayrr but which is derepressed by spase inhibition . The natural physiological role of the pathway may be to support spase function at times when elevated secretion is required and the capacity of spase has been exceeded . Alternatively, it may have evolved to facilitate survival in the presence of an spase inhibitor . The latter possibility is consistent with the fact that spase is the only component of the general secretory pathway that is exposed on the outer surface of the cytoplasmic membrane, making it uniquely susceptible to extracellular inhibitors . The evolution of four different families of arylomycins (10) further suggests that that spase inhibition may have represented a significant selection pressure, as does the existence of multiple, redundant spases in many gram - positive bacteria . Hints as to the mechanism by which ayrabc mediates the release of proteins from their attachment to the cell may be gleaned from an analysis of the individual genes . It is possible that the abc transporter functions as part of the translocation process, perhaps augmenting or even replacing other components of the general secretory pathway . However, bacterial abc transporters have been identified that extract lipophilic moieties from the cytoplasmic membrane (24), and ayrbc may function after sec translocation to extract intact preproteins or proteins after intramembrane cleavage . The role of ayra, which encodes a membrane protein of unknown function, is even less clear, but it is interesting to speculate that it could be a protease responsible for intramembrane cleavage, if one is required, which is consistent with its predicted membrane localization . It is also interesting that ayrr homologs in other species appear to control the regulation of proteases (25, 26). A more complete understanding of the pathway awaits detailed analysis of these proteins as well as the signal responsible for derepression of the operon, which is in progress . Undoubtedly, understanding the mechanistic details of this alternate terminal step of the sec pathway will lead to a greater understanding of secretion and the response to secretion stress . The stop and start codons of ayrr and ayra overlap, as do those of ayrb and ayrc, while the stop and start codons of ayra and ayrb are separated by only 24 bp (fig . Using reverse transcription - pcr (rt - pcr), we examined transcription of ayrrabc in the arylomycin - resistant strain arc0001, which harbors a nonsense mutation in ayrr (15), and its parental wild - type strain n315 . As expected, no differences in transcript levels were observed for control genes (sa0334 to sa0336 and gyrb), but transcript levels of ayrr, ayra, ayrb, and ayrc in arc0001 were each increased ~8-fold compared to those in the wild type (fig . 1c). Moreover, analysis of our previously published rna - seq data (15) reveals reads that when overlapped align continuously across the entire ayrrabc locus, including the intergenic sequences (see fig . Collectively, these results suggest that ayrrabc is an operon and demonstrate that the mutation in ayrr that renders s. aureus resistant to spase inhibition results in operon derepression . Ayrr encodes a helix - turn - helix motif protein annotated as an xre family transcriptional regulator with homology to the phage cro repressor . Sequence analysis revealed an almost perfect 22-nucleotide (nt) palindrome, tttgacaaatatagttgtcaaa, upstream of ayrr which overlaps the 35 promoter element (fig . Such palindromes commonly compose the binding site of transcriptional regulators (16), and cro regulates its own transcription by binding such a palindrome (17, 18). To determine if ayrr binds its upstream palindrome, a gel shift assay was employed using various dna fragments (fig . To test the functional significance of this binding, we constructed the transcriptional reporter plasmid parc1, which harbors the intergenic region between sa0336 and ayrr upstream of the genes encoding luciferase (luxcdabe) (19). Wild - type n315 and a strain lacking the entire region from sa0336 to ayrc (n315sa0336-ayrrabc) were then transformed with either the empty luxcdabe vector or parc1, and luminescence was assayed in actively growing cultures (fig . Luminescence was significantly lower for n315 harboring parc1, consistent with repression by the genomically encoded ayrr protein . As an additional control, the ability of spase inhibition to release repression was tested by conducting the same experiment in the presence of a subinhibitory concentration of arylomycin m131 (0.5 mic). Two hours after arylomycin addition, the luminescence signal observed with wild - type n315 cells harboring parc1 was 7-fold higher than that observed in the absence of arylomycin (fig . These results confirm that ayrr acts as a repressor of ayrrabc and that spase inhibition induces derepression . Ayra encodes a putative membrane protein, predicted to possess a duf3169 domain of unknown function . Proteins in the duf3169 family are found in both staphylococcus and streptococcus species, with one homolog present per genome in sequenced strains, and while they share only ~30% sequence identity, six predicted transmembrane domains and a d - e(a / g)e motif located in the loop connecting the fourth and fifth predicted transmembrane segments are highly conserved . Interestingly, each ayra homolog identified appears to be immediately downstream of a gene that is homologous to ayrr . The downstream genes ayrb and ayrc are predicted to encode the two domains of a type 2 family abc transporter, with ayrc encoding the transporter domain and ayrb encoding the atp - binding cassette domain (conserved domain cd03230). Although ayra could be deleted in an otherwise wild - type strain (n315), we were unable to delete ayra in arc0001 . Thus, to test whether ayra is required to tolerate spase inhibition, we attempted to evolve arylomycin resistance in n315ayra . As a control, we constructed and analyzed a strain lacking sa0336, which is predicted to encode a hypothetical protein and which is not part of the ayrrabc operon . High - level arylomycin m131 resistance (> 16 g / ml) was evolved in n315sa0336 with frequencies indistinguishable from the wild - type parental strain (2.0 10) (15) (see table s2 in the supplemental material). In addition, sequencing revealed that 8 out of 8 of the resistant strains examined contained mutations in ayrr (see table s3 in the supplemental material), similar to the behavior observed with wild - type cells (15). In contrast, n315ayra developed high - level resistance with a significantly reduced frequency (2.9 10) (see table s2), and for the single resistant clone isolated, sequencing revealed a wild - type ayrr . Deletion of ayrbc in arc0001 (arc0001ayrbc) resulted in full resensitization to arylomycin m131, demonstrating that the abc transporter is required to tolerate spase inhibition (see table s2 in the supplemental material). Moreover, deletion of ayrbc in n315 (n315ayrbc) results in the same reduced frequency of resistance to arylomycin m131 that was observed with n315ayra (see table s2), and again, sequencing revealed wild - type ayrr in the single resistant mutant isolated . Collectively, these results demonstrate that each member of the ayrrabc operon is required to tolerate spase inhibition . Because derepression of ayrrabc in s. aureus confers high - level resistance to spase inhibition, we speculated that it may render spase nonessential . Using the same chromosomal integration approach, we attempted to delete the spase gene spsb in arc0001 and its parental wild - type strain, n315 . Not surprisingly, attempts to delete spsb in the wild - type strain resulted in the recovery of the wild - type sequence . In addition to confirming the absence of spsb at its chromosomal locus via sequencing, we demonstrated that spsb was not present elsewhere in the genome of arc0001spsb via pcr and rt - pcr of genomic dna and rna, respectively, using primers internal to the spsb gene (see table s4 in the supplemental material). Deletion of spsb was also verified phenotypically via an examination of the susceptibility of n315 and arc0001spsb to gentamicin and the -lactam antibiotics cefoxitin, oxacillin, and penicillin g. previously, we and others demonstrated that the activity of these antibiotics is uniquely synergistic with that of the arylomycins (13, 20). As controls, we also examined susceptibilities to cccp, vancomycin, daptomycin, erythromycin, trimethoprim, and tetracycline . For each control antibiotic tested, the observed susceptibilities of n315 were virtually identical to those observed for n315 and arc0001 (15). However, the susceptibility of arc0001spsb to gentamicin and each -lactam was 8- to 64-fold greater than that of n315 or arc0001 (see table s5 in the supplemental material), consistent with the absence of spase activity . We next evaluated protein secretion in n315, arc0001, and arc0001spsb using one - dimensional (1d) sds - page (fig . 2). Identical patterns of secretion were observed for the wild type and arc0001, which suggests that arc0001 still employs spase even though the ayrrabc operon is derepressed . In contrast, a small but clearly significant alteration in the pattern of secretion was apparent for arc0001spsb . To confirm that this altered pattern of secretion was the result of the loss of spase activity, secretion was examined in the presence of arylomycin m131 (4 mic). As expected, wild - type cells showed drastically reduced levels of secretion in the presence of the arylomycin . However, treatment of arc0001 cells with the arylomycin did not reduce secretion and resulted in a secretion pattern indistinguishable from that of arc0001spsb, clearly revealing that the altered pattern does indeed result from the absence of spase activity . Moreover, the pattern and quantity of secreted proteins observed with arc0001spsb were unchanged by the addition of the arylomycin, confirming that secretion did not depend on spase function . The extracellular proteomes of n315, arc0001, and arc0001spsb in the absence and presence of arylomycin m131 . To minimize cell lysis, strains were grown until wild - type n315 reached an od590 of 4.0, corresponding to late exponential growth . Strains were then balanced to an od590 of 4.0, pelleted by centrifugation, and washed twice with medium before resuspension in fresh medium . Strains were then grown in the presence or absence of 4 mic of arylomycin m131 (n315) for 20 min . Proteins in the supernatant were precipitated overnight with 10% (wt / vol) tca, washed with acetone, resuspended in 1 loading dye, and visualized by sds - page . Arrows denote bands where significant differences are observed in the extracellular proteomes of wild - type n315 and arc0001spsb . The secreted proteomes of arc0001spsb and n315 were next compared in more detail by isolating proteins from the medium, digesting with trypsin, using reductive dimethylation (redime) to label the wild - type fragments with heavy isotopes and label the arc0001spsb fragments with light isotopes, and then analyzing the combined samples via multidimensional mass spectrometry (mudpit) (21, 22). In total, tryptic peptides corresponding to 38 proteins that are encoded with signal peptides were identified (table 1), which is consistent with previous studies of the s. aureus secretome (6). The quantity of secreted proteins was then determined from the ratio of parent ion peak areas, which demonstrated that while tryptic peptides for the same proteins were detected from each strain, several were detected at different levels (table 1). Sixteen of the 38 secreted proteins were detected in both strains at similar levels (within 2-fold), while 20 were detected at 2- to 8-fold - reduced levels (for example, the proteases staphylokinase and sspp, the complement inhibitor sak, and a hypothetical surface protein sa2285). Two proteins (sa0663 and sdre) were detected at 6- to 8-fold - elevated levels in the spsb strain, while prsa was detected at a 16-fold - elevated level . Prsa is a peptidyl - prolyl isomerase involved in protein folding of secreted proteins in the extracellular environment (23), and we previously demonstrated that it is upregulated at the transcriptional level by spase inhibition . Examination of signal sequences revealed no obvious correlation with observed differences in secretion . Comparison of the secreted proteomes of wild - type n315 and arc0001spsb reported as the average for all detected tryptic peptides for a given protein . . Underlined amino acids correspond to tryptic peptides detected in arc0001spsb which contain part of the spase signal sequence . As expected for secretion mediated by spase, none of the tryptic peptides isolated from the wild - type sample contained any part of the signal peptide; however, peptide fragments for eight of the proteins were found with n termini that precisely matched the predicted spase cleavage site (see fig . In contrast, with arc0001spsb, tryptic peptides containing portions of the n - terminal leader peptides of 12 of the 38 proteins were detected (table 1; also, see fig . These data suggest that ayrabc mediates secretion of the same proteins as does spase but either via cleavage at a more n - terminal site or perhaps by extricating the entire intact preprotein from the cytoplasmic membrane . Spase functions at the terminal step of the general secretory pathway by releasing translocated proteins from the cytoplasmic membrane at a defined cleavage site . We have provided evidence of an inducible alternative terminal step in s. aureus encoded by the ayrrabc operon, which under normal conditions is repressed by ayrr but which is derepressed by spase inhibition . The natural physiological role of the pathway may be to support spase function at times when elevated secretion is required and the capacity of spase has been exceeded . Alternatively, it may have evolved to facilitate survival in the presence of an spase inhibitor . The latter possibility is consistent with the fact that spase is the only component of the general secretory pathway that is exposed on the outer surface of the cytoplasmic membrane, making it uniquely susceptible to extracellular inhibitors . The evolution of four different families of arylomycins (10) further suggests that that spase inhibition may have represented a significant selection pressure, as does the existence of multiple, redundant spases in many gram - positive bacteria . Hints as to the mechanism by which ayrabc mediates the release of proteins from their attachment to the cell may be gleaned from an analysis of the individual genes . It is possible that the abc transporter functions as part of the translocation process, perhaps augmenting or even replacing other components of the general secretory pathway . However, bacterial abc transporters have been identified that extract lipophilic moieties from the cytoplasmic membrane (24), and ayrbc may function after sec translocation to extract intact preproteins or proteins after intramembrane cleavage . The role of ayra, which encodes a membrane protein of unknown function, is even less clear, but it is interesting to speculate that it could be a protease responsible for intramembrane cleavage, if one is required, which is consistent with its predicted membrane localization . It is also interesting that ayrr homologs in other species appear to control the regulation of proteases (25, 26). A more complete understanding of the pathway awaits detailed analysis of these proteins as well as the signal responsible for derepression of the operon, which is in progress . Undoubtedly, understanding the mechanistic details of this alternate terminal step of the sec pathway will lead to a greater understanding of secretion and the response to secretion stress . The ayrrabc operon corresponds to the region from position 343,418 to 345,827 in the nctc8325 chromosome . The major transcriptional start site corresponds to coordinate 434,400, which is 18 nt upstream of the gtg start codon . A small number of reads corresponded to coordinate 343,248 suggesting a secondary minor promoter region further upstream . Overlapping rna reads were detected for the entire ayrrabc locus, suggesting that they are cotranscribed . Download figure s1, tif file, 0.8 mb solid - state synthesis of ayrr . The entire ayrr protein was synthesized using standard methods for boc solid - state peptide synthesis employed in our laboratory (r. adhikary, j. zimmermann, j. liu, p. e. dawson, f. e. romesberg, j. phys ., the peptide was synthesized on 0.4 mmol of p - methyl - benzhydrylamine hcl (mbha) resin using 2.2 mmol of protected amino acid, 4 ml 0.5 m of the peptide coupling reagent hctu (2.0 mmol), and 700 l n, n - diisopropylethylamine (diea) (~4.1 mmol), with a coupling time of 20 min . The first amino acid, boc - lys(fmoc)-oh, was coupled onto the mbha resin, and the fmoc group was removed by reaction with 20% piperidine - dimethylformamide (vol / vol); boc groups were deprotected with neat trifluoroacetic acid (tfa) for 2 min . An extra lysine residue was added at the c terminus of ayrr to conjugate biotin with the side chain of lysine . The biotinyl - n - hydroxysuccinimide ester was generated by first dissolving 2 mmol n - hydroxysuccinimide and 2 mmol biotin in 5 ml dimethyl sulfoxide (dmso) and then adding 2 mmol n, n-diisopropyl carbodiimide . After 30 min, the mixture was added to the resin and coupled for 1 h, followed by the addition of 2 mmol diea, which was coupled for an additional hour . The peptide was cleaved from the dry resin support using anhydrous hf with 10% (vol / vol) anisole for 1 h at 0c . The resulting polypeptide was purified via reverse - phase high - performance liquid chromatography (hplc) using a linear gradient of solvent a (0.1% [vol / vol] aqueous tfa) and solvent b (90% [vol / vol] acetonitrile, 10% [vol / vol] water, 0.1% [vol / vol] tfa). Purity was assessed by analytical hplc (a) and mass spectrometry (b and c). The observed mass of the pure biotinylated peptide is 8,027.0 da (calculated: 8,027.3 da); shown are the mass spectrum (b) and reconstructed mass spectrum (c). Download figure s2, tif file, 0.1 mb a representation of tryptic peptides detected from the secreted proteome . A schematic of a secreted protein depicting its signal peptide (grey) and mature protein (white) with the location of 3 tryptic peptides is shown: (1) tryptic peptides identified in the arc0001spsb strain which contain amino acids located in the signal peptide and which overlap the spase cleavage site; the spase cleavage site is denoted by a dash in the amino acid sequence; (2) tryptic peptides found in wild - type n315 that correspond to the n terminus of the mature spase processed secreted protein; and (3) representative tryptic peptides from the mature protein . Below the schematic are parent ion chromatograms of three representative proteins (immunoglobulin g - binding protein a [spa sa0107], the putative surface protein sa2285, and the staphylococcal complement inhibitor sa1754), detected from the secreted proteome of wild - type (blue trace) or arc0001spsb (red trace) s. aureus . Download figure s3, tif file, 0.3 mb primers used in this study . Table s1, doc file, 0.01 mb mics and frequencies of resistance to arylomycin m131 . Table s2, doc file, 0.03 mb ayrr mutations that evolve to arylomycin m131 in n315sa0336 . Table s3, doc file, 0.01 mb amplification of spsb from rna and genomic dna . Table s4, doc file, 0.01 mb mics of n315, arc0001, and arc0001spsb table s5, doc file, 0.03 mb
Research on the etiology and natural history of asthma has identified a web of predisposing factors (e.g., genetics, atopy), causal factors that may sensitize the airways (e.g., animal dander, dust mites, workplace allergens) and contributing factors (perinatal events such as mode of delivery, exposure to cigarette smoke during pregnancy, gestational age and childhood respiratory infections, air quality, socioeconomic status, and pollution) [16]. Conclusions that can be drawn from this research outline a myriad of exposures and complex interactions in the etiology of pediatric asthma [710]. Given the complex nature of pediatric asthma etiology, factors in the perinatal period that would predispose individuals to asthma are of particular interest [11, 12]. Corticosteroid (cs) therapy, administered during labour and delivery to accelerate fetal lung maturation, has not been fully examined as a potential risk factor for the development of asthma in humans . Cs therapy has been shown to alter the development of the fetal lung and has been linked, in animal studies, to changes in brain chemistry and subsequent hypertension later in life [1428]. Complex time - dependent relationships between cs therapy and subsequent lung function in animals has been noted . The use of cs therapy, in canada, among preterm infants before 34 weeks' gestation has increased in the last 15 years from less than 25 percent to approximately 60 percent [30, 31]. This increase of cs therapy follows from randomized controlled trial evidence indicating increased infant survival among preterm infants exposed to antenatal cs therapy . A limited number of previous studies have investigated lung function (not asthma) in childhood among those previously exposed to cs therapy [3336]. The small sample sizes and convenience samples of these studies do not provide sufficient evidence for or against an association between cs therapy and asthma . Therefore, a large population - based cohort study where confounding by indication can be controlled is warranted . This study was designed to test the hypothesis that fetal exposure to corticosteroids in the antenatal period is an independent risk factor for the development of asthma in childhood . Further, the risk of asthma is hypothesized to be greater in the early childhood years and attenuated in later childhood . A population - based cohort study of all pregnant women who resided in nova scotia, canada and gave birth to a singleton fetus between january 1, 1989, and december 31, 1998, and lived to discharge was undertaken . The mchd is a longitudinal population - based database of all mothers and infants delivered while resident in nova scotia that is linked to health services utilization data . Four independently collected linked databases comprise the mchd used in this study including data from the nova scotia atlee perinatal database (nsapd), the nova scotia physician visits medical services insurance file database, the nova scotia vital statistics database and the nova scotia portion of the canadian institute for health information hospital admissions database . Pregnancies that resulted in the birth of more than one infant (twins, triplets, quadruplets) differ from singleton pregnancies both physiologically and obstetrically and therefore were not eligible for inclusion . Mothers who suffer from thyroid conditions (due to higher levels of hormones already present) and mothers experiencing active asthma during the gestational period (due to increased risk of obstetrical complications and the possibility of fetal exposure to steroids because of the pharmaceutical interventions used to manage the mothers asthma) were also excluded [3739]. Previous work in canada has established that health service administrative records can be used to identify and describe children with asthma [4043]. The definition of asthma employed in this study was based on this previous work, determined by examining physician visits or hospitalizations where the primary diagnostic field was for asthma or asthma - like conditions (asthma: icd-9-cm code 493, bronchitis: icd-9-cm code 490, or bronchiolitis: icd-9-cm code 466). Subjects who experienced at least two health care interactions in any 365 day period, starting at 156 weeks post birth (3 years), where the diagnosis was asthma or an asthma - like condition and at least one visit was not in the winter period (december to march inclusive), or experienced one hospitalization specifically for asthma (icd-9-cm code 493) were considered to be asthmatic . This definition captures all levels of asthma severity . A second more conservative definition of asthma was developed to examine the potential that the primary definition would be too encompassing and therefore have a high level of misclassification . The conservative definition of asthma was similar to the primary definition except that only interactions where asthma (icd - cm code 493) was the primary diagnostic field were considered . The first dose of the first course of betamethasone or dexamethasone coupled with the timing of the administration is recorded . For this study, infants were considered exposed to cs therapy if either of these drugs was administered regardless of timing . Potential confounders considered in this analysis included the infant's sex, gestational age at birth, maternal age, one minute apgar score, administration of surfactant, infant's birth weight, delivery by caesarian section, maternal smoking during the gestational period, socio - economic status (ses), hyaline membrane disease, bronchopulmonary dysplasia, maternal insulin dependant diabetes mellitus, gestational diabetes, number of siblings, and year of birth . The infant's gestational age at birth, in completed weeks, was examined both continuously and as a dichotomous variable indicating full term / preterm, with full term being 37 or more completed weeks of gestation . Infants born at an early gestational age are at increased risk of poor lung function and preterm labour is often used as an indication for cs therapy [30, 4547]. Control for confounding by indication was achieved in this study given not all infants who are preterm or low birth weight were exposed to cs therapy and a portion of infants who are full - term or full birth weight will have been exposed . There is a portion of infants in each year of the cohort where the therapy is clinically indicated, but for various reasons not administered (e.g., where mothers presented to the hospital too late). Also, there is a portion of infants in each year of the cohort where the therapy is clinically indicated and administered, but for various reasons delivery was able to be delayed until the infant was delivered full term . Therefore, each year a proportion of preterm and low birth weight infants are unexposed and a portion of full - term infants are exposed to cs therapy allowing for differences in the development of childhood asthma to be examined . Given maternal age has been shown to be related to preterm birth and related to obstetrical complications leading to the administration of cs, maternal age was only considered as a confounder in this analysis [31, 48]. The infants one minute apgar score (examined in three categories, 03, 46, 710) has been shown to be an independent risk factor for childhood asthma . Delivery by caesarean section was examined as a dichotomous variable where, regardless of other obstetrical interventions during delivery (e.g., the use of forceps prior to surgery), if the infant was ultimately born by caesarean section it was considered a caesarean section . Delivery by caesarean section is related to cs therapy and cs therapy has been shown to be associated with poor lung function in the infant [49, 50]. Cigarette exposure during the gestational period has been shown to be related to the development of childhood asthma and risk of obstetrical complications (including prematurity) that are related to the administration of cs [47, 5156]. Differences in the complication rate and the potential for threatened preterm labour is increased when the mother is diagnosed with either type 1 diabetes mellitus or gestational diabetes . Hyaline membrane disease is a respiratory condition in the infant that is often associated with preterm delivery and subsequent morbidity . However, the administration of surfactant to alleviate various respiratory difficulties in the infant after birth, such as hyaline membrane disease, has been shown to be related to both long term lung function and the use of cs therapy [5760]. Due to the declining incidence of asthma over the time period under study, the year of birth was used to control for birth cohort effects (temporal effects). A greater number of older siblings in a household has been shown to be associated with reduced risk of childhood asthma and forms part of the basis for the hygiene hypothesis [6163]. Also related to the hygiene hypothesis is the socioeconomic status of the infants family [64, 65]. Mothers who are of low socioeconomic status are also more likely to have various pregnancy complications that include threatened preterm labour . Therefore, two socioeconomic status measures were used in this study . Marital status at the time of delivery (married or common law versus single, widowed, divorced, or separated) and quintiles of neighbourhood income (based on the postal code of residence at the time of delivery). Extended cox proportional hazards regression models were used to analyse the data . Subjects were censored the week they were identified as having asthma, died or were lost to follow - up . Given the outcome of asthma was not considered before 156 weeks the minimum amount of follow - up time required to be included in this analysis was therefore 156 weeks . A cox proportional hazards regression stratified by antenatal steroid therapy was utilized to assess the time dependant hazard comparing subjects that received and did not receive steroid therapy . A kernel smoothing method was utilized to remove the extreme variability in the time - specific hazard estimates [68, 69]. Given that one mother may give birth to more than one infant in the cohort an examination of robust variance estimates was undertaken using a sandwich estimator . The nonindependence altered the variance estimates by less than 5 percent so no consideration of this nonindependence was given in subsequent analysis . To quantitatively assess if the hazard ratio for the association between exposure to antenatal corticosteroids and the subsequent development of asthma changes with time, an extended cox proportional hazards regression with time - dependant covariates was used . The effect of steroid administration through time was described by three hazard ratios; one when time was between 156 and 260 weeks, one when time was between 261 and 364 weeks and one when time was greater than 364 weeks . These time periods were selected based on an examination of the smoothed figure derived from the stratified cox model . All potential confounders were examined with nested models with the time - dependant covariates for steroid administration . Each potential confounder's effect on the parameter estimates for antenatal steroid therapy (early, mid and late) was assessed by examining the percentage difference in the parameter estimates from a model with and without the potential confounder . Differences larger than 10 percent in any one of the parameter estimates was considered evidence of confounding . Potential cofounders that did not demonstrate evidence of confounding were not retained in the final models . Thus, the statistical model for the final analysis was as follows: (1)h(t, x(t))=h0(t)exp [1(steroid)g1(t)+2(steroid)g2(t) + 3(steroid)g3(t)+x], where (2)g1(t)={1if 156<t260 weeks,0if t<260 weeks, g2(t)={1if 261t364 weeks,0if t<261 or t>364 weeks, g3(t)={1,if t365 weeks,0,if t<365 weeks, x={x other covariates . There were a total of 113,145 births between january 1, 1989, and december 31, 1998, in nova scotia . Exclusions included 1,325 fetal, neonatal or infant deaths that occurred before 1 year of age, 2,408 infants who were delivered from a multiple gestation, 4,958 infants who were born to mothers experiencing active asthma during the gestational period and a further 89 infants were excluded because they were born to mothers experiencing endocrine abnormalities . There were 104,365 infants eligible for record linkage . Birth records for 18,627 infants could not be successfully linked to the health care follow - up data due to errors or missing information with the provincial unique identifier . Due to deaths, migration, and other events an additional 3,484 infants did not have 156 or more weeks of follow - up time . A further 1,671 infants did not have either a birth weight or gestational age recorded (which is required for the analysis), 135 infants were greater than or less than three standard deviations from their mean sex - specific birth weight for gestational age and therefore were excluded . Both a row and column percentage is provided to assist in the interpretation of the relationships . The asthma incidence among birth cohorts peaked in 1989 and has been on a decline since . The number of siblings, income, marital status, mean birth weight, mean gestational age and mean maternal age at birth are similar between asthmatics and nonasthmatics . Infants born preterm had an elevated incidence of asthma over the follow - up period . As expected, children who developed asthma had higher prevalence of caesarean section, surfactant administration, maternal smoking, hyaline membrane disease and bronchopulmonary dysplasia compared to children who did not develop asthma . The median overall follow - up time was nearly 8.5 years (440 weeks). On average asthma developed in this cohort at approximately 5.5 years of age (287 weeks). Table 2 provides the rates of antenatal steroid therapy administration and preterm birth by year of birth . The use of antenatal steroid therapy increased 3-fold over the 10-year time period of the study, from a rate of 7.5 in 1989 to 23.7 per 1,000 births in 1998 . The preterm birth rate increased approximately 30 percent over the same period, from a rate of 40.5 in 1989 to 52.7 per 1,000 live births in 1998 . Figure 1 provides the smoothed adjusted hazard function over time, stratified by antenatal steroid therapy . Adjustments were made for the infant's sex, gestational age at birth and year of birth . Given the smoothing algorithm used a bandwidth of 75 weeks, the tales of the curves have not been estimated . The figure provides evidence that the hazard for developing asthma differs by antenatal steroid therapy dependant on time . The difference is greatest in the early period, diminishing over the middle period with little differences noted beyond 7 years (400 weeks). Table 3 contains the estimated adjusted hazard ratios for the development of asthma for antenatal steroid therapy from 3 to 5, 6 to 7 and 8, or greater years of age along with the estimates for all other confounders in the model . Adjustments were made for infant's sex, gestational age at birth and an indicator for perterm birth, one minute apgar score, administration of surfactant, infant's birth weight, delivery by caesarian section, maternal smoking during the gestational period, hyaline membrane disease, bronchopulmonary dysplasia, number of siblings and year of birth . The effect of antenatal steroid therapy on the development of asthma is seen to be larger in early childhood (hr = 1.19, 95% ci: 1.03, 1.39) with no effect noted in mid childhood (hr = 1.06, 95% ci: 0.86, 1.30) and potentially a protective effect in late childhood (hr = 0.74, 95% ci: 0.54, 1.03). When considering the more conservative definition of asthma the prevalence of asthma dropped from 25.4% to 18.0% . The estimated adjusted hazard ratios for the development of asthma for antenatal steroid therapy from 3 to 5, 6 to 7 and 8 or greater years of age were hr = 1.37 (95% ci: 1.15, 1.62), hr = 1.17 (95% ci: 0.93, 1.49) and hr = 0.92 (95% ci: 0.67, 1.26), respectively . Antenatal steroid therapy appears to be an independent risk factor for the development of childhood asthma after controlling for confounding . The risk appears to be time - dependent with the highest risk early in childhood and diminishing as the child ages . The reasons for this increase risk are not obvious but many animal studies have established latent effects of corticosteroid exposure in utero . Although a mechanistic link between antenatal corticosteroid therapy and the onset of asthma in childhood has not been fully established, wide ranging effects of corticosteroid therapy have been demonstrated . The alteration in brain chemistry and hypothalamic - pituitary - adrenal (hpa) axis, the shift in immune function and other wide - ranging latent effects such as changes in kidney development and subsequent hypertension all provide biological plausibility for a link to childhood asthma . Time - dependent complications associated with antenatal corticosteroid therapy have been demonstrated [15, 16]. Corticosteroids have been shown to affect hpa development in primates, sheep and to a limited extent in humans [1720]. The hpa axis is important in regulating the physical growth and organ development of the neonate . The timing of exposure to corticosteroids within a species, late in gestation, has been shown by matthews to be critical with regard to the effect on the hpa axis [17, 21]. Published and unpublished work by clifton in australia indicates that corticosteroid exposure not only alters the hpa axis but strengths of this study are rooted in the large population cohort that were assembled and followed for extended lengths of time . Control for confounding by indication was also inherent in this study design providing considerable methodological strength . Errors in the primary reason for admission in the cihi hospital discharge data may occur when several competing causes of admission are present in any one individual . Errors of this sort, with both databases, would have a conservative effect on the estimate of association generated in this study . When considering the more conservative definition of asthma the pattern of early childhood risk for the development of asthma after antenatal exposure to corticosteroids remains the same as with the main asthma definition . As expected with this definition the risk estimate, particularly in the early childhood period this sensitivity analysis lends credence to the hazard ratio estimates using the main asthma definition . Limitations in the measure of exposure exist, in that there is no measure of the cs that crosses the placenta and is biologically available to each fetus . Information that pertains to multiple exposure to cs therapy and the actual amount of medication administered is not recorded in the nsapd . With all data limitations outlined, bias would only result if there was a systematic difference between those exposed to cs therapy and those not exposed . There is no indication that physician billing or hospital discharge records would be different based on exposure . Given that this study is focussed on mothers and infants from the province of nova scotia, canada, any findings from this current study would need to be replicated in other populations to strengthen external validity . Although no factors specifically related to the biological effects of cs therapy are postulated to differ between this population and others, other factors related to childhood asthma may differ . The trend in asthma incidence by birth year for this population appears to be different than that of other populations and therefore replication of these findings in different populations is warranted . Continued research into the perinatal factors in the etiology of childhood asthma is warranted . Information continues to surface that draws attention to the potential long term consequences of exposures in utero and subsequent disease risk . Cohort studies that examine the time - dependant effects are crucial in establishing periods of disease susceptibility . Although only a small, but significant, elevated risk for childhood asthma and antenatal steroid exposure was demonstrated in the current study, further research into the smallest possible dose required for the steroid to achieve the desired post - natal effect could be undertaken potentially limiting the long term consequences such as childhood asthma.
During the past decade, radiofrequency catheter ablation has emerged as an effective therapy for ventricular tachycardia (vt) in patients with arrhythmogenic right ventricular cardiomyopathy (arvc). One of the major challenges in mapping and ablation is vt non - inducibility . Although conventional vt induction protocols including extra - stimuli and low rate (<250 beats / min), incremental stimulation were widely used, a considerable number of patients were still non - inducible ., the anatomic and electrophysiologic substrates of vt vary widely in different diseases, which may lead to inconsistent results of vt induction . Fast rate (250 beats / min) burst stimulation is rarely used for vt induction in structural heart diseases for security consideration . In particular, few study focused on this field in arvc due to the low prevalence up to now . This study aimed to evaluate the value of fast rate (250 beats / min) right ventricular burst stimulation for vt induction in arvc, which was by no mean to deny the conventional vt induction protocols but just wanted to contribute an additional tool . From november 2005 to july 2013, 91 consecutive arvc patients with clinical sustained monomorphic vt who underwent electrophysiological study were enrolled . The exclusion criteria included: (1) patients who had previous vt ablation history; and (2) patients whose vts were spontaneous or induced by mechanical stimulation during the catheterization . Sustained vt was defined as continuous vt for at least 30 s or that required an intervention for termination such as cardioversion . At the beginning of the study, the diagnosis of arvc was based on the criteria set by the task force of the working group of myocardial and pericardial disease of the european society of cardiology and of the scientific council on cardiomyopathies of the international society and federation of cardiology . The 2010 revised task force criteria was employed to enroll new patients after published, and the previous enrolled patients were re - checked according to the 2010 revised task force criteria, patients who were not meet the revised criteria were excluded . All antiarrhythmic drugs were discontinued at least five half - lives prior to electrophysiological study except amiodarone . Surface electrocardiogram and bipolar endocardial electrogram were continuously monitored and recorded (bard electrophysiology, lowell, ma, usa). The pacing stimuli were 2 ms in duration and twice diastolic threshold current in strength . The stimulation protocol was implemented stepwise at both right ventricular apex and outflow tract as follows: first, up to double extra - stimuli (step a). Patients who failed to induce received low rate (<250 beats / min) incremental stimulation (step b). In patients whose vt was not induced, fast rate (250 beats / min) burst stimulation was performed (step c). Finally, step d was given in patients whose vt were still failed to induce, which repeated all steps above sequential during an intravenous infusion of isoproterenol . The isotproterenol was infused at a rate of 16 g / min in order to increase the heart rate by at least 30% . All steps above were performed only once . For step a, single extra - stimuli (s2) were introduced with an initial coupling interval of 400 ms after eight driven beats at a cycle length of 500 ms . Double extra - stimuli (s2, s3) were performed with s2 initially positioned 30 ms beyond ventricular refractoriness, and initially positioned 300 ms beyond s2 . The coupling intervals of extra - stimuli were shortened by 10 ms decrements . For step b, incremental stimulation was performed at rate of 150240 beats / min for 10 s, and for step c, stimulation were performed for 10 beats and increased by 10 beats / min increments . The endpoints were: (1) sustained vt was induced; and (2) the ventricular refractoriness was reached . The morphology and rate of the induced vt was compared with the electrocardiogram of the clinical vt . The definition of a fragmented qrs complex was deflections at the beginning of the qrs complex, on top of the r - wave, or in the nadir of the s - wave similar to the definition previously used in coronary diseases . All electrocardiogram analyses were performed by the agreement of two independent readers blinded to patient characteristics and induction results . Statistical analyses were performed using spss 19.0 software (spss inc, chicago, illinois, usa). Comparisons of continuous variables between groups were performed with the student's t - test or wilcoxon test . Chi - square analysis was used to compare the categorical variables between groups . For the multivariate logistic regression analysis, the following variables including sex, age, amiodarone use, baseline qrs duration under sinus rhythm, vt history, number and rate of clinical vt, left ventricular ejection fraction (lvef) and right ventricle (rv) dilatation were analyzed to evaluate in association with vt inducibility . All tests were two - tailed and statistical significance was established at a p <0.05 . From november 2005 to july 2013, 91 consecutive arvc patients with clinical sustained monomorphic vt who underwent electrophysiological study were enrolled . The exclusion criteria included: (1) patients who had previous vt ablation history; and (2) patients whose vts were spontaneous or induced by mechanical stimulation during the catheterization . Sustained vt was defined as continuous vt for at least 30 s or that required an intervention for termination such as cardioversion . At the beginning of the study, the diagnosis of arvc was based on the criteria set by the task force of the working group of myocardial and pericardial disease of the european society of cardiology and of the scientific council on cardiomyopathies of the international society and federation of cardiology . The 2010 revised task force criteria was employed to enroll new patients after published, and the previous enrolled patients were re - checked according to the 2010 revised task force criteria, patients who were not meet the revised criteria were excluded . All antiarrhythmic drugs were discontinued at least five half - lives prior to electrophysiological study except amiodarone . All patients received local anesthesia with lidocaine . Surface electrocardiogram and bipolar endocardial electrogram were continuously monitored and recorded (bard electrophysiology, lowell, ma, usa). The pacing stimuli were 2 ms in duration and twice diastolic threshold current in strength . The stimulation protocol was implemented stepwise at both right ventricular apex and outflow tract as follows: first, up to double extra - stimuli (step a). Patients who failed to induce received low rate (<250 beats / min) incremental stimulation (step b). In patients whose vt was not induced, fast rate (250 beats / min) burst stimulation was performed (step c). Finally, step d was given in patients whose vt were still failed to induce, which repeated all steps above sequential during an intravenous infusion of isoproterenol . The isotproterenol was infused at a rate of 16 g / min in order to increase the heart rate by at least 30% . All steps above were performed only once . For step a, single extra - stimuli (s2) were introduced with an initial coupling interval of 400 ms after eight driven beats at a cycle length of 500 ms . Double extra - stimuli (s2, s3) were performed with s2 initially positioned 30 ms beyond ventricular refractoriness, and initially positioned 300 ms beyond s2 . The coupling intervals of extra - stimuli were shortened by 10 ms decrements . For step b, incremental stimulation was performed at rate of 150240 beats / min for 10 s, and for step c, stimulation were performed for 10 beats and increased by 10 beats / min increments . The endpoints were: (1) sustained vt was induced; and (2) the ventricular refractoriness was reached . The morphology and rate of the induced vt was compared with the electrocardiogram of the clinical vt . The definition of a fragmented qrs complex was deflections at the beginning of the qrs complex, on top of the r - wave, or in the nadir of the s - wave similar to the definition previously used in coronary diseases . All electrocardiogram analyses were performed by the agreement of two independent readers blinded to patient characteristics and induction results . Statistical analyses were performed using spss 19.0 software (spss inc, chicago, illinois, usa). Comparisons of continuous variables between groups were performed with the student's t - test or wilcoxon test . Chi - square analysis was used to compare the categorical variables between groups . For the multivariate logistic regression analysis, the following variables including sex, age, amiodarone use, baseline qrs duration under sinus rhythm, vt history, number and rate of clinical vt, left ventricular ejection fraction (lvef) and right ventricle (rv) dilatation were analyzed to evaluate in association with vt inducibility . All tests were two - tailed and statistical significance was established at a p <0.05 . From november 2005 to july 2013, 127 arvc patients with clinical sustained monomorphic vt underwent electrophysiological study in our institution . Among which, 22 patients had previous vt ablation history and 14 patients whose vts were spontaneous or induced by mechanical stimulation during the catheterization . The remaining 91 patients were enrolled (72 male; age 40.5 12.3 years). The mean age at first presentation of symptoms was 36.0 12.3 years (range: 866 years), and 27 (29.7%) patients had more than one type of vt . A total of 76 (83.5%) patients had inducible vts, among which 49 were induced by step c, 15 were induced by step b whereas 8 and 4 by step a and d, respectively . In details, only 8 (8.8%) patients had inducible vts by step a. however, in those patients who were failed to induce by step a and b, 49 (72.1%) had inducible vts by step c. eighteen patients who failed to induce by step a, b, and c were performed with step d, while vt was induced in four patients (figure 1). Data are presented as n (%), mean sd, or median (range) unless other indicated . * rv dilatation was defined as rv transverse diameter 40 mm in four - chamber view in the end - diastolic . Lvef: left ventricular ejection fraction; rv: right ventricle; vt: ventricular tachycardia . Data are presented as n (%), or median (range) unless other indicated . * clinical vts were induced in 60 (65.9%) patients, which accounted for 78.9% of the total number of the study cohort . The proportions of clinical vts were 7/8, 12/15, 38/49 and 3/4 in step a, b, c and d, respectively, significant statistical difference was not found among each step (p = 0.928). Of note was that vts induced by step c were faster than those by step a and b (p = 0.001). For step b and step c, the median induction conditions were 215 and 280 beats / min, respectively . It was worth mentioning that most vts were easily induced when burst stimulation rates were 40 to 60 beats / min faster than clinical vts (table 2). The median baseline qrs duration under sinus rhythm was 110 ms in the inducible group and 96 ms in the non - inducible group (table 1). Significant statistical difference was found in baseline qrs duration between the two groups (p = 0.006). Multivariate analysis showed that a narrower baseline qrs duration under sinus rhythm was independently associated with vt non - inducibility (or: 1.1; 95% ci: 1.01.1; p = 0.019). Two spontaneously ceased ventricular fibrillations were induced in two patients by step c (320 and 330 beats / min at right ventricular apex) without long - term sequelae . From november 2005 to july 2013, 127 arvc patients with clinical sustained monomorphic vt underwent electrophysiological study in our institution . Among which, 22 patients had previous vt ablation history and 14 patients whose vts were spontaneous or induced by mechanical stimulation during the catheterization . The remaining 91 patients were enrolled (72 male; age 40.5 12.3 years). The mean age at first presentation of symptoms was 36.0 12.3 years (range: 866 years), and 27 (29.7%) patients had more than one type of vt . The outcomes of vt induction were summarized in table 2 . A total of 76 (83.5%) patients had inducible vts, among which 49 were induced by step c, 15 were induced by step b whereas 8 and 4 by step a and d, respectively . In details, only 8 (8.8%) patients had inducible vts by step a. however, in those patients who were failed to induce by step a and b, 49 (72.1%) had inducible vts by step c. eighteen patients who failed to induce by step a, b, and c were performed with step d, while vt was induced in four patients (figure 1). Data are presented as n (%), mean sd, or median (range) unless other indicated . * rv dilatation was defined as rv transverse diameter 40 mm in four - chamber view in the end - diastolic . Lvef: left ventricular ejection fraction; rv: right ventricle; vt: ventricular tachycardia . Data are presented as n (%), or median (range) unless other indicated . * the specificity represented the proportion of induced clinical vt counts in total induced vt . Clinical vts were induced in 60 (65.9%) patients, which accounted for 78.9% of the total number of the study cohort . The proportions of clinical vts were 7/8, 12/15, 38/49 and 3/4 in step a, b, c and d, respectively, significant statistical difference was not found among each step (p = 0.928). Of note was that vts induced by step c were faster than those by step a and b (p = 0.001). For step b and step c, the median induction conditions were 215 and 280 beats / min, respectively . It was worth mentioning that most vts were easily induced when burst stimulation rates were 40 to 60 beats / min faster than clinical vts (table 2). The median baseline qrs duration under sinus rhythm was 110 ms in the inducible group and 96 ms in the non - inducible group (table 1). Significant statistical difference was found in baseline qrs duration between the two groups (p = 0.006). Multivariate analysis showed that a narrower baseline qrs duration under sinus rhythm was independently associated with vt non - inducibility (or: 1.1; 95% ci: 1.01.1; p = 0.019). Two spontaneously ceased ventricular fibrillations were induced in two patients by step c (320 and 330 beats / min at right ventricular apex) without long - term sequelae . This study provided results of sequential vt induction protocols in detail and showed that fast rate right ventricular burst stimulation was feasible in a large number of arvc patients . Application of the fast rate right ventricular burst stimulation protocol would to be a useful supplement for vt induction in order to facilitate ablation . Although conventional vt induction protocols including extra - stimuli and incremental stimulation (<250 beats / min) had been found to increase the yield of inducible vt in a group of diseases, a number of variables including different numbers of extra - stimuli, drive cycle length, current strength and sites of stimulation have been performed, lack of control of those variables has led to a significant variability in reported sensitivity, specificity and reproducibility ., furthermore, there were still a considerable number of patients who were non - inducible despite combined a number of protocols in patients with arvc . This study showed that fast rate right ventricular burst stimulation was an effective and safe supplementary protocol for vt induction in arvc . The multi - formity in anatomic and electro - physiologic substrates contributed to different mechanisms of vt . In ischemic cardiomyopathy, the border zone of infarcted myocardium were shown to be an area of relatively slow conduction with highly heterogeneous recovery of excitability and activation times, which provided the substrate for the formation of functional block lines that could promote reentry. Vt in ischemic cardiomyopathy could be reproducibly induced by extra - stimuli, which were considered as a hallmark of reentrant arrhythmia . Although previous studies showed vt in arvc was characterized by areas of slow conduction within the diseased myocardium which allow continuous electrical activity in an expanded area, creating a circuit pathway . Our study showed that the majority of clinical vts could be induced easily by fast rate burst stimulation, which might support that localized mechanism including micro - reentry and triggered activity might be the main mechanism of vt in arvc . Previous studies found that fragmented diastolic potentials were recorded in 94% of arvc patients, and the rate of inducible vts was up to 93% by extra - stimuli, comparing to 3% in patients with idiopathic vt whose main mechanism was triggered activity ., o' donnell, et al . Found that 82% of the vt was induced by up to five extra - stimuli, and the mean cycle length of induced vt was 310 ms in arvc patients, which was slower than by fast burst stimulation in our study . The differences indicated that the mechanisms of vt in arvc patients were diverse, extra - stimuli might be effective to induce relatively slower macro - reentry vt in arvc, while fast rate burst stimulation might be useful for fast vt due to mechanism of micro - reentry or triggered activity . The pharmacological mechanisms indicate that isoproterenol was useful for the induction of exercise - induced vt which mainly based on triggered activity . Freedman, et al . Found that facilitation of vt induction by isoproterenol might be up to 20% in a group of diseases . Josephson suggested that isoproterenol has a low yield (less than 5%) in coronary disease patients with sustained vt, because the main mechanism of ischemic cardiomyopathy vt is macro - reentry, which does not depend on catecholamine concentrations ., philips, et al . Reported a high degree of association between premature ventricular contractions at baseline and the vts induced during the use of high dose isoproterenol in patients with arvc, which indicated that high dose isoproterenol infusion could contributes to induce vt in arvc . A recent study found that ventricular arrhythmogenicity during isoproterenol testing had a high sensitivity of 91.4% for the diagnosis of arvc, particularly in its early stages . Although the design of this study might underestimate the true yield of induced vt with isoproterenol, the results supported that isoproterenol was useful for vt induction in arvc . One possible explanation was that isoproterenol could enhance the automaticity of vt triggers . In addition to the specific methodological features of stimulation protocols, some clinical characteristics might associate with vt induction . This study showed that a narrower qrs duration under sinus rhythm was a risk factor of vt non - inducibility . The reasonable explanation was that wider qrs duration might represent more severe myocardial fibrosis, which could predict fatal and nonfatal arrhythmic events . However, it was worth mentioning that there was a considerable overlap in the duration of qrs complex in patients who were inducible and non - inducible, the predictive value of the qrs duration should be carefully interpretated . The design of this study precluded any conclusions regarding the true yield of induced vt with each pacing modality . Furthermore, this study only evaluated up to two extra - stimuli, whereas many electrophysiologists would use three extra - stimuli . However, although three extra stimuli were used, there were still a considerable number of patients who are non - inducible . This study was mainly to provide a supplemental method to those difficult cases, and was the first one that confirmed fast rate ventricular burst stimulation is feasible in arvc . Fast rate (250 beats / min) right ventricular burst stimulation was often been considered unreliable and unsafe but few study validated it in patients with arvc . Our results showed its safety and it should be a supplementary method for vt induction in arvc patients, especially in patients who were non - inducible by conventional vt induction protocols . This method will not only facilitate catheter ablation of vt in arvc, but also add more knowledge to arvc vt mechanism research . The design of this study precluded any conclusions regarding the true yield of induced vt with each pacing modality . Furthermore, this study only evaluated up to two extra - stimuli, whereas many electrophysiologists would use three extra - stimuli . However, although three extra stimuli were used, there were still a considerable number of patients who are non - inducible . This study was mainly to provide a supplemental method to those difficult cases, and was the first one that confirmed fast rate ventricular burst stimulation is feasible in arvc . Fast rate (250 beats / min) right ventricular burst stimulation was often been considered unreliable and unsafe but few study validated it in patients with arvc . Our results showed its safety and it should be a supplementary method for vt induction in arvc patients, especially in patients who were non - inducible by conventional vt induction protocols . This method will not only facilitate catheter ablation of vt in arvc, but also add more knowledge to arvc vt mechanism research.
A 49-year - old man (body weight: 86 kg, height: 169 cm) who was diagnosed as having hepatocellular carcinoma with liver cirrhosis due to hepatitis b virus (child - pugh grade c, meld score 14) presented for ldlt . His past medical history revealed diabetes mellitus that was well controlled with an oral hypoglycemic agent . The abnormal results of preoperative laboratory testing were as follows: aspartate transminase (ast): 59 u / l, total bilirubin: 3.1 mg / dl, albumin: 2.4 g / dl, platelets: 91,000/ml . Preoperative transthoracic echocardiography (tte) showed diastolic dysfunction grade 2 with left atrial enlargement . Anesthesia was induced with thiopental sodium 5 mg / kg and vecuronium 0.1 mg / kg and it was maintained with desflurane in air and oxygen . The routine vascular access consisted of a 9 fr introducer (advanced venous access) on the right internal jugular vein, a pulmonary artery catheter and two arterial catheters on the radial artery and femoral artery respectively, and a femoral venous catheter . After anesthetic induction, the vital signs were stable and the intraoperative course was unremarkable during the preanhepatic phase (table 1). The anhepatic phase began 3 hours 30 minutes after anesthetic induction, and hemodynamic instability abruptly developed . The hemodynamic instability improved with a large volume of fluids and blood products (crystalloid solution 10,500 ml, colloid solution 1,200 ml, cell saver autotransfusion 978 ml and leukocyte - depleted red blood cells (ldrbcs) 2 units for 2 hours) and catecholamine infusion (dopamine 5 g / kg / min and norepinephrine 0.1 g / kg / min) (table 1). Reperfusion began 2 hours 20 minutes after portal vein clamping, and the hemodynamic instability further deteriorated . We administered a large volume of fluids and blood products (crystalloid solution 3,400 ml, cell saver autotransfusion 500 ml and fresh frozen plasma (ffp) 2 units in 1 hour) and increased the doses of catecholamines (norepinephrine 0.3 g / kg / min) (table 1). Two hours and 30 minutes after reperfusion, the endtidal co2 was suddenly decreased and the st segment of the ecg was elevated; the hemodynamic instability did not improve despite intravascular volume replacement and increased doses of catecholamines (dopamine 10 g / kg / min, norepinephrine 0.1 g / kg / min and epinephrine 0.1 g / kg / min) (table 1). As there was preoperative diastolic dysfunction, we inserted a tee to accurately evaluate the cardiac function at 3 hours and 30 minutes after reperfusion . The tee evaluation revealed signs of dynamic lvoto, including systolic anterior motion (sam) and eccentric mild - to - moderate mitral regurgitation . Based on the tee findings, the epinephrine infusion (0.1 g / kg / min) was immediately discontinued, esmolol infusion (30 g / kg / min) was initiated and several 100 g boluses of phenyleprine and aggressive volume resuscitation were performed (crystalloid solution 1,400 ml, colloid solution 500 ml, cell saver autotransfusion 1,071 ml, ffp 2 units for 1 hour). Afterward, the vital signs gradually became stable and the operation ended uneventfully (table 1). The total infused volume included crystalloid solution: 26,400 ml, colloid solution: 2,650 ml, cell saver autotransfusion: 4,071 ml, ldrbcs: 5 units, ffp: 8 units and cryoprecipitate: 9 units . Postoperatively, large volumes of fluids and blood products were necessary to stably maintain the hemodynamics and the decreasing the dose of vasopressors was not possible . On second postoperative day, the tte revealed the sam with dynamic lvoto (fig . He was discharged on postoperative day 42 . A 45-year - old man (body weight: 84 kg, height: 180 cm) was diagnosed with liver cirrhosis due to hepatitis c virus (child - pugh grade c, meld score 40) and he presented for ldlt . He had oliguric acute renal failure caused by hepatorenal syndrome, and this required continuous renal replacement therapy . U / l, alkaline phosphatase 35 u / l, ggt (gamma()-glutamyl transferase): 20 u / l, total bilirubin: 12.5 mg / dl, albumin: 2.6 g / dl, bun (blood urea nitrogen): 31.7 mg / dl, creatinine: 3.75 mg / dl, platelets: 35,000/ml and the prothrombin time (inr): 2.71 however, the chest x - ray showed pulmonary edema on the bilateral lung fields . The preoperative tte revealed asymmetric septal hypertrophy (the thickness of the interventricular septum and left ventricular posterior wall was 29 mm and 9 mm, respectively) with lvoto . He experienced occasional hypotension (mean arterial pressure: 40 mmhg) accompanied by drowsy mentality, dizziness, blurred vision and chest discomfort caused by aggravation of the lvoto due to volume depletion . Anesthesia was induced with etomidate 0.2 mg / kg and atracurium 0.5 mg / kg and it was maintained with desflurane in air and oxygen . A 9 fr introducer (advanced venous access) was placed on the right internal jugular vein, and a pulmonary artery catheter and two arterial catheters were placed on the radial artery and femoral artery, respectively, and a femoral venous catheter were placed for routine vascular access . The tee probe was gently inserted and there was no variceal bleeding . There were no noticeable changes of the lvoto and sam compared to the preoperative findings . The vital signs were stable during induction, but the blood pressure began to decrease 20 minutes later . The favorable response to phenylephrine 100 g bolus was confirmed, and then continuous infusion of norephinephrine was started at 0.05 g / kg / min and this was adjusted in the range of 0.05 - 0.2 g / kg / min along with maintaining the intravascular volume . After bolus phenylephrine 100 g and increasing the norephnephrine dose to 0.25 g / kg / min, the anhepatic phase began and a portocaval shunt was created to mitigate the venous pooling . The patient's intermittent hypotension was treated with intravascular volume expansion and bolus phenylephrine, and the norephinephrine dose was increased to 0.3 g / kg / min . The infused fluid and blood products were crystalloid solution 1,900 ml, half saline 5,600 ml, colloid solution 2,450 ml, cell saver autotransfusion 1,355 ml, ldrbcs 2 units, ffp 2 units and cryoprecipitate 6 units until 1 hour after the anhepatic phase . There were no noticeable changes of the lvoto and sam compared to the initial findings after induction . The rest of the operation was relatively uneventful with maintaining the intravascular volumeand continuous infusion of norepinephrine at 0.4 - 0.6 g / kg / min . The total administered volume included crystalloid solution 7,700 ml, half saline 7,300 ml, colloid solution 2,970 ml, 5% dextrose water 200 ml, cell saver autotransfusion 3,701 ml, ldrbcs 5 units, ffp 6 units, leukocyte - depleted platelet concentrates 6 units and cryoprecipitate 6 units for 11 hours 30 minutes . On postoperative day 1, the tte revealed moderate mitral regurgitation and dynamic lvoto with sam . The patient's postoperative course was complicated by septic shock due to the cellulitis of both thighs . Doppler ultrasonography showed sluggish flow of the hepatic vein, portal vein and hepatic artery . A 49-year - old man (body weight: 86 kg, height: 169 cm) who was diagnosed as having hepatocellular carcinoma with liver cirrhosis due to hepatitis b virus (child - pugh grade c, meld score 14) presented for ldlt . His past medical history revealed diabetes mellitus that was well controlled with an oral hypoglycemic agent . The abnormal results of preoperative laboratory testing were as follows: aspartate transminase (ast): 59 u / l, total bilirubin: 3.1 mg / dl, albumin: 2.4 g / dl, platelets: 91,000/ml . Preoperative transthoracic echocardiography (tte) showed diastolic dysfunction grade 2 with left atrial enlargement . Anesthesia was induced with thiopental sodium 5 mg / kg and vecuronium 0.1 mg / kg and it was maintained with desflurane in air and oxygen . The routine vascular access consisted of a 9 fr introducer (advanced venous access) on the right internal jugular vein, a pulmonary artery catheter and two arterial catheters on the radial artery and femoral artery respectively, and a femoral venous catheter . After anesthetic induction, the vital signs were stable and the intraoperative course was unremarkable during the preanhepatic phase (table 1). The anhepatic phase began 3 hours 30 minutes after anesthetic induction, and hemodynamic instability abruptly developed . The hemodynamic instability improved with a large volume of fluids and blood products (crystalloid solution 10,500 ml, colloid solution 1,200 ml, cell saver autotransfusion 978 ml and leukocyte - depleted red blood cells (ldrbcs) 2 units for 2 hours) and catecholamine infusion (dopamine 5 g / kg / min and norepinephrine 0.1 g / kg / min) (table 1). Reperfusion began 2 hours 20 minutes after portal vein clamping, and the hemodynamic instability further deteriorated . We administered a large volume of fluids and blood products (crystalloid solution 3,400 ml, cell saver autotransfusion 500 ml and fresh frozen plasma (ffp) 2 units in 1 hour) and increased the doses of catecholamines (norepinephrine 0.3 g / kg / min) (table 1). Two hours and 30 minutes after reperfusion, the endtidal co2 was suddenly decreased and the st segment of the ecg was elevated; the hemodynamic instability did not improve despite intravascular volume replacement and increased doses of catecholamines (dopamine 10 g / kg / min, norepinephrine 0.1 g / kg / min and epinephrine 0.1 g / kg / min) (table 1). As there was preoperative diastolic dysfunction, we inserted a tee to accurately evaluate the cardiac function at 3 hours and 30 minutes after reperfusion . The tee evaluation revealed signs of dynamic lvoto, including systolic anterior motion (sam) and eccentric mild - to - moderate mitral regurgitation . Based on the tee findings, the epinephrine infusion (0.1 g / kg / min) was immediately discontinued, esmolol infusion (30 g / kg / min) was initiated and several 100 g boluses of phenyleprine and aggressive volume resuscitation were performed (crystalloid solution 1,400 ml, colloid solution 500 ml, cell saver autotransfusion 1,071 ml, ffp 2 units for 1 hour). Afterward, the vital signs gradually became stable and the operation ended uneventfully (table 1). The total infused volume included crystalloid solution: 26,400 ml, colloid solution: 2,650 ml, cell saver autotransfusion: 4,071 ml, ldrbcs: 5 units, ffp: 8 units and cryoprecipitate: 9 units . Postoperatively, large volumes of fluids and blood products were necessary to stably maintain the hemodynamics and the decreasing the dose of vasopressors was not possible . On second postoperative day, the tte revealed the sam with dynamic lvoto (fig . A 45-year - old man (body weight: 84 kg, height: 180 cm) was diagnosed with liver cirrhosis due to hepatitis c virus (child - pugh grade c, meld score 40) and he presented for ldlt . He had oliguric acute renal failure caused by hepatorenal syndrome, and this required continuous renal replacement therapy . The abnormal results of the preoperative laboratory testing were as follows: ast: 56 u / l, alkaline phosphatase 35 u / l, ggt (gamma()-glutamyl transferase): 20 u / l, total bilirubin: 12.5 mg / dl, albumin: 2.6 g / dl, bun (blood urea nitrogen): 31.7 mg / dl, creatinine: 3.75 mg / dl, platelets: 35,000/ml and the prothrombin time (inr): 2.71 sodium 129 meq / l . However, the chest x - ray showed pulmonary edema on the bilateral lung fields . The preoperative tte revealed asymmetric septal hypertrophy (the thickness of the interventricular septum and left ventricular posterior wall was 29 mm and 9 mm, respectively) with lvoto . He experienced occasional hypotension (mean arterial pressure: 40 mmhg) accompanied by drowsy mentality, dizziness, blurred vision and chest discomfort caused by aggravation of the lvoto due to volume depletion . Anesthesia was induced with etomidate 0.2 mg / kg and atracurium 0.5 mg / kg and it was maintained with desflurane in air and oxygen . A 9 fr introducer (advanced venous access) was placed on the right internal jugular vein, and a pulmonary artery catheter and two arterial catheters were placed on the radial artery and femoral artery, respectively, and a femoral venous catheter were placed for routine vascular access . The tee probe was gently inserted and there was no variceal bleeding . There were no noticeable changes of the lvoto and sam compared to the preoperative findings . The vital signs were stable during induction, but the blood pressure began to decrease 20 minutes later . The favorable response to phenylephrine 100 g bolus was confirmed, and then continuous infusion of norephinephrine was started at 0.05 g / kg / min and this was adjusted in the range of 0.05 - 0.2 g / kg / min along with maintaining the intravascular volume . After bolus phenylephrine 100 g and increasing the norephnephrine dose to 0.25 g / kg / min, the anhepatic phase began and a portocaval shunt was created to mitigate the venous pooling . The patient's intermittent hypotension was treated with intravascular volume expansion and bolus phenylephrine, and the norephinephrine dose was increased to 0.3 g / kg / min . The infused fluid and blood products were crystalloid solution 1,900 ml, half saline 5,600 ml, colloid solution 2,450 ml, cell saver autotransfusion 1,355 ml, ldrbcs 2 units, ffp 2 units and cryoprecipitate 6 units until 1 hour after the anhepatic phase . There were no noticeable changes of the lvoto and sam compared to the initial findings after induction . The rest of the operation was relatively uneventful with maintaining the intravascular volumeand continuous infusion of norepinephrine at 0.4 - 0.6 g / kg / min . The total administered volume included crystalloid solution 7,700 ml, half saline 7,300 ml, colloid solution 2,970 ml, 5% dextrose water 200 ml, cell saver autotransfusion 3,701 ml, ldrbcs 5 units, ffp 6 units, leukocyte - depleted platelet concentrates 6 units and cryoprecipitate 6 units for 11 hours 30 minutes . On postoperative day 1 the patient's postoperative course was complicated by septic shock due to the cellulitis of both thighs . Doppler ultrasonography showed sluggish flow of the hepatic vein, portal vein and hepatic artery . Dynamic lvoto in a structurally normal heart has been described in the medical literature [3 - 5], aniskevich et al . Reported that a dynamic lvoto with sam developed intraoperatively in a patient with a normal stress echocardiogram preoperatively and who underwent lt . In addition, although performing preoperative dobutamine stress echocardiography for cardiac evaluation seems to be valuable to predict adverse cardiovascular events after lt, the incidence and prognosis of dynamic lvoto during lt are still unknown . Further, the preoperative degree of the peak pressure gradient in a patient with lvot does not necessarily predict the outcome and the presence of dynamic lvoto is not a contraindication for lt . In addition, lt is associated with further decreases in the left ventricular preload secondary to intraoperative bleeding or surgical interruption of the inferior vena cava (the preanhepatic and anhepatic period) and the decrease of systemic vascular resistance after recirculation of the donor liver (the postreperfusion period). Therefore, the patients undergoing lt often have several risk factors for dynamic lvoto and this may be precipitated by hypovolemia in combination with a hyperdynamic state that is induced by catecholamine administration . In case 1, hemodynamic instability developed immediately after the start of the anhepatic period and the dynamic lvoto might have been triggered by hypovolemia, as evidenced by the low cvp and rvedv, which are the parameters of the intravascular volume status (table 1). The anesthetic management of hypovloemia is aggressive volume therapy, and the left ventricular end - diastolic volume can be improved with aggressive intravascular volume loading to increase the preload . Yet the diastolic dysfunction in case 1 deterred us from performing aggressive volume therapy for fear of congestive heart failure until the exact volume status of the heart was confirmed by tee . Moreover, in the case of a small graft volume - to size when performing ldlt, excessive volume during reperfusion could also damage the sinusoids in the grafted liver . These reasons make the fluid management more delicate and it had to be done in a sophisticated manner . For case 1, the cardiac evaluation with tee guided us to change the therapeutic directions by discontinuing the epinephrine infusion and then start fluid loading . We next used phenylephrine to increase the systemic vascular resistance and esmolol (an ultrashort acting beta - blocker) to improve the degree of obstruction by decreasing both the chronotropic and inotroic state of the left ventricle and inhibiting the profound beta - adrenergic mediated splanchnic vasodilation that is common in patients with end stage liver disease . For case 2, since we noticed the lvoto before operation, we chose norepinephrine instead of other inotropics and we created portocaval shunt at the early stage of the anhepatic period to minimize the mesenteric venous pooling . As a result, the hemodynamic stability was maintained throughout the surgery . The tee played an important role in diagnosing and managing the dynamic lvoto in our cases . The choice of drug and the amount of fluid challenge were guided by the findings of tee . During the reperfusion period, which is the most critical period of lt, the cardiac output measured by the thermodilution technique can be inaccurate due to thermal gradients in the venous flow from the cold liver and also due to the rapid changes in cardiac function with time constants that are shorter than that for calculating the cardiac output . In contrast, tee provides continuous monitoring of the cardiac function and it facilitates appropriate management . The presence of esophageal varices was previously thought to be a contraindication for tee, but several studies demonstrated the safe use of tee in these patients . Based on these studies, tee appears to be safe in patients with varices and its use must be strongly considered in patient with cardiac problems . In conclusion, dynamic lvoto should be ruled out in patients with refractory hemodynamic instability during lt although no evidence of lvoto is found on the screening echocardiography . Moreover, intraoperative tee is highly useful for confirming dynamic lvoto and to provide the surgeon with therapeutic guidance.
To report a case of bilateral spontaneous anterior lens dislocation associated with retinitis pigmentosa (rp). A 45-year - old male with rp presented with elevated intraocular pressure (iop) in the right eye and was treated with laser iridotomy (li). Surgical intervention, including anterior vitrectomy, intracapsular cataract extraction (icce), and iol scleral fixation was performed . Two years later, the same episode occurred in his left eye and a similar treatment was done . This is the first report of bilateral spontaneous anterior lens dislocation in a rp patient . A 45-year - old korean male presented on march 27, 2003 with right ocular pain . Right and left iop, measured by a goldman applanation tonometer, was 66 and 11 mmhg, respectively . Right and left eye best corrected visual acuity (bcva) was 20/70 with -4.50 + 1.00165 and 20/100 with -3.50 + 1.00180, respectively . Right eye slit lamp examination showed steamy opaque cornea and a central anterior chamber depth of 2 corneal thickness (ct)., bone - spicule pigmentation sparing macula, and pale optic disc was noted in the right eye (fig . The patient was given intravenous 15% mannitol (300 ml), oral acetazolamide (diamox) (500 mg), oral 50% glycerin (glycerol) (50 cc), carbonic anhydrase inhibitor (cosopt), and 0.2% brimonidine (alphagan) eye drops . One day after treatment right eye iop decreased to 50 mmhg, the cornea became clear, and the central anterior chamber depth remained 2 ct . Six days later, the cornea was clear, the anterior chamber was deep and clear bilaterally, and the right eye li opening was patent . Using the humphrey visual field (model 750, humphrey instruments, inc, dublin, california; c24 - 2 program) 2), and electroretinogram findings were prolonged in a and b amplitudes (fig . The right eye li opening was patent, and the anterior chamber was deep and clear . Six months later, on december 19, 2003, the patient presented with right ocular pain . For the surgical procedure, pupillary constriction was achieved using 4% pilocarpine, administered 3 times in 5 minute intervals . A 7 mm wide scleral tunnel was made 1.5 mm posterior to the limbus at the superotemporal side . Healon gv was injected into the anterior chamber to create space between the posterior lens capsule and iris . Icce was done successfully using capsule forceps . Prolapsed vitreous in the anterior chamber was removed by vitrector . A posterior chamber lens (alcon cz70bd) was introduced and sutured to the sclera by the ab interno method . Three days postoperatively, the iol was well positioned and no abnormal ocular findings were noted . Two years later, on june 8, 2005, the patient presented with left ocular pain lasting one day . Right and left bcva was 20/50 with -1.50 + 2.00165 and 20/160 with -3.50 + 1.0010, respectively . Slit lamp showed anterior lens dislocation with mild inflammatory cells, and there were no abnormal findings in the right eye (fig . 4). Anterior vitrectomy, icce, and iol scleral fixation was done on the left eye . Four weeks post - operatively, left bcva and iop was 20/200 with -1.50 + 1.2515 and 10 mmhg, respectively . The causes of ectopia lentis are trauma, pseudoexfoliation syndrome, spontaneous dislocation, and various heredofamilial diseases including marfan syndrome, homocystinuria, and rp.4,5 in rp patients, hayashi et al . Has suggested that zonular weakness can cause ectopia lentis6 and namiki et al . Showed unilateral zonular dehiscence.7 we report the first case of anterior lens dislocation in an rp patient following high iop . After glaucomatous attack, mid - dilated pupil occurred with a weakened iris and ciliary body; these structure caused anterior segment lens dislocation . The mechanisms of glaucoma associated with a dislocated lens are pupillary block, lens degenerative changes, and concomitant anterior chamber angle damages.8 allingham et al . These include pupillary block by subluxating the lens posterior to the iris, lens incarceration directly within the pupil, and complete lens dislocation into the anterior chamber.9 in this case, iop elevation was due to posterior pupillary block, which responded dramatically to laser iridotomy . If the lens is displaced into the anterior chamber, it may be possible to relieve the condition by dilating the pupil and allowing the lens to reposition into the posterior chamber . However with a totally dislocated lens in the anterior chamber, it is better to constrict the pupil and surgically remove the lens . Choi et al . Have treated anteriorly dislocated lens by lensectomy, vitrectomy, phacoemulsification, and ab externo scleral iol fixation.10 for scleral fixation, young et al . Used a scleral tunnel to relieve iol fluctuation and to maintain anterior chamber depth.11 in this case, the anteriorly dislocated lenses were removed using a 7 mm wide scleral tunnel, combined with an anterior vitrectomy and ab interno scleral fixation . This is the first case of bilateral spontaneous anterior lens dislocation in an rp patient reported in the literature.
Messori and colleagues are to be congratulated on their interesting report on the italian observational study of antithrombin iii (at iii) use in intensive care units . Audits of this sort are difficult and time consuming to conduct . Observational studies are also difficult to analyse and interpret because of the heterogeneity of real - life patient populations, the lack of standardised treatment regimens, the lack of standardised indications for treatment, and the lack of predefined endpoints for assessing survival (e.g. 28 day survival or in - hospital mortality). For these reasons we take issue with messori and colleagues over the very strong conclusion they draw on the basis of their survey . In our opinion, this nonrandomised observational study is not able to provide scientific evidence to support the authors' statements . As pointed out by pocock and elbourne, observational studies have one crucial deficiency: the design is not an experimental one . Experimental design involves randomisation, the use of entry criteria, and the rigorous use of standard definitions of index medical conditions such as disseminated intravascular coagulation and septic shock . None of these are to be found in the survey by messori and colleagues . In the absence of rigorous experimental methodology, it is not possible to be sure that the findings of an observational study are predictive of the results of a randomised, controlled trial (rct). The published finding that observational studies usually agree with rcts comes from studies in indications unrelated to sepsis and septic shock . Furthermore, it is a logical fallacy to suggest that agreement in one direction implies prediction in the other direction . Observational studies are not scientifically capable of proving or disproving any hypothesis . With this in mind, it is interesting to note that one of the main observations of messori and colleagues' survey (n = 56 for sepsis) is at variance with the results from a very much larger rct reported in the same indication by warren and colleagues (n = 2341 for sepsis). Whereas warren and colleagues' rct found significantly lower mortality in a prespecified subgroup analysis of at iii - treated patients not receiving heparin (n = 352) compared with placebo - treated patients not receiving heparin (n = 346), messori and colleagues' survey observed an increase in mortality in patients not receiving heparin . This casts serious doubt over the scientific value of a survey reporting outcomes in a treatment that was not randomised to patients, with only 56 patients with sepsis treated with at iii . Such a type of study is not scientifically or statistically capable of proving causality or supporting statements . Other crucial weaknesses of messori and colleagues' survey lie in its observational nature and the lack of any standardisation of definitions or outcomes . Controlled studies usually indicate the 28 day mortality whereas observational studies indicate the hospital mortality, which normally is higher than the 28 day mortality . In addition, hospital mortality may depend on local factors affecting clinical practice such as when to discharge patients from hospital, which varies among countries . Messori and colleagues also report the results of a meta - analysis of four studies of at iii in sepsis . It is probably unwise, however, to place too much weight on meta - analyses of studies conducted in critical care settings because the spectacular failure of meta - analyses to predict the outcomes of subsequent large - scale rcts is well known . Furthemore, if confidence intervals overlap or p> 0.05, this does not prove that there is no difference; on the contrary, there is still a possibility that the observation suffers from a type ii error (failure to detect a true difference due to inadequate sample size). This is particularly likely to occur when the number of patients studied is small, as in the phase ii trials on at iii . In this context, it is misleading of messori and colleagues to use figure 1 of their paper to suggest, with reference to the confidence intervals, that there is no difference between the studies or that their observational study has a very similar result to the other studies cited . In the same way, potentially misleading claims are made about the subgroup analysis of the effects of coadministered heparin . In the results section it is suggested, erroneously, that no differences exist between results on the basis of p values alone . Surveys and audits are very important ways of documenting routine clinical practice and of confirming the implementation of guidelines based on the results of rcts . Surveys and audits are also useful for generating hypotheses, but they are not a substitute for rcts when proving or disproving hypotheses . Messori and colleagues have raised interesting concerns but they have not provided answers to those concerns . The author has participated as a clinical investigator in the kybersept trial and served as coordinator in a basic research program of the university of innsbruck on anti - inflammatory mechanisms of antithrombin, which was in part supported by a grant from aventis behring gmbh (marburg, germany).
The world report on disability in 2012 estimated there are 360 million people with hearing loss representing, approximately 5.3% of the world population . In recent years, aging has become one of the most common causes of hearing loss and the prevalence of hearing loss among adults over 65 years of age is five times higher than that among adults under 65 years of age . According to the world health organization, an 18 - 50 percentage increase in 65 years of age and over is expected from 2010 - 2020, which will lead to an increase in the hearing impaired . As the number of people with hearing impairments has increased, treatments for hearing loss have become an important issue . Hearing aids are one of the most widely - used treatment options for the hearing impaired . For the proper use of hearing aids, hearing aid fitting management (hafm) is a crucial issue for manufacturers, dispensers, and service providers, and especially for hearing aid users . Optimal outcomes of hearing aids are supported by comprehensive hearing aid fitting protocols and the impact of " poor fit and comfort " is particularly notable for hearing aid return rates . Accordingly, the whole process of hearing aid fitting needs to be normalized for its efficiency and transparency, user satisfaction, and cost effectiveness . Two issues are expected to influence hearing aid markets and stakeholders, and lead to the exploration of the standardization of hafm in this study . First, the term' hearing aid fitting' is prevalently used among service and industry sectors with its comprehensive procedures not systematically explicated . Second, a variety of non - normalized guidelines for hearing aid fitting has led to non - uniform care, outcome variability, and dissatisfaction of the use of hearing aids . The main purpose of the present study is to suggest a general framework of standardized practice for hafm including the pre- and post - fitting stages . To establish the framework, this study investigated various guidelines and essential components for hearing aid fitting around the world . The management framework centers on its fitting process with its prior steps of assessment as well as its posterior steps of follow - up, thereby eliminating diverging interpretations and nonuniform practices . The outcomes of this study are expected to improve potential benefits such as quality of hearing aid fitting, user satisfaction, and cost effectiveness, for all relevant stakeholders . The establishment of hafm relies on the extraction and unification of fundamental components from pre - existing hearing aid fitting guidelines for both adults and children . Table 1 summarizes the essential components of hearing aid fitting guidelines globally (one standard and 15 guidelines) consisting of 11 components: counseling, audiometry, hearing aid evaluation, hearing aid selection, ear impression, adjustment, verification, orientation, auditory training, outcome measures, and comprehensive report . It is evident that most of these components are common across the guidelines although each guideline includes slightly different components . The hafm in this study is defined as a systematic process targeting hearing aid fitting with its prior and posterior stages to help the hearing impaired recognize and interpret sounds better with hearing aids by providing auditory training as well as optimizing audibility and comfort . 1 shows the general framework of hafm based on the documentary data of table 1 with three inclusive aspects: functional, relational, and procedural . The functional aspect includes the use of hearing aids for helping people with hearing impairments by improving their hearing ability and overall satisfaction . 1, with three modules: 1) assessment, 2) fitting, and 3) follow - up . The procedural aspect is modeled by a potential flow occurring in the process of hafm and this model is simply depicted below: 1) start at assessment, 2) go to fitting, 3) go to follow - up, 4) no further action required if satisfied, 5) go to fitting if unsatisfied . At the pre - fitting stage, the assessment module focuses on identifying hearing loss, functional problems, and hearing aid candidacy and selecting appropriate hearing aids . This module encompasses several components including counseling, audiometry testing, hearing aid evaluation, hearing aid selection, and ear impression . The next stage of hafm involves the fitting module which establishes optimal audibility and physical comfort with hearing aids while ensuring good sound quality . Ideally, the fitting module efficiently restores hearing loss based on types, degrees, and configurations of individual hearing by adjusting hearing aids . Then, optimal sound quality and comfort are verified with subjective and objective tests . Use and care instructions for hearing aids are also provided at this module . At the post - fitting stage, the follow - up module focuses on maximizing and validating hearing aid benefits through auditory training and outcome measures, respectively . Auditory training attempts to maximize various effects associated with hearing ability, speech perception, and hearing functions of users . Outcome measures validate acoustic and psycho - social performances of users after a specified period of time of hearing aid adoption with or without auditory training . Finally, a comprehensive report for the whole hafm is necessary for current and future references . As depicted in fig . 1, the fitting module may be repeated if the follow - up module indicates unsatisfactory outcomes . Taken together, the general framework of hafm emphasizes three factors: 1) importance of three main modules, 2) interactive relations between the modules, and 3) systematic approaches to improve outcomes with hearing aids . Fig . 2 illustrates the assessment module consisting of five components: 1) counseling, 2) audiometry, 3) hearing aid evaluation, 4) hearing aid selection, and 5) ear impression . The whole process of this module is simply depicted below: 1) start at counseling, 2) go to audiometry, 3) go to hearing aid evaluation, 4) go to hearing aid selection, 5) go to ear impression if a customized type of hearing aid is selected, 6) do not take ear impression if noncustomized type of hearing aid is selected . The assessment module serves as a framework for investigating the pre - fitting stage that consists of preparatory components described below . It is generally accepted that counseling identifies individual needs, concerns, and abilities and establishes realistic expectations . Audiometry primarily evaluates hearing loss of potential hearing aid users by measuring hearing thresholds and speech recognition scores with various tests such as pure tone audiometry . In most cases, hearing aid evaluation, pre - treatment trial, is performed with a loaner hearing aids and it attempts to establish realistic expectations by providing typical experiences of wearing hearing aids before making a decision of users' own hearing aids . Hearing aid selection includes decision making processes associated with multiple features of hearing aids by considering device features, and anatomic characteristics and cosmetic factors of potential hearing aid users . Specifically, it determines earmold, style, vent, and other physical and acoustical properties of hearing aids . Then, ear impression is taken if a customized type of hearing aid is selected, such as shell or behind - the - ear type . However, taking an ear impression is not necessary for a non - customized type of hearing aid . 3 illustrates the fitting module consisting of three components: 1) adjustment, 2) verification, and 3) orientation . The whole process of this module is simply depicted below: 1) start at adjustment, 2) go to verification, 3) go to orientation if verified, 4) go to adjustment if failed verification . Physical adjustment aims for maintaining the physical integrity and comfort in wear by modifying and checking physical features of hearing aids such as modification of earmolds or tubes and evaluation of physical adequacy . Electro - acoustic adjustment targets optimal establishments of electro - acoustic characteristics of hearing aids based on types, degrees, and configurations of individual hearing loss . Main contents of adjustment are: 1) quality control: analysis of electro - acoustic specification, 2) selecting a fitting formula based on individual audiogram, 3) checking physical and electro - acoustic comfort (e.g., acoustic feedback, occlusion), 4) modifying physical and electro - acoustic characteristics . Verification, is a process evaluating physical, electro - acoustic, and psycho - acoustic aspects with hearing aids by presenting signals to hearing aids in the test box or a real - ear and evaluating subjective responses using questionnaires . Main contents of verification are: 1) coupler measurements, 2) real ear measurements, 3) sound field tests, 4) psycho - acoustic checking associated with loudness comfort / discomfort, 5) questionnaires focusing on acoustic aspects . Orientation, the final component of the fitting module, mainly provides the information relevant to operational techniques, maintenance strategies including trouble shootings, realistic performance expectations, and other resources needed for hearing aid users . 4 presents the follow - up module consisting of three components: 1) auditory training, 2) outcome measures, and 3) comprehensive report . The whole process of this module is simply depicted below: 1) start at auditory training, 2) go to outcome measures, 3) go to comprehensive report if satisfied, and 4) go to auditory training if unsatisfied . The follow - up module serves as a framework for investigating the components of a post - fitting stage described below . Auditory training is an action to aid the capability of hearing perception through hearing aids and to maximize performance with hearing aids by focusing on various rehabilitative aspects including auditory perception, communication strategies, and individual needs . Although entire training sessions depend on the capability and need of individual hearing aid users, it is generally accepted that auditory training consists of consecutive training sessions for a certain amount of time . Outcome measures validate multidimensional aspects of hearing aid usage by measuring the outcomes associated with acoustic and psycho - social aspects of hearing aid users . Some common outcome domains include measures of speech recognition performance and objective benefits with hearing aids and subjective measures of sound quality, listening efforts, hearing aid usage, and satisfaction . As previously mentioned in fig . 1, if the results from outcome measures with hearing aids are unsatisfactory, the fitting module may be repeated . Comprehensive report reflecting substantial information occurred during the whole hafm process is filed for informative reports and future references at the final stage of the module . Additionally, it is important to note that documentations for each module and component are also needed although this framework does not specifically address . Fig . 2 illustrates the assessment module consisting of five components: 1) counseling, 2) audiometry, 3) hearing aid evaluation, 4) hearing aid selection, and 5) ear impression . The whole process of this module is simply depicted below: 1) start at counseling, 2) go to audiometry, 3) go to hearing aid evaluation, 4) go to hearing aid selection, 5) go to ear impression if a customized type of hearing aid is selected, 6) do not take ear impression if noncustomized type of hearing aid is selected . The assessment module serves as a framework for investigating the pre - fitting stage that consists of preparatory components described below . It is generally accepted that counseling identifies individual needs, concerns, and abilities and establishes realistic expectations . Audiometry primarily evaluates hearing loss of potential hearing aid users by measuring hearing thresholds and speech recognition scores with various tests such as pure tone audiometry . In most cases, hearing aid evaluation, pre - treatment trial, is performed with a loaner hearing aids and it attempts to establish realistic expectations by providing typical experiences of wearing hearing aids before making a decision of users' own hearing aids . Hearing aid selection includes decision making processes associated with multiple features of hearing aids by considering device features, and anatomic characteristics and cosmetic factors of potential hearing aid users . Specifically, it determines earmold, style, vent, and other physical and acoustical properties of hearing aids . Then, ear impression is taken if a customized type of hearing aid is selected, such as shell or behind - the - ear type . However, taking an ear impression is not necessary for a non - customized type of hearing aid . Fig . 3 illustrates the fitting module consisting of three components: 1) adjustment, 2) verification, and 3) orientation . The whole process of this module is simply depicted below: 1) start at adjustment, 2) go to verification, 3) go to orientation if verified, 4) go to adjustment if failed verification . Physical adjustment aims for maintaining the physical integrity and comfort in wear by modifying and checking physical features of hearing aids such as modification of earmolds or tubes and evaluation of physical adequacy . Electro - acoustic adjustment targets optimal establishments of electro - acoustic characteristics of hearing aids based on types, degrees, and configurations of individual hearing loss . Main contents of adjustment are: 1) quality control: analysis of electro - acoustic specification, 2) selecting a fitting formula based on individual audiogram, 3) checking physical and electro - acoustic comfort (e.g., acoustic feedback, occlusion), 4) modifying physical and electro - acoustic characteristics . Verification, is a process evaluating physical, electro - acoustic, and psycho - acoustic aspects with hearing aids by presenting signals to hearing aids in the test box or a real - ear and evaluating subjective responses using questionnaires . Main contents of verification are: 1) coupler measurements, 2) real ear measurements, 3) sound field tests, 4) psycho - acoustic checking associated with loudness comfort / discomfort, 5) questionnaires focusing on acoustic aspects . Orientation, the final component of the fitting module, mainly provides the information relevant to operational techniques, maintenance strategies including trouble shootings, realistic performance expectations, and other resources needed for hearing aid users . Fig . 4 presents the follow - up module consisting of three components: 1) auditory training, 2) outcome measures, and 3) comprehensive report . The whole process of this module is simply depicted below: 1) start at auditory training, 2) go to outcome measures, 3) go to comprehensive report if satisfied, and 4) go to auditory training if unsatisfied . The follow - up module serves as a framework for investigating the components of a post - fitting stage described below . Auditory training is an action to aid the capability of hearing perception through hearing aids and to maximize performance with hearing aids by focusing on various rehabilitative aspects including auditory perception, communication strategies, and individual needs . Although entire training sessions depend on the capability and need of individual hearing aid users, it is generally accepted that auditory training consists of consecutive training sessions for a certain amount of time . Outcome measures validate multidimensional aspects of hearing aid usage by measuring the outcomes associated with acoustic and psycho - social aspects of hearing aid users . Some common outcome domains include measures of speech recognition performance and objective benefits with hearing aids and subjective measures of sound quality, listening efforts, hearing aid usage, and satisfaction . As previously mentioned in fig . 1, if the results from outcome measures with hearing aids are unsatisfactory, the fitting module may be repeated . Comprehensive report reflecting substantial information occurred during the whole hafm process is filed for informative reports and future references at the final stage of the module . Additionally, it is important to note that documentations for each module and component are also needed although this framework does not specifically address . The general framework of hafm with its modules presented in this study includes common practices for practitioners recommended by various pre - existing guidelines . The main purpose of proposing this framework is to make it more explicit and transparent so that its related tasks, including professional services, administration, and financial aspects can be systematized without creating any misunderstandings among stakeholders . Explicitly systemizing the framework of hafm provides substantial benefits of engaging with various stakeholders: hearing aid users, service providers, manufacturers, and public health administrators . For instance, by modularizing the whole procedure of hafm, service providers systematically identify and administer comprehensive components and outlines of hafm leading to the improved service quality and user satisfaction . This contributes to enhance patient - centered services, patient' self - efficacy, and adherence rates with hearing aids (e.g., consistently using hearing aids and attending follow - up appointments). Additionally, systematized services with this framework improve sound quality and comfort of hearing aid users . As a result, it contributes to increase hearing aid adoption rates or decrease hearing aid return rates . Above this, the cost calculation based on each module or each component is applicable to improve public health funding system for the people with hearing impairments as well as clarify payment claim . Another possible application is to develop professional training programs of audiology students and hearing health providers as well as experts in hafm . This framework based on existing guidelines for hearing aid fitting provides the first insight toward the international standardization of hafm.
The 6 principles of integrative nursing are used to frame the comparison of the two perspectives on whole - person / whole - systems health and wellbeing . These principles include (1) human beings are whole systems, inseparable from their environments; (2) human beings have the innate capacity for health and wellbeing; (3) nature has healing and restorative properties that contribute to health and wellbeing; (4) integrative nursing is person - centered and relationship - based; (5) integrative nursing is informed by evidence and uses the full range of therapeutic modalities to support / augment the healing process, moving from least intensive / invasive to more, depending on need and context; and (6) integrative nursing focuses on the health and wellbeing of caregivers as well as those they serve . In din teachings, to live in a state of hzh requires conscious awareness of the collective and interrelated relationships between self and others . Others are broadly defined and include the universal whole: (1) the elements of nature, including animals and insects; (2) the creator; (3) the din holy people; (4) spirits; (5) mother earth; (6) father sky; and (7) the distinct characteristics and cycles within nature and the universe such as seasons, night, day, sun, moon, stars, time . The conscious awareness of this inseparable interrelatedness between human beings and their environment establishes an ownership with responsibility to honor and respect each aspect of ourselves and our inseparable connections to nature and the universe . The inseparable nature between humans and the universal whole lends to the reality of a pre - existing network of internal, external, and existential support for each human being . These relational systems (self, relatives, community, healthcare, healing systems, healers, nature and the environment, culture, and spirits) support human beings on physical, mental, emotional, spiritual, and existential planes to meet our biopsychosocialspiritual needs . Recognizing the existence and availability of these supportive entities is critical to establishing and maintaining health and wellbeing . Hzh is built on positive certainties of the innate capacity of each individual to achieve wholeness, happiness, health, balance, harmony, and ultimate wellbeing through attentive conscious practice of the hzh attributes . The hzh teachings emphasize the utmost sacredness of the collective human body - mind - spirit that is naturally situated within the healing resources of the environment (eg, family, community, spirits, nature, animals). Hzh becomes disrupted as a result of irresponsible thoughts, speech, or behavior, resulting in disharmony, or hchx, which is manifested as chaos, ignorance, evil, sadness, grief, disharmony, imbalance all that is contra to hzh . However, the din belief in the human being's innate ability and capacity to heal empowers those experiencing hchx with the hope of re - establishing individual, community, and environmental health through conscious participation in the process to attain hzh . A din restorative healing ceremony based on the hzh teachings is the hzhji (blessing way) ceremony . The hzhji ceremony is performed when one needs additional support from a healer, the extended family, spirits, nature, and the din holy people to re - establish a state of hzh . Hzh recognizes and honors the healing energy and restorative properties of nature and the universal whole . The hzh teachings insist on a high regard for animals, living creatures, nature, and elements of the earth because of their influence (both negative and positive) on the health and wellbeing of human beings . Recognizing that animals, all living creatures, and elements of nature (water, minerals, plants, air) serve as sources of food, shelter, spiritual guidance, and spiritual healing, the din people hold them as sacred . Din and american indian (ai) healing ceremonies incorporate elements of nature (plants, herbs, air, fire, water, earth), living animals and living creatures, and nonliving animal essence (feathers, hides, bones, and tissue) to cleanse, purify, and restore optimal body - mind - spirit wellbeing . Likewise, the din make sacred offerings to animals and nature to honor their interdependent relationship and express gratitude for their support, ensuring day - to - day opportunities for the healing exchanges and interactions . The teachings of hzh stress the importance of nurturing and sustaining relationship with self and others through respectful thought, speech, and behavior . Relationship with self is viewed as most critical, requiring full understanding of the sacred nature of the collective body - mind - spirit . When the sacred miracle of self is fully embraced, the person operates from a grounded, person - centered perspective and is prepared to extend the honor to external relationships that are not limited to other human beings; the din often extend relationships to animals, spirits, and elements of nature . Constant practice of respect for self and others helps a person master the teachings of hzh and an awareness of the differences between positive, nurturing relationships and negative, destructive relationships . Recognizing the sacredness of self enables recognition of the sacredness of others, thus establishing the essential, interdependent, dynamic nature of relationships that are viewed as sources of stability and support . Ai cultural teachings imply that relationships are everlasting, undergoing only temporary disruptions at times of separation, death, or physical absence . When a relationship becomes disrupted, the disruption impacts the collective unified whole, including friends and distant relatives, pets and animals, the collective environment, and perhaps even spiritual ancestors and future generations . Hzh teachings remind us of the often forgotten relationships between living beings, elements of nature, and inanimate objects, such as the relationship one has with his or her home or with sacred items such as protective stones and arrowheads . The din honor their relationship with the home by blessing and showing gratitude for the home . Recognizing the sacred protective and sheltering properties of homes, the din bestow honor and respect on the home through song and action . Homes are often adorned with objects that protect the home and the people, animals, inanimate objects, and spirits within . The effort of maintaining relationships is constant, a grounding force that promotes individual, spiritual, and collective health and strength . Generosity and reciprocity are significant in hzh philosophy and precious gem (white shell, turquoise, abalone, and jet) and corn offerings are symbolic of the honored relationships between humans and mother earth, father sky, lightning, the sun, the moon, the creator, and the holy people . Likewise, reciprocal exchanges of support, food, gifts, love, compassion, and respect are viewed as a means of maintaining relationship . Honoring, preserving, sustaining, and nurturing positive and healthy interdependent relationships may be the richest source of internal, external, and existential health protection attained through the practice of din hzh . Integrative nursing is informed by evidence and uses the full range of therapeutic modalities to support / augment the healing process, moving from least intensive / invasive to more, depending on need and context . The survival and transfer of the hzh philosophy across generations of din serve as evidence of the value and significance of these teachings for the health and wellbeing of the din . Because hzh philosophy embraces all mechanisms that lead to wellbeing, the full range of therapeutic healing modalities (eg, western medicine, ai medicine, ai spiritual healing, ai healing ceremonies, complementary and alternative health options) that support the attainment of physical, mental, spiritual, emotional, social, and environmental wellbeing align with the goal of hzh . There remains limited western scientific evidence to confirm the health protective benefits of ai cultural wisdom; however, emerging scientific literature does support and recognize the validity of indigenous ways of knowing as a source of rigorous and relevant knowledge relevant for the improved health of indigenous people . The ways in which american indians and indigenous people view and approach knowledge differ from the ways in which western scientific, evidence - based knowledge is viewed and approached . Indigenous knowledge is shared, transmitted orally, role - modeled, and often received interpersonally or through spiritual processes, ceremony, interpretation of symbols and divination, and through dreams and visions . Indigenous knowledge in the form of culture, stories, chants, song, and prayer is also thought to be alive, and the sharing of this knowledge requires levels of care and respect in the exploration and transmission of the knowledge . Harvey emphasizes the need for a constructive synthesis of differing worldviews, ideologies, and technologies through the application of indigenous wisdom as a means to improve the health of indigenous populations . The evidence supporting hzh as a health - sustaining philosophy is apparent in the survival of the din as well as in the survival of the oral teachings and patterned behaviors of the din . These patterns of din thought, speech, and behavior have endured for generations, despite the historical traumatic events of the past . Yet to improve the health of the din, the tenets of hzh philosophy must be explored, understood, correctly translated, and conveyed to the people using methods such as storytelling, talking circles, or broadcasting via older (radio) and contemporary (social networking) media communicating the significant benefits of the ancient, time - honored hzh wisdom to all generations of din as well as all individuals interested in sharing in this valuable health - sustaining knowledge . When the din seek and receive healing in the form of ceremony, prayer, or medical care, hzh principles require them to show gratitude for the healing by presenting a token, a gift, or a ceremonial offering to the healer, which may include the caregiver, nature, holy people, or the creator . This offering not only represents gratitude for the healing received but also serves as a protective shield of energetic intention; the recipient of the healing provides a method to ensure the safety and protection of the healer, whose service is sometimes hazardous and exhausting . This offering demonstrates the recipient's genuine concern for the wellbeing of the healer or caregiver; the health of the individual seeking healing and the health of the healer providing the healing service are both valued within the hzh philosophy . In din teachings, to live in a state of hzh requires conscious awareness of the collective and interrelated relationships between self and others . Others are broadly defined and include the universal whole: (1) the elements of nature, including animals and insects; (2) the creator; (3) the din holy people; (4) spirits; (5) mother earth; (6) father sky; and (7) the distinct characteristics and cycles within nature and the universe such as seasons, night, day, sun, moon, stars, time . The conscious awareness of this inseparable interrelatedness between human beings and their environment establishes an ownership with responsibility to honor and respect each aspect of ourselves and our inseparable connections to nature and the universe . The inseparable nature between humans and the universal whole lends to the reality of a pre - existing network of internal, external, and existential support for each human being . These relational systems (self, relatives, community, healthcare, healing systems, healers, nature and the environment, culture, and spirits) support human beings on physical, mental, emotional, spiritual, and existential planes to meet our biopsychosocialspiritual needs . Recognizing the existence and availability of these supportive entities is critical to establishing and maintaining health and wellbeing . Hzh is built on positive certainties of the innate capacity of each individual to achieve wholeness, happiness, health, balance, harmony, and ultimate wellbeing through attentive conscious practice of the hzh attributes . The hzh teachings emphasize the utmost sacredness of the collective human body - mind - spirit that is naturally situated within the healing resources of the environment (eg, family, community, spirits, nature, animals). Hzh becomes disrupted as a result of irresponsible thoughts, speech, or behavior, resulting in disharmony, or hchx, which is manifested as chaos, ignorance, evil, sadness, grief, disharmony, imbalance all that is contra to hzh . However, the din belief in the human being's innate ability and capacity to heal empowers those experiencing hchx with the hope of re - establishing individual, community, and environmental health through conscious participation in the process to attain hzh . A din restorative healing ceremony based on the hzh teachings is the hzhji (blessing way) ceremony . The hzhji ceremony is performed when one needs additional support from a healer, the extended family, spirits, nature, and the din holy people to re - establish a state of hzh . Hzh recognizes and honors the healing energy and restorative properties of nature and the universal whole . The hzh teachings insist on a high regard for animals, living creatures, nature, and elements of the earth because of their influence (both negative and positive) on the health and wellbeing of human beings . Recognizing that animals, all living creatures, and elements of nature (water, minerals, plants, air) serve as sources of food, shelter, spiritual guidance, and spiritual healing, the din people hold them as sacred . Din and american indian (ai) healing ceremonies incorporate elements of nature (plants, herbs, air, fire, water, earth), living animals and living creatures, and nonliving animal essence (feathers, hides, bones, and tissue) to cleanse, purify, and restore optimal body - mind - spirit wellbeing . Likewise, the din make sacred offerings to animals and nature to honor their interdependent relationship and express gratitude for their support, ensuring day - to - day opportunities for the healing exchanges and interactions . The teachings of hzh stress the importance of nurturing and sustaining relationship with self and others through respectful thought, speech, and behavior . Relationship with self is viewed as most critical, requiring full understanding of the sacred nature of the collective body - mind - spirit . When the sacred miracle of self is fully embraced, the person operates from a grounded, person - centered perspective and is prepared to extend the honor to external relationships that are not limited to other human beings; the din often extend relationships to animals, spirits, and elements of nature . Constant practice of respect for self and others helps a person master the teachings of hzh and an awareness of the differences between positive, nurturing relationships and negative, destructive relationships . Recognizing the sacredness of self enables recognition of the sacredness of others, thus establishing the essential, interdependent, dynamic nature of relationships that are viewed as sources of stability and support . Ai cultural teachings imply that relationships are everlasting, undergoing only temporary disruptions at times of separation, death, or physical absence . When a relationship becomes disrupted, the disruption impacts the collective unified whole, including friends and distant relatives, pets and animals, the collective environment, and perhaps even spiritual ancestors and future generations . Hzh teachings remind us of the often forgotten relationships between living beings, elements of nature, and inanimate objects, such as the relationship one has with his or her home or with sacred items such as protective stones and arrowheads . The din honor their relationship with the home by blessing and showing gratitude for the home . Recognizing the sacred protective and sheltering properties of homes, the din bestow honor and respect on the home through song and action . Homes are often adorned with objects that protect the home and the people, animals, inanimate objects, and spirits within . The effort of maintaining relationships is constant, a grounding force that promotes individual, spiritual, and collective health and strength . Generosity and reciprocity are significant in hzh philosophy and precious gem (white shell, turquoise, abalone, and jet) and corn offerings are symbolic of the honored relationships between humans and mother earth, father sky, lightning, the sun, the moon, the creator, and the holy people . Likewise, reciprocal exchanges of support, food, gifts, love, compassion, and respect are viewed as a means of maintaining relationship . Honoring, preserving, sustaining, and nurturing positive and healthy interdependent relationships may be the richest source of internal, external, and existential health protection attained through the practice of din hzh . Integrative nursing is informed by evidence and uses the full range of therapeutic modalities to support / augment the healing process, moving from least intensive / invasive to more, depending on need and context . The survival and transfer of the hzh philosophy across generations of din serve as evidence of the value and significance of these teachings for the health and wellbeing of the din . Because hzh philosophy embraces all mechanisms that lead to wellbeing, the full range of therapeutic healing modalities (eg, western medicine, ai medicine, ai spiritual healing, ai healing ceremonies, complementary and alternative health options) that support the attainment of physical, mental, spiritual, emotional, social, and environmental wellbeing align with the goal of hzh . There remains limited western scientific evidence to confirm the health protective benefits of ai cultural wisdom; however, emerging scientific literature does support and recognize the validity of indigenous ways of knowing as a source of rigorous and relevant knowledge relevant for the improved health of indigenous people . The ways in which american indians and indigenous people view and approach knowledge differ from the ways in which western scientific, evidence - based knowledge is viewed and approached . Indigenous knowledge is shared, transmitted orally, role - modeled, and often received interpersonally or through spiritual processes, ceremony, interpretation of symbols and divination, and through dreams and visions . Indigenous knowledge in the form of culture, stories, chants, song, and prayer is also thought to be alive, and the sharing of this knowledge requires levels of care and respect in the exploration and transmission of the knowledge . Harvey emphasizes the need for a constructive synthesis of differing worldviews, ideologies, and technologies through the application of indigenous wisdom as a means to improve the health of indigenous populations . The evidence supporting hzh as a health - sustaining philosophy is apparent in the survival of the din as well as in the survival of the oral teachings and patterned behaviors of the din . These patterns of din thought, speech, and behavior have endured for generations, despite the historical traumatic events of the past . Yet to improve the health of the din, the tenets of hzh philosophy must be explored, understood, correctly translated, and conveyed to the people using methods such as storytelling, talking circles, or broadcasting via older (radio) and contemporary (social networking) media communicating the significant benefits of the ancient, time - honored hzh wisdom to all generations of din as well as all individuals interested in sharing in this valuable health - sustaining knowledge . When the din seek and receive healing in the form of ceremony, prayer, or medical care, hzh principles require them to show gratitude for the healing by presenting a token, a gift, or a ceremonial offering to the healer, which may include the caregiver, nature, holy people, or the creator . This offering not only represents gratitude for the healing received but also serves as a protective shield of energetic intention; the recipient of the healing provides a method to ensure the safety and protection of the healer, whose service is sometimes hazardous and exhausting . This offering demonstrates the recipient's genuine concern for the wellbeing of the healer or caregiver; the health of the individual seeking healing and the health of the healer providing the healing service are both valued within the hzh philosophy . Recognizing that the 566 federally recognized american indian / alaska native (ai / an) tribes in the united states are strikingly different in their own culture, geographic region, language, dress, food, ceremonies, philosophies, beliefs, and teachings, we believe that the din hzh wellness philosophy provides an important example of one tribal philosophy on wellness . Sharing the din hzh philosophy re - introduces this philosophy to the din while informing other ai / an nations, global indigenous cultures, and even nonindigenous people of the world who may benefit from these health - sustaining lifeways . Though this wisdom comes from the din, it is offered as a prompting framework for other ai / an tribes, villages, pueblos, and indigenous people of the world to explore, reexamine, and bring forth their own distinct and innate wellness philosophies as mechanisms for health promotion . For the indigenous populations who may have lost elements or the entirety of their own distinct cultural wisdom due to historical adversities of the past, the 6 tenets of the hzh wellness philosophy are intertribally relevant because of the vast cultural similarities of our people . In addition, the attributes of the hzh philosophy are universally relevant to all individuals and communities or agencies that may be interested in novel or ancient ways to promote the health and wellbeing of the people of the world . The hzh wellness philosophy and the hzh resilience model (figure) also serve as frameworks through which more biomedically focused healthcare providers can become acquainted with cultural world - views of ai / an people . It is important to keep in mind that these frameworks provide only a brief glimpse into a differing cultural worldview; integrate this ai / an cultural wisdom with care and caution . Remember, the ai / an worldview honors knowledge, stories, and cultural wisdom as sacred entities; when we fold it into our clinical settings, we should do so with honor and respect by including ai / an cultural experts as part of the planning and implementation process . This cautionary approach communicates our understanding and our respect of the cultural values and ensures accurate implementation of unique culturally tailored approaches for wellbeing and health . The stunning alignment of integrative nursing principles and the hzh wellness philosophy illustrates the power of the integrative nursing meta - theoretical perspective and how we can transform healthcare delivery, moving us from parallel healthcare systems (indigenous and biomedical) to a healthcare system that is inclusive and responsive to the needs of our varied populations . Ai cultural wisdom is essential to the well - being of the din and all indigenous populations who experience considerable health disparities that have not been adequately addressed by current biomedical efforts . The collective literature on ai / an culture consistently emphasizes the health - protective nature of shared intertribal cultural wisdom across ai / an tribes, villages, and pueblos . And it is critical that we embrace the benefits of cultural wisdom while recognizing the inherent wholeness that can be used as a valuable alternative to or in combination with biomedicine . Removing someone's cultural wisdom is destructive and leaves an existential hole in the body - mind - spirit, creating vulnerabilities to physical, mental, and social ills . Ongoing refusal of our health - care system to acknowledge the need for and benefit of our cultural practices and health beliefs continues to inflict the trauma of our past, adding to the chronic stress that has resulted from historical traumas and attempts to eradicate our culture . Integrative nursing offers the opportunity to re - introduce cultural wellness wisdom, such as the din hzh philosophy, as a means to improve the health of individuals . Integrative nursing, through the acceptance and validation of indigenous health - sustaining wisdom, contributes to the delivery of effective, authentic, culturally tailored, whole - person and whole - system patient - centered, relationship - based healthcare . This article began with an excerpt from the the fundamental law of the navajo nation, which reminds us of the diversity of the world while also reminding us of the importance of the individual yet distinct needs of each culture . As individuals, we must never forget how we embody the origins of our cultures and that it is only natural that our healing mechanisms should embody the wealth of rich and diverse healing wisdom . Integrative nursing offers the scientifically based platform that will bring forth increased collaboration among healers and healthcare providers and will transform healthcare delivery that is focused on the health and wellbeing of the biopsychosocialspiritual beings of the world . Hzh nahasdl: happiness, health, harmony, peace, wellbeing, beauty, and all that is good restored.
Bridging integrator-1 breast cancer 1 and 2 cyclin - dependent kinase cyclin - dependent kinase inhibitor human homolog of double minute 2 poly(adp - ribosyl)ation poly(adpribose) polymerase 1 retinoblastoma 1 protein the discovery of gleevec (sti-571), a tyrosine kinase inhibitor, was a milestone achievement in clinical oncology and this inhibitor has demonstrated remarkable efficacy in philadelphia chromosome - positive (ph) chronic myeloid leukemia . Since then, mechanism - based approaches have been used to specifically target various kinases and/or downstream oncogenic pathways that are critically involved in cell cycle progression and tumorigenesis . However, in addition to this approach, a more recent and novel use of small - molecule inhibitors has emerged as a promising endeavor in the field of cancer chemotherapy . Here, we briefly review the mechanistic basis of restoration of a tumor suppressor and its potential complications for cancer therapy . The tumor suppressor function mediated by the retinoblastoma 1 protein (rb1) is principally attributed to its interaction with the e2f transcription factor 1 (e2f1). The rb1/e2f1 complex represses a number of e2f1-dependent transcriptional target genes that are required for the transition from g1 to s phase in the cell cycle . Because rb1 is inactivated by phosphorylation mediated by the g1 cyclin - dependent kinases 4 and 6 (cdk4 and cdk6), restoring rb1 function by inactivating cdk4/6 is theoretically an obvious approach . Although structural similarities among a number of cdk family members hampered the development of a cdk4/6-specific inhibitor for many years, some agents, including palbociclib (pd-0332991), have recently demonstrated promising results in phase i / ii clinical trials for human malignancies, including breast cancer . Above and beyond rb1, another tumor suppressor that is critical for numerous growth inhibitory pathways the abundance of wild - type p53 protein is massively reduced as a result of ubiquitin - dependent and human homolog of double minute 2 (hdm2)-mediated degradation of p53 . Therefore, dissociation of p53 from the p53/hdm2 complex is a reasonable strategy for rescuing p53 function . Based on the crystallographic structure of the p53/hdm2 peptide complex, small p53 peptides that mimic the region of p53 sufficient for hdm2 binding and small - molecule hdm2 antagonists have been shown to disrupt the p53/hdm2 interaction in vitro and in vivo . Some of these, including mi-219, nutlin-3, and rg7112, have been found to be effective preclinically and have consequently moved into phase i / ii clinical trials although proteasome inhibitors such as bortezomib (ps-341) may not be as specific for stabilizing p53 as these hdm2 inhibitors, other growth - inhibitory gene products, including the cyclin - dependent kinase inhibitor 1b (cdkn1b or p27, kip1) protein, can also be degraded in an ubiquitin - dependent manner . Therefore, it may be advantageous to re - establish a broad spectrum of growth - inhibitory functions by blocking the proteasome pathway . Although the approach of re - establishing tumor suppressor function in tumors as a therapeutic option is mechanistically intriguing, there are potential dilemmas associated with the systemic restoration of tumor suppressor function . Tumor suppressor genes are frequently mutated or deleted in cancer patients, and given that some of the mutant genes acquire oncogenic potential, this approach may simply reboot a mutant (i.e., oncogenic) tumor suppressor . Even if a tumor suppressor gene is intact, its function should not depend on other cancer - susceptible proteins . For example, the cyclin - dependent kinase inhibitor 2a (cdkn2a) gene is not frequently deleted in cancer cells, but is inactivated by dna methylation . However, epigenetic reactivation of the cdkn2a gene may not be an effective approach if rb1 and/or p53 are deficient, because the tumor suppressor functions of the products of the 2 alternative reading frames of cdkn2a p16 and p14 proteins largely depend on rb1 and p53, respectively . Therefore, for the tumor suppression approach to be fully effective, it will be important to identify a non - mutated (or non - deleted) tumor suppressor whose function does not rely on other tumor suppressors that might be already mutated or deleted . Bridging integrator-1 (bin1) was originally identified as a c - myc oncoprotein - interacting tumor suppressor . The bin1 gene itself is rarely mutated or deleted, but is frequently silenced in human cancer cells moreover, bin1 acts as a tumor suppressor in vitro and in vivo in the absence of rb1 and p53 . We recently demonstrated that bin1, whose gene promoter is activated by e2f1, directly interacts with e2f1 and represses its transcription, implying that a negative - feedback loop regulates bin1 gene expression . Interestingly, we found that e2f1 is poly(adp - ribosyl)ated by poly(adp - ribose) polymerase 1 (parp1) and that parp1 inhibition unlocks the e2f1bin1 negative - feedback loop to vigorously activate the bin1 gene, which induces g2/m arrest in the cell cycle and/or apoptosis . Synthetic lethality, parp inhibitors have been actively used for clinical trials in breast cancer 1 and 2 (brca1/2)-deficient breast and ovarian cancers . However, it was unclear why parp inhibitors alone also show therapeutic efficacy, even in cancer cells expressing wild - type brca1/2 . Based on our recent data, the restoration of bin1 by parp inhibitors may offer a mechanistic rationale for expanding the clinical usage of parp inhibitors over a wider range of tumor types, regardless of the status of rb1, tp53, and brca1/2 genes (fig . 1). Figure 1.small-molecule inhibitors used in cancer therapy restore the functions of various tumor suppressors in malignant cells . The tumor suppressor functions of retinoblastoma 1 protein (rb1), tumor protein p53 (tp53, known as p53), and bridging integrator-1 (bin1) can be pharmacologically restored by small - molecule inhibitors, such as palbociclib (pd-0332991), mi-219, and olaparib, respectively . In the case of p53 restoration, a human homolog of double minute 2 (hdm2), named hdm4 (also known as hdmx), might interfere with the effectiveness of hdm2 antagonists, probably because of overlapping functions between hdmx and hdm2 . P, phosphorylation; par, poly(adp - ribosyl)ation; dp1 (or tfdp1), transcription factor dp1; e2f1, e2f transcription factor 1; cdk4/6, cyclin - dependent kinase 4 and 6; g1/s: transition between g1 (growth1/gap1) phase to s (dna synthesis) phase in the cell cycle; g2/m, transition between g2 (growth2/gap2) phase to m (mitosis) phase in the cell cycle . Small - molecule inhibitors used in cancer therapy restore the functions of various tumor suppressors in malignant cells . The tumor suppressor functions of retinoblastoma 1 protein (rb1), tumor protein p53 (tp53, known as p53), and bridging integrator-1 (bin1) can be pharmacologically restored by small - molecule inhibitors, such as palbociclib (pd-0332991), mi-219, and olaparib, respectively . In the case of p53 restoration, a human homolog of double minute 2 (hdm2), named hdm4 (also known as hdmx), might interfere with the effectiveness of hdm2 antagonists, probably because of overlapping functions between hdmx and hdm2 . P, phosphorylation; par, poly(adp - ribosyl)ation; dp1 (or tfdp1), transcription factor dp1; e2f1, e2f transcription factor 1; cdk4/6, cyclin - dependent kinase 4 and 6; g1/s: transition between g1 (growth1/gap1) phase to s (dna synthesis) phase in the cell cycle; g2/m, transition between g2 (growth2/gap2) phase to m (mitosis) phase in the cell cycle . Chemotherapy and radiotherapy are conventional treatments for eradicating tumors, but cancer often develops therapeutic resistance over time . Given that many tumor suppressors are proapoptotic in response to dna damaging agents, it would be clinically pertinent to increase the chemo- and radiosensitivities of cancer by combining standard treatments with agents that can restore the activity of silenced tumor suppressors, provided they are not mutated or deleted, in human malignancies . This work was partially supported by a grant from the us national institutes of health (nih) (r01ca140379) (to d.s . ).
Pulmonary sequestration (ps) is a rare developmental disorder characterized by the presence of a nonfunctioning lung parenchyma that lacks normal tracheobronchial tree connection and is supplied by a systemic artery.1 congenital diaphragmatic hernia (cdh) is a malformation characterized by a diaphragmatic defect through which the abdominal viscera migrate into the chest during fetal life.2 the association between ps and cdh has been previously described.1 2 3 4 5 6 7 we report the case of a neonate affected by prenatally diagnosed cdh treated by thoracoscopy . During the procedure, we detected an associated extralobar ps, which was preoperatively undiagnosed, and removed it . A female infant was delivered by elective cesarean section at 35 weeks of gestation and was referred to our department for prenatal diagnosis of left diaphragmatic hernia detected at 32 weeks of gestation by ultrasounds and characterized by a lung - to - head ratio (lhr) of 1.6 . Weight at birth was 3,400 g, and apgar index was 9 at 1 minute and 10 at 5 minutes, respectively . At birth, the newborn underwent nasotracheal intubation and high - frequency oscillatory ventilation, positioning of a nasogastric tube, and chest x - ray that confirmed prenatal diagnosis . During the first day of life, the patient maintained clinical stability, so we decided for surgical correction through thoracoscopic approach on the second day of life . A 5-mm trocar was positioned in the fifth intercostal space over the middle axillary line, and two further operative trocars (3 mm, reusable) were placed in the sixth intercostal space over the anterior and posterior axillary line, respectively . Capnothorax was created with a 6mmhg pressure and 0.5l / min flow, and maintained with intermittent insufflation to reduce side effects of co2 absorption . Once entered in the pleural cavity, we noted a diaphragmatic hernia with a sac . A basal extralobar sequestration was also detected and removed after ligation of the feeding artery with two endoloops (fig . The diaphragmatic defect was repaired with an interrupted 3/0 braided polyester suture (ethibond) (figs . 2 and 3). The patient underwent first follow - up chest x - ray after 2 months, and she is currently asymptomatic without radiologic signs of recurrence (6-month follow - up). Ps includes two forms: intralobar ps (within the normal parenchyma), which is the most common form, and extralobar pulmonary sequestration (eps).1 eps has its own pleural covering and can be located inside or outside the thorax . The associations are more common in the extralobar type and include congenital pulmonary airway malformation, configuring a lesion termed as hybrid lesion, congenital heart disease, and cdh in up to 15 to 40% of cases.7 8 9 10 a possible explanation is the interference related to the presence of a sequestered lung mass with the fusion of diaphragmatic components (mechanical obstacle).7 11 in recent years, there has been speculation on the prognosis associated with the two simultaneous defects (cdh and ps).7 ps can be associated with pulmonary hypoplasia, and being a space - occupying lesion, it could constitute a further obstacle to lung development.3 8 indeed, the prognosis of ps combined with cdh remains unclear, and although there is not sufficient evidence regarding the influence of ps on cdh prognosis, some authors hypothesized that ps acts as a protective factor during pregnancy and probably improves the prognosis of patients with cdh.7 two possible explanations for this improved outcome may be given: (1) ps acts as an anatomical barrier to the rise of the abdominal viscera; (2) ps causes a mediastinal shift leading to an underestimation of lhr, thus distorting the prognosis that seems poorer.7 prenatal identification of both cdh and ps is possible, and frequently occurs.7 in our case, prenatal diagnosis of concurrent cdh and ps was not possible probably because herniated organs obscured the presence of a sequestered lung mass . The treatment of choice of a symptomatic eps in addition to a cdh is surgical excision.1 2 however, some authors recommend observation of asymptomatic eps, as these lesions rarely produce complications.12 we believe that resection should be the treatment of choice in all cases since there are hybrid lesions misdiagnosed as pure sequestrations and one can never be sure that the lesion will remain asymptomatic.4 the most frequent argumentation for removal is the prevention of recurrent infections and malignancy . Malignancy has been reported to be associated to cystic lesions, in particular to type i congenital cystic adenomatoid malformation in 1% of cases.12 although the risk is quite low, in case of recurrent infections or neoplastic evolution, the previous wait and see approach would be difficult to justify . Regarding the surgical approach, minimally invasive alternatives to the traditional surgical technique have been recently proposed, although the best approach to cdh is controversial.2 recently, hypercapnia and acidosis during thoracoscopy in newborns have been reported.13 14 in our case, we used co2 insufflation at the beginning of the procedure to facilitate reduction of herniated organs only for brief periods to improve visibility . The distinction between a diaphragmatic hernia with sac and a congenital localized eventration may be difficult . Sac removal seems to be associated with a lower recurrence rate,15 but during thoracoscopy the presence of a sac can facilitate reduction, can lessen visceral trauma, and keeps the two cavities separate.2 according to a recent survey among the international pediatric endosurgery group members,16 22% of surgeons declared to leave the sac in place during the thoracoscopic repair of cdh, so this approach is quite accepted even if in a minority of cases . Accurate closure of this vessel is mandatory to avoid postoperative bleeding, which can be life - threatening.17 in case of eps, vessel sealing can be achieved with different devices such as ligasure, clips, and ligation . In this case, since the eps was of limited size, we closed the feeding artery with two endoloops and cut the vessel with cold scissors to avoid thermic damage of the ligatures . We would probably have missed the ps using a different surgical approach (classical laparotomy), as during the laparotomic approach the posterior position of the diaphragmatic defect makes it hard to inspect the entire hypoplastic lung that is atelectatic and displaced in the upper part of the chest . The thoracoscopic approach for cdh offers the advantage to easily diagnose and treat associated lung lesions . The case report describes the usefulness of thoracoscopy as diagnostic and therapeutic tool in case of diaphragmatic hernia associated with undiagnosed lung malformation . Thoracoscopy allows to explore thoracic cavity looking for lung lesions that are associated to diaphragmatic hernia in 1540% of cases and to remove them.
Diarrhea, including that of parasitic origin, remains one of the most common illnesses among children and, as reported by the world health organization, is one of the major causes of infant and childhood mortality in developing countries . Intestinal opportunistic parasitic infections are important causes of diarrhea which is a serious health problem in tropical regions . Are common parasitic causes of human diarrhea with the prevalence rate of 1%3% in the industrialized world and 4%-17% in developing countries . In developed countries, massive cryptosporidium food borne and waterborne outbreaks have been reported because cryptosporidium oocysts are not killed by conventional disinfectants and chlorination . In developing countries, cryptosporidium affects mostly children under five years of age and has been reported to be more common in malnourished than in well - nourished children[4, 5]. C. parvum, a protozoan parasite of the phylum apicomplexa, is an enteric pathogen that causes an acute as well as chronic diarrhea in humans . The human host range is broad and includes people with immunodeficiency, children in developing countries, and outbreaks among immuno - competent individuals . In a healthy adult, infection often results in a self - limited diarrheal illness, however, in children of developing countries, cryptosporidiosis can result in persistent diarrhea leading to malnutrition and developmental delays . Indeed, cryptosporidium is still an under - diagnosed cause of diarrheal illness in children . Few reports with relatively small sample sizes are available on the prevalence and clinical presentation of cryptosporidium infection in both immuno - compromised and immune - competent children in iran[713]. The purpose of this study was to determine and compare the prevalence of c. parvum infection in children with an acute or persistent diarrhea in isfahan city, central iran . This cross - sectional study was prospectively conducted in three university hospitals in isfahan from august 2007 to june 2008 . Children aged one month to 10 years presenting with acute or persistent diarrhea were visited in each hospital by one of the researchers (pediatrician) of the study and were included consecutively . Diarrhea was defined as the passage of abnormally liquid or unformed stools at an increased frequency and further defined as acute if lasted <2 weeks and persistent if lasted for 2 to 4 weeks before admission . Children who had used antiparasitic or antibiotic drugs before admission and also those with known hematologic diseases and immuno - compromised children were excluded from the study . Assuming the average prevalence of c. parvum to be 5% according to previous reports, and a margin error of 2% (difference between estimated and reported prevalence) and 95% confidence, a sample size of 456 was calculated for determining the overall prevalence of c. parvum . Fecal samples were collected in sterile plastic bottles and were transported without preservative and arrived in the central laboratory of isfahan city within 24 hours of collection . The oocyst of c. parvum was tested using a modified acid - fast staining method which is a sensitive and specific approach for the identification of cryptosporidium and coccidian oocysts in stools . In this technique, the oocysts appear as pink to red, spherical to ovoid bodies on a blue or purple background . The study protocol was approved by the ethical committee of isfahan university of medical sciences and consent was obtained from the parents . The chi - square test was used to compare relative frequency of cryptosporidium infection between children with acute and persistent diarrhea . Independent t - test was used to compare the mean age between infected and non - infected children . During the study period, 606 children aged one month to 10 years were presented with acute or persistent diarrhea to three university hospitals in isfahan . There were 254 (41.9%) males and 352 (58.1%) females with the mean age of 42.430.0 months . Acute or persistent diarrhea was present in 422 (69.6%) and 184 (30.4%) of the children, respectively . Cryptosporidium oocysts were detected in 12.5% (23/184) of the children with persistent compared to 1.2% (5/422) of the children with acute diarrhea; p<0.001 . Most (89.2%) of the infected children were under 5 years of age, however, the age difference between the infected and non - infected children was not statistically significant; p=0.1 . Also, there was no significant sex difference between the infected and non - infected children; p=0.5 (table 1). Comparison of infected and non - infected children the age difference between children with acute and persistent diarrhea was not statistically significant (43.933.4 vs 38.919.6 months, p=0.06) and there was no significant difference between the two sexes (248/174 vs 104/80 female / male ratio, p=0.6). Intestinal parasites are very common in developing countries and cryptosporidium has revealed to be one of the most common parasites . Cryptosporidium infests the small intestinal epithelium, thereby, results in an accelerated loss of villous enterocytes, leading to severe villous atrophy and a malabsorptive and secretory diarrhea which is the most pronounced clinical feature of the infection . Other symptoms include dehydration, weight loss, stomach cramp, or pain, fever, nausea, and vomiting . The infectious dose is low and feeding studies have demonstrated that the ingestion of as few as 1030 oocysts can cause infection in healthy persons . Infected persons have been reported to shed 1010 oocysts in a single bowel movement and to excrete oocysts for up to 50 days after cessation of diarrhea[16, 17]. There are discrepancies in the prevalence of cryptosporidiosis in children among surveys in different parts of iran . The reported prevalence is from 1.5% in rasht (northern city of iran) to 21.4% in shiraz (southern city of iran)[713]. The difference in the reported prevalence may be attributed to differences in study population (considering age range), diagnostic methods, environmental risk factors (public water supply), time of the study (summer vs winter), nutritional status of the children, and other risk factors[6, 18, 19]. In other countries, the rates of cryptosporidiosis are 1%-2% in europe, 0.6%4.3% in north america, and 3%-10.2% in asia, australia, africa, and central and south america . Several methods are available for identification of cryptosporidial oocysts in fecal specimens including modified acid - fast staining which detects oocyst wall, fluorescein - conjugated monoclonal antibody - based detection of oocyst wall antigen, enzyme - linked immunosorbent assay (elisa) which detects cryptosporidial antigen and most recently polymerase chain reaction (pcr) which detects crytosporidial dna . Modified acid - fast stain of a fecal smear has been the gold standard for detecting cryptosporidium oocysts in stool . Although the concentration and staining procedures are time- consuming and also require an experienced microscopist to read the slides, it is inexpensive and allows the detection of other parasites (eg, isospora and cyclospora) at the same time . However, the sensitivity for the detection of cryptosporidium is substantially better with elisa formats or immunofluorescent assays . Pcr method is sensitive, but the cost and tedious methodology have limited its use in the clinical microbiology laboratory . Regardless of the method used, it is extremely important that the physician remembers to request cryptosporidial testing when stool from a child with diarrheal illness is to be examined for ova and parasites . The results of the present study showed that children with cryptosporidiosis are more likely to present with persistent diarrhea, compared to acute diarrhea . In a report from southeastern iran, hamedi and however, khalili and colleagues in their study on 171 children found no relationship between type of diarrhea and infection . Although our study did not show significant age difference between the infected and non - infected children, most of the infected children, as expected, were below 5 years old . Though humans are susceptible at any time in their lives, the prevalence of cryptosporidiosis is higher in younger children mostly in children aged below 5 years[8, 10, 11]. The occurrence of high infection rates in younger children might be attributed to their weak immune responses and/or risk of contact with the oocysts . However, more studies are needed to confirm this subject[12, 18]. Although the sample size of the study was large enough, sampling was consecutive and not random . However, the study was multicenter and we conducted the study through one year, so it is unlikely that season or selection bias affected our results . Also we applied the modified acid - fast staining and although it is a sensitive and specific approach for the identification of cryptosporidium, elisa and immunofluorescent assays are more sensitive in this regard . We recommend evaluating children for important risk factors for infection with cryptosporidium, such as nutritional and socioeconomic status, for future studies . The prevalence of cryptosporidiosis in children presenting with persistent diarrhea is considerable and we suggest routine stool examination for cryptosporidium in these children for early diagnosis and treatment to prevent possible complications . Also, for control and prevention of this infection, strengthening water quality standards and improving sanitary practices are required.
Ventilator - associated pneumonia (vap) is a dangerous complication in patients who need mechanical ventilation (mv). Vap is the most common infection among patients undergoing mv and has been associated with an increased mortality rate (approaching 50%), an increased morbidity rate, the length of intensive care unit (icu) stay, and the duration of mv . Despite advances in preventive strategies, such as the implementation of the vap bundle and the centers for disease control and prevention's (cdc) recommendations for critical care, type 2 diabetes mellitus (t2 dm) is a condition that has been linked to alterations of immune response and is often found in critical care patients . However, the impact of t2 dm as a risk factor for vap in critically ill patients has not been sufficiently studied . There are limited data concerning selected populations of critically ill patients, which indicate that diabetic patients have a higher probability of developing icu - acquired infections compared to nondiabetic subjects while some studies involving critically ill patients in the icu found no association between t2 dm and the development of vap infection . The purpose of this study was to determine the risk of vap for diabetic and nondiabetic patients with normal glycemic control who underwent mv for at least 48 h. this is a secondary analysis from a prospective, observational, cohort survey conducted in three general icus in the 1,000-bed imam khomeini medical center located in mazandaran, iran . Approval of the institutional review board was obtained for the study . Also, informed consent in the case of conscious patients was obtained from the patients themselves and in unconscious patients, from the proper surrogate decision - maker . Informed consent for the present study was obtained regardless of the consent obtained for the main study . This prospective study was conducted between september 22, 2012 and september 23, 2013 to determine the incidence, risk factors, and outcome of vap in the icus . All traumatic patients aged> 18 years without pneumonia at icu admission and who then required at least 48 h of mv were included in this study . Patients who were undergoing mv before admission to the icu or those who died within 48 h of starting mv were excluded . A group of attending physicians and nurses prospectively collected data on all patients who underwent mv . Moreover, the clinical review included the clinical pulmonary infection score (cpis) records for diagnosis of vap . Patients were assessed every day (in the morning; once every 24 h) during the entire length of the study . Our study's primary focus was the rate of diagnosis of vap . Only the first episode of vap was evaluated . All the patients undergoing mv routinely received stress ulcer prophylaxis [ranitidine, 50 mg via intravenous (iv) three times a day] and intravenous antibiotic prophylaxis for 24 - 36 h. administered antibiotic prophylaxis was based on hospital routine, including cefalotin (1 g, divided into four doses a day) for mechanically ventilated adult trauma patients . In all patients, the blood glucose was controlled with insulin therapy (infusion or subcutaneous) within a range of 80180 mg / dl for the duration of icu stay . In diabetic and nondiabetic patients, if it rose up to 200 mg / dl, 2 units of insulin subcutaneously was prescribed for per 20 mg / dl blood glucose, higher than 200 mg / dl . Infusion insulin therapy with rate of 0.52 units was commenced in diabetic patients with blood glucose up to 350 mg / dl according to blood glucose per 1 h. the baseline data collected included demographic data [sex, age, body mass index (bmi), date of admission to the icu], primary diagnosis, underlying illness, type of tracheal intubation (elective / emergency), history of hypertension, chronic obstructive pulmonary disease, and t2 dm, limitation in positional changes, enteral nutrition (gavage feeding), icu stay and length of hospital stay, duration of intubation, glasgow coma scale (gcs) with ventilatory support and with or without sedation, and duration of mv . Diagnosis of vap was according to original cpis after at least 48 h of mv . Cpis was developed in 1991 and it is including a new chest x - ray infiltrate persistent for 48 h or more, a body temperature of more than 38.58c or less than 35.08c, changes in white blood cell count (wbc) as a leukocyte count of more than 10,000/ll or less than 3,000/ll, worsening hypoxia (arterial oxygenation, pao2/fraction of inspired oxygen, fio2 ratio 240 without acute respiratory distress syndrome (ards), and ards), purulent tracheal secretions, and microorganisms isolated from at least one of the following samples: bronchoalveolar lavage (bal) 10,000 cfu / ml), endotracheal aspirate (eta) 100,000 cfu / ml, or sputum . Semi - quantitative eta or bal samples of suspected cases of vap were collected from icu patients in this study . The cpis ranges from zero to 12 and scores higher than 6 indicate vap . Validity and reliability of the persian version of the cpis is confirmed, and it is used widely in research studies to appraise suspected vap . A cumulative survival curve for each patient was calculated using the kaplan meier method and was compared by use of log - rank tests . All statistically marginally significant prognostic factors identified by univariate analysis (p <0.2) (sex, age, limitation in positional changes state, gcs score) were entered into a cox proportional hazard (ph) model with forward stepwise (likelihood ratio) to identify independent predictors of vap event . Only variables with statistically significant effect were kept in the final model . The cox ph model assumes that the hazard ratio (hr) for any two specifications of predictors is constant over time . For all analyses, p values were two - sided and p <0.05 was considered to be statistically significant . All statistical analyses and graphics were performed using the statistical package for the social sciences (spss) software package (version 16.0, spss inc ., chicago, il, usa) and stata statistical package (version 10, stata, college station, tx). This is a secondary analysis from a prospective, observational, cohort survey conducted in three general icus in the 1,000-bed imam khomeini medical center located in mazandaran, iran . Approval of the institutional review board was obtained for the study . Also, informed consent in the case of conscious patients was obtained from the patients themselves and in unconscious patients, from the proper surrogate decision - maker . Informed consent for the present study was obtained regardless of the consent obtained for the main study . This prospective study was conducted between september 22, 2012 and september 23, 2013 to determine the incidence, risk factors, and outcome of vap in the icus . All traumatic patients aged> 18 years without pneumonia at icu admission and who then required at least 48 h of mv were included in this study . Patients who were undergoing mv before admission to the icu or those who died within 48 h of starting mv were excluded . A group of attending physicians and nurses prospectively collected data on all patients who underwent mv . Moreover, the clinical review included the clinical pulmonary infection score (cpis) records for diagnosis of vap . Patients were assessed every day (in the morning; once every 24 h) during the entire length of the study . Our study's primary focus was the rate of diagnosis of vap . Only the first episode of vap was evaluated . All the patients undergoing mv routinely received stress ulcer prophylaxis [ranitidine, 50 mg via intravenous (iv) three times a day] and intravenous antibiotic prophylaxis for 24 - 36 h. administered antibiotic prophylaxis was based on hospital routine, including cefalotin (1 g, divided into four doses a day) for mechanically ventilated adult trauma patients . In all patients, the blood glucose was controlled with insulin therapy (infusion or subcutaneous) within a range of 80180 mg / dl for the duration of icu stay . In diabetic and nondiabetic patients, blood glucose was checked every 6 h and 24 h, respectively . If it rose up to 200 mg / dl, 2 units of insulin subcutaneously was prescribed for per 20 mg / dl blood glucose, higher than 200 mg / dl . Infusion insulin therapy with rate of 0.52 units was commenced in diabetic patients with blood glucose up to 350 mg / dl according to blood glucose per 1 h. the baseline data collected included demographic data [sex, age, body mass index (bmi), date of admission to the icu], primary diagnosis, underlying illness, type of tracheal intubation (elective / emergency), history of hypertension, chronic obstructive pulmonary disease, and t2 dm, limitation in positional changes, enteral nutrition (gavage feeding), icu stay and length of hospital stay, duration of intubation, glasgow coma scale (gcs) with ventilatory support and with or without sedation, and duration of mv . Diagnosis of vap was according to original cpis after at least 48 h of mv . Cpis was developed in 1991 and it is including a new chest x - ray infiltrate persistent for 48 h or more, a body temperature of more than 38.58c or less than 35.08c, changes in white blood cell count (wbc) as a leukocyte count of more than 10,000/ll or less than 3,000/ll, worsening hypoxia (arterial oxygenation, pao2/fraction of inspired oxygen, fio2 ratio 240 without acute respiratory distress syndrome (ards), and ards), purulent tracheal secretions, and microorganisms isolated from at least one of the following samples: bronchoalveolar lavage (bal) 10,000 cfu / ml), endotracheal aspirate (eta) 100,000 cfu / ml, or sputum . Semi - quantitative eta or bal samples of suspected cases of vap were collected from icu patients in this study . The cpis ranges from zero to 12 and scores higher than 6 indicate vap . Validity and reliability of the persian version of the cpis is confirmed, and it is used widely in research studies to appraise suspected vap . A cumulative survival curve for each patient was calculated using the kaplan meier method and was compared by use of log - rank tests . All statistically marginally significant prognostic factors identified by univariate analysis (p <0.2) (sex, age, limitation in positional changes state, gcs score) were entered into a cox proportional hazard (ph) model with forward stepwise (likelihood ratio) to identify independent predictors of vap event . Only variables with statistically significant effect the cox ph model assumes that the hazard ratio (hr) for any two specifications of predictors is constant over time . For all analyses, p values were two - sided and p <0.05 was considered to be statistically significant . All statistical analyses and graphics were performed using the statistical package for the social sciences (spss) software package (version 16.0, spss inc ., chicago, il, usa) and stata statistical package (version 10, stata, college station, tx). Basic and clinical characteristics of patients according to diabetic state table 1 shows that there was no significant difference in the sex, age, bmi, hypertension (htn), chronic obstructive pulmonary disease (copd), limitation in positional changes, tracheal intubation, gcs, and gavage feeding between diabetic and nondiabetic patients . Results show that the mean age was 47.81 years (21.7); the mean duration of admission, icu stay, and intubation were 17.16 days (13.34), 16.2 days (13.19), and 11.71 days (7.56), respectively . The vast majority of the patients were males of 3065 years of age and overweight [table 2]. Median (95% ci) time to the progression of vap (kaplan meier method) in icu patients according to the possible prognostic factors during the study period, 186 patients required mv for more than 48 h out of whom, 41 developed vap (17 cases (11%) in nondiabetic patients and 24 cases (75%) in diabetic patients), corresponding to an average of 18.82 (95% ci: 13.8625.57) vap cases per 1,000 days of intubation [50 (95% ci: 33.5174.6) and 10.01 (95% ci: 6.2216.1)] vap cases per 1,000 days of intubation in diabetic and nondiabetic patients, respectively). The median time from hospitalization to vap was 29.09 days (95% ci: 26.2731.9) and this in relation to patient characteristics is shown in table 2 . As shown in table 2, median time from hospitalization to vap occurrence was higher in females, middle - aged patients, nondiabetics, patients with no limitation in positional changes, and patients with higher gcs . The cox ph model revealed that after adjusting other variables, diabetic patients have a higher hazard of progression to vap than nondiabetic patients (hr: 10.12; 95% ci: 5.120.2, p <0.0001) [table 3]. Table 3 shows that aging patients versus patients less than 30 years of age (3.12; 95% ci: 1.566.25; p = 0.001), and thin (5.1; 95% ci: 1.124.57; p = 0.04) and overweight (2.75; 95% ci: 1.415.37; p = 0.003) patients versus patients of normal weight have a higher hazard of progression to vap . This assumption of the cox ph model was evaluated with the schoenfeld residuals method and we saw that the plot of the residuals were horizontal and close to 0 . Association between prognostic factors and progression to vap in cox proportional hazard regression multivariate analysis icu mortality rates in diabetic and nondiabetic patients in our study were 37.5% and 28.6%, respectively (p = 0.32). Icu mortality increased by 66.6% (p <0.0001) when diabetic patients developed vap, remaining an independent predictor of mortality [hazard ratio (hr) 18.18; 95% ci: 4.76100] [table 3]. A survival curve of the 186 patients, some with and some without t2 dm, is shown in figure 1 . Survival curves of 186 patients with different baseline characteristics of t2 dm as shown in figure 1, the median time from hospitalization to vap occurrence in diabetic patients was lower than in nondiabetic patients [9.34 (95% ci: 7.3911.29) and 34.32 (95% ci: 31.8136.83) days, respectively; p <0.0001]. We conducted a study to determine the relationship between t2 dm and suspected vap in mechanically ventilated adult trauma patients admitted to icus . A major finding of this secondary analysis of the cohort study that is contrary to the findings of previous studies was that the critically ill trauma patients with t2 dm had a significantly higher incidence of vap compared to patients who did not have t2 dm . Hence, t2 dm is probably associated with an increased risk of vap in intubated critically ill trauma patients undergoing mv . This study also demonstrated that limitation in positional changes, age, and being overweight were also risk factors for the development of vap . There are a few studies that evaluate the association between personal history of t2 dm and the occurrence of vap in critically ill patients . A prospective observational study on patients admitted to the general icu showed that the overall icu incidence of vap was 26%, and t2 dm and hba1c were not associated with increased risk of vap . Similarly, a review study that surveyed 84 studies sought to identify the risk factors for hospital - acquired pneumonia (hap). Cohort, case - control, and observational studies were included in this work, which revealed that t2 dm was not a risk factor for the development of hap . In relation to lung infections in diabetic patients, of the 354 patients with a positive sputum culture, 125 patients (35.5%) were diabetics . Results of this study revealed that the diabetic patients were generally older with a male predominance compared to nondiabetics . It is possible that the older age of these diabetic patients is one of the reasons for the increased rate of pneumonia in this population . Tamayo et al . Conducted a prospective cohort study that included 1,610 postoperative cardiac surgery patients after cardiopulmonary bypass (cpb). It observed that 124 patients (7.7%) developed vap . After performing the cox multivariate analysis adjustment, t2 dm was identified as one of the important independent mortality risk factors (hr: 1.90). In another prospective study by falguera results of this study showed that patients with t2 dm were significantly associated with the evidence of pleural effusion and higher mortality . We speculate that t2 dm might affect the risk of vap through immune dysfunction and age effects in intubated critically ill patients . The observed predisposition of diabetic critically ill patients to vap could be partly due to significant changes within their immune system, particularly changes related to pulmonary dysfunction, and alterations of pulmonary host defenses . Although the rate of t2 dm in critically ill patients is high, the impact of t2 dm as a risk factor for vap in these patients has not been adequately evaluated . Our findings demonstrate that t2 dm in critically ill trauma patients is associated with the increased risk of vap in the icu . Well - designed prospective cohort or case - control studies in diabetic critically ill patients could provide more information about the correlation between t2 dm and vap in the icu . In agreement with several previous studies, this study showed that the limitation of positional changes, increase in age, and also being overweight are risk factors of developing vap . Although the data in this analysis were prospectively collected, this study was a secondary analysis and therefore, may have had some limitations consistent with a retrospective approach . Because of these limitations, our results need confirmation, and future cohort studies investigating the relationship between t2 dm and vap in mechanically ventilated adult trauma patients in the icu are needed . This study was a secondary analysis and thus, was not specifically designed for the given research question . Patients with formerly undiagnosed t2 dm and subjects with fasting blood sugar or impaired glucose tolerance may have been neglected, because identifying patients with t2 dm was based on a previous history . Also, no data were presented for all diabetic patients concerning the duration of t2 dm and the severity of organ damage in these patients . Moreover, some variables including icu scoring such as the simplified acute physiology score (saps) ii score and the sequential organ failure assessment (sofa) score were not recorded for analysis . Likewise, mortality in diabetic patients with vap was not compared to nondiabetic subjects . In our study, we used semi - quantitative eta more than bal because of its cost and ease . Some studies have showed that there was a total agreement in bacteriology between eta and bal in sensitivity for vap diagnosis . Unfortunately, because of the inadequate sample size of patients, we also did not test the association between early- and late - onset vap and t2 dm in this study . Thus, this study lacks baseline data that will need to be addressed in the next generation of studies . In conclusion, it seems that a diabetic condition in critically ill patients is an important baseline characteristic that can be associated with various aspects of critical illness such as infection . Our results indicate that t2 dm is significantly associated with increased vap risk during icu stay for mechanically ventilated adult trauma patients . The results suggest that greater care in the prevention of vap should be considered for this important population . This study was funded by the research deputy of islamic azad university, sari branch, iran . All authors declare no conflict of interest; no conflict of interest exists for any of the authors associated with the manuscript, and the entire study was performed without external funding . The funding organization had no role in the design and conduct of the study, or in the collection, analysis, and interpretation of the data . This study was funded by the research deputy of islamic azad university, sari branch, iran . All authors declare no conflict of interest; no conflict of interest exists for any of the authors associated with the manuscript, and the entire study was performed without external funding . The funding organization had no role in the design and conduct of the study, or in the collection, analysis, and interpretation of the data . Study concept and design: hdkh, aez, mon, and af . Statistical analysis and interpretation of data: aa . Critical revision of the manuscript for important intellectual content: hdkh, aez, mon, af, and aa.
While adipose tissue has in the past been viewed as a depot involved in passive storage of energy, it is increasingly clear that it is in fact a highly active tissue . It is an endocrine organ, secreting various cytokines and regulating metabolism of other organs . Adipose tissue affects not only body weight, but also insulin sensitivity, atherogenesis, blood pressure, and other physiological functions . The body has several depots of white adipose tissue, of which the major two categories are subcutaneous and visceral, although visceral fat can be divided into subcategories of intraperitoneal, retroperitoneal, and pelvic . Most studies have shown relatively little difference in the fatty acid composition of the various fat stores including fat in serum . However, there is suggestion that this may not be the case in obese individuals and kishino et al . Reported that visceral fat thickness was positively correlated to levels of palmitic and saturate fatty acids in serum, but negatively correlated with levels of linoleic and polyunsaturated fatty acids . There is a growing body of evidence that many of the cytokines and growth factors have depot - specific distribution in human adipose tissue [57]. It is known that visceral fat correlates better with the development of insulin insensitivity and the metabolic syndrome than does subcutaneous fat, and this may be related to the demonstrations that there is greater glucose uptake and insulin metabolism in visceral fat . Persistent organic pollutants (pops) are highly lipophilic chemicals that bioaccumulate in animal and human fats . Pops includes chemicals such as dioxins / furans, polychlorinated biphenyls (pcbs), chlorinated pesticides, brominated flame retardants, and perfluorinated compounds . Because of chlorine, bromine, or fluoride groups on the hydrocarbon rings or chains, these substances are resistant to degradation both in the environment and in the human body . The half - lives of substances such as dde, the major metabolite of ddt, and some pcb congeners are of the order of 510 years, and because of their presence in almost all animal fat consumed with the diet, the concentrations in the human body tend to increase with age because rates of intake exceed those of metabolism and excretion . While the manufacture and use of most of these chemicals has been prohibited in the us through the toxic substances control act of 1976 and their manufacture and use on a global scale is outlawed by the stockholm convention of the united nations which entered into force in 2004, these toxic chemicals remain present in the bodies of most humans . Pops are toxic to human health, being probable human carcinogens, having immunosuppressive activity, causing neurotoxicity, increasing risk of diabetes, cardiovascular disease, and hypertension, and being endocrine disruptive chemicals [11, 12]. Different pops often have somewhat different health outcomes, and most studies have focused on only single chemicals . However, since essentially all humans are exposed to many different pops, but perhaps in different relative concentrations, it is important to consider the health effects of chemical mixtures . Multiple chemicals may have additive, less than additive, or synergistic effects . Because pops are stored in adipose tissue and because the various adipose tissues of the body have somewhat different endocrine functions, it is important to know whether or not pops distribute equally throughout all adipose tissues and ultimately to determine how levels of these chemicals influence the local adipose tissue . Have reported that pcb 77 but not pcb 153 induces adipocyte differentiation and expression and release of proinflammatory cytokines . This preliminary study of analysis of pcbs and chlorinated pesticides in various adipose tissues obtained from seven human subjects is a first attempt to determine whether the distributions of several pcb congeners and several chlorinated pesticides are the same in different adipose tissues . Seven human subjects scheduled for elective surgery for either benign lesions or cancer provided informed consent for the surgical team to obtain blood and fat biopsies from several different fat compartments at the time of surgery for analysis of ten pcb congeners and 22 chlorinated pesticides . Four subjects (3 males and 1 female) presented with renal cell carcinoma, 1 male had a benign renal cyst, 1 male had a benign small intestinal tumor, and 1 male had prostate cancer . All subjects had preoperative cat scans of the abdomen and pelvis, bone scans, and complete blood metabolic studies . After blood samples were drawn, the surgeon excised samples of 5 grams or greater from the subcutaneous peripheral fat as well as from one or more intra - abdominal fat stores, including visceral, retroperitoneal, and pelvic fat as well as from the kidney or prostate that was removed at surgery . Visceral fat commonly refers to adipose tissue within the peritoneal cavity . In some subjects, visceral fat from three different locations was obtained, the mesenteric fat from around the small and large intestines, omental fat from the folds of the peritoneum that connect the stomach with other abdominal organs, and fat from the falciform ligament that attaches part of the liver to the diaphragm and abdominal wall . Retroperitoneal fat lies posterior to the peritoneal compartment and is separated from it by the posterior peritoneum . Retroperitoneal fat surrounds the adrenal glands, pancreas, kidneys, and the great vessels as well as large lymphatic channels . The pelvic fat compartment is an inferior extension of the retroperitoneal fat and surrounds the iliac vessels, rectum, bladder, and prostate in males . Figure 1 shows the location of the various fat stores as well as the major abdominal organs . Serum and fat samples were analyzed by accu - chem laboratories, richardson, tx . Pcbs and chlorinated pesticides were extracted into hexane after the addition of an internal standard (2,2,3,3,4,5,5,6-octachlorobiphenyl). The solvent was then evaporated to dryness and the sample reconstituted and analyzed on a gas chromatograph (hewlett packard 5890) equipped with an electron capture detector using a hewlett packard 5970 mass spectrometer and a db5 capillary column . Three sets of standard references were used, one from the national institute of standards and technology and two commercially available reference sets . The level of detection was 0.15 ng / ml and the level of quantitation was 0.3 ng / ml . Twenty three chlorinated pesticides were measured, including hexachlorobenzene (hcb), endrin,,,, and hexachlorocyclohexane (hch), heptachlor, heptachlor epoxide,, and chlordane, oxychlordane, trans - nonachlor, endosulfan i and ii, endosulfan sulfate, aldrin, dieldrin, dichlorodiphenyltrichloroethylene (dde), dichlordiphenyltrichlorethane (ddt), dichlorodiphenyldichloroethane (ddd), methoxychlor, mirex and endrin aldehyde . Ten pcb congeners were analyzed including pcbs 47 (2,2,4,4), 105 (2,3,3,4,4), 118 (2,3,4,4,5), 137 (2,2,3,4,4,5), 138 (2,2,3,4,4,5), 153 (2,24,4,5,5), 170 (2,23,34,4,5), 171 (2,23,3,4,4,6), 180 (2,23,4,45.5), and 183 (2,23,4,4,5,6). All results are reported as wet weight values, based in part on the basis of the demonstration [15, 16] that lipid adjustment increases the bias in the measurement . The limits of detection were 0.3 ppb in serum, 10 ppb in fat and kidney cancer tissue, and 35.7 ppb in cancerous tissue . Table 1 gives the results of pesticide levels in the various samples from the seven subjects, while table 2 provides similar information for pcb congeners . Of the 23 pesticides monitored, none of the samples showed levels of aldrin, and chlordane, and hch, endosulfan ii, endosulfan sulfate, ddd, ddt, endrin aldehyde, or methoxychlor above the level of quantitation, while for pcb congeners levels of pcbs 47 and 171 were not found in any tissue at concentrations above the levels of quantitation . Figure 2(a) shows the average levels of selected pesticides in serum and various adipose tissues . Figure 2(b) shows the same data without dde, which is present at much higher concentrations than any other pesticide . This figure clearly shows that there are some differences in the patterns, most striking in the one individual from whom a pelvic sample was obtained . The differences are less clear for those tissues for which more samples were obtained . Figure 4 shows a plot of oxychlordane concentration in visceral versus subcutaneous adipose tissues and demonstrates that there is a very close relationship with an r of 0.939, p = .0003 . The results of this investigation show a much more complex picture for the distribution of pesticides and pcbs in various adipose tissue pools than expected . It is usually assumed that in a fasting person (all of these surgical patients were fasting), pops will be equally distributed in all adipose tissue sites, and consequently one can obtain use serum samples to monitor body burden of lipophilic substances . While serum usually contains somewhere between 14% lipid, the assumption is that the pops concentrations in the serum lipid fraction reflect all adipose tissue . In the past, results were usually reported as lipid adjusted concentrations although reports that lipid - adjustment creates bias have resulted in more reports of wet weight values or using serum lipids as a covariate . Unfortunately, the sensitivity used in the serum analyses in these subjects was not sufficiently sensitive so as to provide much basis for comparison, but there is valuable data from the different adipose tissue sites . It is clear that levels of individual pops in any one individual reflect both the degree and source of exposure, the time since that exposure occurred, genetic differences among individuals in rates of metabolism, body mass index and, if female, number of pregnancies and whether or not the child was breast fed . Most metabolism of pops is a result of activity of cytochrome p450s, and the ability to metabolize individual pops varies with structure and genetic susceptibility . For pcbs, for example, congeners with fewer chlorines are in general more easily metabolized than those with more chlorines . Thus, an individual with major exposure in the distant past will show a congener pattern dominated by higher chlorinated congeners . However one would assume that the same pattern would be found in the various fat stores . For pcbs, there is reasonably good correlation between serum levels and those in subcutaneous fat for gk, hm, and as but not ms, go, and jt . When comparing the different adipose tissues, there is a clear tendency for visceral and retroperitoneal to have a greater percentage of higher chlorinated congeners than subcutaneous fat for gk, go and jt . However, for ml subcutaneous and visceral patterns were similar but concentrations higher than in retropertoneal fat . Ms showed relatively similar patterns, but with concentrations in mesenteric fat considerably greater than in subcutaneous fat, while as showed higher concentrations in subcutaneous fat . Hm, the only subject with pelvic fat, showed relatively similar concentrations for pcbs 118, 138, and 170, but high levels of 153 in pelvic but not subcutaneous and of 180 in subcutaneous but not pelvic fat . Dde was present in the highest concentration in most samples, but was below the detection limit in subcutaneous fat in gk and jt in spite of being present at high concentrations in serum and other adipose tissues . In all other subjects, dieldrin, heptachlor epoxide, oxychlordane, and transnonachlor were present in most adipose samples and had relatively similar distributions in all fat stores . Hch was present in comparable amounts in most samples that contained measureable levels, and hch was found in only a few samples . Mirex was found in serum at a relatively high concentration in gk, but was not present in any adipose tissue, and similar situation was found with heptachlor in ml . These results suggest a very recent exposure to these two pops that has not yet equilibrated with adipose tissue . When comparing general levels of pcbs to those of the pesticides, distributions were similar for ml, hm, and go . However for as and jt, pcbs were at higher concentrations in subcutaneous fat, but pesticides were at higher concentration in visceral and retroperitoneal fat . For ms levels of pcbs were greater in subcutaneous than visceral fat, but levels of pesticides were approximately equal in the two stores . Only one cancerous tissue (kidney) gave detectible levels of pops, and it contained pcb 153 and oxychlorodane at relatively low concentrations, but no other pops . These two pops were present in the highest concentration in subcutaneous fat . Since the lipid content of a tumor would be expected to be significantly less than adipose tissue, the relative wet weight results are not surprising . It is unlikely that the variable results seen in levels of both pcb and pesticides are due to chance or to faulty chemical analysis . There is always some variability in analysis of pops, but usually this is not more than 20% . There are reports that there are large differences in the average fatty acid composition between individual subjects, as well as some differences in composition of different adipose tissue sites within subjects . If this is the case, it is possible that the lipid composition of the various adipose tissue stores is reflected in different solubility of individual pops in different depots even in the same individual . Even subcutaneous fat taken from different parts of the body has been found to show between 210% variation [2, 20]. There is some information suggesting that subcutaneous and visceral fats differ more, with saturated fats higher and monounsaturated fat lower in visceral than subcutaneous fat . Since p450 enzymes are found in almost all cells, not just liver, it is conceivable that there is different metabolism in different fat depots . Certainly there is evidence that different depots express different endocrine factors . However, unless the pops were bound in some fashion, it seems unlikely that even if there were differences in local metabolism that there would not be a relatively rapid redistribution . They reported six pops where serum levels correlated relatively well with those in adipose tissues, but no clear relationship with the other nine pops . The mean adipose / serum ratios varied from 2.85 for endosulfan - ether to a high of 150.68 for dde and 159.47 for endosulfan ii . They questioned the common assumption that serum concentrations are indicative of those in adipose tissues . Our results are consistent with their observations and indicate that the same surprising relationship may also occur with different pcb congeners . There are major limitations to this study because of small sample size, lack of measurement of the lipid content of tissues, and the relatively low sensitivity of the analytical methods . However, the difficulty in obtaining samples and the lack of any comparable study elsewhere in light of the overall importance of the question of how different pops distribute in different adipose tissue depots is justification for publishing these results . Replication is certainly needed to confirm the overall conclusion that pops do not always distribute equally among various lipid compartment in the human body . Contrary to expectations that patterns of various pesticides and pcb congeners were not as similar as expected when comparing levels in serum, subcutaneous, visceral, retroperitoneal, and pelvic fat . These results indicate that care should be taken when assuming that serum levels of pops reflect patterns found in lipid stores throughout the body.
Following exposure of a population to radiation, concern about the risk of radiation - induced cancer can be a major source of anxiety,1,2 particularly for parents concerned about the future health of their children.3 despite the obvious importance of preventing accidental radiation exposure and reducing exposure levels, it is not currently known whether there is a practical way to specifically mitigate radiation - induced cancer risk once exposure has occurred.4,5 post - exposure measures to increase survival from acute radiation are available for individuals needing intensive care after high - dose exposure; however, for large populations exposed to low radiation doses, there are no medical interventions which are currently advised to reduce the probability of a radiation - induced malignancy later in life . Research into chemical and biologic radio - protectors has been ongoing for many decades,6 and while some agents have been found to confer a certain degree of protection against acute effects when delivered within a short period after irradiation, many need to be administered prior to exposure in order to be effective . In the absence of a specific medical countermeasure, a consensus regarding other strategies proven to be effective in minimizing radiation - induced cancer risk and/or cancer risk more generally, would likely be a valuable public health tool . In addition to direct effects on cancer burden, empowering individuals in exposed populations by providing safe, proven methods to lower their cancer risk could assist in decreasing anxiety and improving coping skills.7 one candidate for such an approach is caloric restriction (cr). Controlled dietary intake has long been studied in terms of its effects on increasing longevity and reducing cancer incidence.8 cr has also been investigated for its potential as an adjuvant cancer treatment, to slow the growth of existing tumors.9,10 although many specific mechanisms of cr have been documented, it has effects on a wide range of physiological and cellular systems, not all of which are understood . In fact, the levels of cr required to produce the greatest longevity effects in experimental animals are perhaps beyond what could be reasonably maintained by most people, and certainly would not be advised for children or young people during their developmental years.11 yet, understanding the kinetics of how cancer preventative / suppressive approaches can be instigated long after the time of irradiation affects the risk of radiation - induced cancer can help us to determine the utility of such approaches, and whether they can specifically prevent or merely offset radiation - induced cancer risk . Seven weeks after 1-week - old mice were subjected to 3.8 gy irradiation, they were fed either a diet equivalent to their ad libitum calorie intake (95 kcal / mouse / week) or switched to a nutritionally - balanced diet limited to approximately 1/3 fewer calories (65 kcal / mouse / week).12 the results showed that irradiation alone decreased tumor - free lifespan, cr alone increased tumor - free lifespan, and initiating cr after irradiation was able to partially mitigate radiation - induced cancer . Separating the data by cause of death revealed that different tumor types / sites responded differently to cr, with radiation - induced lymphomas showing little to no response, while a clear effect could be seen for late - occurring solid tumors . Here, we analysed the pathology results further to identify cases of lethal solid tumors and applied a multistage mathematical model of carcinogenesis to the data13 in order to gain mechanistic insight into the protective effect of cr, specifically as it relates to radiation - induced cancers . We used lifespan data from male b6c3f1 mice published by shang et al.12 briefly, mice were either irradiated with 3.8 gy of x - rays at 1 week of age, or were sham - irradiated . This radiation dose was chosen because it is known to efficiently induce both haematopoietic and various solid tumors in the b6c3f1 mouse strain, providing the opportunity to examine whether cr would have differential effects by tumor type . At 7 weeks of age, irradiated and unirradiated mice were switched from an ad libitum diet to either a diet limited to 95 kcal / week / mouse (equivalent to ad libitum) or to a nutrient - balanced but calorically restricted diet of 65 kcal / week / mouse (a caloric reduction of approximately one - third). At this age, mice are fully developed adults but have not yet reached their peak body weight, allowing the mice to physiologically adapt to long - term cr without interrupting normal development or inducing a sharp decline in body weight . The four groups were monitored over their natural lifespan, with detailed autopsy and pathology analysis for each mouse . Tumor spectrum and latency, and their effects on overall lifespan, were used as measures of the interaction between radiation - induced carcinogenesis and cr . In the original study, lifespans were compared based on tumors that the mice harboured at the time of autopsy . However, as such tumors could be either incidental or lethal, the age of the mouse at the time of death may not have been directly related to any one of the tumors discovered at autopsy.14 thus, we elected to use data only from mice with tumor(s) that were diagnosed as lethal, with the autopsy and pathology records re - evaluated by a veterinary pathologist . This approach allowed us to compare the age - specific tumor mortality in a manner analogous to that used in human cancer epidemiology . Since cr was not observed to have a significant effect on leukaemia / lymphoma, we limited our analyses to deaths due to any solid tumor (except sarcoma, which was rare), and then further analysed deaths due to lethal hepatocellular carcinoma (hcc), lethal lung tumor, or lethal hemangioma . We used a simple implementation of the armitage - doll model,13 which postulates that a normal single cell must undergo several critical steps, such as mutations or other rate - limiting events, to become a malignant cell . Armitage and doll used this model to explain the temporal variation in the death rate for solid cancer, and it has since been used across a large number of epidemiological studies to estimate the stages of cancers at many sites.15,16 the mortality rate of cancer at age t, i(t) (tumor deaths / mouse - day), is related to the probabilities of the transition between each step, p (transitions per unit time). Because the transitions must proceed in a unique, sequential order, i(t) is expressed as shown in equation (1), below, where k is the number of critical stages in the course of carcinogenesis a cell needs to pass through before becoming fully malignant, t is the age of the mice, and pi is the probability of a transition from the (i1) to the i change (i k). (1)i(t)=p1p2pk(k-1)tk-1the logarithm of i(t) is directly proportional to the logarithm of age, as shown in equation (2), where a is a constant representing the product of the transition (pk) rates divided by (k1)!, and t (in our analysis) is the mid - point of age divided into 200-day increments . (2)log i(t)=a+(k-1)logtby plotting the log of age - specific mortality rate, i(t), against the log of age (t) obtained from the lifespan and pathology data, we used a linear fit (estimated using the non - linear least squares method with r software version 3.1.3) to calculate the intercept (a) and slope (k1) for each of the three site - specific tumors and all solid tumors to derive the model parameters pk and k. an estimate of the per - stage transition rate, pk, was calculated from the product of the transition rates, pk . Figure 1 shows the model fit for all solid tumors for the four treatment groups, with the model parameters for all solid tumor data and for the three site - specific analyses shown in table 1 . The data clearly show a more rapid increase in the mortality rate with age in the two 65 kcal groups (cr) compared to the mortality rate of mice on the 95 kcal diet . This is consistent with the increased lifespan associated with cr reported in the original data, since, although there is a stronger effect of age on the increase in the mortality rate, the mortality rate starts at a lower point and remains lower for most of the animals lifespans, exceeding that of the standard diet only at the most advanced ages, by which time few animals remain alive . The increase in the slope and thus in the model parameter k with cr is significant for all solid tumors, hcc, and lung tumor deaths (table 2), but is not significant for hemangioma . For all solid tumors and hcc, the increase in k associated with cr was equivalent in irradiated and unirradiated mice . On the contrary, irradiation did not change the estimate of k, with no significant change in the slopes of the graphs of solid tumor deaths or any of the site - specific data . For each of the four treatment groups, the age - specific mortality data for all solid tumors shows an increase with age that is consistent with the power function characteristic of the simple armitage - doll model (r> 0.98). Hcc represented between 27% to 56% of the diagnoses for solid tumor death in each group, and the model for the hcc data (r> 0.93) was very consistent with the all - solid tumor model . Although the model fits were reasonable (r> 0.88), there were too few cases of lung cancer to provide a model fit for each group, and the model fit was poor for hemangioma (r> 0.38). Both the pooled solid tumor and hcc models showed a significant increase in k with cr . An increase in k of around 3 reflects a more rapid increase in the mortality rate in aging mice on the lower calorie diet, albeit starting from a lower baseline mortality rate . According to the tenets of the model the carcinogenic process which delay the onset of tumors but also allow for the accumulation of cells in the penultimate stages of tumor formation . The reduction in the product of the transition rates with cr could be the result of an overarching effect common to all of the steps (such as a decrease in the spontaneous mutation rate), or the result of a specific effect which alters the probability of one particular transition . The results presented here confirm that solid tumor mortality is indeed lower when mice are subjected to cr for most of their lifespan, and excludes artefacts due to variations in tumor stage or accelerated discovery of incidental tumors due to earlier death caused by another tumor type . The similarity in the effect of cr on both parameters between the irradiated and unirradiated groups may suggest that there is no or little interaction between the detrimental effects of irradiation and the beneficial effects of the dietary regimen . It is thus possible that cr offsets both spontaneous and radiation - induced carcinogenesis in a similar fashion . This could mean that existing data on the optimal levels, duration, and timing of cr may be applicable to individuals who have previously been exposed to radiation . Interestingly, although the products of the transition rates did increase, the modelled values of k were not significantly lower in the irradiated mice, consistent with the notion that the probability of mutations was altered without concomitant alterations in the oncogenic pathway . It may be that a radiation - induced dna mutation, such as inactivation of a tumor suppressor gene, might instantaneously move cells one or more steps forward through a multistage progression towards carcinogenesis, effectively removing the step(s). Although not significant, the effect of irradiation on the values of k were estimated to be in the range of a loss of a single step, and, thus, such an explanation is not excluded by our data . Since radiation may act on the carcinogenic process by inducing genomic instability, stimulating tissue regeneration following radiation - induced apoptosis, and other physiological changes, as well as introducing dna mutations, it is plausible that the overall effect of irradiation on age - specific mortality would affect both the number of steps and the transition rate . Here, the effects of radiation and cr were not included in the model itself, since a sufficient quantity of data are not currently available to formalize the relationship . However, having demonstrated an effect of cr on both the parameters of this simple model, we can begin to consider the types of experimental data that would be valuable for incorporating radiation exposure and cr directly into the model . The most pertinent questions pertaining to cr would be: what is the relationship between the level of cr and the induction of protective effects, and is there a threshold? What is the effect of the interval between radiation exposure and the onset of cr? What is the effect of the duration of cr? What is the relationship between the level of cr and the induction of protective effects, and is there a threshold? What is the effect of the interval between radiation exposure and the onset of cr? What is the effect of the duration of cr? In one study, adult trp53 mice (which are highly predisposed to cancer development) were assigned to either a calorie - restricted diet or a one - day - a - week fasting regimen, and both diets were associated with a delay in cancer - related death.17 as discussed above, if radiation - mediated cancer induction and cr - mediated cancer suppression are indeed independent of one another, existing data on cr regimens could be used to formulate a model to examine optimal strategies for offsetting both radiation - induced and spontaneous cancer risk . Such approaches have been used to model lung cancer risk after smoking cessation to understand the competing effects of smoking levels, duration, and time since quitting.18 analogously, comparing the effectiveness of long - term / lifetime cr versus short - term intense cr after irradiation might provide insight into the nature of the additional steps implied by the model discussed here, and such animal experiments are now being undertaken . Ultimately, the impractical nature of extreme cr gives rise to the question of whether complementary stressors or mimetics could be used instead . Much research has been undertaken to find cr mimetics,19 and candidates which prove effective will likely be applicable to post - radiation risk mitigation . We are investigating the effect of addition of dietary supplements (such as the plant phenol resveratrol, one candidate cr mimetic20) to the standard laboratory diet of mice . We are also undertaking experiments to determine whether radiation - induced tumors arising in calorie - restricted and -unrestricted animals show any differences in key carcinogenic pathways at the molecular level . Any differences in tumor phenotype may indicate pathways where cr interacts with the accumulation or selection of mutations . In parallel, we have established experiments utilizing environmental enrichment for laboratory mice (play equipment, increased housing space, nesting materials) following radiation exposure to explore whether other simple lifestyle changes may mitigate radiation - induced cancer . It is not expected that any one of these measures alone will have a dramatic impact on cancer incidence, but lifestyle changes that are safe, simple, and beneficial represent low - hanging fruit that might be included in post - disaster counselling and public health campaigns . Human populations invariably have additional cancer risk factors,21 such as smoking, alcohol consumption, sedentary lifestyles, and associated obesity, which are difficult to reproduce in experimental animals . However, it is likely that existing public health recommendations for reducing cancer risk will be similarly effective in offsetting or mitigating low dose radiation - induced cancer risk . Ultimately, at the radiation exposure levels relevant to large populations in the wake of environmental contamination, the increased risk to the individual is small compared to the background risk of cancer in the population . Simple lifestyle changes which can be proven to help offset both spontaneous and radiation - induced risk might provide significant public health benefits, both in terms of decreased cancer burden and improved mental health and well - being.
Histological evaluation of a peripheral nerve is often the final diagnostic work - up for a neuropathy of unknown origin, and is most informative when the nerve is clinically affected . A distal sensory nerve, including the sural, superficial peroneal, or superficial radial nerve, is usually biopsied, and a motor nerve branch is rarely considered [2, 3, 4]. Here, we report a case with painful unilateral neuropathy in the upper arm . In this case, a biopsy of a pronator teres motor branch provided useful information in consideration of the pathogenesis . A previously healthy 19-year - old japanese woman noticed a skin rash on her lower limbs . After 4 months, the rash spread to her hands and forearms . In february 2013 (9 months from the onset of the exanthema), she was admitted to our hospital because of sudden paresis and pain in her left upper limb . At the first presentation, she showed livedo reticularis in all four limbs, with a temperature of 36.5c, a pulse of 65 beats per minute, and a blood pressure of 112/62 mm hg . Examination showed decreased sensation in the radial side of her left forearm and dorsum of the hand . Manual muscle testing was uncompleted in her left forearm because of severe neuralgia, although it was normal in the left proximal arm and lower limbs . Laboratory findings were normal for sedimentation rate (8.0 mm / h), leukocyte counts (5,880/mm), serum c - reactive protein (0.01 mg / dl), serum creatinine (0.49 mg / dl), and urinalysis . Tests for serum antibodies, including antinuclear, anti - dna, anti - rnp, anti - ssa and anti - ssb, igg anticardiolipin, and anti - beta-2-glycoprotein i, were negative . Serum antineutrophil cytoplasmic antibody was negative in both perinuclear and cytoplasmic staining patterns, and antimyeloperoxidase and proteinase 3 specificity were not detected . The results of the serological tests for acute infection with epstein - barr virus, herpes simplex virus, varicella - zoster virus, cytomegalovirus, human parvovirus b19, and mycoplasma pneumoniae were all negative . Antibodies to human immunodeficiency virus (hiv) and hepatitis b and c viruses were also negative . Cerebrospinal fluid analysis showed a normal cell count of 1/mm (mononuclear cells) with normal total protein concentration (26 mg / dl, normal <40 mg / dl). Mri scans of the brain, whole spine, left upper limb, cervical nerve roots, and brachial plexus and whole - body computed tomography scans showed no abnormal findings . Skin biopsy specimens from the livedo reticularis in her left lower limb showed no specific findings of vasculitis or thrombophlebitis . In neurophysiological studies, the patient's motor nerve conduction in the median, ulnar, and radial nerves was within the normal range; however, the compound muscle action potential and conduction velocity in the left radial nerve (8.4 mv, normal> 7.0 mv; 64.4 m / s, normal> 60 m / s, respectively) were decreased in comparison with the right radial nerve (10.6 mv and 91 m / s, respectively). The result of the sensory nerve conduction study was normal . Whilst we were unable to record the motor unit potentials during voluntary contraction of the left forearm muscles because of the patient's severe pain, ultrasound examination of the ulnar, median, and radial nerve in the left forearm showed no abnormalities . Livedo reticularis and severe neuralgia pointed to vasculitic neuropathy as the most likely pathological condition in this patient . Due to the acute progression of the motor impairment, a single course of intravenous immunoglobulin (400 mg / kg body weight daily for 5 consecutive days) with methylprednisolone pulse therapy (intravenous methylprednisolone 1 g daily for 3 consecutive days) was commenced . Improvement of the patient's neuralgic pain and motor dysfunctions was observed within a few days of commencement of the therapy . However, severe tenderness along the median nerve in her left forearm and muscle weakness mainly in the left median nerve territory remained . Manual muscle testing showed muscle weakness in the left median nerve territory: flexor digitorum profundus (second finger: 3/5; third finger: 4/5; intact in fourth and fifth finger), flexor digitorum superficialis muscle (second finger: 2/5; third finger: 2/5; fourth finger: 4/5; intact in fifth finger), and pronator teres muscle (2/5). A second mri of the left upper limb showed new abnormal signal intensities in the median nerve at the forearm and muscles innervated by the radial and median nerve, including flexor pollicis longus, flexor digitorum profundus, flexor digitorum superficialis, extensor carpi ulnaris, and extensor digitorum (fig . 1). These findings were suggestive of the presence of mononeuropathy multiplex involving the left median and radial nerves (predominant in the former). Despite vasculitic neuropathy being the most likely causative pathological condition in this patient, histological confirmation was required in order to justify the length of her treatment with potentially cytotoxic medications . A superficial radial nerve biopsy was not encouraged because there were no apparent findings of its involvement . We performed a combined biopsy of the left pronator teres muscle and a motor branch of the median nerve to the pronator teres . We found that her left pronator teres motor nerve had two branches, one proximal branch going to the superficial (humeral) head and the other distal branch going to the deep (ulnar) head of the pronator teres muscle . We biopsied the latter, which was a very thin nerve branch, to reduce potential motor complications of the procedure . The specimens from a motor branch, that contained only one nerve bundle, showed severe loss of myelinated fibers with a sectional distribution, myelin ovoids with many foamy macrophages, and marked edema in the perineurium and subperineurium (fig . The specimens from the pronator teres muscle showed typical neurogenic features including small group atrophy with small - angulated muscle fibers (fig . As expected, vasculitis seemed to be the most probable cause of the condition, although direct findings (e.g., fibrinoid necrosis of wall vessels) were not seen in these specimens . As a result of these histological findings, two cycles of a 3-day regimen of high - dose intravenous methylprednisolone (1 g / day) were initiated, followed by oral prednisolone at 0.5 mg / kg / day . The patient was discharged in april 2013, and returned to full - time housekeeping with no notable neurological deficits . She is currently continuing treatment with low - dose oral prednisolone under active follow - up . The original technique of diagnostic biopsy of the pronator teres and a motor branch of the median nerve was described at academic meetings [5, 6] and reported in a retrospective review of 20 patients as a safe and useful procedure to differentiate between motor neuropathy and motor neuron disease in patients with weakness of the upper limbs . A biopsy of the motor branch of the pronator teres muscle nerve may be considered a valuable diagnostic option in selected cases with neuropathy affecting the upper limb, when performed in cooperation with neurologists and orthopedic surgeons.
2009 saw the first influenza pandemic of the 21st century, when an h1n1 influenza a virus spread to over 200 countries, resulting in more than 18 000 deaths globally . The wide spread of the virus resulted from its efficient human - to - human transmission . Transmission requires binding of the virus surface envelope protein, hemagglutinin (ha), to the host surface glycan receptors containing terminal sialic acid (sa). The h1n1 human flu virus preferentially binds to receptors with a 2,6 linkage between sa and galactose (gal). These 2,6-linked sialic acids (2,6-sa) are predominantly found in the epithelial cell surface of the human upper respiratory tract . In contrast, the has of avian - adapted virus preferentially bind to 2,3-sa, which are found in the digestive and respiratory tracts of birds, and on human lung alveolar cells . Although the overall case fatality rate of 2009 h1n1 was not much different from seasonal flu, a specific mutation in ha at position 222 (h1 numbering) from aspartic acid to glycine (d222 g) was more likely to be associated with severe or fatal cases . The presence of this d222 g mutation in both the ha from 1918 h1n1 and 2009 h1n1 viruses was reported to enhance ha binding to 2,3-sas, potentially causing the severe cases observed early in the 2009 pandemic . The results also showed that the d222 g mutation significantly weakened binding to 2,6-sa for ha from 1918 h1n1, but only modestly affected the 2009 h1n1 virus . To explain this discrepancy, we previously performed computer simulations for the 2,6-sa analog 6sln binding with has from the 1918 h1n1 (a / south carolina/1/18, sc18) and 2009 h1n1 (a / netherlands/602/2009, nl602) viruses with and without the d222 g mutation . For wild type sc18 (sc18-wt), two binding modes for these modes differ in the interactions formed between 2,6-sa and ha, which alter the position of the galactose sugar within the receptor binding pocket . Structures with shorter distances between the ligand and the residue at the 224 position are classified as mode 1, and those with longer distances are identified as mode 2 . By definition, the ligand in mode 1 is buried deep inside the pocket, while in mode 2 it is situated higher up . Nl602-wt was also observed to adopt two similar binding modes alongside many intermediate structures . For sc18 carrying the d222 g mutation (sc18d222 g), almost no binding mode 2 was observed, while for nl602d222 g, although a decrease in binding mode 2 was seen, it was not entirely absent . This contrasting stability is a result of the additional interaction between 2,6-sa and the side chains of 130k, 142k, and 224e, which can stabilize 2,6-sa binding to ha . We suggested that these interactions allow nl602d222 g to maintain binding to 2,6-sa, explaining the fitness of this mutant virus strain . The binding pocket region distal to the viral membrane is highlighted for one monomer . (b) schematic illustration of the two binding modes; the 6sln glycan in mode 1 and mode 2 is colored blue and green, respectively . The distance between the -c of 224a and the o3 atom of gal (denoted as d224) other efforts have also been made to understand how the d222 g mutation changes the binding preference, in both experiments and computer simulations . In 2013, zhang et al . Reported the crystal structures of ha from both h1n1 a / california/04/2009 and sc18 flu virus bound to 2,6-sa and 2,3-sa analogs . They suggested that the d222 g mutation results in a missing salt bridge between 222d and 219k, loosening the 220-loop and enabling the key residue 223q to interact with the avian receptor, switching the binding preference . Using nmr and molecular dynamics (md) simulation, elli et al . Also reported stable structures of 2,6-sa binding to ha from 1918 flu before and after d222 g mutation . The results suggested that the interaction between 2,6-sa and the 220-loop in wild type ha no longer exists after the mutation, which allows the galactose sugar to move away from the protein surface . Also performed md simulations investigating the binding of sialyldisaccharides to various has . Using neu5ac(26)gal as the analog of 2,6-sa and ha from the a / swine / iowa/30 virus, they also observed two binding modes . Due to the difference of the analog and ha model, these two binding modes are not exactly the same as those reported in 2010, most likely due to differences in the ha model and glycan analogue employed . Structures of all initial end points . The names of key residues in sc18-wt binding mode 1 are labeled . The previous work described above mainly focused on the differences between static structures having identified alternative binding modes, one can investigate not only their kinetic stability, both in terms of on / off rates and the free energy of binding, but also how readily they interconvert . This information, especially the transition mechanism, the barriers, and the key intermediates, could be helpful in identifying additional therapeutic targets and provide a more complete understanding of the effect of a particular mutation on viral fitness . In the present work, we investigate interconversion pathways between two binding modes to elucidate the contrasting affinity of the two different h1n1 virus mutants for 2,6-sa . A new intermediate network scheme is introduced to accelerate the search for kinetically relevant paths in the potential energy landscape . By comparing the distribution of energy barriers and energy changes of key rearrangements, we find that the d222 g mutation exchanges the energetic preference of the two binding modes in both sc18 and nl602 has and that the effect is more dramatic in sc18 . By analyzing the shape of the binding pocket, we have identified differences between the two binding modes for the 1918 and 2009 strains . Our results indicate that changes at positions 183 and 224 generate a smaller pocket in nl602 than in sc18, permitting greater flexibility in the 2,6-sa transformation between the two modes in nl602 . The amber ff99sb molecular mechanics force field in conjunction with the gb generalized born implicit solvent method (igb = 2) was used to represent the protein . The potential was modified to remove a discontinuity at the nonbonded interaction cutoff, which was set to 16 . The trisaccharide, neuac2,6-gal14glcnac1 (6sln), with a methyl cap attached to the o1 atom of the terminal sugar, was used to represent 2,6-sa in our simulations, since it has been seen experimentally to bind to a range of h1n1 flu viruses . Parameters for the three sugars that comprise this analog (neuac, gal, and glcnac) were obtained from the glycam06 g force field, as in our previous study, where the binding modes were first identified . The initial structure of the ha in nl602-wt was based upon the crystal structure in the sc18 h1n1 . The order parameter used as the criterion to choose initial structures was the separation of the center of mass of the backbone atoms of residue 224 and the center of mass of the o2 and o3 atoms of the 2,6-sa galactose sugar . This order parameter was chosen because it was found to demarcate two distinct binding modes, which had been observed in md snapshots from earlier work . Once these snapshots had been sorted into one of these binding modes (or intermediate structures), we selected representatives based on the following criteria: (i) structures temporally separated from an observed transition between modes and (ii) structures clearly assigned as mode 1 or mode 2 . These choices ensure that the structures are indeed representative, prior to minimization . For sc18-wt and nl602-wt, we then manually interpolated between the two end points, removing those differences in binding mode 2 far away from the pocket, which do not affect interconversion behavior . For sc18d222 g, binding mode 2 was entirely absent from the md trajectory reported previously . Hence, in the current work, a structure for mode 2 was generated using the ligand from sc18-wt, superimposing it into the pocket of sc18d222 g, incorporating residue moves around the pocket corresponding to binding mode 2, and finally performing a local minimization . For nl602d222 g, although some of the structures seen in previous work exhibit features of binding mode 2, the total number is very small . In the present work, binding mode 2 was also generated using the ligand from nl602-wt mode 2, superimposing it into the binding pocket, then minimizing . A detailed description can be found on our website and in the supporting information . The doubly nudged elastic band (dneb) method, as implemented in the optim program, was used to generate likely candidate structures for transition states along the pathway between a pair of end point structures . The two minima that each transition state connects were identified by calculating approximate steepest - descent paths . All the minima and transition states characterized during the connection of each pair of end points were collected in the initial database generated by the intermediate network scheme, as described in the following section . The databases were then expanded using the discrete path sampling (dps) framework, with several shortcut, shortcut barrier, and untrap runs, as implemented in the pathsample program . Shortest - path algorithm with appropriate edge weights was used to determine the discrete path that makes the largest contribution to the rate constant between the end points of interest (the fastest path). Efficiently finding a connected pathway between two significantly different end points can be very difficult if attempted in a single step due to limitations in how intelligently we can interpolate between them . We found that the main differences between the two end points are mainly rotations of 6sln and the side chains of residues on the edge of the binding pocket . The main body of the complex, including the position of the center of mass of 6sln in the pocket, is almost the same . This observation inspired a new intermediate network interpolation technique to optimize refinement of the kinetic transition network and ensure that we have included as many paths as possible in the initial database . This scheme is illustrated in figure 3 and consists of the following steps:(1)for two end points a and b, identify all the differences of local conformations and label these differences as ai and bi, (i = 1, n). The end point a can then be denoted as the set a = {a1, a2,..., an}. For example, in figure 3, the molecular fragment in question is represented by the blue square, which should be similar for a and b. four differences are identified and labeled with small circles, which are white in a and red in b. (2)new configurations are generated for end point a by transplanting the different local b conformations into the original a minimum . We adopt the notation i to denote the minimum obtained by relaxing the structure after the local conformation replacement, where is a binary string, identifying the origin of each local configuration . Those originally from a and b are denoted as 0 and 1, respectively . The leftmost digit represents a1 or b1 . For example, in figure 3, i0110 = {a1, b2, b3, a4} corresponds to the minimum obtained by relaxing the structure with the b2 and b3 conformations replacing a2 and a3 . Hence i00 = a. we note that i11 can differ in detail from b because the local relaxations after transplanting conformations can produce minor changes in nearby side chains . In principle the total number of ia is 2, including a. however, some minima obtained after relaxation may be the same, because certain substitutions may have to follow a particular sequence to be favorable. (3)double - ended connection attempts are then run for all the possible i and i corresponding to a single local conformation change between and . Here we require the pathway search algorithm to locate multiple transition states, ensuring that any given intermediate pairs will eventually be connected, as implemented in the missing connection approach, which is coded in optim . In figure 3, we have shown several possible connections . In principle, the total number of connections covered by this network could be n 2 + 1, where the extra + 1 corresponds to the case when i11 b. the number will be less than this upper bound if some of the minima generated in step 2 are the same . For two end points a and b, identify all the differences of local conformations and label these differences as ai and bi, (i = 1, n). The end point a can then be denoted as the set a = {a1, a2,..., an}. For example, in figure 3, the molecular fragment in question is represented by the blue square, which should be similar for a and b. four differences are identified and labeled with small circles, which are white in a and red in b. new configurations are generated for end point a by transplanting the different local b conformations into the original a minimum . We adopt the notation i to denote the minimum obtained by relaxing the structure after the local conformation replacement, where is a binary string, identifying the origin of each local configuration . Those originally from a and b are denoted as 0 and 1, respectively . For example, in figure 3, i0110 = {a1, b2, b3, a4} corresponds to the minimum obtained by relaxing the structure with the b2 and b3 conformations replacing a2 and a3 . Hence i00 = a. we note that i11 can differ in detail from b because the local relaxations after transplanting conformations can produce minor changes in nearby side chains . In principle the total number of ia is 2, including a. however, some minima obtained after relaxation may be the same, because certain substitutions may have to follow a particular sequence to be favorable . Double - ended connection attempts are then run for all the possible i and i corresponding to a single local conformation change between and . Here we require the pathway search algorithm to locate multiple transition states, ensuring that any given intermediate pairs will eventually be connected, as implemented in the missing connection approach, which is coded in optim . In figure 3, we have shown several possible connections . In principle, the total number of connections covered by this network could be n 2 + 1, where the extra + 1 corresponds to the case when i11 b. the number will be less than this upper bound if some of the minima generated in step 2 are the same . Schematic illustration of the intermediate network scheme . The blue square represents end points a and b. four small circles represent the variable fragments in a and b, which are white (a1a4) in a and red (b1b4) in b (b i1111). We consider this procedure to be converged when pathways have been found between all of the states i. one advantage of the scheme is that all the minimizations in step 2 and the connection attempts in step 3 can be run in parallel independently . The corresponding pathways can have multiple transition states with additional local minima stabilized by nonlocal interactions . However, they are precisely the paths that are likely to have the lowest barriers and make the largest contribution to the overall rate constant . For the ideal case that i11 = b, and any pair of intervening minima have only one possible connection, corresponding to a single transition state, then in principle, this network will cover all the possible paths that connect the two end points . In practice, we may have to group more than one residue into a single fragment so that one single connection in step 3 still contains several elementary steps and alternative possibilities . Hence, it is still worthwhile to further refine the initial database that includes all the minima and transition states from the intermediate network connection searches . The glycan binding pocket is located on the top of each monomer of the ha trimer . As shown in figure 1b, it consists mainly of three secondary structure features: the 190-helix (residues 187 to 195), 220-loop (residues 218 to 225), and 130-loop (residues 132 to 135). The aspartic acid residues located at the 222 and 187 positions contribute most to the binding of 2,6-sa . By definition, the fastest path makes the largest contribution to the rate constant between the end points of interest . Analyzing this path helps us to understand how displacements of individual residues contribute to the overall interconversion mechanism between the two binding modes . The fastest paths in each of the four strains all involve loss of the interaction between neuac and 187d and formation of a new hydrogen - bond between gal and the 220-loop . Several motions are shared by the fastest paths of all four strains, namely (1) the rotation of the 4-hydroxyl of gal toward the backbone of 222d / g (g4 movement), (2) the 3-hydroxyl of gal binding to the side chain of 222d of wild type has, or moving to where it would have bound to the side chain of 222d in mutant has with 222 g (g3 movement), and (3) the rotation of the side chain of 187d toward the outside of the binding pocket (r187 movement). For the fastest path in sc18-wt, we can distinguish four sets of movements . Neuac first loses interactions with 187d by rotating the 7-hydroxyl and forms new intramolecular hydrogen - bonds, after which the g4 and g3 movements occur in succession . The 3-hydroxyl of gal points toward the 2-hydroxyl of gal in the binding mode 1, which is unique to the sc18-wt strain . These two steps together involve a highest barrier of 7.8 kcal / mol and make the largest contribution to the distance change between the -c of 224a and the o3 atom of gal (denoted as d224), which distinguishes the two binding modes . The final change is the r187 motion, where 187d loses most of its interactions with the ligand, leaving only one hydrogen - bond with glcnac . Further details of the fastest path can be found in the supporting information . In sc18d222 g, the position of the ligand is first changed by the rotation of the 8-hydroxyl of neuac, which breaks its hydrogen - bond with 187d and forms new hydrogen - bonds within neuac itself . This step has a high barrier of 14.1 kcal / mol and is endothermic by 11.7 kcal / mol . Next are the g4 and g3 movements, which are similar to those in sc18-wt, except that the 3-hydroxyl of gal interacts with the backbone of 222 g in binding mode 1 . The g3 movement still results in the largest change in d224 but the corresponding energy changes are much smaller than those in sc18-wt . The first steps involve breaking the hydrogen - bond between 224e and the 2-hydroxyl of gal, followed by conformational changes of the 224e side chain . This step does not exist in sc18 since the 224a side chain in sc18 is too short to interact with the ligand . The 9-hydroxyl of gal then rotates, forming a hydrogen - bond with 224e, which shifts the whole 220-loop toward the ligand . The g4, g3, and r187 movements that follow have two interesting differences from previous pathways: (1) for both sc18-wt and sc18d222 g strains, we observed translational motion of the ligand toward the 220-loop when the g4 and g3 movements happen, which is not seen in nl602-wt . (2) after the r187 motion, the ligand in sc18-wt and sc18d222 g moves away from the base of the pocket, losing interactions with 187d, while in nl602-wt, most of the hydrogen - bonds remain after the r187 displacement . The r187 displacement comes first, together with hydrogen - bond breaking between 224e and the 2-hydroxyl of gal . As discussed for nl602-wt, then, following the g4 and g3 movements, d224 increases from 5.2 to 6.4 . The final two steps correspond to motion of residues 180 h and 191l toward the base of the pocket . By comparing the fastest path that connects the two binding modes in each case, we find that the shift of the 3-hydroxyl of gal, either binding to the side chain of 222d of wild type has, or moving to where it would have bound to the side chain of 222d in mutant has with 222 g (the g3 movement), is the key elementary step differentiating the two binding modes although the g3 displacement is common to all four cases, the fastest transition paths exhibit systematic differences . In sc18-wt, the 3-hydroxyl does not bind to the backbone first before binding to the side chain of 222d . In nl602-wt this change, and the binding of the 4-hydroxyl of gal to the backbone of 222d, occur simultaneously . To compare further we collect all the transition states corresponding to steps where the 3-hydroxyl of gal either binds to the side chain of 222d in wild type has, or moves to the place where it would have bound to the side chain of 222d in mutant has with 222 g . The barrier and energy change distributions for the four cases are illustrated in the contour plots shown in figure 4 . In sc18-wt, the peak value of the distribution is around (0.7, 1.7), corresponding to an energy barrier of 0.7 kcal / mol and an energy difference of 1.7 kcal / mol . The pathways that contribute to this peak all involve breaking the hydrogen - bond between the 3-hydroxyl of gal and the backbone of 222d . The transition states that contribute to the shoulder peak around (0.5, 2.5) are broadly similar, with insignificant differences in nearby side chains, for example, the position of the 7-hydroxyl of neuac . The step in which the 3-hydroxyl of gal breaks hydrogen - bonds with non-222d residues mainly contributes to the small peak at (3.9, 1.2), as we observed in the fastest path . Contour plots illustrating the distribution of the barriers and the energy changes corresponding to the 3-hydroxyl of gal, either binding to the side chain of 222d in (a) sc18-wt and (c) nl602-wt, or moving to where it would have bound to the side chain of 222d in (b) sc18d222 g and (d) nl602d222 g with 222 g . The x axis corresponds to the height of the barrier and the y axis corresponds to the energy difference . In sc18d222 g, 126 out of 3475 transition states in the database correspond to the key elementary step . The main peak shifts to (0.6, 0.5), which still mainly correlates with interactions of the 3-hydroxyl of gal and the backbone of 222 g in the initial state . The energy barrier remains almost the same as in the wild type ha, while the overall energy difference increases by at least 2.2 kcal / mol, as a result of the d222 g mutation . The small peak located at around (2.8, 2.3) corresponds to cases where the 3-hydroxyl of gal is not bound to the backbone of 222 g in the initial state . The structure of the corresponding initial state is very similar to the one reported by zhang et al ., where the 3-hydroxyl binds to 219k and the 4-hydroxyl of gal binds to the backbone of 222 g . In nl602-wt, 332 out of 3719 transition states in the database correspond to the 3-hydroxyl motion . The main peak in figure 4c is around (0.1, 3.4) and represents paths where the 3-hydroxyl breaks the hydrogen - bond to the backbone of 222d, while simultaneously binding to the side chain of 222d . Compared to sc18-wt, this step is significantly faster and more exothermic in nl602-wt . The same mechanism also contributes to the broad peak ranging from (2.7, 5.5) to (3.6, 4.9), and in these paths, the 4-hydroxyl of gal is already bound to the backbone of 222d in the initial state . The 4-hydroxyl subsequently binds to the backbone of 222d, as in the fastest path . Another small peak located at around (0.1, 1.0) corresponds to paths where the 3-hydroxyl of gal does not interact with the backbone of 222d in the initial state . In nl602d222 g, only one peak located at (0.3, 0.1) can be seen in figure 4d, corresponding to paths with the 3-hydroxyl of gal bound to the backbone of 222 g in the initial state . For wild type has, we see that binding the 3-hydroxyl of gal to the side chain is generally an energetically favorable process . When the hydroxyl shifts from interacting with the backbone to the side chain, the barrier is always lower than 1 kcal / mol . As a result, mode 2 corresponds to an energy minimum in this transformation, increasing the equilibrium population and making it accessible during md simulations, as observed in previous work . For mutant has, mode 2 is generally energetically unfavorable compared to mode 1 . In particular, for sc18d222 g mode 2 is so unfavorable that the reverse process (mode 2 to mode 1) has a barrier of only 0.1 kcal / mol . According to the energy profile, other processes, such as the rotation of the 187d residue, are also more endothermic in sc18d222 g than in nl602d222 g . As a result, the equilibrium occupation probability of mode 2 in sc18d222 g is rather low, which is consistent with the previous observation that mode 2 is rarely observed during md simulations . For nl602d222 g, the paths are similar, but the energy difference is lower . Hence, binding mode 2 can still be observed in the nl602d222 g mutant ha, albeit with decreased probability . A more detailed analysis of this key elementary step will be addressed in future work one reason why the binding of the 3-hydroxyl of gal to the side chain of 222d is more exothermic and has a lower barrier in nl602 may be the differences in the shape of the binding pocket . When the ligand binds to 222d / g or unbinds from 187d in sc18, we always observe a significant shift of the gal and glcnac sugars toward the 222 position . We examined the shape of the pocket for the four strains using the povme program and illustrate the differences observed in figure 5 . Structures were aligned based on the 190-helix, 130-loop, and 220-loop using vmd . Two major differences between sc18-wt and nl602-wt can be observed in figure 5a: (1) the pocket in sc18-wt is larger than that in nl602-wt on the side of 220-loop and 190-helix, where the majority of the interactions between ligand and pocket are . (2) the pocket in nl602-wt is larger on the side of the 130-loop and also between the 220-loop and 130-loop . Comparing d222 g mutants in figure 5b, the blue region is larger than that in figure 5a . However, the extra volume is located mainly at the top of the pocket, which may arise due to measurement error, since the pockets are open at the top . Pocket shape differences between (a) sc18-wt (cyan) and nl602-wt (pink) and (b) sc18d222 g (cyan) and nl602d222 g (pink). The blue and red regions correspond to areas where the pocket in sc18 is larger and than that in nl602, respectively . To analyze the pocket shape differences further, in figure 6 we show a triangular region defined by 187d, 219k, and 222d / the shape of this region provides a simple representation of the 220-loop side of the binding pocket, where the majority of the rearrangements between the two binding modes are localized . We find that the 219k residue in sc18 is about 12 further away from 187d but slightly closer to 222d / g compared to nl602 . As a result, 224a interacts with the 4-hydroxyl of gal in binding mode 1 and 224e in nl602 interacts with the 3- and 4-hydroxyl of gal . Hence, 224a / e mainly determines how far to the left (from the viewpoint of figure 6) and how deep 2,6-sa binds within the pocket . In sc18, the distance between 224a and the line connecting 187d and 222d / g is 2.8 in the wild type ha and 2.5 in the 222 g mutant ha . In nl602, the position of 224e is close to the line connecting 187d and 222d / g . The distance between 224e and the line connecting 187d and 222d / g is 1.1 in the wild type ha and 1.4 in the mutant ha, and the distance between 224a and 187d in sc18 is also longer than that in nl602 . Since 222d protrudes slightly from the edge of the pocket, the position of 224a in sc18 allows the ligand to bind deeper and closer to the left side . The variations in the distance between the 220-loop and 190-helix confirm the shape differences observed from the povme measurement: the pockets in sc18-wt and sc18d222 g are larger than for nl602-wt and nl602d222 g on the side of the 220-loop . (a d) triangle formed by 187d, 219k, and 222d / g in mode 1 of (a) sc18-wt, (b) sc18d222 g, (c) nl602-wt, and (d) nl602d222 g, respectively . The distances between residues are calculated between the backbone nitrogen atoms, and the values in brackets are the corresponding distances in mode 2 . Binding pocket changes during the interconversion between the two modes in four different strains . The blue and red regions correspond to volumes lost and gained after a particular displacement, respectively . (c) rotation of the 8-hydroxyl of gal and g4 and g3 shifts in sc18d222 g (m1m9 in figure s3). (d) r187 movement in sc18d222 g (m9m11 in figure s3). (e) rotation of the 9-hydroxyl of gal and g4 shifts in nl602-wt (m4m6 in figure s5). (h) g4, g3, 180h, and 191l movement in nl602d222 g (m7m11 in figure s7). We also used povme to identify changes in pocket shape during the interconversion . For sc18-wt, the first nontrivial shape change of the pocket results from the rotation of the 4-hydroxyl of gal (figure 7a), which shrinks the volume between the 220-loop and the 130-loop . This reduction in size is caused by both the conformational change of the 223q side chain and the movement of the 220-loop toward the 130-loop . Another significant shape change results from the rotation of 187d (figure 7b), which reduces the size of the region in front of 187d . The observations in the case of sc18d222 g are similar to those for sc18-wt . The first few movements, including the rotation of the 8-hydroxyl of gal and the movement of the 4- and 3-hydroxyl of gal, contribute to the shrinking of the pocket between the 220- and 130-loops, as shown in figure 7c . The side chain rotation of 187d then removes the region in front of 187d (figure 7d). The changes in pocket shape for nl602-wt differ greatly from those for sc18-wt . The rotation of the 9-hydroxyl of gal shrinks the pocket between the 220-loop and 190-helix and enlarges the volume between the 190-helix and 130-loop, as shown in figure 7e . The rotation of 187d, however, contributes less to the shape change than it does for sc18-wt, which is consistent with the previous observation that the rotation of 187d does not remove many interactions with the ligand . The rotation of 187d slightly shrinks the region in front of 187d, and enlargement of the pocket next to 224e occurs via movement of the 224e side chain (figure 7 g). The next few steps, including the movement of the 4- and 3-hydroxyl and residues 180 h and 191l, slightly shrink the pocket between the 220-loop and 190-helix, as for nl602-wt . In summary, the two strains from 1918 south carolina are more flexible between the 220-loop and 130-loop but more rigid between the 220-loop and 190-helix when compared to the 2009 netherlands strains . This observation is consistent with the distance changes among key residues (figure 6). The maximum distance variation in sc18-wt is only 0.3 for 187d and 222d . However, in nl602, the distance from 187d to residues 219k, 222d, and 224a is reduced by roughly 1 in binding mode 2 . Since the ligand mainly interacts with the 190-helix and the 220-loop, the flexibility of this region plays an important role in the two binding modes . When the transformation between the two modes occurs, due to the shape and the size of the pocket in nl602, the 220-loop can adapt its position to reflect the position of the ligand, which allows most interactions with the edge of the pocket to be conserved . However, for sc18, because the pocket is larger, the ligand must move from one side to the other during the transformation to maintain these interactions, and the 220-loop cannot adapt to the ligand position as flexibly as for nl602 . As a result, the binding of 2,6-sa to sc18 relies more heavily on the side chain of 222d than in nl602, since the ligand has limited access to the edge of the pocket . The additional interactions between the ligand and the pocket in nl602 that were reported previously can also been explained in the light of this observation . Key residues in (a) sc18 and (b) nl602 that determine the relative positions of the 190-helix and 220-loop . Interestingly, as shown in figure 8a, the junction structures between the 190-helix and 220-loop, which determine the relative position of the two regimes, are different in the two has . In sc18 183p is in front of the 190-helix and does not interact with other residues nearby . The hydrogen - bonds between 215a and 182p, together with 217r and 224a, determine the relative positions of the 190-helix and the 220-loop . The residues at the junction between the 190-helix and 220-loop change from 183p and 224a in sc18, to 183s and 224e in nl602 . Due to these differences, the shape of the pocket in nl602 is affected by the interaction between 183s and 213e, as well as the interaction between 183s, 224e and 226r, as shown in figure 8b . There have been several experimental results reported since 2010 that the s183p mutation and the e224a mutation with or without other mutations, are correlated with the pathogenesis and transmission changes observed for h1n1 . Since the majority of the shape difference between sc18 and nl602 during the binding mode interconversion occurs between the 190-helix and 220-loop, the accumulated mutations at positions 183 and 224 may explain the differences in pocket shape flexibility during binding mode interconversion observed here . In the present work, we have investigated the transformation mechanism between two binding modes for 2,6-sa in both the wild type and mutant h1n1 hemagglutinin proteins from 1918 and 2009 pandemic flu using the 6sln ligand . We introduced an intermediate network scheme to accelerate the search process of pathways connecting the two binding modes . The advantage of this procedure is that searches can be systematically conducted in parallel for all the single changes in identifiable local conformational states . By comparing the paths predicted to dominate the kinetics, we have identified the key elementary step that distinguishes the two binding modes, namely the 3-hydroxyl of gal either binding to the side chain of 222d in wild type has or moving to the place where it would have bound to the side chain of 222d in mutant has with 222 g . We find that the different residues at the 183 and 224 positions in the two has change the connecting pair between the 190-helix and 220-loop, and hence the shape of the pocket . The smaller size of the binding pocket in nl602 allows the ligand to interact with more residues in nl602 than in sc18 and also makes the 220-loop more flexible . The d222 g mutation exchanges the energy preference between the two binding modes in both sc18 and nl602 hemagglutinins . However, the transition between mode 1 and mode 2 raises the energy more in sc18 than in nl602, probably because of the different shape of the binding pocket . The different shape, the flexibility of the pocket during binding mode interconversion, and the resulting energy profile changes provide insight into the contrasting pathogenicities induced by the d222 g mutation in the sc18 and nl602 viruses . These results may help to explain the difference between the impact of the 1918 and 2009 h1n1 strains . A change in preference from 2,6- to 2,3-linked sialic acid is associated with a shift in pathogenicity . However, in the 2009 d222 g mutant, the 2,6 affinity is not weakened as much as for the corresponding 1918 mutant . Understanding such distinctions at an atomic level of detail should be helpful in predicting the potential emergence of strains that may pose serious threats to human health.
Breast cancer is the most common cancer among women in the usa and western europe . World incidence was 1 050 000 cases during the year 2000 with a mortality rate of 373 000 1 . Despite earlier diagnosis and improvement in adjuvant therapies then, the disease is incurable and the median of survival is 18 to 24 months 2 - 3 . The use of systemic therapies such as hormonal therapy, chemotherapy or new biological treatment is to reduce tumour masses, improve survival and preserve quality of life . Whatever the initial efficacy of the treatment undertaken in metastatic setting, almost every patient will relapse . The main goal is to improve progression free survival (pfs). To achieve this, the type of chemotherapy, the optimal duration of chemotherapy, the benefit of maintenance chemotherapy, the benefit of maintenance hormonal treatment are debatable . The present study was conducted to identify the factors which influence progression - free survival after the first line of chemotherapy . Among them, the present study focuses on the impact of hormonal maintenance therapy and constitutes the largest retrospective study on this subject . This study included 934 patients treated for metastatic breast cancer in 4 french cancer centres . A total of 772 patients received first - line chemotherapy 4 . Because the present analysis focuses on the impact of hormonal treatment beyond first line chemotherapy, we included only patients with positive tumour hormonal receptor status established on the primary tumour . When early disease progression occurs at first chemotherapy response assessment or within 3 months after the first cycle of chemotherapy in metastatic disease, one can consider that it is a failure of chemotherapy . It will not be relevant to search in this subset for factors which influence progression - free survival (pfs). Those cases were excluded from the present analysis . In total, 560 patients were studied to detect predictive factors to the duration of pfs after first - line chemotherapy and among those factors the impact of hormonal treatment given as maintenance therapy was analysed . The duration of pfs is defined as the time from the beginning of first line chemotherapy treatment to the date of progressive disease or death . Metastatic survival is defined as the time from the diagnosis of a metastasis to date of death or last follow - up . Proportional hazards cox model was used to identify which factors could influence the duration of pfs . Each significant variable in univariate analysis was included in multivariate analysis . The adjusted hazard ratio (ad - hr) the following variables were included in the cox model: menopausal status (pre- versus post- menopause); nodal involvement of the primary tumour (positive versus negative); hormonal receptor (hr) status (positive if estrogen receptors and/or progesterone receptors are positive versus negative); initial surgery (partial versus radical mastectomy); adjuvant chemotherapy (yes versus no); adjuvant hormonal therapy (yes versus no); complementary radiotherapy (yes versus no); disease free interval (dfi) between the date of diagnosis of breast cancer and the date of first diagnosis of metastatic disease (under or above two years); metastatic site (bone and/or node and/or skin and/or pulmonary versus liver); number of metastatic sites (single versus multiple); type of first - line chemotherapy (anthracycline- and/or taxane - containing regimen versus other); previous line of hormonotherapy administered in metastatic setting before the first - line chemotherapy (no versus yes); best response to first - line chemotherapy (complete (cr) or partial response (pr) versus stable disease (sd) defined according to recist criteria); maintenance hormonal therapy (yes versus no). This study included 934 patients treated for metastatic breast cancer in 4 french cancer centres . A total of 772 patients received first - line chemotherapy 4 . Because the present analysis focuses on the impact of hormonal treatment beyond first line chemotherapy, we included only patients with positive tumour hormonal receptor status established on the primary tumour . When early disease progression occurs at first chemotherapy response assessment or within 3 months after the first cycle of chemotherapy in metastatic disease, one can consider that it is a failure of chemotherapy . It will not be relevant to search in this subset for factors which influence progression - free survival (pfs). Those cases were excluded from the present analysis . In total, 560 patients were studied to detect predictive factors to the duration of pfs after first - line chemotherapy and among those factors the impact of hormonal treatment given as maintenance therapy was analysed . The duration of pfs is defined as the time from the beginning of first line chemotherapy treatment to the date of progressive disease or death . Metastatic survival is defined as the time from the diagnosis of a metastasis to date of death or last follow - up . Proportional hazards cox model was used to identify which factors could influence the duration of pfs . Each significant variable in univariate analysis was included in multivariate analysis . The adjusted hazard ratio (ad - hr) was provided for each significant variable . The following variables were included in the cox model: menopausal status (pre- versus post- menopause); nodal involvement of the primary tumour (positive versus negative); hormonal receptor (hr) status (positive if estrogen receptors and/or progesterone receptors are positive versus negative); initial surgery (partial versus radical mastectomy); adjuvant chemotherapy (yes versus no); adjuvant hormonal therapy (yes versus no); complementary radiotherapy (yes versus no); disease free interval (dfi) between the date of diagnosis of breast cancer and the date of first diagnosis of metastatic disease (under or above two years); metastatic site (bone and/or node and/or skin and/or pulmonary versus liver); number of metastatic sites (single versus multiple); type of first - line chemotherapy (anthracycline- and/or taxane - containing regimen versus other); previous line of hormonotherapy administered in metastatic setting before the first - line chemotherapy (no versus yes); best response to first - line chemotherapy (complete (cr) or partial response (pr) versus stable disease (sd) defined according to recist criteria); maintenance hormonal therapy (yes versus no). The hormonal treatment was tamoxifen (94), aromatase inhibitors (153), fulvestran (47) and megesterol acetate (14). The median duration of first line chemotherapy was 4.4 months (ranges: 3 9.7). The factors identified to improve the duration of pfs in multivariate analysis were: number of metastatic sites (p = .01), metastatic sites (p = .02), disease free interval (p = .001), previous hormonal therapy (p = .03), response to first line chemotherapy (p <.0001) and an administration of maintenance hormonal therapy (p <.0001). The factors related to an increase of os duration in multivariate analysis were: number of metastatic sites (p = .01), metastatic sites (p = .04), disease free interval (p <.0001), response to first line chemotherapy (p = 0.0001) and an administration of maintenance hormonal therapy (p = .001). Tables 2 and 3 list the significant predictive factors for the duration of pfs and os after first - line chemotherapy . Figures 1 and 2 show the patients' pfs and os from the first line of chemotherapy according to maintenance hormonal status . The search for prognostic and predictive factors that could influence the survival of patients treated for metastatic breast cancer has already been the subject of several studies . The first category is related to the primary characteristics such as initial histological grade, hormonal receptor status . The second category is linked to the metastatic characteristics: proliferation index reflected by the length of disease - free interval, type and number of metastatic sites involved . On the other hand, some prognostic factors are linked to the treatments undertaken, stressing their impact on the natural outcome of the disease: type of hormonotherapy, type of chemotherapy, type of response achieved by treatment 5 - 12 . The optimal duration of chemotherapy in patients who respond or have stable disease is not identified . In 1987, coates compared continuous chemotherapy (until progression or toxicity) versus intermittent chemotherapy (stop after three cycles and re - treatment at the time of disease progression) 13 . Patients receiving continuous therapy had superior response rates, time to progression, and quality - of - life scores, but no improvement in survival was observed . A similar trial conducted by the piedmont oncology association randomly assigned patients who had responding or stable disease after six cycles of caf to either cmf or observation . In the observation subset time to progression was three times longer in patients under continuous therapy than for those with interrupted treatment (9.4 vs. 3.2 months, respectively), but overall survival in both groups was similar . Falkson et al randomly assigned 141 patients whose measurable disease showed a complete response after six cycles of caf to receive either maintenance chemo - hormonal therapy or observation 15 . Time to disease progression was 19 months in patients who received the maintenance treatment versus 8 months in patients under observation but again the overall survival curves were similar in both groups . The french epirubicin study group study, gregory trial and nooij study lead to the same results when they compared interrupted with prolonged chemotherapy regimen: continuous therapy tends to improve duration of response and progression - free survival without a significative impact on overall survival 16 - 18 . In total, a chemotherapy holiday is associated with a shorter time - to - progression but no adverse effect on survival . While in some studies, continuous chemotherapy seemed not to affect the quality of life 13, 18, several studies showed increased rates of adverse effects 14, 15, 17 . Definitively, the major limit to the use of prolonged regimens of chemotherapy is related to their toxicity, all the more so as they are cumulative (cardiac toxicity of anthracyclins, neurologic toxicity of taxanes, haematological cumulative toxicities with any chemotherapy). The proposition to give hormonal treatment to prolong therapy in hormonal - positive tumors is another possible option . In the literature, only one prospective randomised study published by kloke et al in 1999 is available 19 . In this phase - iii trial, 90 patients with a disease controlled after 6 cycles of anthracyclin- and ifosfamide - containing regimen were randomised to receive or not maintenance therapy by medroxyprogesterone acetate . A longer median time - to - progression was reported among patients who were treated by maintenance hormonotherapy (4 . 9 versus 3 . Two retrospective studies found hormonal maintenance therapy as a significant factor among several prognostic factors for disease - free survival and overall survival after first line chemotherapy . In 1997, berruti et al analysed the factors influencing response rate and overall survival among 207 patients treated by epirubicin, followed or not by maintenance hormonotherapy 20 . The patients who received maintenance hormonotherapy survived significantly longer than those submitted to observation in uni- and multivariate analysis . The positive impact of maintenance hormonal therapy is impressive and deserves confirmation in randomized studies . Montemurro et al studied 109 patients receiving high - dose chemotherapy and analysed the factors which improve its efficacy 21 . The maintenance hormonal treatment improved the progression - free survival from 19, 2 to 31, 1 months (p = 0, 022). The influence of the type of response achieved by first line chemotherapy is well established 11 . Strikingly, in the present study, hormonal treatment administered after response or stabilisation with first - line chemotherapy seemed related to a better outcome with 7.8 to 16.3 months for the duration of pfs (p <0 . 0001) and from 30 to 48.1 months for the overall duration of metastatic survival (p <0 . This benefit was observed independently of the type of response achieved by first line chemotherapy . One can object that the choice of patient / tumour characteristics for who would or would not receive the maintenance hormonal therapy was not random, or controlled in any way . We cannot know whether we are observing natural history or impacting it in such a trial . Nevertheless the major impact obtained by maintenance hormonal treatment after the first line chemotherapy might indicate that this strategy should be recommended in patients with an er or pgr positive tumour . Based on the amplitude of the benefit observed, it may be ethically debatable to conduct a prospective randomized study . Moreover, randomized trials which assess the benefit of a new chemotherapy regimen should allow the possibility to give maintenance hormonal treatment.
The generation of neurons in the developing central nervous system requires a number of precisely orchestrated steps, whereby proliferating neural progenitors become committed to the neuronal fate, exit cell cycle, and undergo a long and complex program of migration and differentiation (kriegstein and alvarez - buylla, 2009). Proneural transcription factors (tfs) of the bhlh family, such as ascl1/mash1, are the main regulators of neurogenesis in the mammalian brain, and gain and loss - of - function analyses have shown that they are both required and sufficient to promote neurogenesis (bertrand et al ., 2002, wilkinson et al ., 2013). Accordingly, while genetic ablation of proneural genes in mice results in neural developmental defects associated with reduced neurogenesis, overexpression of proneural factors in neural progenitors induces a full neuronal differentiation program (berninger et al ., 2007b, casarosa et al ., 1999, geoffroy et al ., 2009). In addition to its pivotal role in development, ascl1 has been extensively used in protocols to reprogram somatic cells, including fibroblasts, astrocytes, and pericytes, into induced neurons (berninger et al ., 2007a, karow et al ., 2012, vierbuchen et al ., 2010), renewing interest in understanding the neurogenic activity of this proneural factor . Previously, we characterized the transcriptional program of ascl1 in the ventral telencephalic region of the embryonic mouse brain by combining gene expression profiling with chromatin immunoprecipitation (chip), followed by hybridization to promoter oligonucleotide arrays (chip - chip). This work resulted in the identification of a set of ascl1 target genes with various biological roles at distinct stages of the differentiation program, raising intriguing questions concerning the molecular basis for such temporal pattern (castro et al . In addition, it led to the identification of a novel function for ascl1 in maintaining cell proliferation, mediated by the direct activation of genes that promote cell cycle progression . This resulted in a model whereby this proneural factor sequentially promotes the proliferation and differentiation of progenitor cells along the neuronal lineage, reconciling the classical view of this proneural protein as a differentiation factor with the fact that it is mostly expressed in cycling progenitors . Moreover, a recent study has shown that these two opposing activities are associated with distinct modes of ascl1 expression, with oscillating or sustained ascl1 promoting proliferation or differentiation, respectively (imayoshi et al ., 2013). In spite of the significant progress made on the characterization of its transcriptional targets, little is still known about how ascl1 regulates gene expression . In particular, the relationship between ascl1 binding, regulation of the chromatin landscape, and gene transcription is poorly understood . It was recently shown that during neuronal reprogramming, ascl1 can access its cognate sites in nucleosomal - dna when ectopically expressed in fibroblasts, defining it as a pioneer tf (wapinski et al ., 2013). However, it remains to be seen whether ascl1 works as a pioneer factor in a neurogenic context and whether binding of ascl1 results in alterations to the chromatin landscape at its target regions, as it has been shown for some, but not all, other pioneer tfs (zaret and carroll, 2011). Mammalian neurogenesis is not a synchronized process at a cell population level and studies to investigate the mechanistic basis of ascl1 function at a genome - wide scale are difficult to perform in the developing embryo or in the adult brain . An alternative is the use of adherent cultures of neural stem (ns) cell lines derived from embryonic stem cells or embryonic neural precursors (conti et al . These cultures provide us with reliable models to study neurogenesis in culture, without the confounding effects of cellular heterogeneity, characteristic of other cellular models such as neurospheres . In proliferating culture conditions, endogenous ascl1 regulates a progenitor program that functions to maintain cell proliferation (castro et al ., 2011), whereas overexpression of ascl1 leads to efficient cell cycle exit and neuronal differentiation . Here we investigate how ascl1 activity is restricted by and impacts the chromatin landscape, when driving neuronal differentiation . We combined expression profiling with genome - wide mapping of ascl1 binding sites (chip - seq) (park, 2009), and dnase i hypersensitivity sites (dnase - seq) (song and crawford, 2010), in a cellular model of neurogenesis driven by overexpressed ascl1 . We identify a large number of genes directly regulated by ascl1 and characterize widespread changes in chromatin accessibility during differentiation . Ascl1 binding correlates with activation of gene transcription, targeting not only regions of accessible but also of closed chromatin . In addition, binding of ascl1 to dna precedes a local increase in chromatin accessibility at the regulatory regions of its target genes, providing the first direct link between ascl1 regulation of gene expression and local changes in chromatin landscape . Overexpression of ascl1 promotes cell cycle exit and neuronal differentiation of neural progenitor cells (castro et al ., 2006). To study this process in controlled conditions, we established a cellular model of ascl1-driven neurogenesis by expressing an inducible version of ascl1 in ns cells in culture (ns5 cell line) (pollard et al ., 2006). Fusion of full - length ascl1 to the modified ligand binding domain of the estrogen receptor (ert2) renders ascl1 activity dependent on the presence of 4-hydroxytamoxifen (herein referred to as tamoxifen) (bergstrom et al ., 2002,, ascl1-ert2 induces the transcriptional activation of an enhancer of the ascl1 target gene dll1 (castro et al ., 2006) in an inducible manner to levels that are similar to its wt counterpart (figure s1a). In order to test for the ability of the inducible ascl1 protein to promote neuronal differentiation, we transduced ns cells in culture with a retrovirus vector co - expressing ascl1-ert2 and green fluorescent protein (gfp). In the vast majority of transduced cells, drastic morphological changes characterized by the extension of long processes and associated with the expression of the neuronal marker tuj1 were observed only upon the addition of tamoxifen, confirming the ability of ascl1-ert2 to activate the neurogenic differentiation program in an inducible manner (figure s1b). Although activation is associated with the nuclear translocation of a small fraction of ascl1-ert2, most of the protein is already nuclear prior to addition of tamoxifen (as previously reported with other cases of tfs fused to ert2) (burk and klempnauer, 1991, roemer and friedmann, 1993), and additional mechanisms must therefore contribute to the inducibility of ascl1-ert2 (figure 1a). To obtain large numbers of synchronized differentiating neural progenitors, we used antibiotic selection following the retroviral delivery of the ascl1-ert2 transgene . Quantification of immunofluorescence levels upon immunohistochemistry estimated the total ascl1 protein level in transduced cells to be 8- to 9-fold higher when compared with endogenous ascl1 expressed in embryonic ventral telencephalic progenitors (figure 1b). Ns cells expressing ascl1-ert2 plated at low density differentiate with great efficiency upon addition of tamoxifen (figure 1c). Four days after induction, immunocytochemical analysis shows expression of the rate limiting enzyme for gaba synthesis glutamic acid decarboxylase gad65/67 (15.1% 0.3%) (figure 1d). Whole - cell patch - clamp recordings of retrovirus - transduced cells 14 days after onset of tamoxifen treatment show that these cells consistently exhibit electrophysiological properties of neurons (figure 1e). Moreover, their action potential discharges pattern in response to step current injections is highly reminiscent of low - threshold burst spiking interneurons and very similar to those generated from medial ganglionic eminence progenitors of the ventral telencephalon (figure 1d) (martnez - cerdeo et al ., 2010). To characterize the transcriptome changes in our model, we performed expression profiling at various time points 4, 12, 24, and 50 hr after the onset of differentiation . A large number of genes (1,508) are differentially regulated at least at one time point (fold change> 1.5, p <10; see figure 1f for a breakdown of numbers), as expected upon induction of a cellular differentiation program (table s1). Functional annotation of differentially expressed genes by gene ontology shows considerable enrichment in terms associated with distinct phases of neurogenesis, describing both early events (e.g., notch signaling, cell division) and later steps of the differentiation program (e.g., cell migration, axon guidance) (figure 1 g). Overall, these results show ascl1 drives an extended program of neuronal differentiation in our model . To understand how the observed changes in gene expression relate to ascl1 function, we mapped ascl1 binding sites genome - wide by chip followed by massive parallel sequencing (chip - seq) (wapinski et al ., 2013), using an antibody against the ha tag of ascl1-ert2 . This was performed 18 hr after the onset of differentiation, at a time point when ns cells have already undergone large transcriptional changes . Using input chromatin as control, we defined an extended list comprising 21,582 ascl1 binding sites, with fdr <0.5% and p <10 (table s2). In order to validate binding events (bes) mediated by the ascl1-ert2 protein, we used chip - pcr against wt ascl1, with chromatin extracted from embryonic e14.5 ventral telencephalic progenitors, or wt ns5 cells (figure s2a). We reasoned that most bes in differentiating ns cells should be identified in at least one of the chromatin samples . Indeed, this is the case for all chip - seq peaks associated with p <10 (with the majority of bes validated in both chromatins), indicating a good match between ascl1-ert2 and wt ascl1 . Most of the 11,782 ascl1 bes defined by this high - confidence list (p <10) are located within intergenic regions (63%) and at long distances from the nearest identified transcription start site (tss). Less than one third of ascl1 binding occurs inside genes (30%) or their promoter regions (7%) (figures 2a and 2b), suggesting that ascl1 binds predominantly to distal enhancer regions . We next used a hidden - markov model to characterize the chromatin states at ascl1-bound regions, using genome - wide profiles of histone modifications generated from proliferating and differentiating ns cells (before or 24 hr after addition of tamoxifen, respectively) (ernst and kellis, 2012). Ascl1 bes fall mostly within regions of chromatin highly co - enriched for h3k4me1 and h3k27ac, characteristic of active enhancers (same result for p <10; data not shown). Moreover, the same association with this chromatin state is found in proliferating ns cells, indicating that during differentiation ascl1 binds predominantly to regions that are already marked as active enhancers in proliferating ns cells (figures 2c, 2d, and s2b). To determine the dna sequence mediating ascl1 binding, we searched for motifs enriched within the 20 nucleotides region centered under the ascl1 peak summits . A strong preference is found for the hexamer sequence cagctg, corresponding to the e - box type sequence previously associated with ascl1 binding (castro et al ., 2011) and in agreement with its role in mediating direct dna binding (figure 2e). Although it is generally believed that proneural factors function by activating gene transcription, some reports have challenged this view (alvarez - rodrguez and pons, 2009, rheinbay et al ., 2013). In order to investigate this issue, we started by performing an in depth cross - comparison between the location analysis and expression profiling results, annotating each ascl1 be to its nearest tss . Direct comparison with the set of deregulated genes previously described shows that only a small fraction of bes are considered to be regulatory using nearest gene annotation (figure 3a). Nonetheless, we find a positive correlation between peak height (which is proportional to significance of binding, or p value) and its potential to upregulate gene expression (top peaks 20%). In contrast, the fraction of regulatory peaks that can be associated to repressed genes is invariant with changes in peak size, suggesting no direct relation between ascl1 binding significance and downregulation of gene expression . Therefore, mere binding of ascl1 to chromatin does not predict a regulatory event; however, the size of an ascl1 peak may be correlated with its potential to activate gene transcription . To have a global view of the association between the data sets of genes bound by ascl1 and regulated during differentiation, we determined the frequency at which deregulated genes (grouped in bins of increasing thresholds of fold change of expression) are associated with ascl1 bes (grouped in bins of increasing p value cutoff) and therefore considered to be directly regulated by ascl1 (figure 3b, left). The statistical significance of the overlaps was assessed by a similar comparison against 100 randomly generated control chip - seq data sets of identical size (figure 3b, right). The resulting heat maps indicate a strong association between ascl1-bound genes and those upregulated during differentiation and a much less pronounced association with downregulated genes (figure 3b, top left versus bottom left). Moreover, the fraction of downregulated genes that are considered ascl1 targets is very similar to that expected by chance (figure 3b, left versus right). In addition, this function is independent of the progress of differentiation during the first 50 hr, as shown with similar analyses for the different time points considered in the transcription profiling (figures 3c and s3). Importantly, restricting the analysis to the ascl1 bes located within 5 kb of a tss, producing a more stringent gene annotation, results in a similar conclusion (figure s4). To integrate the transcriptional profiling data sets collected at distinct time points, we applied a fuzzy c - means clustering algorithm, resulting in the identification of seven distinct temporal clusters (clusters 14 comprised of upregulated genes, clusters 57 of downregulated genes) (figure s5; table s3). Clusters of genes that are upregulated during differentiation are all strongly associated with ascl1 bes (p 1.3 10, 8.0 10, 1.1 10, and 7.9 10 for clusters 1, 2, 3, and 4, respectively), whereas clusters of downregulated genes show no such association (figure 3d), further indicating that ascl1 binding correlates with transcriptional activation . The relative size of each cluster (figure s5b) and their enrichment for bes indicate that most ascl1 target genes fall within a cluster characterized by an early activation profile (cluster 2), while a smaller but significant number is upregulated at mid or late time points (clusters 3 and 4, respectively). We then compiled a high - confidence list of 272 ascl1 direct targets comprising genes from clusters 2, 3, and 4 that are associated with at least one ascl1 be with high regulatory potential (p <10) (table s4). A search for terms describing biological process, molecular function, and pathways (panther classification system) revealed the segregation in each temporal cluster of ascl1 target genes encoding proteins of distinct classes and functions, suggesting the control of various subprograms at distinct developmental stages (figure s5c). Genes with the earliest onset of activation (belonging to cluster 2) are associated with signal transduction and cell communication, while targets with a mid - onset of activation (cluster 3) encode mostly proteins with a function related to transcription or notch signaling . Genes with latest onset (cluster 4) are enriched for generic terms associated with neural development and relate to late events in the differentiation program, from cell migration to axonal growth and guidance (figure s5c). In summary and in agreement with our previous study performed on a smaller scale, the identification of ascl1 target genes in our neurogenesis model demonstrates the direct control of distinct components of the neurogenic differentiation program by ascl1 . It was previously shown that ascl1 target genes display different onsets of expression at distinct stages of the neuronal lineage (borromeo et al ., 2014, castro et al ., 2011). Such temporal patterning could be associated with distinct accessibilities of ascl1 to its binding sites (e.g., due to differences in the chromatin landscape). To investigate this, we compared the binding profile of overexpressed ascl1 at two distinct stages by extending the previous chip - seq analysis to a time point at the onset of differentiation (30 min upon addition of tamoxifen) (table s9). Surprisingly, visual inspection of the genomic distribution of ascl1 peaks finds a remarkable similarity between ascl1 bes at t = 30 min and t = 18 hr (figure 4a). Comparison of the two ascl1 binding profiles in a bin - by - bin approach within the confidence limits defined by the previous chip - pcr validation (figure 4b, dashed line) shows an overlap of 86% between the two stages (figure 4b). Furthermore, the presence of normalized sequencing signal across both samples in genomic regions centered at putative sample - specific bes suggests that the overlap of occupancies may have been underestimated by peak calling conditions (figure 4c). Overall, our results indicate that the accessibility of ascl1 to the full complement of its binding sites remains largely identical throughout differentiation . We next asked what impact ascl1 may have on the chromatin landscape when it promotes neuronal differentiation of ns cells . We started by characterizing the chromatin landscape of proliferating ns cells and of ns cells undergoing differentiation by ascl1, using a dnase i hypersensitivity assay coupled to massive parallel sequencing (dnase - seq). Open chromatin) on a genome - wide scale, which correspond mostly to active regulatory elements such as promoters, enhancers, insulators, and silencers (boyle et al ., 2008, the density of mapped reads for each genome position was computed to generate a comprehensive list of dnase i hypersensitivity sites (dhss). Using a constant threshold of p <10, we identified 87,000 and 94,000 dhss in proliferating and differentiating ns cells, respectively (tables s5 and s6), numbers with a magnitude consistent with those obtained in other cell types (song et al ., 2011). Although the majority of these sites is shared by both experimental conditions, each cell state exhibits a specific set of 20,000 dhss (figure 5a). Since ascl1 functions as a transcriptional activator, we focused on the dhss induced during differentiation (table s7), as these are more likely to provide new insights into ascl1 function . To validate these dhss, we used formaldehyde - assisted isolation of regulatory elements (faire), an alternative method to identify regions of open chromatin . Although distinct preferences have been described for each technique in the identification of distal or proximal regulatory regions, the two techniques have been shown to yield largely overlapping results on a genome - wide basis (song et al .,, 9 of 13 tested regions presented more than 2-fold enrichment by faire - pcr across samples collected before and 24 hr after addition of tamoxifen, as opposed to control regions (figure 5b). Next, we investigated how regions of chromatin newly opened during differentiation may be associated with the observed changes in gene expression . We find a statistically significant enrichment of differentiation - induced dhss in the vicinity of upregulated genes (the sum of clusters 14; p <1.3 10), in sharp contrast with downregulated genes (clusters 57) (figure 5c). Moreover, a large fraction of all upregulated genes (413 of 760) is associated with at least one differentiation - induced dhs, suggesting the importance of these putative regulatory regions in activating gene expression during neurogenesis . Notably, upregulated genes associated with differentiation - induced dhss are either not expressed in proliferating ns cells or expressed at a low level when compared with upregulated genes near constant dhss (figure 5d). This is well exemplified by the induction of neurod4, a pro - differentiation tf only expressed in post - mitotic precursors (ohsawa et al ., 2005), and which is associated with newly open dhss (figure 6b). Overall, our results indicate that activation of differentiation genes is strongly associated with the appearance of new dhss . In order to investigate which dna - binding tfs may be associated with the appearance of newly open chromatin regions, we took advantage of the high resolution potential of digital genomic footprinting (dgf) applied to dnase - seq data to determine the position of sites occupied by tfs within differentiation - induced dhss (figure 5e) (piper et al ., 2013). Dgf identified almost 17,000 genomic sites (table s8) with significant occupancy levels (p <10), and the corresponding sequences were then subject to a search for frequency of motif occurrence . The most abundant motif found corresponds to the consensus sequence of ctcf and is likely to reflect its frequent binding to insulator regions identified by dnase - seq (figure 5e) (thurman et al ., 2012). Three additional motifs reveal binding by tfs of the bhlh or nfi families, with the most frequent one corresponding to the e - box consensus sequence, which mediates ascl1 binding . This observation establishes ascl1 as a prime candidate to promote the opening of chromatin structure and induce new dhss during differentiation . To understand how ascl1 binding may relate to the observed changes in chromatin compaction, we compared the genomic distribution of ascl1 bes with that of dhss before and after neuronal differentiation (figure 5f). Ascl1 chip - seq and dnase - seq data sets show a high degree of overlap (e.g., 80.3% of ascl1 bes with p <10 at t = 30 min fall within dhss in proliferating cells), indicating that a large fraction of bes, but not all, occur in regions of open chromatin in proliferating ns cells (figure 5f). Strikingly, this overlap increases significantly under differentiation conditions (to 91.05% after differentiation, in the aforementioned case; figure 5f). Altogether, a comparison between the ascl1 chip - seq and dnase - seq suggests that (1) the ascl1 binding profile remains constant at the two stages analyzed, (2) a large fraction of ascl1 bes occurs in regions of open chromatin in proliferating cells, with a subset falling within closed chromatin, and (3) binding of ascl1 to closed chromatin precedes the appearance of new dhss during differentiation . Visualization of aligned ascl1 chip - seq and dnase - seq data suggests that many differentiation - induced dhss overlap with ascl1 bes (figure 6a), as exemplified by those in the vicinity of genes activated during differentiation such as neurod4, ap3b2, mcf2l, and nrxn3 (figure 6b). Validation of changes in chromatin compaction at the selected ascl1 binding sites was performed by faire - pcr using chromatin extracted from ns5 cells expressing full - length ascl1 under the regulation of a doxycycline inducible promoter, before and after ascl1 induction (figure 6c). Although not all dhss that arise de novo during differentiation are associated with an ascl1 be, as not all ascl1 bes overlap with changes detected by dnase - seq, the vast majority of ascl1 bes fall within dhss in differentiating cells (91.05% or 91.27% with p <10, at t = 30 min or t = 18 hr, respectively; figure 5f). To investigate the correlation between ascl1 binding and changes in chromatin compaction on a large scale, we quantified the dnase - seq signal from both time points centered on ascl1 peak summits . Confirming previous observations, the overall dnase - seq profile at ascl1 peak summits is consistent with many bes occurring in regions of already open chromatin at t = 30 min (figure 6d). Nevertheless, a significant increase is observed when comparing profiles before and after differentiation (2.33 increase of median read count) at ascl1 peak summits genome - wide (figure 6d). This increase is amplified when focusing our analysis on ascl1 peaks that fall within differentiation - induced dhss (4.37 increase of median read count; figure 6d). Ascl1 bes co - localizing with differentiation - induced dhss are strongly associated with gene activation and not with gene repression (p <6.8 10; figure 6e), targeting a total number of 98 genes during differentiation . Overall, the integration of ascl1 location analysis, dnase - seq results, and expression profiling demonstrate ascl1 promotes genome - wide changes in chromatin compaction at its target sites during neuronal differentiation and the appearance of new dhss associated with activation of gene expression . Our results are in agreement with the observation that ascl1 can bind to nucleosomal dna when overexpressed in fibroblasts (wapinski et al ., 2013). To verify that ascl1 can bind to closed chromatin in a physiological context, we focused on the ascl1-bound regulatory region identified immediately downstream the neurod4 promoter (bs2 in figure 6a), a target gene of ascl1 in our cellular system that is not expressed in ventral telencephalon . We reasoned that binding to closed chromatin to neurod4 may still occur in this embryonic brain region at the neurogenic period, even in the absence of gene expression . Indeed, we detected strong binding of ascl1, as compared with a negative control region within the neurod4 locus by chip - pcr in chromatin extracted from mouse ventral telencephalon (figure 6f). However, in contrast to our previous findings with differentiating ns cells in culture, a lack of enrichment for non - nucleosomal dna assessed by faire - pcr shows this ascl1 target site is found within closed chromatin in ventral telencephalon (figure 6 g, compare with ascl1 site in dll1). Thus, our results with the neurod4 regulatory region demonstrate that endogenous ascl1 can bind to closed chromatin in an in vivo context . Finally, we wished to analyze the accessibility of chromatin at ascl1 target sites in the absence of ascl1 expression . For this, we used chromatin extracted from ventral telencephalon of ascl1-null embryos and probed ascl1-bound regions associated with previously validated ascl1 target genes (dll1, fbxw7, and stk33) in this embryonic structure (castro et al ., 2006). Ascl1 null embryos show a very significant reduction of faire - pcr signal relative to wt chromatin at all tested ascl1 binding sites, as compared with pairwise control regions, which is consistent with the results above and in support of a role for ascl1 in regulating chromatin compaction in vivo (figure s6). In spite of its pivotal role during neurogenesis, little is known on how ascl1 functions to regulate gene expression . Here we investigated the reciprocal interactions between ascl1 and the chromatin landscape when promoting the neuronal differentiation of ns cells in culture . We found that ascl1 can bind to and promote the opening of closed chromatin regions in its native context . Below we discuss our findings and their implications to the mechanisms that govern the temporal progression of the ascl1 transcriptional program . Our work resulted in a fine - grained characterization of a differentiation program driven by ascl1 . In spite of displaying a wide range of biological roles, the first consists of genes encoding transcriptional regulators, an observation consistent with the master regulator function of ascl1 in this developmental process . Importantly, the activation of some tfs may define feedback loops that modulate ascl1 activity . This is the case of id1, an inhibitor of bhlh activity that functions by sequestering e - proteins (ruzinova and benezra, 2003), and cbfa2t2/mtgr1, a zinc - finger protein that has been shown to function as a co - repressor of proneural proteins, including ascl1 (aaker et al ., 2010). Other examples, suggesting positive feedback mechanisms, are tfs previously linked to a relief of notch inhibition by counteracting the activity of hes1 (hes6), repressing notch1 (prox1), or by as yet unknown mechanisms (myt1) (bellefroid et al ., 1996, gratton et al . These regulatory events define a less well - understood role of ascl1 in modulating notch signaling cell autonomously, of putative importance to the progression of differentiation . The abundance of this group of genes is likely to result from its recognized role in many cellular functions, from signal transduction (where it may serve as a scaffold for components of signaling pathways) to cell shape change and locomotion, activities that are essential to the neurogenic differentiation program (forgacs et al ., 2004). It was recently shown that ascl1 promotes sequentially the proliferation and differentiation along the neuronal lineage, with the concomitant activation of distinct target genes (castro et al ., 2011). Our study focused on the transcriptional program activated by ascl1 when promoting neuronal differentiation . Comparing the identity and molecular determinants for target gene specificity associated with both cellular functions will likely require the characterization of ascl1 binding profile in an oscillatory versus sustained mode of expression . Nevertheless, the observation that overexpressed ascl1 can readily access the full complement of its differentiation sites at the onset of differentiation suggests these may already be accessible to this tf in proliferating cells, an important observation in the context of the dynamics of ascl1 function along the neuronal lineage . It was previously shown that overexpressed ascl1 can bind to closed chromatin in fibroblasts (as assessed by faire - pcr), defining it as a pioneer tf (wapinski et al ., 2013), results that the present study validates for the first time in a neural context . Moreover, our analysis of binding of ascl1 to the neurod4 locus in ventral telencephalic progenitors that do not express this gene allowed for the dissociation between binding and opening of chromatin, revealing binding of endogenous ascl1 to closed chromatin in vivo . Altogether, our study supports the idea that ascl1 displays its pioneer activity when regulating gene expression in its native context . Our analysis of chromatin states suggests that most ascl1 binding in our differentiation model occurs within enhancer regions that are already active in proliferating ns cells as shown by their enrichment for both h3k4me1 and h3k27ac . This may be due to the fact that many genes upregulated by ascl1 during differentiation are already expressed in proliferating ns cells . Alternatively, it may reflect the trivalent mark (h3k4me1, h3k27ac, and h3k9m3) identified at ascl1 binding sites in various cell types permissive to ascl1-mediated reprogramming (wapinski et al ., 2013). Most importantly, ascl1 promotes dnase i accessibility to regions of closed chromatin at a smaller but significant number of its target sites associated with the activation of a differentiation - specific component of its transcriptional program . Our results cast light into a previously unknown function of ascl1 and establish the first link between the chromatin landscape at ascl1-bound regulatory regions and the temporal pattern of the ascl1 program along the neuronal lineage . Moreover, they suggest that the hierarchical model recently proposed for the reprograming paradigm (wapinski et al ., 2013) may also be representative of the neurogenesis process and that opening of chromatin regions at ascl1 target sites may be important to allow subsequent binding of other tfs in differentiating cells . Finally, it is tempting to speculate that the broad effect of ascl1 in promoting chromatin accessibility described in this study will be important for the reprogramming capacity of this tf upon its ectopic expression in various cell types . Ascl1-ert2 encodes full - length mouse ascl1 in frame with the modified ligand binding domain of the human estrogen receptor (ert2) and an n - terminal flag / ha tag sequence . For virus production, ascl1-ert2 was subcloned into pmx - ires - gfp or pbabe - ires - gfp - puro . Replication - incompetent retroviruses were produced from hek293 t cells transiently transfected with retroviral, viral envelope-, and vsvg - pseudotyping plasmids . Retroviral particles were concentrated from supernatant by ultracentrifugation at 90,000 g for 90 min . Viral titers were typically 1010 infectious particles / ml . Ns5 cells were cultured as previously described (conti et al ., 2005) using laminin as media supplement (20 g / ml). Ns5 ascl1-ert2 cells were generated upon infection with pbabe - ascl1-ert2-ires - gfp - puro, followed by selection in complete medium containing 1-g / ml puromycin (calbiochem). Ns5 ascl1-ert2 cells were plated at 28,000 per cm of density and induced with 50-nm 4-hydroxytamoxifen, while reducing egf concentration to 5 ng / ml . All experiments were carried out upon approval and following the guidelines of the ethics committee of instituto gulbenkian de cincia . Quantification of ascl1 (endogenous + ascl1-ert2) done by parallel immunostaining with anti - ascl1 antibody in acutely cultured primary ventral telencephalon progenitors from e14.5 mouse embryos and ns5 ascl1-ert2 cells (three replicates each). A total of 939 and 751 cells for embryonic and ns5 progenitors, respectively, was imaged with identical exposure and images thresholded and normalized mean fluorescence per cell x (mean fluorescence of all cells in picture / mean fluorescence of all cells per sample)to define positive cells . Extraction of mrna and cdna synthesis performed as previously described (castro et al ., 2011). Quantitative real - time pcr (qrt - pcr) was performed according to the manufacturer using sybr green super - mix (quanta biosciences) on an abi7500 machine (applied biosystems). For microarray analysis, quality of total rna from biological triplicates was assessed using agilent bioanalyser, and samples were processed according to the illumina whole - genome gene expression direct hybridization assay guide, using illumina totalprep-96 rna amplification kit (life technologies). Quality control was performed on labeled crna, and 750 ng of labeled crna was hybridized to mouse ref-8v2 arrays and scanned by beedstudio v.3.1.3 (quantile normalization with background correction). Further details of data processing, including gene clustering, are described in supplemental information . For ascl1 chip - seq, ns5 ascl1-ert2 cells were fixed sequentially with 2 mm di(n - succimidyl) glutarate and 1% formaldehyde in pbs and then lysed, sonicated, and immunoprecipitated with anti - ha antibody, as previously described (castro et al ., 2011). Dna libraries were prepared from 10 ng of immunoprecipitated dna according to the standard illumina chip - seq protocol and sequenced with illumina gaiix . Raw reads were mapped to the mouse genome (ncbi37/mm9) with bowtie 0.12.7 (langmead et al ., 2009). Uniquely mapped reads data (11.5 million for both ascl1 chip and input chromatin samples) were then subsampled before peak calling with macs 1.4.1 (zhang et al ., 2008). For histone marks chip - seq, ascl1-ert2 cells were fixed in 1% formaldehyde in pbs, prepared as described above and immunoprecipitated with anti - h3k27ac and anti - h3k4me1 antibodies . Raw reads were mapped as above (25 million uniquely mapped reads for all samples) and data processed with macs 2.1.0 . For chromatin state characterization, we used a hidden markov modeling of chromatin enrichment software chromhmm (ernst and kellis, 2012). Chromatin preparation was done with a single fixation with 1% formaldehyde, from ns5 ascl1-ert2 cells, or when indicated with ns5 cells infected with a doxycycline inducible lentivirus expressing wt ascl1 (wapinski et al ., three rounds of phenol / chloroform extraction were followed by isopropanol precipitation of the dna . Quantification of genomic regions was done using a standard curve generated with decross - linked input chromatin by qrt - pcr as above . Dnase - seq samples from differentiating ns5 ascl1-ert2 cells or proliferating ns5 cells were prepared as previously described (song et al ., 2008, song and crawford, 2010) with a slight modification to the linkers to increase ligation efficiency (song et al ., 2011) and then sequenced with illumina gaiix . Mapping was done as for chip - seq, resulting in 165.9 and 168.7 millions of unique reads for proliferating and differentiating conditions, respectively . Genomic location of dhss was identified with macs version 1.4.1, following a protocol described in supplemental information . For oligonucleotides used in this study, designed research and performed experimental work, except electrophysiology tests performed by b.b . D.s.c . And a.a.s.f.r.
Tooth whitening is a highly desirable esthetic treatment, as the tooth color is one of the most important factors related to the patients' satisfaction with their appearance . Dental bleaching treatments are mainly based on the action of hydrogen peroxide, which is able to penetrate the tooth structure and release free radicals, oxidizing the chromophore molecules . Such molecules are mainly organic, although inorganic molecules can also be affected by these reactions . Nevertheless, the free radical reaction is not specific and it may also alter the organic component of enamel . Since the organic content contributes to the integrity of enamel, different adverse effects on both mineral and organic parts of bleached enamel have been observed . Alterations in enamel surface morphology [36], chemical composition [710], and microhardness values [3, 11, 12] after bleaching were previously reported . Furthermore, some changes in bleached enamel were also described as slight erosive effects promoted by the bleaching agent [6, 13]. Nevertheless, some authors claim that the erosive pattern on the surface of bleached enamel only occurs when bleaching gels with low ph are used [14, 15]. Attempts to minimize the adverse effects of bleaching treatments by increasing enamel remineralization have been conducted, however, the results are contradictory . De oliveira et al . Observed no significant increase of bleached enamel microhardness when calcium and fluoride were added to 10% carbamide peroxide gel . On the other hand, borges et al . Observed a significant increase of enamel microhardness after bleaching with 35% hydrogen peroxide agent with the addition of calcium and fluoride . Chen et al . Also reported a less distinct erosion pattern on the surface of enamel bleached with fluoridated gels . The association between the bleached enamel surface alterations and the subsequent susceptibility to erosive lesions resulting from the contact of bleached enamel with demineralizing solutions has been discussed . In a previous study, in other studies, at - home bleaching technique did not increase the susceptibility of enamel to erosion [18, 19]. However, the effect of at - office bleaching agent (35% hp) on the enamel susceptibility to erosion has not been properly discussed . Considering the possibility that bleaching gels with high concentration of hp could increase the susceptibility of enamel to erosion, the addition of remineralizing ions into bleaching gels would be beneficial for preventing a further enamel demineralization . Therefore, the objective of this in vitro study was to evaluate the effect of bleaching agents based on 35% hydrogen peroxide modified or not by the addition of calcium or fluoride on the susceptibility of enamel to erosion . The null hypotheses tested were (1) 35% hp bleaching agent does not increase the susceptibility of enamel to erosive challenges and (2) there is no difference in erosion susceptibility of enamel that has been treated with hydrogen peroxide compared to enamel treated with hydrogen peroxide supplemented with calcium or fluoride . Freshly extracted nondamaged and intact bovine incisors were stored in a 0.1% thymol solution and refrigerated at 4c, until required . Cylindrical enamel samples (3 mm in diameter and 1 mm height) were prepared from the labial surface of the tooth using a trephine mill (dentoflex, so paulo, sp, brazil). The enamel surface was ground flat and polished with water - cooled silicon carbide (sic) paper discs (1200, 2500, and 4000 grit; fepa - p, panambra, so paulo, sp, brazil), thereby removing approximately 200 m of the outermost layer as verified with a micrometer (micromar 40exl, mahr - goettingen, germany). The specimens were immersed in deionized water and placed in an ultrasonic bath for 10 min (ultrasonic cleaner, odontobras, ribeirao preto, brazil) for the removal of all waste, and then stored in thymol solution at 0.1% for rehydration . After polishing, the enamel specimens were selected from the average of the surface microhardness measured using a microhardness tester (fm-700, future - tech, tokyo, japan - knoop tip, average of three indentations, under 25 g - load for 10 s) with an allowable variation within 20% of the mean . Microhardness average of each specimen was used for stratified allocation among 4 groups (n = 15), so that the average microhardness for each group was similar . In order to maintain the reference surfaces for lesion - depth determination (profilometry), 2 layers of nail varnish were applied on 2/3 of the surface of each sample (1/3 of each side) exposing a central band area (see figure 1). The groups were divided as follows: c (control)-nonbleached, hp - bleached with 35% hydrogen peroxide gel without addition of remineralizing agents (ph = 6.35), hp + ca - bleached with 35% hydrogen peroxide gel with the addition of 2% calcium gluconate (ph = 7.99), and hp + f - bleached with 35% hydrogen peroxide gel modified by the addition of 0.6% sodium fluoride (ph = 8.11). The experimental groups were subjected to 35% hydrogen peroxide - based bleaching agents and modified by the manufacturer (based on whiteness hp blue formulation - fgm dental products, joinville, sc, brazil). The ph of the agents was measured using a ph meter (digimed dm-20-digicrom analtica ltda ., so paulo, brazil) fitted with an electrode (dme - digimed cv8) that was calibrated using solutions with ph 4.01 and 6.86 . A layer of approximately 2 mm of the bleaching gel was applied on the enamel surface for 40 minutes . After this period, the specimens were rinsed with deionized water to remove the bleaching agent (20 s). After the bleaching procedures, the enamel samples were immersed in artificial saliva for 2 h and then subjected to erosive challenges for 5 days . The erosive cycles consisted of immersion in unstirred soft drink (20 ml / sample, sprite zero, companhia fluminense de refrigerantes, porto real, rj, brazil), ph 2.8, four times per day for 2 min each time, followed by a remineralizing period of 2 h between erosive challenges (immersion in unstirred artificial saliva, ph 7.0, 20 ml / sample), at room temperature in small containers . The composition of the artificial saliva was the same as used by ghring et al . : hydrogen carbonate (22.1 mmol / l), potassium (16.1 mmol / l), sodium (14.5 mmol / l), hydrogen phosphate (2.6 mmol / l), boric acid (0.8 mmol / l), calcium (0.7 mmol / l), thiocyanate (0.4 mmol / l), and magnesium (0.2 mmol / l). The beverage was replaced at the end of each challenge, and artificial saliva was renewed daily . Profiles were obtained from the enamel surfaces with a profilometer (maxsurf xt 20, mahr - goettingen, germany) after the erosive challenge . To determine the change in the surface profile after the experiment, the nail varnish was carefully removed with a spatula and a solution of acetone (1: 1-acetone: water) to clean the surface . The diamond stylus moved from the first reference to the exposed area and then over to the other reference area (2.5 mm long and 1.0 mm wide). The vertical distance between the horizontal line drawn on the reference areas and the horizontal line drawn on experimental area was defined as tooth wear using the software (software mahr surf xt20, 2009). Five profile measurements were performed for each specimen at intervals of 0.25 mm and the values were averaged (m). A schematic representation of the sample preparation and analysis is presented in figure 1 . Assumptions of normal distribution of error and equality of variance (kolmogorov - smirnov and bartlett's tests) were checked for all the variables tested . Since the assumptions were justified, one - way anova followed by post hoc tukey's test were used to compare the different bleaching agents in relation to the susceptibility of enamel to erosive wear . Statistical analysis was performed with the software statistica for windows (statsoft, tulsa, ok, usa), with a significance level of 5% . One - way anova revealed significant differences between the groups (p = 0.009). The bleaching groups hp, hp + f, and hp + ca, presented similar enamel wear values among them (n.s . ). However, specimens from group hp + ca had significantly less mean enamel wear compared to the control after the erosive challenges, while the groups hp and hp + f did not differ from the control . Although in - office bleaching is considered a short - term treatment, it should not represent an additional risk for subjects susceptible to erosion . In the present study, the exposure of 35% hp - bleached enamel to the acid beverage did not cause higher enamel surface wear compared to control group (unbleached). It may be suggested that possible microstructural changes caused by the bleaching treatment did not predispose the enamel to greater erosive wear . These microstructural changes may have been repaired by the adsorption and precipitation of salivary calcium and phosphate, when immersed in artificial saliva for 2 h before the erosive challenge . The ability of saliva to reharden and replenish lost minerals from bleached enamel has been demonstrated previously [22, 23]. Although the adverse effects of bleaching agents on enamel are mainly attributed to the concentration and ph of the bleaching gel [14, 24], some surface alterations are reported even with low - concentrated and nonacidic agents [4, 5, 9]. In fact, the demineralization of bleached enamel can occur as a result of mineral and organic content alterations [4, 5, 25]. Even though these factors are considered reversible with exposure to saliva, it might be speculated that when the bleached enamel is exposed to acid, these minimal changes could promote a greater spread of erosive agents inside enamel, leading it to a more pronounced demineralization . Previous studies have shown that bleached enamel exposed to 37% phosphoric acid, which is applied before bonding procedures, presented a higher acid dissolution, with increased amount of ca extracted from enamel and an uneven etched surface, compared to the unbleached enamel [26, 27]. Nevertheless, other authors only reported this increased decalcified effect when high - concentrated hydrogen peroxide was used . Pretty et al . Applied 20 cycles of bleaching (2 h) using carbamide peroxide gels, from 10 to 22% concentration, and brushing (2 min), on enamel surface . The specimens were then immersed in 0.1% citric acid for a total of 14 h. the authors found no increased risk of enamel to acid dissolution . Engle et al . Also failed to demonstrate a significant increase in the susceptibility of bleached enamel to erosion / abrasion after a 5 days - bleaching treatment with 10% carbamide peroxide (10 h / day) associated with erosive and abrasive challenges . Although the bleaching agent tested in this study was more concentrated, which could increase the adverse effects, the unique application time was shorter (40 min) and, thus, similar results were achieved compared to control . This study simulated only one session of bleaching treatment, however, multiple appointments are needed for obtaining optimal bleaching outcomes . Therefore, the subsequent erosive wear of bleached enamel could be more evident after successive bleaching, as more severe chemical alterations have been observed in enamel bleached with high - concentrated hydrogen peroxide for long periods of exposure . On the other hand, it might be speculated that the alterations provoked by the bleaching agents could be relevant only for the susceptibility of the enamel to initial erosive lesions (erosion phase), in which enamel softening, but not wear, is seen, as observed previously . This experiment was conducted to simulate the effect of the intake of soft drinks (sprite zero that has no potential to stain the bleached enamel) after a session of in - office bleaching treatment using a high - concentrated hydrogen peroxide gel . The use of bovine enamel is considered a convenient substitute for human enamel in erosion studies, as they are easier to obtain, to handle, and standardize . Nevertheless, it has to be considered that these two substrates can behave in different physical and chemical manners and it was shown that the demineralization occurs faster in bovine enamel, due to its greater porosity [3133]. The addition of potentially remineralizing agents in bleaching gels was also investigated in this study . The calcium and fluoride were added to the thickener phase of the gel, resulting in a concentration of 2% and 0.6%, respectively, after the final mixture, as these concentrations were compatible with the chemical formulation of the gel, without impairing its thickness . It would be conceivable that the bleaching gels containing remineralizing agents could act not only as whitening agents but also as remineralizing agents . Supplements of fluoride and calcium in the bleaching gels have been shown to minimize the deleterious effects on enamel . It is supposed that the saturation of these ions in the gel allow their incorporation into the enamel apatite, increasing the resistance to demineralization [12, 35]. It was previously reported that the application of fluoride gel after bleaching resulted in increased resistance of the enamel against erosive attacks, compared to bleached / unfluoridated enamel . However, when fluoride was added to the bleaching gel, the protection against demineralization was reported to be limited compared to fluoride only, since peroxide reduced the fluoride uptake by bleached enamel . In the present study, the erosive challenges tested were frequent (4 cycles of 2 min each per day, for 5 days); therefore, the presence of fluoride into gel might have not been able to provide protection against enamel wear . As a reduced formation of koh - soluble fluoride was observed in enamel bleached with fluoridated agents, it can be speculated that any calcium fluoride - like precipitation was readily dissolved by the subsequent acid challenges . In addition, it has to be considered that fluoride dentifrices are frequently used in the daily practice and this additional source of fluoride can eventually contribute remineralizing the enamel surface after the bleaching procedures and erosive challenges . Nevertheless, the fluoride dentifrice was not included in this experimental model as this study attempted to analyze the effect of remineralizing agents added to the bleaching gel . Furthermore, fluoride dentifrice application is usually associated with a brushing procedure, which may increase the enamel erosive wear . Due to the fact that the focus of this study was only on enamel susceptibility to erosion, brushing with fluoride dentifrice in contrast to the effect of adding fluoride into the bleaching gel, the addition of calcium gluconate to the bleaching gel resulted in a protective effect against the erosion; thus, the second null hypothesis was rejected . In a previous study, deposits of calcium on the surface of the enamel bleached with calcium - added agent were shown using scanning electron microscope analysis . These deposits may have acted as a physical barrier, minimizing the contact of the acid to enamel, or providing additional mineral to be dissolved during the acid challenge before the underlying enamel was attacked . Another forms of calcium combined with bleaching agents have been previously investigated, such as the casein phosphopeptide - amorphous calcium phosphate (cpp - acp) [39, 40] and calcium chloride, resulting in increased mechanical properties of bleached enamel . Nevertheless, future studies should be conducted to investigate the solubility of different presentation forms of calcium salts, as well as their interaction with enamel surface, so that the mechanism of action of calcium in improving the resistance of bleached enamel to erosion can be established in different phases of its development (erosion and erosive wear). Considering the experimental design, it can be concluded that the application of 35% hp - based bleaching agents did not alter the enamel susceptibility to erosion, and the addition of calcium to the bleaching gel improved erosion resistance of the bleached enamel.
Transversetesticular ectopia (tte) is a rare form of testicular ectopia in which one of the testes crosses midline and occupies contralateral side of hemiscrotum . Tte with persistent mullerian duct syndrome usually presents as undescended testis with contralateral inguinal hernia . Laparoscopy is being increasingly used for diagnostic as well as therapeutic management of undescended testis . A 4-month - old male child was brought to uswith complaint of pain in the left inguinal region for 3 days . Ultrasonography showed funiculitis of the left cord structures and 1.5 1 cm testis - like structure present just above the left testes . After recovery of funiculitis, we posted the patient for diagnostic laparoscopy followed by definitive repair . During the procedure, a 5 mm port was inserted at umbilicus forthe camera and two working ports were inserted 5 cm away on either side of the umbilical . Right - sided testicular vessels crossed from right to left, entering in left internal ring [figure 1]. We divided the uterus in midline with help of bipolar cautery [figure 3]. After mobilization of right testicular vessels we found adequate length to bring it out through right - sided internal ring . Both testes brought out and fixed in the subdartous pouch . Left - sided hernial sac closed with intracaropreal suturing . Photograph showing right - sided testicular vessels crossing midline to left and entering into internal ring photograph showing uterus in between two testis photograph showing division of uterus in midline we successfully managed transverse testicular ectopia with persistent mullerian duct syndrome with hernia laparoscopicaly . On 6 months of follow - up, transverses testicular ectopia is a rare form of testicular ectopia in which testis is present in contralateral side of hemiscrotum . The first case is reported by lenhossek in 1886, he found it during an autopsy of adult man . Gupta suggested that adherence of developing wolffian duct with mullerian duct causes descend of both testis on the same side . Three types of tte are described: (1) tte with hernia (4050%), (2) tte with pmds (30%), (3) tte with hypospadias, pseudohermaphrodite, or some complexity (20%). Classified pmds into three groups: group a - the testes are in the position of the normal ovaries (female type); group b - one testis is found in a hernia sac orscrotum along with the uterus and tubes (male type); group c - both testes are found in the same hernia sac with associated tubes and the uterus (male type). Our patient belongs to group c. most common presentation is the absence of testis on the one side with inguinal hernia on opposite side . Ultrasonography and magnetic resonant imaging helps to diagnose this condition but most of them are diagnosed at the time of surgery . Various methods to manage this condition have been described, like herniotomy with trans - septal fixation of testis, that is, ombredanne technique, fixation on opposite side through suprapubic subcutaneous tunnel, and staged procedure . Dean and shah reported laparoscopically assisted correction of tte . In 2007, evans reported total laparoscopic correction of tte . Uniqueness of our case is that our case was having tte with hernia and pmds, which was totally managed by laparoscopy . Division of uterus in midline and mobilization of cord and vessels carried out by laparoscopy.
Due to the inherent limitations of the 12-lead surface ecg to provide accurate information on the precise location and activation sequence of cardiac arrhythmias within the human heart, non - invasive electrocardiographic imaging (ecgi) was developed . Electrocardiographic imaging is based on non - invasive numerical reconstruction of cardiac activation from unipolar body surface potentials combined with data on anatomy and position of the body surface electrodes from ct or mri . The use of ecgi may facilitate rapid non - invasive diagnosis of atrial or ventricular arrhythmias or the detection of ventricular dyssynchrony ., non - invasive mapping of both epi- and endocardial surfaces applying novel numerical algorithms was reported . The aim of the present study was to validate the mapping accuracy of a novel non - invasive epi- and endocardial electrophysiology system (neees). Prior to the electrophysiology study, a maximum of 224 unipolar body surface mapping electrodes compatible with mri or c imaging were placed onto the patient's torso (figure 1), followed by same - day ecg - gated contrast mri (magnetom avanto, 1.5 t, siemens ag) or ct (somatom definition 128, siemens ag) scanning of the heart and thorax . In the electrophysiology laboratory, custom - built mri - compatible body surface mapping electrodes were connected to the multichannel ecg amplifier as part of the neees (ep solutions sa). Figure 1non - invasive endo- and epicardial electrophysiology study procedure (see text for details). Non - invasive endo- and epicardial electrophysiology study procedure (see text for details). During ct imaging two scans were performed in a craniocaudal direction . The first scan examined the anatomy of the torso, including all body surface electrodes applied to the patient's upper body . The scan of the torso was processed using a slice thickness of 5 mm and reconstruction steps of 3 mm . The high - resolution ecg - gated scan of the heart was performed thereafter using automated intravenous injection of a non - ionic contrast bolus to a maximum of 2 ml / kg body weight during breath hold . Data reconstruction was performed using a slice thickness of 3 mm and reconstruction steps of 1.5 mm . Magnetic resonance imaging scanning followed the same general principles applied to ct imaging with regard to resolution, scanning direction, patient position, and breath hold . Localizer scan was performed initially to determine upper and lower margins of the torso such as to keep all body surface electrodes in the field of view . Between 7.5 and 20 ml of gadolinium - based contrast agent was injected, maintaining optimal contrasting during the initial 515 min following injection, depending on the agent used . The main scanning series was completed without ecg - gating utilizing the scanner sequences that allowed saving data in the form of axial section sets (slices) to implement volume reconstruction . Radiographic data imported in dicom format and coordinates of all unipolar ecg electrodes were automatically processed by the neees, to render a realistic three - dimensional reconstruction of the torso geometry and surface electrodes positions (figure 1). The three - dimensional heart model was plotted semi - automatically and segmentation optimized using a three - dimensional editor program as part of the neees . A tailored approach to three - dimensional a similar technique was used except for the difference in custom - built body surface mapping electrodes utilized . Following heart and torso segmentation and volume reconstruction, the surfaces of the torso and cardiac chambers were triangulated and polygonal meshes constructed . The surface triangulation procedure included construction of the primary surface mesh using the marching cubes method . The three - dimensional tetrahedral mesh was constructed using the advancing front approach . In order to display cardiac activation at a given point during the cardiac cycle, multichannel ecg recordings 1530 s in duration the imported ecg files were processed by neees inverse problem solution software in combination with heart and torso anatomy data . The resulting endo- and epicardial isopotential maps were reconstructed on the three - dimensional heart model and displayed as individual frames or as activation movies (figure 2a and b). Isopotential maps were analysed to determine the earliest activation zone, i.e. The potential minimum during early depolarization . Figure 2(a) non - invasively reconstructed endocardial isopotential map with earliest activation zone (first negative potential breakthrough) at the region of the right ventricular apex during pacing from the right ventrical apex in a patient with a cardiac resynchronization device . (b) non - invasively reconstructed epicardial isopotential map with earliest activation zone (first negative potential breakthrough) along the posterolateral left ventricular wall during pacing from the posterolateral wall of the left ventricle in a patient with a cardiac resynchronization device . (a) non - invasively reconstructed endocardial isopotential map with earliest activation zone (first negative potential breakthrough) at the region of the right ventricular apex during pacing from the right ventrical apex in a patient with a cardiac resynchronization device . (b) non - invasively reconstructed epicardial isopotential map with earliest activation zone (first negative potential breakthrough) along the posterolateral left ventricular wall during pacing from the posterolateral wall of the left ventricle in a patient with a cardiac resynchronization device . See also supplementary material online, video s2 . In order to assess the accuracy of the neees, a two - step approach was used . During step 1, patients scheduled in the device clinic for regular check - up and device optimization were included and stimulation from the atrial or ventricular pacemaker leads was performed at a rate of 80 b.p.m . In a subgroup of patients, pacing was completed during breath holding in order to assess the impact of breathing on the accuracy of non - invasive reconstruction . During pacing, a minimum number of six consecutive pacing stimuli were analysed in order to average the impact of potential confounding factors such as ecg signal noise . The earliest activation as denoted on the neees three - dimensional heart model was compared with the true anatomic location of the pacing electrode . The precise position of the pacemaker lead was visualized in the neees using the same ct or mri data to reconstruct the three - dimensional cardiac model and applying a semi - transparent myocardial mode (figure 3). The position of the pacing electrode was annotated and transferred onto the three - dimensional non - invasive cardiac surface map (figure 4a). Next, the site of earliest activation on the non - invasive cardiac surface map was marked and the distance between both was measured to validate the accuracy of the neees (figure 4b). In order to assess for data reproducibility, analysis was performed for each patient at least six times per pacing site . Figure 3computed tomography - based volume rendering, three - dimensional heart reconstruction, and marking of distal tip of the coronary sinus, right atrial, and right ventricular pacemaker lead . (b) three - dimensional heart reconstruction, volume rendering after segmentation using the non - transparent mode (distal tip of coronary sinus lead is marked). (c) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . This mode allows simultaneous visualization of the epicardial coronary sinus lead and the endocardial right atrial and ventricular pacing leads . (a) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . Position of the tip of the coronary sinus lead is marked with a white diamond . (b) non - invasive reconstruction of an epicardial isopotential map superimposed on the three - dimensional heart model demonstrating earliest activation along the distal tip of the coronary sinus lead . The anatomical position of the distal tip of the coronary sinus lead is marked with a white diamond, reconstructed pacing site is marked by red dot . Computed tomography - based volume rendering, three - dimensional heart reconstruction, and marking of distal tip of the coronary sinus, right atrial, and right ventricular pacemaker lead . (b) three - dimensional heart reconstruction, volume rendering after segmentation using the non - transparent mode (distal tip of coronary sinus lead is marked). (c) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . This mode allows simultaneous visualization of the epicardial coronary sinus lead and the endocardial right atrial and ventricular pacing leads . (a) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . Position of the tip of the coronary sinus lead is marked with a white diamond . (b) non - invasive reconstruction of an epicardial isopotential map superimposed on the three - dimensional heart model demonstrating earliest activation along the distal tip of the coronary sinus lead . The anatomical position of the distal tip of the coronary sinus lead is marked with a white diamond, reconstructed pacing site is marked by red dot . During step 2, the neees was used in concert with a three - dimensional electroanatomical mapping system (carto 3, biosense webster inc .) In patients referred for catheter ablation of atrial fibrillation . A point - by - point electroanatomical map was created followed by pacing from the distal tip of the ablation catheter (thermocool navistar, biosense webster inc .) During sinus rhythm at a rate of 8090 b.p.m . Either at the onset or at the end of the ablation procedure . The operator selected each pacing site and annotated its position on the three - dimensional electroanatomical map (figure 5), while those team members acquiring the neees body surface ecg and processing the neees non - invasive results were blinded with regards to the location of the individual pacing sites . Analysis of the non - invasively reconstructed isopotential maps allowed locating the earliest site of cardiac activation . This site was termed the reconstructed pacing site and marked on the mri or ct - based three - dimensional heart model (figure 5). Figure 5merging the invasive three - dimensional electroanatomical map (carto 3, biosense webster inc .) With the mri - based non - invasively reconstructed model using the neees . Non - invasively reconstructed pacing sites with earliest activation are annotated in red . Merging the invasive three - dimensional electroanatomical map (carto 3, biosense webster inc .) With the mri - based non - invasively reconstructed model using the neees . Following completion of the procedure, each electroanatomical carto map was exported as a polygonal mesh, including all marked reference pacing site coordinates and processed along with data from the neees containing the mri or ct - based three - dimensional heart model as a polygonal mesh and all annotated reconstructed pacing sites . The three - dimensional heart models generated by the carto system and the neees were merged utilizing a special software integrated into the neees (figure 5). If the volume of the space between the superimposed three - dimensional carto and neees mesh surfaces exceeded 10% of the volume of the carto map, the merged maps were exempt from further data analysis . The nearest point on the neees mesh model (ref ps) was determined for each carto pacing site (carto ps) previously marked on the carto mesh model (see supplementary material online, figure s1). Analysis was based on ref ps in order to compensate for the insufficient specificity of the carto mesh model constructed from a limited number of mapping points compared with the high - resolution mri or ct - derived heart models . The distance between ref ps and reconstructed pacing sites on the neees mesh model was measured using the ruler function integrated with the neees software (see supplementary material online, figure s1). For each pacing site, at least six consecutive pacing stimuli were analysed in order to average the influence of random factors such as patient breathing or ecg signal noise . The validation study in patients with implanted devices was performed at almazov federal research medical center in st petersburg, russia . The validation study using the carto 3 mapping system was performed at asklepios klinik st georg in hamburg, germany and at bakoulev research center for cardiovascular surgery in moscow, russia . The mean distances between reference sites and reconstructed pacing sites, as well as the standard deviations from the mean distances were calculated for all pacing sites and cardiac cycles . The results are shown in the corresponding tables as mean distances standard deviation and are expressed as absolute values . The student's t - test (unpaired) was used to evaluate mean differences between statistical data groups . Prior to the electrophysiology study, a maximum of 224 unipolar body surface mapping electrodes compatible with mri or c imaging were placed onto the patient's torso (figure 1), followed by same - day ecg - gated contrast mri (magnetom avanto, 1.5 t, siemens ag) or ct (somatom definition 128, siemens ag) scanning of the heart and thorax . In the electrophysiology laboratory, custom - built mri - compatible body surface mapping electrodes were connected to the multichannel ecg amplifier as part of the neees (ep solutions sa). Figure 1non - invasive endo- and epicardial electrophysiology study procedure (see text for details). Non - invasive endo- and epicardial electrophysiology study procedure (see text for details). During ct imaging two scans were performed in a craniocaudal direction . The first scan examined the anatomy of the torso, including all body surface electrodes applied to the patient's upper body . The scan of the torso was processed using a slice thickness of 5 mm and reconstruction steps of 3 mm . The high - resolution ecg - gated scan of the heart was performed thereafter using automated intravenous injection of a non - ionic contrast bolus to a maximum of 2 ml / kg body weight during breath hold . Data reconstruction was performed using a slice thickness of 3 mm and reconstruction steps of 1.5 mm . Magnetic resonance imaging scanning followed the same general principles applied to ct imaging with regard to resolution, scanning direction, patient position, and breath hold . Localizer scan was performed initially to determine upper and lower margins of the torso such as to keep all body surface electrodes in the field of view . Between 7.5 and 20 ml of gadolinium - based contrast agent was injected, maintaining optimal contrasting during the initial 515 min following injection, depending on the agent used . The main scanning series was completed without ecg - gating utilizing the scanner sequences that allowed saving data in the form of axial section sets (slices) to implement volume reconstruction . Radiographic data imported in dicom format and coordinates of all unipolar ecg electrodes were automatically processed by the neees, to render a realistic three - dimensional reconstruction of the torso geometry and surface electrodes positions (figure 1). The three - dimensional heart model was plotted semi - automatically and segmentation optimized using a three - dimensional editor program as part of the neees . A tailored approach to three - dimensional a similar technique was used except for the difference in custom - built body surface mapping electrodes utilized . Following heart and torso segmentation and volume reconstruction, the surfaces of the torso and cardiac chambers were triangulated and polygonal meshes constructed . The surface triangulation procedure included construction of the primary surface mesh using the marching cubes method . In order to display cardiac activation at a given point during the cardiac cycle, multichannel ecg recordings 1530 s in duration were logged and exported into the neees . The imported ecg files were processed by neees inverse problem solution software in combination with heart and torso anatomy data . The resulting endo- and epicardial isopotential maps were reconstructed on the three - dimensional heart model and displayed as individual frames or as activation movies (figure 2a and b). Isopotential maps were analysed to determine the earliest activation zone, i.e. The potential minimum during early depolarization . Figure 2(a) non - invasively reconstructed endocardial isopotential map with earliest activation zone (first negative potential breakthrough) at the region of the right ventricular apex during pacing from the right ventrical apex in a patient with a cardiac resynchronization device . (b) non - invasively reconstructed epicardial isopotential map with earliest activation zone (first negative potential breakthrough) along the posterolateral left ventricular wall during pacing from the posterolateral wall of the left ventricle in a patient with a cardiac resynchronization device . (a) non - invasively reconstructed endocardial isopotential map with earliest activation zone (first negative potential breakthrough) at the region of the right ventricular apex during pacing from the right ventrical apex in a patient with a cardiac resynchronization device . (b) non - invasively reconstructed epicardial isopotential map with earliest activation zone (first negative potential breakthrough) along the posterolateral left ventricular wall during pacing from the posterolateral wall of the left ventricle in a patient with a cardiac resynchronization device . In order to assess the accuracy of the neees, a two - step approach was used . During step 1, patients scheduled in the device clinic for regular check - up and device optimization were included and stimulation from the atrial or ventricular pacemaker leads was performed at a rate of 80 b.p.m . In a subgroup of patients, pacing was completed during breath holding in order to assess the impact of breathing on the accuracy of non - invasive reconstruction . During pacing, a minimum number of six consecutive pacing stimuli were analysed in order to average the impact of potential confounding factors such as ecg signal noise . The earliest activation as denoted on the neees three - dimensional heart model was compared with the true anatomic location of the pacing electrode . The precise position of the pacemaker lead was visualized in the neees using the same ct or mri data to reconstruct the three - dimensional cardiac model and applying a semi - transparent myocardial mode (figure 3). The position of the pacing electrode was annotated and transferred onto the three - dimensional non - invasive cardiac surface map (figure 4a). Next, the site of earliest activation on the non - invasive cardiac surface map was marked and the distance between both was measured to validate the accuracy of the neees (figure 4b). In order to assess for data reproducibility, analysis was performed for each patient at least six times per pacing site . Figure 3computed tomography - based volume rendering, three - dimensional heart reconstruction, and marking of distal tip of the coronary sinus, right atrial, and right ventricular pacemaker lead . (b) three - dimensional heart reconstruction, volume rendering after segmentation using the non - transparent mode (distal tip of coronary sinus lead is marked). (c) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . This mode allows simultaneous visualization of the epicardial coronary sinus lead and the endocardial right atrial and ventricular pacing leads . (a) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . Position of the tip of the coronary sinus lead is marked with a white diamond . (b) non - invasive reconstruction of an epicardial isopotential map superimposed on the three - dimensional heart model demonstrating earliest activation along the distal tip of the coronary sinus lead . The anatomical position of the distal tip of the coronary sinus lead is marked with a white diamond, reconstructed pacing site is marked by red dot . Computed tomography - based volume rendering, three - dimensional heart reconstruction, and marking of distal tip of the coronary sinus, right atrial, and right ventricular pacemaker lead . (b) three - dimensional heart reconstruction, volume rendering after segmentation using the non - transparent mode (distal tip of coronary sinus lead is marked). (c) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . This mode allows simultaneous visualization of the epicardial coronary sinus lead and the endocardial right atrial and ventricular pacing leads . (a) three - dimensional heart reconstruction, volume rendering after segmentation using the semi - transparent myocardial mode . Position of the tip of the coronary sinus lead is marked with a white diamond . (b) non - invasive reconstruction of an epicardial isopotential map superimposed on the three - dimensional heart model demonstrating earliest activation along the distal tip of the coronary sinus lead . The anatomical position of the distal tip of the coronary sinus lead is marked with a white diamond, reconstructed pacing site is marked by red dot . During step 2, the neees was used in concert with a three - dimensional electroanatomical mapping system (carto 3, biosense webster inc .) In patients referred for catheter ablation of atrial fibrillation . A point - by - point electroanatomical map was created followed by pacing from the distal tip of the ablation catheter (thermocool navistar, biosense webster inc .) During sinus rhythm at a rate of 8090 b.p.m . Either at the onset or at the end of the ablation procedure . The operator selected each pacing site and annotated its position on the three - dimensional electroanatomical map (figure 5), while those team members acquiring the neees body surface ecg and processing the neees non - invasive results were blinded with regards to the location of the individual pacing sites . Analysis of the non - invasively reconstructed isopotential maps allowed locating the earliest site of cardiac activation . This site was termed the reconstructed pacing site and marked on the mri or ct - based three - dimensional heart model (figure 5). Figure 5merging the invasive three - dimensional electroanatomical map (carto 3, biosense webster inc .) With the mri - based non - invasively reconstructed model using the neees . Non - invasively reconstructed pacing sites with earliest activation are annotated in red . Merging the invasive three - dimensional electroanatomical map (carto 3, biosense webster inc .) With the mri - based non - invasively reconstructed model using the neees . Following completion of the procedure, each electroanatomical carto map was exported as a polygonal mesh, including all marked reference pacing site coordinates and processed along with data from the neees containing the mri or ct - based three - dimensional heart model as a polygonal mesh and all annotated reconstructed pacing sites . The three - dimensional heart models generated by the carto system and the neees were merged utilizing a special software integrated into the neees (figure 5). If the volume of the space between the superimposed three - dimensional carto and neees mesh surfaces exceeded 10% of the volume of the carto map, the merged maps were exempt from further data analysis . The nearest point on the neees mesh model (ref ps) was determined for each carto pacing site (carto ps) previously marked on the carto mesh model (see supplementary material online, figure s1). Analysis was based on ref ps in order to compensate for the insufficient specificity of the carto mesh model constructed from a limited number of mapping points compared with the high - resolution mri or ct - derived heart models . The distance between ref ps and reconstructed pacing sites on the neees mesh model was measured using the ruler function integrated with the neees software (see supplementary material online, figure s1). For each pacing site, at least six consecutive pacing stimuli were analysed in order to average the influence of random factors such as patient breathing or ecg signal noise . The validation study in patients with implanted devices was performed at almazov federal research medical center in st petersburg, russia . The validation study using the carto 3 mapping system was performed at asklepios klinik st georg in hamburg, germany and at bakoulev research center for cardiovascular surgery in moscow, russia . During statistical data processing, the mean distances between reference sites and reconstructed pacing sites, as well as the standard deviations from the mean distances were calculated for all pacing sites and cardiac cycles . The results are shown in the corresponding tables as mean distances standard deviation and are expressed as absolute values . The student's t - test (unpaired) was used to evaluate mean differences between statistical data groups . Twenty - nine patients (23 men, mean age 62 11 years) with previously implanted devices seen for regular check - up at the pacemaker clinic were enrolled . Twenty - one of 29 (72%) patients had a history of ischaemic cardiomyopathy, 7 of 29 (24%) patients had a history of dilated cardiomyopathy, and 1 of 29 (4%) had a history of restrictive cardiomyopathy . The type of implanted device, predominant rhythm at the time of study, and type of underlying conduction disturbance are summarized in supplementary material online, table s1 . Pacing was performed from a total of 76 pacing sites: 21 in the right atrium, 29 in the right ventricle, and 26 in the left ventricle through the coronary sinus, respectively . The different pacing lead positions are detailed in supplementary material online, tables s2 and s3 . The mean distance from the non - invasively predicted pacing site to the anatomic reference site was 10.8 5.4 mm for the right atrium, 7.7 5.8 mm for the right ventricle, and 7.9 5.7 mm for the left ventricle activated via the coronary sinus lead (table 1). No significant difference in accuracy was found for the left vs. right ventricular sites (p = 0.815), while the difference in accuracy was significant comparing ventricular and atrial sites (p <0.001). Table 1accuracy according to paced cardiac chamber and number of pacing attempts analysed in implanted device study cohortheart chamberra, endocardium (n = 126)rv, endocardium (n = 174)lv, epicardium (n = 156)ra + rv + lv (n = 456)mean sd (mm)10.8 5.47.7 5.87.9 5.78.6 5.8mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events.ra, right atrium; rv, right ventricle; lv, left ventricle . Accuracy according to paced cardiac chamber and number of pacing attempts analysed in implanted device study cohort mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events . The influence of breathing on the accuracy of non - invasive reconstruction was studied in a subgroup of six patients . Breath hold during inspiration (see supplementary material online, figure s2) as compared with free breathing or breath hold on expiration resulted in highest accuracy, that is, smallest difference in the mean distance from non - invasively predicted pacing site to reference pacing site (table 2). Table 2effect of breathing on the accuracy of the neeesheart chamberra (n = 24)rv (n = 36)lv (n = 36)ra + rv + lv (n = 96)spontaneous breathing, mean sd (mm)8.0 1.86.3 1.86.4 1.56.8 1.9 (p <0.001 vs. inspiration and expiration)inspiration breath hold, mean sd (mm)5.9 1.34.7 1.35.9 0.001 vs. spontaneous breathing and expiration)expiration breath hold, mean sd (mm)9.5 1.98.1 1.78.5 1.18.6 1.6 (p <0.001 vs. spontaneous breathing and inspiration)pacing during spontaneous breathing and during breath hold on inspiration and expiration . Mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events.ra, right atrium; rv, right ventricle; lv, left ventricle . Effect of breathing on the accuracy of the neees pacing during spontaneous breathing and during breath hold on inspiration and expiration . Mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events . Five patients (four with paroxysmal and one with persistent atrial fibrillation, four males, mean age 65 4 years) referred for pulmonary vein isolation aided by three - dimensional electroanatomical mapping underwent electrical stimulation from a total of 412 different intracardiac positions . The mean distance between non - invasively reconstructed pacing site and the reference pacing site was 7.4 2.7 mm for the right atrium, 6.9 2.3 mm for the left atrium, 6.5 2.1 mm for the right ventricle, and 6.4 2.2 mm for the left ventricle, respectively (table 3). There was a statistically significant difference in accuracy comparing the mean distance for both ventricles vs. the left and right atria (p = 0.004), while no statistically significant difference in accuracy was found for the left vs. right ventricle (p = 0.864) or left vs. right atrium (p = 0.143). Table 3results for the carto pacing study according to anatomical site pacedheart chamberra (n = 92)la (n = 167)rv (n = 61)lv (n = 92)ra + la + rv + lv (n = 412)mean distance sd (mm)7.4 2.76.9 2.36.5 2.16.4 2.26.8 2.4mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events.ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; sd, standard deviation . Results for the carto pacing study according to anatomical site paced mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events . Ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; sd, standard deviation . Twenty - nine patients (23 men, mean age 62 11 years) with previously implanted devices seen for regular check - up at the pacemaker clinic were enrolled . Twenty - one of 29 (72%) patients had a history of ischaemic cardiomyopathy, 7 of 29 (24%) patients had a history of dilated cardiomyopathy, and 1 of 29 (4%) had a history of restrictive cardiomyopathy . The type of implanted device, predominant rhythm at the time of study, and type of underlying conduction disturbance are summarized in supplementary material online, table s1 . Pacing was performed from a total of 76 pacing sites: 21 in the right atrium, 29 in the right ventricle, and 26 in the left ventricle through the coronary sinus, respectively . The different pacing lead positions are detailed in supplementary material online, tables s2 and s3 . The mean distance from the non - invasively predicted pacing site to the anatomic reference site was 10.8 5.4 mm for the right atrium, 7.7 5.8 mm for the right ventricle, and 7.9 5.7 mm for the left ventricle activated via the coronary sinus lead (table 1). No significant difference in accuracy was found for the left vs. right ventricular sites (p = 0.815), while the difference in accuracy was significant comparing ventricular and atrial sites (p <0.001). Table 1accuracy according to paced cardiac chamber and number of pacing attempts analysed in implanted device study cohortheart chamberra, endocardium (n = 126)rv, endocardium (n = 174)lv, epicardium (n = 156)ra + rv + lv (n = 456)mean sd (mm)10.8 5.47.7 5.87.9 5.78.6 5.8mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events.ra, right atrium; rv, right ventricle; lv, left ventricle . Accuracy according to paced cardiac chamber and number of pacing attempts analysed in implanted device study cohort mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events . The influence of breathing on the accuracy of non - invasive reconstruction was studied in a subgroup of six patients . Breath hold during inspiration (see supplementary material online, figure s2) as compared with free breathing or breath hold on expiration resulted in highest accuracy, that is, smallest difference in the mean distance from non - invasively predicted pacing site to reference pacing site (table 2). Table 2effect of breathing on the accuracy of the neeesheart chamberra (n = 24)rv (n = 36)lv (n = 36)ra + rv + lv (n = 96)spontaneous breathing, mean sd (mm)8.0 1.86.3 1.86.4 1.56.8 1.9 (p <0.001 vs. inspiration and expiration)inspiration breath hold, mean sd (mm)5.9 1.34.7 1.35.9 0.001 vs. spontaneous breathing and expiration)expiration breath hold, mean sd (mm)9.5 1.98.1 1.78.5 1.18.6 1.6 (p <0.001 vs. spontaneous breathing and inspiration)pacing during spontaneous breathing and during breath hold on inspiration and expiration . Mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events.ra, right atrium; rv, right ventricle; lv, left ventricle . Effect of breathing on the accuracy of the neees pacing during spontaneous breathing and during breath hold on inspiration and expiration . Mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events . Five patients (four with paroxysmal and one with persistent atrial fibrillation, four males, mean age 65 4 years) referred for pulmonary vein isolation aided by three - dimensional electroanatomical mapping underwent electrical stimulation from a total of 412 different intracardiac positions . The mean distance between non - invasively reconstructed pacing site and the reference pacing site was 7.4 2.7 mm for the right atrium, 6.9 2.3 mm for the left atrium, 6.5 2.1 mm for the right ventricle, and 6.4 2.2 mm for the left ventricle, respectively (table 3). There was a statistically significant difference in accuracy comparing the mean distance for both ventricles vs. the left and right atria (p = 0.004), while no statistically significant difference in accuracy was found for the left vs. right ventricle (p = 0.864) or left vs. right atrium (p = 0.143). Table 3results for the carto pacing study according to anatomical site pacedheart chamberra (n = 92)la (n = 167)rv (n = 61)lv (n = 92)ra + la + rv + lv (n = 412)mean distance sd (mm)7.4 2.76.9 2.36.5 2.16.4 2.26.8 2.4mean distance between reference pacing site and reconstructed pacing site and standard deviation for n pacing events.ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; sd, standard deviation . Results for the carto pacing study according to anatomical site paced mean distance between reference pacing site and reconstructed pacing site and standard deviation for ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle; sd, standard deviation . The present study demonstrates that non - invasive reconstruction of individual pacing sites using a novel neees is feasible and accurate . The cardiac structure from which stimulation was performed was recognized correctly in all cases . While stimulation from the permanent coronary sinus lead results in early activation of the epicardial layer of the ventricular myocardium, pacing from the right atrial and right ventricular lead causes the activation front to spread from endo- to epicardium . Applying a similar principle than previously described for non - invasive electrocardiographic imaging systems for obtaining endocardial electrophysiological data, the neees adds the option to simultaneously map the endo- and epicardial surfaces and was equally effective in delineating epi- or endocardial pacing sites . The current results are in line with a study by cucculich et al . Assessing the accuracy of a non - invasive electrocardiographic imaging system in patients with atrial fibrillation . Spatial accuracy within the left atrium was 6.3 3.9 mm compared with a three - dimensional electroanatomical mapping system . Analysed the accuracy of a non - invasive electrocardiographic imaging system in the ventricle reporting an accuracy of 210 mm . The present study indicates that respiration influences the accuracy of the non - invasive reconstructed pacing site location . Respiration may result in cyclic changes of the anatomical relationship between heart and torso and may alter the electrical conductivity of the lungs . The highest accuracy for reconstructed pacing sites this may simply be explained by the fact that pre - procedural ct imaging is also routinely being performed during breath hold on inspiration . Compared with other available non - invasive mapping systems relying merely on epicardial surface information, the neees is capable of mapping both the endo- and epicardial surface . Extrapolating the findings from the current study into clinical practice, the use of the neees may facilitate accurate non - invasive identification of focal atrial or endo- or epicardial ventricular arrhythmias and proper delineation of the re - entry wavefront . Furthermore, the neees may aid in non - invasive optimization of resynchronization therapy . In addition to its non - invasive nature, the neees offers the ability to simultaneously reconstruct activation patterns of all four cardiac chambers during a single cardiac cycle . Comparing the neees to the carto electroanatomical mapping system may have introduced a source of error due to the inherent differences in the geometries created . However, the use of the carto system was advantageous, in that pacing could be performed from various atrial and ventricular sites, while statistical processing compensated to some extent for geometrical differences . In contrast, the stimulation protocol utilizing implanted pacemaker leads to verify the accuracy of the neees permitted only a limited number of pacing locations to be selected in each patient . However, the exact position of each pacing lead could be integrated into the neees with high accuracy using the ct data set . While team members acquiring and processing the neees data were blinded during the validation study using the carto three electroanatomical mapping system, this was not the case for the validation study using implanted devices . The latter may have introduced a source of bias in the validation of the neees . Comparing the neees to the carto electroanatomical mapping system may have introduced a source of error due to the inherent differences in the geometries created . However, the use of the carto system was advantageous, in that pacing could be performed from various atrial and ventricular sites, while statistical processing compensated to some extent for geometrical differences . In contrast, the stimulation protocol utilizing implanted pacemaker leads to verify the accuracy of the neees permitted only a limited number of pacing locations to be selected in each patient . However, the exact position of each pacing lead could be integrated into the neees with high accuracy using the ct data set . While team members acquiring and processing the neees data were blinded during the validation study using the carto three electroanatomical mapping system, this was not the case for the validation study using implanted devices . The latter may have introduced a source of bias in the validation of the neees . The novel neees was able to correctly identify the site of pacing from various endo- and epicardial sites with high accuracy . Future studies are indicated to assess the feasibility of the neees to correctly diagnose the origin of focal or re - entry arrhythmia within the human heart . This study was in part funded by a grant from the russian science foundation, project #14 - 15 - 01097, noninvasive electrocardiographic imaging for diagnostics and catheter ablation guidance of atrial fibrillation and atrial flutter: a feasibility study . Funding to pay
Fossa navicularis strictures following radical prostatectomy are rare . Unlike other anterior urethral strictures, fossa strictures are usually inflammatory or procedure - induced, arising from urethral trauma caused by endoscopic procedures, catheterizations, or subsequent infections . In the literature we describe the incidence of fossa stricture in our rlp series to determine its likely etiologic factors . The data of 424 men undergoing rlp from june 2002 to may 2006 by a single surgeon were prospectively entered into an electronic database . Prior to surgery, all men were evaluated, and the following data entered into the database: age, height, weight, american urological association (aua) symptom score, urinary bother score, prostate - specific antigen (psa), and pertinent medical history . Urinary and functional outcomes were determined on the basis of self - administered questionnaires, including the seven - item international prostate symptom score, and selected questions from the 26-item expanded prostate cancer instrument composite, at the routine 3-month and 9-month follow - up visits . The questionnaire asked whether they wore pads, how many weeks did it take to not need pads, how many weeks needed to return to work, and how many weeks to return to baseline energy levels . A nonclinical research associate (d.w.s .) Collected the follow - up information . Complications were defined by the need for prolongation of hospitalization, the need for a secondary procedure, or rehospitalization within 30 days . All statistical comparisons between the stricture and nonstricture groups as well as between the 18 and 22 french (f) catheter groups were two - sided using fisher s exact test and student s t - test for means (sas ver . Multivariate analysis was also performed with a stepwise logistic regression which used the preoperative continuous variables, body mass index (bmi), psa, estimated blood loss (ebl), aua symptom score, bother score, age, and prostate weight (table 1) as independent variables in the prediction of a positive stricture . Table 1demographic, clinical, and perioperative data for men reporting strictures versus stricture - free menvariable no stricture standard error (se)stricture se p - valuepatients (n)41410age (year)61.4.0 (4379)0.363.8 (5171)2.40.29bmi27.0 (20.634.9)0.226.3 (24.234.0)0.70.52aua symptom score8.6 (032)0.47.4 (127)1.80.59urinary bother score1.7 (06)0.11.6 (06)0.50.86preoperative psa (ng / ml)6.9 (0.162.0)0.36.2 (1.818.0)1.50.71prostate size (g)53.1 (12.5163.0)1.446.1 (20.069.1)3.30.41ebl (ml)107 (25350)2.867.5 (25400)16.30.03 bmi, body mass index; aua, american urological association; psa, prostate - specific antigen; ebl, estimated blood loss values are the mean, with the range given in parenthesis two - sided fisher s exact test demographic, clinical, and perioperative data for men reporting strictures versus stricture - free men bmi, body mass index; aua, american urological association; psa, prostate - specific antigen; ebl, estimated blood loss values are the mean, with the range given in parenthesis two - sided fisher s exact test all rlps were performed in the same manner with the da vinci surgical system (intuitive surgical, mountain view, calif . ). Patients are positioned supine in an extreme trendelenburg position and undergo abdominal insufflation with a pneumoperitoneum of 15 mmhg . The standard protocol consists of inserting a silastic urethral catheter at the beginning of the case that is utilized throughout the dissection until completion of the anastomosis . At this point a new 18 f silastic catheter is put in place, and this is generally removed 7 days postoperatively . At case number 35, however, we switched from an 18 to a 22 f silastic urethral catheter during the dissection as an aid to prevent stapling the urethra . We use a stapling device instead of suturing the dorsal venous complex based on findings from a previous study . In that study, the data demonstrated that the stapler technique provided a more defined apical dissection and a statistically significant reduction in positive margins in patients with organ - confined disease . We switched back to an 18 f catheter at case number 166 due to concerns regarding strictures in the fossa navicularis . Fossa strictures were diagnosed based on acute onset of obstructive lower urinary symptoms and bougie a boule calibration . Obstructive voiding symptoms included a decreased force of stream, dribbling or splaying, and prolonged voiding . Table 1 presents the demographic, clinical, and perioperative data for men reporting strictures versus stricture - free men . The mean follow - up was 26.4 months for stricture patients versus 22.1 months for stricture - free patients and 22.8 months (range: 3.645.6 months) for all patients . The groups were comparable for the standard clinical factors, such as age, bmi, aua symptom score, urinary bother score, and preoperative psa level . There was also no difference between the groups in reference to operative time, prostate size, and cardiovascular disease . The stricture - free group, however, had a higher ebl than the stricture group (p = 0.03). In addition, none of the patients in the stricture group had any history of a previous endoscopic procedure . Multivariate analysis also demonstrated that none of the preoperative continuous variables predicted the presence of strictures (all p> 0.18). Table 2 outlines the demographic, clinical, and perioperative data for men stratified by catheter size . The 18 and 22 f catheter groups were also comparable for age, bmi, baseline aua symptom score, urinary bother score, preoperative psa, operative time, prostate size, ebl, and complications . The 22 f catheter group, however, had a significantly higher rate of stricture formation . A total of 6.9% of patients developed a stricture using a 22 f catheter versus 0.3% of patients using an 18 f catheter during the intra - operative dissection (p <0.01). The mean length of urethral catheterization was 7.1 days (range: 78 days) in the stricture group, with a mean time to stricture development of 50 days (range: 3493 days). One patient in the stricture group developed a bladder neck contracture and another went into acute urinary retention . Table 2demographic, clinical data, and perioperative data for 18 and 22 f catheter groupsvariable 18 f se22 f se p - valuepatients (n)293131age (year)61.1(4679)0.462.2 (4378)0.70.14bmi27.0 (20.640.0)0.226.80.30.49aua symptom score8.7 (035)0.48.2 (031)0.60.54urinary bother score1.7 (06)0.11.7 (06)0.10.93preoperative psa (ng / ml)6.7 (1.131.0)0.37.3 (0.162.0)0.60.30prostate size (g)54.1 (12.5163.0)1.850.5 (15.0135.0)1.90.21ebl (ml)109.3 (25350)3.298.9 (25400)5.50.10fossa stricture190.0002 bmi, body mass index; aua, american urological association; psa, prostate - specific antigen; ebl estimated blood loss values are the mean, with the range given in parenthesis two - sided fisher s exact test demographic, clinical data, and perioperative data for 18 and 22 f catheter groups bmi, body mass index; aua, american urological association; psa, prostate - specific antigen; ebl estimated blood loss values are the mean, with the range given in parenthesis two - sided fisher s exact test in addition, urethral reconstruction was required in two of nine patients (22%) who developed strictures with the larger 22 f catheter . Only one subject developed a stricture using an 18 f catheter; he required only soft dilation as treatment . In the present series, all comparisons made by the student s t - test were also examined using the nonparametric wilcoxon rank sum test, with similar conclusions . A thorough review of the literature revealed an absence of papers specifically focused on fossa strictures; most information relating to these strictures are a secondary thought to more common strictures of the bulbar urethra . It has been reported that, in general, strictures involving the fossa have three distinct etiologic factors: inflammatory, procedure - related, or catheter - induced . Inflammatory conditions include balanitis xerotica obliterans (bxo), lichen sclerosis, and an inflammatory variant of vitiligo [1, 36]. Procedure - related causes are most commonly associated with transurethral resection of the prostate, which has a reported rate of approximately 2.6% . However, we could find no information suggesting that a larger caliber resectoscope was responsible for a greater risk of fossa strictures . The incidence of urethral stricture following cardiovascular surgery in the early 1980s prompted much investigation and was thought to arise from catheter toxicity and urethral ischemia [9, 10]. However, these strictures usually affected the anterior urethra and none solely involved the fossa navicularis . Abdel - hakim and elhilali [11, 12] found some evidence for urethral ischemia in stricture cases utilizing strain gauge plethysmography to determine penile - brachial indexes . In addition, nacey demonstrated a significantly increased incidence of urethral strictures following catheterization with silastic catheters compared to silicone catheters in a controlled randomized prospective study of patients undergoing elective cardiac surgery . Other experimental and clinical studies demonstrated that latex catheters were more toxic than non - latex catheters [1418]. As noted above, there were essentially no published reports of fossa strictures following rlp and very little information regarding its etiology . We initially looked at factors such as age, medical diseases, bmi, ebl, operative length, among others . Although ebl was noted to be significantly higher in stricture - free men, its association with stricture formation is unclear since the ebl between the two different catheter groups was essentially the same . With no obvious factor we were concerned that the electrical current leaving the monopolar tip might potentially be transmitted down the catheter in the urethra and injure the fossa . In response to this potential source of injury, the grounding pad was moved from the upper thigh (i.e., adjacent to the penis) to the thorax . However, the fossa strictures continued . We next turned to the possibility that the 22 f caliber of the urethral catheter might be responsible . At case number 35 we had switched from our standard 18 f catheter to a 22 f caliber in an attempt to avoid inadvertent stapling of the urethra . At case number 166 remarkably, our data suggest and continued experience supports the data that the 22 f silastic urethral catheter is the likely etiology . The use of a 22 f silastic urethral catheter during the intra - operative dissection would appear to have increased the risk for fossa stricture 20-fold relative to the 18 f catheter . However, definitive empirical proof that the larger sized catheter is the etiological factor is absent; it may be that in addition to catheter size, the combination of extreme trendelenburg position and the pneumoperitoneum associated with rlp are other contributing factors . This combination of factors may account for the lack of reports on similar stricture experiences among the large volume of articles published on rlp and cystectomy . The mean time to fossa the strictures were not equally distributed over time, with the majority of strictures diagnosed by postoperative day 50 . We do not have an explanation why some men developed strictures after one month and others after 3 months . As table 1 demonstrates, there were no other obvious differences between the two groups . Two of ten strictures (both in the 22 f group) subsequently required open reconstruction, whereas 80% resolved with soft dilations continued over a 3- to 6-month interval following the development of the fossa stricture . In summary, an 18 f catheter is sufficient to drain the bladder safely . A larger urethral catheter size during the intra - operative dissection in rlp appears to increase the risk for fossa navicularis stricture.
Similar experience is seen in malaysia with breast cancer being the most common cancer among women . Advances in detection and treatment modalities have improved the survival rate of women with breast cancer . In malaysia, the 5-year survival rate among breast cancer patients has seen improvements over the past decades; studies revealed a 5-year observed survival from 58.4% (ci 0.540.63) to 75.7% (ci 0.730.79). Because of its high prevalence and relatively good prognosis, the increase in numbers of survivors forms a growing area of clinical interests and research . A diagnosis of cancer can alter a person's perspective on health and life itself . Female breast cancer survivors are often weighed down by issues of physical lethargy, pain, breast sensitivity, and difficulty to concentrate which were associated with diminished physical functioning and emotional well - being . Their psychological well - being was found to be affected by fear of cancer spread, recurrence, distress from surgery, fear of second cancer, and future tests . Cancer survivors face a wide range of problems during and after their primary treatment which often persists in a chronic, long - term manner . During this period of survivorship, these survivors have multitude of needs which require attention and identification . Recognizing these needs early in the cancer care continuum therefore, needs assessment should be carried out as it offers three advantages: (i) patient's perceived needs are directly assessed, (ii) the level of need can be identified as well, and (iii) individuals or patient subgroups with higher level of needs can be identified . Furthermore, understanding unmet needs among cancer survivors, across different age groups, gender, regions, cancer types, stages, survival durations, and various other factors, is in line with making healthcare patient - centered . With the growing number of breast cancer survivors in the milieu of limited healthcare resources, there is a pressing demand to uncover the unmet needs of these survivors so that health systems may prioritize its service delivery in order to be more effective and efficient . Therefore, this study aims to describe the prevalence of unmet needs among breast cancer survivors in kuching, sarawak, and to assess relationship between their unmet needs and various associated factors . This descriptive cross - sectional study was conducted among breast cancer survivors recruited from the community - based nongovernmental organization (ngo) sarawak breast cancer support group (sbcsg) in kuching, sarawak . Prior permission was obtained from the center in writing and all survivors in the support group were invited to participate in the study . Written informed consent was obtained prior to the interview assisted survey, which was conducted at the center from january 2014 to june 2014 . This community - based approach, in which respondents were recruited, ensured that breast cancer survivors, whether they received treatment from public, private, or mixture of health facilities, were captured in this study . The supportive care framework by fitch recognizes that about 20% of cancer patients within the healthcare system have unmet needs . Recent studies showed that cancer patients in taiwan and hong kong had prevalence of supportive care needs between 1733% and 1846%, respectively . Therefore, in order to estimate the 20% prevalence of unmet supportive care needs (moderate to high needs) with a confidence interval of 95% and a 10% margin of error, an estimate sample size of 62 was required as per the equation of n = zpq / d, where z = 1.96, p = 0.2, q = 1 p, and d = 0.1 . The eligibility criteria for inclusion in the study were (i) adult malaysian females aged 18 years and above, (ii) diagnosed with breast cancer (all stages), and (iii) physically and mentally able to participate . Sociodemographic characteristics of respondents such as age, ethnicity, marital status, cohabitation status, household income, education, and employment status were included in the survey . Medical characteristics such as age at diagnosis, duration of survivorship, cancer stage at time of diagnosis, and current treatment status were collected as well . The 34-item short - form supportive care needs survey (scns - sf34) was adapted and employed in this study to identify the unmet supportive care needs among survivors . Respondents have reported their preference for the scns questionnaire over other health - related quality of life questionnaires . Furthermore, the scns - sf34 maintained the same constructs which were (i) psychological, (ii) physical and daily living, (iii) sexuality, (iv) health system and information, and (v) patient care and support, while preserving the psychometric properties of the original long - form scns . Validation studies have shown that the cronbach's alpha of the english version was 0.860.96 . The 34-item supportive care needs survey (scns - sf34) offered an assessment of needs in cancer patients via five analytically derived domains: (i) psychological (10 items), (ii) physical and daily living (5 items), (iii) sexuality (3 items), (iv) health system and information (11 items), and (v) patient care and support (5 items). For each item, the respondents were required to indicate their level of need for help over the past one month in relation to having cancer . The level of need would be scored on a five - point likert scale: 1, no need, not applicable; 2, no need, satisfied; 3, low need; 4, moderate need; and 5, high need . For each item, the respondents are dichotomized to as having moderate to high level of need if they scored options 4 or 5 or no to low level of need if they scored options 1, 2, or 3 . The items with highest percentage of respondents reporting the domain score was obtained by summing up the responses to each of the items within the domain and dividing the sum by the number of items in the domain . For the purpose of analysis, the sociodemographic and medical characteristics of respondents were organized and compared with the supportive care needs domain mean scores using independent t - test and one - way analysis of variance (anova). The collected data was then entered and analyzed using the ibm statistical product and service solutions (spss) statistics program version 20 . Inferential statistics were generated to answer the study objective based on p value of less than 0.05 (p <0.05). This study was conducted with approval from the medical ethics committee, faculty of medicine and health science, universiti malaysia sarawak (unimas), malaysia [unimas / tnc(aa)-03.02/06 - 11 jld . A total of 101 female breast cancer survivors participated in this study . The average age of respondents was 57.9 (sd 9.53) years . Most of the respondents stay with 2 to 4 other persons at home (56.4%) and have an average household income of between rm 3001 to rm 5000 (43.6%). Majority of the respondents were unemployed (70.3%) which include survivors who were housewives and retired from employment . The mean age at first diagnosis of breast cancer among the respondents in this study was 49.7 (sd 8.45) years with 54.5% of the respondents diagnosed at the age 50 years and older . Nearly two - thirds (63.4%) of the respondents had a survival duration of more than 5 years . Approximately 80% of the respondents had early stage (stages i and ii) breast cancer at time of diagnosis . Among the respondents, 72.3% were no longer undergoing any active treatment at time of study and were only on annual outpatient department follow - ups . The other information on the sociodemographic and medical characteristics of the respondents is depicted in table 1 . Domain mean scores are identified by summing up the responses to each of the items within the domain and dividing the sum by the number of items in the domain . A higher score (maximum 5.00, minimum 1.00) would indicate higher level of needs in the domain . Table 2 revealed that health systems and information domain was found to have the highest mean score (2.48; 95% ci, 2.322.64), followed by psychological domain (2.01; 95% ci, 1.912.12) and patient care and support domain (1.93; 95% ci, 1.832.03). Meanwhile, the sexuality domain has the lowest mean score (1.57; 95% ci, 1.441.70). The top 10 items that respondents have indicated a moderate to high level of need for help are as depicted in table 3 . Overall, 9 out of the top 10 items of unmet needs were from the health systems and information domain with only one item from the psychological domain . The highest ranked items were having one member of hospital staff with whom you can talk about all aspects of your condition, treatment, and follow - up (34.7%), being given explanations on those tests about which you would like to get explanations (29.7%), and having access to professional counseling (e.g., psychologist, social worker, counselor, and specialist nurse) if you, family, or friends need it (27.7%). The ten items with lowest moderate to high level unmet needs among the respondents are as shown in table 4 . Overall, the listed items had only less than 5% of the respondents indicated moderate to high level of need for help . Five out of 10 of these items are from the psychological domain, followed by sexuality domain (2 items), patient care and support domain (2 items), and physical and daily living domain (1 item). Items with the lowest percentage of respondents reporting needs were feeling down or depressed (1%), feelings of sadness (1%), changes in sexual feelings (1%), changes in your sexual relationships (1%), and more choice about which hospital you attend (1%). The supportive care needs (scns) domain mean scores are compared to the sociodemographic and medical characteristics of the respondents in table 5 . Respondents age below 60 years (n = 57, 56.4%) reported significantly higher mean score across physical and daily living domain (p = 0.009), psychological domain (p = 0.002), sexuality domain (p <0.001), and patient care and support domain (p = 0.038) compared to survivors aged 60 years and older (n = 44, 43.6%). Ethnic malays and sarawak indigenous group recorded higher mean score in the physical and daily living domain (p = 0.017) and sexuality domain (p = 0.028) compared to the chinese respondents . Survivors who are married reported higher mean score in the sexuality domain (1.69, 95% ci: 1.541.84) compared to those who are never married / widowed / divorced / permanently separated (1.11, 95% ci: 0.971.25) (p <0.001). Respondents with primary education level reported a significantly lower domain mean score compared to those with postsecondary or tertiary education level across the domains of physical and daily living (1.71 versus 2.19, p = 0.012), sexuality (1.33 versus 1.95, p = 0.007), patient care and support (1.76 versus 2.21, p = 0.014), and health system and information (2.37 versus 3.00, p = 0.035). Respondents who were unemployed indicated a higher mean score in both the psychological domain (2.18 versus 1.94, p = 0.042) and the patient care and support domain (2.13 versus 1.85, p = 0.034) compared to those who were still employed . Meanwhile, when comparing the scns domain mean scores to the medical characteristics and disease stages of the respondents in table 5, significantly higher mean scores were reported by respondents with survival duration of 5 years or less compared to those who have survived more than 5 years in the physical and daily living domain (p <0.001), psychological domain (p <0.001), sexuality domain (p = 0.019), and patient care and support domain (p = 0.008). Treatment status among respondents showed significant difference in mean scores across all 5 domains with survivors undergoing active treatment indicating a higher mean score compared to those who are not undergoing any current active treatment (p value range from <0.001 to 0.019). There were no significant differences in supportive care needs domain mean scores among respondents with various cohabitation statuses, household income level, and age at diagnosis and between respondents who had early stage cancer compared to those with late stage cancer . The present study is a cross - sectional analysis of the perceived needs among breast cancer survivors in kuching, sarawak, malaysia . The survivors recruited were generally less than 60 years old, were mainly chinese, were married, received at least secondary level education, and were unemployed (table 1). The age at diagnosis (mean age 49.7) and staging (early stage, 80.6%) were closely similar to various studies done in malaysia . The mean duration of survivorship which was 8.2 years and was higher in this study compared to 6.7 years in another local study; this could be attributed to the fact that there were more later stage cases recruited from the hospital - based medical records in that study . The current study (table 2) revealed that survivors indicated a higher mean score (higher scores representing a higher level of unmet needs in the domain, range 1.005.00) in the health system and information domain (2.48, 95% ci: 2.322.64), followed by psychological domain (2.01, 95% ci: 1.912.12), and patient care and support domain (1.93, 95% ci: 1.832.03). Meanwhile, sexuality domain was ranked lowest (1.57, 95% ci: 1.441.70). This finding is consistent with recent research among breast cancer survivors in singapore, hong kong, and korea which ranked the health system and information domain with the highest mean score . This tendency towards informational needs has been studied and established, with recent systematic reviews suggesting that asian women reported higher informational needs compared to western women . Furthermore, the current finding could suggest a dissatisfaction among survivors with information received during follow - up care . Based on individual item ranking (table 3), there is a greater need expressed within the health system and information domain compared to the other domains such as psychological domain . These findings however were in contrast to multiple australian - based research outcomes, in which cancer survivors ranked the psychological domain as area with highest level unmet needs [11, 19]. Such difference in prevalence has been established in recent systematic review and could be a reflection of underlying cross - continental cultural difference between survivors of asian compared to australian origin . Researchers discovered a shift in perceived needs from informational needs to psychological needs among cancer survivors, citing improvements in information delivery over the years, which have led to the shift . Assuming this trend holds true for cancer survivors worldwide, the current trend in this study indicates much needs to be done in addressing the informational needs of our survivors . Nevertheless, a less emphasis on psychological domain in this study sample could be influenced by the fact that the study population was from a community - based breast cancer support group, in which their psychological and emotional needs would probably have been addressed . This finding highlights the importance of proper support group among cancer survivors, as well as having breast cancer survivors as support members . Younger respondents aged below 60 years old reported higher level of unmet needs across all 5 domains compared to their older counterparts (table 5), with significant differences seen in the domains of sexuality, psychological, physical and daily living, and patient care and support . This observation is congruent with studies by researchers in singapore, which demonstrated that the level of need is higher among survivors aged below 60 years . Similarly australian cancer patients aged 3160 years endorsed higher levels of unmet needs in sexuality, psychological, patient care and support, and health system and information domains compared to those who are 70 years or older . Comparable conclusions were drawn from a qualitative study which explored the psychosocial needs of breast cancer survivors in which younger women reported a higher level of needs in terms of support, psychological, practical, physical and information needs . Such disparity may reflect the difference in attitudes between younger adults who are more vocal of their unmet needs than older adults who believe that they should have better coping capacity and thus keep things to themselves . Additionally, a diagnosis of cancer in younger adults may impart a sense of perceived loss in all areas of life, such as physical, psychosocial, emotional, sexuality, and the need for support, which give rise to higher magnitude of needs compared to older adults who may well have adjusted to changes in their life as they age . There were, however, no significant differences (p = 0.643) in mean scores within the health system and information domain which may suggest that, across all age group, the informational need is ever present once a person is diagnosed with cancer . Significant difference in mean domain scores was found between malays and sarawak indigenous groups compared to ethnic chinese survivors in both the physical and daily living (2.18 versus 1.84, p = 0.017) and sexuality (1.81 versus 1.48, p = 0.028) domains . This variation could be influenced by the fact that almost 75% of the respondents in this study were of ethnic chinese group, and the number of malays and sarawak indigenous groups were inadequate to be representative . In the absence of adequate research information, this finding is inconclusive . There may be an element of cultural background and variation in health beliefs which influence the level of needs in these domains . Further exploration into this area is needed to understand these variations . Comparison with other studies involving ethnic chinese survivors revealed similar lower scores in sexuality domain [9, 14] and physical and daily living domain . Lower sexuality domain scores could be due to underreporting of sexuality needs, as it is regarded as an intimate matter and not for discussion within the asian chinese community . Specific cultural norms among the chinese could also account for lower physical and daily living needs, as it is believed that personal problems or outcry for help reflects badly on the family and may bring shame to the family name; therefore, this may lead to a lower report for need in this domain among the chinese - majority sample of this study . Higher level of unmet needs in the sexuality domain was endorsed by survivors who are married compared to others (p <0.001). This was consistent with a korean study whereby married patients (p = 0.03) were significantly more likely to indicate the need for help in this domain . Furthermore, this finding makes intuitive sense, given that married survivors have spouse in whom they need to confront their sexuality needs more frequently, hence giving rise to needs . Higher education attainment was associated with greater level of unmet needs among the respondents (table 5) with mean domain scores being significantly different between respondents with primary level compared to postsecondary or tertiary level education in psychological, sexuality, patient care and support, and health system and information domains . This finding is in agreement with other studies which concluded that education level was found to be predictor of higher level of unmet needs among breast cancer survivors [25, 26]. Survivors with higher level of education are more aware and receptive of their underlying conditions, this has led to their need to actively seek ways to improve their current state of health, and the barriers which they face would be reflected in the domain scores . Current study revealed that survivors who were unemployed reported a higher domain mean score across all 5 domains compared to those who were employed (table 5) with significant differences reported in the psychological and patient care and support domains . Could be complications from treatment of breast cancer with residual deficits, which would hamper their ability to work and thus be reported as unemployed . Such disadvantage may increase their needs in the psychological and patient care and support domain . In this study, shorter duration of survivorship was significantly associated with higher domain mean score across all supportive care needs domains except the health system and information domain (table 5). This is in agreement with recent systematic review which suggests shorter time since diagnosis predicts higher level of needs . These findings can be attributed to the fact that newly diagnosed cancer patients had greater physical and emotional needs compared to those who were already receiving posttreatment follow - up care . This universal observation illustrates the need for concerted effort at addressing unmet needs across various domains early in cancer survivorship . Furthermore, independent of survival duration, informational needs among current survivors are persistent throughout the continuum of survivorship care with no differences seen between younger or older survivors . This could be an indication highlighting the gap in information flow between providers and patients . Closing this gap, a systematic approach in information delivery across the period of survivorship with focus on different aspect of needs and its change in relation to survival duration being under active treatment is significantly associated with higher mean scores across all 5 domains in this study (p value range <0.001 to 0.019) (table 5). This finding is consistent with other studies whereby patients under treatment reported high level of unmet needs while those in remission reported fewer unmet needs in the psychological, information, daily living, and patient care domains . Additionally, recent findings among breast cancer survivors showed that those who were not in remission were associated with unmet needs in health system and information and patient care and support domains suggesting that these groups of survivors are likely to be receiving intermittent treatment and had to deal with symptom management . Greatest difference in mean scores was observed in the physical and daily living domain (2.33 versus 1.77, p <0.001). This difference can be attributed to the fact that survivors receiving active treatment were still coping with physical side effects of treatment modalities such as surgery and chemotherapy and hence had much greater need in this domain compared to those who were not on any active treatment any more . There was no significant difference in mean scores across all 5 domains in relation to cohabitation status of the survivors (p = 0.0970.866) (table 5). This finding is different from other studies which concluded that generally cancer survivors living alone reported higher level of unmet needs, and breast cancer survivors living alone reported significantly more needs in area of patient care domain (p = 0.044). This could be influenced by the fact that the sampled survivors were recruited from a community - based breast cancer support group, and these survivors being part of this group have had some of their needs satisfied . Household income level did not show any significant difference in any of the 5 domains mean scores . This finding differs from a korean study which concluded that less income predicted higher needs in physical and daily living domain (p = 0.019). It was suggested that having less income leads to financial barriers in terms of ability to satisfy daily personal care and physical needs . Majority of these survivors receive their initial management and follow - up care from the public funded government health facilities which are considered to be relatively free, and therefore the income level within the current healthcare system does not make much impact . Current study revealed that, in relation to cancer stage at time of diagnosis, there was no significant difference in mean scores across all domains (p value range 0.627 to 0.964). This however was different from other studies which reported that tumor size was significant in predicting needs in the psychological and health system and information domains . Nevertheless, this study suggests that, independent of cancer staging at time of diagnosis, there are still unmet needs to be addressed, with highest mean score in the health system and information domain, early stage (2.48, 95% ci: 2.302.66) and later stage (2.42, 95% ci: 1.982.85). The present finding may highlight a lack of understanding among survivors about the differences of cancer staging which also is indirectly reflected in the high level of unmet needs in the health system and information domain . On the other hand, there may be some form of underlying passive coping mechanism in play, whereby survivors choose to keep themselves occupied and not to think too much about their condition; therefore the differences in staging (early or later state) do not weigh in on their unmet needs mean scores . First, it was conducted among survivors in a community - based support group, whereby participation was based on voluntary basis and representation from late stage (stages iii and iv), that survivors may be less than expected . This could be attributed to the fact that late stage survivors have shorter life span and might not be in the best health to participate regularly in support group activities . Second, the chinese - majority ethnic representation in this study, despite not being the majority population in the country, concurs with other local studies and characterizes the higher breast cancer incidence rate among the chinese in malaysia . Third, the inherent limitation of being a cross - sectional study means that cause - effect relationships could not be assessed, and changes in unmet needs could not be evaluated over time . Despites these limitations, this study has its strength in the fact that samples were drawn from community - dwelling breast cancer survivors which is more likely to represent their needs in the actual lived - in environment compared to samples obtained purely from hospitals . In conclusion, this study provided valuable insights into the characteristics of breast cancer survivors in kuching, sarawak, and their associated unmet supportive care needs with gaps in the informational domain emphasized . Efforts should include the systematic delivery of health information which is targeted, culturally sensitive, and linguistically appropriate, especially to breast cancer survivors who were younger, had higher education attainment, were unemployed, had survivorship of 5 years or less, and were undergoing active treatment . Future research should recruit samples from both community - based support group and survivors receiving treatment . A longitudinal exploration of the supportive care needs relative to survival duration is also recommended.
We have recently described mixed ti / m oxo clusters with divalent metals m (ca, sr, zn, cd). These clusters have a basic hexanuclear structure that varies according to the size and coordination number of the m ions.1 in another series of ti / m oxo clusters with divalent metals (m = sr, pb), crown ether type structures were obtained.2 the clusters were prepared by the reaction of ti(or)4 with the corresponding metal acetate [m(oac)2] and methacrylic acid (mcoh). The carboxylic acid not only provides carboxylate ligands [equation], but also acts as an in situ source of water through its esterification with the eliminated alcohol [equation (1b)]. Methacrylic acid was initially used to obtain carboxylate - substituted metal oxo clusters that can subsequently polymerize to yield hybrid materials.3 it turned out, however, that methacrylic acid is particularly well suited to obtaining crystalline clusters . We therefore used methacrylic acid in this work as well, although no subsequent polymerizations were intended . The charges on the metal atoms as well as the total number of coordination sites must be balanced by the ligands to obtain a stable cluster.4 for this reason, clusters of different compositions are expected if the metal charge is varied . Carboxylate - substituted tim oxo clusters with trivalent metals are only known for y. in previous work, three y / ti clusters of the general composition ti4y2o4(omc)12x2l2 (x = -omc or och2ch2ome; l = meoch2ch2oh or mcoh) were obtained upon treating ti(oipr)4 and y(och2ch2ome)3 with methacrylic acid, among them y2ti4o4(omc)14(homc)2 (y2ti4).5 because the variance of the ion size of trivalent rare - earth ions is much smaller than that of the divalent metals mentioned above, much more detailed information on the dependence of ion size of a specific cluster structure can be expected . The mixed ti / ln clusters were prepared according to [equation (1c)]. Size - dependent structural changes have been found, for example, for ln(omes)3 (omes = 2,4,6-trimethylphenolate), with the dinuclear species ln2(omes)6(thf)4 with two bridging omes ligands observed for the larger ions la and nd, whereas the mononuclear compounds ln(omes)3(thf)3 were found for sm, tb, er, yb and y.6 the metal atoms are six - coordinate in both structures . A few ln / ti oxo / alkoxo clusters are known, but none of them contains ligands other than oxo and alkoxo groups . The structure of k3eu3tio2(otbu)11(ome / oh)(tbuoh) is based on a k3eu3o octahedron capped by a k3tio tetrahedron on the k3 face,7 and the metal atoms in sm4tio(oipr)148 and (tb0.9er0.1)4tio(oipr)149 form a trigonal bipyramid with an encapsulated 5-oxygen atom . Although the work reported here involves only structural issues, the mixed ln / ti clusters could be interesting precursors for mixed - oxide materials.10 in the course of this work we found that y2ti45 is also formed when y(oac)3 is used as the y precursor instead of y(och3ch3ome)3 . Isomorphous and isostructural clusters were obtained from lanthanide acetates that have ln ion radii similar to that of y (1.109). Centrosymmetric clusters ln2ti4o4(omc)14(homc)2 (ln2ti4, figure1) were thus synthesized from ln(oac)3 (ln = sm, eu, gd, ho), ti(oipr)4 and methacrylic acid with the ratio of ln(oac)3/ti(oipr)4 ranging from 2:1 to 1:2 . The bond lengths and angles of sm2ti4, eu2ti4, gd2ti4 and ho2ti4 (as well as y2ti4) are almost the same; therefore, only those of eu2ti4 are discussed exemplarily . The ln atom in the ln2ti4 structures shows positional disorder with 66:34 occupancy, eu1a is shifted relative to the eu1 position by 0.247(7). Selected bond lengths [] and angles []: eu1o1 2.319(3), eu1o1 2.457(4), eu1o2 2.491(3), eu1o3 2.368(3), eu1o5 2.464(3), eu1o7 2.330(4), eu1o9 2.341(4), eu1o11 2.382(4), ti1o1 1.718(2), ti1o2 2.022(2), ti1o4 2.019(2), ti1o6 1.958(2), ti1o8 1.992(2), ti1o13 2.128(2), ti2o2 1.730(2), ti2o10 1.987(3), ti2o12 1.981(3), ti2o14 1.991(3), ti2o15 2.140(2), ti2o17 1.974(3); eu1o1eu1 111.06(11), eu1o1ti1 108.87(12), eu1o1ti1 133.59(12), eu1o2ti1 93.46(10), eu1o2ti2 126.19(12), ti1o2ti2 137.75(12). The basic structural motif of ln2ti4 is a zigzag chain of two central [lno8] dodecahedra and two terminal [tio6] octahedra (ti1) that share edges . Two additional [tio6] octahedra (ti2) are condensed onto the main chain at both ends of the zigzag chain through shared corners . Thus, ti1 is bonded to two 3-oxygen atoms, with ln, ln and ti1 (denotes symmetry - related atoms) are connected through o1 and ln, ti1 and ti2 connected through o2 . The oxygen atom o1 in eu2ti4 is slightly unsymmetrically located between the two eu atoms [eu1o1 2.319(3), eu1o1 2.457(4)]; the distance of eu1 to the other 3-oxygen atom (o2) is only slightly lengthened [eu1o2 2.491(3)]. The ti o distances of the oxygen atom that connects ti2 to the main chain [ti2o2 1.733(3)] are in the same range as that of ti1o1 [1.718(2)], whereas ti1o2 is significantly lengthened [2.022(2)]. In the case of ln2ti4, the total metal charge is + 22, and the number of coordination sites is 40, assuming six - coordinate ti atoms and eight - coordinate ln atoms . Thus, 14 monoanionic ligands are necessary to balance the charges of the ln2ti4o4 core, and 28 coordination sites must still be occupied . Apparently this is not possible for steric reasons, and two omc ligands coordinate only in an manner; to occupy all the available coordination sites two neutral mcoh ligands are additionally coordinated to the same ti atom (ti2). Both interact with each other through a strong hydrogen bond [o16o18 2.454(8) in eu2ti4]. Because the ti2o15 distance is significantly longer than that of ti2o17 [ti2o15 2.140(2), ti2o17 1.974(3)] we pointed out earlier that the combination of an and a neutral proton - donating ligand connected through a hydrogen bond (scheme 1) is structurally equivalent to a monoanionic bidentate ligand.11 due to the octahedral coordination of ti, chelating carboxylate ligands are extremely rare . -carboxylate ligand stabilized by a coordinated rcooh (left, as in ln2ti4) or roh molecule (right, as in ln2ti6). Ln1 and ti1 as well as ti1 and ti2 are bridged by one omc ligand, while ln1 and ti2 as well as ln1 and ti1 are bridged by two omc ligands . Clusters with the composition ln2ti6o6(omc)18(hoipr)2 (ln2ti6) were obtained (ln = nd, ce, la) with a larger ln ion radius (figure2). This type of cluster was also obtained for ln = sm (sm2ti6) when the reaction mixture with a ti / sm precursor ratio of 2:1 was heated at 80 c . The centrosymmetric clusters la2ti6, ce2ti6, nd2ti6 and sm2ti6 are again isomorphous and isostructural; the structural parameters will therefore only be discussed for la2ti6 . In this cluster, the ln atoms also show positional disorder with a 85:15 occupancy and an la1la1a distance of 0.27(3). Selected bond lengths [] and angles []: la1o1 2.538(3), la1o1 2.552(3), la1o2 2.526(3), la1o3 3.045(3), la1o4 2.447(4), la1o6 2.482(4), la1o8 2.496(4), la1o10 2.545(4), la1o12 2.458(4), ti1o1 1.705(3), ti1o2 1.987(3), ti1o5 2.004(3), ti1o7 1.977(3), ti1o9 1.990(3), ti1o14 2.185(3), ti2o2 1.743(3), ti2o3 1.976(3), ti2o11 1.954(3), ti2o15 1.974(3), ti2o16 2.023(3), ti2o18 2.101(3), ti3o3 1.742(3), ti3o13 1.944(4), ti3o17 2.010(4), ti3o19 2.011(4), ti3o20 1.924(4), ti3o22 2.158(4); la1o1la1 112.93(12), la1o1ti1 107.50(14), la1o1ti1 131.54(15), la1o2ti1 99.27(12), la1o2ti2 116.61(15), ti1o2ti2 142.62(17), la1o3ti2 90.89(11), la1o3ti3 130.38(16), ti2o3ti3 130.7(2). The structures of ln2ti6 and ln2ti4 are based on the same central structural element, namely a zigzag chain of two central [lno8] dodecahedra and two terminal [tio6] octahedra (ti1) that share edges . Four additional [tio6] octahedra are condensed onto this ln2ti2 core in ln2ti6, however, instead of the two in ln2ti4 . Two of the four [tio6] octahedra (ti2) share edges with the [lno8] dodecahedra, and the other two (ti3) share a corner with the polyhedra of ln and one of the ti atoms . The metal atoms are connected through six 3-oxygen atoms, one (o1) connecting ln, ln and ti1, the second ln, ti1 and ti2, and the third ln, ti2 and ti3 . The la1o3 distance is relatively long [3.045(3) compared with la1o1 2.538(3), la1o1 2.552(3), la1o2 2.526(3)], and therefore it can be debated whether o3 is a 3-oxygen atom with a very long la o distance (and nine - coordinate ln) or a 2-oxygen atom with a short la each ti atom has one very short bond to a core oxygen atom [ti1o1 1.705(3), ti2o2 1.743(3), ti3o3 1.742(3)], but the ti1o2 and ti2o3 bonds are more than 0.2 longer [ti1o2 1.987(3). Ti2o3 1.976(3)]. The same calculation as above shows that 18 mononegative bidentate ligands are required to balance the 30 positive charges of the metal atoms and the 36 remaining coordination sites of the ln2ti6o6 core (again assuming six - coordinate ti atoms and eight - coordinate ln atoms). As discussed above, and for the same reason, two of the omc ligands (at ti3 and ti3) are only -coordinated and are stabilized by a hydrogen - bonded neutral ligand [oo 2.653(7)]. This is hoipr (o22) in the case of ln2ti6 (scheme 1, right). The long ti3o22 distance of 2.158(4) indicates that the hydrogen atom is closer to the oipr group . Each of the other omc ligands in ln2ti6 bridges two metal atoms . The lanthanide ion and each ti atom of the same asymmetric unit are bridged by one omc ligand and by two omc ligands to ti1 of the other asymmetric unit [la1o4, la1o10 2.545(4), la1o12 2.458(4), ti1o5 2.004(3), ti2o11 1.954(3), ti3o13 1.944(4), ti1o7 1.977(3), ti1o9 1.990(3)]. The reaction of ln(oac)3 (ln = la, ce), ti(oipr)4 and methacrylic acid with a higher proportion of ti(oipr)4 than that used for the preparation of la2ti6 or ce2ti6 resulted in the formation of lnti4o3(oipr)2(omc)11 (lati4 and ceti4, figure3). The clusters are isomorphous and isostructural, and therefore the bond lengths and angles will only be discussed for lati4 . Crystals of lnti4 contain two independent clusters in the asymmetric unit (atom labels la1/ti1ti4 and la2/ti5ti8, respectively). One of them shows disorder of the central ln atom and one ti atom (70:30 occupancy). La2a is shifted by 0.262(5) from the original la2 position towards ti8, which is also disordered [ti8ti8a 0.412(8)]. This also affects the omc ligands bridging la2/ti8 and ti5/ti6, as well as the terminal oipr group on ti8 and the second oipr group on ti5 . Selected bond lengths [] and angles []: la1o1 2.6410(18), la1o2 2.4793(17), la1o3 2.6228(18), la1o4 2.5455(18), la1o6 2.447(2), la1o8 2.5447(19), la1o10 2.558(2), la1o12 2.533(2), la1o14 2.548(2), ti1o1 1.9688(18), ti1o5 1.927(2), ti1o7 1.977(2), ti1o16 2.038(2), ti1o18 2.051(2), ti1o26 1.782(2), ti2o1 1.7904(18), ti2o2 1.8249(18), ti2o9 1.981(2), ti2o17 2.056(2), ti2o19 2.019(2), ti2o20 2.0826(19), ti3o2 1.8409(18), ti3o3 1.7786(18), ti3o11 1.975(2), ti3o21 2.084(2), ti3o22 2.041(2), ti3o24 2.035(2), ti4o3 1.9702(19), ti4o13 1.925(2), ti4o15 1.938(2), ti4o23 2.029(2), ti4o25 2.046(2), ti4o27 1.774(2); o1la1o2 60.33(6), o2la1o3 60.40(6), la1o1ti1 126.53(8), la1o1ti2 100.45(7), ti1o1ti2 132.00(10), la1o2ti2 105.50(7), la1o2ti3 105.34(8), ti2o2ti3 149.13(10), la1o3ti3 101.80(8), la1o3ti4 123.24(8), ti3o3ti4 132.39(10). Contrary to the structures of ln2ti4 and ln2ti6, the cluster core of lnti4 contains only one ln atom, which is surrounded by a semicircle of four [tio6] octahedra (figure3). The la distances to the two terminal, edge - sharing ti atoms ti1 and ti4 are much longer than those to the central, face - sharing ti atoms [la1ti1 4.1282(6), la1ti4 4.0530(6), la1ti2 3.4489(5), la1ti3 3.4571(7)]. Each of the three 3-o atoms link two [tio6] octahedra to the central ln atom . All the metal atoms and core oxygen atoms are nearly coplanar . Although the ti o bond lengths of the two central ti atoms ti2 and ti3 are the shortest in the structure [ti2o1 1.790(2), ti2o2 1.825(2), ti3o2 1.841(2), ti3o3 1.779(2)], those of ti1 and ti4 are significantly longer [ti1o1 1.969(2), ti4o3 1.970(2)]. The same applies for la, with la1o2 [2.479(2)] about 0.2 shorter than la1o1 [2.641(2)] and la1o3 [2.623(2)]. The coordination polyhedron can be described as a square - faced monocapped antiprism (saprs-9).12 the terminal ti atoms ti1 and ti4 are additionally coordinated by four bridging omc ligands, two of which connect ti1 and ti4 to the ln atom and the other two to the central ti atoms ti2 and ti3 . Ti2 and ti3 are additionally connected to each other by an omc bridge and to ln by one bridging omc ligand each . The latter ligands are arranged on opposite sides of the plane of the cluster core, almost perpendicular to the plane . 2.545(2), la1o10 2.558(2), ti2o9 1.981(2), ti3o11 1.975(2)]. The structure can also be derived from the previously described clusters sr2ti8o8(oipr)2(oac)2(omc)16 and pb2ti8o8(obu)2(omc)18(buoh)2, in which the two central sr / pb atoms are surrounded by a ring of eight ti atoms . Lnti4 can be seen as half of the sr / ti or pb / ti cluster . Alternatively, it may be considered half of the ln2ti6 cluster core, in which an additional [tio6] octahedron is condensed to the ln atom instead of there being a dimerization of the lnti3 unit by condensation of two [lno8] polyhedra . The cluster structures described in this article show a clear correlation with the ionic radii of the trivalent metal atom (see table1). Clusters with the composition ln2ti4o4(omc)14(homc)2 (ln2ti4) were formed with the smaller ions (ion radii 1.021.08), and clusters with the composition ln2ti6o6(oipr)2(omc)16(homc)2 (ln2ti6) were formed with the larger ions (ion radii 1.081.16 for coordination number 8). Sm with a radius of 1.08 is a borderline case as both clusters can be formed . The increasing ion radii are also reflected in the increasing m o bond lengths . The comparison shows that small variations in the ion radii of the second metal may result in different structures of the m / ti mixed - metal clusters . The larger ions allow coordination of an additional [tio6] unit (with concomitant partial rearrangement of the ligand sphere). An increase in the ion radii is of course closely associated with the ability to increase the coordination number of the metal . Although the ln atom in ln2ti4 is clearly eight - coordinate, that in ln2ti6 is between eight- and nine - coordinate (eight stronger ln o bonds and one weak interaction). This is also seen for the alternatively formed clusters lati4o3(oipr)2(omc)11 with the largest ln ions (lati4 and ce2ti4) in which the coordination number of the ln atom is nine . An interesting side aspect of this work is that no acetate and only methacrylate ligands are incorporated into the clusters . General: all experiments were carried out under ar by using standard schlenk techniques . Y(oac)3xh2o, la(oac)3xh2o, ce(oac)3xh2o, eu(oac)3xh2o, sm(oac)3xh2o and gd(oac)3xh2o, were obtained from aldrich . The acetates were dried in a vacuum chamber at 130 c overnight, and the removal of water was monitored by ir spectroscopy . All solvents used for nmr spectroscopy (eurisotop) were degassed prior to use and stored over molecular sieves . H and c nmr spectra in solution were recorded with a bruker avance 250 spectrometer [250.13 mhz (h), 62.86 mhz (c)] equipped with a 5 mm inverse - broadband probe head and a z - gradient unit . Ir spectra were recorded with a bruker tensor 27 spectrometer under ambient conditions with 32 scans at a resolution of 4 cm on a diamond atr unit . X - ray crystallography: crystallographic data were collected with a bruker axs smart apex ii four - circle diffractometer with geometry at 100 k by using mo - k (= 0.71073) radiation . The data were corrected for polarization and lorentzian effects, and an empirical absorption correction (sadabs) was employed . Saint plus software (bruker analytical x - ray instruments, 2007) was used to integrate the frames . The symmetries were then verified by using the platon program.13 the structures were solved by charge flipping (jana2006). Refinement was performed by the full - matrix least - squares method based on f (shelxl9713) with anisotropic thermal parameters for all non - hydrogen atoms . Hydrogen atoms bound to carbon atoms were inserted at calculated positions and refined by using a riding model . Hydrogen atoms bound to oxygen atoms were identified on the difference electron density map, and the o crystal data, data collection parameters and refinement details are listed in tables 2, 3 and 4 . Although all structures of the ln2ti4 clusters could be refined to satisfying values despite the disorder described before, there was still some unresolved electron density . The distances of this peak to any of the atoms or other unresolved electron density maxima do not fit with any chemical entity . When the data were cut at lower degrees, the electron density of this maximum decreased . It was nevertheless observed in all x - ray experiments on the ln2ti4 clusters, although different crystals were investigated . Such a residual electron density was not mentioned in the literature for the isostructural y2ti4.5 therefore, the cluster was prepared again with y(oac)3 as precursor . Hence the unresolved electron density in the ln2ti4 clusters is most probably a feature related to the ln ions and not caused by some disorder or partial substitution of ligands . Examination of the diffraction pattern showed only separated and well - defined reflections . As was observed for both lati4 and ceti4, this appears to be a feature of this structure . Ccdc-1014141 (for sm2ti4), -ccdc-1014142 (for eu2ti4), -ccdc-1014143 (for gd2ti4), -ccdc-1014144 (for ho2ti4), -ccdc-1014145 (for la2ti6), -ccdc-1014146 (for ce2ti6), -ccdc-1014147 (for nd2ti6), -ccdc-1014148 (for sm2ti6), -ccdc-1014149 (for lati4) and -ccdc-1014150 (for ceti4) contain the supplementary crystallographic data for this paper . These data can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif . [a] = 1/[(fo) + (xp) + yp], in which p = (fo + 2fc)/3 . [a] = 1/[(fo) + (xp) + yp], in which p = (fo + 2fc)/3 . [a] = 1/[(fo) + (xp) + yp], in which p = (fo + 2fc)/3 . General synthetic procedure: ti(oipr)4, the corresponding water - free metal acetate and an excess of methacrylic acid were mixed . Y2ti4o4(omc)14(homc)2 (y2ti4): y(oac)3 (1 mmol, 0.266 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (13.5 mmol, 1.162 g). Ir: $\tilde {\nu}$= 2979 (w), 2961 (w), 2928 (w), 1697 (w), 1643 (w), 1563 (s), 1454 (m), 1385 (s), 1368 (s), 1232 (s), 1007 (m), 938 (m), 852 (w), 826 (s), 760 (s), 654 (m) cm . Sm2ti4o4(omc)14(mcoh)2 (sm2ti4): sm(oac)3 (2 mmol, 0.955 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (18 mmol, 1.55 g). The solution was stirred for 1 d. pale - yellow crystals were isolated after 2 weeks . The same cluster was obtained when sm / ti / mcoh molar ratios of 2:1:13.5 and 1:2:13.5 were employed . H nmr (cd2cl2, 250 mhz): = 1.521.80 (m, 6 h, ch3), 1.97 (br . S, 30 h, ch3), 2.192.70 (m, 12 h, ch3), 5.225.58 (m, 5 h, ch2), 5.70 (br ., s, 10 h, ch2), 5.886.05 (m, 3 h, ch2), 6.25 (br . S, 10 h, ch2), 6.487.09 (m, 4 h, ch2), 8.67 (br . Ir: $\tilde {\nu}$= 2978 (w), 2927 (w), 1698 (w), 1643 (w), 1562 (s), 1454 (m), 1364 (s), 1231 (s), 1122 (w), 1007 (m), 937 (m), 851 (m), 825 (m), 739 (s), 656 (w), 615 (m) cm . Eu2ti4o4(omc)14(homc)2 (eu2ti4): eu(oac)3 (1 mmol, 0.329 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (13.5 mmol, 1.162 g). The solution was stirred for 1 d. colourless crystals of eu2ti4 were isolated after 1 week . The cluster was also obtained when eu(oac)3 (1 mmol, 0.329 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and heated at about 80 c for 2 h, followed by cooling to room temperature and addition of methacrylic acid (13.5 mmol, 1.162 g). Ir: $\tilde {\nu}$= 2979 (w), 2927 (w), 1697 (w), 1643 (w), 1560 (s), 1454 (m), 1384 (s), 1367 (s), 1232 (s), 1006 (m), 937 (m), 851 (w), 826 (s), 754 (s), 653 (w), 601 (s) cm . Gd2ti4o4(omc)14(homc)2 (gd2ti4): gd(oac)3 (1 mmol, 0.334 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (13.5 mmol, 1.162 g). The solution was stirred for 1 d. pale - orange crystals were isolated after 1 week . This cluster was also obtained with gd / ti ratios of 1:1 and 2:1 in the precursor mixture . Ir: $\tilde {\nu}$= 2979 (w), 2927 (w), 1698 (w), 1664 (w), 1644 (w), 1561 (s), 1454 (m), 1384 (s), 1367 (s), 1232 (s), 1129 (w), 1006 (m), 937 (m), 852 (w), 825 (s), 758 (s), 653 (m), 601 (s) cm . Ho2ti4o4(omc)14(homc)2 (ho2ti4): ho(oac)3 (1 mmol, 0.342 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (13.5 mmol, 1.162 g). This cluster was also obtained with ho / ti ratios of 1:1 and 2:1 in the precursor mixture . Ir: $\tilde {\nu}$= 2979 (w), 2961 (w), 2929 (w), 1697 (w), 1643 (w), 1561 (s), 1530 (s), 1455 (m), 1388 (s), 1371 (s), 1231 (s), 1006 (m), 942 (m), 868 (m), 851 (w), 829 (s), 761 (m), 658 (m), 601 (s) cm . La2ti6o6(omc)18(hoipr)2 (la2ti6): la(oac)3 (2 mmol, 0.632 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (18 mmol, 1.550 g). The same cluster was obtained with an la / ti ratio of 2:1 in the precursor mixture . H nmr (cd2cl2, 250 mhz): = 1.171.28 (m, 12 h, ch3, hoipr), 1.622.09 (m, 54 h, ch3, omc), 4.02 (sept ., 2 h, ch, hoipr), 5.295.67 (m, 18 h, = ch2, omc), 5.896.23 (m, 18 h, = ch2, omc) ppm . C nmr (cd2cl2, 250 mhz): = 17.618.0 (ch3, omc), 25.0 (ch3, hoipr), 64.364.6 (ch, hoipr), 127.4 (ch2, omc), 138.4 (c, omc), 172.8 (coo, omc) ppm . Ir: $\tilde {\nu}$= 3098 (w), 2976 (w), 2957 (w), 2927 (w), 1697 (w), 1643 (w), 1563 (s), 1543 (s), 1503 (w), 1453 (m), 1385 (s), 1363 (vs), 1327 (m), 1232 (s), 1121 (w), 1045 (w), 1007 (m), 940 (m), 849 (w), 827 (m), 807 (m), 745 (s), 656 (m), 612 (s) cm . Ce2ti6o6(omc)18(hoipr)2 (ce2ti6): ce(oac)3 (1 mmol, 0.317 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (13.5 mmol, 1.162 g). The same cluster was also obtained with ce / ti ratios of 1:1 and 2:1 in the precursor mixture . H nmr (cd2cl2, 250 mhz): = 1.171.28 (m, 12 h, ch3, hoipr), 1.622.08 (m, 54 h, ch3, omc), 4.01 (sept, 2 h, ch, hoipr), 5.335.68 (m, 18 h, = ch2, omc), 6.056.22 (m, 18 h, = ch2, omc) ppm . C nmr (cd2cl2, 250 mhz): = 17.6 (ch3, omc), 25.0 (ch3, hoipr), 64.467.8 (ch, hoipr), 124.4127.3 (ch2, omc), 136.0138.9 (c, omc), 172.5 (coo, omc) ppm . Ir: $\tilde {\nu}$= 2975 (w), 2927 (w), 1698 (w), 1645 (m), 1601 (m), 1577 (s), 1516 (m), 1454 (m), 1414 (s), 1385 (s), 1365 (s), 1235 (m), 1162 (w), 1123 (m), 1104 (m), 936 (m), 857 (s), 825 (m), 741 (m), 659 (m) cm . Nd2ti6o6(omc)18(hoipr)2 (nd2ti6): nd(oac)3 (1 mmol, 0.321 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (13.5 mmol, 1.162 g). Ir: $\tilde {\nu}$= 2976 (w), 2927 (w), 1697 (w), 1643 (w), 1532 (s), 1541 (s), 1454 (m), 1363 (s), 1329 (s), 1230 (s), 1122 (w), 1007 (w), 939 (m), 849 (w), 828 (m), 808 (m), 739 (s), 657 (m) cm . Sm2ti6o6(omc)18(hoipr)2 (sm2ti6): sm(oac)3 (1 mmol, 0.327 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and heated at 80 c for 2 h. the mixture was cooled to room temperature, and then methacrylic acid (13.5 mmol, 1.162 g) was added . H nmr (cd2cl2, 250 mhz): = 1.191.34 (m, 12 h, ch3, hoipr), 1.69 (s, 3 h, ch3, omc), 1.762.36 (m, 51 h, ch3, omc), 4.604.80 (m, 2 h, ch, hoipr), 5.345.92 (m, 18 h, = ch2, omc), 6.316.80 (m, 18 h, = ch2, omc) ppm . Ir: $\tilde {\nu}$= 2973 (w), 2926 (w), 1697 (w), 1641 (w), 1594 (w), 1527 (s), 1454 (s), 1403 (s), 1389 (s), 1367 (s), 1241 (m), 1206 (w), 1047 (w), 1024 (w), 1006 (m), 932 (m), 852 (w), 832 (m), 692 (m), 659 (w) cm . Lati4o3(oipr)2(omc)11 (lati4): la(oac)3 (1 mmol, 0.316 g) was mixed with ti(oipr)4 (2 mmol, 0.568 g) and methacrylic acid (13.5 mmol, 1.162 g). The solution was stirred for 1 d. dark yellow crystals were isolated after 2 weeks . H nmr (cd2cl2, 250 mhz): = 1.191.28 (m, 12 h, ch3, oipr), 1.622.08 (m, 33 h, ch3, omc), 4.02 (sept, 2 h, ch, oipr), 5.295.68 (m, 11 h, = ch2, omc), 5.906.13 (m, 11 h, = ch2, omc) ppm . Ir: $\tilde {\nu}$= 2978 (w), 2928 (w), 1680 (w), 1640 (w), 1542 (s), 1453 (m), 1403 (s), 1385 (s), 1366 (m), 1326 (m), 1231 (m), 1208 (w), 1053 (w), 1006 (w), 936 (m), 863 (w), 829 (m), 759 (w), 675 (m), 656 (m) cm . Ceti4o3(oipr)2(omc)11 (ceti4): ce(oac)3 (1 mmol, 0.317 g) was mixed with ti(oipr)4 (6 mmol, 1.705 g) and methacrylic acid (28 mmol, 2.712 g).
Percutaneous nephrostomy (pcn) and percutaneous nephrolithotomy (pnl) are established minimally invasive renal interventions . Improvement in understanding of vascular and renal calyceal anatomy, refinement of the techniques and technology, and increasing experience in the last two decades have led to increased safety and efficacy of these procedures . However, complications still do happen; perioperative hemorrhage is one of the most dreadful complications . Fortunately, most of the hemorrhage is venous in origin and conservative measures are adequate . Arterial bleeding, although rare, needs angioembolization with a high success rate of 93 - 95% . Recurrent hemorrhage after angioembolization is unusual and is mostly because of failure to demonstrate bleeding vessels at initial angiography or due to migration of embolizing material . We encountered unusual findings of bleeding from new lesions away from initial intervention site on repeat angiography with recurrent bleeding after initial angioembolization . Clinical, microbiological, and radiological findings of all the patients who underwent renal angiography for post - renal biopsy, post - pcn, or post - pnl bleed for a period of 5 years were reviewed . Preoperative renal function, presence of any coagulopathy, renal diversion if any, microbiological status of urine, site of puncture, number of puncture, per - operative bleeding complications, postoperative hematuria requiring blood transfusion, need of embolization, recurrence of bleeding, and their management and outcome were analyzed . Totally 29 patients underwent angiography for post - percutaneous renal intervention bleeding and 28 of them required embolization of the bleeding vessels; post - embolization check film confirmed successful control of hemorrhage . In one patient, initial angiography did not demonstrate any vascular lesion and the bleeding site . Six patients had recurrence of bleeding and hematuria for which they underwent repeat angiography . In two patients, repeat angiography revealed bleeding from the site of previous puncture; in one of them, bleeding was due to migration of embolizing material and in the second patient previous angiography had not revealed the bleeding site . In the remaining four patients, recurrence of bleeding was due to new pseudoaneurysms located well away from the site of pcn / pnl puncture and the site of previous bleeding [figures 1 - 3]. Suitable antibacterial and antifungal agents (fluconazole) were administered and selective embolization of the bleeding vessel was performed in view of continued bleeding . None of them had recurrence of bleeding during follow - up [table 1]. Patient 1: left renal angiogram digital substraction (dsa) shows pseudoaneurysm (arrow) near the inferior pole of kidney, in relation to lower division of anterior middle segmental artery (a). Post - embolization check dsa shows no filling of the pseudoaneurysm (hollow arrow) (b) patient 1: repeat left renal angiogram (dsa) following recurrence of bleeding shows appearance of multiple small peripherally placed aneurysms (arrows) in relation to the anterior middle segmental artery (a). Post - embolization check dsa shows (hollow arrow) successful embolization (b) repeat left renal angiogram in patient 3 shows multiple peripheral aneurysms (arrows). Note the coils of previous successful embolization (hollow arrow). (b) shows multiple aneurysms (thin arrows) and av fistula (bold arrow) in relation to upper pole . Note previous embolized vessel in (hollow arrow) relation to lower pole pcn site demographic profiles and clinical details of the patients pcn has become an increasingly acceptable therapeutic alternative, both for high - risk patients and for patients in whom the procedure offers delay or avoidance of major surgery . Lower incidence of major morbidity, early palliation of symptoms, and convalescence are its major advantages . Pnl is a relatively safe and reliable operative technique even for large, multiple, and staghorn renal calculi . However, this invasive procedure is associated with a complication rate of 3 - 18% . One of the most serious complications is renal hemorrhage with a transfusion rate of 3 - 23% . Venous bleeding can be controlled with conservative measures, such as clamping the nephrostomy, hydration and diuretics, hemostatic medications, and balloon tamponade . Arterial injury can cause severe intraoperative bleeding and may lead to formation of pseudoaneurysm or arteriovenous (av) fistula later . Their rupture can cause delayed bleeding and in the majority (more than 90% of cases) can be managed by superselective embolization . This is mostly because of failure to demonstrate some bleeding vessels at initial angiography, as was observed in one of our cases . Use of papaverine during angiography has reduced the chances of missing any injured artery because of spasm . Recurrent bleeding is almost always from lesions at the site of previous renal puncture . Finding recurrence of bleeding from aneurysms located away from the site of puncture is unusual . All of our four patients in whom recurrence of bleeding was detected from aneurysm located away from the site of intervention had initial successful embolization of the bleeding artery documented on post - embolization check angiography . All these patients had single puncture during the procedure, and therefore does not explain the development of new lesions well away from the site of intervention . Despite extensive search of english literature, we are intrigued by noticing such incidences and have tried to find explanations for the same . All these patients had some common findings: chronic kidney disease (ckd; baseline serum creatinine 2.5 - 5.0 mg / dl)prolonged urinary diversion (with pcn in three and dj stent in one)polymicrobial urinary tract infection (escherichia coli in two, pseudomonas in two, and proteus in one)candiduria (in three patients)none of them had recurrence of bleeding after repeat angioembolization and treatment with antibiotic and antifungal agents . Chronic kidney disease (ckd; baseline serum creatinine 2.5 - 5.0 mg / dl) prolonged urinary diversion (with pcn in three and dj stent in one) polymicrobial urinary tract infection (escherichia coli in two, pseudomonas in two, and proteus in one) candiduria (in three patients) none of them had recurrence of bleeding after repeat angioembolization and treatment with antibiotic and antifungal agents . Patients with chronic renal failure (ckd) are known to have coagulopathy and are prone to have more bleeding after any surgery . It has been shown that the risk of peri - operative bleeding increases with the degree of renal insufficiency . Patients with advanced renal insufficiency [estimated glomerular filtration rate (gfr) of <40 ml / min] have a sixfold increase in the risk of severe bleeding postoperatively, and even those with mild levels of renal insufficiency (estimated gfr of 61 - 80 ml / min) have a twofold increase in the risk of serious postoperative bleeding as compared to patients with normal renal function, after adjusting for coagulation status and platelet function . However, ckd per se is not known to cause vascular lesions like pseudoaneurysm and av fistula . Moreover, in our patients, these lesions developed acutely after initial angiography . Polymicrobial colonization of urinary tract of our patients can be attributed to both ckd and prolonged diversion with dj stent / pcn tube . Infection can occur in several ways: (1) lodgment of septic emboli, most commonly at points of branching, sites of rapid tapering, on sharp bends in a normal vascular tree, and with resultant destruction of the media and weakening of the arterial wall; (2) lodgment of bacteria in vasa vasorum or on diseased intima, such as atherosclerotic plaques, with resultant weakening of the wall; (3) contiguous spread from a localized area of inflammation or infection, with destruction of the arterial wall and formation of a pseudoaneurysm; and (4) injury to an artery, with concomitant contamination and subsequent formation of an infected pseudoaneurysm . Once the infection is established, progression depends mainly on the virulence of the organism and the rapidity of destruction of the arterial wall . Mycotic aneurysms have a very high incidence of rupture, with life - threatening hemorrhage as a direct consequence . All our patients had predisposing conditions for formation of mycotic aneurysm: longstanding foreign body (pcn / dj stent), resistant polymicrobial infection, fungal colonization, and immune - compromised status . However, in all these patients, no vascular lesions were seen other than at the site of puncture during the first angiography and angioembolization . All of them had rapid development (4 - 9 days) of small, often multiple, eccentric, peripherally placed pseudoaneurysms away from the site of previous puncture after first embolization . Whether introduction of micro - organism during angiographic intervention (injection site, catheters, contrast or embolizing materials) was additionally the cause of these aneurysms is difficult to comment upon . Hence, in the light of above discussion, there is a strong possibility of mycotic aneurysms developing in the contaminated system with immuno - compromised patient . After second embolization, these patients received long course of bactericidal and fungicidal antimicrobials and did not have recurrence of hemorrhage . The limitation of this study was lack of histopathologic or direct microbiological evidence in favor of our infective or therefore, the diagnosis of mycotic aneurysm remains only circumstantial and logical, thus speculative . Nevertheless, despite these limitations, we do believe that in such high - risk infective setting, possibility of mycotic aneurysm should be considered and timely introduction of appropriate antimicrobials is prudent . Development of multiple aneurysms at sites away from punctures in patients with ckd following percutaneous renal intervention is very unusual . Its causation including infection with bacteria and fungus and resultant angiopathy needs to be explored.
Natural contamination of cockroaches with wide range of pathogenic organisms including about 40 species of bacteria, nearly 12 species of pathogenic helminthes, the second largest group of vertebrate pathogens, and also viruses, protozoa and fungi affecting man and other vertebrate animals have been reported by numerous studies (le guyader et al . 1997, cochran, 1999, eggleston and arruda 2001, savoldelli and luciano 2005). Often their movement between waste and food materials led to acquire, carry, and mechanically transfer of these pathogens . As proven or suspected carries, cockroaches play a prominent role in caring and distributing of organisms causing ddiarrhea, dysentery, cholera, leprosy, plague, typhoid fever, and viral diseases such as poliomyelitis are carries by cockroaches . They also carry the parasitic worms such as taenia, shistosoma, ascaris and may cause allergic reactions, including dermatitis, itching, swelling of the eyelids, and more serious respiratory conditions (stankus et al . 1990, savoldelli and luciano 2005). The brown - banded cockroach, supella longipalpa known previously as s. supellectilium as a nearly cosmopolitan cockroach, has recently become a hygiene problem in the city of ahvaz, southwestern iran and it seems to be the dominant species of dwellings particularly in apartments (vazirianzadeh et al . This is small cockroach measuring 1014 mm in length with pronotum of male is rather uniformly dark with lateral edges and definitely lacks of the two parallel stripes of blatella germanica . The adult males appear to be very slender with its wing extending beyond the tip of the abdomen . The common name derives from presence of two dark - colored transverse bands on mesonotal and abdominal terga (cochran 1999). 1989), and is a vector of pathogenic bacteria in urban environments (rivault et al . It is also reported as an allergen source (eggleston and arruda 2001, savoldelli and luciano 2005). Twenty nine bacterial species were isolated from s. longipalpa caught in the hospital (le guyader et al . Its movement from one department to the others, inside the hospital increases potential bacterial contamination risks, for some of the species such as acinetobacter and pseudomonas are dangerous for some kind of patients (le guyader et al . The cockroach infestations are common particularly in dwellings without proper ventilation in warm climates, hospitals, and restaurants and in business establishments with a relatively high ambient temperature and humidity (schal et al . The brown - banded cockroach needs nearly much less relative humidity to complete its life cycle and spread the infestation . It may be the main reason for more distribution of this cockroach species in ahwaz city, as a warm area, in recent years compared with same species, the german cockroach . Conventional insecticides are used as main tool to control cockroach infestations but there are many concerns about the harmful side - effects of these chemical compounds . Also the insecticide use is restricted in places such food preparation areas, restaurants, storage buildings and apartments . These restrictions of chemical insecticide application increase demand for safer alternatives against cockroach infestations (savoldellis and suss 2005, phillips and appel 2010). Different level of resistance to many compounds of chemical insecticides including organochlorine, organophosphorus and carbamat insecticides have been documented in many field - collected strains of cockroaches from iran . So, application of these insecticides should be stopped and replaced with other safer compounds (nasirian et al . Essential oils, as secondary plant compounds responsible for the aromatic characteristics of plants, present the potential alternative to conventional insecticides (isman 2000, 2006, omara et al . Plant extracts and essential oils are reported to have a wide range of activity against insect and mite pests, plant pathogens, fungi and nematodes (isman 2006). Recent reports have highlighted antimicrobial, antifungal, anti - cancer and insecticidal properties of plant essential oils (isman 2000, 2006). They have fumigants, antifeedant and repellent effects as well as inhibiting the reproduction in cockroaches and other insects (omara et al . The repellent effect of essential oils has been reported against many insect pests such as cockroaches, termites, mosquitoes, ticks, ants and houseflies (chen et al . Numerous studies have demonstrated the toxicity and repellency of essential oils against cockroaches (ahmad et al . 2010, phillips and appel 2010, zhu et al . 2012, manzoor et al . Consider to cockroaches role in transmission and distribution of many human pathogens, their resistance to many chemical insecticides, the side - effects of insecticide usage in human dwellings, high distribution of the brown - banded cockroach in ahvaz city during recent years, and eventually in order to finding a safe alternative for chemical insecticides, the present study was done to evaluate toxicity and repellency of following five essential oils, eucalyptus oil (eucalyptus sp), mint oil (mentha piprita), yarrow oil (achillea millefolium), oregano oil (origanum vulgare) and rosemary oil (rosmarinus officinalis) against this cockroach species . The brown - banded cockroaches were reared in plexiglas containers and maintained at 272 c, 505% rh, and the photoperiod of 12:12 (l: d) h. they were fed with dry crumbled biscuits, bread and water . The study was done in the laboratory of medical entomology, department of medical entomology and vector control, ahvaz jundishapure university of medical sciences during april 2012 to september 2013 . Five essential oils including, eucalyptus sp (eucalyptus oil), mentha piprita (mint oil), achillea millefolium (yarrow oil), origanum vulgare (oregano oil) and rosmarinus officinalis (rosemary oil) used in the tests were extracted from fresh or dried plant foliage by the hydrodistillation method using clevenger apparatus in herbal and natural product research center of ahvaz jundishpapur university of medical science . Plant material was placed in a 2-liter round bottomed flask with distilled water (100 ml for 75 g dry material and 400 ml for 200 g fresh material) and the essential oil was extracted by water distillation . The distillation period was 1 h for fresh samples and 1 h 15 min for dried samples (charles and simon 1990). Contact toxicity: the bioassay method of who (world health organization) (1975) was used to determine susceptibility or resistance of the cockroaches to essential oils . Essential oils were prepared in acetone as the solvent (v / v) at concentrations of 2.5%, 5%, 10%, 15% and 30% . Glass jars (600 ml) were treated uniformly with 2.5 ml of each concentration and left under room conditions to dry . The top inner surfaces of jars were smeared with a thin layer of butter to restrict cockroach movement within the jars . Groups of thirty 3 and 4 nymph instars were anesthetized by chilling and transferred to plastic cups . After recovery, they were transferred to treated jars and left to continuous exposure to treated surface . Fumigant toxicity: groups including thirty 3 and 4 nymphs instars were released in 1-lit glass jars with a 1-cm diameter cotton ball treated with 50 l pure essential oil . To prevent direct contact of cockroaches with essential oil, repellency test: the method applied that of ferrero (2007) and manzoor (2012) with some modifications . Circular white filter paper no 1 (15 cm diameter, whatman) was divided in to two approximately equal pieces . One half was treated with 1 ml essential oil solution using a micro sampler and the other half was left untreated . The oils were assayed at the following 5 concentrations: 2.5, 5, 10, 15 and 30% (v / v). After solvent evaporation, filter paper was used to cover the floor of cylindrical plexiglas jars . Thirty nymphs (3 and 4 instars) were released into the center of each jar and distribution of the nymphs was calculated 24 h after exposure . For the control group, one half of the filter paper was treated with acetone and the other half was left untreated . Means of mortality percentages and standard errors in contact and fumigant toxicity were calculated using spss 16 software . Repellency values (rv) were determined using this formula: repellency (%) = 100(t100)/n where t stands for the number of cockroaches located in the treated area and n stands for the total number of cockroaches used (thavara et al . Analyses by anova and comparison of mortality and repellency percentage means was done by tukey's test (p <0.05), using spss software (chicago, il, usa). The brown - banded cockroaches were reared in plexiglas containers and maintained at 272 c, 505% rh, and the photoperiod of 12:12 (l: d) h. they were fed with dry crumbled biscuits, bread and water . The study was done in the laboratory of medical entomology, department of medical entomology and vector control, ahvaz jundishapure university of medical sciences during april 2012 to september 2013 . Five essential oils including, eucalyptus sp (eucalyptus oil), mentha piprita (mint oil), achillea millefolium (yarrow oil), origanum vulgare (oregano oil) and rosmarinus officinalis (rosemary oil) used in the tests were extracted from fresh or dried plant foliage by the hydrodistillation method using clevenger apparatus in herbal and natural product research center of ahvaz jundishpapur university of medical science . Plant material was placed in a 2-liter round bottomed flask with distilled water (100 ml for 75 g dry material and 400 ml for 200 g fresh material) and the essential oil was extracted by water distillation . The distillation period was 1 h for fresh samples and 1 h 15 min for dried samples (charles and simon 1990). Contact toxicity: the bioassay method of who (world health organization) (1975) was used to determine susceptibility or resistance of the cockroaches to essential oils . Essential oils were prepared in acetone as the solvent (v / v) at concentrations of 2.5%, 5%, 10%, 15% and 30% . Glass jars (600 ml) were treated uniformly with 2.5 ml of each concentration and left under room conditions to dry . The top inner surfaces of jars were smeared with a thin layer of butter to restrict cockroach movement within the jars . Groups of thirty 3 and 4 nymph instars were anesthetized by chilling and transferred to plastic cups . After recovery, they were transferred to treated jars and left to continuous exposure to treated surface . Fumigant toxicity: groups including thirty 3 and 4 nymphs instars were released in 1-lit glass jars with a 1-cm diameter cotton ball treated with 50 l pure essential oil . To prevent direct contact of cockroaches with essential oil, repellency test: the method applied that of ferrero (2007) and manzoor (2012) with some modifications . Circular white filter paper no 1 (15 cm diameter, whatman) was divided in to two approximately equal pieces . One half was treated with 1 ml essential oil solution using a micro sampler and the other half was left untreated . The oils were assayed at the following 5 concentrations: 2.5, 5, 10, 15 and 30% (v / v). After solvent evaporation, filter paper was used to cover the floor of cylindrical plexiglas jars . Thirty nymphs (3 and 4 instars) were released into the center of each jar and distribution of the nymphs was calculated 24 h after exposure . For the control group, one half of the filter paper was treated with acetone and the other half was left untreated . Means of mortality percentages and standard errors in contact and fumigant toxicity were calculated using spss 16 software . Repellency values (rv) were determined using this formula: repellency (%) = 100(t100)/n where t stands for the number of cockroaches located in the treated area and n stands for the total number of cockroaches used (thavara et al . Analyses by anova and comparison of mortality and repellency percentage means was done by tukey's test (p <0.05), using spss software (chicago, il, usa). Concentrations of 30% and 15% of the five essential oils caused 100% mortality against the cockroach nymphs using continuous exposure method . Mortality rates were 100%, 62.2%, 45%, 36.2% and 5.2% respectively at the concentration of 2.5% for the essential oils of rosemary, oregano, yarrow, eucalyptus and mint after 24 h (table 1) which were significantly different (p <0.0001). Furthermore, significantly difference was noted between effectiveness of the essential oils with comparison of the total means of mortality (p <0.0001) against the brown - banded cockroach . Contact toxicity of essential oils against supella longipalpa at different concentrations at 24 h after recovering period . (department of medical entomology, ahvaz jundishapure university of medical sciences during april 2012 to september 2013 rosemary oil was the most toxic oil against s. longipalpa because it caused 100% mortality of the cockroach nymphs at all concentrations (table 1). The next most effective oils were oregano and eucalyptus oils because they killed 100% of the nymphs at the concentration range of 530% . At the concentration of 2.5% nymph mortality means were reduced to 62.2% and 36.2% with oregano and eucalyptus oils respectively which showed significantly difference (p <0.0001). So, oregano oil was more effective than eucalyptus oil for the brown - banded cockroach (table 1). Mortality means of the cockroach nymphs varied from 45% to 100% by yarrow oil which were significantly different (p <0.0001). Mint oil caused 100% mortality at concentrations range of 1030% but mortality reduced to 24.7% and 5.2% at the treatments of 5% and 2.5% respectively . So, mint oil was clearly determined as the least effective oil against the cockroach at lower concentrations compared to the other tested oils but its effect was similar to that of other oils at higher concentrations . All nymphs in the control group remained live 24 h after exposure, and even after a week . Fumigation of all the five essential oils caused 97.2% to 100% mortality in the brown - banded cockroach nymphs at 24 h after exposure or even earlier . Mint oil exhibited lower fumigant effect compared to the other essential oils and it caused a mean mortality rate of 97.2% in nymph population that was not significantly different with the other oils (p> 0.05). All tested essential oils showed high repellency against the brown - banded cockroach at the prepared concentrations (table 2). The highest and the lowest repellent effects were recorded in oregano oil and eucalyptus oil respectively compared with the control group . Repellent effect of oregano oil at different concentrations ranged from 96.5 to 99.1% which were not significantly different together (p> 0.05), while the highest repellency (99.1%) was observed at the lower concentration of 2.5% (table 2). In the repellency test with oregano oil, nearly all the released cockroaches were concentrated at the same place in untreated area and they were not close to the treated area even after a week . Repellency effects of plant essential oils against the brown - banded cockroach, s. longipalpa at 24 h after recovery period (department of medical entomology, ahvaz jundishapure university of medical sciences during april 2012 to september 2013 . Eucalyptus oil was determined as the least repellent compound, it caused 27.749.8% repellency at different concentrations against the brown - banded cockroach compared to the other oils . Its repel effects were a little significantly different (p <0.05) at different concentrations . Mint oil caused 59.168.8% repellency against the brown - banded cockroach and that repellency of this oil at lower concentration (2.5%) was not significantly different with higher concentrations (p> 0.05). Repellent effect of yarrow oil was varied from 79.3 to 92.8% that was not significantly different (p> 0.6). The most repellent activity was related to the concentration of 5% that was 92.8% but decreased to 79.3% at the concentration of 2.5% while the different between repellency at the concentration of 2.5% and 5% was not significantly (p= 0.149). The repellent effect of rosemary oil was tested with only two concentrations . While the highest effect was recorded at the lower concentration of 2.5% (94.3%) but it was not differ significantly with the concentration of 5% (86.2%). The difference was significant between the repellency of the essential oils with control group (p <0.0001). Concentrations of 30% and 15% of the five essential oils caused 100% mortality against the cockroach nymphs using continuous exposure method . Mortality rates were 100%, 62.2%, 45%, 36.2% and 5.2% respectively at the concentration of 2.5% for the essential oils of rosemary, oregano, yarrow, eucalyptus and mint after 24 h (table 1) which were significantly different (p <0.0001). Furthermore, significantly difference was noted between effectiveness of the essential oils with comparison of the total means of mortality (p <0.0001) against the brown - banded cockroach . Contact toxicity of essential oils against supella longipalpa at different concentrations at 24 h after recovering period . (department of medical entomology, ahvaz jundishapure university of medical sciences during april 2012 to september 2013 rosemary oil was the most toxic oil against s. longipalpa because it caused 100% mortality of the cockroach nymphs at all concentrations (table 1). The next most effective oils were oregano and eucalyptus oils because they killed 100% of the nymphs at the concentration range of 530% . At the concentration of 2.5% nymph mortality means were reduced to 62.2% and 36.2% with oregano and eucalyptus oils respectively which showed significantly difference (p <0.0001). So, oregano oil was more effective than eucalyptus oil for the brown - banded cockroach (table 1). Mortality means of the cockroach nymphs varied from 45% to 100% by yarrow oil which were significantly different (p <0.0001). Mint oil caused 100% mortality at concentrations range of 1030% but mortality reduced to 24.7% and 5.2% at the treatments of 5% and 2.5% respectively . So, mint oil was clearly determined as the least effective oil against the cockroach at lower concentrations compared to the other tested oils but its effect was similar to that of other oils at higher concentrations . All nymphs in the control group remained live 24 h after exposure, and even after a week . Fumigation of all the five essential oils caused 97.2% to 100% mortality in the brown - banded cockroach nymphs at 24 h after exposure or even earlier . Mint oil exhibited lower fumigant effect compared to the other essential oils and it caused a mean mortality rate of 97.2% in nymph population that was not significantly different with the other oils (p> 0.05). All tested essential oils showed high repellency against the brown - banded cockroach at the prepared concentrations (table 2). The highest and the lowest repellent effects were recorded in oregano oil and eucalyptus oil respectively compared with the control group . Repellent effect of oregano oil at different concentrations ranged from 96.5 to 99.1% which were not significantly different together (p> 0.05), while the highest repellency (99.1%) was observed at the lower concentration of 2.5% (table 2). In the repellency test with oregano oil, nearly all the released cockroaches were concentrated at the same place in untreated area and they were not close to the treated area even after a week . Repellency effects of plant essential oils against the brown - banded cockroach, s. longipalpa at 24 h after recovery period (department of medical entomology, ahvaz jundishapure university of medical sciences during april 2012 to september 2013 . Eucalyptus oil was determined as the least repellent compound, it caused 27.749.8% repellency at different concentrations against the brown - banded cockroach compared to the other oils . Its repel effects were a little significantly different (p <0.05) at different concentrations . Mint oil caused 59.168.8% repellency against the brown - banded cockroach and that repellency of this oil at lower concentration (2.5%) was not significantly different with higher concentrations (p> 0.05). Repellent effect of yarrow oil was varied from 79.3 to 92.8% that was not significantly different (p> 0.6). The most repellent activity was related to the concentration of 5% that was 92.8% but decreased to 79.3% at the concentration of 2.5% while the different between repellency at the concentration of 2.5% and 5% was not significantly (p= 0.149). The repellent effect of rosemary oil was tested with only two concentrations . While the highest effect was recorded at the lower concentration of 2.5% (94.3%) but it was not differ significantly with the concentration of 5% (86.2%). The difference was significant between the repellency of the essential oils with control group (p <0.0001). There are many studies on evaluation of essential oils against other species of cockroaches, but this is the first time to evaluate the efficacy of these components against the brown - banded cockroach, s. longipalpa . The tested essential oils showed contact toxicity, fumigant toxicity and repellent activity against the brown - banded cockroach compared to control group during this study . Results of continuous contact toxicity showed considerable effect at different concentrations, but yarrow oil was determined as the most toxic oil . Comparison contact toxicity of rosemary, oregano, yarrow, eucalyptus and mint oils at the lowest concentration (2.5%) showed mortality rates of 100%, 62.2%, 45%, 36.2% and 5.2% respectively at 24 h after treatment . These mortality rates were significantly different together and with control group and confirmed that rosemary oil was the most toxic oil against the brown - banded cockroach nymphs . The highest repellency was related to oregano oil and the most repellent activity was observed at lower concentrations of 2.5% that was 99.1% with a residual effect lasting at least a week after treatment . Oregano and rosemary oils were more efficient at the concentration of 2.5% with 99.1% and 94.5% repellency . Yarrow, mint and eucalyptus oils showed the most repellent effects at the concentration of 5% but the differences were not significant with the repel activity of 2.5% (p values were 0.15 and 0.97 and 0.99 for yarrow, mint and eucalyptus oils respectively). Repellency of these oils was 92.8%, 68.8% and 51.7% at the concentration of 5% and it was 79.3%, 63.3% and 49.8% at the concentration of 2.5% . So it could be concluded that the essential oil concentration of 2.5% is the favorite concentration to recommend for repellency against the brown - banded cockroach for further evaluation . Evaluation of repellency and fumigant toxicity of clove (syzygium aromaticum) and sesame (sesamum indicum) oils against the american cockroach (periplaneta americana) showed complete repellency (100%) against first nymph at concentration of 2% for clove oil and 6% for sesame oil . Same result was obtained against fourth nymph at concentration of 10% of sesame oil after 48 h. while clove oil completely repelled all fourth nymphs after 24 h at concentration of 8% . For adult stage, the greatest repellency percentages were recorded by clove oil (90.005.77%) and sesame oil (83.333.33%) after 48 h at a concentration of 10% (omara et al . The repellency value of clove oil at concentration of 8% against the fourth instar nymphs of the american cockroach is nearly close to our repellency value of oregano oil at concentration ranges of 2.510% which repel 96.0399.1% of the brown - banded cockroach nymphs after 24 h and event lasted for a week . Both essential oils have considerable repellency against cockroaches but the cockroach species, essential oil type led to a little difference in the obtained results . Toxicity and repellent evaluation of eucalypltus citriodora, mentha arvensis and cymbopogen citratus against p. americana . C. citratus exhibited the maximum toxicity and repellency with 20% to 100% toxicity between 2 to 24 h intervals, 100% repellency and 70100% fumigation after 24 h exposure . Minimum repellency ranged from 1167% was observed from eucalyptus oil against p. americana (manzoor et al . The minimum repellency against s. longipalpa was also recorded for eucalyptus oil in our study that ranged from 27.751.7% . Differences in the obtained result could be probably related to difference in cockroach species . In another study, the essential oil of citrus hystrix exhibited 100% repellency effect against p. americana and blatella germanica, and also about 87.5% against neostylopyga rhombifolia under laboratory conditions . The essential oil caused an 86% reduction in cockroaches with a residual effect lasting a week after treatment in the field (thavara et al . These results are also similar to repellency values of oregano oil that continued for at least a week or even 10 days after treatment . Besides, all five essential oils showed nearly complete fumigant toxicity (97.2 100%) against the brown - banded cockroach at the concentration of 50 l per 1 lit air of pure oil . (2001) investigated that fumigant toxicity of mint oil at the concentration of 50 l per 1 lit air of pure oil killed 100% of both p. americana and b. germanica after 24 h and caused 92.3100% repellent effect on both cockroach species during each day of the 14-day experiment (appel et al . Repel activity of mint oil ranged from 59.168.8% against s. longipalpa with the concentration of 2.530% and the fumigant toxicity was 97.2% at 24 h after exposure in our study . It seems that the fumigant toxicity of mint oil in the mentioned concentration is not significantly difference against the three cockroach species whereas the repellent activity shows considerable difference . Clove oil provided highly fumigant toxicity against nymphs and adults of p. americana after 24 and 48 h, respectively . Complete mortality (100%) was recorded at a concentration of 7.5 l / l of air for first nymph, 10 l for fourth one and 17.5 l for adults after 48 h of fumigation (omara et al . 2013). In the another study, the fumigant toxicity of 12 essential oil components including: carvacrol, 1,8-cineole, trans - cinnamaldehyde, citronellic acid, eugenol, geraniol, s-()-limonene, ()-linalool, ()-menthone, (+) -alpha - pinene, ()-beta - pinene, and thymol was determined against adult male, adult female, gravid female, and large, medium, and small nymphs of the german cockroach, b. germanica . 1,8-cineole was the most toxic essential oil component to adult males and females, gravid females, and large nymphs, with lc50 values of 6.8, 8.4, 5.3, and 11.4 mg / liter air at 24 h, respectively (phillips and appel 2010). Evaluation of fumigant toxicity of some plant essential oils by tunza et al . Determined that allyl isothiocyanae (monoterpen oil component) was the most toxic compound, followed by essential oil of allium sativum against b. germanica (tunza et al . While all five essential oils exhibited complete fumigant toxicity against s. longipalpa which is comparable with the findings of other studies but it is necessary to test lower concentrations . The findings of current study are confirmed by all the mentioned studies in that essential oils can be favorite alternative to conventional chemical insecticides to control cockroach infestations especially in situations where the use of chemical insecticides has many harmful side effects . Differences in the obtained results could be probably related to difference in cockroach species, type of essential oil, bioassay method and exposure time . Plant essential oils offer the potential repellent agents to cockroach infestations in hidden and hard - to - reach areas and to eliminate such infestations (steltenkamp et al . This strategy would increase the efficacy of non - repellent, insecticide treated areas because cockroaches in the hidden areas will be get out and they will exposed to such treated areas and finally killed (steltenkamp et al ., essential oils could be applied as a safe treatment and used to treat surfaces for food preparation in order to deter cockroach infestations (steltenkamp et al . 1992). Essential oil applications may also be effective around cockroach infested places that cannot be treated with chemical insecticides, such as food stores, sensitive equipment, clothes lockers and in beds (koehler et al . The repellent effects of essential oils could be applied as a flushing agent during inspections of cockroach infestation in order to determine the degree of an infestation . Given that s. longipalpa can be found in all parts of the buildings such as clothes lockers, beds, furniture and cabinets and that ootheca stick in hard - to - reach or obscure places, application of chemical insecticide spraying in such places is hard and unacceptable by the people . However, the use of essential oils presents a safe alternative for indoor application against cockroaches because of less toxicity to humans and no toxic residues . Oregano oil showed considerable repellent effect against s. longipalpa, so, it could be recommended as a potential repellent compound for further evaluation on a larger scale and under field conditions . Contact and fumigant toxicity of the five selected essential oils were considerable but additional study is required for the practical application and developing favorable formulation of them against the brown - banded cockroach and also other cockroach's species.
Patients experience preventable harm from medical errors, mistakes and teamwork failures (1). It is a guiding factor in daily care and a central issue in healthcare (3, 4). For example, in iran, it is not taught to medical personnel, traditionally (6). However, preventable adverse events are common in developed and developing countries (7). There has been a growing awareness of the measures and cost of incidence in two decades ago (8). For example, childbirth s mortality and morbidity highlight the importance of learning from events (10). It is necessary to revisit patient safety, changes, remained challenges, emerging new problems, and effect of health care in the quality and safety of healthcare (11). Patients perspective is patients beliefs and attitudes in the field of preventing and controlling errors and the risk of error occurring (13). A patient engaging with safety is the most benefit of strengthening a relationship with physicians and nurses (14). Patients misperceptions result in emerging obstacles in the environment that patients themselves have to manage (13). The patients negative perspective into hospital s patient safety may reversely conduce to incongruity in referrals and follow - up, petitions and allegations concerning jeopardizing patient s life . There are many individual, environmental and organizational factors for not accurately evaluating healthcare, quality and patient safety level in hospitals (16). Acording to the findings of australian patient safety report (2001), public hospitals survey in kerman, iran (2006), educational hospital survey in izmir, turkey (2006), patient safety survey in urmia, iran (2010), urmia patient safety survey (2012), patient safety survey in isfahan, iran (2012) and iranian patient safety survey (2012), the premier factors are as follows: age, gender, marital status, education, insurance coverage, employment status, period of referring to physician and the date of latest hospitalization (17). Even so, patients perspective have excluded in the construction of clinical governance and health - care reforms . It requires decreasing the imbalance of information and power between patients and healthcare professionals . Addressing this problem therefore, it is necessary to notify, realize, modify or remove the cause and effect of the negative perspective (18). The general aims of this study were: - to determine patient safety level in tehran university of medical sciences general hospitals from patients perspective- to determine the contributory factors on patients perspective . - to determine patient safety level in tehran university of medical sciences general hospitals from patients perspective - to determine the contributory factors on patients perspective . This study had a cross - sectional design carried out in a period of six - month from may 2011 to november 2011 . The study populations were inpatients in the clinical wards that had the experience of hospitalizing . First step: the sample size was calculated by using the following formula: n = z1-/22p(1-p)d2on the ground that no researches have been done so far, the favorable perception of involvement in treatment decisions and patient safety was considered 50% (p=0.5). The sample size for each treatment decision and perceptions of safety were calculated 180 in the first step considering to the confident interval 95% (=0.05) and maximum deviation 7.5% (d=0.075). Second step: according to clark s study (2001), the minimum r that influenced various factors was 0.6 (19). Therefore, the desirable sample size was calculated 300 . In the spring of 2011, the list of six general hospitals affiliated to tehran university of medical sciences (tums) clinical wards were prepared . The numbers of clinical wards in each hospital were 120 as follows: - imam hospital: 39 wards (icu & ccu: 13, surgery, transplant, obstetrics and gynecology: 13, internal medicine, infectious diseases, ear, nose and throat, general: 13)- baharlou hospital: 16 wards (icu & ccu: 5, surgery, transplant, obstetrics and gynecology: 5, internal medicine, infectious diseases, ear, nose and throat, general: 6)- shariati hospital: 37 wards (icu & ccu: 12, surgery, transplant, obstetrics and gynecology: 12, internal medicine, infectious diseases, ear, nose and throat, general: 13)- ziaian hospital: 13 wards (icu & ccu: 4, surgery, transplant, obstetrics and gynecology: 4, internal medicine, infectious diseases, ear, nose and throat, general: 5)- amiralam hospital: 7 wards (icu & ccu: 1, surgery, transplant, obstetrics and gynecology: 1, internal medicine, infectious diseases, ear, nose and throat, general: 5)- sina hospital: 8 wards (icu & ccu: 3, surgery, transplant, obstetrics and gynecology: 3, internal medicine, infectious diseases, ear, nose and throat, general: 2). - imam hospital: 39 wards (icu & ccu: 13, surgery, transplant, obstetrics and gynecology: 13, internal medicine, infectious diseases, ear, nose and throat, general: 13) - baharlou hospital: 16 wards (icu & ccu: 5, surgery, transplant, obstetrics and gynecology: 5, internal medicine, infectious diseases, ear, nose and throat, general: 6) - shariati hospital: 37 wards (icu & ccu: 12, surgery, transplant, obstetrics and gynecology: 12, internal medicine, infectious diseases, ear, nose and throat, general: 13) - ziaian hospital: 13 wards (icu & ccu: 4, surgery, transplant, obstetrics and gynecology: 4, internal medicine, infectious diseases, ear, nose and throat, general: 5) - amiralam hospital: 7 wards (icu & ccu: 1, surgery, transplant, obstetrics and gynecology: 1, internal medicine, infectious diseases, ear, nose and throat, general: 5) - sina hospital: 8 wards (icu & ccu: 3, surgery, transplant, obstetrics and gynecology: 3, internal medicine, infectious diseases, ear, nose and throat, general: 2). Eventually, the stratified random sampling method was used . The 120 wards were divided into three groups as: - group 1: intensive care unit (icu and ccu) (38 wards);- group 2: surgery, transplant, obstetrics and gynecology (38 wards);- group 3: internal medicine, infectious diseases, ear, nose and throat, general (44 wards).300 samples were proportionally divided between 3 groups . The numbers of samples in each group were as: - group 1: 95- group 2: 95- group 3: 110 - group 1: intensive care unit (icu and ccu) (38 wards); - group 2: surgery, transplant, obstetrics and gynecology (38 wards); - group 3: internal medicine, infectious diseases, ear, nose and throat, general (44 wards). The numbers of wards in each group were: - group 1: 5 wards- group 2: 5 wards- group 3: 6 wards . Finally, the sample was selected as follows: - group 1: imam ccu, baharlou ccu, shariati ccu, general imam icu, ziaian ccu and post ccu.- group 2: imam surgery, amiralam surgery, sina general surgery, shariati general surgery, ziaian surgery.- group 3: imam internal, amiralam internal, baharlou internal, shariati internal pulmonary, shariati internal, ziaian internal . - group 1: imam ccu, baharlou ccu, shariati ccu, general imam icu, ziaian ccu and post ccu . - group 2: imam surgery, amiralam surgery, sina general surgery, shariati general surgery, ziaian surgery . - group 3: imam internal, amiralam internal, baharlou internal, shariati internal pulmonary, shariati internal, ziaian internal . Library and internet research the questionnaire was developed by clark in 2001 (17) to assess patients opinions about patient safety issues, medical error, and event reporting . It includes 37 items in 4 sections as follows: demographic characteristics (eight questions), general information regarding the doctors and hospitals (six questions), participation in treatment decisions (12 questions) and patient safety (11 questions). The five - level likert scale was employed for the responses as follows: strongly disagree (1 score), disagree (2 score), neither (3 score), agree (4 score) and strongly agree (5 score). Therefore, the participation s level was evaluated as follows: - 1236 score: low- 3660 score: high . Therefore, the patient safety s level was as follows: - 2641 score: intermediate the questionnaire was translated into farsi . Then, both the translated questionnaire and the original one were handed to some experts in order to revise it . After that, the comprehensibility of the survey was tested on 20 patients from the study population who had not been included in our sample . The reliability coefficient of the questionnaire was calculated 0.78 . According to patients perspective, we prepared the final version of the farsi questionnaire after altering some questions and eliminating irrelevant questions . The questionnaire was developed by clark in 2001 (17) to assess patients opinions about patient safety issues, medical error, and event reporting . It includes 37 items in 4 sections as follows: demographic characteristics (eight questions), general information regarding the doctors and hospitals (six questions), participation in treatment decisions (12 questions) and patient safety (11 questions). The five - level likert scale was employed for the responses as follows: strongly disagree (1 score), disagree (2 score), neither (3 score), agree (4 score) and strongly agree (5 score). Therefore, the participation s level was evaluated as follows: - 1236 score: low- 3660 score: high . Therefore, the patient safety s level was as follows: - 2641 score: intermediate the questionnaire was translated into farsi . Then, both the translated questionnaire and the original one were handed to some experts in order to revise it . After that, the comprehensibility of the survey was tested on 20 patients from the study population who had not been included in our sample ., we prepared the final version of the farsi questionnaire after altering some questions and eliminating irrelevant questions . Participants demographic characteristics were as follows: 60% were female, and the rests (40%) were male . 20%, 23% and 20% were 2418, 4435 and 55 years - old people, respectively . The ethnicity of 32%, 24.3% and 21% was azeri, kurd and fars, sequentially . Health insurance, social security insurance and other insurances coverage were 37%, 23% and 25.7%, sequentially . Patient safety was evaluated high, intermediate and low by 60%, 14% and 26% of patients, respectively . Patient safety variables included: demographic variables (including age, gender, education, ethnicity, marital status, insurance type, employment status and income statues), period of referring to family physician or general practitioner, the date of latest medical consultation, the number of hospitalizations in the past year, the date of latest hospitalization, hospital s type, exposure to adverse events and patient participation in treatment decisions . Tables 1 and 2 show the results of linear regression and multivariate logistic regression, respectively . According to the table 1, patients socio - demographic, medical and hospitalized variables affecting on the hospital s patient safety score were as follows: age, gender, ethnicity, marital status, education, insurance coverage, employment statues, period of referring to family physician, or general practitioner, the date of latest hospitalization, the date of latest exposure to adverse events, participation in treatment decisions (p<0.1). For adjusting possible problematical factors and achieving independent factors, affecting variables imported in the final multivariate logistic regression model by a stepwise method . The most important variables affecting on the hospital s patient safety score were as follows: education, employment status and marital status . Education, employment status and marital status decreased odds ratio of dedicating high score to hospital s patient safety, 0.014, 0.32 and 0.19 times, respectively . It was meaning that the unmarried or educated or employed individuals tended to score patient safety lower than others . According to the findings, patient safety was evaluated high by the most of patients (60%) hospitalized in tums s general hospitals . Considering to their perspective, much attention has been paid to patient safety in health care, the registration and the examination of safety incidents, particularly in hospitals . It is in concordance with sheikh beiklou s survey in urmia s public and private hospitals . According to the findings, patient safety was ranked high (56%) from the patients perspective (17). Poor attention to patient safety results in errors, low quality of care, and increases the length of stay (5).there are a number of risk areas in which errors and risks are more likely to occur . Therefore, patients perspective provides a rich source of data in looking at how patient s power impacts upon safety in organizational contexts (3). Education, employment status and marital status are the premier factors affecting on evaluating patient safety in hospitals . It is in concordance with florin s survey in 2006 . According to the findings, the disparate perspective was pertinent to age, marital and social status including example education, employment (21). It is consistent with the findings of other surveys: clark in australia s private and general hospitals (19), larsson et al . (22), in kerman s public hospitals, iran (23), in izmir s educational hospitals, turkey (24), and schwappach and et al . Nowadays, the consumers are more conscious in healthcare market . Because educated people have more risk - assessment ability they rapidly conceive whether providers perform their duties, the services are concordant, effective and patient - centered, there is do it right culture in healthcare organization or not . It is consistent with the findings of other reviews: oskouiee and zare in tabriz s educational hospitals, iran (17), leventhal (25), ozdemir et al . In izmir s educational hospitals, turkey (24), and ghaffari and rakhshande in iran (16) it is consistent with the findings of other reports: clark in australia s general and private hospitals (19) and ozdemir and et al in izmir s educational hospitals, turkey (24). According to the findings of patient safety survey in urmia hospitals, mousavi et al . Emphasized that patients perspective is the most important criterion for appraising patient safety in hospitals . It is pertinent to some of patients characteristics such as marital status (26). It is because of communicating with others, increasing awareness and understanding issues such as safety . The employed or married patients inquire into disease, medical process and healthcare organizations from communicating or consulting with their colleagues, friends and family members . Patients perspective is the most important catalyst to the emergence of a safety movement in healthcare over the last decade (3). Understanding the patients perspective is a pivotal way of generating knowledge about the processes involved in harm . It must nt ignore in the adoption of a no - blame culture in patient safety . However, tums s general hospitals are enough safe from patients perspective, patient safety should be improved . The contributory factors, such as education, employment and marital status, were conclusive to claim that these factors were predicting patients perspective in safety matters . In clinical governance, contributing patients perspective to the improvement of patient safety reforms is critical in generating new models of good practice . Health care organizations can go beyond mainstream frameworks for quality and patient safety improvement by create higher value for patients perspective . This study has two research methods also have limitations: the lack of cooperation of some hospitals to do research and patients unwillingness to fill the questionnaire . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
Thalidomide has been successful use in patients with refractory crohn disease (cd) in recent years . We collected the data of a postoperative cd patient who was prescribed thalidomide to induce remission and reviewed the relevant literatures . A 51-year - old female was diagnosed as cd after an urgent terminal intestinal resection and presented endoscopic recurrence despite the prophylactic treatment with azathioprine (aza). Fortunately, she achieved mucosal healing (mh) at a low dose of thalidomide for 15 months . Crohn disease (cd) is a progressive and destructive disease, over 70% cd patients require intestinal resection at some time during their life time . The rate of endoscopic recurrence in cd patients 1 year after operation ranges from 30% to 70%, increasing to 50% to 100% after 3 years . Despite evolving prophylactic treatment algorithms, treatment of postsurgical recurrence is still required for a proportion of patients . Here presents a case of postoperative endoscopic cd recurrence that achieves mucosal healing (mh) on thalidomide . In march 2014, a 51-year old female with a history of 3-year gastrointestinal bleeding, underwent the ileocolonoscopy which revealed some terminal ileal longitudinal ulcers and spontaneous hemorrhage (fig . The colon was normal . As the bloody stool was out of control with the decreased level of hemoglobin (hb) from 113 to 85 g / l within 24 hours, she subsequently had an urgent resection of the affected terminal intestine, and the remaining intestine were detected to be normal . Four weeks later, the disease was clinically quiescent with no bloody stools (hb: 112 g / l), which gave a crohn disease index activity (cdai) score of 144 . And she was prescribed azathioprine (aza) 100 mg / d because of the high risk of recurrence . Three months later, however, the patient experienced endoscopic recurrence as the colonoscopy (fig . 2b) both revealed the anastomotic ulcers which gave a rutgeerts score of i2 with cdai score of 102 . Considering the high cost of biological agents, she refused to take infliximab (ifx) and started on thalidomide at 50 mg / d . Six months after initiation of thalidomide therapy, the patient again developed complaints of abdominal pain and bloody stool (cdai score: 185) and noted no improvement of ulcers (fig . The cdai reached 101 and the rutgeerts score declined to i1 after 9-month adjusted therapy (fig . A telephone conversation confirmed that until may 2016 the patient insisted on thalidomide 100 mg / d with no adverse effect and had been asymptomatic . (a) longitudinal ulcers were observed in the terminal ileum and (b) spontaneous hemorrhage before surgery . (c) the histopathological examination revealed noncaseating granulomas in the submucosal layer (arrow). Before treatment with thalidomide . (a) no improvement was noted after 6 months therapy with thalidomide 50 mg / d . (b) disappearance of anastomotic ulcers with only a few aphthae was revealed after 9-month therapy with thalidomide 100 mg / d . Surgery is not a cure for cd patients, and postoperative recurrence is frequent . Among the risk factors for the postoperative recurrence, smoking, perforating disease, perianal disease, extension, and urgent indication for surgery thioprine was associated with a significantly reduced risk of clinical and severe endoscopic recurrence when compared to placebo . There are promising data that targeting tnf- therapy prevents postoperative recurrence . Nevertheless, its routine prophylactic use after surgery to prevent recurrence is debatable taking into account its high medical cost and the potential side effects . Postoperative management should be personalized based on the estimated probability of cd recurrence . In our case, aza (100 mg / d) was started as early as 4 weeks after the surgery to prevent recurrence taking into account the high risk (urgent procedure). The consensus on the medical paradigm of postoperative cd recurrence has not yet been achieved . Sorrentino et al conducted a pilot study and prescribed ifx to 13 patients and mesalamine to 11 patients who experienced postoperative recurrence, at week 54, 0/11 patients treated with mesalamine and 7/13 patients treated with ifx had endoscopic remission (p = 0.01). The study showed that anti - tnf agents may be efficient in treating postoperative cd recurrence . Thalidomide as a tnf inhibitor was prescribed to our patient other than ifx secondary to the expenses . Thalidomide was originally prescribed to a pregnant woman as its sedative property, and was withdrawn from the market in 1961 because of its teratogenic side effects, particularly phocomelia . During the last decades, it has been reintroduced as a potent antiinflammatory and immunosuppressive drug and shown to have a role in some diseases such as erythema nodosum leprosum, sarcoidosis, behcet syndrome, and cd . Thalidomide may shift the th1 pattern to th2 with the inhibition of tnf-, ifn-, and il-12, stimulate il-4 and il-5 and block nf-b activation . Since the first report of the efficacy of thalidomide treatment in a complicated cd patient, several studies have demonstrated the role of thalidomide in active cd . Two studies both demonstrated the long - term clinical outcome of thalidomide in refractory cd . Simon et al conducted a recent multicenter observational study of 77 patients with active cd, refractory to conventional immunosuppressive therapies, 54% of the patients were in clinical remission after thalidomide treatment within the first year . Of the 7 cd patients complicated with fistulae who had failed anti - tnf biologic treatment and received thalidomide as rescue therapy, 5 had complete fistula closure during the follow - up period . Fifteen intractable cd patients were started on thalidomide (100 mg / d) after they had responded to ifx (5 mg / kg infusions), remission rates were 92%, 83%, and 83% at 3, 6, and 12 months . Thalidomide also appears to be successful use in refractory esophageal cd and patients who developed ifx - induced delayed hypersensitivity action . In recent years, mh is recommended to be the therapeutic goal associated with a reduced risk of relapse, fewer surgeries, fewer hospitalization, and steroid tapering . Mh in ibd could be achieved by several drugs, such as aza, biological agents, corticosteroids, and thalidomide . Scribano et al reported the efficacy of thalidomide in 3 patients with moderate - to - severe cd who failed to biological anti - tnf agents, all the 3 patients at a low dose of thalidomide (50150 mg / kg) for 9 to 36 months achieved clinical remission and mh . In our case, the recent consensus guidelines from european crohn's and colitis organization / european society for pediatric gastroenterology hepatology and nutrition (ecco / espghan) commended that thalidomide therapy could be alternative for anti - tnf agent responders who do not tolerate or lost response to biological anti - tnf agents . Zheng et al in a retrospective study evaluated the effects of thalidomide in 6 chinese refractory pediatric cd, all patients clinical symptoms improved remarkably during the follow - up time . A multicenter randomized clinical trial of 56 children and adolescents with refractory cd also has shown that clinical remission was achieved in significantly more children treated with thalidomide (1.52.5 mg / kg) than with placebo at week 8 (46.4% vs 11.5%). Few studies have focused on the efficacy of thalidomide in the surgical cd . In 2004, hershfield et al presented a 29-year - old case affected by cd, he was seen for persistent bleeding and anemia after 1 year of the reoperation procedure, and the colonoscopy revealed terminal ileal ulcers . His problem was persistent with the use of methotrexate, corticosteroids, and aza, then the patient was placed on thalidomide at 50 to 100 mg / d . Twelve months later, the colonoscopy demonstrated remarkable improvement of the appearance of the terminal ileum with only a few small ulcers remained . In our case, the aza therapy failed to prevent postoperative recurrence, and the low dose of thalidomide succeeded in achieving the mh of the anastomotic ulcers . No clear correlation between neuropathy and cumulative thalidomide dose were established in inflammatory bowel disease (ibd). In a long - term study on thalidomide in cd patients, during the follow - up for a median of 58 months, an adverse event occurred in 68% of the patients, over a third (13/37) of patients experienced neuropathy with a median cumulative dose of 11.1 g (range: 0.35225.5 g), and all but 3 patients resolved with dose reduction or discontinuation . Lazzerini et al performed a retrospective study of 28 children and adolescents with chronic refractory ibd treated with thalidomide, 7 patients experienced reversible neuropathy with all cumulative doses over 28 g. in our case, the cumulative dose of thalidomide on the patient was over 28 g, until the mh was achieved, she did not experience any adverse events . In conclusion, thalidomide is effective to induce mh in the postoperative cd endoscopic recurrence in our case . To our knowledge thalidomide, the anti - tnf agents, may be an alternative therapy regimen for the postoperative cd endoscopic recurrence.
Patterns of tumor metastases are dependent on the recipient organ microenvironment, and each component of the metastatic tumor microenvironment plays distinct and unique roles in disease progression . To better understand organ - specific metastases and to ultimately develop more effective therapeutics, the biology of key cellular components in the microenvironment must be clearly determined . Among many types of solid tumors, prostate, the normal physiology of the skeletal system requires a tightly - regulated balance between osteoblasts and osteoclasts . Briefly, the migrated breast cancer cells express bone - modulatory factors such as parathyroid hormone - related peptide (pthrp), leading to up - regulation of receptor activator of nuclear factor b (rankl) and monocyte - colony stimulating factor (m - csf) in adjacent osteoblasts . Subsequently, osteoclastogenesis occurs, followed by osteolysis and the release of bone matrix - embedded growth factors (e.g., transforming growth factor [tgf-]). The homeostasis of the human skeletal system is maintained by the coupling of bone resorption (by osteoclasts) and new bone formation (mainly by osteoblasts). Alterations in the bone homeostasis result in many types of bone diseases such as fractures and osteoporosis . In addition to the vicious cycle hypothesis described above, increasing lines of evidence now clearly support that alterations in the bone homeostasis contribute to the progression of bone metastases . Among the many cell types in the metastatic bone microenvironment, including osteocytes, osteoblasts, osteoclasts, mesenchymal stem cells, and hematopoietic bone marrow cells in diverse stages of differentiation, the majority of research data concentrate on osteoclasts, and thus the current therapeutic approaches targeting the metastatic bone microenvironment are mostly osteoclast inhibitors (e.g., bisphosphonates, denosumab, and cathepsin k inhibitors). On the other hand, numerous research groups have demonstrated that other types of cells, particularly osteoblasts, are also important in the metastatic bone microenvironment, and also that osteoblasts play distinct and essential roles in metastatic progression . Accordingly, this review paper will summarize and highlight the recent publications supporting the role of osteoblasts in bone metastasis . Hedgehog (hh) signaling is important in development, regulating cell growth, body pattern formation and organogenesis . Mammalians have three types of hh proteins, i.e., sonic, indian, and desert hh with sonic hh (shh) being the most thoroughly investigated in development and pathology . Several lines of evidence suggest that hh is involved in organ - specific metastasis to bone, especially in breast and prostate cancers, as well as in bone tumors such as osteosarcomas . . Demonstrated that shh up - regulates rankl expression in bone stromal cells and osteoblasts, leading to osteoclast formation . Subsequently, an increase in osteoclasts contributes to tumor - induced osteolysis and ultimatelys to increased tumor growth in bone . Recently, shimo et al . Analyzed human oral squamous cell carcinoma patient samples and identified that shh is expressed in bone - invasive tumor cells while patched receptor and gli2 (a downstream transcription factor of hh signaling) are expressed in osteoclast progenitor cells, indicating that tumor - derived shh stimulated osteoclast formation and bone resorption in tumor - induced jaw bone destruction . The above examples clearly show that hh is a paracrine factor in the tumor microenvironment . Accordingly, metastatic tumor - derived hh proteins stimulate the bone stromal cells to promote metastatic tumor growth . Indeed, hh proteins reregulate rankl expression in adjacent osteoblasts and bone marrow stromal cells, leading to osteoclastogenesis via activation of extracellular signal - regulated kinases (erk), p38 mitogen - activated protein kinases (mapk), increased expression of nuclear factor of activated t cells, cytoplasmic 1 (nfatc1). Chan et al . Demonstrated that osteoblast - specific hh expression increased osteosarcoma development in p53 mutant mice . Furthermore, inhibition of hh signaling reduced the expression of yes - associated protein (yap1), a potent oncogene, in osteosarcoma cells . They also showed that the long non - coding rna h19, a highly relevant candidate for osteosarcoma development, is aberrantly expressed and induced by up - regulated hh signaling and yap1 overexpression, suggesting a molecular mechanism of hh - dependent osteosarcoma progression from osteoblasts . Briefly, in the absence of hh ligands, the patched (ptch1) receptors accumulate and inhibit the function of smoothened (smo) protein, leading to degradation of the gli transcription factor by proteasome . Upon activation, the ptch1 receptors are degraded and thus gli is activated, leading to its nuclear translocation and function as a transcriptional activator . Johnson et al . Demonstrated that bone matrix - derived tgf- increased the expression of gli2 and pthrp in metastatic breast cancer cells, leading to further bone destruction . Furthermore, the authors showed that gli activity was not dependent on canonical hh signaling, because mda - mb-231 breast cancer cells do not express smo . . Demonstrated that hh pathway is important in osteoblast maturation, particularly in the presence of breast cancer cells . Hh - mediated gli2 stimulated secretion of bone morphogenetic protein (bmp2), a potent osteogenic differentiation factor, as well as cyclin d, osteopontin, and insulin - like growth factor, suggesting that hh is an important factor of osteoblastic functions . Collectively, the hh proteins are important mediators between tumor and bone, and the signaling is bi - directional (i.e., tumor - to - stroma and stroma - to - tumor). Runt - related transcription factor 2 (runx2) is also known as core - binding factor subunit- 1 (cbf-1) and serves as a key transcription factor in osteoblast differentiation and gene expression . Runx2 binds to cognate dna elements with the consensus nucleotide sequence 5-accaca in the promoters / enhancers of target genes . Franceschi and xiao extensively summarized the data supporting that runx2 is important in the responsiveness to multiple signal transduction pathways in osteoblasts . Indeed, the franceschi group demonstrated that functional runx2 is essential to fibroblast growth factor 2 (fgf2)- and parathyroid hormone - induced osteocalcin expression in osteoblasts, and bmp2-stimulated osteoblast differentiation . In this context, runx2 was shown to be a critical regulator of osteoblasts in the bone metastatic tumor microenvironment . For example, zong et al . Showed that the runx2-phtrp - rankl pathway contributes to the progression of an experimental breast cancer bone metastases . Furthermore, li et al . Demonstrated that runx 2 increased integrin -like 1 expression, leading to up - regulation of tgf- in the bone metastatic breast cancer stroma . Runx2 has been classically considered an osteoblast - specific transcription factor, but more recently, increasing evidence supports that runx2 is not only important in osteoblasts but also in tumor cells in the bone microenvironment . For example, li et al . Recently showed that runx2 promotes breast cancer bone metastasis by increasing integrin 5-mediated colonization . In addition, ge et al . Provided pivotal evidence that phospho - runx2 is proportionately expressed during the progression of prostate cancer bone metastasis with the highest expression in metastatic lesions using tissue microarray and in vitro prostate cancer cell line models . The authors also concluded that phospho - runx2 has a prognostic value for prostate cancer patients . Runx2 is also involved in tumor - induced osteolysis by transcriptional suppression of tssc1 via the bmp - tgf-, wnt, pthrp, and src pathways . More specifically, expression of runx2 is associated with osteolysis, and up - regulated runx2 mediated direct interactions between the tgf-/bmp and smad signaling pathways, contributing to the formation of osteolytic- and osteoblastic - mixed bone lesions that are related with prostate cancer cells . Moreover, tumor growth and distant metastasis of prostate cancer occur via the runx2-smad complex . The runx2-smad complex has functional activity that mediated tgf- and bmp signaling through smid (i.e., smad interaction domain). For example, vascular endothelial growth factor (vegf), matrix metalloproteinases, interleukin 8 (il-8), and pthrp are up - regulated by runx2 in tumor cells, and mediated tumor growth and organ - specific metastasis to bone . Vegf and il-8 are both potent angiogenic factors and strong pro - tumorigenic factors . Pthrp is an important mediator of the vicious cycle of bone metastasis and also plays diverse roles in tumor progression . Runx2 physically and functionally interacts with the receptors for androgens and estrogens, which are the targets for many prostate cancer and breast cancer drugs . In short, overexpression of runx2 is observed in the stromal compartment as well as in tumor cells and its phosphorylation is important when the tumor cells metastasize to bone . The runx2-smad complex regulates the activation of tgf-/bmp signaling, stimulating osteolysis and bone destruction . Lastly, runx2 inhibits the expression of tssc1, a tumor suppressor that inhibits bone metastasis by blocking the promoter of the tssc1 gene . Micrornas (mirnas) are non - coding rnas composed of very short nucleotides sequences up to 22 nucleotides . Mirnas down - regulate mrna - dependent translation of target genes by producing short hairpin rnas . One mirna can affects dozens of mrnas, and mirnas have an important role as post - transcription regulators in bone formation . Mirnas potentially contribute to the proliferation and metastasis of tumor cells by regulating target gene expression . Many mirna function as tumor suppressors; however, some mirnas aid in tumor growth and metastasis . For example, mir103/107 contributes to tumor progression by down - regulating dicer, which has an important role in processing mirna precursors . Up - regulated dicer promotes the expression of mirnas that inhibit tumor progression and metastasis, and therefore mir103/107 inhibits the expression of dicer, contributing to tumor progression . Mir-154, mir-379, and mir-409 - 3p/5p work as oncogenes in bone metastasis of prostate cancer . Mir-154, mir379 and mir-409 - 3p/5p are members of -like 1 homolog - deiodinase, iodothryonine3 (dlk1-dio3), and are activated in the epithelial to mesenchymal transition (emt) of prostate cancer cells . The mirnas of the dlk1-dio3 cluster are up - regulated in bone metastatic prostate and breast cancer cells . Among those, mir-379 and mir-154 located in the dlk1-doi3 cluster are specifically up - regulated in prostate cancer cells with the mesenchymal phenotype (i.e., more metastatic). Mir-379 and mir-154 are expressed by embryonic stem cells and pluripotent stem cells, and these mirnas regulate bone - specific prostate cancer cells by inducing emt . Second, mir-154 regulates the expression of emt- and stemness - related genes to mediate downstream convergent signal pathways . When mir-154 is inhibited, e - cadherin expression is up - regulated, suggesting decreased invasion and metastasis . In addition, mir-379 is located in the upstream of the mir-154 gene, and has a similar role as mir-154 . In bone metastatic prostate cancer cells, mir-409 - 3p/5p inhibits tumor suppressor genes in prostate cancer cells, for example stromal antigen 2 (stag2), ras suppressor protein 1 (rsu1), retinoblastoma - like 2 (rbl2), and nitrogen permease regulator - like 2 (nprl2). Mir-409 - 3p is predicted to activate the ras signaling pathway and the hypoxia inducible factor-1 pathway, as well as regulate polycomb group proteins and osteoblastic pathways . Mir-409 - 5p is predicted to activate the e2f pathway, ras signaling pathway, akt pathway, and aneuploidy . Taken together, multiple lines of evidence demonstrate that mir-409 - 3p/5p is elevated in the bone metastatic emt cell models and this microrna functions by repressing several tumor suppressor genes . Osteoblasts are derived from mesenchymal stem cells in bone marrow, and serves as the major cell type essential to bone anabolic processes . Differentiation of osteoblasts is determined by multiple steps via many growth factors, receptors, and transcriptional factors such as hh / ptch, bmp, fibroblast growth fators (fgfs), parathyroid hormone (pth), runx2, wnt, and tgf--smad . The pathways have a high degree of cross - talk among them, with runx2 being a major point of convergence . Osteons, a functional unit of osteoblasts, mediate the bone anabolic reaction by producing organic matrices of bone tissue including cross - linked collagen, and osteocalcin, osteopontin . Subsequently, the matrices become mineralized, forming hard tissue . The roles of osteoblasts in the tumor microenvironment have been a focus of extensive research efforts thus far, with the vicious cycle hypothesis being the most well investigated by dr . The hypothesis was later clinically and experimentally proven to be legitimate and provided a solid scientific rationale for the development of multiple bone - targeted agents . However, the over - simplified cycle comprised of tumor, osteoblasts, osteoclasts, and tgf-, does not perfectly explain the complex interactions among the extremely diverse stromal cells in the bone microenvironment or the pathologic progression of bone metastasis . For example, we previously demonstrated that megakaryocytes, previously considered to have no clear roles in bone metastasis, are the first cell type that encounter extravasating tumor cells in the bone sinusoidal vessel architecture, and induce apoptosis of tumor cells thus protecting from bone metastasis . Provided pivotal evidence supporting that the osteoblastic hematopoietic stem cell niches are the main sites of tumor cell localization, and metastatic tumor cells compete with stem cells for the occupancy of the stem cell niche . Most notably, we demonstrated that primary tumor - derived pthrp circulates to stimulate osteoblasts, leading to up - regulation of myeloid - derived suppressor cells (mdsc). These pre - activated mdscs in the tumor hosts then enter the systemic circulation to increase primary tumor growth and angiogenesis . 1 demonstrates that osteoblasts are stimulated to grow (bracket) and form new woven bones (solid arrow), indicating that osteoblasts are one of the first responder cells in bone metastasis, potentially playing critical functions other than expressing osteoclastic factors (rankl and m - csf). This review paper summarizs the current evidence supporting how osteoblasts are regulated and what roles they play in the progression of bone metastasis . In particular, we examined the three most significant mediators including hh and patched receptor signaling (i.e., an extracellular stimulant and the cell surface receptor), the runx2 transcription factor and mirnas (i.e., post - transcriptional level regulation). All of this evidence clearly warrants extensive further research, potentially providing an important foundation for novel and advanced therapeutics specific to bone metastasis . Beyond the conventional concept of tumor - initiative and bone - responsive process, recent data are focused on how the cells in the bone environment (i.e., osteocytes, osteoblasts, and osteoclasts) initiate the dormant disseminates tumor cells in bone . The current evidence clearly demonstrates that tumor cells engage with osteoblastic lineage cells on the endosteal surface and enter the long - term dormancy . On the other hand, osteoclast - mediated bone remodeling that is activated by estrogen withdrawal can release dormant tumor cells from the active control of the endosteal niche, facilitating reactivation and tumor growth . Instead of osteoclasts passively responsing to tumor - derived products in this point of view, the osteoblastic niche can be a potential therapeutic target for the treatment of bone metastasis . Further intensive studies focusing on bone cell responding to tumor cells are required to overcome bone metastasis, one of the leading causes of cancer mortality.
Influenza c virus remains poorly studied compared with influenza a and b, although it has been shown to cause upper respiratory tract (urt) and lower respiratory tract (lrt) infections of varying severity similar to those associated with the other respiratory viruses . Symptoms include fever, cough, rhinorrhea, and lrt illness such as pneumonia, bronchitis, and bronchiolitis.1, 2 influenza c virus has been shown to be a significant cause of urt illness in children less than 6 years old, and the risk of complications with lrt illness is particularly high in children less than 2 years old,2, 3 resulting in more severe illness and hospitalization.4, 5 in adult volunteer studies, the disease caused by influenza c can vary from an asymptomatic to a mild upper respiratory tract infection.6 a case of acute encephalopathy associated with influenza c has also been reported.7 influenza c virus has been documented as the etiological cause of several outbreaks in schools and the community8, 9, 10, 11, 12, 13; hence, its role in adding to the overall burden of respiratory illness should not be underestimated . High rates of seroprevalence have been reported for influenza c, suggesting that the virus is circulating widely in the population.1, 14, 15, 16 historically, the underdiagnosis of influenza c has resulted from the difficulty in culturing this virus and the lack of readily available monoclonal antibodies for detection by direct florescence microscopy . Although mdck and hmvii cells have been used for the isolation of influenza c,17, 18, 19, 20 the rate of recovery is low . Amniotic inoculation of embryonated hen's eggs remains the most sensitive technique to isolate this virus; however, few clinical laboratories have this capability . Molecular assays for detection of this virus have been few and essentially based on endpoint or twostep realtime reversetranscriptase pcr (rt rtpcr).21, 22, 23, 24, 25 here, we report on the development of a onestep rt rtpcr assay and its application to the detection of influenza c in a selected panel of respiratory samples . A subset of samples collected during hospital visits from children less than 10 years of age and from respiratory outbreaks (n = 47 from 19 outbreaks) submitted to the provincial laboratory for public health (provlab) for respiratory virus testing between sept 1, 2010 and april 30, 2011 were included in the study . Pediatric samples were randomly selected for influenza c screening to include similar number of samples per month based on availability (approximately 55); multiple samples from the same patient were excluded . All specimens collected from the study patients had tested negative for influenza a and b, respiratory syncytial virus (rsv), human metapneumovirus, parainfluenza virus types 14, coronaviruses 229e, oc43, nl63, and hkui, adenovirus, entero / rhinovirus using a realtime rtpcr for influenza a and b26 and the respiratory viral panel (rvp) assay from luminex molecular diagnostics . The numbers and types of specimens tested included: nasopharyngeal / nasal (n = 431), throat (n = 24), bronchoalveolar lavage (n = 4), sputum (n = 1), endotrachael tube (n = 2), and the collection site was not provided for 12 specimens . Respiratory samples were extracted from an input volume of 200 l into an elution volume of 110 l using the easymag automated extractor (biomrieux), according to the manufacturer's instructions . All available matrix (m) gene sequences from genbank were aligned to design primers (infcmfor / infcmrev) and a minor groove binding probe (infcmprobe) labeled with fam as the reporter dye to amplify and detect a 64bp region of the matrix gene of influenza c virus . Infcclonefor and infcclonerev were designed for amplification of the m gene to generate a plasmid clone with the detection region . Additional primers were designed for sequencing the m and hemagglutininesterase (he) genes, sequences and locations of all oligos are provided in table 1 . Primers and probes used for the detection and characterization of influenza c virus he, hemagglutininesterase . Nucleotide positions for the primers and probes targeting the matrix gene are as for segment 6 of influenza c (c / johannesburg/1/66) genbank am410042.1 and the hemagglutininesterase gene are as for segment 4 of influenza c (c / johannesburg/66) genbank m17868.1 . A onestep rtpcr method was used for the amplification and detection of influenza c virus . The taqman fast virus onestep rtpcr master mix (abi) was used with 08 m each of the sense and antisense primers and 02 m of the probe . Five microliters of the extracted rna was combined with 5 l of the master mix, and the rt step was performed at 50c for 5 minutes followed by incubation at 95c for 20 seconds . Amplification included 45 cycles of denaturation at 95c for 3 seconds, followed by annealing, extension, and data acquisition at 60c for 30 seconds on the 7500 fast realtime pcr system (abi). Primers infcclonefor and infcclonerev (table 1) were designed to amplify 1032 bp of the m gene from influenza ctype strain (c / taylor/1233/47, kindly provided by dr yan li, national microbiology laboratory, winnipeg, canada). The pcr products were cloned using the topo ta cloning dual promoter kit (life technologies, california, usa). The plasmid dna was linearized using restriction enzymes hind iii and transcribed using the t7 ribomax express (promega, madison, wi, usa) to synthesize negativestrand rna in vitro . Tenfold serial dilutions ranging from 45 108 to 45 10 copies / reaction of quantified in vitro transcribed rna were tested to determine assay sensitivity . The specificity of the assay was determined by testing high copy number samples of common respiratory pathogens including different strains of influenza virus a and b, parainfluenza virus 1, 2, 3, 4a and 4b, rsv a and b, human coronaviruses 229e, nl63, hku1 and oc43, human bocavirus, coxsackievirus a16 and b6, echovirus 2, human metapneumovirus, rhinovirus serotype 1b, adenovirus serotype 4, legionella pneumophila, mycoplasma pneumoniae, bordetella bronchiseptica, b. holmseii, b. parapertussis, b. pertussis, hemophilus influenzae, neisseria meningitidis, and streptococcus pneumoniae . The reproducibility of the influenza c rtpcr was evaluated using serial dilutions of positive patient specimens made in a negative nasopharyngeal matrix at ct values of 2110, 2765, and 3408 and in negative auger suctions at ct values of 2036, 2789, and 3094 . The primers described in table 1 were used to amplify the m and he genes from positive samples . Sequences were analyzed using seqscape v2.6 and clustalx multiple sequence alignment program (version 1.81). Phylogenetic analysis was conducted using treecon.27 a total of 1806 bases of the hemagglutininesterase (he) gene (from base pair 24 to 1830 based on numbering of c / taylor/1233/47; genbank m11637.1) from six influenza c positive samples were used for sequence comparison . The he gene of influenza c viruses has been classically divided into six lineages, represented by c / taylor/1233/47, c / aichi/1/81, c / sao paulo/378/82, c / kanagawa/1/76, c / yamagata/26/81, and c / mississippi/805, 11, 28, 29; these sequences in addition to representative sequences from different continents of the world and one sequence from a porcine isolate were included for comparison . A total of 963 bases of the matrix (m) gene (from base pair 67 to 1029 based on numbering of the influenza c / taylor/1233/47; genbank d26546.1) from 10 influenza c positive samples were used for sequence comparison . Influenza c viruses have been divided into three lineages based on the m gene.30 lineage i consists of viruses with the he gene from the c / yamagata/26/81related lineage, lineage ii consists of viruses with he gene of either c / aichi/1/81 or c / mississippi/80related lineage, and lineage iii consists of viruses with c / aomori/74 . The sequence of influenza c viruses belonging to the three lineages based on the m gene, six lineages based on the he gene, and a porcine isolate were used for comparison . All he (jx080409jx080414) and m (jx133150jx133159) gene sequences were submitted to genbank . A subset of samples collected during hospital visits from children less than 10 years of age and from respiratory outbreaks (n = 47 from 19 outbreaks) submitted to the provincial laboratory for public health (provlab) for respiratory virus testing between sept 1, 2010 and april 30, 2011 were included in the study . Pediatric samples were randomly selected for influenza c screening to include similar number of samples per month based on availability (approximately 55); multiple samples from the same patient were excluded . All specimens collected from the study patients had tested negative for influenza a and b, respiratory syncytial virus (rsv), human metapneumovirus, parainfluenza virus types 14, coronaviruses 229e, oc43, nl63, and hkui, adenovirus, entero / rhinovirus using a realtime rtpcr for influenza a and b26 and the respiratory viral panel (rvp) assay from luminex molecular diagnostics . The numbers and types of specimens tested included: nasopharyngeal / nasal (n = 431), throat (n = 24), bronchoalveolar lavage (n = 4), sputum (n = 1), endotrachael tube (n = 2), and the collection site was not provided for 12 specimens . Respiratory samples were extracted from an input volume of 200 l into an elution volume of 110 l using the easymag automated extractor (biomrieux), according to the manufacturer's instructions . All available matrix (m) gene sequences from genbank were aligned to design primers (infcmfor / infcmrev) and a minor groove binding probe (infcmprobe) labeled with fam as the reporter dye to amplify and detect a 64bp region of the matrix gene of influenza c virus . Infcclonefor and infcclonerev were designed for amplification of the m gene to generate a plasmid clone with the detection region . Additional primers were designed for sequencing the m and hemagglutininesterase (he) genes, sequences and locations of all oligos are provided in table 1 . Primers and probes used for the detection and characterization of influenza c virus he, hemagglutininesterase . Nucleotide positions for the primers and probes targeting the matrix gene are as for segment 6 of influenza c (c / johannesburg/1/66) genbank am410042.1 and the hemagglutininesterase gene are as for segment 4 of influenza c (c / johannesburg/66) genbank m17868.1 . A onestep rtpcr method was used for the amplification and detection of influenza c virus . The taqman fast virus onestep rtpcr master mix (abi) was used with 08 m each of the sense and antisense primers and 02 m of the probe . Five microliters of the extracted rna was combined with 5 l of the master mix, and the rt step was performed at 50c for 5 minutes followed by incubation at 95c for 20 seconds . Amplification included 45 cycles of denaturation at 95c for 3 seconds, followed by annealing, extension, and data acquisition at 60c for 30 seconds on the 7500 fast realtime pcr system (abi). Primers infcclonefor and infcclonerev (table 1) were designed to amplify 1032 bp of the m gene from influenza ctype strain (c / taylor/1233/47, kindly provided by dr yan li, national microbiology laboratory, winnipeg, canada). The pcr products were cloned using the topo ta cloning dual promoter kit (life technologies, california, usa). The plasmid dna was linearized using restriction enzymes hind iii and transcribed using the t7 ribomax express (promega, madison, wi, usa) to synthesize negativestrand rna in vitro . Tenfold serial dilutions ranging from 45 108 to 45 10 copies / reaction of quantified in vitro transcribed rna were tested to determine assay sensitivity . The specificity of the assay was determined by testing high copy number samples of common respiratory pathogens including different strains of influenza virus a and b, parainfluenza virus 1, 2, 3, 4a and 4b, rsv a and b, human coronaviruses 229e, nl63, hku1 and oc43, human bocavirus, coxsackievirus a16 and b6, echovirus 2, human metapneumovirus, rhinovirus serotype 1b, adenovirus serotype 4, legionella pneumophila, mycoplasma pneumoniae, bordetella bronchiseptica, b. holmseii, b. parapertussis, b. pertussis, hemophilus influenzae, neisseria meningitidis, and streptococcus pneumoniae . The reproducibility of the influenza c rtpcr was evaluated using serial dilutions of positive patient specimens made in a negative nasopharyngeal matrix at ct values of 2110, 2765, and 3408 and in negative auger suctions at ct values of 2036, 2789, and 3094 . The primers described in table 1 were used to amplify the m and he genes from positive samples . Sequences were analyzed using seqscape v2.6 and clustalx multiple sequence alignment program (version 1.81). Phylogenetic analysis was conducted using treecon.27 a total of 1806 bases of the hemagglutininesterase (he) gene (from base pair 24 to 1830 based on numbering of c / taylor/1233/47; genbank m11637.1) from six influenza c positive samples were used for sequence comparison . The he gene of influenza c viruses has been classically divided into six lineages, represented by c / taylor/1233/47, c / aichi/1/81, c / sao paulo/378/82, c / kanagawa/1/76, c / yamagata/26/81, and c / mississippi/805, 11, 28, 29; these sequences in addition to representative sequences from different continents of the world and one sequence from a porcine isolate were included for comparison . A total of 963 bases of the matrix (m) gene (from base pair 67 to 1029 based on numbering of the influenza c / taylor/1233/47; genbank d26546.1) from 10 influenza c positive samples were used for sequence comparison . Influenza c viruses have been divided into three lineages based on the m gene.30 lineage i consists of viruses with the he gene from the c / yamagata/26/81related lineage, lineage ii consists of viruses with he gene of either c / aichi/1/81 or c / mississippi/80related lineage, and lineage iii consists of viruses with c / aomori/74 . The sequence of influenza c viruses belonging to the three lineages based on the m gene, six lineages based on the he gene, and a porcine isolate were used for comparison . All he (jx080409jx080414) and m (jx133150jx133159) gene sequences were submitted to genbank . The assay was sensitive, specific, reproducible, and precise for the detection of a range of influenza c viral loads from patient samples . Six specimens with a range of viral loads were tested in triplicate on different runs . These samples gave mean crossing threshold (ct) values of 2036 10, 2110 04, 2765 04, 2789 01, 3094 02, 3408 05 . For the study period (sept 1, 2010 to april 30, 2011), 427 respiratory specimens from individual patients and 47 specimens from 19 respiratory outbreaks were screened for influenza c virus using this assay . Analysis of the data shows that 11 specimens obtained from individual patients were positive for influenza c virus giving a detection rate of 258% . Influenza c virus was detected in eight nasopharyngeal and three auger suction specimens, none of the outbreak samples were positive . The age of patients ranged from 2 days to 97 years, and positive cases were detected in patients 7 months to 7 years old (figure 1). Also indicated is the percentage of positive samples detected in each age group . For the study period (sept 1, 2010 to april 30, 2011), positives cases were detected between december and april . The seasonal distribution of the number of samples tested and positives detected is indicated in figure 2, illustrating an increased circulation of this virus during the winter months . The number of samples tested, positives detected, and percentage of positive samples in each month is indicated . A total of 1806 bp of the he gene from influenza c positive isolates was compared with the six classically defined lineages described above . One sample isolated from our population clustered with the c / sao paulo/378/82 lineage and five samples clustered with the c / kanagawa/1/76 lineage (figure 3) suggesting that the two lineages were cocirculating during the same period . Of the five sequences that clustered with the c / kanagawa/1/76 lineage, four samples (ab494111, ab292111, ab1016111, and ab475311) had greater than 99% sequence identity, sample ab308711 was 99% identical to these sequences . The closest match for these sequences as compared with the ncbi database was c / catalonia/1754/2009.28 sample number ab350211 clustered with the sao paulo lineage, and the closest match was c / catalonia/1318/2009 . Ab350211 was about 93% identical to the five samples belonging to the c / kanagawa/1/76 lineage . Phylogenetic tree showing the relationship between influenza c viruses based on partial hemagglutininesterase (he) gene sequence . The phylogenetic tree includes the six classically different lineages of influenza c (representatives in bold), positive samples from this study, representative sequences from different parts of the world and one sequence from a pig isolate for comparison . The genbank numbers for sequences used in the alignment are as follows: taylor_47 = c / taylor/1233/47 (m11637.1), aichi_81 = aichi/1/81 (d28970), sao paulo_82 = c / sao paulo/378/82 (ab035364.1), kanagawa_76 = c / kanagawa/1/76 (d63470), and yamagata_81 = yamagata/26/81 (d28971.1), aichi_99 = c / aichi/1/99 (ab182357), catalonia_2009 = c / catalonia/1457/2009 (hm748633.1), england_83 = c / england/892/83 (m11642.1), fukuoka_2004 = c / fukuoka/2/2004 (ab252164.1), georgia_69 = c / georgia/1/69 (ab035359.1), greece_79 = c / greece/1/79 (ab035363), johannesburg_66 = c / johannesburg/66 (m17868), kyoto_79 = c / kyoto/1/79 (d63472), miyagi_92 = c / miyagi/3/92 (ab219076), mississippi_80 = c / mississippi/80 (m11640), paris_67 = c / paris/1/67 (ab035357), kansas_79 = c / kansas/2/79 (ab035361.1), yamagata_93 = c / yamagata/1/93 (ab035365.1), singapore_2006 = c / singapore / dso050530/2006 (gq853455.1), yamagata_2004 = c / yamagata/3/2004 (ab252153.1), yamagata_98 = c / yamagata/6/98 (ab064402.1), pigbeijing_81 = c / pig / beijing/115/81 (m11644.1), yamagata_88 = c / yamagata/3/88 (d63473.1), new jersey_76 = c / new jersey/1/76 (ab035362.1), sapporo_71 = c / sapporo/71 (d63468.1). A total of 963 bp of the matrix (m) gene from 10 influenza c positive isolates was compared with the classically defined lineages based on the m and he genes . Phylogenetic clustering based on the he and m genes was different, showing that the viruses were reassortants . All the positive samples from this study clustered with miyagi/92 (d87384.1) in the m gene (figure 4). As previously suggested, clustering in the m gene was different from that in the he gene.30 all 10 samples were over 98% identical . The group of viruses from samples ab2110010, ab475311, ab292111, ab1016111, ab494111, and ab219311 were over 99% identical with only two base pair changes . The group of viruses from samples ab350211, ab261611, and ab440611 were over 99% identical with a total of seven base pair changes . Ab308711 was slightly different with 11 base pair changes as compared with ab219311 (989%) and 15 changes as compared with ab350211 (984%). The predicted amino acid sequence comparison included 7 changes (218%) of which two changes were included in the m1 region and five in the m2 region . Phylogenetic tree showing the relationship between influenza c viruses based on partial m gene sequence . The phylogenetic tree compares the matrix gene from the lineages defined based on the m and hemagglutininesterase (he) genes (representatives in bold), representative sequences from different parts of the world and one sequence from a pig isolate for comparison . The genbank numbers for sequences used in the alignment are as follows: pig_beijing_81 = c / pig / beijing/115/81(ab000722.1); mississippi_80 = c / mississippi/80 (ab000720.1); kyoto_79 = c / kyoto/1/79 (ab000609.1); england_83 = c / england/83 (ab000725.1); yamagata_81 = c / yamagata/26/81(ab000721.1); kanagawa_76 = c / kanagawa/1/76 (ab000606.1); johannesburg_66 = c / johannesburg/1/66 (am410041.1); greece_79 = c / greece/79 (ab099602.1); newjersey_76 = c / newjersey/76 (ab099600.1); taylor_47 = c / taylor/1233/47 (d26546.1); yamagata_88 = c / yamagata/1/88 (d16261.1); miyagi_92 = c / miyagi/2/92 (d87384.1); sao paulo_82 = c / sao paulo/378/82 (ab035372.1); yamagata_93 = c / yamagata/1/93 (ab035373.1); aichi_81 = c / aichi/1/81 (d16260.1); aichi_99 = c / aichi/1/99 (d16260.1); kansas/1/79 = c / kansas/1/79 (ab099601.1); aomori_74 = c / aomori/74 (d16259.1). The assay was sensitive, specific, reproducible, and precise for the detection of a range of influenza c viral loads from patient samples . Six specimens with a range of viral loads were tested in triplicate on different runs . These samples gave mean crossing threshold (ct) values of 2036 10, 2110 04, 2765 04, 2789 01, 3094 02, 3408 05 . For the study period (sept 1, 2010 to april 30, 2011), 427 respiratory specimens from individual patients and 47 specimens from 19 respiratory outbreaks were screened for influenza c virus using this assay . Analysis of the data shows that 11 specimens obtained from individual patients were positive for influenza c virus giving a detection rate of 258% . Influenza c virus was detected in eight nasopharyngeal and three auger suction specimens, none of the outbreak samples were positive . The age of patients ranged from 2 days to 97 years, and positive cases were detected in patients 7 months to 7 years old (figure 1). For the study period (sept 1, 2010 to april 30, 2011), positives cases were detected between december and april . The seasonal distribution of the number of samples tested and positives detected is indicated in figure 2, illustrating an increased circulation of this virus during the winter months . The number of samples tested, positives detected, and percentage of positive samples in each month is indicated . A total of 1806 bp of the he gene from influenza c positive isolates was compared with the six classically defined lineages described above . One sample isolated from our population clustered with the c / sao paulo/378/82 lineage and five samples clustered with the c / kanagawa/1/76 lineage (figure 3) suggesting that the two lineages were cocirculating during the same period . Of the five sequences that clustered with the c / kanagawa/1/76 lineage, four samples (ab494111, ab292111, ab1016111, and ab475311) had greater than 99% sequence identity, sample ab308711 was 99% identical to these sequences . The closest match for these sequences as compared with the ncbi database was c / catalonia/1754/2009.28 sample number ab350211 clustered with the sao paulo lineage, and the closest match was c / catalonia/1318/2009 . Ab350211 was about 93% identical to the five samples belonging to the c / kanagawa/1/76 lineage . Phylogenetic tree showing the relationship between influenza c viruses based on partial hemagglutininesterase (he) gene sequence . The phylogenetic tree includes the six classically different lineages of influenza c (representatives in bold), positive samples from this study, representative sequences from different parts of the world and one sequence from a pig isolate for comparison . The genbank numbers for sequences used in the alignment are as follows: taylor_47 = c / taylor/1233/47 (m11637.1), aichi_81 = aichi/1/81 (d28970), sao paulo_82 = c / sao paulo/378/82 (ab035364.1), kanagawa_76 = c / kanagawa/1/76 (d63470), and yamagata_81 = yamagata/26/81 (d28971.1), aichi_99 = c / aichi/1/99 (ab182357), catalonia_2009 = c / catalonia/1457/2009 (hm748633.1), england_83 = c / england/892/83 (m11642.1), fukuoka_2004 = c / fukuoka/2/2004 (ab252164.1), georgia_69 = c / georgia/1/69 (ab035359.1), greece_79 = c / greece/1/79 (ab035363), johannesburg_66 = c / johannesburg/66 (m17868), kyoto_79 = c / kyoto/1/79 (d63472), miyagi_92 = c / miyagi/3/92 (ab219076), mississippi_80 = c / mississippi/80 (m11640), paris_67 = c / paris/1/67 (ab035357), kansas_79 = c / kansas/2/79 (ab035361.1), yamagata_93 = c / yamagata/1/93 (ab035365.1), singapore_2006 = c / singapore / dso050530/2006 (gq853455.1), yamagata_2004 = c / yamagata/3/2004 (ab252153.1), yamagata_98 = c / yamagata/6/98 (ab064402.1), pigbeijing_81 = c / pig / beijing/115/81 (m11644.1), yamagata_88 = c / yamagata/3/88 (d63473.1), new jersey_76 = c / new jersey/1/76 (ab035362.1), sapporo_71 = c / sapporo/71 (d63468.1). A total of 963 bp of the matrix (m) gene from 10 influenza c positive isolates was compared with the classically defined lineages based on the m and he genes . Phylogenetic clustering based on the he and m genes was different, showing that the viruses were reassortants . All the positive samples from this study clustered with miyagi/92 (d87384.1) in the m gene (figure 4). As previously suggested, clustering in the m gene was different from that in the he gene.30 all 10 samples were over 98% identical . The group of viruses from samples ab2110010, ab475311, ab292111, ab1016111, ab494111, and ab219311 were over 99% identical with only two base pair changes . The group of viruses from samples ab350211, ab261611, and ab440611 were over 99% identical with a total of seven base pair changes . Ab308711 was slightly different with 11 base pair changes as compared with ab219311 (989%) and 15 changes as compared with ab350211 (984%). The predicted amino acid sequence comparison included 7 changes (218%) of which two changes were included in the m1 region and five in the m2 region . Phylogenetic tree showing the relationship between influenza c viruses based on partial m gene sequence . The phylogenetic tree compares the matrix gene from the lineages defined based on the m and hemagglutininesterase (he) genes (representatives in bold), representative sequences from different parts of the world and one sequence from a pig isolate for comparison . The genbank numbers for sequences used in the alignment are as follows: pig_beijing_81 = c / pig / beijing/115/81(ab000722.1); mississippi_80 = c / mississippi/80 (ab000720.1); kyoto_79 = c / kyoto/1/79 (ab000609.1); england_83 = c / england/83 (ab000725.1); yamagata_81 = c / yamagata/26/81(ab000721.1); kanagawa_76 = c / kanagawa/1/76 (ab000606.1); johannesburg_66 = c / johannesburg/1/66 (am410041.1); greece_79 = c / greece/79 (ab099602.1); newjersey_76 = c / newjersey/76 (ab099600.1); taylor_47 = c / taylor/1233/47 (d26546.1); yamagata_88 = c / yamagata/1/88 (d16261.1); miyagi_92 = c / miyagi/2/92 (d87384.1); sao paulo_82 = c / sao paulo/378/82 (ab035372.1); yamagata_93 = c / yamagata/1/93 (ab035373.1); aichi_81 = c / aichi/1/81 (d16260.1); aichi_99 = c / aichi/1/99 (d16260.1); kansas/1/79 = c / kansas/1/79 (ab099601.1); aomori_74 = c / aomori/74 (d16259.1). In this study, we report the detection of influenza c in respiratory samples from alberta, canada . There have been no other reports of the occurrence of this virus in canada; however, detection in respiratory samples has been previously reported from different countries.1, 5, 12, 15, 16, 23, 28 the prevalence of antibodies to influenza c has been shown to range from 60 to 100%.1, 15, 16 influenza c has been shown to cause a spectrum of symptoms2, 3, 4, 5, suggesting that diagnosis of influenza c should be considered in the range of viral etiologies that cause respiratory illness . As a preliminary study, our data shows that influenza c circulates in the community causing respiratory infections severe enough to require medical intervention as all the samples that tested positive for influenza c were collected during hospital visits and were also negative for all other commonly tested respiratory viruses . However, as the samples tested were restricted to hospitalized patients, the prevalence of influenza c respiratory illness in the community is unclear . Others studies have reported the presence of influenza c in nonhospitalized patients.28 similar to previous observations, this study detected influenza c infections primarily in children less than 10 years of age2, 3, 5, 31; however, influenza c infections in older patients have also been reported.28 using our limited sample size, the peak of illness occurs in late winter and early spring; studies spanning more respiratory seasons will be required to understand whether influenza c infections are endemic or cyclical . The absence of influenza c positive samples in the outbreaks is likely due to the small number of specimens available . Other studies have reported on the detection of influenza c in samples collected from outbreaks.8, 13 it will be interesting to determine whether there is a relationship between the incidence of influenza c infections in the community and the numbers of outbreaks in residential facilities caused by this virus . As more laboratories incorporate molecular assays into their test menus, the availability of a sensitive realtime rtpcr will make it easier to implement surveillance studies for influenza c. this will allow us to understand the burden of influenza c infections in the community both as single and mixed infections with other respiratory viruses . Mixed infections in children are not uncommon, with various studies showing that coinfection rates range from 5 to 65%,32 and have a higher likelihood of being admitted to a pediatric icu.33 the participating role of influenza c as a sole or mixed infection in this vulnerable patient category deserves further study . Another area of interest is patients with haematopoietic dysfunctions, such as stem cell transplants, who are subject to respiratory infections that can persist for a long period of time . Given the relative frequency of influenza c from studies in the literature, it is likely that some of these infections could be caused by this virus; presently the proportion is unknown . Recent studies32 show that human metapneumovirus and parainfluenza virus in this group of patients have a significantly higher rate of mortality, and defining the frequency and outcome of influenza c infections in this group of patients is worthy of study . In our study, we found that strains from two lineages (c / sao paulo and c / kanagawa) were cocirculating, and other publications have also reported the cocirculation of different influenza c lineages.28 while there have been few studies on the epidemiology of this virus in north america, data from japanese studies show that up to five lineages can cocirculate, resulting in strain replacement and frequent reassortment, allowing the virus to persist and spread in the human population.34, 35, 36 genetic drift in the he gene of influenza c has been shown to be independent of the year of isolation and nucleotide changes do not appear to accumulate with time . This suggests that epidemiologically dominant variants of influenza c viruses do not emerge successively.37 in summary, sensitive nucleic acidbased assays will make it possible to study the disease burden and epidemiology of influenza c viruses . Further studies are planned to look at a number of research questions in the hospitalized and community patients.
Alopecia areata (aa) is a chronic inflammatory disease of the hair follicles and nails, its etiology is unknown, probably multifactorial with evident autoimmune and genetic components . The first clinical description of aa is attributed to celsus (14 - 37 b.c .) And the designation aa was given by sauvages . The importance of genetic factors in aa is underlined by the high frequency of a positive family history in affected individuals . In most reports, these range from 10% to 20% of cases . A study in monozygotic and dizygotic pairs found a concordance rate of 55% for alopecia amongst monozygotic twins with no concordance among the dizygotic pairs . Though it can affect any age group, demographical data on aa is still lacking . Two brothers aged 5 and 7 years presented to the outpatient department with complaints of asymptomatic patchy loss of scalp hair for the past 2 and 4 months respectively . There was no similar complaint in any other family member, siblings and no history suggestive of systemic involvement, drug intake, trauma, pus filled lesions or any other skin eruptions . Mucocutaneous examination revealed multiple smooth patches of alopecia ranging from 2 cm 2 cm to 4 cm 6 cm over occipital and vertex area of elder child and multiple patches over occipital and temporal region measuring 2 cm 2 cm to 2 cm 4 cm of the younger one [figure 1a and b]. (a) multiple areas of nonscarring alopecia involving the occipital and vertex area in a 7-year - old child (b) multiple areas of nonscarring alopecia involving the occipital and temporal area in sibling 5-year - old surface of the patches was smooth with no apparent changes . Histopathological examination of the biopsy sample from the alopecia patch from the scalp confirmed the diagnosis, which showed inflammatory infiltrate in and around the bulbar region of hair follicle . Alopecia areata is t cell mediated autoimmune disease with genetic predisposition resulting in partial and total nonscarring alopecia . Scalp is the predominant site of involvement with the most common clinical pattern involving multiple areas of patchy hair loss . Most patients develop aa before the age 40 years with 11 - 20% of all cases occurring in children . The ratio of male: female in aa in pediatric age group is 1:1 where as in adolescent and adult it is more common in female . Human leukocyte antigen (hla)-dq3 and hla - dqb1fnx0103 alleles appear to be marker for genetic susceptibility to aa with the latter serving as a special genetic marker for more severe variants . The diagnostic pathologic feature is peribulbar lymphocytic inflammation (swarm of bees) predominately cd4 + cells along with cd8 + t cells affecting anagen follicles or follicles in early catagen . Short, easily extractable broken hairs, known as exclamation mark hairs, are often seen at the margins of the bald patches during active phases of the disease . The scalp is the first affected site in most cases, but any hair - bearing skin can be affected . The term alopecia totalis is applied to total or almost total loss of scalp hair, and alopecia universalis is the loss of all body hair . Children with aa demonstrate an increased levels of activated t cells leading to various autoimmune diseases like vitiligo, thyroiditis, connective tissue disorders, lichen planus, type 1 diabetes, and pemphigus foliaceous . Geometric and superficial pits are typical of aa whereas deep and irregularly distributed pits are seen in psoriasis and atopic dermatitis . Familial aggregation of aa has been studied and it has been found that estimated lifetime risk has been 7.1% in siblings, 7.8% in parents, 5.7% in offspring . Aa has been also reported after allogenic bone marrow transplantation from an affected, hla matched sibling . Menon and kiran reported a case of concomitant occurrence of aa in the sibling with emphasis on environmental precipitating factors . Evidence based management of aa in children is limited due to the lack of well controlled randomized studies . Reassurance should be given to patients with limited disease as spontaneous remission occurs in 80% of patients . Various treatment options tried in aa are topical, intralesional, and systemic corticosteroids, topical immunomodulators, and topical irritants like dithranol, psoralen plus ultraviolet a therapy, excimer laser therapy.
Mllerian anomalies are congenital defects of the female reproductive tract resulting from failure in the development of the mllerian ducts and their associated structures symptoms appear principally during adolescence or early adulthood, and affect the reproductive capacity of these women . When clinically suspected, investigations leading to diagnosis include imaging methods such as hysterosalpingography, ultrasonography, magnetic resonance imaging, and cystoscopy . A 25-year - old nulliparous lady, presented to the gynecology opd with complaints of inability to conceive . She was married for 7 years, cohabiting since 5 years . At 16 years of age, she consulted a local doctor in her village for primary amenorrhea and cyclical pain in abdomen, where some operative procedure was done . Since then the patient has passage of blood mixed urine periodically every month for 2 to 3 days with suprapubic abdominal pain . External urethral meatus appeared dilated, and there was blindly ending vagina approximately 3 cm in length . On per rectum examination, uterus appeared normal in size and there was no bulge felt below it to suggest any collection . On investigations, the ultrasound showed normal uterus with endometrial thickness 9.5 mm, ovaries were normal, and bilateral kidneys were also normal . Mri of abdomen showed normal uterine body and cervix with small amount of fluid in endometrial cavity, endocervical cavity, and in upper part of vagina; below that there was a transverse vaginal septum of about 3.5 cm in thickness . [figure 1] on cystoscopy, a fistulous opening of approximately 2 mm was seen below the internal sphincter of urethra through which menstrual blood was seen coming out; the bladder and ureteric orifices were normal . Vaginoplasty and repair of fistulous communication between the genital tract and the urethra was planned for the patient . The mri shows uterus with cervix and upper 2 cm of vagina containing some blood products . There is a septum between the upper and lower part of vagina (the lower part identified by tampon in vaginal canal) the woman was subjected to operation using abdominoperineal approach . On laparotomy, uterus was normal in size, bilateral tubes were present, and ovaries were normal . The methylene blue dye was injected into the fundus of the uterus; dye was visualized coming out of urethra by the side of the indwelling catheter . From the perineal approach, a transverse incision was made through the vault of the short vagina, the dissection was done upward in the connective tissue between the bladder above and rectum below and vaginal space was created . After sharp and blunt dissection, cervix was visualized as dye was coming through it . The urethrovaginal fistulous tract was identified and repair was done in two layers . Over the raw area of vagina, the mould change was done after a week; the amnion graft had successfully taken up . The patient was found to be fine at follow - up after a month, with no hematuria and normal menstruation . She was explained about the fertility period and asked to come at 3 monthly follow - up . When a mullerian duct becomes obstructed, the patient may present with an abdominal mass and dysmenorrhea . If the patient is not treated in a timely fashion, the consequences can be severe, extending even to infertility . In this case, there was transverse vaginal septum in the upper part of vagina leading to hematocolpus and amenorrhea . An iatrogenic fistulous tract was probably created which led to cyclical hematuria . Magnetic resonance imaging (mri) is the mainstay in imaging for the evaluation of mullerian agenesis and is considered a very useful diagnostic tool . In our case, mri determined the precise mullarian anomaly giving us the details regarding the presence of normal cervix with precise length of vaginal septum to help us judge the prognosis and success of the operative procedure, but we got no information about the fistulous communication as it was quite fine and narrow . The site of the opening of fistulous tract in the urethra was apparent on cystoscopy and led to the assumption that the other end tract opened in the vagina . Therefore, apart from mri, cystoscopy played a major role in determining the anomaly in this case . Upon literature review of cases with genitourinary fistula the fistula in that case, however, was congenital, unlike our case in which it was iatrogenic in all probability . It also presented with cyclical hematuria with no incontinence of urine due to imperforate hymen . Congenital vesicouterine, cervicovesical, and vesicovaginal fistula associated with other mullarian anomalies has also been reported in literature. [46] iatrogenic fistulous communication between the genital tract and the urinary tract, known to occur as a complication of cesarean section, has been described in literature as youssef's syndrome . It is characterized by cyclical hematuria, absence of vaginal bleeding, and complete urinary continence . In all these reports in which there was a fistulous communication between the urinary and the genital tract, cystoscopy, hysterosalpingography, and dye test were required for confirmatory diagnosis and only computerized tomography (ct) or mri did not give the full clinical picture . This report highlights the diagnostic and therapeutic challenge faced by the clinician in dealing with a fistulous genital and urinary tract communication with obstructing transverse vaginal septum . Increasing awareness of this rare entity calls for more meticulous evaluation before any surgical intervention in patients with complex genitourinary anomalies.
Acquired brain injuries (abi) are a crucial issue for healthcare and social services . In 2000 and 2005 two consensus conferences in italy [1, 2] produced a set of recommendations to improve patients healthcare . In 2009, to improve the continuum of care for acquired brain injuries in tuscany, italy (3,500,000 million inhabitants). For the 20032007 study period, a total annual number of 465770 incident cases of abi with residual serious disability was estimated on the basis of integrated computerized administrative databases . In 2008, a multidisciplinary workgroup drafted a technical document designing a model of care based on: five fundamental steps of care, rigorous criteria for appropriate transfers between different steps, subgroups of patients requiring different combinations of steps according to their clinical conditions . The above - mentioned model of care is now being implemented throughout the region, addressing some readjustments of hospital beds, shared adoption of clinical protocols and integration between social and health care, different professional skills and subsequent levels of care . The strengths of this model are its flexibility and circularity: according to their clinical needs, patients belonging to different subgroups can move along the clinical pathway in several ways: through subsequent steps, bypassing intermediate steps or being readmitted to previous steps.
Moreover, a clinical investigation demonstrated that more than 20% of patients with subaxial cervical dislocations were combined with traumatic disc herniation (tdh). A surgical realignment, decompression, and reconstruction is usually needed for the instable cervical spine due to disc disruption, facet locking, etc . . There are several surgical management strategies, including one - stage anterior, posterior, and combined approaches for subaxial cervical facet dislocation, but the optimal surgical approach is still controversial [4,59]. For examples, it was preliminarily shown that anterior and posterior surgical approaches for patients with subaxial cervical spine facet dislocations exhibited little difference with regard to long - term neurologic status, quality - of - life, medical adverse events, and rates of instrumentation failure and infection, except that anterior approach might bring about better sagittal alignment . On the other hand, it was reported that the anterior approach was effective and safe for the treatment of subaxial cervical facet dislocation with mild or without spinal cord injury, and after the surgery the cervical spines were well recovered, with intervertebral fusion, but without redislocation or symptoms of spinal cord injury . Generally it is recommended that an initial closed reduction should be conducted, followed by next step, which is based on whether or not the closed reduction succeeded . However, since a considerable proportion of patients simultaneously suffered from incomplete neurological deficit, initially manual closed reduction may not be the best option for the potential impairment of spinal cord or nerve root . In this study, we reported our surgical treatment experience for a series of 52 cases of subaxial cervical facet dislocation patients with incomplete or without neurological deficit . This study will provide a rational alternative to select surgical strategy for the treatment of subaxial cervical facet dislocations with incomplete or without neurological deficit . This study was approved by the ethical committee of the affiliated hospital of nantong university (nantong, china). From september 2009 to august 2014, 77 consecutive acute subaxial cervical dislocations with locked facets were admitted to the department of spinal surgery, affiliated hospital of nantong university . Patients with acute subaxial cervical dislocations with locked facets, aged 1875 years, with incomplete or without spinal cord injury (american spinal injury association, asia grade b patients with complete spinal cord injury (asia grade a), multiple injuries, or without consciousness were excluded . All the patients underwent anterior - posterior and middle - lateral view computed tomography (ct) and magnetic resonance image (mri) examinations of the cervical spine . Traumatic disc herniation (tdh) was determined by mri examination, which was defined as the presence of an extruded disc pressing the thecal sac or the nerve root, and being behind the line between the postero - inferior corner of cranial vertebrae and the postero - superior corner of caudal vertebrae . On admission, patients received gardner - wells tong skull traction for cervical spine with a force of one - tenth of patient s body weight . Mannitol (250 ml) and small doses (40 mg) of methylprednisolone were administered to patients except those with high neurological intact (asia grade e). A preventative antibiotic cefazolin (5 g) was administrated 30 minutes before the surgery and within 72 hours following the surgery . All the surgical procedures were supervised by two senior spinal surgeons (yc and fz). The closed and open reductions were performed under the control of an x - ray image intensifier and a spinal cord monitoring device . The selection of surgical plans and approaches were determined based on whether or not the patients with subaxial cervical facet dislocations were simultaneously combined with tdh and whether or not the initial anterior closed reduction was successful . For subaxial cervical facet dislocations without tdh, closed reduction was manually performed using skull traction before the operation, starting in a flexion supine position and turning to a slight extension position once a satisfactory reduction was achieved . If closed reduction was successful, anterior cervical discectomy and fusion (acdf) was then carried out (single anterior approach). If it failed, a posterior open reduction and fixation and subsequent acdf procedure (posterior - anterior approach, p - a approach) was performed instead . In brief, between the first and second turn over of the body, the patienst were in a prone position and posterior open reduction was obtained by distracting the two dislocated spinous processes with two bone - holding forceps, and if necessary, facets was simultaneously poked with a narrow osteotome . For some difficult cases, when the reduction was satisfactorily achieved, the cervical spine was moved into a slight extension position, and the dislocated spinous process was then fastened by a titanium cable . For subaxial cervical facet dislocations with tdh, patients received anterior discectomy and decompression first . After the herniated disc was certainly moved out, an anterior closed reduction was manually performed by an assistant staff when the patients were in a supine position . The successful cases continued treatment with an acdf surgery, which is also called single anterior approach, while the failed cases received a posterior open reduction in a prone position and cable fixation between the first and second turn over of the body, followed by anterior fusion (anterior - posterior - anterior approach, a - p - a approach). The kyphosis of the dislocated segments was measured from the angles between the superior endplate of the upper vertebra and the inferior endplate of the inferior vertebra in the middle - lateral view radiographs (supplementary figure 1). Body function and neurologic status, reflecting the neurologic function, were measured according to the neck disability index (ndi) and asia classification, respectively . Fusion was defined as one of the following three situations: (1) the formation of the bridging trabecular bone at the interface of graft - vertebral body, (2) less than 50% radiolucency around the border of the bone graft, and (3) no implant failure signs . Patients were followed - up by regular outpatient visits . At 12, 26, and 52 weeks after the surgery, patients were asked to respond to the vas and ndi - related questionnaires at the outpatient service center of the department of spine surgery of our hospital, and also to undergo special physical examination and receive anterior - posterior and lateral x - ray checks . Data were expressed as mean standard derivation, and analyzed using graphpad prism software (version 5.01; graphpad, san diego, ca, usa). Clinical and radiographic characteristics immediately after, and at 12 and 52 weeks, respectively after the surgery were compared with those before the surgery by using a t - test . A value of p<0.05 was considered statistical significance . This study was approved by the ethical committee of the affiliated hospital of nantong university (nantong, china). From september 2009 to august 2014, 77 consecutive acute subaxial cervical dislocations with locked facets were admitted to the department of spinal surgery, affiliated hospital of nantong university . Patients with acute subaxial cervical dislocations with locked facets, aged 1875 years, with incomplete or without spinal cord injury (american spinal injury association, asia grade b patients with complete spinal cord injury (asia grade a), multiple injuries, or without consciousness were excluded . All the patients underwent anterior - posterior and middle - lateral view computed tomography (ct) and magnetic resonance image (mri) examinations of the cervical spine . Traumatic disc herniation (tdh) was determined by mri examination, which was defined as the presence of an extruded disc pressing the thecal sac or the nerve root, and being behind the line between the postero - inferior corner of cranial vertebrae and the postero - superior corner of caudal vertebrae . On admission, patients received gardner - wells tong skull traction for cervical spine with a force of one - tenth of patient s body weight . Mannitol (250 ml) and small doses (40 mg) of methylprednisolone were administered to patients except those with high neurological intact (asia grade e). A preventative antibiotic cefazolin (5 g) was administrated 30 minutes before the surgery and within 72 hours following the surgery . All the surgical procedures were supervised by two senior spinal surgeons (yc and fz). The closed and open reductions were performed under the control of an x - ray image intensifier and a spinal cord monitoring device . The selection of surgical plans and approaches were determined based on whether or not the patients with subaxial cervical facet dislocations were simultaneously combined with tdh and whether or not the initial anterior closed reduction was successful . For subaxial cervical facet dislocations without tdh, closed reduction was manually performed using skull traction before the operation, starting in a flexion supine position and turning to a slight extension position once a satisfactory reduction was achieved . If closed reduction was successful, anterior cervical discectomy and fusion (acdf) was then carried out (single anterior approach). If it failed, a posterior open reduction and fixation and subsequent acdf procedure (posterior - anterior approach, p - a approach) was performed instead . In brief, between the first and second turn over of the body, the patienst were in a prone position and posterior open reduction was obtained by distracting the two dislocated spinous processes with two bone - holding forceps, and if necessary, facets was simultaneously poked with a narrow osteotome . For some difficult cases, when the reduction was satisfactorily achieved, the cervical spine was moved into a slight extension position, and the dislocated spinous process was then fastened by a titanium cable . For subaxial cervical facet dislocations with tdh, patients received anterior discectomy and decompression first . After the herniated disc was certainly moved out, an anterior closed reduction was manually performed by an assistant staff when the patients were in a supine position . The successful cases continued treatment with an acdf surgery, which is also called single anterior approach, while the failed cases received a posterior open reduction in a prone position and cable fixation between the first and second turn over of the body, followed by anterior fusion (anterior - posterior - anterior approach, a - p - a approach). The kyphosis of the dislocated segments was measured from the angles between the superior endplate of the upper vertebra and the inferior endplate of the inferior vertebra in the middle - lateral view radiographs (supplementary figure 1). Body function and neurologic status, reflecting the neurologic function, were measured according to the neck disability index (ndi) and asia classification, respectively . Fusion was defined as one of the following three situations: (1) the formation of the bridging trabecular bone at the interface of graft - vertebral body, (2) less than 50% radiolucency around the border of the bone graft, and (3) no implant failure signs . Patients were followed - up by regular outpatient visits . At 12, 26, and 52 weeks after the surgery, patients were asked to respond to the vas and ndi - related questionnaires at the outpatient service center of the department of spine surgery of our hospital, and also to undergo special physical examination and receive anterior - posterior and lateral x - ray checks . Data were expressed as mean standard derivation, and analyzed using graphpad prism software (version 5.01; graphpad, san diego, ca, usa). Clinical and radiographic characteristics immediately after, and at 12 and 52 weeks, respectively after the surgery were compared with those before the surgery by using a t - test . A value of p<0.05 was considered statistical significance . Fifty - two patients with subaxial cervical facet dislocation with incomplete deficit or without neurological deficit were enrolled in this study, including 37 males and 15 females . The injury mechanisms included falling from height (20 cases), motor - vehicle accident (16 cases), sport activity (nine cases) and head hitting (seven cases). Spines were affected at c3/4 (two cases), c4/5 (six cases), c5/6 (20 cases), c6/7 (21 cases), and c7/t1 (three cases). There were 17 cases with unilateral facets dislocation and 35 cases with bilateral facets dislocation . Particularly, there were 13 cases with tdh, and the other 39 without tdh . There were five patients with asia grade b, 19 grade c, 19 grade d, and nine grade e. five patients with tdh and 17 with non - tdh were successfully treated by closed reduction and fusion (single anterior approach). Twenty - two non - tdh patients were considered failed in closed reduction and were then treated by a p - a approach . Another eight tdh patients received anterior decompression and releasing but failed to achieve manual closed reduction; they were finally treated with an a - p - a approach . A representative case of a 62-year - old male patient with tdh (asia grade e) who received single anterior approach treatment is shown in figure 2; a case of a 38-year - old male patient without tdh (asia grade b) who received posterior - anterior approach treatment is shown in figure 3; and a case of a 50-year - old female patient with tdh (asia grade d) who received anterior - posterior - anterior approach treatment is shown in figure 4 . Herniated discs in all the tdh patients were completely resected . The mean blood loss for single anterior, p - a, and a - p - a approaches was 7830 ml, 14253 ml, and 18944 ml, respectively . The mean prone position time was 0, 4215 minutes and 3918 minutes, respectively . The mean total operation time was 6619 minutes, 11244 minutes, and 13637 minutes, respectively . The mean hospital stay was 7.12.3 days, 11.24.1 days, and 12.74.6 days, respectively (table 2). No severe adverse events occurred during the operations, except one patient presented splitting of posterior incision when taking out the stitches for hypoproteinemia, but was finally healed by suture and supporting therapy . The patients were followed for a mean of 2333 (range 1461) months . For shoulder obstruction kyphosis with a preoperative 10.75.4 disappeared in all the patients after the surgery and did not recur during the follow - up . Lordosis was increased to 5.32.9 after the surgery and maintained over 3 during the follow - up . This result showed the deformity of patients was obviously rectified after the surgery and the angle of lordosis was well maintained until fusion . Postoperative pain was significantly decreased immediately after and at 12 and 52 weeks, respectively, after the surgery compared with preoperative pain (p<0.05). Ndi was significantly decreased at 12 and 52 weeks, respectively, after the surgery compared with before the surgery (p<0.05, table 3). The number of patients with higher asia grade (b, c) was decreased but that of patients with lower asia grade (d, e) was increased (table 3). This result indicated that the vas and patients neurologic function (shown by ndi and asia data) were significantly improved, which were better during the follow - up (table 3). No implant failure was observed and dislocated segments were fused in all patients within one year after the surgery . Particularly, improvement of kyphosis, lordosis, vas, and ndi between before, immediately after, and at 12 and 52 weeks after the surgery for single anterior, p - a, and a - p - a approaches are shown in table 4 . The decision - making for these patients is mainly affected by patients neurologic status, presentation, or absence of disc herniation, dislocation of the facet joint, etc . . For subaxial cervical dislocation patients with incomplete or without neurological deficit, the possibility of recovery is relatively higher . In this case, protection of the remaining neurologic functions from further damage appears to be more important, which needs to be considered during the surgery for these patients . In addition, tdh often is accompanied by subaxial cervical dislocations . For example, according to rizzolo et al ., there was a 22% rate of tdh in cases of unilateral subaxial cervical dislocations and 40% tdh in cases of bilateral subaxial cervical dislocations . Nakashima et al . Showed 29.2% of patients with subaxial cervical dislocations presented with tdh . Consistently, in our patient cohort, 25% (13/52) presented tdh revealed by preoperative mri examination . Based on the situation of initial closed reduction and presence / absence of tdh, in this study we planned effective surgical strategies, including single anterior, posterior - anterior, and anterior - posterior - anterior approaches, and achieved satisfactory outcomes in patients with subaxial cervical dislocations with incomplete or without neurologic compromise . Several management strategies have been reported for the treatment of lower cervical dislocations with incomplete neurological deficit . Manually closed reduction is usually initially employed, which is a basis on which the next steps are based . Reported a high success rate 80.5% (33 of 41 cases) of closed reduction and designed an anterior open approach reduction manner for eight patients who failed in closed reduction . Unfortunately, one patient was found to have neurologic deterioration after reduction maneuver . In our study, 22 of 52 patients (42.3%) successfully received closed reduction, and no neurologic deterioration was found . Study and our study may be related to the affected segments of cervical spine, the presence of tdh or not, the extent of neurological deficit (asia grade), and the presence of unilateral or bilateral facets dislocation in patients . Nakashima et al . Reported that using posterior open reduction and posterior spine arthrodesis in a series of 40 patients with cervical dislocations with tdh obtained favorable reduction without neurologic deterioration . However, 62.5% (25 out of 40) patients had complete neurological deficit cases (asia grade a) in their series; in contrast, patients in our study had incomplete or had no spinal cord injury, placing more importance on protecting the remained neurologic functions from further damage . Therefore, this management strategy used by nakashima et al . May not be suitable for patients with incomplete or without spinal cord injury, such as the patients included in our study . Park et al . Reported a single - stage posterior approach, i.e., open reduction and pedicle screw fixation with the removal of herniated disc fragments via a postero - lateral approach, for patients with subaxial cervical facet dislocations with tdh . Although all the patients were improved neurologically and segmental angles were significantly corrected, patients had to suffer a prolonged time in the prone position . Different from these studies, we initially tried closed reduction so as to decrease the risks of open reduction, followed by the next steps based on whether or not the closed reduction was successful and whether or not tdh was presented . Moreover, spinal surgeons prefer removing a prolapsed disc via an anterior procedure versus a posterior procedure . Treated 21 cases of lower cervical facet dislocations with tdh by combined approaches, i.e., anterior decompression and bone grafting and posterior fixation . Although good kyphosis correction was achieved after surgery and optimal neurologic recovery was observed at least one year later, the long levels of posterior rigid fixation sacrificed the mobility of adjacent normal segments . Previously, anterior reduction was commonly used for patients, regardless of whether or not tdh was present . For patients with tdh, herniated discs might be displaced backward and even dropped into the spinal canal during the anterior reduction, which may then aggravate the spinal cord injury . Moreover, anterior open reduction usually needs excessive distraction of the dislocated segments, which is technically demanding with a potential risk of iatrogenic neurologic injury . Therefore, in our study, before the anterior closed reduction procedure, the herniated disc was resected by anterior discectomy to decrease the risks of anterior reduction . Our results showed no neurologic deterioration was noted postoperatively in all of the tdh patients . As to the cases without successful anterior closed reduction in our series, posterior approach had to be used for reduction and temporary stabilization . We used titanium wires, with the advantage of time saving (averaged 45 minutes) and flexibility, for posterior inter - process fixation, which allowed secondary anterior reposition . In addition, a biomechanical test and our follow - up data indicated that anterior fusion could effectively reconstruct and well maintain the stability of index segments . Although different surgical approaches have been previously reported for patients with subaxial cervical dislocation with incomplete neurologic defect [4,711], how to select an optimal treatment strategy for a specific individual has not been clear . To try to utilize the advantages of anterior closed reduction and simultaneously avoid its potential risk as well as ensure the efficacy of reduction, we prospectively designed an effective surgical treatment strategy to gain satisfactory outcomes . This strategy was basis of the observation of the situation of the initial anterior closed reduction and determination of the presence / absence of tdh . An important limitation of this study was that the follow - up period was not long enough . Future studies with more related cases that are treated and analyzed during a longer follow - up period need to be carried out to verify these results . For subaxial cervical dislocation with incomplete or without neurological deficit, a prospectively designed surgical treatment strategy based on the situation of initial anterior closed reduction and presence / absence of tdh is effective, with satisfactory short - term clinical outcomes . Cervical vertebral kyphosis of the dislocated segments was measured from the angle between the superior endplate of the upper vertebra and the inferior endplate of the inferior vertebra in the middle - lateral view radiographs.
Prosite, initially a signature or pattern database, was created in 1988 by amos bairoch . It was first distributed through pc / gene, the sequence analysis software suite he was developing at the time . The first release of prosite was made available in pc / gene in march 1988 and contained 58 patterns . Each pattern was accompanied by an abstract that described the corresponding protein family or domain (1). Prosite was developed in parallel with swiss - prot and both databases benefited from each other . Many patterns were identified by annotating protein families in swiss - prot (often before any description in the literature). Prosite generated an immediate interest and it then grew regularly to reach 1000 entries 6 years later . Such regions are typically enzyme catalytic sites, prosthetic group - attachment sites (haem, pyridoxal phosphate, biotin, etc . ), metal ion - binding amino acids, cysteines involved in disulfide bonds or regions involved in binding a molecule . But this syntax is very sensitive to any sequence exception, whether due to a bona fide divergence or to a sequencing error . Patterns are thus not adapted to identify less - conserved regions or whole domains . In 1994, all profile methods are more or less statistical descriptions of the consensus of a multiple sequence alignment . They use position - specific scores for amino acids and position - specific penalties for opening and extending an insertion or deletion . Generalized profiles, compared to previous profiles (3), use a more rigorous syntax for insertion, deletion and match states . Generalized profile scores can be mapped to hmm parameters used by hmmer (4). It is thus possible to convert an hmm profile into a generalized profile format and several prosite profiles are in fact hmm profiles that were converted with the pftools program htop (5)., prosite has provided extensive documentation and detailed annotation of domains, families and functional sites . This information was mainly stored in free text and used by biologists who read the various documents and made their own decisions on the function of their protein according to the prosite matches . But with the rapid growth of sequence databases during the last 10 years, there was an increasing need for a reliable tool that could generate automatically precise and accurate functional annotation in standard format . In 2005, we decided to group some functional information stored in prosite in a database of rules that can easily be read by a program and applied on proteins that are recognized by prosite profiles (6). The main characteristic of prorule is that it generates conditional annotation: the annotation is dependent on the presence of given amino acids at precise positions, on the occurrence of other domains or on taxonomic specificity . For example, an enzymatic active site is annotated only if the correct amino acid is found at the required position (for an example of prorule, see figure 1). As prorule uses prosite profiles that are mainly directed against protein domains, it is well adapted to annotate modular proteins . The swiss - prot group has also developed a complementary database of rules (hamap), which uses the same format of rules but which is specific for well - conserved bacterial protein families (7). Niceview format from the prosite web page showing the different types of annotation that can be generated by prorule . The rule pru00298 is used to annotate proteins matched by the animal peroxidase profile (ps50292) on the scanprosite web page . It can annotate comment lines, kw, go terms and various ft lines . In this rule, catalytic activity is generated only if the condition ftgroup(2) is fulfilled (a h in the sequence must align with position 96 of the profile and a r with position 235). To column in the features tables correspond to specific columns in the profile (for more details on the prorule format see: ftp://ftp.expasy.org/databases/prosite/unirule.pdf). Niceview format from the prosite web page showing the different types of annotation that can be generated by prorule . The rule pru00298 is used to annotate proteins matched by the animal peroxidase profile (ps50292) on the scanprosite web page . It can annotate comment lines, kw, go terms and various ft lines . In this rule, catalytic activity is generated only if the condition ftgroup(2) is fulfilled (a h in the sequence must align with position 96 of the profile and a r with position 235). To column in the features tables correspond to specific columns in the profile (for more details on the prorule format see: ftp://ftp.expasy.org/databases/prosite/unirule.pdf). A pattern or regular expression is a qualitative descriptor: it either matches or it does not . It does not produce a score that can help to estimate the significance of a match . There are currently various quantitative methods producing scores that are more efficient than regular expressions (8), but patterns are still very popular because of their intelligibility for users, and because, when used to scan protein databases, they are not cpu expensive compared to quantitative methods . We thus still maintain patterns, but to make them more accurate, we have developed a new method to estimate the significance of their matches . A profile has been constructed and associated to each pattern and used to assign a status to pattern matches . When a pattern matches a protein, the corresponding profile is run on this protein and, if it is also identified by the profile, the match is tagged as true positive, otherwise the status is unknown . The advantage of this approach is that it does not influence the rapidity of the search algorithm and it keeps the user - friendly format of patterns . For each pattern, prosite maintains a manually curated match - list, in which a status is assigned to each pattern match on uniprotkb / swiss - prot entries . The status can be true positive (tp), unknown (? ), false positive (fp), false negative (fn) or potential (p). For each pattern, we have extracted the sequence of each tp match . As the efficiency of a motif descriptor is partly dependent on the size of the sequences in the seed alignment (9), we have increased the length of each tp matched fragment . The average size of prosite patterns is 20 amino acids, and we noticed that it is difficult to construct good profiles smaller than 50 amino acids . We also did not want to increase too much the size of the profile as the scanning time of a profile is proportional to its size . Starting with multiple sequence alignments of an average size of 60 amino acids is thus a good compromise . The sequences were then aligned with t - coffee (10), the alignment was used to construct a profile for each pattern (hereafter miniprofile) and the miniprofile was then calibrated on a randomized protein database . If a prosite profile was already associated with a pattern and was of better quality than the automatically generated one, we used the prosite profile to assign the status . Each automatically generated miniprofile was then tested on the corresponding pattern match - list and the cutoff calculated to recover all tp and no fp, and the maximum of fn . When a miniprofile did not recover all tp it was reconstructed with different profile construction parameters or different multiple sequence alignment programs (clustalw and probcons) (11,12). When it was not possible to recover all tp, the cutoff was set just above the highest fp . The rule is that none of the miniprofile should recover uniprotkb / swiss - prot proteins tagged as fp with the corresponding pattern and indeed 95% of the profiles recover 100% of the tp and only 65 profiles do not recover all tp . Since a miniprofile is always run on a subset of the database (proteins matched by the pattern) the e - value is always very low (<0.001). Even though miniprofiles were not designed to be scanned alone, and must be run on proteins matched by a pattern, 80% of the miniprofiles produce only matches with e - value bellow 0.01 (n_score = 9) on uniprotkb . This subset of miniprofiles is thus safe enough to be run alone on a database of proteins . In our search algorithm, miniprofiles are run only on proteins matched by patterns, and this procedure does not drastically impact the calculation time . It takes about 10 min on 1 cpu (pentium 4, 3.4 ghz) to run all the prosite patterns on the whole escherichia coli proteome (4339 protein sequences). It is also possible to download the different tools to use it locally from the prosite ftp site (ftp://ftp.expasy.org/databases/prosite/tools/). The sensitivity and specificity of descriptors can be enhanced by taking into account some contextual information, such as the co - occurrence of other domains, the position of a match in a protein, the taxonomic distribution, etc . Such information can be used to promote some weak matches or to demote some irrelevant strong matches . Prosite profiles normally use two cutoff levels, a reliable cutoff (level = 0) and a low confidence cutoff (level = 1). The low - level cutoff covers the twilight zone where few true positives that cannot be separated from false positives, might be present . By default, only matches higher than the reliable cutoff are shown . We have added a post - processing step in our scanning procedure, which allows the display of weak matches or the masking of strong ones according to the occurrence of other specific features in the protein . The required features for the post - processing step are stored in a new line type in the profile (pp). We have defined three types of post - processing: when at least two matches overlap, only the one that has the highest score is reported . This is mainly used when two or more families are very closely related, like the different types of hth dna - binding domains, or when it makes sense to define subfamilies of a larger protein family; for example the abc transporter family was subdivided into subfamilies to predict the type of transported substrate . The format is: pp /competes_hit_with: ps - accession;a weak match is promoted by the presence of another prosite profile in the protein . This may happen, for example, when two domains are known to be frequently associated, like for example the atp - binding helicase domain (ps51192) and the c - terminal helicase domain (ps51194). The format is: pp /promoted_by: ps - accession;a strong match is demoted by the presence of another domain in the protein . The format is: pp /demoted_by: ps - accession; when at least two matches overlap, only the one that has the highest score is reported . This is mainly used when two or more families are very closely related, like the different types of hth dna - binding domains, or when it makes sense to define subfamilies of a larger protein family; for example the abc transporter family was subdivided into subfamilies to predict the type of transported substrate . The format is: pp /competes_hit_with: ps - accession; a weak match is promoted by the presence of another prosite profile in the protein . This may happen, for example, when two domains are known to be frequently associated, like for example the atp - binding helicase domain (ps51192) and the c - terminal helicase domain (ps51194). The format is: pp /promoted_by: ps - accession; a strong match is demoted by the presence of another domain in the protein . The format is: pp /demoted_by: ps - accession; in a given entry, different pp line types can be combined . Prosite can now be browsed by taxonomic scope, by prorule description, by the number of positive hits or by matched proteins . We have reorganized the data presentation, which are now grouped into five different sections (scanprosite, prorule, documents, downloads and links) besides the home page . A new prorule section has been created, which allows the visualization of the different rules that are used to generate annotation on the the ftp site has also been reorganized, and new files can now be downloaded . As we have introduced prosite version numbers for each pattern / profile entries, we now distribute the old versions of prosite to allow the recovery of previous versions of entries . We also distribute a file that contains all the multiple sequence alignments of matched regions by patterns and profiles on uniprotkb / swiss - prot database . We have made available to prosite users our tool that generates domain images to represent protein architectures . For a given protein, the user can enter in a web form the size of the protein, the positions of the domains, their names and, for each domain, the colour and the shape of the wanted image . The web form returns an image in portable network graphics (png) format that can be integrated into any publication (figure 2). The tool is accessible at the following address: http://www.expasy.org / tools / mydomains/. Figure 2.the web form and the output of the prosite a very simple syntax allows the user to define the shape, colour, size and name of one or several domains . A very simple syntax allows the user to define the shape, colour, size and name of one or several domains . The scanprosite tool can be accessed programmatically through a simple web http service where the naked data (without any message exchange envelope) is retrieved directly as the content of an http query response (low rest service). When a client sends an http get or post query to the service; the response content will contain the results in xml or in the lightweight data - interchange format json (javascript object notation). For details see http://www.expasy.org/tools/scanprosite/scanprositerest.html . Note: to avoid timeout problems with long jobs, we will soon introduce a queuing system . It is also possible to access prosite entries in raw text / plain format using the following url: http://www.expasy.org/cgi-bin/get-prosite-raw.pl?pdoc-accession or prosite alignments: http://www.expasy.org/cgi-bin/aligner?psa=ps-accession.
Ureteric injury from herniorrhaphy is unusual . We present a case of ureteric injury complicating an inguinal herniorrhaphy for a huge right inguino - scrotal hernia . Case reports are important as they describe rare clinical conditions, highlight uncommon challenges or therapeutic adaptations . They can, however, draw attention to uncommon sequel of standard treatment to serve as caution in future practice . Ureteric injury following herniorrhaphy is rare .this is probably the first reported case from nigeria . We present a 66-year - old male farmer with right inguino - scrotal swelling noticed 10 years earlier which became irreducible 2 years prior to presentation . He had dysuria, urgency, urge incontinence, and feeling of incomplete emptying of the urinary bladder . He had a chronic dry cough and smoked two packets of cigarette per day in last 15 years . His respiratory rate, pulse rate and blood pressure were 22 cycle per minute, 100 beats per minute and 200/120 mmhg respectively . A provisional diagnosis of lower urinary tract infection (uti), incarcerated right inguino - scrotal hernia with bladder outlet obstruction due to prostatic enlargement was made . The complete blood count, fasting blood sugar, serum electrolytes, urea, and creatinine, prostate - specific antigen, urine microscopy, culture and sensitivity, chest x - ray, and elecrocardiography done were all normal . Ultrasonography revealed a thickened urinary bladder wall, bilateral pelvi - calyceal dilatation of kidneys but the ureters could not be visualized . Scrotal ultrasound reported a large cystic mass on the right extending into the groin and surrounded by loops of bowel . Intraoperative findings were: a right pantaloon hernia sac (the indirect sac containing an inflamed appendix about 16 cm in length, while the direct sac component contained grossly normal cecum and the urinary bladder). The right ureter was inadvertently transected during dissection which necessitated laparatomy and conversion from spinal anesthesia to general anesthesia . The patient had bassini - type right inguinal herniorrhaphy and reimplantation of the transected right ureter into the urinary bladder over a transvesical ureteric stent (figure 1 and 2) an urologist at the same session . Surgery lasted about 3 h. he developed features of systemic inflammatory response syndrome (sirs) which progressed to multiple organ failure culminating in death by the fourth day postsurgery . . Probable cause of death was septicemia with multiple organ failure . Showing the transected ureter held with a clamp . Showing the insertion of size 5 feeding tube as a stent postureter - neocystostomy . Groin hernia surgery demands sound knowledge of the anatomy of the region as it does the principles of good perioperative care . Complications may arise from groin hernia repair; these commonly include: hemorrhage, surgical site infection, numbness of groin, chronic pain, and abnormal scar . Injuries to gut and bladder can occur during repair of complicated hernias (incarcerated, obstructed or ischemic / gangrenous) and in hernia - en - glissade . The latter type was noted in our patient . Generally ureteric injuries are either caused by external violence, usually penetrating missiles, or more commonly from surgical misadventure . . The most common procedures are hysterectomy, salpingo - oophorectomy, vesico - urethral suspension, ureteroscopy, endo - pyelotomy, and uretero - lithotomy and surgical procedures on the great vessels and colon as well as retroperitoneal tumor excision . Lately, laparoscopic procedures have become a common cause of ureteric injuries . It is likely to occur if the ureter forms part of the sac or is contiguous with the spermatic cord as was seen in this patient . Ureteric herniation can either have a peritoneal covering (para - peritoneal herniation, 80%), or lacks peritoneal covering (extraperitoneal, 20%). Para - peritoneal ureteric hernia is common on the right, in males between fourth and sixth decades of life, and occurring in association with a sliding indirect hernia . The patient also had uti, symptomatic prostatic enlargement, and hypertension; all of which could promote adverse surgical outcome . Ureters within the hernia sac or contiguous to it can be identified as a thickened cord which exhibits peristalsis especially when stimulated, and contain fluid (urine) on test aspiration . The lower urinary symptoms (luts) noted in the patient could have been from the prostatic enlargement or from the involvement of the bladder in the hernia process or from the uti . The presence of prostatic enlargement with dilatation of proximal urinary tract as seen in our patient was previously reported by kate et al . . Preoperative diagnosis of groin hernia containing the ureter or bladder can be made with a micturating cystogram or an intravenous urogram . Early detection at time of injury and primary repair is said to confer better prognosis . Intraoperative localization of point of ureteric injury can be achieved using intravenous 5 ml of indigo carmine intravenously and observing its extravasations into the periureteric tissues . The diagnosis of a ureteric injury generally is not made until many days after the injury has occurred . Most common signs and symptoms seen in these patients are flank pain, fever, anuria (if bilateral), uretero - vaginal fistula, and uretero - cutaneous fistula . In these conditions, an excretory urogram or computerized tomography (ct) if the patient is scheduled for exploration or the presence of an injury is in doubt, then a retrograde ureterogram is strongly advised . Uretero - neocystostomy can be used to repair distal ureteric injuries that occur so close to the bladder thus precluding need for a psoas hitch or boari procedure . Mortality is said to be worse in the elderly, following emergency procedure and in the face of comorbid diseases . The mortality recorded may be due to his high preoperative cardio - pulmonary risk status (long - term smoking with chronic cough, and severe hypertension); long duration of surgery in an elderly patient who had spillage of potentially infected urine in to the surgical wound . The resultant septicemia from the latter coupled with the metabolic response to surgery in an already challenged cardio - pulmonary system could be enough to tip him into sirs and finally death from multiple organ failure . We cannot categorically state the primary cause of death; an autopsy would have helped elucidate this . Complete inguinal hernia may contain the lower urinary tract especially if the patient presents with luts . Preoperative radiological evaluation is advised in such cases to avoid injury to the ureter and/or bladder.
Serial analysis of gene expression (sage) is a powerful method for obtaining comprehensive and quantitative gene expression profiles from cell populations under selected physiological conditions . Sage counts polyadenylated transcripts by sequencing a short 14 bp tag at the gene's 3 end, adjacent to the last restriction site . The tag counts are than archived electronically for future analysis and comparisons . Since the first publication introducing sage (1), computational tools (27) and statistical methods (820) have been developed to correctly perform the analysis of a sage experiment . Because sage determines absolute expression levels, comparisons between different sage libraries are relatively easy to perform . For this reason, sage has been selected as the major platform technology for the cancer genome anatomy project (cgap). For 5 years, a large number of sage libraries, generated from diverse cancer and normal tissues in many laboratories, have been accessible via the national center for biotechnology information website (). The sage genie website () was constructed recently and is equipped with additional search and presentation tools (7). These libraries were all constructed by using the same anchoring enzyme, thus all yielding tags likely to be 10 bp downstream from the 3 most nlaiii site in the transcript . The aim of most sage studies is to identify genes of interest by comparing the number of specific tags found in two different sage libraries . One of the most attractive applications of transcript profiling is to address the question of expression differences between normal and cancer samples or cancer and metastasis samples in order to define new diagnostic markers and therapeutic targets . Over the past few years, several methods have been reported for determining the statistical significance of gene expression difference provided by the sage experiments . Most of these methods have been incorporated into public database systems and analysis programs (24,68,1013,1820). If within two types of cells y and z, a particular mrna species has unknown respective concentrations y and z and a total of a tags are sequenced from cell type y and b tags from cell type z, and among these, a and b tags, respectively, correspond to the mrna of interest, the question is: what inference may be made about the relative size of the actual concentrations, y and z? They consider the null hypothesis h0 that y = z, and the alternative that y z, and derive formulas based upon the observed data for rejecting h0 with various degrees of confidence . If a / a and b / b differ significantly, one rejects h0, concluding that the expression levels y and z are unequal . We have used their statistical approach and developed a useful and flexible tool (websage) that analyse and compare a large number of sage tags . The novelty of our tool compared with those available online (3,4) consists of the visualization of the results of the comparison of two sage libraries in a scatter plot . One of the libraries is presented on the x - axis and the second one on the y - axis . More than two libraries cannot be visualized using our tool unless to pool libraries composed by the sum of all libraries from one group such as cancer group and all libraries from normal group . However, vncio et al . (20) showed clearly that this summing is an error and proposed a statistical analysis that accounts for the within class variability . In our tool, full sage data are very comprehensively represented in plots, which correspond to one or a set of tags, having the same p - value and the same apparition in two libraries . Moreover, websage gives not only the identification but also the function of the gene . To gain insights into the molecular basis for metastasis, we compared the global gene expression profile of metastatic pancreatic cancer with that of primary cancer . The two cell lines were established from a 60-year - old woman with surgically removed pancreatic carcinoma that presented simultaneously metastatic liver lesion (21). We isolated total cellular rna from pancreatic cancer cells and metastatic liver cells and constructed each sage library, using sage protocol version 1.0c (1). Afterwards, tags were extracted from the raw sequence data and repeated ditags were excluded using the sage software (version 1) developed by kinzler and co - workers (1). From the two sage libraries 12 090 tags were obtained, enabling us to compare the expression levels of 4807 unique transcripts . At this step, the question of interest is whether one sample has a significant change in expression relative to the other sample for each transcript . Over the past few years, several methods have been developed for determining the statistical significance of gene expression difference derived from the sage experiments . Zhang et al . (17) used a simulation approach to determine the probability of obtaining the observed difference and more extreme ones . Other statistical approaches of the number of specific tags found in sage can be envisioned . (19) used a test for differences in tag numbers found in two experimental conditions, seemingly based on poisson distribution statistics . Their approach suffers from the disadvantage that it can only be used reliably on tag libraries of similar size . The second approach is to look at the number of copies of a specific mrna per cell as a fraction or proportion of the total number of mrna molecules in that cell . The same proportion (p) of specific tags should be present in the sage library of all sequenced tags . In this approach, the number of tags found is binomially distributed because it is the result of the p of each tag to be identified as being the specific mrna or not . A bayesian method has been used by audic and claverie (8) and vncio et al . Comparison of this test and the test based on poisson - distributed tag counts used by madden et al . (19) shows a difference in sensitivity, the test of madden et al . Being more conservative (10). To quantify the transcripts of the two cell line, we have used the significance test () of audic and claverie (8) and developed a tool to visualizing the analysis of differentially expressed tags in a scatter - plot shape (log / log coordinates). Websage is the software that enables a rapid and comprehensive analysis of the sage data and integrates statistical data analysis methods using a database system . Websage enables simple and rapid analysis of a file of tags and the determination of differentially expressed tags (p - values). To provide tag - to - gene links, three files from the ncbi ftp site (sage genie) were stored in database (mysql) and used at each analysis, to get the tag - to - gene assignment, gene accession number (unigene and genbank) and gene function information (kegg, biocarta and gene ontology databank), respectively . Sage genie provides the best match between gene name and tag, although there are options for manually viewing an alternate tag's expression data . Furthermore, the information is stored in a table and visualized in a scatter plot . Figure 1 shows an example of websage analysis of our file of tags . The total number of tags analysed included 5257 tags from pancreatic cancer cell line and 6833 from cell line derived from liver metastasis . Websage computed a p - value and classified the tags into three groups: green, p 0.01 (significant); red, p> 0.05 (not significant); and yellow, p in] 0.01; 0.05] (require further analysis). The information computed for the query is stored in a temporary table during the session . Next, the data are exported in the csv format (compatibility with all spreadsheets). Our application is a web tool, developed in the php language using jpgraph library () that has been modified ., websage is a useful web service that performs statistical analysis on the sage data, identifies the tags differentially expressed and shows the results in a scatter plot . Moreover, the user can query with a specific tag and get a tag - to - gene assignment and gene function from kegg, biocarta and gene ontology databank . A total of 4809 tags from pancreatic cancer lines and an isogenic liver metastasis cell line were analysed . Each plot corresponds to one tag or a set of tags, having the same p - value and the same apparition on the two libraries . . Once clicked, a web page is displayed with a list of tags and their identification . The identification consists of the name of the tag and the corresponding gene, the p - value and the unigene and genbank accession number.
Leprosy is a well - known chronic infectious disease that is caused by mycobacterium leprae (m. leprae). The incidence of leprosy has declined worldwide after the introduction of multidrug therapy (mdt) by the world health organization (who) in 1982;1 however, it is still a public health problem in many countries, which have a high rate of endemic infection.2 this was an indicator to modify or change who mdt, aiming at a more effective, safe, compliant, and shorter - duration regimen that is free from the fear of the emergence of resistant lepra bacilli.3 advances in, and deep understanding of, immunology, pathogenesis, and genetics of leprosy could improve the ability to fight against this potentially devastating infectious disease and may lead to the development of better protocols for treatment and prevention of the disease.4 the status of dermal vasculature was considered an important underlying pathogenic factor in leprosy, and the study of dermal vascular changes in leprosy was a matter of interest in previous reports.5 it was observed that there is narrowing, tortuosity, dilatation, or occlusion of the skin s blood vessels of lepromatous leprosy patients, especially those located in the extremities.6 it was proposed that there are two microvascular architectural patterns observed in cutaneous lesions of leprosy: a dense and tortuous mesh of microvessels among the granulomatous infiltrate in lepromatous leprosy and a microvessel network restricted to the periphery of the granulomas in tuberculoid leprosy.7 moreover, the proliferation and migration of endothelial cells result in the formation of new blood vessels from pre - existing vessels, a process that is known as angiogenesis, which has also been studied in leprosy . It was proposed that there is a significant correlation between angiogenesis and bacterial load in cutaneous lesions of lepromatous patients.8 in this study we assessed angiogenesis in lepromatous leprosy patients before and after treatment with mdt alone and in combination with minocycline, a drug known to have an antiangiogenic effect . Moreover, we correlated the changes in angiogenesis with the changes in bacterial density (bd) in the same cutaneous lesions . A multicenter study was carried out on 40 lepromatous leprosy patients recruited from the outpatient clinics of the dermatology departments in al - hussein, tanta, and al- zahraa university hospitals, egypt . All the patients included were newly diagnosed, and none of them had received any treatment for leprosy prior to the commencement of the study . The diagnosis of leprosy was based on the clinical presentation of bilateral, symmetrical, nonscaly erythematous nodular skin lesions, which was confirmed by the detection of m. leprae in direct nasal or slit skin smear . Exclusion criteria included pregnancy, lactation, known hypersensitivity to any of the therapeutic drugs, and serious illness or bad general condition . Written consent was signed by each patient after full explanation of the nature of the study, expected benefits of treatment, and possible side effects . Group a (n = 20) received who mdt in the form of monthly observed doses of rifampicin (600 mg) and clofazimine (300 mg) and an unobserved daily dose of dapsone (100 mg) and clofazimine (50 mg). Group b (n = 20) received who mdt therapy in addition to a monthly observed dose of minocycline (100 mg). A 4 mm punch biopsy was taken from each patient before treatment and 6 months after . The first biopsy was usually taken from the largest nodular lesion, and the second biopsy was taken from nearly the same site . From each specimen, one for routine hematoxylin and eosin staining for diagnosis and evaluation of the granulomatous reaction, one for modified ziehl - neelsen stain (wade - fite method) to record the bd (assessed by bacterial index [bi]), and two for immunohistochemical staining with anti - cd31 and anti - cd34 monoclonal antibodies to assess microvascular density (mvd). Immunohistochemical staining was performed using an avidin - biotin peroxidase complex method on formalin - fixed, paraffin - embedded tissue sections,9 using a 1/50 dilution of monoclonal cd31 and cd34 antibodies (dako, glostrup, denmark). Briefly, tissue sections were mounted on 3-aminopropyl - triethoxysilane - coated slides and dried overnight at room temperature . Subsequently, they were dewaxed in xylene and rehydrated in graded ethanol . After being rinsed with phosphate buffered saline, they were immersed in 0.01 mol / l citric acid titrated to ph 6.0 and heated twice for 10 minutes in a microwave oven . Diaminobenzidine was used as a chromagen, and the slides were counterstained with mayer s hematoxylin . Microvessel density was assessed by immunostaining for cd31 and cd34 according to weidner.10 areas with higher vascularity (so - called hot - spots) were located at low magnification (40) and then were counted at 400 magnification . Each positive endothelial cell or group of cells in contact with a spot was counted as an individual vessel . The mean vessel count from three fields was used as cd34 microvessel density or cd31 microvessel density . The assessment of both bd and mvd was performed only in the dermal granuloma without assessment of the stromal changes . Bd in each wade - fite stained specimen was assessed by bi following ridley s logarithmic scale (from 1 to 6). The number of solid staining (viable) lepra bacilli was visually calculated in ten different high power fields (hpf), and the mean standard deviation (sd) was recorded . For quantitative data, the mean and sd were calculated . Follow - up of the patients was done monthly for observation of the monthly doses of treatment and recording of any side effects, complications, or lepra reaction . We reported two patients in group a who developed erythema nodosum leprosum during the first month of treatment (2 and 3 weeks, respectively). The treatment was continued in both patients with the addition of oral corticosteroids (40 mg daily for 15 days then gradually withdrawn). All the patients included were newly diagnosed, and none of them had received any treatment for leprosy prior to the commencement of the study . The diagnosis of leprosy was based on the clinical presentation of bilateral, symmetrical, nonscaly erythematous nodular skin lesions, which was confirmed by the detection of m. leprae in direct nasal or slit skin smear . Exclusion criteria included pregnancy, lactation, known hypersensitivity to any of the therapeutic drugs, and serious illness or bad general condition . Written consent was signed by each patient after full explanation of the nature of the study, expected benefits of treatment, and possible side effects . Group a (n = 20) received who mdt in the form of monthly observed doses of rifampicin (600 mg) and clofazimine (300 mg) and an unobserved daily dose of dapsone (100 mg) and clofazimine (50 mg). Group b (n = 20) received who mdt therapy in addition to a monthly observed dose of minocycline (100 mg). A 4 mm punch biopsy was taken from each patient before treatment and 6 months after . The first biopsy was usually taken from the largest nodular lesion, and the second biopsy was taken from nearly the same site . From each specimen, one for routine hematoxylin and eosin staining for diagnosis and evaluation of the granulomatous reaction, one for modified ziehl - neelsen stain (wade - fite method) to record the bd (assessed by bacterial index [bi]), and two for immunohistochemical staining with anti - cd31 and anti - cd34 monoclonal antibodies to assess microvascular density (mvd). Immunohistochemical staining was performed using an avidin - biotin peroxidase complex method on formalin - fixed, paraffin - embedded tissue sections,9 using a 1/50 dilution of monoclonal cd31 and cd34 antibodies (dako, glostrup, denmark). Briefly, tissue sections were mounted on 3-aminopropyl - triethoxysilane - coated slides and dried overnight at room temperature . Subsequently, they were dewaxed in xylene and rehydrated in graded ethanol . After being rinsed with phosphate buffered saline, they were immersed in 0.01 mol / l citric acid titrated to ph 6.0 and heated twice for 10 minutes in a microwave oven . Diaminobenzidine was used as a chromagen, and the slides were counterstained with mayer s hematoxylin . Microvessel density was assessed by immunostaining for cd31 and cd34 according to weidner.10 areas with higher vascularity (so - called hot - spots) were located at low magnification (40) and then were counted at 400 magnification . Each positive endothelial cell or group of cells in contact with a spot was counted as an individual vessel . The mean vessel count from three fields was used as cd34 microvessel density or cd31 microvessel density . The assessment of both bd and mvd was performed only in the dermal granuloma without assessment of the stromal changes . Bd in each wade - fite stained specimen was assessed by bi following ridley s logarithmic scale (from 1 to 6). The number of solid staining (viable) lepra bacilli was visually calculated in ten different high power fields (hpf), and the mean standard deviation (sd) was recorded . Follow - up of the patients was done monthly for observation of the monthly doses of treatment and recording of any side effects, complications, or lepra reaction . We reported two patients in group a who developed erythema nodosum leprosum during the first month of treatment (2 and 3 weeks, respectively). The treatment was continued in both patients with the addition of oral corticosteroids (40 mg daily for 15 days then gradually withdrawn). Both patients showed significant relief of symptoms within 3 weeks . In group b, no significant side effects or complications were encountered during the treatment period . Their ages ranged from 25 years to 53 years with a mean of 32 4.5 years . Their ages ranged from 28 years to 49 years with a mean of 34 2.5 years . The duration of the disease ranged from 3 weeks to 11 months with a mean of 14 3 weeks . In 29 patients (72.5%) skin lesions were located on the trunk and extremities, whereas in eleven patients (27.5%) the lesions were distributed mainly on the extremities . Facial involvement was reported in 13 patients (32.5%) and nerve thickening in 27 patients (67.5%). M. leprae were detected in 16 patients (40%) by nasal smear and in 24 patients (60%) by slit skin smears . In group a, the mvd (number of vessels [v]/hpf) ranged from 35 v / hpf to 43 v / hpf with a mean of 39.1 3.1 before treatment, whereas after treatment the mvd decreased to 16.5 2.7 (ranged from 13 v / hpf to 19 v / hpf). This reduction was statistically highly significant (p <0.001) (table 1). In group b, the mvd ranged from 35 v / hpf to 46 v / hpf with a mean of 38.3 2.5 before treatment, which decreased to 7.6 1.9 after treatment (ranged from 5 v / hpf to 10 v / hpf). This reduction was statistically highly significant (p <0.001) (table 2). In group a, the mean mvd significantly decreased from 42.2 3.1 before treatment (figure 1a) to 18.8 2.4 after treatment (figure 1b) (table 1). In group b, the mean mvd significantly decreased from 43.7 2.3 before treatment (figure 2a) to 11.5 1.6 after treatment (figure 2b) (table 2). There was a statistically significant higher reduction (p <0.001) in angiogenesis evaluated by both cd31 and cd34 markers in group b compared with in group a (table 3). In group a, bi ranged from 4 (10100 bacilli / hpf) to 6 (> 1000 bacilli / hpf) with a mean of 5.1 0.4 before treatment, which was significantly decreased to 2.3 0.4 after treatment (table 1). In group b, the mean bi significantly decreased from 4.9 0.3 before treatment (figure 3a) to 1.4 0.2 after treatment (figure 3b) (table 2). There was a statistically significant higher reduction (p <0.001) in bd in group b compared with in group a (table 3). Their ages ranged from 25 years to 53 years with a mean of 32 4.5 years . Their ages ranged from 28 years to 49 years with a mean of 34 2.5 years . The duration of the disease ranged from 3 weeks to 11 months with a mean of 14 3 weeks . In 29 patients (72.5%) skin lesions were located on the trunk and extremities, whereas in eleven patients (27.5%) the lesions were distributed mainly on the extremities . Facial involvement was reported in 13 patients (32.5%) and nerve thickening in 27 patients (67.5%). M. leprae were detected in 16 patients (40%) by nasal smear and in 24 patients (60%) by slit skin smears . In group a, the mvd (number of vessels [v]/hpf) ranged from 35 v / hpf to 43 v / hpf with a mean of 39.1 3.1 before treatment, whereas after treatment the mvd decreased to 16.5 2.7 (ranged from 13 v / hpf to 19 v / hpf). This reduction was statistically highly significant (p <0.001) (table 1). In group b, the mvd ranged from 35 v / hpf to 46 v / hpf with a mean of 38.3 2.5 before treatment, which decreased to 7.6 1.9 after treatment (ranged from 5 v / hpf to 10 v / hpf). This reduction was statistically highly significant (p <0.001) (table 2). In group a, the mean mvd significantly decreased from 42.2 3.1 before treatment (figure 1a) to 18.8 2.4 after treatment (figure 1b) (table 1). In group b, the mean mvd significantly decreased from 43.7 2.3 before treatment (figure 2a) to 11.5 1.6 after treatment (figure 2b) (table 2). There was a statistically significant higher reduction (p <0.001) in angiogenesis evaluated by both cd31 and cd34 markers in group b compared with in group a (table 3). In group a, the mvd (number of vessels [v]/hpf) ranged from 35 v / hpf to 43 v / hpf with a mean of 39.1 3.1 before treatment, whereas after treatment the mvd decreased to 16.5 2.7 (ranged from 13 v / hpf to 19 v / hpf). This reduction was statistically highly significant (p <0.001) (table 1). In group b, the mvd ranged from 35 v / hpf to 46 v / hpf with a mean of 38.3 2.5 before treatment, which decreased to 7.6 1.9 after treatment (ranged from 5 v / hpf to 10 v / hpf). This reduction was statistically highly significant (p <0.001) (table 2). In group a, the mean mvd significantly decreased from 42.2 3.1 before treatment (figure 1a) to 18.8 2.4 after treatment (figure 1b) (table 1). In group b, the mean mvd significantly decreased from 43.7 2.3 before treatment (figure 2a) to 11.5 1.6 after treatment (figure 2b) (table 2). There was a statistically significant higher reduction (p <0.001) in angiogenesis evaluated by both cd31 and cd34 markers in group b compared with in group a (table 3). In group a, bi ranged from 4 (10100 bacilli / hpf) to 6 (> 1000 bacilli / hpf) with a mean of 5.1 0.4 before treatment, which was significantly decreased to 2.3 0.4 after treatment (table 1). In group b, the mean bi significantly decreased from 4.9 0.3 before treatment (figure 3a) to 1.4 0.2 after treatment (figure 3b) (table 2). There was a statistically significant higher reduction (p <0.001) in bd in group b compared with in group a (table 3). The major role of angiogenesis was reported in relation to neoplastic conditions and malignancies that need an ongoing blood supply to grow and expand.11 abnormal angiogenesis was also implicated in other conditions such as rheumatoid arthritis, inflammation, and degenerative eye conditions, in addition to many biological processes such as development, reproduction, and wound repair.12 in dermatological disorders, angiogenesis was implicated mainly in psoriasis,13 and to a lesser extent in skin aging and photoaging.14 drugs that inhibit angiogenesis were used primarily in the treatment of malignancy . The role of these antiangiogenic or antivascular therapies in the treatment of cancer became important after the establishment of the relationship between angiogenesis and tumor growth.15 inhibition of angiogenesis growth factors and transfer of antiangiogenesis genes were proposed as basic mechanisms of antiangiogenic drugs.16 the target receptors of antiangiogenic drugs include ptdins- 4,5-p2 that regulate vessel stability,17 vascular endothelial growth factor receptor key proteins,18 and cap43 calcium - inducible genes.19 there are certain drugs which have an antiangiogenic effect and are used in the treatment of some skin diseases, such as chloroquine, which was suggested to have a beneficial effect in the treatment of discoid lupus erythematosus due to its antiangiogenic properties.20 thalidomide also has an antiangiogenic effect and is currently used in the treatment of different dermatological conditions including lepra reaction,21 sclerodermatous cutaneous reaction of graft versus host disease,22 and jessner s lymphocytic infiltration of the skin.23 recently, rifampicin, which is a component of mdt for leprosy, was suggested to have promising antiangiogenic effects that may enable its use as an effective antitumor agent.24 minocycline was also reported as one of the drugs that inhibit angiogenesis,25 and it is known to have an important role in the treatment of leprosy as a main component of rifampicin, ofloxacin, and minocycline (rom) therapy.26 however, the mechanism of action of both drugs in the treatment of leprosy remains unclear . Is it due to their antimicrobial effect only or due also to their antiangiogenic effect? To our knowledge, no previous studies have evaluated the efficacy of these antiangiogenic drugs on angiogenesis in cutaneous lesions of lepromatous leprosy patients, but a few studies monitor angiogenesis in the spectrum of leprosy . Bhandarkar et al8 studied angiogenesis in 32 leprosy patients using cd31 as a marker for microvessel density . The mvd of lepromatous leprosy lesions was (44.0 9.8 v / hpf). We found a lower mvd value with a cd31 marker in lepromatous leprosy (39.1 3.1 in group a and 38.3 2.5 in group b), and with a cd34 marker we found a higher value (42.2 3.1 in group a and 43.7 2.3 in group b), which was close to that encountered by bhandarkar et al . This may suggest the benefit of using more than one marker (cd31, cd34) in the assessment of angiogenesis . Kim et al27 also found a significant increase of mvd in lepromatous leprosy lesions compared with in normal skin, with an overall increase in the mean number of vessels from tuberculoid leprosy through borderline (tuberculoid and lepromatous) to lepromatous leprosy lesions . The mvd values in lepromatous leprosy were higher (104.40 27.71) compared with our results . This can be explained by the use of a different vascular marker (factor viii - related antigen) or a different counting system . These studies suggested that angiogenesis could be evaluated by using different vascular markers . In the current study we preferred to use two markers (cd31 and cd34) in the assessment of angiogenesis for proper evaluation of mvd and to minimize false positive results . Both cd31 and cd34 are known as effective markers in identifying blood vessels and were proposed also to be good prognostic markers of neoangiogenesis.28,29 the significant reduction of mvd and bd in group a, which was treated with mdt, may prove the possible anti - angiogenic effect of rifampicin in addition to its antimicrobial effect . Rifampicin was suggested to exert its antiangiogenic effect through inhibition of the expression of angiogenesis - associated genes with downregulation of proangiogenic genes and inhibition of microvascular endothelial cell proliferation.24 in a manner comparable with the action of endostatin, the endogenous angiogenesis inhibitor is known to downregulate a variety of growth and angiogenesis - related genes in a wide range of endothelial lineage cells.30 in group b, minocycline 100 mg, which is known to have antiangiogenic properties, was added to mdt as a monthly dose, aiming to potentiate the antiangiogenic efficacy of the classic mdt regimen . Although minocycline has different mechanisms of action from its antimicrobial properties, such as inhibition of collagenase activity,31 and its anti - inflammatory effect through reduction of cytokines and pro - inflammatory protein expression,32 it also possesses inhibitory properties of angiogenesis.25 this antiangiogenic effect of minocycline may contribute to the direct inhibition of matrix metalloproteinase activity and also to the inhibition of vascular endothelial growth factor - induced smooth muscle cell migration.33 in the present work the reduction of mvd was significantly higher in group b than in group a using both markers . These results may suggest the presence of a synergistic effect of both minocycline and rifampicin as antiangiogenic drugs in addition to their antimicrobial effect . Moreover, the absence of any side effects or lepra reaction in group b compared with in group a may add a new benefit for the combination of mdt with minocycline . Another important finding in this work is the significantly higher reduction in bi in group b compared with in group a. this may suggest that the addition of minocycline to the mdt can cause a more rapid elimination of lepra bacilli from cutaneous lesions of lepromatous leprosy patients . This rapid elimination of lepra bacilli from cutaneous lesions and also from mucous membranes is helpful in decreasing the transmission of the disease and maintaining the standard target of elimination of leprosy . To our knowledge, this is the first work that assesses angiogenesis in leprosy patients after treatment with antiangiogenic drugs . The synergistic antiangiogenic effect of both minocycline and rifampicin was found to be promising and effective in the treatment of leprosy . Moreover, this combination showed an advantage of rapid elimination of m. leprae from the cutaneous lesions of lepromatous leprosy patients and decreased the possibility of the occurrence of lepra reaction . We hope that this regimen will be considered in wide - scale studies, especially in endemic areas of leprosy.
Toxicity from chloroform can include central nervous system depression, cardiac arrhythmias, hepatic injury, and hepatic cancer . Most cases of chloroform toxicity are the result of inhalational exposures, and very few reports involve oral ingestions . Since this route of exposure is exceedingly rare, information regarding the clinical course and management dilemmas of chloroform ingestions is limited . We describe the successful treatment of an acute chloroform ingestion with intravenously administered n - acetylcysteine (nac) despite having one of the highest reported serum chloroform levels in a survivor . A 19-year - old african - american man was found unconscious in his car with a suicide note and a half - empty bottle of chloroform he had ordered on the internet from overseas (fig . 1). He was brought to the emergency department obtunded and required immediate endotracheal intubation and mechanical ventilation . It was later determined that he had drank about 75 ml of the chloroform prior to his being discovered . His initial vital signs included a heart rate of 75 beats / min and a blood pressure of 113/60 mm hg; he was mechanically ventilated at a rate of 16 breaths / min with an oxygen saturation of 100% on 100% fio2 . The patient had an ecg performed, which did not reveal any signs of ischemia with a qrs of 86 ms and qt of 408 ms . Blood drawn on arrival revealed a glucose of 135 mg / dl, ast of 18 iu / l, alt of 10 iu / l, total bilirubin of 1.3 mg / dl, and direct bilirubin of 0.1 mg / dl; pt was 13.6 s (inr 1.16) and had normal serum electrolytes, bun, and creatinine . His admission serum ethanol concentration was 15 mg / dl, and serum salicylate and acetaminophen concentrations were undetectable . Fig . 1a half - empty bottle of chloroform a half - empty bottle of chloroform the poison center was notified by ems upon finding the patient with bottle of chloroform, allowing us to evaluate the patient at bedside upon arrival in the emergency department . In light of chloroform s hepatotoxicity, therapy with nac was recommended . The patient was treated with intravenously administered nac 150 mg / kg over 1 h, followed by 50 mg / kg over 4 h, and then was started on an intravenous drip at a rate of 6.25 mg kg h. on the evening of the second hospital day, the patient had regained consciousness and was successfully extubated . Prior to extubation, the patient completed 21 h of intravenously administered nac finishing the infusion the pharmacy had prepared . Because the patient was still intubated when the first infusion of nac was completed, the decision was made to continue nac at 6.25 mg kg h. we had planned on stopping the intravenously administered nac when the second pre - mixed infusion was finished; however, on hospital day 3, his total bilirubin began to rise although his ast and alt remained normal . Since information on the clinical course of chloroform ingestions is limited, we continued nac therapy . On hospital day 4, his serum ast and alt began to rise . His total serum bilirubin and pt both peaked on hospital day 4 at 16.3 mg / dl and 21.3 s (inr 2.25), respectively (fig . 2). On hospital day 5, his ast and alt had peaked at 224 and 583 at that time, his total serum bilirubin had fallen to 4.1 mg / dl, and his pt had declined to 17.7 s (inr 1.87). The following day, his serum bilirubin, ast, and alt all began to decline, and intravenously administered nac therapy was stopped . By hospital day 8, the patient s ast had fallen to 47 iu / l, his alt had fallen to 231 iu / l, his total serum bilirubin was 1.1 mg / dl, and his pt had normalized . His serum chloroform concentration on admission was later determined to be 91 g / ml . Fig . Like all halogenated hydrocarbons, chloroform exerts its anesthetic effects through activation of the gabaa receptor, causing an influx of chloride ions, hyper - polarizing the neuron and thereby clinically inducing sedation . Most fatalities are believed to be the result of anoxia secondary to deep sedation and impairment of airway reflexes and respiratory drive [1, 3]. Other observed effects of these agents can include sensitization of the myocardium to catecholamines, ventricular arrhythmias, and delayed hepatotoxicity resulting from free radical damage to the centrilobular hepatocytes [1, 3]. Besides the above clinical findings, oral exposure to chloroform can also cause direct irritation of the oral, esophageal, and gastro - intestinal mucosa . Associated symptoms include chest pain, abdominal pain, nausea, and vomiting . If aspirated, chloroform can also cause symptoms of acute pneumonitis and acute lung injury . Intravenously administered nac therapy is commonly used for intubated patients in our institution . After the decision to use intravenously administered nac, we had limited information on ingestions of chloroform to predict the duration of hepatotoxicity . His significant elevation of bilirubin on day 4 was surprising given that fact that his transaminases had just started to rise . The nac regimen used in this case was the identical protocol used in acetaminophen toxicity . Due to continued abnormalities with his liver function testing, we continued the regimen of 6.25 mg kg h of intravenously administered nac therapy through hospital day 6 until the hepatotoxicity had clearly improved . Case reports of oral ingestions of chloroform have been previously described [1, 3 - 5]. Other than the direct mucosal irritant effects described above, oral chloroform toxicity is believed similar to that of inhaled chloroform [1, 3, 4]. These previous case reports demonstrated average blood concentrations in fatal chloroform poisonings to have ranged between 33 and 64 g / ml . These levels are significantly lower than that of our patient who went on to have a complete recovery [1, 6]. The utilization of nac for chloroform - induced hepatotoxicity has demonstrated successful outcomes in cases with mild hepatotoxicity [5, 7]. However, none of these previous cases utilized the intravenous formulation of nac . Because chloroform is believed to cause hepatic damage by free radical injury and nac is known to replete glutathione and scavenge free radicals, it is logical that both formulations of nac may decrease hepatic injury secondary to chloroform exposure [8, 9]. A recent report describes a case of both chloroform and dichloromethane ingestion who survived without nac therapy . In this case, chloroform levels were not reported, but liver function tests peaked with an ast of 1,617 iu / l and alt of 2,677 iu / additionally, the total bilirubin peaked at 7 mg / dl, and they report that liver function tests took 4 weeks to return within normal limits . Without knowing the chloroform level and further clinical information, it is difficult to quantify the effects of nac therapy . However, our patient presented with a chloroform level well above reports of previous fatalities, and his ast and alt peaked at only 224 and 583 iu / l, respectively . Considering that nac therapy has such low risk and high theoretical benefit, it should be considered for use in chloroform and other halogenated hydrocarbon - induced hepatotoxicity . In summary, chloroform can cause injury resulting in central nervous system depression, respiratory depression, direct mucosal irritation, myocardial sensitization, and hepatotoxicity . Aggressive treatment with supportive care, with emphasis on protecting the airway and ventilation, as well as the use of nac therapy to treat hepatic injury, can result in complete recovery even with significant elevated chloroform levels.
In 2006, national institute for health and clinical excellence (nice) recommended laparoscopic surgery as an alternative to open surgery for colorectal resections . The bowel cancer report commissioned by the cancer services coordinating group revealed that in wales only 9.5% of colorectal resections were performed laparoscopically between 2005 and 2007 . In 2007, the association of laparoscopic surgeons of great britain and ireland set up the national training programme (lapco), intended to train existing consultant surgeons . Although the welsh assembly government did set aside some funds for laparoscopic colorectal training in wales in 2008, this was used mainly to develop an immersion course for existing consultants and a laboratory based training scheme for registrars, delivered from one centre only . There was no proposal for an outreach preceptorship style programme to deliver training and support at the point of service delivery . At this time, setting up such a service would require team training with a focus on developing not only the operating surgeon, but also the rest of the team including the anaesthetist and nurses . A single experienced surgeon (preceptor) introduced a preceptorship programme aimed at supporting existing consultants (preceptees) wishing to develop laparoscopic colorectal surgery (lcs) services at their centre . Preceptees were expected to have attended relevant courses, workshops (including live animal laboratory) and be able to demonstrate management support for a new service development . The programme consisted initially of a minimum 1 day master class at the preceptor's operating theatre attended by the preceptee and his team where they would observe 2 - 3 laparoscopic colorectal resections . This was to focus primarily on team development and to facilitate rapport between the preceptor and preceptee . This was followed by several visits by the preceptor to the preceptee's hospital to assist in cases . At the first outreach visit the preceptor was accompanied by his own middle grade and theatre nurse . This was to ensure competent assistance and to supervise and support the nursing team, leaving the preceptor free to concentrate on training the consultant surgeon during the case . The preceptor and his team were all contracted on an honorary basis by the preceptee's hospital for each visit . The number of outreach visits and supervised cases was dependent on the individual preceptee's progress and stage in their own learning curve . Each case was performed on the basis of a standard step - wise approach to each resection . Patient safety and duration of procedure were paramount considerations and were maintained under control by the preceptor . Preceptorship was tapered at each visit, varying from the preceptor being scrubbed for the entire procedure to providing verbal support only . Case selection was discussed prior to each visit, including a review of relevant pre - operative investigations by the preceptor and the initial cases were standard right hemicolectomy and high anterior resection with progression to more challenging cases as the preceptee gained confidence . This programme was funded through an educational sponsorship from johnson and johnson (ethicon endosurgery). A single experienced surgeon (preceptor) introduced a preceptorship programme aimed at supporting existing consultants (preceptees) wishing to develop laparoscopic colorectal surgery (lcs) services at their centre . Preceptees were expected to have attended relevant courses, workshops (including live animal laboratory) and be able to demonstrate management support for a new service development . The programme consisted initially of a minimum 1 day master class at the preceptor's operating theatre attended by the preceptee and his team where they would observe 2 - 3 laparoscopic colorectal resections . This was to focus primarily on team development and to facilitate rapport between the preceptor and preceptee . This was followed by several visits by the preceptor to the preceptee's hospital to assist in cases . At the first outreach visit the preceptor was accompanied by his own middle grade and theatre nurse . This was to ensure competent assistance and to supervise and support the nursing team, leaving the preceptor free to concentrate on training the consultant surgeon during the case . The preceptor and his team were all contracted on an honorary basis by the preceptee's hospital for each visit . The number of outreach visits and supervised cases was dependent on the individual preceptee's progress and stage in their own learning curve . Each case was performed on the basis of a standard step - wise approach to each resection . Patient safety and duration of procedure were paramount considerations and were maintained under control by the preceptor . Arrangements for clinical governance considerations at each hospital were met by the preceptee . Preceptorship was tapered at each visit, varying from the preceptor being scrubbed for the entire procedure to providing verbal support only . Case selection was discussed prior to each visit, including a review of relevant pre - operative investigations by the preceptor and the initial cases were standard right hemicolectomy and high anterior resection with progression to more challenging cases as the preceptee gained confidence . This programme was funded through an educational sponsorship from johnson and johnson (ethicon endosurgery). The aim of our study was to evaluate the early experience of this programme with regards to the clinical outcome as well as preceptee satisfaction . A prospective database was maintained to record cases performed as a part of master class or opp . The programme remains on - going, but for the purpose of this paper data from the start of the programme (may 2008) until july 2010 has been analysed . Follow - up data including mortality, morbidity and length of stay was obtained through postal / telephone questionnaire . In addition, two neutral observers (the first and second authors) used a structured questionnaire to interview each preceptee surgeon either in person or through the post, to obtain some feedback about the programme . Between may 2008 and july 2010, 11 consultants were inducted and 66 cases (20 in master class, 46 as an outreach service) were performed as a part of this programme . Right hemicolectomy was the most commonly performed operation (n = 24), but cases also included complex major procedures such as panproctocolectomy and total colectomy for consultants who had already been performing some lcs prior to the programme [table 1]. Cases mix in the master class and outreach service distribution of preceptees with number of cases and preceptor's visits in total there were 12 intra - operative events (18%) that could have led to conversion [table 3]. All but one of these events however were managed laparoscopically, therefore conversion rate was low at 1.5% (1/66). This one conversion seen in our series to date was due to difficulty identifying the left ureter during an anterior resection . Significant intra - operative events with possibility of conversion overall 30-day mortality was 1.5% [table 4]. This was due to a delayed anastomotic leak followed by multi - organ failure in a patient who had a low anterior resection after neo - adjuvant therapy . There were three anastomotic leaks, one of which occurred at day 20, resulting in the mortality mentioned above . Another patient required a secondary hartmann's procedure and the third patient was clinically well and managed conservatively . There were two cases of post - operative bleeding, one (part of the master class) managed with a laparoscopic washout and the other by a laparotomy (part of the opp). Morbidity and mortality during master class and opp median length of hospital stay was 6 days (range: 2 - 30). All the patients who underwent lcr for cancer had clear margins with a median lymph node harvest of 11.5 (range: 2 - 23). One patient undergoing a low anterior resection had a rectal perforation, the site of which was well clear of the tumour . To evaluate the programme, 11 of the preceptee consultant surgeons from five hospitals all the respondents found the master class and outreach service to be well organised and all would recommend the service to their colleagues . The four consultants who were already practising some lcs felt that the programme had improved their practice with regards to safety and confidence . Following the programme, all preceptored consultants have performed laparoscopic cases independently . When the respondents were asked to rate various courses available for developing lcs skills on a visual analogue scale (0 - 10), the median score for animal workshops (5.5), master class alone (7.0), websurg (7.0), immersion course (8.0) were all less than the score for the opp (8.5). All the 11 respondents found the master class to be well organised, with a good case - mix and felt that the preceptor demonstrated all relevant steps and answered all their queries . They found the direct observation and interaction on a one - to - one basis extremely beneficial . All 11 respondents felt that the preceptor was approachable for discussing case selection and was flexible with dates of visit . They also found the preceptor to be supportive, encouraging them to perform as much of each procedure as safely possible . 9 of the 11 preceptored consultants felt the number of outreach visits to be the right amount, while two felt it was less than they required . One of the two surgeons is very senior and close to retirement, hence the uptake of the laparoscopic approach has been relatively slow . All the other preceptee surgeons have been undertaking standard right and left side resections routinely and some have progressed to complex, benign and multi - segmental resections, there was unanimous agreement amongst the preceptored consultants that the structured programme had helped improve their practice and all would recommend this programme to their colleagues . Between may 2008 and july 2010, 11 consultants were inducted and 66 cases (20 in master class, 46 as an outreach service) were performed as a part of this programme . Right hemicolectomy was the most commonly performed operation (n = 24), but cases also included complex major procedures such as panproctocolectomy and total colectomy for consultants who had already been performing some lcs prior to the programme [table 1]. Cases mix in the master class and outreach service distribution of preceptees with number of cases and preceptor's visits in total there were 12 intra - operative events (18%) that could have led to conversion [table 3]. All but one of these events however were managed laparoscopically, therefore conversion rate was low at 1.5% (1/66). This one conversion seen in our series to date was due to difficulty identifying the left ureter during an anterior resection . Significant intra - operative events with possibility of conversion overall 30-day mortality was 1.5% [table 4]. This was due to a delayed anastomotic leak followed by multi - organ failure in a patient who had a low anterior resection after neo - adjuvant therapy . There were three anastomotic leaks, one of which occurred at day 20, resulting in the mortality mentioned above . Another patient required a secondary hartmann's procedure and the third patient was clinically well and managed conservatively . There were two cases of post - operative bleeding, one (part of the master class) managed with a laparoscopic washout and the other by a laparotomy (part of the opp). Morbidity and mortality during master class and opp median length of hospital stay was 6 days (range: 2 - 30). All the patients who underwent lcr for cancer had clear margins with a median lymph node harvest of 11.5 (range: 2 - 23). One patient undergoing a low anterior resection had a rectal perforation, the site of which was well clear of the tumour . To evaluate the programme, 11 of the preceptee consultant surgeons from five hospitals were interviewed . All the respondents found the master class and outreach service to be well organised and all would recommend the service to their colleagues . The four consultants who were already practising some lcs felt that the programme had improved their practice with regards to safety and confidence . Following the programme, all preceptored consultants have performed laparoscopic cases independently . When the respondents were asked to rate various courses available for developing lcs skills on a visual analogue scale (0 - 10), the median score for animal workshops (5.5), master class alone (7.0), websurg (7.0), immersion course (8.0) were all less than the score for the opp (8.5). All the 11 respondents found the master class to be well organised, with a good case - mix and felt that the preceptor demonstrated all relevant steps and answered all their queries . They found the direct observation and interaction on a one - to - one basis extremely beneficial . All 11 respondents felt that the preceptor was approachable for discussing case selection and was flexible with dates of visit . They also found the preceptor to be supportive, encouraging them to perform as much of each procedure as safely possible . 9 of the 11 preceptored consultants felt the number of outreach visits to be the right amount, while two felt it was less than they required . One of the two surgeons is very senior and close to retirement, hence the uptake of the laparoscopic approach has been relatively slow . All the other preceptee surgeons have been undertaking standard right and left side resections routinely and some have progressed to complex, benign and multi - segmental resections, there was unanimous agreement amongst the preceptored consultants that the structured programme had helped improve their practice and all would recommend this programme to their colleagues . All the 11 respondents found the master class to be well organised, with a good case - mix and felt that the preceptor demonstrated all relevant steps and answered all their queries . They found the direct observation and interaction on a one - to - one basis extremely beneficial . All 11 respondents felt that the preceptor was approachable for discussing case selection and was flexible with dates of visit . They also found the preceptor to be supportive, encouraging them to perform as much of each procedure as safely possible . 9 of the 11 preceptored consultants felt the number of outreach visits to be the right amount, while two felt it was less than they required . One of the two surgeons is very senior and close to retirement, hence the uptake of the laparoscopic approach has been relatively slow . All the other preceptee surgeons have been undertaking standard right and left side resections routinely and some have progressed to complex, benign and multi - segmental resections, there was unanimous agreement amongst the preceptored consultants that the structured programme had helped improve their practice and all would recommend this programme to their colleagues . Since nice recommended laparoscopic surgery as an alternative to open surgery for colorectal cancer, there has been a steady increase in the uptake of lcs across the uk . The absence of formal training and hence the initial slow uptake of lcs in wales has been addressed by this preceptorship programme . The unique points of this programme are its flexibility to provide preceptees the opportunity to be trained in selected cases, in their own hospital environment and with their own team . Various recommendations have been made to maintain operator competence, such as a frequency of at least two procedures per month or 24 annually . A questionnaire survey of association of coloproctology of great britain and ireland (acpgbi) members in 2008 revealed that only 25% of colorectal cancers were being treated laparoscopically . This percentage is variable however with some centres reporting figures as high as 90% for elective laparoscopic colorectal resections . The concept of preceptorship itself is not new . Even in the context of lcs in the uk, this was established in 2004 under the auspices of the specialist associations of laparoscopic surgery and coloproctology . The model of the programme we describe in this paper is similar in its structure except for the avoidance of any compulsion on the preceptee on achieving specific case numbers . The authors also believe the hierarchical guidelines developed by the council of the colorectal surgical society of australia and new zealand to be too rigid, with limitations imposed by case selection etc . This not only prolongs the learning curve but also limits the ability to tailor training depending on an individual surgeon's ability . This programme, therefore, allows specific one to one tutoring, facilitating the progress of each individual preceptee at his / her own pace . Although in- house preceptorship is ideal, having been shown to improve lcs workload, very few units in the uk can offer such opportunity at the present time . As per the acpgbi / association of laparoscopic surgeons of great britain and ireland programme, a preceptor should have performed a minimum of 100 laparoscopic resections . We however felt that though this may be sufficient to train registrars, a preceptor may need greater experience before taking on the training of consultant peers . Registrar training in a consultant's own operating theatre is quite different to training a consultant colleague in an alien environment with unfamiliar staff, necessitating a different skillset including a greater level of confidence and good people management skills . Immersion courses are aimed at training the surgeon to operate in a controlled environment at the trainers' centre of excellence . Even though some immersion courses offer team training, it remains different to training at the point of service delivery . This paper also highlights the fact that even during the early stages of a preceptee's learning curve; intra - operative problems can be surmounted satisfactorily with appropriate guidance from a skilled preceptor as demonstrated by a relatively low rate of conversion of 1.5% . Our preliminary evaluation of this structured training programme reveals that lcs can be developed with acceptable levels of morbidity, mortality and conversion rates . This finding is confirmed with a previous publication by araujo et al . Which has shown comparable outcomes between case matched series of operations performed by preceptors independently and preceptees being trained . Our survey has demonstrated that all the preceptees valued the presence of experienced theatre staff accompanying the preceptor at the first outreach visit . Not all anaesthetists (2/11) attended the master class and hence it is not surprising that most surgeons (7/11) felt the programme had not materially changed anaesthetic practice in their hospital . The fact that all of the preceptored consultants were satisfied with the programme and would recommend it to colleagues is very encouraging . The number of visits varied between individuals with the median number of visits per hospital being five . The lapco programme has specified that a minimum of 20 cases (an arbitrary number) would be required to reach a level of competence . Our programme is one of facilitation and not accreditation, which reflects the difference in case numbers performed by each preceptee and also highlights its flexibility to accommodate consultants with varying degrees of expertise . Lcs includes a range of operations with different levels of technical difficulty as well as wide ranging complexity due to different pathological processes such as inflammatory bowel disease, complicated diverticular disease and malignancy . Hence, traditional indicators such as the number of cases operated, conversion rates, mortality and morbidity are not reliable indicators for the assessment of competence . The programme described in this paper has the flexibility to offer subsequent visits by the preceptor as and when the preceptee starts undertaking more complex cases and indeed, some of the preceptees in this series have already requested a second round of preceptorship . The most important aspect of this programme is that training is not continuous but intermittent . Once a preceptee feels confident to perform simpler colorectal resections, he can then be preceptored to perform more complex cases . As seen from our results, one preceptee has performed a laparoscopic panproctocolectomy as part of this programme . In our programme, the median number of cases per preceptee was only five, which is not enough to gain competency in lcs . The purpose of this programme was to try and combine the existing skills of consultant colleagues in general laparoscopic as well as open colorectal surgery . Competence (by providing data regarding their lcs workload, conversion rates, complications etc .) As such an analysis would be a breach of the principles underpinning this programme, which was established as one of facilitation and not aimed at achieving this programme is an excellent example of partnership between the national health service (nhs) and a private provider (johnson and johnson) with the funding serving to backfill the preceptor's absence from his own nhs commitments and to support research and educational activities in the preceptor's department . In our programme, the median number of cases per preceptee was only five, which is not enough to gain competency in lcs . The purpose of this programme was to try and combine the existing skills of consultant colleagues in general laparoscopic as well as open colorectal surgery . Competence (by providing data regarding their lcs workload, conversion rates, complications etc .) As such an analysis would be a breach of the principles underpinning this programme, which was established as one of facilitation and not aimed at achieving this programme is an excellent example of partnership between the national health service (nhs) and a private provider (johnson and johnson) with the funding serving to backfill the preceptor's absence from his own nhs commitments and to support research and educational activities in the preceptor's department . This opp has been shown to be effective in achieving safe transference of skills to surgeons wishing to develop a service for lcs in their own departments, with acceptable levels of morbidity and mortality . Such a programme, delivering one - to - one training at the point of service delivery, may play a vital role in making safe lcs available widely.
Acute - onset postoperative endophthalmitis is characterized by marked inflammation of intraocular fluids and tissues . Gram - negative bacteria are less commonly isolated than gram - positive bacteria in patients with acute - onset postoperative endophthalmitis . Gram - negative organisms have been isolated in 26%42% of patients with cataract surgery related to endophthalmitis in developing countries,1,2 as compared with 5.9%11.8% in developed countries.3 the more common gram - negative organisms causing endophthalmitis include species of pseudomonas, haemophilus, proteus, and klebsiella . There have been reports of multidrug - resistant strains of gram - negative bacteria being isolated from patients with endophthalmitis.4 endophthalmitis caused by klebsiella pneumoniae is associated with generally poor visual outcomes, despite treatment with appropriate antibiotics.5 three cases of acute postoperative endophthalmitis due to multidrug - resistant k. pneumoniae are reported . Three patients developed acute - onset endophthalmitis within 13 days following cataract surgery (table 1). All the patients were in good general health and were not known to be suffering from any known systemic illness in the pre and postoperative periods . Presenting visual acuity ranged from 20/50 to light perception . Hypopyon was observed in all patients and corneal infiltrate was observed in two patients who presented with visual acuity of light perception . One patient underwent vitreous tap with intraocular injection of vancomycin 1 mg/0.1 ml, amikacin 400 g/0.1 ml, and dexamethasone 400 g/0.1 ml . The other two patients underwent pars plana vitrectomy with intravitreal injection of the aforementioned antibiotics and dexamethasone . Gram - negative bacilli were isolated and confirmed to be k. pneumoniae by vitek 2 compact (biomrieux, france). The organism that was isolated in case 3 was also found to be sensitive to ceftazidime and colistin . Additional procedures in the form of pars plana vitrectomy with intravitreal injection of imipenem 50 g/0.1 ml were carried out in all patients . Since there was no improvement 2 days after the second intervention, all patients were reinjected with intravitreal imipenem . At last follow - up, visual acuity was poor in all the patients, there was light perception in two patients, and no light perception in the third patient . Multidrug resistance is defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories.6 incidences of multidrug - resistant gram - negative bacilli have been increasing in tertiary care hospitals.7 it was reported that 5.2% of microbiological isolates from endophthalmitis patients were multidrug resistant, and 78.6% of those isolates were gram - negative bacteria.4 the various mechanisms contributing to virulence of k. pneumoniae are the presence of capsular k1/k2 serotypes, hypermucoviscosity, and the presence of the maga gene and the blandm-1 gene, which is a carbapenemase -lactamase.79 these bacteria can be resistant to all currently available antimicrobial agents or remain susceptible only to older, potentially more toxic agents such as the polymyxins, leaving limited and suboptimal options for treatment.6 intravitreal imipenem has been studied previously in rabbit eyes and has been reported to be nontoxic to retina.10 it acts by inhibiting cell wall synthesis of various gram - positive and gram - negative bacteria . It is stable to hydrolysis by the common plasmid - mediated -lactamases produced by various bacteria and lacks cross - resistance with penicillins and third - generation cephalosporins.11 one case series reported use of intravitreal imipenem in the treatment of endophthalmitis caused by acinetobacter baumannii . The authors reported that despite the use of multiple intravitreal injections of imipenem, the visual outcomes were poor.12 based on reported antimicrobial sensitivity, 50 g/0.1 ml intravitreal imipenem was utilized in all patients in the current series . K. pneumoniae has been commonly associated with endogenous endophthalmitis . In reported series, most patients have poor visual outcome despite appropriate antibiotic therapy, and the outcomes are usually phthisis and blindness.5,13 this may be attributed to the highly virulent organism as well as to delayed recognition of antibiotic resistance and susceptibility . The outcome of patients in the current case series was either phthisis or blindness, even after the best possible treatment.
Tinnitus affects about 16% of the adult population [1, 2] and is strongly associated with hearing loss . There is as yet no cure and so clinical management typically involved education and sound - based therapies . It is associated with pathological neural activity in the central auditory system, possibly resulting from suboptimal or maladaptive response to a peripheral lesion (hearing loss) within a certain frequency range resulting from ageing or noise exposure . While the loss of cochlear hair cells causes elevated thresholds in that frequency range, at the same time increased spontaneous firing rate and spike - timing dependent plasticity facilitate increased synchrony of firing within and across neural assemblies; both hyperactivity and increased synchrony are proposed as neural correlates of tinnitus [7, 8]. Although it is difficult to separate contributions of the two mechanisms based solely on experimental data, a recent review of physiological evidence suggests that changes in spontaneous firing alone are unlikely to generate a tinnitus percept . In the intact brain, transient synchronization of neuronal oscillatory activity is thought to be the mechanism that facilitates functional connectivity between different regions, which binds the activity from distributed neural ensembles into a coherent representation of cognitive and sensory functions . Conversely, altered synchronization has been observed in a number of psychiatric and cognitive conditions, including schizophrenia, alzheimer's disease, and attentional deficits . Likewise, in tinnitus, a number of studies have reported tinnitus - related functional connectivity between disparate regions of the brain based on brain oscillations; for example, connectivity between the anterior cingulate cortex (acc) and both the right frontal lobe and right parietal lobe strongly correlates with tinnitus distress [1517]. Studies typically attempt to correlate brain oscillations and their interregional statistical interdependencies with observed changes in perception and behaviour . More recently, a number of studies take an alternative approach in that they attempt to alter brain oscillations by means of brain stimulation to observe changes in behaviour . This is based on the assumption that modulating brain rhythms may affect cognitive performance and thus can be used to treat neurological disorders . Numerous studies over many decades indicate that low - intensity electrical currents can modulate network dynamics noninvasively in humans . Electrical stimulation has also been applied invasively in humans, either on the cortical surface or as deep implanted electrodes . However, the precise neural mechanism by which changes occur at both local and network levels is not fully understood (for a review of the animal literature see reato et al . ). It is not clear how the effects at the single neuronal level influence the network activity both spatially and temporally . Moreover, with regard to noninvasive techniques, it is not known which precise location of the brain is being stimulated . Nevertheless, studies report changes in perception during or following electrical stimulation of the brain . To date, a small number of neurostimulation techniques for tinnitus have been the subject of either a systematic review and meta - analysis (transcranial direct current stimulation (tdcs), cochlear implantation [21, 22]) or a scoping review (tdcs). Here the objective was to scope the literature on approaches to electrical stimulation of the ear, head, cranial nerve, or cortex for tinnitus which are more experimental insofar as they have not yet been the subject of a scoping or systematic review . All study types were included in this scoping review . Inclusion was determined according to the implementation of an intervention of interest and the measurement of an outcome of interest . Interventions included in this review used noninvasive electrical stimulation of the ear, transcranial alternating current stimulation (tacs), vagal nerve stimulation (vns), transcutaneous vagal nerve stimulation (tvns), procedures involving direct cortical stimulation, or paired electrical and acoustic stimulation . In terms of outcomes, of interest was whether the intervention had any potential clinical effect on symptom severity, measured as the global score on a self - report measure of tinnitus, generalised anxiety, depression, or quality of life, and equally whether there were adverse effects of the intervention . Our second interest was the potential neurophysiological mechanisms of any effects, for example, change in neurophysiological function as measured by magnetoencephalography (meg) or electroencephalography (eeg). Database searches were conducted in december 2015 using the search terms tinnitus, or tinnit, and (transcutaneous electric nerve stimulation) or (vagus nerve stimulation) or (electric stimulation) or (electric stimulation therapy) or (tacs or vns or tvns) or (neuromodulat or neurostim) or (vagus or vagal or transcutaneous or transcranial or transdermal or percutaneous or cutaneous or ac near stimul) or (alternating near current near stimul) or (electrostimul or neurostimulation or electro - stimul or neuro - stimul or electro stimul or neuro stimul) or (electrical stimul or electric stimul). To identify ongoing or unpublished trials we searched clinicaltrials.gov and conducted a hand search for recently published study protocols relevant to this review . From initial 642 search records, 46 studies were selected for full - text review by dh . Nineteen human studies met our criteria for inclusion and are reviewed here by class of intervention . These included three studies using noninvasive electrical stimulation of the ear (reported 2013 - 2014), two studies of transcranial alternating current stimulation (tacs) (reported 2013 - 2014), one pilot study and one case study using vagal nerve stimulation (vns) (reported 2014 - 2015), five studies using transcutaneous vagal nerve stimulation (tvns) (reported 20122015), one study examining vestibulocochlear nerve stimulation (reported in 2007), and seven studies involving different forms of direct cortical stimulation (reported 20042015). Suppression of tinnitus by electrical stimulation of the preauricular skin, mastoid, eardrum, promontory, and round window and within the cochlea has been employed for many years [24, 25]. Efficacy of the electrical stimulation reported by some early studies varies from 7 to 82% of patients perceiving benefit for their tinnitus [2435]. A more recent study by mielczarek et al . Assessed the effectiveness of electrical stimulation of the hearing organ on tinnitus (severity not measured) in patients with cervical spine degeneration, also examining any additive effect of cervical spine kinesitherapy (therapy involving muscle movement) on tinnitus . The frequency of the stimulation, delivered through the electrode placed in the ear canal targeting the cochlea, was adapted according to tinnitus frequency . The treatment involved 15 applications of 4 min electrical stimulation 3 - 4 times a week . After the treatment in both groups the number of ears (location not specified) with permanent tinnitus decreased from 88% in the group with electrical stimulation only and 90% in the group with combined electrical stimulation and kinesitherapy to 43 and 54%, respectively . In 37% of ears after electrical stimulation and 28% of ears after combined treatment tinnitus disappeared completely . In addition self - reported and audiometric improvement of hearing was noted in both groups, in 34% and 26% of ears, respectively . This study was followed by a double - blind placebo - controlled study in 120 patients using the same method of electrical stimulation of the ear . Similar to their earlier study, proportion of ears with permanent tinnitus decreased from 89% before treatment to 49% immediately after treatment . In 34% of ears interestingly, in the placebo group, the proportion of ears with tinnitus also decreased immediately after the control from 86% to 71% . However, the effect was reported as stable in the treatment group after 30 and 90 days; the placebo effect was not maintained . Moreover, in the treatment group, there was a statistically significant improvement in audiometric thresholds, with patients also reporting subjective improvement in 30% of ears . Whilst impressive, tinnitus was not measured in these studies using a multi - item tinnitus questionnaire so the results have to be considered with caution . Investigated effectiveness of transcutaneous electrical nerve stimulation (tens) applied to the external pinna in 65 participants with moderate severity tinnitus (randomized such that 45 were in the treatment arm and 20 received sham stimulation). Only two participants had a long - term improvement after treatment with tens (effects maintained at three months) and it was noted that both had low frequency tinnitus and milder hearing losses . No studies of tens for tinnitus were identified as ongoing, and a systematic review is not indicated at present . Tacs involves the delivery of alternating current (constant polarity changes) between electrodes placed on the skin over cortical regions of interest . It is hypothesised to affect upregulation and downregulation of synapses and may have an effect on oscillatory cortical activity, indicating it for tinnitus . In the first study of its kind vanneste et al . Compared single session tacs, tdcs, and transcranial random noise stimulation (trns; equating to tacs modified to have a 0 ma offset applied at random frequencies; frequencies ranged from 0.1 to 100 hz). The main outcomes were tinnitus loudness and annoyance, measured before and after treatment on a 10-point numeric rating scale . Only the group receiving trns showed significant within and between group effects of the intervention, on both rating scales, with the authors concluding this to be the most clinically effective for transient tinnitus suppression within a single session . It was suggested that the lack of an effect of tacs might have been due to the rather weak current used (1.5 ma for 20 min), a consideration for future studies . Also compared tinnitus suppression using single sessions of individual alpha - modulated tacs or tdcs in a randomized placebo - controlled study . Fifty participants were randomly allocated to receive either real stimulation (up to 10 ma and lasting 20 minutes) or sham stimulation (current was turned off after 30 seconds). Again, the main outcomes were tinnitus loudness and annoyance, measured on a 10-point numeric rating scale . The authors reported a positive effect of tdcs ratings of tinnitus loudness or annoyance but no significant effect of tacs compared to sham stimulation . Most recently, in a retrospective analysis of 228 patients who received therapy for tinnitus, claes et al . Examined the effects of single or repeated sessions of alpha - modulated tacs (ramped to 2.0 ma) and trns as described earlier . Effects on tinnitus loudness and annoyance were measured using simple 10-point numeric rating scales . In both interventions stimulation was applied to the auditory cortex in sessions lasting 20 minutes . In this study, tacs had no effect on tinnitus . Trns however was associated with reduced tinnitus loudness, both within a single session and additively across sessions . Trns was also associated with an improvement after a single session; multiple sessions had no additive effect . The study of tacs is therefore at an early stage, although there are no strong indicators for further study, and no studies were identified as ongoing . Current protocols need replication to discount them, and alternative protocols should explore the optimal conditions of tacs and whether any subgroups of tinnitus patient show benefit . As discussed by claes et al ., potential influencing variables including age, sex, and tinnitus characteristics need to be considered as variables in future work . Langguth and colleagues are conducting a safety / feasibility / efficacy study (nct01965028) with 30 participants, all receiving high - frequency trns daily for two weeks, with a primary outcome of number of participants with a 5-point tinnitus questionnaire (tq) score at long - term follow - up . This contrasts with previous studies where only acute effects were measured and simple rating scales as opposed to clinical questionnaire tools were used . The same group are also examining the safety / feasibility / efficacy of daily low frequency trns (nct02615600) using the same protocol . One of the postulated ways of reversing the pathological plastic changes in the auditory system associated with hearing loss and tinnitus is through targeted auditory stimulation or auditory discrimination training . However, to date, the clinical benefit of auditory stimulation approaches to treating tinnitus has been limited and only temporary relief has been demonstrated . It is postulated that auditory stimulation alone is not enough to affect plastic changes in the brain and so research attention has shifted to the forebrain cholinergic and noradrenergic systems that play a significant role in modulating cortical plasticity . Studies in animals looking at the electrical stimulation of the cholinergic nucleus basalis provided evidence for pronounced and long - lasting changes in cortical reorganization and led to the idea that pairing auditory stimulation with electrical stimulation could be used to treat tinnitus . However, stimulation of the nucleus basalis is an invasive procedure and therefore not a viable option for widespread use in tinnitus patients . In 2011, engineer and colleagues published a study looking to obtain similar effect to nucleus basalis stimulation but using much less invasive electrical stimulation of the vagus nerve (10th cranial nerve; vns) in noise - exposed rats . The vns approach was considered promising as previous clinical studies showed it can alleviate epilepsy and depression; it is now a food and drug administration approved treatment for drug - resistant epilepsy and depression . Used adult rats with noise - induced hearing loss presumed to have a related tinnitus in the region of 810 khz as verified with the startle reflex paradigm . Noise - exposed rats showed a significant cortical map distortion, decreased frequency selectivity, increased tone - evoked response, and increased spontaneous neural activity in the auditory cortex and increased cortical neural synchrony . A regime consisting of acoustic stimulation composed of multiple tones at frequencies outside the tinnitus region, paired with vns, was associated with reversal of both behavioural and physiological markers of tinnitus, except for increased spontaneous activity which was still observed . The authors postulated that while acoustic stimuli are essential to activating particular neural populations within the auditory cortex (to be able to target neuroplastic change), the vns component of the treatment was essential to promoting those plastic changes through what engineer et al . Specifically, it was postulated that acetylcholine and muscarinic receptors are involved in vns mechanism of action [48, 52]. Several studies explored the safety and efficacy of the vns paired with acoustic stimulation in humans . De ridder and colleagues conducted a pilot study with 10 participants with chronic moderate - to - catastrophic tinnitus severity tinnitus . The patients heard tones, excluding the tinnitus - matched frequency, paired with brief electrical stimulation of the vagus nerve . Whole group analysis revealed a decrease in tinnitus handicap inventory (thi) score of 11% immediately after the treatment and 12% at four - week follow - up relative to baseline (not a clinically significant score change). Exploratory analyses suggested that taking psychoactive medications such as antidepressants may inhibit the benefit to be had from the treatment . The mean decrease in thi scores for nondrug group was 29% immediately after treatment and 26% at follow - up, while for the drug group the change was minimal . Authors also reported a more pronounced reduction of minimum masking levels (mmls) in the nondrug group (18.8 db reduction) than in the drug group (4.3 db reduction). From egg recordings (performed in 7 participants only due to technical problems) they concluded that vns decreased band power in the delta (14 hz) and theta (48 hz) bands in subjects who responded to therapy, with increased band power in those bands observed in subjects who did not respond to the therapy . For both delta and theta bands the difference in band power change between vns and sham trials strongly correlated with their thi scores (r 0.74). Only one participant had a clinically significant improvement (20 or more points) on the thi, 6 had an improvement that was not clinically meaningful, and 3 had a slightly worse thi score after treatments . As the study was uncontrolled placebo effects it is worth noting too that all participants in the study had undergone between 3 and 8 previous treatments for tinnitus with medication, neurofeedback therapy, hearing devices, tinnitus masker devices, various other noninvasive forms of electrical stimulation, or cortical implantation . A recent case report from the same research group compared the effects of vns to the effects of sham stimulation in a 59-year - old man with refractory tinnitus . It is not clear if the patient was one of the participants in the previous study . The methods used were the same as before with the exception that for 2 months (washout period) after paired vns treatment the patient received sham stimulation (only tone presentation without the simultaneous electrical stimuli). After four weeks of vns treatment the patient showed a reduction in thi scores of 48% and tinnitus reaction questionnaire (trq) scores by 68% . In contrast, sham stimulation led to a 27% increase in thi and 15% increase in trq scores . Again, vns was observed to induce changes in the resting state oscillatory brain activity as measured with eeg . A significant decrease in relative power in the delta, theta, alpha, beta, and gamma frequency band was observed after vns, suggestive of desynchronization; no changes were observed as a result of the sham stimulation . It is worth noting that implantation of the vagal nerve stimulator is a full surgical procedure performed under general anaesthetic . While authors stated that no major adverse events were observed in either study, several adverse effects of either the surgery or stimulation were listed, including infection of the extension lead requiring removal of the lead and electrode, inflammation at the abdominal surgical site, hoarseness during stimulation, transient left vocal cord hypomobility, and temporary increases in tinnitus symptoms . Have recently completed recruitment to a pilot study (nct01962558) involving 30 participants randomized to receive either (1) vns paired with tones or (2) vns and unpaired tones hypothesised to be ineffective for tinnitus . An alternative noninvasive approach using transcutaneous stimulation of the vagus nerve (tvns) is considered in parallel . Several studies examining the safety and efficacy of tvns either alone or paired with acoustic stimulation have been conducted in recent years . Two consecutive reports from kreutzer and colleagues [57, 58] describe the results of the single - arm pilot study looking at the tvns without acoustic stimulation . The study was conducted in two phases, with different groups of participants with tinnitus in each phase . The first phase of the study was terminated early due to two cardiac events and was followed by detailed analysis of the ecg data collected throughout the study in all participants . Authors concluded it was very unlikely that the events were caused by the tvns device and the study progressed to phase 2 . Analysis of the results from both phases showed that tvns was suitable for long - term use, but without the use of paired acoustic stimuli it appeared to have no effect on tinnitus . Reductions in tq scores were minimal (4 8 in phase i and 3 9 in phase ii) and dropout was high (10 out of 23 patients in phase i and 6 out of 21 in phase ii). This suggests that the treatment protocol, consisting of several hours of stimulation over 6 months, if effective at all might only be feasible for some patients . In addition, a number of adverse events were associated with the treatment including tingling sensation, dysesthesia, skin redness and pressure marks at the stimulation sites, painful stimulation, dyspnea of low intensity, headaches, hoarseness, nausea, and transient subjective hearing impairment . Overall it was concluded that tvns was safe for prolonged use in patients without a history of cardiac disease . The treatment resulted in improvements in mood as measured with the who 5-point well - being questionnaire . The mean well - being score increased from 56 points at baseline to 76 points after treatment . Thi (severe at baseline) and mini - tq questionnaire score decreased significantly, and subjective loudness and annoyance, measured with visual analogue scales (vas), were also reduced after treatment . The auditory n1 m response to the tinnitus frequency, measured by magnetoencephalography (meg), was compared as tvns on versus the tvns off conditions; tvns decreased the amplitude of the auditory n1 m response, while the peak latencies did not change . Authors stressed the importance of these findings as a first demonstration of acute neuromodulative effects of tvns on evoked auditory cortical responses . Compared cortical oscillatory power (power spectral density, psd) and synchronization (phase lag index, pli) in people with moderately severe tinnitus and normally hearing controls using meg . Specifically, baseline activity when the tvns was not activated was compared between groups as well as differences in tone - evoked (tinnitus frequency in participants with tinnitus and 1 khz in controls) brain activity between tvns not activated and tvns activated conditions in the tinnitus group . The results revealed higher psds in gamma band at baseline in tinnitus than in control group and higher plis particularly in beta and gamma bands in tinnitus patients compared to controls . Tvns had different effects on synchronicity in people with tinnitus and controls . In the tinnitus group, whole - head alpha activity, occipital alpha, and right temporal gamma activity increased during stimulation, and occipital theta activity decreased in response to tvns . Higher whole - head beta (r = 0.74), low temporal delta (r = 0.74), and low temporal theta (r = 0.71) were associated with higher thi scores when the tvns was not activated . In addition, thi scores also correlated positively with the tvns induced change in normalized psd values for multiple frequency bands and brain regions . Tvns induced changes in synchrony correlated strongly with thi scores, at whole - head beta (r = 0.86), whole - head gamma (r = 0.95), and frontal gamma bands (r = 0.95). Li et al . Are conducting a randomized, single - blind, controlled clinical study (chictr - trc-14004940) comparing (1) tvns only, (2) tvns paired with sound stimuli, (3) nonvagus electrical stimulation of the outer ear, and (4) an electrical acupuncture group . They aim to recruit 120 participants and are using the thi as their primary outcome measure . As this questionnaire was also used in both previous studies of tvns, the outcome of this study can be usefully compared or synthesised with existing data . An interesting alternative to the vns is chronic electrical vestibulocochlear nerve stimulation (vcns), where the stimulation electrode is surgically placed around the vestibulocochlear nerve and connected to a pulse generator placed under the skin . In this approach bartels and colleagues investigated safety and efficacy of this method in six participants with unilateral, severe, chronic refractory tinnitus and severe hearing loss in the tinnitus ear . Two participants dropped out, one due to the neurostimulation being unsuccessful after a period of three months and one due to other health and psychiatric problems not due to tinnitus, leaving four participants for long - term evaluation (42.5 months on average). Mean thi scores reduced from 77 points at the baseline to 55 points at 3 months and 38 points at long - term follow - up . This equated to a large clinical effect size (d = 1.75) at follow - up . For two participants, thi score was reduced after 3 months and for the other two participants thi scores were reduced after about 6 months of treatment . Self - rating of tinnitus severity was also reduced on a 10-point scale from a mean of 8 to 3 at long - term follow - up . As described by authors, while none of the participants' tinnitus disappeared during the treatment, all patients reported that the neurostimulation system transformed intrusive combination of noises into a single, pleasantly - perceived noise the earliest report of invasive electrical stimulation in tinnitus was provided by de ridder et al . Who demonstrated suppression of tinnitus in a single patient following focal electrical stimulation of the primary auditory cortex . The patient, a 32-year - old woman, suffered from severe left - sided tinnitus following total loss of hearing in the left ear . Tinnitus was rated 9 on a 10-point vas, and loudness was matched to an 80 db external tone and between 8 and 9 khz . Initially, tms was applied to the right auditory cortex following functional magnetic resonance imaging (fmri) to identify heschl's gyrus . Subsequently, an extradural octopolar electrode was implanted onto the right auditory cortex for electrical stimulation via a neurostimulator which generated and delivered 0.5 msec pulses at a rate of 40 pps and 2.7 ma, to which the patient's tinnitus disappeared completely . However, three weeks after the operation, high - pitched tinnitus returned . The authors suggested this was due to cortical plasticity in response to constant stimulation at the high - frequency areas but could not explain how electrical stimulation of the cortex eliminated tinnitus . . Examined the effect of implanted electrodes in a group of patients with moderate - to - severe tinnitus . Based on the similarity of tinnitus with pain, and the assumption that the sound is generated in the central nervous system, the authors argued that at least some forms of tinnitus are caused by neural plasticity and the reorganization of the central nervous system involving the auditory cortex . In addition, they assume that neural hyperactivity within the auditory system underlies the tinnitus abnormality . They thus propose that tinnitus can be treated by suppressing this hyperactivity by means of implanted electrodes in both the primary and secondary auditory cortices . They selected 12 participants who reported their tinnitus to be suppressed by at least 50% on a visual analogue scale by application of tms . Primary and secondary auditory cortices were first localised using fmri and frequency specific sounds (matched to tinnitus pitch), supposed to improve accuracy of finding the tinnitus generator in the auditory cortex . Two - pole electrodes were positioned such that one was intradural, on the primary auditory cortex, and the other was extradural, overlaying the secondary auditory cortex . The results were as follows: stimulation through the extradural electrode resulted in tinnitus suppression of 26% for patients with white noise tinnitus and 97% for those with pure tone tinnitus . As for the intradural electrodes, percentage suppression was not reported but only 2 patients failed to report an improvement in tinnitus, which suggests stimulation of primary auditory cortex provides more lasting suppression . Moreover, in patients with white noise and pure tone tinnitus, electrical stimulation suppressed the pure tone tinnitus, while the white noise tinnitus remained unchanged . Importantly, tinnitus reappeared at some later time beyond the main study but it is not clear how long after the operation their tinnitus returned to its preoperative levels . The authors suggest that the intensity of the perceived tinnitus is related to the degree of cortical reorganization; however, this has since been shown not to be the case by a number of studies (e.g., see sereda et al . ). Moreover, the assumption that tinnitus is generated within the auditory cortex, which guided the implantation loci, has not been established . Their reasoning was based on the thalamocortical dysrhythmia model [66, 67] which suggests that tinnitus emerges as a result of peripheral damage and deafferentation, leading to reduced neural firing rates in corresponding thalamocortical columns . Consequently, this leads to hyperactivity in the neighbouring regions (edge effect) and characteristic 40 hz oscillations as the correlate of tinnitus . They therefore attempted to generate a therapeutic benefit by introducing an electrical signal in the vicinity of this reorganized auditory cortex . In two patients with chronic severe tinnitus, they first attempted to identify the tonotopic maps in heschl's gyrus using meg and fmri, before implanting electrodes within areas showing increased activity . Electrode arrays were advanced into the brain tissue in heschl's gyrus and in proximity to the area localised noninvasively . In one patient with bilateral, high - pitched tinnitus, loudness decreased significantly in both ears as the pulse generator was activated . In follow - up evaluations, this patient reported that tinnitus has remained significantly reduced (visual analogue scale rating). In the second patient with unilateral left - sided high - pitch narrow band tinnitus, several attempts at stimulation treatments over a period of two years did not diminish tinnitus . The authors suggest that while electrical stimulation is clearly beneficial, perhaps in some patients areas such as the amygdala or hippocampus should be stimulated instead of the auditory cortex . Most recently, de ridder and vanneste report the case of a single tinnitus patient who experienced beneficial effect of an auditory cortex implant . In this study auditory cortex stimulation and c2 nerve stimulation were combined using implanted electrodes overlying the auditory cortex and subcutaneous electrodes modulating the c2 dermatome . It has been found that auditory deafferentation induces compensatory increases of somatosensory influences on the auditory pathways at the level of the dorsal cochlear nucleus and that c2 electrical stimulation can suppress or enhance responses to sound via a pattern of inhibition and excitation of the principal cells in the ventral and dorsal division of the cochlear nucleus . It was hypothesised therefore that cross - modal convergence or interaction of multisensory neurons, whereby the modulation of activity evoked by one modality modulates the activity evoked by another, could be beneficial . The participant had suffered from severe unilateral right - sided tinnitus for 1.5 years prior to testing . Subjective tinnitus loudness score was 8.5/10 on a 10-point scale, matched to a 6 khz with an audiometric threshold that also dipped at 6 khz . Fmri was performed to identify the auditory cortex and subsequently an electrode was implanted overlying the secondary auditory cortex . The authors report a complete suppression of the pure tone component of the tinnitus; however, the noise - like component was only suppressed to 4 on a 10-point scale, which worsened again . Subsequently, transcutaneous electrical nerve stimulation (tens) at c2 reduced the noise from 7 to 1 - 2/10 consistently over 4 weekly sessions . The patient used the tens on a daily basis with good result but after 3 months the effect diminished . A permanent subcutaneous electrode was then inserted which activated the auditory cortex electrode . As a result, noise - like tinnitus reduced to 4/10 and remained so for 5 years, while pure tone tinnitus remained absent . Despite this, moreover, given that only a single patient was examined, it is not possible to speculate on its general effectiveness . The method requires application in a larger group of patients as it is possible that patients with different tinnitus characteristics will respond differently to this form of treatment . Based on the similarity of pain and tinnitus pathways, acc and anterior insula have been suggested to be a core network for the noise cancelling system . Fluctuations of activity in the acc and anterior insula determine whether a near threshold pain stimulus is consciously perceived or not . A number of anatomical and functional studies indicate that tinnitus distress may be related to a cortical network involving the acc, anterior insula, and the parahippocampal area [72, 73]. In particular, self - reported tinnitus distress may be related to increased activity in the dorsal acc and insula . Report the results of a study on two patients who underwent implantation of electrodes in the dorsal acc . Both patients suffered from chronic tinnitus subjectively rated 10/10 on a scale for loudness and highly on questionnaire measures of distress . Initially, fmri was used to identify target zones for neurostimulation using sound stimuli matched to the tinnitus frequency . Tms was applied to various regions including the auditory cortices and the prefrontal regions over repeated sessions, but this did not produce lasting benefits to either patient . One patient reported dramatic improvement in both tinnitus severity and loudness after one week, which halved from the preoperative stage . Additional stimulation after four weeks resulted in further reduction of tinnitus loudness and distress ratings . Conversely, the second patient did not report any change as a result of the implant which was trialled at various stimulation configurations . The authors found increased functional connectivity between dorsal acc, pregenual acc, parahippocampal area, and auditory cortex in the patient who reported improvements in their tinnitus . Clearly, while the results of this study offer possibilities for tinnitus relief, the hypotheses need further evaluation with a wider pool of tinnitus patients, particularly with regard to assessment of connectivity . Whether the assessment of functional connectivity in the specified network is predictive of postoperative success cannot be ascertained from this . It is clear that not all patients with tinnitus benefit from neurostimulation via implanted electrodes . De ridder and vanneste have shown that functional connectivity analysis based on eeg resting state activity between the auditory cortex and parahippocampus may determine whether a tinnitus patient will respond to a cortical implant . This might be a necessary initial step prior to neurosurgery given the various risks attached with the latter . Deep brain stimulation (dbs) involves the implantation of a neurostimulator within the brain to deliver electrical pulses . It has been used with varying degrees of success to treat a number of neurological disorders, most notably movement and affective disorders such as parkinson's disease but also pain . For tinnitus, dbs aims to disrupt the communication between various parts of the brain involved by modifying or preventing the abnormal neural signal from reaching the auditory cortex . A number of studies have assessed the therapeutic merits of dbs to affect mainly subcortical (but also cortical) structures which are thought to be involved in tinnitus including the thalamus and the inferior cortex . The first dbs study in tinnitus was a case series reported by shi et al . . They collected self - rating and psychoacoustic measures of tinnitus loudness in seven patients who were implanted for movement disorders but also reported having tinnitus . Electrodes were placed unilaterally or bilaterally in the ventral intermedius nucleus of the thalamus for control of involuntary tremors . Decreases in matched tinnitus loudness were found in two patients exceeding the range of normal test - retest variations (2 db), which agreed with their subjective rating of their dbs - related tinnitus changes . For the two tested patients whose tinnitus responded to dbs, tinnitus remained suppressed for 15 to 20 minutes after stimulation was turned off . None of the patients reported stimulation - related changes in hearing and audiometry confirmed there were no changes in hearing thresholds . The results indicate that changes in activity within nonauditory system may be involved in the maintenance of tinnitus . The ventral medial nucleus is involved in motor functions and mainly receives input from the cerebellum and the globus pallidus . It is the site of dbs for motor - related movements such as multiple sclerosis and parkinson's disease . However, it is not clear why stimulation of motor centres affects the auditory system, and given that hearing thresholds were not affected, it is unlikely that the stimulation spreads to other areas of the thalamus that comprise the auditory pathways . In the two patients who did not respond to stimulation, tinnitus had existed for more than 20 years, compared to less than 10 years in responders, which the authors suggest possibly indicating decreased plasticity in factors related to tinnitus perception over very long periods of time (p.286). In another study, cheung and larson applied dbs to a locus of caudate neurons (area lc), a subsite of striate nucleus and a part of basal ganglia . It was assumed that stimulation of the basal ganglia would disrupt attention to auditory cortical representations of tinnitus . Six patients with chronic tinnitus were recruited who were primarily being treated for parkinson's disease or tremors . Three patients had a preoperative and postoperative audiogram; none showed any significant change postoperatively . The authors speculate that while the lc is not part of the classical auditory pathway, it may play a role in integration of phantom sensations generated from the central auditory system with areas involved in perceptual awareness . The inferior colliculus (ic) and its various subdivisions are a major relay centre for auditory signals arriving from the cochlear nucleus . It is situated within the midbrain and plays a significant role in auditory signal integration and frequency and pitch processing . Found abnormal activity in ic of patients with tinnitus . In order to assess the potential effects of ic stimulation on tinnitus, offutt et al . Compared the extent of suppression and facilitation elicited in the central ic by different stimulation paradigms . Using a guinea pig model, they found that stimulation of dorsal ic induces both suppressive and facilitatory changes across the central ic which can occur during stimulation and that these remain after stimulation . Interestingly, stimulation of dorsal ic induced greater suppression when paired with broadband noise stimulation . The study, while not using tinnitus induced animals, demonstrates that stimulation of the dorsal ic can induce plasticity within the central ic and concomitant changes in activity, which may be applicable in treating tinnitus . Recently, shekhawat et al . Showed that a combination of noise and cortical stimulation using tms may be more effective in suppressing tinnitus than either one alone . The study by offutt and colleagues can guide future tms studies to target the appropriate regions of the brain for more optimal therapeutic results . It must be noted that the studies attempting dbs are principally aimed at alleviating other coexisting conditions (parkinson's disease, tremor). It is not clear how the minute effects of weak currents exert an effect on isolated neuron membrane potentials and lead to significant changes in network dynamics at the cellular (e.g., timing) and/or network levels . It is also not clear what frequency of oscillations and intensities are necessary to induce neural changes that improve tinnitus therapy in patients with differing clinical manifestations . Such a framework for neuromodulation is currently lacking . Moreover, it is intriguing that, in most of the reported studies, hearing function (audiometric thresholds) is not affected given that tinnitus is an auditory sensation that primarily involves the auditory system . Therefore, any changes in the tinnitus perception would be expected to also affect other aspects of audition; but none are reported in these studies . Van den berge and van dijk are planning a safety / efficacy study (nct02630589) involving an auditory brainstem implant for tinnitus . Ten participants with severe intractable tinnitus will be neurosurgically implanted, whereby an electrode array is inserted into the lateral recess of the fourth ventricle and placed on the cochlear nucleus . This study will add to the slowly growing body of evidence for the use of invasive techniques to treat intractable tinnitus . Each tinnitus patient represents a unique combination of tinnitus characteristics, aetiology, pathophysiology, and psychoperceptual factors . Therefore, it is unlikely that any treatment will provide relief for all tinnitus sufferers, as indeed is demonstrated by the results of the studies reviewed here . While their precise mechanism may not be known, noninvasive methods are worth exploring as they seem to provide comparable benefits to invasive methods, although this will not be the case for every individual patient . It is possible that structures involved in neuromodulation are not involved in generation and maintenance of tinnitus in all but only a subtype of patients . Therefore, neuromodulation offers another venue for identifying the subtypes of tinnitus, which is the goal of a current european - funded cooperation in science and technology (cost action) program for a tinnitus research network (tinnet; http://tinnet.tinnitusresearch.net/index.php/2015-10-29-10-22-16/wg-3-neuroimaging) to identify subtypes of tinnitus and their neural correlates and thus develop innovative hypothesis driven treatment approaches.
Endoscopic treatment of advanced biliary disorders and their complications following surgical procedures is often difficult especially after billroth ii gastric resection . In such cases, endoscopic access to the papilla and neopapilla thus, percutaneous cholangiodrainage or re - operation is therefore indicated when endoscopic access to the papilla failed . But both of those methods are much more invasive and more likely to be followed by complications than use of a purely endoscopic approach . The present case report describes endoscopic access to the papilla with both push enteroscopy and double - balloon enteroscopy (dbe, push - and - pull enteroscopy) in a female patient after billroth ii resection with bile leakage following cholecystectomy . Successful closure of the bile duct fistula could be achieved via an unusual abdominal - biliary - jejunal cannulation way after several attempts of modern enteroscopy and in this way re - operation was avoided . Modern enteroscopy by experienced investigators using push - and - pull enteroscopes can provide access to the papilla even in complex postoperative anatomic rearrangements . Push - and - pull enteroscopes offer a further option for successful cannulation of the papilla and therapeutic interventions via additional stabilization with balloons and the modern enteroscopic approach by push - and - pull enteroscopy appears to provide more patient comfort, requires less analgo - sedation and examination time and in cases with intra - abdominal drainage this external access may be used as an additional aid for exploration and intervention in complex individual cases with extremely difficult treatable bile duct injuries . Endoscopic treatment of advanced biliary disorders and their complications (e.g. Benign, malignant strictures, bile duct trauma, bile duct leakage etc) following surgical procedures is often difficult after billroth ii gastric resection, whipple s operation or extensive resection of the small intestine . In such cases, endoscopic access to the papilla and neopapilla is often low (525%), and access to the choledocho- or hepaticojejunostomy is often hampered by jejunal loops of different lengths, awkwardly positioned braun s anastomoses or even by postoperative alterations (e.g. Adhesions) [15]. Thus, percutaneous cholangiodrainage (ptcd) or re - operation is therefore indicated when endoscopic access to the papilla failed . But ptcd is regarded as much more invasive and more likely to be followed by complications than use of a purely endoscopic approach, especially in persons who have undergone billroth ii resection with a long afferent loop or a roux - en - y construction . The present case report describes endoscopic access to the papilla with both push enteroscopy and double - balloon enteroscopy (dbe, push - and - pull enteroscopy) in a female patient after billroth ii resection with bile leakage following cholecystectomy . Successful closure of the bile duct fistula could technically only be achieved via an unusual abdominal - biliary - jejunal cannulation way after several attempts of modern enteroscopy and in this way re - operation was avoided . A 79-year - old female patient was transferred from the surgical unit of an external hospital to the department of medicine 1 of the university erlangen because of an ouvert persisting bile duct leakage after open cholecystectomy . Cholecystectomy was required to treat acute cholecystitis with choledocholithiasis, since endoscopic stone removal by gastroscope and side - viewing duodenoscope (ercp) failed due to lack of access to the papilla after bilroth ii resection . At admission, the patient presented with an intraabdominal drain secreting approximately 500 ml of bile per day, both into the abdominal cavity and externally via an easy - flow drainage . At the time of admission the patient was awake and her cardiovascular system stable, but she presented with dementia . Her current schedule of medication was metoprolol 47.5 mg / d and thiamazol 10 mg / d . The physical examination revealed poor general health, a slightly adipose nutritional status; blood pressure was 110/80 mmhg with 100 heartbeats / min, body temperature 37.6c . There was a vesicular respiratory sound and a respiration rate of 16/min . A well - healed median laparotomy scar was found after open cholecystectomy and normal peristaltic sounds in all quadrants . At the time of admission the woman had a bile collection bag due to persisting leakage in the right upper abdomen; an intra - abdominal drainage tube was still in place . Laboratory tests showed the following values: crp slightly elevated at 18 mg / l (<5 mg / l), got elevated at 77 u / l (<31 u / l), gpt 65 u / l (<34 u / l), cholestasis with gamma - gt 580 u / l (<38 u / l), alkaline phosphatase 674 u / l (53141 u / l), bilirubin 1.5 mg / dl (<1.0 mg / dl, of which 0.7 mg / dl was direct bilirubin and 0.8 mg / dl indirect bilirubin). Billroth ii gastric resection had been carried out in 1982 because of peptic ulcer disease . Access to the braun s anastomosis was achieved 20 cm distally from gastro - jejunal anastomosis (58 cm) by means of push enteroscopy (sif q140, olympus, japan) and the afferent loop with a further length of 42 cm could be traversed only by enteroscopy . At the first examination, the papilla was found at the end of the afferent loop at 120 cm depth, approximately 1.5 cm away from the unattached, closed end; however, the papilla could only be viewed tangentially . Following the difficult adjustment of focus on the papilla with a sif q140 push - enteroscope (olympus, japan), contrast medium was applied and showed an unobstructed bile duct system; however, the proximal common bile duct was clearly pulled out of shape in a v - shaped curve to the left (figures 1 and 2 for a postoperative view). Contrast medium was clearly seen to emerge from the v - shaped bend of the bile duct approximately 3 cm proximally from the papilla, with rapid formation of a 2.41.0 cm large depot of contrast medium (figure 1) nearby the surgically placed abdominal drainage . The surgically emplaced robinson drainage tube laid since the time of cholecystectomy (postoperative day 68). Although the bile duct leakage could endoscopically be illustrated during pro - grade ercp, it remained impossible to selectively cannulate the distal bile duct using the push enteroscope (sif q140, olympus, japan), since the bile duct tended to bend sharply at an angle of 90 along the course of the distorted bile duct s curve (figures 1, 2). At the end of this unsuccessful examination, the afferent loop was marked with sterile ink (gi - supply co., sold by mandel & rupp co., erkrath, germany). A second attempt using a sif q140 push - enteroscope (olympus, japan) and a lateral - viewing enteroscope (tjf 160, olympus, japan) failed and thus, a third examination was performed by push - and - pull - enteroscopy (dbe based ercp), since a balloon - assisted course of action held out the promise of greater stability for positioning the endoscope in relation to the papilla . Although it proved possible to cannulate the biliary system now for the first time, the insertion of a more rigid endoprosthesis failed at this moment . At a second dbe based prograde, technically very demanding ercp (en-450t5, fujinon, japan), it appeared initially again impossible to introduce more rigid instruments into the cranial parts of the bile duct due to postoperatively altered anatomy . Thus, in order to avoid an open re - operation, an unusual abdominal - biliary cannulation way was tried during enteroscopy and fluoroscopy by advancing a wire into the bile duct via the abdominal drain which was already in place (figures 2, 3). Thereupon it was found that the position of the abdominal drain was apparently intraductal, since the passageway was immediately explorable with a flexibe j - terumo guidewire (figure 3). The abdominal drain was therefore probed percutaneously with the flexible terumo wire, making it possible to reach the distal common bile duct and then to advance the terumo wire into the intestinal passageway . This guidewire was then grasped with a biopsy forceps and pulled to the outside (figure 3). While the terumo guidewire was held in place at the abdominal drain, the ercp catheter was introduced endoscopically over the guidewire straight into the distal bile duct . Even then, however, it proved impossible to reach the cranial bile duct due to the disadvantageous vertical position in relation to the bile duct wall . A maxforce balloon (4 mm) was then introduced percutaneously via the abdominal drain, advanced to the v - shaped pucker of the proximal common bile duct, and successfully plugged the bile duct leak (fig . 4). Now at last, it was possible to advance the ercp catheter straight ahead and probe the cranial bile duct system, since the wire, when advanced in a straight line, bounced off the balloon which had plugged the leakage, and moved toward the hilus (figures 4, 5). Finally, the bile duct system cranially to the hilus could be selectively reached, allowing insertion of a rigid blackwire guidewire for stable position . Implantation of a 7 fr double - pigtail prosthesis in segment ii (figure 5) for decompression of the biliary tract was achieved by using a 5 fr nasobiliary probe as pusher . Interestingly, three days after implantation of the 7 fr double - pigtail prosthesis, secretion of bile from the abdominal drain had already come to a full stop . Antibiotic therapy was discontinued after removal of the abdominal drain prior to the patient s release from our hospital . Bile duct injury has been reported to be less common after open cholecystectomy (0.20.5%) than after laparoscopic cholecystectomy (02.7%). Small bile leaks are more commonly found after laparoscopic cholecystectomy (1015%) and are in most cases clinically asymptomatic . Large, clinically relevant leaks manifest as biliary fistulas, biliary ascites, peritonitis etc . And are potentially fatal if left untreated . Although endoscopic treatment of bile duct leakage by performing papillotomy, insertion of nasobiliary probe or stent placement is deemed as first line therapy of choice, these interventions and the endoscopic approach may be technically very difficult or impossible in postsurgical patients with altered abdominal anatomy as described here in a female patient with injured bile duct and status after billroth ii resection with roux - en - y loop . Although conventional endoscopy using gastroscope and side - viewing duodenoscope was repeatedly unsuccessful, enteroscopy with both a push enteroscope and a push - and - pull enteroscope was able to gain access to the papilla at 120 cm insertion depth, to illustrate the biliary tree and to recognize exactly the position of the bile duct trauma . In order to avoid open re - operation, several enteroscopic attempts had to be undertaken until a successful complex enteroscopic and radiological intervention using the intraabdominal drainage for an unusual bile duct cannulation could be performed . After cannulation of the injured bile duct via the abdominal drainage to the bile duct leakage and then to the afferent jejunal loop by a flexible guidewire, intermittent closure of the bile duct leakage was then required by a 4 mm max force balloon to achieve selective cannulation of the proximal and intrahepatic ducts . A permanent closure of this technically very demanding, v - shaped bile duct injury during prograde enteroscopy based erc was finally achieved after insertion of a 7fr double - pigtail endoprosthesis . As to our knowledge, the above mentioned unusual cannulation way via the abdominal drainage to the bile leakage and then through the papilla to the jejunum with intermittent closure of the bile leakage by a balloon has not yet been described in the literature for treatment of bile duct injuries up to now . Interestingly, push - and - pull enteroscopy provided a much better and more stable position at the papilla due to use of a balloon on the overtube and the enteroscope tip than push enteroscopy . By inflating the balloon on the tip of the enteroscope the very tangentially position of the enteroscope in front of the papilla could sometimes be compensated, which allowed first successful guidewire cannulation . But apart from the postoperative endoscopic route to the papilla, the situation was much more complicated, however, by the v - shaped angle of the bile duct, the size of the leakage and the extremely unfavorable postoperative anatomy of the biliary system, requiring further innovative interventional steps like usage of the abdominal drainage as cannulation way, extraction of the guidewire out of the enteroscope, intermittent closure of the bile leakage from the external access by an appropriate high - pressure balloon allowing finally the permanent decompression of the biliary system with an effective endoprosthesis therapy . This case report illustrates the following points: 1) modern enteroscopy by experienced investigators using push - and - pull enteroscopes can provide access to the papilla even in complex postoperative anatomic rearrangements . 2) push - and - pull enteroscopes offer a further option for successful cannulation of the papilla and therapeutic interventions via additional stabilization with balloons and 3) the modern enteroscopic approach by push - and - pull enteroscopy appears to provide more patient comfort, requires less analgo - sedation and examination time and 4) in cases with intra - abdominal drainage this external access may be used as an additional aid for exploration and intervention in complex individual cases with extremely difficult treatable bile duct injuries . Admission of such patients to experienced endoscopic centers for checking, examination and trying of conservative interventional endoscopic- or enteroscopic - radiological therapy is thus warranted before performing bile duct re - operation in such patients . Concerning the proceeds and cost situation of the endoscopic therapy the effective amount for this therapy was 5287,91 according to the german accounting system (drg; primary / leading diagnosis: h41a). The comparable surgical therapy (open revision of the biliary tract and surgical placement of a stent) would have resulted in costs of 7165,57 (primary / leading diagnosis: h05z). All in all, endoscopic therapy represents a more conservative and more cost - effective method to treat complex postoperative bile duct leakage, most notably by use of modern enteroscopy in experienced centers.
The rapidly aging population drives an increase in the incidence and prevalence of cardiovascular disease (cvd), which in turn contributes to an explosion of vascular dementia (vad) and alzheimer s disease (ad). Many vascular risk factors such as atherosclerosis, stroke and cardiac disease could result in cerebrovascular dysfunction and trigger ad pathology (rocchi et al . 2009). Many patients with vad have clinical features of ad coupled with vascular risk factors and associated strokes . The overlap between ad and cerebrovascular disease therefore produces a disorder that may be amenable to therapeutic approaches based on either mechanism (kotze et al . Research efforts are increasingly focused on elucidation of the underlying cause of ad, which has resulted in significant progress in understanding the wide range of both genetic and environmental risk factors that converge into the development and progression of this common neurodegenerative disease . Prospective follow - up studies performed in nearly 10 000 californians over a period of 27 years have shown that the same risk factors that lead to the development of cvd in middle age also significantly increase the risk of dementia in old age (whitmer et al . Overweight individuals had a 35% increased risk, while obesity was associated with a 74% increased risk of dementia compared with normal - weight individuals . High serum cholesterol levels, hypertension, diabetes and smoking between the ages of 4044 years were associated with a 2040% increased risk of dementia in old age . Treatment of hypertension was shown to reduce the risk of dementia by 50% (poon 2008) and represents one of several cvd risk factors that can be targeted to protect against cognitive decline . (2011) investigated whether abnormal lipid metabolism was associated with ad pathology (neuritic plaques and neurofibrillary tangles), and found higher lipid measurements, including cholesterol and triglycerides, in subjects with neuritic plaques . Excessive intake of dietary carbohydrates and particularly high fructose corn syrup, may lead to oxidative damage and ultimately apoptosis of brain cells (seneff et al . 2011). These findings are in accordance with loss of lipid asymmetry and consequent neurotoxicity as demonstrated in the ad brain using a mouse model (bader lange et al . Phosphatidyldserine asymmetry was significantly altered in an age - dependent manner as a result of oxidative stress and/or apoptosis . The same disease mechanism may apply to other neurodegenerative diseases that share many properties with ad . In an effort to define dietary patterns that promote cognitive health, bowman et al . (2012) examined the cross - sectional relationships between nutrient status and psychometric and imaging indices of brain health in 104 dementia - free elders . Distinct nutrient biomarker patterns analysed in plasma were shown to account for a significant degree of variance in both cognitive function and brain volume . More favourable cognitive and mri measures were significantly associated with two nutrient biomarker patterns: one high in plasma vitamins b (b1, b2, b6, folate and b12), c, d and e, and another high in plasma marine omega-3 fatty acids . A high trans fat pattern was consistently associated with worse cognitive performance and less total cerebral brain volume . Trans fats may replace docosahexaenoic acid (dha) in neuronal membranes and high intake increases the risk of cvd, systemic inflammation, and endothelial dysfunction that could explain the deleterious effect on cognitive decline (lopez - garcia et al . 2005; schaefer et al . Depression attenuated the relationship between the omega-3 fatty acids and white matter hyperintensity volume (bowman et al . 2012) and should be addressed as part of a comprehensive cvd and ad risk reduction strategy . A diet protective against cvd and ad includes reduced intake of animal products, especially red and organ meats and high - fat dairy and avoidance of trans fats, and high intake of fish, fruit, dark and green leafy vegetables and cruciferous vegetables (morris et al . 2007; gu et al . 2010). However, large clinical trials failed to show the benefit of vitamin e, b vitamins or dha (aisen et al . 2012) despite several previous studies in favour of the important role of certain nutrients in the prevention of ad . Given the differences in dietary patterns between populations, and the interactive nature of nutrient action and metabolism, contradictory findings are not surprising . Individual genetic differences in the absorption and utilisation of nutrients in the diet could also affect the risk of disease development and progression . This underscores the rationale for an integrative genomic healthcare approach that collectively incorporates the influence of multiple genetic and environmental risk factors in relation to protective nutrients in a new integrative medical model for optimising vascular and brain health . In this review the genetic link between cvd and ad is discussed in relation to single nucleotide polymorphisms (snps) in the apo e, mthfr, hfe and fto genes implicated in both conditions . These genes are involved in the metabolism of fat and cholesterol, folate and homocysteine, and iron dysregulation in the liver that is closely related to glucose secretion . We provide the rationale for use of a pathology supported genetic testing (psgt) approach towards healthy aging . The aim is to 1) diagnose treatable genetic subtypes of complex diseases as early as possible, 2) facilitate the prevention of cumulative risk and 3) formulate intervention strategies tailored to the needs of the individual . Psgt is applied with use of the gknowmix database (https//:www.gknowmix.com) that matches known genetic and environmental risk factors identified in patients with their medical history and biochemical measurements, allowing for gene expression and response to treatment to be determined and monitored as part of routine clinical care . Although serum and brain cholesterol are two separate pools (bjorkhem 2006) high serum total cholesterol in midlife is associated with an increased risk of both ad and vad (solomon et al . Based on the finding that even moderately elevated cholesterol increases the risk of dementia, these authors recommended early intervention before underlying disease(s) or symptoms appear . Prior to the development and implementation of a treatment plan in patients with high cholesterol levels, the extent to which genetic risk factors may play a role needs to be determined . An accurate diagnosis is a prerequisite for optimal treatment and the appropriateness of genetic testing should only be considered after careful documentation of other potential risk factors . These include a family history of hypercholesterolaemia and cvd, personal medical history and current health status, as well as environmental risk factors such as smoking and body mass index (bmi) known to influence cholesterol levels . Distinction of patients with familial hypercholesterolaemia (fh) from those with less severe forms of dyslipidaemia is very important in the south african population, where the prevalence of this lipid disorder is increased 510 times compared to most other populations due to a founder effect (kotze et al . While nutrition and lifestyle modifications are sufficient to normalise cholesterol levels in most hypercholesterolaemics, fh patients additionally require long - term drug treatment to reduce the risk of premature heart attacks . Lessons learned from extensive study of fh in the genetically distinct populations of south africa have led to a modified approach to risk management of cvd, which culminated in the psgt approach . Nearly 20 years ago we demonstrated for the first time that the same fh mutation could be associated with variable clinical expression, ranging from occurrence of a myocardial infarction at an early age (<50 years) to good health into advanced age (> 80 years) in the same family, depending on gene - gene and gene - environment interaction (kotze et al . The type or severity of the gene defect furthermore affects serum cholesterol levels differentially, although not sufficiently to explain clinical variability in coronary events (kotze et al . 1993b). Based on the knowledge that high - risk environments and modifier genes affecting disease expression in monogenic conditions such as fh would also increase the risk of cvd in the general population, a comprehensive cvd multi - gene test performed in conjunction with a medical and lifestyle assessment was developed (kotze et al . 2003; kotze and thiart 2003). This cvd testing approach summarised in table 1 can be applied in patients at risk of both cvd and ad (kotze et al . The mutations or functional polymorphisms included in the cvd assay was based on their phenotypic effect, allele frequencies in the local population and availability of appropriate intervention or treatment options . The genotypes being tested (i) affect the function or level (expression) of the gene products, (ii) affect biological processes involved in cvd and related disorders, and (iii) have apparent metabolic / clinical implications, either alone or in combination with other genetic or environmental risk factors.table 1different test options for cardiovascular risk management, indications for referral and clinical applicationstest optionsindication of referralapplicationfh testfh diagnosis high pre - treatment total (tc) and ldl - cholesterol levels in the presence of a strong family history of early - onset (<55 years) coronary heart disease and/or xanthomas (tc usually> 7.5 mmol / l with normal triglyceride levels)population - specific diagnose fh or confirm clinical diagnosis of fh in the index case . A positive test enables family screening for pre - clinical diagnosis in at - risk family memberscvd multi - gene testcvd risk reduction normal or abnormal lipid profile, typically moderate - high pre - treatment serum cholesterol levels, low hdl - cholesterol and/or high triglycerides . Elevated lp(a), hs - crp, homocysteine, glucose, ferritin and/or transferrin saturation levels . Medical conditions associated with increased cvd risk (e.g. Obesity, hypertension, type ii diabetes, non - alcoholic fatty liver disease)global application determine genetic contributors to disease development / severity and/or genetic basis of metabolic impairments and treatment response for risk management of cvd and related conditions affected by overlapping genes (e.g. Metabolic syndrome, dementia, thrombophilia, depression and pregnancy complications)fh & cvd testsdiagnosis and risk management same as above with elevated pre - treatment serum cholesterol levels (tc> 6 mmol / l). A strong family history of early - onset (<55 years) coronary heart disease and/or xanthomassame as above, with focus on distinguishing fh from other forms of dyslipidaemia . Alternatively, to identify additional risk factors in fh patients aimed at the prevention of a cumulative effect that may lead to the development or progression of cvdfh familial hypercholesterolaemia; cvd cardiovascular disease; lp(a) lipoprotein (a); hs - crp high - sensitivity c - reactive protein.in addition to the use of genetic markers abnormalities reported or detected in relevant biochemical parameters are highlighted and taken into account during interpretation of the genetic results . Different test options for cardiovascular risk management, indications for referral and clinical applications fh familial hypercholesterolaemia; cvd cardiovascular disease; lp(a) lipoprotein (a); hs - crp high - sensitivity c - reactive protein . In addition to the use of genetic markers abnormalities reported or detected in relevant biochemical parameters are highlighted and taken into account during interpretation of the genetic results . The first test option in table 1 includes eight mutations in the low - density lipoprotein receptor (ldlr) gene (d154n, del197, d200 g, d206e, c356y, g361v, v408 m, p664l) (kotze et al . 2003). These mutations account for fh in the majority of affected patients in the high - risk afrikaner, indian and ashkenazi - jewish populations of south africa . Less than 10% of fh patients in south africa have been correctly diagnosed, despite extensive awareness and publicising of hypercholesterolaemia as a cvd risk factor (marais et al . (2001) have demonstrated that determination of total cholesterol levels in fh families with known mutations fails to provide the correct diagnosis in nearly 30% of individuals when the 95th percentile for age and gender is used, and in 12% of cases when the 80th percentile is used . Of the 6070% of south africans with high cholesterol levels, this distinction is very important for treatment considerations to identify those at highest risk, as male fh patients have a more than 50% risk of coronary heart disease by age 50 years and in females the risk is approximately 30% by age 60 . In the high - risk south african population the average age of death is 45 years in men with fh (marais et al . The second test option, performed in conjunction with a medical and lifestyle assessment, includes multiple mutations of relatively low expression in different cvd - related genes . In addition to the dyslipidaemia - related mutations included in the cvd multi - gene assay, genetic variations involved in folate metabolism (methylation), haemostasis (blood clotting) and iron overload are also assessed (kotze et al . 2003; kotze and thiart 2003). These include functional snps in the apoe, (rs429358 and rs7412), mthfr (rs1801133 and rs1801131), f2 (rs1799963), fv (rs6025) and hfe (rs1800562 and rs1799945) genes . The genetic test results are integrated with clinical indicators of cvd risk (table 1) and lifestyle factors to identify a combination of risk factors that could cause or contribute to disease development, if left untreated . Since a prospective genotype - phenotype correlation study has not been performed to date to illustrate the direct correlation between different levels of the specific combination of biochemical parameters and the conditions listed in table 1, continuous monitoring of health outcomes by participating clinicians are encouraged using a combined service and research approach . (2010) who recommended careful examination of the effectiveness of the intervention strategy in routine clinical practice, as opposed to efficacy trails that examine interventions under more structured ideal conditions . The 2020 goals of the american heart association include a new concept of cardiovascular health that incorporates lifestyle risk factors considered key drivers of disease in genetically predisposed individuals . A multi - disciplinary risk reduction approach is necessary since all the risk factors that may lead to complex diseases such as cvd have not yet been elucidated . The clinical usefulness of this psgt approach was assessed as part of a nutrition intervention study in south african patients with the metabolic syndrome (mets) (van velden et al . Use of the multi - gene cvd assay in these patients could partly explain differential responses to nutrition intervention because of their genetic background . Genetic testing revealed that two of the twelve mets patients included in this pilot study had fh, while four individuals tested positive for the cholesterol - raising e4 allele of the apolipoprotein e (apoe) gene . The apoe polymorphism occurs in 3040% across ethnic groups and is associated with abnormal lipid levels, as also confirmed in the general south african population (kotze et al . The deleterious effects of the apoe polymorphism are mediated by modifiable environmental risk factors such as smoking, diabetes and obesity, which increase the risk of both cvd and ad (whitmer et al . While fh patients require long - term drug treatment with cholesterol - lowering statins, diet and lifestyle modification is the treatment method of choice in hypercholesterolaemics with the apo e4 allele . The psgt approach combines genetic testing with blood biochemistry (pathology) tests to determine gene expression and to monitor the effectiveness of the treatment strategy advised . Identification of a genetic risk factor in the presence of high cholesterol levels would confirm a genetic contribution to the risk profile, and in some patients a treatable genetic subtype such as fh or type iii dysbetalipoproteinaemia may be diagnosed after taking all known risk factors documented into account . When a cholesterol - raising genetic risk factor is identified in the presence of normal cholesterol levels it does not mean that the individual has a disease or will develop cvd, but that a genetic predisposition was identified that may in future turn into disease if a high - risk environment is entered . High cholesterol levels or cvd in the absence of any of the genetic risk factors tested for may furthermore point to lifestyle risk factors such as smoking or obesity as the main causative factors, if present . These clinical features may also be caused by the inheritance of other genetic risk factors not included in the initial genetic analysis . Based on the family history and health status of the individual extended mutation analysis may be recommended in some patients, as part of the diagnostic work - up towards development of a pathology supported gene - based risk reduction plan . According to artinian et al . (2010) a programme of counselling with extended follow - up performed in conjunction with self - monitoring and goal - setting provides the best approach to sustainable lifestyle changes to improve clinical outcome . Our psgt approach requires regular monitoring (e.g. 6-monthly) of blood biochemistry levels, where appropriate, to assess the effectiveness of the intervention strategy . In addition to its role in lipid metabolism, variation in the apoe gene is also associated with inflammation and oxidative stress . This may explain why smoking exacerbates the deleterious effect of the apoe e4 allele on arterial wall thickening (humphries et al . Apoe e4 was shown to be more susceptible to oxidation than the e2 and e3 isoforms and in mice, atherosclerosis could be reversed by dietary vitamin e supplementation (pratico et al ., life - long low dietary fat intake appears to be especially important in individuals with the apoe e4 allele (petot et al . When plasma cholesterol levels are raised, individuals with the apoe e4 allele are most responsive to a low - calorie diet, and less responsive to statin therapy than e2 allele carriers (gerdes et al . 2000). While moderate alcohol intake appears to have a beneficial effect in relation to cvd risk and cognitive decline it may not be the case in apoe e4 allele carriers . (2004) have shown that the risk of dementia increases with increasing alcohol consumption only in those individuals carrying the apoe e4 allele . A cross - sectional study performed in 685 ad patients from three different ethnic groups confirmed the deleterious effect of heavy smoking (one or more packs per day) and alcohol consumption (more than 2 drinks per day) on cognitive function . Detection of the apoe e4 allele, a history of heavy smoking or a history of heavy drinking was each associated with a 23 years earlier onset of ad . In patients with all three risk factors ad was diagnosed on average 10 years earlier than in those with none of the risk factors (harwood et al . (1995), the lifetime risk of developing ad is approximately 15% for persons with no family history and increases to approximately 30% in carriers with at least one apoe e4 allele, compared with less than 10% for those without the e4 allele . In a more recent study it was reported that by the age of 85 years, the lifetime risk of ad without reference to genetic risk factors is approximately 11% in males and 14% in females . However, in the presence of the apoe e4 allele the risk increases to 51% in males and 60% in females homozygous for the apoe e4 allele . In heterozygous carriers the risk was 23% in males and 30% in females, consistent with semi - dominant inheritance of a moderately penetrant gene (genin et al . Estimates were globally similar and reached the highest lifetime risk in some european populations, of up to 68% and 35% for females homozygous or heterozygous for the apoe 4 allele, respectively . Stratification of the data by age groups demonstrated that the apoe e4 allele is a risk factor not only for late - onset but for early - onset ad as well . Several studies support a role of one - carbon metabolism in the development of late - onset alzheimer s disease . The protective effect of omega-3 fatty acids discussed above may be mediated primarily through the vascular system, while cognitive benefit from a plasma profile high in antioxidants appears to affect the rate of total brain atrophy related to an ad type pathology . 2009) and hyperhomocysteinaemia - induced neurotoxicity (troen et al . 2008). Since homocysteine accumulation contributes significantly to the risk of both cvd and ad (seshadri et al . 2002; ravaglia et al . 2005), early detection of genetically increased requirements for folate and other vitamin co - factors are essential for optimal enzyme function and consequently vascular and brain health . Detection of variation in the methylenetetrahydrofolate reductase (mthfr) gene was shown to be most important in this context and provides one of the best examples of gene - diet interaction (nutrigenetics). In a study performed by coppede et al . (2011) the authors demonstrated a significantly increased frequency of the t - allele of the mthfr 677c> t polymorphism and the g - allele of the mtrr 66a> g polymorphism as well as respective heterozygous and homozygous genoptypes in 378 late - onset ad patients compared with 308 matched controls . These findings correlated significantly with increased mean plasma homocysteine levels and decreased serum folate levels . Detection of an interaction between the studied polymorphisms and biochemical biomarkers underlines the importance of a combination approach . It enables evaluation of the effect of both genetic (e.g. Mthfr mutation) and environmental factors (e.g. Folate intake, smoking, alcohol) on accumulation of homocysteine in plasma . Decreased mthfr activity due to genetic variation is of special concern in individuals with high alcohol intake since this may lead to impaired folate status due to malabsorption, increased excretion, or abnormal folate metabolism (halsted et al . The negative effects of low intake of the methyl - related nutrients with high intakes of alcohol are additive, therefore changes in overall dietary patterns are recommended to ensure the consumption of a protective high methyl diet . This is essential not only to reduce cvd risk but also to preserve cognitive function (ravaglia et al . 2005). A meta - analysis performed in 3299 ad patients and 4363 controls has shown that the mthfr t - allele increases the risk for ad in the general population, particularly in asian populations (hua et al . These findings are in accordance with the meta - analysis performed a year earlier including 19 case - control studies (zhang et al . The frequency of the mthfr t - allele was found to be significantly associated with susceptibility to ad in all subjects . In a subgroup analysis of individuals without the apoe e4 allele, the association of the mthfr 677t - allele with ad susceptibility was confirmed in asians but not in caucasians . The mthfr t - allele and mainly the tt genotype, was found to increase the risk of vad in asians (liu et al . Population difference may be explained by the fact that genetic influences on homocysteine levels or disease risk would only be detectable in populations with low folate status . Food fortification and folic acid supplementation represent confounding factors that need to be taken into account in research studies and clinical trials (holmes et al . (2003) in north western russia where the population has limited access to multivitamins and no food fortification, homozygosity for the t - allele of the mthfr 677>t polymorphism was detected in all persons with homocysteine levels above 30 m / l . Amongst carriers with the t allele, thrombotic risk increased 1.9-fold in patients with arterial thrombosis, 1.7-fold for venous thrombosis and 2.5 fold for both conditions . These findings are in accordance with the risk for recurrent venous thrombosis ascribed to the mthfr genotype by keijzer et al . These authors reported a relative risk of between 1.4 and 1.6 in the presence of the t - allele and a combined risk of 18.7 if detected in the presence of the factor v leiden mutation, the most common genetic risk factor for venous thrombosis . Detection of one or more genetic risk factors causing abnormal blood clotting in patients with venous thrombosis represents another preventable cvd subtype identifiable with the multigene assay referred to in table 1 . Individuals with genetic variations in the mthfr gene may require increased amounts of folate above the recommended daily allowance (rda) of 400 g per day, together with adequate amounts of vitamins b6 and b12 . Determination of homocysteine levels and genetic testing performed in conjunction with dietary assessment may therefore serve as useful parameters for improvement of a person s diet . It is not sufficient to perform a biochemical test of plasma homocysteine levels alone, as these levels fluctuate as a consequence of environmental changes (e.g. Medication, diet, alcohol intake). A specific dna test, on the other hand, is performed only once in a lifetime (1) to determine the underlying cause of high homocysteine levels and/or (2) to determine the appropriateness of food supplementation and dosages . As elevated plasma homocysteine is a marker of folate and vitamin b12 deficiency that may lead to many common disorders including cvd and ad, it is important to focus on the prevention of hyperhomocysteinaemia . Before wide implementation of genetic testing could be promoted in the context of cvd, however, consideration had to be given to the uncertainty of whether the association between elevated serum homocysteine levels and cvd is indeed causal . (2002) provided strong evidence for causality, because both genetic and prospective studies (that do not share the same potential sources of error) yielded highly significant results . Lowering of homocysteine concentrations by 3 mmol / l by increasing folic acid intake was predicted to reduce the risk of ischaemic heart disease by 16%, deep vein thrombosis by 25%, and stroke by 24% (wald et al . (2002), individuals with two copies of the most extensively studied 677c> t mutation (t - allele) were found to have a 16% higher risk of chd compared with individuals homozygous for the common allele . The increased risk of stroke associated with the t - allele was furthermore confirmed in a meta - analysis involving 14870 individuals (cronin et al . The benefits of folic acid supplementation are explained largely by favourable endothelial function outcomes after lowering homocysteine levels, as assessed by flow - mediated vasodilation or haemostatic markers (brown and hu 2001). Genetic testing may identify individuals at risk of homocysteine accumulation before damage occurs to dna, arteries and the brain . Vascular risk factors such as high homocysteine levels may modify the neurodegenerative process in patients with mild cognitive impairment (mci) at increased risk of vad and ad . The mthfr 677 tt genotype was shown to promote plasma homocysteine increase which in turn may favour intima - media thickening in patients with cognitive impairment (gorgone et al . 2009). In a one year follow - up study of 55 patients with mild cognitive impairment and 44 controls matched for age, gender and education, vascular risk factors were found significantly more often in the mci group, including the apoe e4 allele (p = 0.018), hyperhomocysteinaemia (p-0.012) and folate deficiency (p = 0.023) (siuda et al . 2009). However, whether these findings translate into significant changes at the clinical level is uncertain as only age and hypertension influenced the progression from mci to dementia within one year of this prospective observation . The influence of the apoe e4 allele on the risk of ad was independent of mthfr mutation status and homocysteine levels (styczyska et al . The importance of iron metabolism extends beyond the field of nutrition, representing a key factor in pathology, cardiology, oncology, neurological and infectious diseases . Brain iron increases with age and is elevated in ad and several other neurodegenerative diseases, correlating with earlier age of onset in men . In a recent study performed by bartzokis et al . (2011) it was shown that higher accumulation of iron in vulnerable gray matter regions may represent a risk factor for accelerated cognitive decline . Variation in numerous genes may interact with each other and the diet to determine iron levels in serum and the brain and age of onset of ad (van rensburg et al . 2000; sampietro et al . (2010) reported that highly prevalent genetic variants in iron metabolism genes can influence brain iron levels in men . Identification of the mechanisms underlying brain iron accumulation may lead to novel ways to offer neuroprotection for age - related neurodegenerative diseases . More than 10 years ago we demonstrated an earlier age of onset in south african patients with ad with both the apoe e4 allele and the transferrin (tf) c2 polymorphism (van rensburg et al the significance of tf c2 in ad has subsequently been confirmed in several studies, including the epistasis project conducted by lehmann et al . The previously reported interaction between the c282y mutation in the haemochromatosis (hfe) gene and tf c2 in northern europeans was confirmed in a comparative study of 1757 patients with ad and 6295 controls . Homozygosity for both the hfe h63d mutation and another variation in the tf gene were also found to be significantly associated with iron loading . Based on these findings, it was concluded that treatment for iron overload may thus be protective in some cases . The tf c2 variant has an increased allele frequency in ad patients compared to controls in some populations (van rensburg et al . 1993; zambenedetti et al . 2003), but not in others, such as in a french population of the bordeaux region (rondeau et al . Since tf c2 is associated with diseases attributed to oxidative damage, it may be hypothesised that diet may have an effect on the expression of the tf c2 variant . In an environment such as bordeaux, where a predominantly mediterranean diet and red wine rich in anti - oxidants are consumed regularly, the deleterious effects of tf c2 variant may be neutralised (van rensburg et al . The deleterious effects of mutations in the hfe gene are usually ascribed to their role in cellular iron overload . However, in the case of mutation h63d found to be over - represented in ad patients, the mutant protein is associated with prolonged stress of the endoplasmic reticulum and chronically increased neuronal vulnerability as demonstrated in an inducible expression cell model (liu et al . While the c282y mutation is more commonly associated with hereditary haemochromatosis (considered a preventable cause of heart disease and other equally important clinical conditions due to potential organ damage), h63d has received more attention for its role in neurodegenerative diseases . Hfe gene variants are considered important in this context due to the association of the hfe h63d mutant protein with iron dyshomeostasis, increased oxidative stress, tau phosphorylation, glutamate release and inflammation at the cellular level (nandar and connor 2011). The importance of assessing genetic risk factors together with relevant blood biochemistry parameters was demonstrated in the study of giambattistelli et al . These authors explored the interconnectedness of genetic variation in the hfe (h63d and c282y) and tf (c2) genes with serum markers of iron status, including iron, ferritin, tf and tf - saturation, as well as liver function (albumin, transaminases) and prothrombin time in 160 ad patients and 79 healthy controls . Statistical analyses revealed that a one - unit tf serum decrease increases the probability of ad by 80%, while a one - unit increase of ast / alt ratio generates a 4-fold probability increase . The hfe h63d mutation was associated with higher levels of iron and lower levels of tf . These results suggest that the presence of hfe mutations and iron abnormalities may increase the probability of developing ad when accompanied by distress of the liver . In contrast to the above studies providing strong evidence that the hfe h63d mutation is a risk factor for ad when evaluated in conjunction with other relevant biomarkers to assess cumulative effects, a meta - analysis of 22 studies performed in 4365 cases and 8652 controls implicated it as a protective factor (lin et al . The previously reported association of hfe c282y with increased risk of ad could also not be confirmed . We believe that the only way to overcome the limitations of low - penetrance genetics is to combine dna tests with relevant pathology or blood biochemistry such as cholesterol, serum iron status, oxidative stress, inflammatory markers and liver function tests as appropriate . For example, biochemical abnormalities associated with low penetrance mutations in the apo e (cholesterol) and hfe (ferritin, transferrin saturation) genes, respectively, are usually not detectable early in life . As the pathology may develop over a long period of time, preventative measures can be implemented based on this knowledge when a genetic risk factor is identified in the presence of known environmental triggers that could be eliminated or targeted during intervention . Application of the psgt approach has been discussed previously in the context of iron metabolism (kotze et al . Hereditary haemochromatosis (hh) provides an excellent example of a complex disease that is best addressed by the psgt approach, which requires that (1) genetic testing is performed within a specific pathology / biochemical and clinical profile that also defines the test selection criteria, (2) the patient report contains both the biochemical and genetic test results for clinical application in the context of relevant documented environmental factors, and (3) gene expression and monitoring of response to treatment are assessed through the accompanying pathology and genetic test parameters . This approach has proved valuable to distinguish patients with hh from those with the dysmetabolic or insulin - resistance iron overload disorder frequently observed in patients with non - alcoholic fatty liver disease (nafld). Nafld is the hepatic manifestation of mets and is considered an independent risk factor for cvd . (2011) have demonstrated that nafld may induce insulin resistance and metabolic disorders associated with age - associated neurodegenerative diseases such as ad . The identification of the spectrum of risk factors underlying iron overload provides a major healthcare opportunity to reduce the burden of dementia, heart disease, cancer, diabetes, arthritis, infertility and many other complications of organ damage in the general population . Recently, variation in the fat mass and obesity - associated (fto) gene was identified as the most significant genetic risk factor for susceptibility of polygenic obesity . With more than 300 million obese persons worldwide it is crucial to understand the clinical implications of genetic variation in the fto gene (ho et al . 2010). A functional snp in intron 1 of the fto gene (rs9939609) (berulava and horsthemke 2010) occurs in nearly 50% of the general population and is associated with an approximately 1.2 kg higher weight, on average, in adults and an approximately 1 cm greater waist circumference (liu et al . 2010a, b). The effect of the fto gene to increase the risk of obesity and diabetes may be mediated through epigenetic changes (almn et al . 2012). Both a high bmi and diabetes linked to the fto gene are vascular risk factors implicated in the development of vad and ad . Much interest is currently focused on the possibility that the fto gene affects brain function and structure as a high bmi correlates with frontal, temporal and subcortical atrophy (bertram and heekeren 2010). Fto is highly expressed in the human brain and primarily affects frontal lobe - dependent cognitive processes (benedict et al . This was shown in a study of verbal fluency performance in 355 elderly men who had no clinically apparent cognitive impairment at the age of 82 years . Obese and overweight but not normal weight individuals with the fto a - allele showed a lower performance on verbal fluency than homozygotes for the t - allele (benedict et al . Generation of 3d maps of regional brain volume revealed a pattern of systematic brain volume deficits in subjects with fto obesity - associated risk alleles when compared with non - carriers (ho et al . The average brain volume difference for the frontal lobes and the occipital lobes were approximately 8% and 12%, respectively, with the greatest effects in individuals with high bmi s . These brain differences were not attributable to the effect of cholesterol, hypertension or the volume of white matter hyperintensities . In a nine - year prospective follow - up study exploring the impact of fto on ad and dementia risk in 1003 persons without dementia, individuals with the fto aa genotype showed a higher risk of cognitive decline after adjustment for age, gender, education, physical activity, bmi, diabetes, cvd and apoe genotype (keller et al . Co - inheritance of both the fto aa genotype and apo e4 allele was associated with a significantly increased risk of dementia, and particularly ad . The fto gene effect acts mostly through interaction with the apo e4 allele, independently of physical activity, bmi, diabetes and cvd . These results are in accordance with an association between fto and reduced brain volume reported by ho et al . (2011) the strong influence of dietary fatty acid distribution on the effect of the rs9939609 polymorphism in the fto gene on obesity risk was clearly demonstrated from childhood . Genotyping of rs9939609 in 354 spanish children and adolescents aged 618 years (49% males), showed a statistically significant interaction between the consumption of saturated fatty acids (sfa) (percentage of total energy) in relation to polyunsaturated fatty acid (pufa) intake and obesity risk linked to the rs9939609 snp of the fto gene . Risk allele carriers consuming more than 12.6% sfa (of total energy) had an increased obesity risk compared with tt carriers . In a similar way, a allele carriers with an intake ratio lower than 0.43 pufa: sfa presented a higher obesity risk than tt subjects . These findings emphasise the importance of early detection of a genetic predisposition for obesity so that preventative measures can be implemented to prevent cumulative risk that may lead to cvd and/or ad . The above - mentioned information highlights the importance of early identification of cvd risk factors and timely intervention before cognitive decline becomes apparent . While current practice is generally to address symptoms as they occur, several studies point out the importance of treating cvd risk factors as early as midlife (solomon et al . According to the world health organisation eight risk factors account for over 75% of cvd, namely hypertension, high body mass index, high cholesterol, high blood glucose, low fruit and vegetable intake, high alcohol consumption and physical inactivity . In a study performed in more than 800 caucasians (szolnoki et al . 2003) it was shown that the presence of the apo e4 allele worsened the unfavourable effects of hypertension, diabetes mellitus, smoking, or drinking on the incidence of ischaemic stroke . Synergistic effects were also found between the mthfr 677tt genotype and alcohol consumption or smoking, while detection of a genetic predisposition for venous thrombosis (e.g. Factor v leiden mutation) in combination with hypertension and diabetes mellitus increased the risk of ischaemic stroke . A study performed by volzke et al . (2005) confirmed the increased risk of atherosclerosis with rising serum ldl - cholesterol concentrations in carriers of the factor v leiden mutation . In a study performed in 12 239 women between the ages of 51 to 69 years, roest et al . (1999) demonstrated that inherited variation in iron metabolism (hfe gene) is involved in cardiovascular death in postmenopausal women, especially in women already carrying classic risk factors such as hypertension and smoking (roest et al . These findings confirm the importance of early detection of a genetic contribution to clinical risk factors for disease prevention . Therapeutic responses may also be genotype specific and this could contribute to the apparent inability of presently available drugs to alter the course of ad for some patients . Approximately 15% of ad cases with adverse response to treatment are associated with a defective cyp2d6 gene which can be assessed genetically (cacabelos 2003). Pharmacogenetic response may be influenced not only by this gene involved in drug metabolism, but also genes involved in the pathogenesis of both cvd and ad . Patients with the apoe e4 allele appear to be the worst responders to both cholesterol - lowering statin treatment and various drugs prescribed to ad patients (cacabelos 2008). This author suggested that the presence of the apoe-44 genotype could convert cyp2d6 extensive metabolisers into poor metabolisers . If confirmed in future studies, gene - gene interaction between the apo e and cyp2d6 genes may have important clinical implications for treatment of a wide range of chronic disorders . The key to disease prevention therefore lies in a better understanding of gene - environment interactions underlying cvd and ad and effective intervention based on this knowledge (fig genetic testing enables the dissection of complex conditions into treatable subtypes; for example to distinguish between fh patients requiring long - term drug treatment and those with the apoe e4 allele who respond well to specific dietary and lifestyle changes without the use of medication.fig . 1co - inheritance of multiple genetic risk factors in the presence of environmental triggers has a significant impact on the development of clinical conditions associated with the development of cardiovascular disease (cvd) and/or alzheimer s disease (ad). Environmental factors that may be either harmful or beneficial (right column) in the context of their direct effect on genetic factors underlying the high - risk clinical conditions highlighted in the circles on the outside, can potentially be manipulated to prevent the conversion of genetic risk factors into disease (reproduced with permission from kotze et al . 2006) co - inheritance of multiple genetic risk factors in the presence of environmental triggers has a significant impact on the development of clinical conditions associated with the development of cardiovascular disease (cvd) and/or alzheimer s disease (ad). Environmental factors that may be either harmful or beneficial (right column) in the context of their direct effect on genetic factors underlying the high - risk clinical conditions highlighted in the circles on the outside, can potentially be manipulated to prevent the conversion of genetic risk factors into disease (reproduced with permission from kotze et al . Genetic testing of cvd risk factors has the potential to translate into improved health outcomes in patients with cognitive impairment and their at - risk family members . Based on the knowledge that assessment and treatment of cvd risk factors in middle age may reduce the risk of heart disease and dementia in old age (kotze et al . 2006), a comprehensive psgt approach was developed . Since the deleterious effects of several genetic risk factors shared between cvd and ad are mediated through diet and lifestyle factors, identification of a genetic predisposition highlights the importance of smoking cessation where relevant and a healthy diet with regular intake of foods that offer neuroprotection . In contrast to severe cvd subtypes such as fh that requires aggressive treatment, simple lifestyle changes can delay the onset and severity of dementia (potocnik et al . Contrary to previous belief, the identification of ad genetic risk factors that may be triggered by environmental factors does not result in increased worrying and depression (romero et al . 2005). Genetic testing for multiple cvd risk factors in conjunction with nutrition and cognitive assessment may empower patients to take the necessary steps to improve their health . Educational campaigns that highlight the link between cvd risk factors and ad may help reduce the impact of both conditions.
To date only two species, mangrovibacter plantisponsor dsm 19579 and mangrovibacter yixingensis kctc 42181 have been described under the genus mangrovibacter (http://www.bacterio.net/mangrovibacter.html). The draft genome of mangrovibacter sp . Strain mfb070, isolated from an aquaculture farm in india has been described recently which is the only available genome sequence within genus mangrovibacter . Members of the genus mangrovibacter have been shown to possess plant growth promoting features such as nitrogen fixation and may provide niche - specific advantage to the plant . In context to invasive weeds, this could provide a better ecological fitness in an invaded territory compared to native vegetation . Thus, understanding the microbiota and role they may play during plant invasion could lead to more directed and sustainable management of weeds . Chilika lagoon (1928-1954n; 8506-8535e) is a brackish water lagoon located in the odisha state of india . P. karka is a large perennial grass of the family poaceae and occupy most of the northern shoreline of lagoon . In order to understand the microbial basis of the invasive success of this weed, a study was undertaken to investigate the culturable diversity of rhizosphere microbiota associated with p. karka . During this study, a gamma - proteobacterium, facultative anaerobic, endophytic nitrogen fixing mangrovibacter sp . Strain mp23 grew at temperatures between 20 c and 40 c with an optimum at 37 c in presence of 1% nacl . The 16s rrna gene phylogenetic analysis showed that strain mp23 was most closely (99.71% similarity) related to m. yixingensis kctc 42181 and m. plantisponsor dsm 19579 indicating that it belong to genus mangrovibacter . Here, we described the draft whole genome shotgun sequence of strain mangrovibacter sp . Strain mp23 (dsm 100250 = kctc 42580), which will provide genetic insights into the nitrogen fixation, stress tolerance, plant niche adaptation, and comparative evolution of this species . The genome of mangrovibacter sp . Strain mp23 was sequenced by a shotgun sequencing method using the illumina miseq sequencing system with a paired - end module . The ngs qc toolkit v 2.3 was used to filter the data for high - quality (hq) vector- and adapter - free reads for genome assembly . A total of 6,895,374 paired end reads were generated out of which 6,702,332 high quality vector - filtered reads were considered, representing approximately 310 fold coverage of the genome . These reads were assembled using masurca v. 3.1.3 and resulted into 50 contigs with a total size of 4,947,475 bp and an n50contig length of 428,946 bp . Annotations of protein - coding genes, as well as other functional genome units were carried out through ncbi prokaryotic genome annotation pipeline (pgap). The genome contained a total of 4592 genes and 4392 protein - coding genes with predicted function . The complete genome of strain mp23 was 4,947,475 bp in length with an estimated g + c content of 49.9% and consists of 77 trna genes and 28 rrna (8 = 5s, 5 = 16s, 15 = 23s) genes (table 1). This whole genome shotgun project has been deposited at ddbj / embl / genbank under the accession number lyrp00000000 . The authors declare that there is no conflict of interests with respect to the work published in this paper.
Aortic valve stenosis (as) is a common valvular disease in the western world . As the geriatric population has grown, so too has the number of patients presenting with symptomatic aortic valve disease because its prevalence increases with age . Surgical aortic valve replacement (savr) has produced significant results that have been well documented for elderly patients, including improved life expectancy, cardiovascular symptoms, and quality of life ., yet, up to one - third of elderly patients with severe as have been excluded from surgery . Not surprisingly, advanced age has been a leading barrier to surgical intervention in elderly patients . This is based on the peri - operative mortality rate which is found to increase with age from 1.3% in patients 70 years old, to about 5% at age 80 - 85 years, and 10% in patients 90 years old ., it is important, though, to realize that age, per se, is not a predictor of poor outcomes of surgery, and those patients usually enjoy the same survival rates within their age and gender matched population . To help bridge this gap in care, medical innovations such as transcatheter aortic valve implantation (tavi) have emerged as a new treatment option which improves the clinical course of as without high surgical risk . Tavi includes the insertion of a prosthetic valve into the stenotic aortic valve through a vascular access without the need for open heart surgery . Two valves are commonly used for this procedure: the edwards sapien valve which is a balloon expandable valve and the core valve which is a self - expanding one . They may also be placed via other arteries such as the axillary artery or directly through the cardiac apex in patients with stenosed iliac and femoral arteries . Tavi is strongly indicated for symptomatic as patients with multiple comorbidities, or high operative risk [i.e., expected mortality> 20% with the european system for cardiac operative risk evaluation score (euroscore) or> 10% with the society of thoracic surgeons score (sts)], as long as life expectancy is a year or more . Pre - procedure multidisciplinary assessment is important for the appropriate selection of patients to undergo tavi . The team usually includes cardiologists, cardiothoracic surgeons, cardiac anesthesiologists, interventional cardiologists and imaging specialists . Cardiac imaging should assess not only the severity of aortic stenosis, but also morphometric characteristics like the diameter of the aortic root, the conformation of the aortic valve and annulus, and area of valve calcification to ensure that the most appropriate valve is implanted, and that the procedure is likely to be successful . Assessment for atherosclerosis of both coronary and peripheral arteries is also important to identify patients at risk for perioperative complications and to select the optimal site for access . Following tavi, an immediate and sustained improvement in aortic valve area, trans - valvular gradient, and left ventricular ejection fraction were reported. These changes were associated with an enhancement in patients' physical and mental functioning, as well as their quality of life ., however, frail elderly patients were found to have smaller gains in functional status after tavi and they may require multidisciplinary interventions, such as nutrition consultation and exercise - training programs, to achieve desired outcomes . Due to the mentioned hemodynamic and functional effects of tavi, the early and midterm survival in patients with as has significantly improved as shown in moat, et al . Study, who reported life expectancy rates of 92.9%, 78.6% and 73.7% at 30 days, 1 year and 2 years after tavi, respectively . Studies have shown that transfemoral approach is associated with better survival rates compared to other vascular accesses . This, however, is still controversial because patients who are referred to transapical or transaxillary approaches usually have higher risk profiles which explains the increased mortality after these procedures . As discussed earlier, both tavi and savr have achieved significant improvements in survival rates as well as other outcomes in patients with severe aortic stenosis . Studies have shown that savr can be performed in elderly patients with acceptable morbidity and mortality rates . The question then becomes, can tavi be trusted as a safe and effective alternative to surgical management in elderly patients? Of the studies that looked at the outcomes of tavi compared to savr, we specifically reviewed the trials where average age of patients who underwent both procedures was 80 years . Only 8 studies were found (5 observational studies and 3 randomized controlled trials) and summarized in (table 1). In these studies, elderly individuals who underwent tavi experienced better in - hospital recovery, and similar short and mid - term mortality compared to those underwent savr (table 1). One of the most important studies is the placement of aortic transcatheter valves (partner) trial which was a multicenter, randomized trial that compared tavi to surgical management and medical therapy in 1,057 as patients . In this study, patients were divided into two cohorts: group a which included those with high surgical risk (the mean age of this group was 84 years old), and group b which included those who were not considered to be appropriate candidates for surgery (the mean age was 83 years old). In the high - risk partner trial cohort a, mortality at 30 days was 3.4% in the tavi group, compared to 6.5% in the savr group (p = 0.07). At the 1-year and 2-year follow - up, mortality was 24.2% and 33.9% with tavi vs. 26.8% and 35.0% in the savr groups, respectively (p = 0.44, p = 0.78, respectively)., partner trial also showed that the incidences of stroke, myocardial infarction, acute kidney injury (aki), endocarditis, and pacemaker placement at one and two years after tavi and savr were comparable ., however, patients who underwent savr experienced more major bleedings, and less major vascular injuries compared to those treated with tavi . The authors concluded that tavi is not - inferior to savr as a treatment of as in high risk surgery patients . Another randomized controlled study was the staccato trial which initially planned to assign 200 patients (age> 75 years) to either tavi vs. savr . However, after the inclusion of 70 patients, the trial was prematurely terminated due to unexpectedly poor outcomes in the tavi cohort . Authors of this trial concluded that current indications for tavi should remain restricted only to surgically inoperable patients . Only a few studies have focused on elderly patients with intermediate or low risk for procedural complications . These include the surgical replacement and transcatheter aortic valve implantation (surtavi) trial and the observant trial . The surtavi investigators prospectively enrolled patients with symptomatic severe aortic stenosis and intermediate surgical risk (sts score between 3% and 8%) to either tavi or savr . All - cause mortality at 30 days (7.8% vs. 7.1%, p = 0.74) and 1 year (16.5% vs.16.9%, p = 0.64) was comparable between the two groups . The italian observant study reported similar 30 days mortality for its intermediate risk, propensity - matched subgroups . Differences in rates of blood transfusion (higher in savr), vascular damage, permanent atrioventricular block, and residual aortic valve regurgitation (all higher in tavi) were reported . F / u: duration of follow up; mo: months; os: observational study; rct: randomized controlled trial; savr: surgical aortic valve replacement; tavi: transcatheter aortic valve implantation . Lastly, while the early and mid - term survival rates seem comparable in tavi and savr in most of the outlined studies, not much is known about long - term outcomes of tavi in elderly patients . Observations by holzhey, et al . Showed that kaplan - meier curves diverged between tavi and savr patients after 6 months in favor of conventional surgery . Follow - up studies, therefore, are necessary to assess whether tavi will remain a comparable approach to savr on long - term basis as well . Given the growing geriatric population in the western countries, more and more of nonagenarians are expected to undergo tavi as the principal treatment for severe as . Yet, relatively few studies have examined the outcomes of this procedure in very old patients . Jabs, et al . Have reported their experience with a 99 year old as patient who underwent tavi after presenting with syncope and progressive dyspnea . Following tavi, aortic valve area improved from 0.6 cm to 1.5 cm and the patient was discharged to a geriatric rehabilitation unit 2 days after the procedure . For the next four years, the patient remained independent, with mild exertional dyspnea, and without recurrent syncopes . Transesophageal echocardiography at 3.5 years follow - up showed aortic valve area of 1.5 cm with persistent optimal positioning of the implanted valve . A recent swedish study reviewed post - procedure mortality in 29 nonagenarian patients who underwent tavi between 2008 and 2011 . One - year, two years, and three years mortality were found as 11%, 28%, and 40%, respectively . The available literature shows only three trials that studied the outcomes of tavi in very old patients comparing to younger patients . All of these studies were observational and no randomized controlled trials were identified as shown in table 2. in a study of 1,386 patients who underwent tavi at ages ranging between 4099 years, the researchers divided the patients into four age groups and analyzed the outcome of tavi in each group . Despite the difference in the groups' characteristics, all groups, including the oldest one, showed immediate and marked improvement of the hemodynamic valve status, functional status, and overall quality of life . Also, each group had similar rates of technical success and 30 day all - cause mortality regardless of age . Havakuk, et al . Evaluated 293 patients who underwent tavi (patients' age ranged between 63 and 98 years and average age was 83 years). The cohort was divided depending on age into 2 groups: younger vs. older than 85 years . Older patients experienced length of in - hospital stay, readmission rates, and 30-day mortality similar to those reported in younger patients . However, overall mortality rate during the entire follow - up (median 480 days, interquartile range 330 to 770 days) was higher in the older age group . In another study, yamamoto et al . Compared the clinical outcomes of tavi in patients 90 years old to patients 90 years old and found similar procedural success in both groups . Although 30-day and 6-month mortality trended to be higher in patients older than 90 years old, the differences in mortality rates were statically insignificant (6% vs. 15%, p = 0.22; and 14% vs. 27%, p = 0.14, respectively). Moreover, the cumulative survival after 13.4 8.0 months of follow - up was comparable between both groups (p = 0.22). Bmi: body mass index; cabg: coronary artery bypass graft; copd: chronic obstructive pulmonary disease; dm: diabetes mellitus; hf: heart failure; icu: intensive care unit; mi: myocardial infarction; nyha: new york heart association; os: observational study; tavi: transcatheter aortic valve implantation . In conclusion, the available literature gives an impression that tavi is a beneficial and tolerable procedure in very old patients with severe as . This hypothesis, however, still needs further testing in randomized controlled trials in order to be confirmed . Although tavi has a high success rate with few surgical complications, it still carries risks . Common procedure complications include vascular injuries, bleeding, stroke, atrioventricular conduction system injuries, aki, and aortic regurgitation . Complication rates differ with operator experience, the size of the device, the site of catheter access, as well as the use of pre - procedural screening . Vascular injuries are the most common complications after tavi, and range from dissection, to perforation, to acute thrombotic occlusion . Yamamoto, et al . Reported more frequent major vascular complications in nonagenarians (19% vs. 5%, p = 0.022)., though, found that only minor vascular injuries increased in older patients (7.5% vs. 16%, p = 0.02), and reported similar rates of major vascular complications in both age groups of their study (4.3% vs. 2.5%, p = 0.41), as shown in figure 1 . Patients also are at risk for cerebrovascular accidents (cva) after tavi . In the high - risk cohort of the partner trial (cohort a), there was a trend toward more strokes in the tavi group at 30 days when compared to the savr group (4.6% vs. 2.4%, p = 0.12) and at 1-year (6.0% vs. 3.2%, p = 0.08). Up to 10% of tavi patients experienced cva, regardless of their age (figure 1). The cva are mostly ischemic, felt to be due to showering of emboli from the aorta or during valve positioning and deployment . To reduce the risk of stroke, aspirin, clopidogrel, or warfarin has been used in tavi patients . In a small retrospective study that included 171 patients (average age 81.6 years), the use of post - tavi dual antiplatelet did not protect patients from stroke, but has been associated with worse bleeding compared to the use of single antiplatelet or warfarin . Durand, et al . Retrospectively compared the use of mono antiplatelet therapy vs. dual antiplatelet therapy in 292 patients underwent tavi (average age was 83.6 years), and found that monotherapy reduced life - threatening and major bleedings without increasing the risk of stroke and myocardial infarction compared to dual antiplatelet therapy . Another study has showed that the safest post tavi regimen was a combination of clopidogrel and vitamin k antagonists . In this study, advanced age increased risk of early bleeding after tavi (or: 5.96, 95% ci: 1.4724.13, p = 0.01). More studies are needed to evaluate the efficacy and risks of different antithrombotic regimens before recommendations can be made regarding stroke prevention in tavi patients . X axis includes age groups of tavi patients as presented in buellesfeld, et al ., havakuk, et al ., and yamamoto, et al ., trials . Represents the youngest group of tavi patients with mean age of 73.4 4.5 years; b: represents the second group of tavi patients with mean age of 80.6 1.1 years; c: represents the third group of tavi patients with mean age 84.5 1.1 years; d: represents the second group of tavi patients with mean age 88.9 2.2 years; e: represents patients who are 85 years old; f: represents patients who are 85 years old; g: represents patients who are 90 years old; h: represent patients who are 90 years old . Tavi: transcatheter aortic valve implantation . Due to the proximity of the aortic valve to the atrioventricular node and bundle of his, tavi is associated with a risk of atrioventricular conduction system injuries and permanent pacemaker implantation . The need of a new pacemaker was reported in partner study in similar rates after both tavi (5% and 6.4%) and savr (6.4% and 7.2%), (p = 0.44, p = 0.69, respectively), at one and two years, respectively . However, this complication was observed more commonly with corevalve implantations presumably because of its longer stent frame, self - expanding nature, or ovoid shape . Advancing age does not increase the risks for atrioventricular nodal block or permanent pacemaker (figure 1). Aki is another potential complication of tavi and is associated with an increase in post - procedure mortality . Pre - existing hypertension, chronic obstructive pulmonary disease, and blood transfusion predicted aki in one study . Surprisingly, neither the amount of intravenous contrast agent nor patient's age, nor pre - procedure chronic kidney disease affected the risk of aki after tavi .,,, paravalvular leak due to incorrect sizing or positioning of the prosthetic valve, or under - expansion of it, can result in aortic regurgitation . This complication was more common in patients undergoing tavi than savr in the partner study (12.0% vs. 0.9%, p <0.001), but did not increase in very old patients when compared to younger patients. Studies found that paravalvular leak is more prevalent with transfemoral approach and in patients received self - expandable valves ., valve leak should be followed closely and managed early because severe aortic regurgitation was found to be an independent predictor of mortality after tavi . Based on the limited published studies, some of the expected complications after tavi are reported more in the oldest patients such as vascular injuries . . However, very old patients may need closer monitoring to avoid further morbidities and mortality . Tavi offers a new treatment approach for tens of thousands of elderly patients with severe as, who otherwise would be treated medically with resultant poor outcomes . As transcatheter technology improves with second generation devices and operator experience increases, indications for tavi may expand to include patients with moderate surgical risk, especially those who are frail . More studies to identify the indications and contraindications for specific prosthetic valves and methods of vascular access would help physicians tailor treatments to patients . Post - tavi morbidities and mortality are expected to decrease significantly with determination of the ideal anticoagulation treatment that can reduce the incidence of stroke without rising bleeding risk . Similarly, since paravalvular leak has a bad impact on survival of patients undergoing tavi, more efforts are made to predict this complication, such as using aortic valve calcium scoring . These efforts may result in better patients' selection for tavi and decrease the expected mortality . Bioprosthetic valves are preferred for elderly patients undergoing savr, yet these tend to fail early, requiring re - operation . Mortality rates for re - operation are high, but theoretically may be lower with valve in valve implantation using the transcatheter technique . Many older adults with severe as are not candidates for savr because of high surgical risk, advanced age, frailty, or comorbidity conditions . Improvements in devices, technical expertise, and clinical experience, along with perioperative patient management may expand the use of tavi for elderly patients at lower surgical risk in the future . More studies, however, are needed to explore a multiplicity of aspects of this treatment especially in very old patients.
Establishment and maintenance of cellular polarization are critical features for development and organ function . Apical - basolateral (a / b) polarity of epithelial cells serves to perform vectorial functions, like transport of fluid or directed secretion of specific proteins . Besides the ubiquitous epithelial a / b polarization, most epithelial tissues present a second polarity axis within the epithelial plane, referred to as pcp . Pcp was initially discovered in insects [24] and studied in drosophila, where all adult cuticular structures display pcp [59]. Many processes and organs requiring pcp have been subsequently discovered in vertebrates ranging from skin and body hair orientation, positioning of primary cilia, and sensory epithelium in the inner ear to directed movement of mesenchymal cell populations during gastrulation among many other processes (for example, [1014]). How individual cells that are hundreds of cell diameters apart acquire the same polarity within the plane of an epithelial organ is a fascinating cell biological problem . Although much progress has been made, the mechanistic aspects of pcp establishment are far from being understood . Initial molecular insights identifying pcp signaling components came from genetic screens in drosophila (table 1) [5,1619]. Functional studies in several tissues placed a set of the pcp regulators into different categories . These consist largely of two groups: (a) the fz - pcp core group and (b) the ft / ds group . Whereas the latter revolves around the heterophilic adhesion of the two protocadherins ft and ds and the associated secreted golgi kinase four - jointed (fj), the so - called fz - pcp core genes historically comprise six proteins that interact with each other inter- and intracellularly to separate into two complexes on opposing sides of each cell, which provides a cell with a planar orientation (see table 1 and figure 1 for molecular details and published reviews for further insight on pathways and regulators associated with these core pcp systems). The transmembrane interacting components of the fz core system are, besides fz itself, the four transmembrane protein van gogh (vang, also known as stbm / strabismus, vangl in vertebrates) and the atypical cadherin flamingo (fmi, also known as stan / starry night, celsr in vertebrates) [79,13]. All drosophila genes have equivalent functional orthologues in vertebrates (often more than one). The homologous vertebrate genes for fw and vhaprr have not yet been functionally characterized in the planar cell polarity (pcp) context . Several other intracellular binding partners exist with yet - to - be - defined functions in pcp . Note that fz and vang act in both the interpretation of the long - range signal(s) wg and dwnt4 - as well as the short - range / local cell - cell transmembrane interactions . The cytoplasmic components dsh, dgo, and pk are essential for the negative feedback loops within individual cells, as they antagonize each other . Dgo, diego; dsh, dishevelled; fmi, flamingo; fw, furrowed; fz, frizzled; pcp, planar cell polarity; pk, prickle; vang, van gogh . The two systems are thought to mediate both long - range and cell - to - cell (short range) interactions in all tissues where pcp has been studied in drosophila . The fz / pcp core group appears fully conserved in vertebrate pcp contexts, although additional components, like receptor tyrosine kinases of the ror family, are involved in vertebrate models . The function of the ft / ds group in vertebrates is less well defined, although there is good evidence in some tissues / organs for their requirement in pcp establishment [2022]. Nevertheless, the study of both pcp systems in vertebrates is complicated by redundancy issues of multiple related proteins for each component . Also, whereas the fz / pcp core group appears fully dedicated to pcp regulation, ft / ds signaling has also been linked to growth control and to hippo signaling as an upstream activator module . Despite these complications, an absolutely key question in pcp regulation is the molecular relationship of the two pcp systems or how their function is integrated within individual cells or groups of cells . Several interaction models have been proposed, but these remain controversial and are an active area of research (see below). Despite the prevalence of studies on the fz / pcp core group and ft / ds signaling in pcp establishment, it is noteworthy that additional cellular mechanisms function in cellular planar polarization contexts that appear to be largely (or fully) independent of these two molecular cassettes . Alone in drosophila, the planar cellular polarization in the embryo that drives germband extension (a process comparable to vertebrate convergence and extension) and that is based on polarized myosin ii localization seems to occur independently of either system . The six components of the fz core group have been known for quite some time, the last of these diego (dgo, related to inversin and diversin in vertebrates)being added in 2001 . However, recent work in drosophila identified two additional factors that might be linked to pcp establishment in a similar manner and thus could also be considered members of the fz core group . These include the vhaprr accessory subunit of the proton pump v - atpase [2830] and furrowed (fw), a drosophila selectin family member . Both affect pcp in a manner similar to the core components in all tissues analyzed (wing, eye, and thorax) and can be co - immunoprecipitated with fz and fmi (vhaprr) or specifically with fz (fw). At least for fw, its function appears to be similar to that of fmi, as it is required to stabilize fz in plasma membrane complexes, and it appears also to promote homophilic cell adhesion . Moreover, fw can mediate an intercellular binding / interaction between fz and vang, again a function related to what fmi is doing, but unlike fmi, fw appears to affect only fz stability and associates only with fz (figure 1). Strikingly, fw and fmi could be partially redundant in mediating an fz - vang interaction or fz membrane association, as the tissues where fmi mutants have a weaker phenotype (thorax) are where fw loss of function (lof) is most severe; for the eye, the opposite is true . Importantly, the pcp defects in fw, fmi double mutants are not more severe than those in fz null flies, suggesting that both require the presence of fz for their function . The role of the vhaprr protein remains to be clarified, but functional studies suggest that it can affect the trafficking of fmi and possibly fz to or from the membrane (or both). Whereas vhaprr can be co - immunoprecipitated with both fz and fmi, it appears to directly physically associate with fmi only . While the function of fw is restricted largely to pcp (with only a mild overgrowth of the eye disc and a potential role in cytoskeletal regulation), vhaprr appears generally required for trafficking, affecting also fz2 (canonical wingless [wg] signaling), notch, and e - cad . The biggest conceptual questions in pcp establishment are how long - range signals might regulate the process and how this is relayed to and reinforced through local cell - to - cell interactions . From early studies with lof mutants, it was clear that cellular orientation was non - random (best visualized in the wing) and that cellular orientation would indeed follow / adjust to the neighbor's patterns, easily observed in fz, ds, ft, fmi, or vang mutants . This behavior was further clarified with clonal analyses and the discovery of non - cell autonomous effects . An example is the non - autonomous behavior of the fz mutant or overexpression clones affecting the polarity of wildtype cells surrounding the clones in the wing, eye, and abdomen . These observations set the stage for analyses of local and global signals to work on pcp orientation: local signals with short - range abilities to communicate on a cell - to - cell basis and global signals that coordinate the process at the organ level over a long range . First, there needs to be spatial restriction or directional signaling for a given signal . Second, a connection between the long - range signal and the cell - cell communication mechanism(s) should exist . Two global clues had been associated with long - range pcp signaling: (a) the ds and fj expression gradients in the ft / ds system and more recently (b) the long - range effects of localized wnt expression on the fz / pcp core factors . How are the ft / ds or fz - pcp core pathways interpreting these signals, and how are these two systems integrating the information? The ft / ds pathway covers both the (long - range) spatial restriction and the (short - range) cell - cell communication features of pcp establishment . Related to long - range effects, two of its key components have a graded / localized expression pattern: in the wing, fj is expressed in an ascending proximo - distal gradient [4547] and ds is expressed at higher levels in the proximal part, opposite to the fj gradient, although its slope is rather steep as compared with other gradients . The third main component, ft, has a rather uniform expression in the wing . Within the current model, ds acts as a ligand for ft, inhibiting its function and creating a distal - to - proximal ft activity gradient (reflected in anisotropic subcellular distribution of ft, along with the fj expression gradient). This model applies also for the eye, where fj is expressed in a graded manner with its peak at the equator, ds forms gradients with peaks at the poles and ft is uniformly expressed . Similarly, in the eye, flat ds expression rescued the ds mutant as long as fj was expressed in a gradient . Next, polarity needs to be translated from the long - range (body) axes to the local (short - range) cell - to - cell communication . How is the long - range ft / ds pathway information transmitted to the short - range cell - to - cell interactions? At the single - cell level, the atypical cadherins ft and ds are localized subapically in epithelial cells near but not directly at the adherens junctions, where they form heterodimers . Fj functions as a kinase at the golgi, affecting the localization of ft and ds and the strength of their interactions . For example, ft mutant tissues induce surrounding cells to point toward the clone, whereas ds clones reorient cells away from the mutant patch, and fj mutant patches also reorient cells toward the clone several unresolved mysteries exist in ft / ds signaling . Heterodimer formation, though seemingly essential for cell - to - cell communication, is itself not required for ft / ds pcp signaling to work, since ds binding to ft is not necessary for ft activity, as shown by the electron capture dissociation (ecd)-ft (ft lacking the extracellular domain). The mirror experiment adds even more mystery, as the ds extracellular region is sufficient to generate pcp defects, whereas its intracellular region is thought to stabilize dachs, an atypical myosin that serves as a good marker for asymmetric ft / ds interactions . The open questions remain (at least in part) because downstream effectors of the ft / ds pathway in pcp are functionally largely not defined, although several proteins are known to bind to ft, for example . Downstream of these complexes, exhibiting a polarized localization (already at the third instar larval stage), with ds recruiting dachs to the membrane and ft antagonizing its membrane localization . Although dachs localization is a very useful marker for this pathway, its function in pcp establishment is still elusive, largely because its lof phenotype is very mild (and this is further complicated by the observation that the intracellular domain of ds, where dachs interacts, seems dispensable for ds pcp signaling). Another downstream player that could provide answers is par-1, which might be the connection between ft / ds and microtubule plus - ends orientation . Again, both pcp systems need to be translated from global / body axis - related polarity to short - range cell - to - cell communication / relay and ultimately to single - cell responses . In the developing wing, each epithelial cell shows polarized core pcp protein complexes of fz and vang early in the prepupal stage or even late third instar, fz / dishevelled (dsh)/dgo on one side and vang / pk on the opposite side, with both complexes also containing fmi . At early stages, the pcp axis is perpendicular to the wing margin, displaying a radial pattern . As mentioned above, the respective complexes are stabilized via feedback loops among themselves (figure 1) in a manner that, disturbing the localization of one component (via either lof or gain of function), affects the localization of all the others, even from the opposite complex (figure 1). Since fmi also interacts with both fz and vang, it had been suggested that homophilic fmi bridges across cell membranes facilitate intercellular fz - vang interactions that are essential to propagate pcp [33,6366]. At the short - range level, cell - cell communication clonal cells lacking fz reorient adjacent cells toward the clone, whereas cells lacking vang reorient neighboring cells to point away; double - mutant clones for fz and vang reorient hairs to point toward the clone . These data suggest that fz is required for sending a polarity signal and vang for its reception nonetheless, cells can and probably do communicate in both directions, possibly also via fmi - fmi bridges . However, although fmi is needed on both sides, the ability of cells to communicate via fmi - fmi bridges depends largely on the presence of fz and vang . Nevertheless, it will be interesting to determine whether other pcp regulators are directly involved in modifying fmi - fmi interactions . It is important to mention that dsh, pk, or dgo mutant clones affect pcp within mutant clones only, suggesting that dsh, pk, and dgo are involved mainly in the intracellular interactions and interpretation of pcp signals . What about long - range regulation? In the wing and eye, pcp axes are orientated toward the prospective margins, the source of wg / dwnt4 expression . Recently, gain - of - function experiments demonstrated that dwnt4 and wg reorient pcp, altering the fz - pcp core complex localization and polarity axis orientation, and thus indicated that these wnts serve an instructive function in defining pcp axes . Moreover, through complicated genetic assays, it was possible to show that wg and dwnt4 are indeed necessary for pcp establishment but that they act redundantly . Like lawrence and colleagues (who mentioned that to understand the machinery, one needs to define its components and then work out what they do), we were able to show that wg / wnt4 modulates the intercellular fz - vang interaction . In agreement with the proposed graded fz activity (instructive capacity to interact with other core pcp components) that directs pcp orientation toward lower fz activity (figure 1), co - overexpression of fz and wnt4 in vivo buffers each other's effects, confirming the cell culture - based modulation of fz - vang interactions by dwnt4 and wg . Thus, the current model for long - range core fz - pcp axis establishment includes wg / dwnt4 as instructive regulators of fz - vang polarization in a graded manner in drosophila imaginal discs (figure 1) (this view applies at least for the wing and eye, but other tissues are more complex). Along these lines, in vertebrates, particularly in zebrafish, wnt5a and wnt11 are involved in pcp establishment, but their mechanism for pcp establishment remains unclear [44,7275]; one mechanism involving wnt5a - mediated vangl2 phosphorylation during digit formation in mouse limbs also involves ror2, a receptor tyrosine kinase that does not appear to have a pcp function in drosophila, suggesting potential additional players within the fz - pcp network . Nonetheless, the drosophila experiments provide strong evidence that wnts serve as an instructive input in regulating fz - pcp axis definition . Although in vertebrates the mechanistic mix is likely to be more complex as wnt5a and wnt11 might employ distinct functions, an instructive role for wnt11 has also been suggested . It is intriguing to think about wg / wnts as the master regulators for pcp: (a) wg itself, through canonical wg signaling, regulates graded fj and ds expression in eyes and wings (in the eye, wg downregulates fj and upregulates ds; in the wing, wg upregulates fj and downregulates ds), and (b) wg / wnt4 regulates fz - pcp activity / polarity axis establishment through regulating the interaction between fz and vang . Strikingly, sagner and colleagues show in drosophila wings that pcp can be affected by morphogenetic organizers and oriented cell divisions . In the larval wing disc, notch and wg signaling sets up the dorso - ventral organizer and hedgehog (hh) and decapentaplegic signaling sets up the antero - posterior organizer . As the morphogens control wing proliferation, size, and axes within the wing, their positional information could provide the earliest cues for pcp orientation . This is a point of view supported by the fact that uniform expression of morphogens during wing development can generate pcp defects . An hh - and - wg / wnt4 interplay has been also demonstrated for pcp establishment in the abdomen and thus might be a more general mechanism (reviewed in). Although it is clear that both ft / ds and fz - pcp pathways are linked to wg / wnt gradients, it remains open as to how these two pcp pathways interact or get integrated at the cellular level . Initial proposed models suggested that ft / ds acts upstream of the fz - pcp core factors, where the ft / ds system would provide global cues that orient the initial polarity of fz - pcp complexes; this was based largely on the observation that, at the cellular level, in ft or ds mutants, fz - pcp core protein complexes remained with polarized distribution in individual cells but with abnormal orientation . Different views emerged when overexpression or lof clones of ft or ds displayed non - autonomy in null mutant backgrounds of fmi or fz . This opened two possibilities, either that ft / ds signals to other tissue - specific modules but not to the core module (called the bypass pathway) or that the ft / ds and fz - pcp systems act in parallel . The latter is supported by many genetic experiments in the fly abdomen and also in the late embryonic / larval abdominal denticle belt orientations . In denticle belts, orientation of denticles is pcp - dependent, but the ft / ds and fz - pcp systems act in a redundant manner . Whereas fz or ft / ds mutants display normal denticle orientations, the double - mutant combinations affecting both systems resulted in marked denticle pcp defects, indicating a clear redundancy in this context . Consistent with two parallel pathways (possibly affecting different cellular features / functions) are the following observations . It was suggested that ds expression was necessary for correct pcp throughout the wing, but rescue experiments of ds mutants with exogenous transgenic constructs suggested that it was not the case, concluding that the ft / ds system was redundant with fz - pcp signaling in much of the wing and pointed out a major phenotypic difference between the two systems: whereas flies carrying mutations in fz / pcp core components exhibit pcp defects throughout the wing, the ft / ds system affects mainly polarity in the proximal wing half (figure 2), the wing area where pcp strongly depends on cellular realignments and rotations during the transition from the radial to the proximo - distal pcp axis (figure 2). In fact, the realignment from the radial to the proximo - distal pcp axis is dependent on ft / ds - induced mechanical forces, and ds in the wing hinge is essential for the wing to respond to the associated anisotropic mechanical stress . Thus, the different pcp systems appear to regulate distinct cellular features, both affecting the final pcp establishment and orientation . The mutations in fz / pcp core components affect the whole wing blade . This is seen in both intercellularly acting factors for example, fmi, the fmi - stan allele (top left)or the cytoplasmic intracellular components for example, pk (bottom left). . Consistently, ds and ft also influence mitotic figure orientations near the hinge at the larval stage . Ds, dachsous; fmi, flamingo; ft, fat; fz, frizzled; pcp, planar cell polarity; pk, prickle; stan, starry night . A potential link between the two pathways might be provided via the polarization / orientation of microtubules . The uemura lab has shown that the ft / ds system can regulate microtubule orientation and that fz and fmi can be seen moving along planar microtubule arrays in pupal wings . Moreover, most recently, it has been suggested that the different protein isoforms of the fz / core group gene pk, pk and sple, provide a different bias toward microtubule, it is noteworthy that recently the two distinct pk - sple isoforms were suggested to provide a link between the fz / pcp core group and the ft / ds system: as the sple isoform specifically binds to ds (and dachs) through its n - terminal extension, it can coordinate (co)localization between the ds and vang complexes (by binding to both), whereas the pk isoform (lacking the n - terminal domain) only physically interacts with vang . As such, the tissues where sple is expressed and genetically more important (eye and leg) would align the vang and ds complexes on the same cellular side, whereas those tissues using mainly pk (wing and thorax) would not align these complexes (allowing fz and ds to cohabit on the same side of cells). Although this requires a more physiological and functional dissection, it provides an intriguing potential mechanism for cross - talk and coordination of the two systems . In conclusion, we support the view that the two main molecular systems regulating pcp establishment act in parallel by using distinct effectors and affecting different cellular responses; as such, they can act together in a non - redundant (imaginal discs) or redundant (embryo) manner . Importantly, such a setup provides a high level of fidelity and potential for corrective measures if needed . Along these lines, the non - autonomous defects associated with mutant clones of fz core components are expanded in ft / ds mutant backgrounds as compared with a wildtype background in wings, arguing that the two pathways can indeed help each other in correcting aberrant cell - to - cell interactions in the respective other system . Such observations and hypotheses are all in favor of two parallel pathways, to provide corrective functions when needed and fidelity to the process . As it appears that both systems act through different effectors, they should converge somewhere downstream . Cortical microtubules align perpendicular to the margin in proximal wing regions, supporting a general role for the orientation of cortical microtubules in pcp and in the realignment of pcp later in development . It will be for future functional dissections to establish the specific intersection and integration points between the two systems.
Chinese anti - cancer association (uscaca,) (http://www.uscaca.org/) is a non - profit professional organiza - tion founded in 2009 . With members from academia, industry and government, uscaca facilitates collaboration among cancer research and physicians in the united states and china . Our current focus is on expediting novel cancer drug development by fostering clinical trial networks, sharing best practices and knowledge of clinical trial, and providing education and training opportunities . Uscaca collaborates with chinese anti - cancer association (caca,), chinese society for clinical oncology (csco), and other professional associations . Yun - guang tong, cedars - sinai medical center / ucla school of medicine michael shi, novartis oncology shi - yuan cheng, northwestern university li yan, peking university and glaxosmithkline roger luo, janssen pharmaceutical co. li - fang hou, northwestern university wei zhang, university of illinois at chicago wei - min qi, biocare medical wei zhang, md anderson cancer center li xu, jiangsu hengrui oncology pascal qian, novartis oncology xiao - xiang chen, boehringer - ingelheim wan - cai yang, xinxiang medical university yun dai, virginia commonwealth university
Quality of diet and physical inactivity have been attributed to the rise in prevalence of obesity leading to type 2 diabetes (the most common form) among developed and developing countries . Prolonged television (tv) viewing time has been associated with increased risk of obesity and type 2 diabetes in men from the health professionals follow - up study (hpfs) and women from the nurses' health study . Average hours of tv viewing, independent of physical activity, was positively associated with low - density lipoprotein cholesterol and inversely associated with high - density lipoprotein cholesterol and apolipoprotein a1 in an eight - year prospective study of 486 men randomly sampled from the hpfs . Several other studies reported tv watching as positively associated with cardiovascular risk factors such as blood pressure, low - density lipoprotein, triglycerides, and insulin resistance [46] and overeating . The average hours of tv viewing was estimated at 2.73 hours per day for all u.s . Civilians and 3.44 hours for those who reported watching tv according to the bureau of labor statistics . The association of tv with poor health outcomes may be partially attributed to the role of television . Although sedentary behaviors such as computer use, reading, board and video game - playing, and driving a car require little physical exertion, tv viewing results in a lower metabolic rate than other sedentary activities . Lower intakes of fruits, vegetables, and whole grains and higher intake of fried foods, processed meat and sugar - sweetened beverages have been associated with tv viewing in adults; and references therein . Exposure to food advertisements has been associated with overeating and choosing foods of poor dietary quality (higher salt, saturated fat, and lower dietary fiber) [1, 2]. Analysis of diet by a dietary index provides a more comprehensive approach to food consumption and health than measurement of single nutrients or diet quantity [12, 13]. Dietary indexes have been developed to measure the degree of adherence to a standard deemed as a dietary index includes the most important dietary components of a particular diet and then scores adherence to those components . The dietary index chosen for this study, the healthy eating index, 2005 (hei-05) (discussed in detail in section 3.2), parallels the dietary recommendations for persons at risk for or with diabetes . Common types of physical activities consist of leisure - time (including sports and social activities), occupational, transport, and at - home or domestic physical work . While physical inactivity is an established risk factor for obesity and diabetes, tv viewing hours has not been established as a risk factor for obesity and diabetes in adult populations . Despite the evidence by prospective studies that high levels of tv viewing was a risk factor for improper dietary intake, poor lipid profile, and type 2 diabetes in both men and women, public health goals target increasing physical activity, but has not set thresholds for sedentary activities . Little or no correlation has been found for average hours of tv viewing and hours of physical activity for large epidemiological studies in men and women; yet, obesity indicators were positively associated with tv hours in these studies . Evidence suggests that tv watching may be an independent risk factor for obesity and type 2 diabetes . The objective of this study was to assess the associations of hours of tv viewing with dietary quality, obesity and physical activity for three ethnic minorities with and without type 2 diabetes . Since median tv viewing was 3 hours and the 75th percentile was 4 hours for the combined sample, high tv viewing was set at more than 4 hours . Those persons with diabetes should be more likely to engage in sedentary behavior such as tv viewing . (1) persons with type 2 diabetes will be more likely to watch more than 4 hours of tv per day as compared to persons without diabetes, independent of ethnicity; (2a) individuals with type 2 diabetes in the lowest quartile of hei-05 (poorest diet) will be more likely to watch over 4 hours of tv as compared to those in the highest quartile, independent of ethnicity; and (2b) participants with the highest quartile of physical activity measured as metabolic equivalents (met) per hour per week (1 kcalkgh) will be less likely to watch more than 4 hours of tv as compared to those in the lowest quartile, independent of diabetes status and ethnicity . Physical activity was estimated by self - reported responses from the modifiable activities questionnaire (further details are provided in section 3.2). Hypothesis 2 was tested with physical activity and diet as covariates so each variable would adjust for the other . A secondary purpose was to assess differences in dietary components of the hei across ethnicities, physical activity level, bmi, and tv viewing by anova models . This was a cross - sectional study of n = 868 participants: cuban american (ca) (n = 361), haitian american (ha) (n = 258), and african american (aa) (n = 249) with (n = 455) and without (n = 410) type 2 diabetes collected from 20082010 . By study design, approximately half of each racial / ethnic minority in this study had type 2 diabetes and the rest were free of diabetes . Sample size was determined by a pilot study of cardiovascular risk factors for ca and was based on 80% power to attain a difference with 95% confidence among 7 subgroups [three ethnicities, two genders and diabetes status (with and without diabetes)], for variations in serum glucose, hemoglobin a1c, and blood lipids . The remaining racial / ethnic minority target goal was lowered to n = 250 . When the target was achieved recruitment efforts were ended . Recruitment was conducted by alternating between selecting potential participants with and then without type 2 diabetes . The lists of addresses were purchased from knowledge base marketing, inc ., richardson, tx, usa this company provided four mailing lists generated from multiple databases of ca and aa, identified as having or not having type 2 diabetes from miami - dade and broward counties, florida, usa during a one year period, approximately 10,000 letters, in english and spanish, explaining the study and containing contact information with an invitation flyer, were mailed to ca individuals with and without type 2 diabetes . Three percent (n = 300) of the letters were returned due to unknown addresses . From the remaining delivered letters, approximately, 6.3% (n = 477) of the letters were returned due to unknown addresses . From the remaining delivered letters, we were not able to recruit ha in the same manner because knowledge base marketing, inc . Did not have a database for the ha community therefore, recruitment of ha participants (n = 258) were from community - based sources . (a) local diabetes educators and community health practitioners in miami - dade and broward counties were contacted: several local diabetes educators who were either former students or in close contact with the department of dietetics and nutrition at florida international university (fiu). Official letters of invitation outlining the study were mailed to the diabetes educators and health professionals in miami - dade and broward counties requesting their cooperation in recruiting individuals . (b) invitational flyers were distributed to all university faculty, staff and students using the university - wide e - mail system explaining the research protocol and requesting their assistance in the study . (c) several residential rental facilities also agreed to help in the recruitment process . (d) print advertisements were placed in local haitian newspapers and principal gathering places of these groups such as churches, supermarkets, and restaurants . When the target recruitment goal was reached for all ethnic groups, efforts to recruit participants were stopped . Interested participants were initially interviewed by telephone, where the study purpose was explained to them . Age, gender, ethnicity, diabetes status and study qualification were determined . To ascertain type 2 diabetes status, each participant who self reported having diabetes participants who did not qualify for the study (n = 28) consisted of those being younger than 35 years old (n = 12), other ethnicity (n = 5), or having other chronic diseases or illnesses (n = 11). If a subject was determined to be eligible, then his or her participation was requested at the human nutrition laboratory at florida international university (fiu). Participants were instructed to refrain from smoking, consuming any food or beverages except water, and engaging in any unusual exercise for at least eight hours prior to their blood collection . The purpose and protocol of the study were explained to the participants, and their written consent either in english, spanish, or creole was obtained prior to the commencement of the study . Laboratory results showed that nineteen participants (ca = 7; ha = 8; aa = 4) who reported not having diabetes were reclassified as having type 2 diabetes according to american diabetes association standards . This study adhered to the institutional review board requirements on the use of human subjects . Trained interviewers who were bilingual in english / creole and english / spanish administered questionnaires . Sociodemographic data was collected by self - report with a questionnaire constructed by the principal investigator . This questionnaire included questions about age, gender, tobacco use, education, income, language preference, years lived in the united states, and health insurance . Physical activity and tv viewing were estimated from the modifiable activity questionnaire (maq). The use of this questionnaire to estimate metabolic cost and perform a met calculation is explained in detail in the compendium of physical activity . The maq assesses leisure activities from a popular list, (such as dancing swimming, bicycling, walking / jogging (outdoor, treadmill), strength / weight training, aerobics (water, dance, step), gardening or yard work) over the past year that were performed more than 10 times . The participants, with the help of a trained interviewer, estimate the number of months and frequency per month and duration of each activity . The number of hours sitting at work and usual mode of transportation to work was recorded for each job . Total physical activity was calculated, summing all leisure and occupational activity hours per week . Activity was expressed as metabolic equivalents (met) per hour per week (1 kcalkgh). Waist circumference to the nearest 0.1 cm was measured horizontally with a nonstretchable measuring tape . The tape was placed midway between the 12th rib and iliac crest at minimal respiration to determine central obesity . Body mass index(es) (bmi) was calculated by weight divided by height (kg / m). Glycosylated hemoglobin (a1c) was measured from a 20 ml sample of venous blood collected from each participant after an overnight fast (810 h) by a certified phlebotomist using standard laboratory techniques . Whole - blood samples for a1c were collected in a tube containing edta and a1c percentages were measured using roche tina quant method by the laboratory corporation of america (labcorp, miami, fl, usa). Dietary intake was measured using the semiquantitative food - frequency questionnaire, version 97gp 2006, developed by walter c. willett (copyrighted at harvard university, boston, ma, usa). The ffq has been extensively validated and standardized in multiethnic population - based prospective and cross - sectional studies [1719]. Participants self - reported average consumption of specific amounts of various foods over the past year, choosing from a frequency range from never to six or more servings per day, based on standardized portion sizes . The participants were shown food models and asked to estimate the number of servings that they consumed for each portion . In addition, this ffq includes questions about type and frequency of vitamin and mineral supplementation, alcohol consumption and details about sugar, salt, and fat used in cooking and as condiments . Open - ended section for ethnically specific foods was included in this version of the ffq . The questionnaire was reviewed by the interviewer with the participant for omissions at the close of the interview . The harvard university food composition database was used as the reference for macro- and micro - nutrient calculations . Daily servings of food groups were calculated by summing frequency factors (which corresponded to the reported consumption frequencies) for all food items . Dietary quality was assessed by the hei-05, which measures adherence of the individual's overall diet to the federal dietary guidelines of the united states department of agriculture (usda). The hei-05 was formulated based on the usda food guide pyramid using the proportions of grains, fruits and vegetables, dairy, meat, and discretionary sweets, fats, and oils . The hei-05 consists of 12 components total fruit, whole fruit, total vegetables, dark green and orange vegetables and legumes, total grains, whole grains, milk, meat and beans, oils, saturated fat, sodium, and energy from solid fats, alcohol, and added sugars (sofaas). Twelve individual components of the hei-05 represent all of the major food groups found in mypyramid (an online, interactive counterpart based on the dga's available to the public). The intakes of foods and nutrients are represented on a density basis, as amounts per 1,000 calories . A minimum score of zero for no intake and a maximum score of either 5 for fruits, vegetables, and grains, 10 for milk and meat / bean products, and 10 for oils was assigned for healthy components . For sugars, saturated the highest score was 20 (representing 20% of energy) and the maximum for saturated fat and sodium was 10 points each . Saturated fat and sodium received a score of 8 for the intake levels that reflect the 2005 and 2010 dga's less than 10% of energy from saturated fat, and 1.1 grams of sodium/1,000 kcal, respectively . Total hei-05 ranges from 0 to 100 points and the higher the score, the more the diet complies with the 2005 dga's, presuming a better diet quality . For this analysis, missing values (n = 32) were assigned to participants with total daily energy (reported dietary intake) of either <500 or> 5000 kcal / day . There were missing values for physical activity and tv (n = 9) leaving n = 827 (224 aa, 246 ha, and 357 ca). There were n = 439 with and n = 388 without type 2 diabetes . Prior to analysis, all continuous variables were tested by the kolmogorov - smirnov test and transformed to achieve normality and linearity if needed . The general characteristics of the study population were compared by diabetes status and ethnicity using the chi - squared test for categorical variables and the student's t - test for continuous variables . To test the hypotheses, a binary variable for high versus normal hours per day of watching television was determined, using the 75th percentile for the combined group and allowing sufficient marginal means to compare by diabetes status . Ethnicity, diabetes status, dietary quality (hei), and physical activity were the major independent variables for this study . Full logistic regression models included 2-way interactions for ethnicity, diabetes status, hei and physical activity . Age, gender, obesity indicators (either waist circumference or bmi due to their collinearity), and smoking were included in the final model due to their clinical significance . A binary variable to indicate risk for diabetes was constructed with a1c = 6% as the cut - off . Education was collapsed from 5 to 3 categories to include sufficient numbers in each category . The most parsimonious model was chosen by hosmer and lemeshow's recommendation for the elimination of adjustment variables (in this case education and marital status) with p values greater than 0.25 . To assess differences in the dietary components of hei across ethnicity, physical activity level, bmi, and tv viewing, all analyses were performed by the statistical package for the social sciences (spss) version 19, (chicago, il, usa), and a p - value <0.05 was considered significant . Aa and ha with type 2 diabetes were significantly older than their counterparts without diabetes, whereas for ca there were no differences in age across diabetes status . As a whole ca had more female participants; however, there were approximately equal numbers of males with and without diabetes within each ethnicity . Participants currently smoking did not differ by diabetes status; conversely, there were significant differences between ethnicities (analysis not shown). There were significant differences in education by ethnicity / race (p <0.001) for the combined sample (with and without diabetes); aa had the highest percent with at least some college (55.1), followed by ca (36.8) and ha (32.9). Ca had the highest percent with less than a high school diploma (49.6), followed by ha (46.3) and aa (16.0) (data not shown). Ha with diabetes had a lower education level than those without diabetes; however, aa and ha had no differences in education by diabetes status (aa and ca with type 2 diabetes had significantly higher bmi than those without diabetes . Aa with type 2 diabetes) had significantly more participants who watched more than 4 hours of tv as compared to those without diabetes . Ha without diabetes had a significantly higher percent of persons at risk for diabetes (56.1) as compared to ca (41.2) and aa (37.5) as indicated by a1c 6.0 (pethnicity = 0.011) (analysis not shown). The likelihood of viewing more than 4 hours of tv / day (high tv) aa were more likely to watch more than 4 hrs of tv as compared to ca [or = 5.30 (3.30, 8.49), p <0.001 and ha [or = 8.55 (4.88, 14.8), p <0.001] (comparison of aa and ha was performed post - hoc by recoding ethnicity). Participants with diabetes were 1.62 (1.12, 2.34) times more likely (odds ratio) to watch more than 4 hours of tv as compared to those without diabetes (p = 0.010). A second model was conducted with a1c to account for differences in this risk factor for persons with and without diabetes . Aa were still more likely to watch more than 4 hours of tv as compared to ca [or = 5.13 (3.18, 8.25), p <0.001]; however, adding a1c to the model negated differences by diabetes status [or = 1.36 (0.85, 2.17), p = 0.199]. The outcome high tv was also explored to test the risk of high a1c by ethnicity stratified by diabetes status . Models, adjusted for age, gender, and bmi quartile, were significant [(10) (n = 433) = 68.4, p <0.001, with diabetes and (10) (n = 386) = 33.9, p <0.001, without diabetes]. Aa were still more likely to watch more than 4 hours of tv as compared to ca [or = 4.98 (2.70, 9.18), p <0.001, with diabetes and or = 3.56 (1.27, 9.94), p = 0.015, without diabetes]. Neither high a1c nor the interaction of high a1c with ethnicity was significant for those with or without diabetes [high a1c: p = 0.878, with diabetes and p = 0.503, without diabetes; high a1c by ethnicity: p = 0.644, with diabetes and p = 0.914, without diabetes] (data not shown). The likelihood of diet score quartile and physical activity level with high aa were 5.27 (3.09, 8.97) times more likely compared to ca and 8.06 (4.48, 14.5) times more likely (odds ratio) to watch more than 4 hours of tv as compared to ha (p <0.001). The comparison of aa with ha was by post - hoc analysis, recoding ethnicity . Diet quality was inversely associated with physical activity level as measured by the chi - squared test and the z - test . The association of dietary quality with physical activity level was examined by the chi - squared test [(9) (n = 826) = 28.1, p = 0.001] and proportions were estimated by the z - test . Of interest were the differences in consumption of each food group of the hei across ethnicities, physical activity, tv viewing, and obesity . First, the results of anova and post - hoc analyses were performed for ethnicities . There were differences across ethnicities in dietary components of the hei for all categories (p <0.001). For most categories, post - hoc analysis showed differences between all three ethnicities from each other . Healthier scores in sofaas and saturated fat were found for ha as compared to ca and aa . Of concern proportionally, average servings from the fruit and vegetable groups were lower than those from the meat and bean group across ethnicities; however, ha had the highest total vegetable intake and the lowest meat and bean consumption as compared to ca and aa . Significant differences were found in all fruit and vegetable groups (total fruit, p <0.001; whole fruit, p <0.001; total vegetables, p = 0.009; whole grains, p = 0.008; and, saturated fat, p = 0.030). Healthier eating patterns with increased pa were found only with the fruits and vegetables, and saturated fat categories, but not for whole grains . As pa increased, fruit and vegetable intake increased . For whole grains and the highest level of pa (q4) as compared to the second level (q2) had a lower consumption of whole grains (0.49, p = 0.008). The second level of pa (q2) had a higher score (lower consumption) for saturated fat as compared to the first level (q1) (0.75, p = 0.036). The third anova and post - hoc analyses evaluated the hei food groups across quartiles of tv viewing . There were differences in all food categories: total fruit, p <0.001; total vegetables, p <0.001; green vegetables, p <0.001, grains p <0.001, milk, p <0.001, oils, p = 0.022; sodium, p = 0.005; and sofaas, p = 0.022, except the meat and bean group (p = 0.337) and the whole fruit group (p = 0.060). Those that watched tv, 3 - 4 hr / day (q3) consumed less total fruits than those that watched the least tv (01.75 hr / day) (0.46, p = 0.023). Total vegetable intake was lower for q3 as compared to q1 (1.20, p <0.001); q4 as compared to q3 (0.059; p = 0.003); and, q4 as compared to q1 (0.61, p = 0.003). Total grain intake was less in q2 as compared to q1 (0.51, p <0.001) and less for q3 as compared to q1 (0.59, p <0.001). Less whole grain consumption was associated with q3 as compared to q1 (0.51, p = 0.003) and q4 as compared to q1 (0.61, p = 0.001). More servings from the milk group were found for q2 (0.92, p = 0.009) and q3 (1.04, p = 0.001) as compared to q1 . Lower intake of oils was associated with q3 as compared to q1 (1.09, p = 0.016). A lower score for saturated fat (higher saturated fat consumption) was associated with q3 and q4 as compared to q1 (1.01, p = 0.001) and (1.26, p <0.001), respectively . For sodium, the highest quartile of tv viewing (q4) had a lower score (higher than recommended intake) as compared q1 (0.79, p = 0.50), q2 (1.00, p = 0.007) and q3 (0.85, p = 0.021); although q4q1 is marginally significant . The third quintile of tv viewing was associated with less adequate scores as compared to the lowest quintile (1.63, p = 0.03) for sofaas . There were no significant differences in any food groups except for the meat and bean group (p <0.001). The trend was as expected, the two highest quartiles of bmi consumed higher mean portions of meat and beans as compared to the two lower quartiles . Participants with and without diabetes who watched more than 4 hours of tv per day were more likely to have poorer quality diets and engage in less physical activity than those who viewed less hours of tv . Poorer quality diets, including higher amounts of processed foods (high calorie, high salt, low nutrition foods such as cold - cuts / lunch meat, sausages, fast food nuggets and burgers) and fried foods, refined grains, sugar, and lower fruit and vegetable intake, were associated with tv watching in adults [1, 2, 11]. The findings of a 6-year follow - up study suggest that participants free of diabetes who engage in prolonged tv viewing may be at risk for developing diabetes . Our results were in agreement with several studies that found persons with type 2 diabetes have been shown not to follow recommended exercise and dietary practices independent of ethnicity . There were no significant differences in physical activity levels and dietary practices between a lower - income sample of approximately equal numbers of white non - hispanics and aa with type 2 diabetes (n = 196) and 75% of both groups did not meet the government recommendations for physical activity and about 50% of both groups reported following recommended dietary practices (at that time was 30 minutes of exercise at least 3 times per week). These results were corroborated in a larger, multiethnic survey (n = 1560 whites, n = 279 blacks, and n = 125 hispanics) of persons with diabetes enrolled in managed care where there were no differences across groups and approximately 55% reported exercising at least 3 times per week . In our study aa with and without diabetes with poorest diet quality (lowest quartile of hei-05) were most likely to watch more than 4 hours of tv as compared to those with better diet quality . Daily tv viewing has been estimated 7 hours for aa as compared to 5 hours for the total population . Similar to the national trend, aa had a higher percent of participants classified in the obese categories . Obesity could be a limiting factor to performing physical activity due to physical and psycho - social factors . Tv programs and commercials may influence eating behavior partly through a stimulus for food and overeating . More commercials for unhealthy foods were shown on tv programs aimed at aa audiences as compared to tv programs for the general population . Our finding that physical activity was inversely associated with tv watching was in agreement with a large male population with type 2 diabetes (n = 37,918; aged 4075 years) which indicated that men who spent more time watching tv were less likely to exercise . Conversely our results were in discord with a population of approximately half caucasian and half african american women (n = 189) as well as a large study of french adults (n = 5028 men aged 4560 and n = 7713 women aged 3560) and a large primarily caucasian study of women (n = 50,277). Our results for no differences in physical activity comparing ethnicities may be inconclusive, since there has been a great variation in self - reported measures of physical activity and indices of cardiorespiratory fitness; and references therein . The authors suggest that these variations may be greater for aa males as compared to other groups; however, they used a modified version of champs physical activity questionnaire among african - americans which assessed moderate and vigorous leisure activities, only . In a us representative population of hispanics, non - hispanic whites, and aa with diabetes, more aa males were exercising regularly as compared to other race and gender groups . Yet, aa men had the lowest met - minutes / week based on the international physical activity questionnaire (ipaq) suggesting discordance with perceptions and performance of exercise . The ipaq is a less - detailed physical activity questionnaire designed for developing countries and may not accurately capture physical activity for aa . Our measure of physical activity included the total of all activities in the past year and the average per week was used for comparison . Accuracy of self - reported measurement of light activities, which occur during leisuretime and employment, may be a confounder in studies of physical activity level and tv viewing . Recreational activities and more intense sports are more accurately recalled than daily or lighter - intensity activities . To its merit, the modifiable activity questionnaire used in this study was designed to assess extreme levels of activity and validated to assess physical activity in populations with diabetes and at high risk for diabetes . Since the majority of our participants engaged in light activity and did not meet the current recommendations for physical activity, a major strength of this questionnaire was the consideration of all types of physical activity performed by the individual including domestic choirs, transportation, employment, and leisure pursuits . The study was cross - sectional and tv viewing cannot be said to cause dietary and physical activity choices . Another possible limitation was nonresponse bias; participants who responded and agreed to be in this study may have different characteristics from those who were not reached or declined . Diet, physical activity and tv viewing were by self - report and the accuracy depends on the participant's ability to recall their behaviors . Future studies are needed to establish prolonged tv watching in adult populations as a detrimental behavior and risk factor for the development of diabetes or diabetes - related complications . Although strategies to reduce tv watching have been proven effective among children, few trials have been conducted in adults . A two - phase (3-week observation and 3-week intervention) randomized trial of 36 obese adults who normally watched a minimum of 3 hours of tv agreed to a 50% reduction in tv viewing per week using a lockout monitor showed promising results . The intervention group showed an increase in energy expenditure without energy intake difference and decrease in bmi . The investigators chose an intervention period of a short duration (3 weeks); albeit, increase in energy expenditure over time may prove to augment weight reduction . The influence of high - calorie, high - fat food advertisements may also be minimized by reducing tv viewing . Persons at risk for or having type 2 diabetes should be targeted for altering tv viewing habits in an effort to increase energy expenditure and reduce the influence of over - eating high - calorie, high - sodium, and high - fat - foods . In addition, strategies to substitute cheaper high calorie dense (low - nutrient) foods with cost - effective healthy foods are needed to reach communities in lower socio - economic neighborhoods.
In the south atlantic ocean is located fernando de noronha archipelago around 540 km of the northeastern brazilian coast . This archipelago is composed by one large island and 20 small adjacent islets that represent a mountain chain top developed along an east - west fracture zone of the ocean floor and was built up by volcanic and subvolcanic essentially alkaline and subsaturated rocks . Most of the investigations carried out since then were taxonomic studies [35]. Also, the families dictyotaceae and sargassaceae of brown algae, the green algae caulerpa verticillata, and the red algae galaxaura spp . Are among the most abundant macroalgae on the rocky and reef shores of the archipelago [5, 6]. Their predominance is probably related to the production of secondary metabolites that inhibit herbivore predation . Also, it has long been established that marine and estuarine macroalgae accumulate metals to levels many times those found in the surrounding waters, and several algae have been used for monitoring concentrations of elements [812]. This study provides baseline information for further investigations of the absorption and accumulation of 20 elements by eleven macroalgae species commonly found in fernando de noronha archipelago . The concentrations of the elements in the seaweeds were determined using synchrotron radiation total reflection x - ray fluorescence analysis (srtxrf). All the reagents were purchased from merck (darmstadt, he, germany) and synth (diadema, sp, brazil). The multielementary solution was prepared using monoelementary solutions purchased from acros organics (geel, ant, belgium and new jersey, nj, usa), and ultrapure (deionized) water was obtained using a deionizer from microtec (ribeiro preto, sp, brazil). Eleven species of seaweeds commonly found in fernando de noronha archipelago were studied: caulerpa verticillata (chlorophyta), asparagopsis taxiformis, dictyurus occidentalis, galaxaura rugosa, g. obtusata, g. marginata (rhodophyta), dictyota cervicornis, dictyopteris justii, dictyopteris plagiogramma, padina gymnospora, and a sargassum sp . (phaeophyta) samples were collected in february and march, 2006 at caieiras beach (35018.8s, 322357.3w) and sueste bay (3521.2s, 322519.7w) which are on the main island (figure 1; table 1). The ibama authorization to collect algae seaweed specimens were collected randomly, that is, some individuals in a population were collect without a rule or defined sequence . Seaweed samples were frozen and sent to the laboratory where they arrived 48 h after harvesting . . Residual sediment, epiphytes, and animals were removed, and the algae were washed with distilled water to remove seawater and air dried in a circulating air oven at 40c for 48 h. after drying, around 5 g of each seaweed species was powdered by a triturating process in a grail after freezing the samples with liquid nitrogen . Samples of 250 mg of each algae species were placed in pyrex test tubes and digested according to a procedure described by ward et al . . Briefly, 6.0 ml of nitric acid (65%) and hydrogen peroxide (30%) were added to each test tube and homogenized . 12 hours) and heated at 130 5c until a translucid, particle - free, and fully digested solution was obtained . 5.00 ml of ultrapure water were transferred to the digested (sample) solution using a pyrex volumetric pipette, and the resulting solution was homogenized . The blank was a mixture of nitric acid, hydrogen peroxide, and deionized water and was done using the same procedure performed to the samples . Then 1.00 ml of this solution was removed using a pyrex volumetric pipette and to this aliquot was added 10 l of ga (1.0 l ml) as an internal standard . Calibration solutions of multielements that emit x - k and x - l rays were prepared, and ga element was added as internal standard as above . 5.0 l of each sample were placed on a perspex support (polished quartz, 28 22 mm). Drying for 1 h under, a 150 w infrared lamp (phillips model 7, amsterdam, nh, netherlands) gave rise to a thin layer of approximately 5 mm diameter . The equipment used was an x - ray fluorescence beamline constructed at the national synchrotron light laboratory lnls (campinas, so paulo state, brazil). For the total reflection of radiation, a series of mirrors the sample and calibration solutions were analyzed (three replicates of each) for 100 s each with a white synchrotron radiation beam using 0.5 mm of al as absorber, 1.0 mm of ta as collimator in the detector, a sample - to - detector distance of 1.1 mm, a height of 1 mm under total reflection conditions, and an angle of incidence of 1 mrad . The characteristic x - rays were detected with the aid of a ge hyperpure semiconductor detector (resolution of 145 ev for energy of 5.9 kev). Energy peaks detected in specters of the calibration samples were determined on the spectrometer, and the energy - emitting elements were identified from their x - ray characteristics (analytical lines). The liquid intensities for characteristic x - rays emitted were calculated with a mathematic adjustment in which the contribution of interfering lines on the analytical line (spectral interference) was considered including the escape peak and the addition peak . The fluorescent intensity of the characteristic line is related to the concentration by the expression i i = s i c i a i, where i i = fluorescent intensity of element i (cps), s i = elemental sensitivity of element i (cps g ml), c i = concentration of element i (g ml), and a = absorption factor . Given the tiny thickness of the prepared samples, the absorption and/or intensification effects (matrix effect) of the analytical line are negligible . Thus, there is no need to consider the absorption factor, and the relation is i i = s i c i. to correct like geometry and x - ray flow variation errors during excitation, ga was used as internal standard . Referencing to internal standard yields the expression (i i / i ga) = (s i / s ga)(c i / c ga), where i ga = fluorescent intensity of ga (cps), s ga = elemental sensitivity of ga (cps g ml), and c ga = concentration of ga (g ml). If we define s i = s i / s ga and r i = c ga (i i / i ga), where s i = relative sensitivity for element i (unidimensional) and r i = product of relative intensity and c ga (g ml), then (1)ri= sici . In the calibration solutions, r i is directly proportional to c i; therefore, the angular coefficient of the calibration curve for the element i is its relative sensitivity . If s i is known for the elements present in the calibration solutions, then the following function is obtained: (2)lnsi=a+bzi+czi2+dzi3, where a, b, c, and d are parameters that can be determinated by variance analyses, and z i is the atomic number for element i. the relative sensitivity for any x - k or x - l ray emitting elements present in the samples can thus be calculated . The sanest program was used to test significance of the parameters at 5% probability for inclusion in the model which was used to determine the above (2). G) for any inorganic element present in different samples were obtained from (1) after obtaining the experimental limits of detection (ld)(3) (3)ldi=3bgi / tcgaigasi, where bgi = background (cps), t = detection time (s), and other variables are defined above [18, 19]. From the calculation of the experimental ld values, it was established that the values of ld are a polynomial function of the atomic number of the elements present, ldi = f(z i). Thus, using this formula, it is possible to calculate the ld for the elements which are not present in the sample . Calibration curves (lns i = f(z i)) with significant parameters at 5% level were obtained for all x - k (table 2, figure 2) and x - l ray (table 3, figure 3) emitting elements through the multielementary calibration solutions . For the srtxrf technique, the maximum s i value obtained is for the internal standard which was the element ga (z = 31) in this experiment . The functions increase for z <31 until z = 31 and decreasing for z> 31 . Theoretically, s ga = 1.0; however, the experimental value of ga was 0.93 . It is important to correct for this difference in the calculation of the other elements . In this case, if the experimental data were used without experimental correction, an error of 6.6% can be observed for all the elements . Some authors attribute the need to perform these corrections to obtain the values close to true net intensities . The minimum detection limits for x - k and x - l ray emitting elements are presented in table 4 . The lowest recorded detection limit for x - k ray emitting elements was ld = 0.01 ppm for zn (z = 30) and ni (z = 28) and the highest detection limit value was ld = 1.72 ppm for k (z = 19). Lowest and highest detection limit values for x - l ray emitting elements were, respectively, 0.01 ppm for cu (z = 29) and 1.57 ppm for mo (z = 42). After determining the experimental detection limits, the results of the analysis of algae (table 5) and the chemical composition of the alkaline rocks where the algae were collected (table 6) were compared . Essential elements ca, fe, k, mn, and zn were found in all algae samples as were relevant species cao, fe2o3, k2o, mno, and zn in the rocks upon which these algae grew . Interestingly, g. marginata had little fe (75.27 ppm) while d. plagiogramma had over 150 times that amount (11936.65 ppm). P. gymnospora (3028.40 ppm) had the least values of ca while g. marginata and a. taxiformis had the highest ca levels detected (82606.32 and 88908.21 ppm, resp . ). Relatively little k was found in c. verticillata (504.13 ppm) while the levels of k in d. occidentalis were almost 100 times this amount (49523.34 ppm). Also, the p. gymnospora had large amounts of zn (274.44 ppm) whereas five algae species had levels of zn in the range of 27 ppm also, sr was found in all rocks analyzed (ca . 9501750 ppm) and was absorbed by all algae species and in relatively large abundance (ca . In contrast, ba was generally abundant in rock samples (ca . 201350 ppm) from where the algae were collected, but ba was only detected in d. justii . While br was detected in several species, it was most concentrated in a. taxiformis (257.10 ppm). The results obtained by srtxrf analysis of algae are comparable to those obtained for algae from other parts of the world using other analytical methods . For example, hou and yan studied elements present in 35 species of marine algae from the coast of china by neutron activation analysis in a miniature neutron source reactor (mnsr). They observed that, in brown, red, and green algae, the levels of individual elements were (averages, resp ., in parentheses): as (159/<0.36/12.2 ppm), ba (76.2/109.6/174 ppm), br (3426/6157/596 ppm), ca (22.7/29.7/11.2 ppt), co (0.93/1.15/1.04 ppm), cr (4.02/4.84/6.33 ppm), cs (1.11/1.02/0.95 ppm), fe (1892/2511/3716 ppm), k (67.5/48.4/29.0 ppt), mn (857/89.4/90.6 ppm), rb (29.4/21.5/25.8 ppm), sr (892/313/161 ppm), and zn (21.7/28.3/23.3 ppm). Besides this study, in another work about 26 marine benthic algae species found in karachi coast, pakistan, the levels (averages for the 26 species are in parentheses) of ca (26.75 ppt), co (5.88 ppm), cr (5.13 ppm), cu (11.87 ppm), fe (2.41 ppt), k (69.5 ppt), pb (13.43 ppm), and zn (53.28 ppm) were established using flame atomic absorption spectrometry (aas). As in the algae in these previous studies, the algae of fernando de noronha were found to contain large amounts of ca, k, and fe (in parts per thousand, ppt) and small or trace amounts of as, ba, co, cr, cs, cu, pb, rb, and zn . Since data on the composition of the surfaces where algae in these previous studies grew was not reported, it is not possible to ascertain the contributions of these surface substrates to the composition of the algae . The similar compositional trends in principle and trace elements present in algae from these previous studies and those of fernando de noronha lends support to the usefulness of the srtxrf technique in the analytical arsenal . Low numbers of algae species contained rb (4 algae species, 1248 ppm), ti (4 species, 109537 ppm), and v (5 species, 1444 ppm) which may be related to geological characteristics of the alkaline rocks present in the study sites . Low concentrations of as (5 species, range 11118 ppm), co (3 species, 1.95.4 ppm), cr (6 species, range 5.354 ppm), cu (3 species, range 1.11.6 ppm), ni (4 species, range 1.915 ppm), and pb (4 species, range 2.027 ppm) were observed in most of the algae species, except for sargassum sp . Which had a higher concentration of as (118 ppm) compared to the other species . These elements may have been absorbed from the seawater through natural weathering and lixiviation of rocks and soil . An interesting finding was the detection of dy which is a rare earth metal present in rocks of both collection sites in a. taxiformis, d. occidentalis, g. rugosa, g. obtusa, g. marginata, and d. plagiogramma . Thus, these algae absorb and store this rare chemical element which is present in rocks in relatively low abundance . The srtxrf technique proved to be adequate for the determination of 20 chemical elements present in eleven species of common macroalgae of fernando de noronha archipelago providing baseline information for the accumulation of metals in two sites . The results indicate a relationship among the metals present in the seaweeds and the rocks present in this area . Besides, the concentrations of common macro- and microelements obtained are comparable to those obtained by other authors using different analytical methods . However, multielement capability in a single analysis, high - sensitivity and precision, short analysis time, and easy sample preparation are some advantages of srtxrf when compared to other elemental determination techniques such as aas or icp - ms.
This study was part of a larger study that looked at several outcomes, including diabetes coping behaviors and glycemic control status of t2 dm patients . The study design was cross - sectional and it was conducted at the national centre for diabetes and endocrinology (ncde) in tripoli, libya, after being approved by universiti kebangsaan malaysia research and ethics committee and the management at the ncde . The ncde, although a referral center, provides diabetes follow - up, medications, and laboratory services for diabetes outpatients . To find out the minimum sample size required for the large study, the largest sample size obtained was considered, and it was based on the sample size needed to test the difference in the prevalence of poor glycemic control across males and females using the fleiss formula (18). The preliminary data used in the calculation included a prevalence of poor glycemic control among males of 45% and among females of 58% (19). A sample size of 498 was needed for a margin of precision () of 5% and a power of 80% . To compensate for low response and rejection due to non - eligibility, 30% was added . Patients were recruited systematically at the laboratory waiting area between october and december 2013, whereby every fourth patient was approached at their coming sequence . When the patient was not eligible, the immediate next patient was invited . The eligibility criteria included being libyan, having t2 dm for at least 1 year, being 18 years old or above, having no cognitive impairment that could interfere with communication or comprehending the questions, being able to read and write in arabic, having no visual impairment that interfered with self - reporting, and being willing to participate in the study . Those who were placed on a diet plan only, pregnant women, and very ill patients were not eligible . The type of diabetes was self - reported, and it was verified whenever applicable by checking the medical follow - up card of each respondent . However, as not all the follow - up cards showed the type of diabetes, further verification of diabetes type was done at the stage of analysis . Those who reported being diagnosed before the age of 40, placed on insulin at diagnosis, and on insulin at the time of the study (20) were considered as having type 1 diabetes and were excluded from the analysis . Out of the 648 addressed patients, 523 responses were considered in the data analysis, which gave a response rate of 80.7% . 1 . Patients were given a briefing about the study, as well as on their participation and withdrawal rights . Data collection was based on a self - reported questionnaire, which gathered information about patients sociodemographic characteristics and disease profile, their diabetes illness perceptions based on the revised illness perception questionnaire (ipq - r), and medication adherence based on the eight - item morisky medication adherence scale (mmas-8). Data on age, sex, marital status, education level, employment status, income, duration of diabetes, type of diabetes medication, presence of comorbidity, and the number of other medications were gathered from the information sheet . It is related to the number of symptoms the patients have experienced since their diabetes diagnosis and that they attribute to their diabetes . The second section comprises 38 items and measures seven illness perception domains (timeline acute / chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations). Each of the 38 items is attached to a five - level (strongly disagree strongly agree) likert - type response . Timeline acute - chronic perception (six items) refers to the perception of the illness duration . A high score indicates a strong perception of diabetes as a chronic (long - term) illness (sample item: timeline cyclical perception (four items) refers to the perception of the pattern of the course of the illness . A high score indicates a strong perception of diabetes course as a cyclical or unstable illness (sample item: i go through cycles in which my diabetes gets better and worse). Consequences perception (six items) refers to the perception of the seriousness of the illness and its impact on life . A high score indicates a perception of diabetes as a serious illness with negative impacts on life, social relationships, or economic status (sample item: my diabetes is a serious condition). Personal control perception (six items) refers to the patient's perception of his or her own ability to manage or control the illness . A high score indicates a high perception of the personal ability to control diabetes (sample item: treatment control perception (five items) refers to the perception of the effectiveness of treatment in controlling the illness . A high score indicates a high perception of the effectiveness of diabetes treatment in controlling diabetes (sample item: my treatment can control my diabetes). Illness coherence (five items) refers to the patient's perception of his or her own understanding of the illness . A high score indicates a high perception of the personal understanding of diabetes and that diabetes is perceived as a clear or unambiguous illness (sample item: i have a clear picture or understanding of my condition). A high score refers to more emotional responses to diabetes and indicates that diabetes affects the patient emotionally and makes him or her emotionally distressed (sample item: the last section of the ipq - r is the cause section, and it comprises 18 disparate items (causes). Each item is attached to a five - level (strongly disagree strongly agree) likert - type response . We dichotomized the responses to each cause item into yes and no, where yes indicated those who strongly agreed or agreed on the item as a cause for their diabetes (22). Only the eight illness perception domains (identity perception, timeline acute - chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations) were defined as independent variables and tested for their contribution to medication adherence, whereas the cause section was only included for description . The scale was translated from english into arabic by two bilingual translators using a forward backward method . The content validity of the scale was assessed by a group of libyan experts, and two items were added to the other 14 generic symptoms in the identity subscale numbness and paresthesia . Then, face validity was pretested on 10 diabetic libyans and the necessary amendments to enhance the clarity were done . It is related to the number of symptoms the patients have experienced since their diabetes diagnosis and that they attribute to their diabetes . The second section comprises 38 items and measures seven illness perception domains (timeline acute / chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations). Each of the 38 items is attached to a five - level (strongly disagree strongly agree) likert - type response . Timeline acute - chronic perception (six items) refers to the perception of the illness duration . A high score indicates a strong perception of diabetes as a chronic (long - term) illness (sample item: my diabetes will last a short time). Timeline cyclical perception (four items) refers to the perception of the pattern of the course of the illness . A high score indicates a strong perception of diabetes course as a cyclical or unstable illness (sample item: i go through cycles in which my diabetes gets better and worse). Consequences perception (six items) refers to the perception of the seriousness of the illness and its impact on life . A high score indicates a perception of diabetes as a serious illness with negative impacts on life, social relationships, or economic status (sample item: my diabetes is a serious condition). Personal control perception (six items) refers to the patient's perception of his or her own ability to manage or control the illness . A high score indicates a high perception of the personal ability to control diabetes (sample item: i have the power to influence my diabetes). Treatment control perception (five items) refers to the perception of the effectiveness of treatment in controlling the illness . A high score indicates a high perception of the effectiveness of diabetes treatment in controlling diabetes (sample item: my treatment can control my diabetes). Illness coherence (five items) refers to the patient's perception of his or her own understanding of the illness . A high score indicates a high perception of the personal understanding of diabetes and that diabetes is perceived as a clear or unambiguous illness (sample item: i have a clear picture or understanding of my condition). A high score refers to more emotional responses to diabetes and indicates that diabetes affects the patient emotionally and makes him or her emotionally distressed (sample item: the last section of the ipq - r is the cause section, and it comprises 18 disparate items (causes). Each item is attached to a five - level (strongly disagree strongly agree) likert - type response . We dichotomized the responses to each cause item into yes and no, where yes indicated those who strongly agreed or agreed on the item as a cause for their diabetes (22). Only the eight illness perception domains (identity perception, timeline acute - chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations) were defined as independent variables and tested for their contribution to medication adherence, whereas the cause section was only included for description . The scale was translated from english into arabic by two bilingual translators using a forward backward method . The content validity of the scale was assessed by a group of libyan experts, and two items were added to the other 14 generic symptoms in the identity subscale numbness and paresthesia . Then, face validity was pretested on 10 diabetic libyans and the necessary amendments to enhance the clarity were done . It is a valid (23, 24) single - dimension measure that comprises eight items (sample item: when you travel or leave home, do you sometimes forget to bring along your medications?). Each item is attached to a yes or no response, except for the last item, which has a five - level likert - type response (23). The short construct of the mmas-8 was useful as data were collected in a busy clinical setting (25). A score of 8 indicates high adherence, a score of 6 and above but less than 8 indicates moderate adherence, whereas a score below 6 indicates low adherence (23, 24). This version was found to be reliable and valid among libyans with t2 dm (26). The internal consistency reliability of the ipq - r and mmas-8 was assessed using cronbach's alpha . A chi - square test, independent t - test, and the mann whitney u test were used to explore the bivariate associations between medication adherence and sociodemographic characteristics, disease profile, and illness perceptions . All variables that showed bivariate associations with medication adherence at a p - value of 0.25 or less were included in the multiple regression analysis . A two - step hierarchical binary multiple logistic regression was run to test the contribution of illness perceptions on low medication adherence . A p - value less than 0.05 was considered in the interpretation of the significance of the findings . This study was part of a larger study that looked at several outcomes, including diabetes coping behaviors and glycemic control status of t2 dm patients . The study design was cross - sectional and it was conducted at the national centre for diabetes and endocrinology (ncde) in tripoli, libya, after being approved by universiti kebangsaan malaysia research and ethics committee and the management at the ncde . The ncde, although a referral center, provides diabetes follow - up, medications, and laboratory services for diabetes outpatients . To find out the minimum sample size required for the large study, the largest sample size obtained was considered, and it was based on the sample size needed to test the difference in the prevalence of poor glycemic control across males and females using the fleiss formula (18). The preliminary data used in the calculation included a prevalence of poor glycemic control among males of 45% and among females of 58% (19). A sample size of 498 was needed for a margin of precision () of 5% and a power of 80% . To compensate for low response and rejection due to non - eligibility, 30% was added . Patients were recruited systematically at the laboratory waiting area between october and december 2013, whereby every fourth patient was approached at their coming sequence . When the patient was not eligible, the immediate next patient was invited . The eligibility criteria included being libyan, having t2 dm for at least 1 year, being 18 years old or above, having no cognitive impairment that could interfere with communication or comprehending the questions, being able to read and write in arabic, having no visual impairment that interfered with self - reporting, and being willing to participate in the study . Those who were placed on a diet plan only, pregnant women, and very ill patients were not eligible . The type of diabetes was self - reported, and it was verified whenever applicable by checking the medical follow - up card of each respondent . However, as not all the follow - up cards showed the type of diabetes, further verification of diabetes type was done at the stage of analysis . Those who reported being diagnosed before the age of 40, placed on insulin at diagnosis, and on insulin at the time of the study (20) were considered as having type 1 diabetes and were excluded from the analysis . Out of the 648 addressed patients, 523 responses were considered in the data analysis, which gave a response rate of 80.7% . 1 . Patients were given a briefing about the study, as well as on their participation and withdrawal rights . Data collection was based on a self - reported questionnaire, which gathered information about patients sociodemographic characteristics and disease profile, their diabetes illness perceptions based on the revised illness perception questionnaire (ipq - r), and medication adherence based on the eight - item morisky medication adherence scale (mmas-8). Data on age, sex, marital status, education level, employment status, income, duration of diabetes, type of diabetes medication, presence of comorbidity, and the number of other medications were gathered from the information sheet . It is related to the number of symptoms the patients have experienced since their diabetes diagnosis and that they attribute to their diabetes . The second section comprises 38 items and measures seven illness perception domains (timeline acute / chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations). Each of the 38 items is attached to a five - level (strongly disagree strongly agree) likert - type response . Timeline acute - chronic perception (six items) refers to the perception of the illness duration . A high score indicates a strong perception of diabetes as a chronic (long - term) illness (sample item: my diabetes will last a short time). Timeline cyclical perception (four items) refers to the perception of the pattern of the course of the illness . A high score indicates a strong perception of diabetes course as a cyclical or unstable illness (sample item: i go through cycles in which my diabetes gets better and worse). Consequences perception (six items) refers to the perception of the seriousness of the illness and its impact on life . A high score indicates a perception of diabetes as a serious illness with negative impacts on life, social relationships, or economic status (sample item: my diabetes is a serious condition). Personal control perception (six items) refers to the patient's perception of his or her own ability to manage or control the illness . A high score indicates a high perception of the personal ability to control diabetes (sample item: i have the power to influence my diabetes). Treatment control perception (five items) refers to the perception of the effectiveness of treatment in controlling the illness . A high score indicates a high perception of the effectiveness of diabetes treatment in controlling diabetes (sample item: my treatment can control my diabetes). Illness coherence (five items) refers to the patient's perception of his or her own understanding of the illness . A high score indicates a high perception of the personal understanding of diabetes and that diabetes is perceived as a clear or unambiguous illness (sample item: i have a clear picture or understanding of my condition). A high score refers to more emotional responses to diabetes and indicates that diabetes affects the patient emotionally and makes him or her emotionally distressed (sample item: the last section of the ipq - r is the cause section, and it comprises 18 disparate items (causes). Each item is attached to a five - level (strongly disagree strongly agree) likert - type response . We dichotomized the responses to each cause item into yes and no, where yes indicated those who strongly agreed or agreed on the item as a cause for their diabetes (22). Only the eight illness perception domains (identity perception, timeline acute - chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations) were defined as independent variables and tested for their contribution to medication adherence, whereas the cause section was only included for description . The scale was translated from english into arabic by two bilingual translators using a forward backward method . The content validity of the scale was assessed by a group of libyan experts, and two items were added to the other 14 generic symptoms in the identity subscale numbness and paresthesia . Then, face validity was pretested on 10 diabetic libyans and the necessary amendments to enhance the clarity were done . It is related to the number of symptoms the patients have experienced since their diabetes diagnosis and that they attribute to their diabetes . The second section comprises 38 items and measures seven illness perception domains (timeline acute / chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations). Each of the 38 items is attached to a five - level (strongly disagree strongly agree) likert - type response . Timeline acute - chronic perception (six items) refers to the perception of the illness duration . A high score indicates a strong perception of diabetes as a chronic (long - term) illness (sample item: my diabetes will last a short time). Timeline cyclical perception (four items) refers to the perception of the pattern of the course of the illness . A high score indicates a strong perception of diabetes course as a cyclical or unstable illness (sample item: i go through cycles in which my diabetes gets better and worse). Consequences perception (six items) refers to the perception of the seriousness of the illness and its impact on life . A high score indicates a perception of diabetes as a serious illness with negative impacts on life, social relationships, or economic status (sample item: my diabetes is a serious condition). Personal control perception (six items) refers to the patient's perception of his or her own ability to manage or control the illness . A high score indicates a high perception of the personal ability to control diabetes (sample item: i have the power to influence my diabetes). Treatment control perception (five items) refers to the perception of the effectiveness of treatment in controlling the illness . A high score indicates a high perception of the effectiveness of diabetes treatment in controlling diabetes (sample item: my treatment can control my diabetes). Illness coherence (five items) refers to the patient's perception of his or her own understanding of the illness . A high score indicates a high perception of the personal understanding of diabetes and that diabetes is perceived as a clear or unambiguous illness (sample item: i have a clear picture or understanding of my condition). A high score refers to more emotional responses to diabetes and indicates that diabetes affects the patient emotionally and makes him or her emotionally distressed (sample item: the last section of the ipq - r is the cause section, and it comprises 18 disparate items (causes). Each item is attached to a five - level (strongly disagree strongly agree) likert - type response . We dichotomized the responses to each cause item into yes and no, where yes indicated those who strongly agreed or agreed on the item as a cause for their diabetes (22). Only the eight illness perception domains (identity perception, timeline acute - chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations) were defined as independent variables and tested for their contribution to medication adherence, whereas the cause section was only included for description . The scale was translated from english into arabic by two bilingual translators using a forward backward method . The content validity of the scale was assessed by a group of libyan experts, and two items were added to the other 14 generic symptoms in the identity subscale numbness and paresthesia . Then, face validity was pretested on 10 diabetic libyans and the necessary amendments to enhance the clarity were done . It is a valid (23, 24) single - dimension measure that comprises eight items (sample item: when you travel or leave home, do you sometimes forget to bring along your medications?). Each item is attached to a yes or no response, except for the last item, which has a five - level likert - type response (23). The short construct of the mmas-8 was useful as data were collected in a busy clinical setting (25). A score of 8 indicates high adherence, a score of 6 and above but less than 8 indicates moderate adherence, whereas a score below 6 indicates low adherence (23, 24). This version was found to be reliable and valid among libyans with t2 dm (26). It is related to the number of symptoms the patients have experienced since their diabetes diagnosis and that they attribute to their diabetes . The second section comprises 38 items and measures seven illness perception domains (timeline acute / chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations). Each of the 38 items is attached to a five - level (strongly disagree strongly agree) likert - type response . Timeline acute - chronic perception (six items) refers to the perception of the illness duration . A high score indicates a strong perception of diabetes as a chronic (long - term) illness (sample item: my diabetes will last a short time). Timeline cyclical perception (four items) refers to the perception of the pattern of the course of the illness . A high score indicates a strong perception of diabetes course as a cyclical or unstable illness (sample item: i go through cycles in which my diabetes gets better and worse). Consequences perception (six items) refers to the perception of the seriousness of the illness and its impact on life . A high score indicates a perception of diabetes as a serious illness with negative impacts on life, social relationships, or economic status (sample item: my diabetes is a serious condition). Personal control perception (six items) refers to the patient's perception of his or her own ability to manage or control the illness . A high score indicates a high perception of the personal ability to control diabetes (sample item: i have the power to influence my diabetes). Treatment control perception (five items) refers to the perception of the effectiveness of treatment in controlling the illness . A high score indicates a high perception of the effectiveness of diabetes treatment in controlling diabetes (sample item: my treatment can control my diabetes). Illness coherence (five items) refers to the patient's perception of his or her own understanding of the illness . A high score indicates a high perception of the personal understanding of diabetes and that diabetes is perceived as a clear or unambiguous illness (sample item: i have a clear picture or understanding of my condition). A high score refers to more emotional responses to diabetes and indicates that diabetes affects the patient emotionally and makes him or her emotionally distressed (sample item: the last section of the ipq - r is the cause section, and it comprises 18 disparate items (causes). Each item is attached to a five - level (strongly disagree strongly agree) likert - type response . We dichotomized the responses to each cause item into yes and no, where yes indicated those who strongly agreed or agreed on the item as a cause for their diabetes (22). Only the eight illness perception domains (identity perception, timeline acute - chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations) were defined as independent variables and tested for their contribution to medication adherence, whereas the cause section was only included for description . The scale was translated from english into arabic by two bilingual translators using a forward backward method . The content validity of the scale was assessed by a group of libyan experts, and two items were added to the other 14 generic symptoms in the identity subscale numbness and paresthesia . Then, face validity was pretested on 10 diabetic libyans and the necessary amendments to enhance the clarity were done . It is related to the number of symptoms the patients have experienced since their diabetes diagnosis and that they attribute to their diabetes . The second section comprises 38 items and measures seven illness perception domains (timeline acute / chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations). Each of the 38 items is attached to a five - level (strongly disagree strongly agree) likert - type response . Timeline acute - chronic perception (six items) refers to the perception of the illness duration . A high score indicates a strong perception of diabetes as a chronic (long - term) illness (sample item: my diabetes will last a short time). Timeline cyclical perception (four items) refers to the perception of the pattern of the course of the illness . A high score indicates a strong perception of diabetes course as a cyclical or unstable illness (sample item: i go through cycles in which my diabetes gets better and worse). Consequences perception (six items) refers to the perception of the seriousness of the illness and its impact on life . A high score indicates a perception of diabetes as a serious illness with negative impacts on life, social relationships, or economic status (sample item: my diabetes is a serious condition). Personal control perception (six items) refers to the patient's perception of his or her own ability to manage or control the illness . A high score indicates a high perception of the personal ability to control diabetes (sample item: i have the power to influence my diabetes). Treatment control perception (five items) refers to the perception of the effectiveness of treatment in controlling the illness . A high score indicates a high perception of the effectiveness of diabetes treatment in controlling diabetes (sample item: my treatment can control my diabetes). Illness coherence (five items) refers to the patient's perception of his or her own understanding of the illness . A high score indicates a high perception of the personal understanding of diabetes and that diabetes is perceived as a clear or unambiguous illness (sample item: i have a clear picture or understanding of my condition). A high score refers to more emotional responses to diabetes and indicates that diabetes affects the patient emotionally and makes him or her emotionally distressed (sample item: the last section of the ipq - r is the cause section, and it comprises 18 disparate items (causes). Each item is attached to a five - level (strongly disagree strongly agree) likert - type response . We dichotomized the responses to each cause item into yes and no, where yes indicated those who strongly agreed or agreed on the item as a cause for their diabetes (22). Only the eight illness perception domains (identity perception, timeline acute - chronic perception, timeline cyclical perception, consequences perception, personal control perception, treatment control perception, illness coherence, emotional representations) were defined as independent variables and tested for their contribution to medication adherence, whereas the cause section was only included for description . The scale was translated from english into arabic by two bilingual translators using a forward backward method . The content validity of the scale was assessed by a group of libyan experts, and two items were added to the other 14 generic symptoms in the identity subscale numbness and paresthesia . Then, face validity was pretested on 10 diabetic libyans and the necessary amendments to enhance the clarity were done . It is a valid (23, 24) single - dimension measure that comprises eight items (sample item: when you travel or leave home, do you sometimes forget to bring along your medications?). Each item is attached to a yes or no response, except for the last item, which has a five - level likert - type response (23). The short construct of the mmas-8 was useful as data were collected in a busy clinical setting (25). A score of 8 indicates high adherence, a score of 6 and above but less than 8 indicates moderate adherence, whereas a score below 6 indicates low adherence (23, 24). This version was found to be reliable and valid among libyans with t2 dm (26). The internal consistency reliability of the ipq - r and mmas-8 was assessed using cronbach's alpha . A chi - square test, independent t - test, and the mann whitney u test were used to explore the bivariate associations between medication adherence and sociodemographic characteristics, disease profile, and illness perceptions . All variables that showed bivariate associations with medication adherence at a p - value of 0.25 or less were included in the multiple regression analysis . A two - step hierarchical binary multiple logistic regression was run to test the contribution of illness perceptions on low medication adherence . A p - value less than 0.05 was considered in the interpretation of the significance of the findings . The mean diabetes duration was 9.4 (sd=7.3) years, and the number of other long - term medications ranged from zero to seven . Sociodemographic characteristics and disease profile (n=523) ld, libyan dinars (conversion rate at the time of the study: 1 ld = 1.32 usd). Table 2 displays the descriptive statistics of diabetes illness perceptions based on the identity and views sections of the ipq - r . All eight illness perception domains displayed a satisfactory internal consistency, with the alpha coefficient ranging from 0.87 for the emotional representations subscale to 0.76 for the consequences perception subscale . Respondents showed low diabetes identity perception, indicating that they attributed few of the symptoms they experienced to their diabetes (mean=4.55, sd=3.72). They showed a high perception of diabetes timeline as a chronic illness (mean=22.08, sd=5.34) and a moderate perception of diabetes course as a cyclical (unstable) illness (mean=12.42, sd=4.80). Respondents had a moderately high perception of the treatment controllability of their illness (mean=19.19, sd=4.48) and their personal control over diabetes (mean=18.93, sd=7.06), while demonstrating a moderate diabetes consequences perception (mean=16.24, sd=6.05) and emotional representations (mean=16.99, sd=7.76). Diabetes illness perceptions based on the revised illness perception questionnaire (ipq - r) scale table 3 shows the descriptive statistics on the perceptions of the cause of diabetes . The five most commonly indicated causes out of the 19 listed items as causes for diabetes were allah's will / fate (86.6%), hereditary it runs in my family (53.0%), my emotional state (44.4%), family problems or worries (40.0%), and diet or eating habits (35.6%). Diabetes cause perceptions (n=523) the cause section of the ipq - r consists of disparate items . Table 4 shows the descriptive statistics on diabetes medication adherence based on the mmas-8 . The mean medication adherence score was 6.06 (sd=1.89), and the prevalence of low medication adherence was 36.1% . Medication adherence based on the mmas-8 use of the mmas is protected by us copyright laws . A license agreement is available from: donald e. morisky, scd, scm, msph, professor, department of community health sciences, ucla school of public health, 650 charles e. young drive south, los angeles, ca 90095 - 1772; dmorisky@ucla.edu . Several sociodemographic variables and disease profile related factors showed statistically significant associations with medication adherence . The frequency of low medication adherers was significantly higher among males than females (=10.247, p=0.001). Respondents with secondary to high education level reported a significantly higher prevalence of low medication adherers than those with primary education (=5.628, p=0.018). A significantly higher frequency of low adherers was also found among employed respondents compared to the unemployed (=17.921, p<0.001) and among the moderate- to high - income group compared to the low income group (=3.925, p=0.048). Respondents who had no comorbidity showed a significantly higher prevalence of low medication adherers compared to those who had comorbidity (=11.466, p=0.001). In addition, there was a significant difference in the number of other long - term medications between low adherence and moderate - high adherence groups (z=2.810, p=0.005). Regarding illness perceptions, a significant difference in the mean treatment control perception score (t=2.366, p=0.018) was found between patients who reported low adherence to their diabetes medications (mean=18.58, sd=4.51) and those who reported moderate - high adherence (mean=19.54, sd=4.44). Bivariate association between sociodemographic factors, disease profile, and illness perceptions with medication adherence t statistic (independent t - test); z statistic (mann whitney u test). Table 6 shows a two - step hierarchical binary logistic regression model . In the first step, all sociodemographic and disease profile related factors that displayed associations with medication adherence at p0.25 in the bivariate analysis were entered into the model . All illness perceptions that displayed crude associations with medication adherence with p0.25 were entered in the second step . In the first step, the model showed a good fit of the included variables as indicated by the non - significant hosmer lemeshow test (p=0.160). Referring to the block omnibus test, the contribution of the model with these variables to low medication adherence variance compared to the model with the constant alone was significant ((df) = 26.997(8), p=0.001). Based on the nagelkerke r, this model contributed to 6.9% of the variance of low medication adherence . In the second step the model showed a good fit of the included variables as indicated by the non - significant hosmer the addition of illness perceptions led to a significant improvement in the model's level of predictivity as indicated by a significant chi - square increment ((df) = 19.867(5), p=0.001). Referring to the nagelkerke r thus, illness perceptions have a unique contribution to medication adherence above and beyond the control variables alone . Hierarchical regression model of low medication adherence predictors, chi - square difference across two subsequent models after adding illness perceptions . The final model showed that two illness perceptions contributed significantly to low medication adherence; these were treatment control perception and illness identity perception . High treatment control perception contributed slightly yet significantly to medication adherence as a protective factor . The odds of having low adherence to medications decreased with the increase in treatment control perception (p=0.044, or=0.95, 95% ci=0.91_0.99). On the other hand, high identity perception predicted lower medication adherence, as the odds of having low adherence to medications increased with the increase in identity perception (p=0.008, or=1.08, 95% ci=1.02=1.14). Two of the control variables also displayed a significant contribution to low medication adherence; these were sex and employment status . Females were 1.5 times less likely than males to be low adherers (p=0.026, or=0.59, 95% ci=0.370.93) and unemployed respondents were half as likely as employed respondents to be low adherers (p=0.008, or=0.45, 95% ci=0.250.81). To the best of our knowledge, this study was the first to explore diabetes perceptions as guided by the csm in the libyan context . Although this might indicate that females were more willing to participate in the study, it may also reflect the sex profile of diabetes in libya . An early study of the epidemiological pattern of diabetes in benghazi, libya, found that the prevalence of t2 dm was significantly higher among females than males (27). The predominance of females over males was also reported in a previous study among diabetics in the same center where our study was conducted (28) and in other libyan diabetes settings (6, 7). The respondents reported a low identity perception, indicating that they attributed few of the symptoms they had experienced after the diagnosis to their diabetes . Regarding personal and treatment control perception domains, the respondents reported a moderately high perception of both their own ability to control their diabetes and the effectiveness of their diabetes treatment . Personal control perception is a close concept to self - efficacy, which is also about the confidence of the patients in their personal ability to control their illness . In line with our finding, a previous study among adult diabetics in libya reported a moderate level of self - efficacy (7). The respondents perceived diabetes as a chronic illness, and moderately perceived the course of diabetes as unstable or cyclical . The respondents reported moderate coherence perception, which reflects a moderate personal understanding of diabetes . This finding shows that many of them perceived diabetes as an ambiguous illness that they did not understand well . Lack of professional information on diabetes, which in turn affects diabetes knowledge, could be one of the reasons . Several previous studies in the libyan diabetes context showed a poor level of diabetes knowledge (6, 7). Therefore, empowering our patients diabetes knowledge through diabetes educational programs could help in optimizing their diabetes coherence or understanding perception . The respondents perceived that diabetes imposed moderate consequences on their life and health and they displayed moderate negative emotional responses to their illness . The islamic religious influences on the constitution of diabetes perceptions were reflected in the perception of the cause of diabetes, where allah's will / fate was the most frequently reported cause for diabetes . We also found that more respondents attributed their diabetes to psychosocial factors like their emotional state and family problems or worries than to their diet or eating habits . For instance, in a study among nepali diabetics, pollution and immunity were frequently reported as the causes of diabetes, whereas emotional state was among the least perceived causes (22). Furthermore, a study in the united states reported heredity, diet and eating habits, and own behavior as the top three perceived causes for diabetes (14). More than one - third of the respondents reported low adherence to their diabetes medications . This is considerable, and it should be taken seriously for its consequences on patients health and its ultimate impacts on the health - care system . Our study confirmed the findings of two previous studies in the libyan diabetes context (6, 7). In a large diabetes care center in benghazi, libya, roaeid and kablan (6) reported that 27.1% of diabetics were not taking their medications regularly . In a more recent study in the same center in benghazi, elkharam et al . (7) assessed adherence to diabetes management advice using hba1c as a distant adherence measure, and their findings also reflected the problem of low adherence levels . Both of these two studies used different adherence measures to that used in this current study . Compared to some middle eastern countries, the reported low medication adherence prevalence in this study is lower than that reported in palestine among t2 dm patients, using the same measure (42.7%) (29), but higher than that reported in jordan (30). In this study our findings added to the preexisting research evidence on the role of the csm illness perceptions in guiding the coping behaviors in illness (1014). Two illness perceptions were found to contribute significantly to medication adherence; these were identity and treatment control perceptions . The respondents who attributed many of the symptoms they had experienced to their diabetes were more likely to be low adherers to their medications . Meanwhile, treatment control perception was a protective factor, as those who reported higher perception of the effectiveness of their treatment were less likely to be low adherers . In line with our findings, it was found that the perceived treatment effectiveness in diabetes control contributed to the use of insulin (31). Although treatment control perception was identified among the predictors of medication adherence among nepali diabetics, unlike in our study, higher perception of treatment effectiveness was associated with lower medication adherence (22). The final model also identified two personal characteristics as predictors of low medication adherence; these were sex and employment status . This was inconsistent with the findings of some studies where no relationship between sex and adherence level was reported (29, 32). This could be related to some barriers that employed diabetics face, such as the lack of time or access to medications while away from home . Consistent with our findings, some other studies found that employment status influenced medication adherence in diabetes (22, 33). Several limitations should be considered in the interpretation of the findings from this study, such as the cross - sectional design, which precludes making causal assumptions, and self - reporting of data, which is subject to recall bias as well as over- and under - reporting bias . Type 2 diabetic libyans who are unable to read and write should be considered in further research that uses face - to - face interviews . As the study was conducted in a single diabetic center in tripoli, the findings could not be generalized to all libyans with t2 dm . However, the findings could be generalized to the study population, which is libyans with t2 dm who attended the center during the study period . The findings may inform local diabetic care providers about how their patients perceive their diabetes . The current study confirmed the suboptimal level of medication adherence reported by some previous studies in the libyan diabetes context . These findings could be used to develop medication adherence promotion approaches, as in the form of psychoeducational interventions . The findings from this study showed that males and employed patients were more likely to be low adherers; therefore, they might represent in - need groups for medication adherence promotion . Thus, further research is needed to identify what other factors could explain low medication adherence among libyan diabetics . In addition, as our study was conducted at a single center, in the capital city of libya, more research in the libyan diabetes context is warranted to support the generalizability of the findings from our study to all libyans with t2 dm.
Thymomas are the most common low - grade malignant tumors that often emerge in the anterior mediastinum in adults . While thymectomy by sternotomy is the traditional method of treating thymoma, recently, video - assisted thoracic surgery (vats) approaches have been often selected . Vats thymectomy is reportedly feasible and safe and is less invasive than transsternal thymectomy [2, 3]. However, we experienced a rare complication of port - site recurrence that would not have occurred with transsternal thymectomy . A 60-year - old woman was referred to our hospital with an anterior mediastinal tumor on computed tomography (ct) performed for follow - up 6 years after a complete response to chemotherapy for malignant lymphoma . Ct revealed a tumor in the right anterior mediastinum with a diameter of 3.5 cm, round shape and demarcation from the neighboring tissue (fig . Nmol / l (within normal range). To confirm the diagnosis and treat the patient, we planned tumor resection via vats . Figure 1:initial ct revealed a tumor in the right anterior mediastinum with a diameter of 3.5 cm, round shape, which was clearly demarcated from the neighboring tissue . Initial ct revealed a tumor in the right anterior mediastinum with a diameter of 3.5 cm, round shape, which was clearly demarcated from the neighboring tissue . The first 15-mm thoraco - port for the videoscope was placed in the sixth intercostal space on the mid - axillary line . The second 15-mm port was placed on the fifth intercostal space on the anterior axillary line . The third 5-mm port was placed in the fourth intercostal space on the anterior axillary line . The tumor was easily identified under the mediastinal pleura as an elevated mass in the thymic tissue . Through the second and third ports, the normal thymus around the tumor was grasped by forceps and cut using an ultrasonic scalpel with marginal tissues . After the resected tumor was placed into a specimen bag, it was removed through the second port . Frozen sections during the operation revealed that the tumor was capsulated type a masaoka stage i thymoma with sufficient marginal tissue . Follow - up ct performed 36 months after the operation revealed two pleural tumors located near the second and third ports (fig . Initially, the operation was performed only by vats with three ports . After removing the severe adhesion between the lung and chest wall given these findings, we diagnosed these tumors as port - site implantations after the first vats . Figure 2:follow - up ct at 36 months after the operation revealed two pleural tumors locating at the second (a) and third (b) ports used in the vats operation . Figure 3:in the operative findings, the tumors were found to have arisen from the intercostal space, and their surfaces were covered with parietal pleura with partial irregularity . Follow - up ct at 36 months after the operation revealed two pleural tumors locating at the second (a) and third (b) ports used in the vats operation . In the operative findings, the tumors were found to have arisen from the intercostal space, and their surfaces were covered with parietal pleura with partial irregularity . With no definitive findings suggesting tumor invasion into the rib we performed mini - thoracotomies just above the tumors and enucleated those two tumors via the mini - thoracotomies using an electric scalpel . To obtain a sufficient surgical margin, we visualized the adequate resected line using a thoracoscope while cutting the intercostal tissue surrounding the tumor . The pathological diagnosis was type a thymoma, which was consistent with port - site metastasis of the initial tumor . After relating these findings and the incomplete resection to the patient, we decided on close follow - up without reoperation . Ishibashi et al . Reported a case of port - site recurrence after a vats operation for type b2 thymoma, to our knowledge, this is the only such previous case report . The current type a and the reported type b2 thymomas are low - grade malignant tumors . Despite their mild biological behavior, implantation at cases of implantation after medical procedures have been reported, not only for malignant tumors, but also for benign tumors . Surgeons should thus be aware that port - site implantation can occur regardless of the tumor's malignant potential . Willard et al . Reported a case that recurred in the small vats port through which partially resected lung cancer had been extracted without a specimen bag . In our case, the implantation developed only at the second and third ports, through which the forceps and ultrasonic scalpel had passed repeatedly . Although we did not detect any tumor exposure during the first operation, we cannot help but assume that the implantation occurred due to contamination with tumor cells from either the ultrasonic scalpel or forceps that had grasped tissue near the tumor . Of further note, implantation did not develop in other pleura in which tumor tissue must surely have been disseminated by contaminated equipment . Taken together, these findings suggest that one of the possible causes of port - site implantation was the rubbing of tumor cells, such as thymoma, which might have a high affinity for pleura, against the vats ports . Surgeons should take extreme care to grasp the tissue as far as possible from the tumor and to use a specimen bag even when tumor exposure is not recognized . Given the lack of any definitive clinical findings that the tumors had invaded the neighbor ribs, we believed that the tumors could be enucleated completely while preserving the ribs . However, the enucleation of the tumors located mainly in the narrow intercostal spaces with a sufficient margin was very difficult . Although no definitive tumor invasion to the skin or subcutaneous tissue was identified, they might nevertheless invade the ribs pathologically . Ishibashi et al . Successfully performed a curative operation in a case of port - site recurrence via wide chest wall resection and reconstruction . In the process a small - extent resection of the skin and wide resection of the chest wall is therefore recommended when performing surgery for port - site recurrence after vats thymectomy.
The austrian stroke prevention study (asps) is a prospective study on the effects of vascular risk factors on brain structure and function in cognitively normal middle - aged and elderly inhabitants of graz, austria; details have been previously described (13,14). Briefly, 2,007 participants were randomly selected from the official community register, and all participants were free of stroke and dementia . The study protocol was approved by the responsible ethics committee . In a first study panel between 1991 and 1994, 509 subjects randomly selected from the entire cohort underwent an extended diagnostic work - up including brain mri and cognitive testing . To enlarge the cohort with imaging and neuropsychological assessments, an additional 567 individuals were randomly selected in a second panel between 1999 and 2003 . Participants of the first and second panels were pooled, which resulted in a total of 1,076 individuals with brain mri and neuropsychological evaluation . The long diagnostic periods resulted from limited research - devoted time slots at the mri center . Clinical history, blood tests, mri, and cognitive assessments were always done in 1 day . A total of 819 subjects had a complete risk factor assessment necessary to determine metabolic syndrome and thus created the current study cohort . Risk factors were determined based on history and measurements at the examination, as previously described (13). Hypertension was considered a history of hypertension with repeated blood pressure readings 140/90 mmhg, treatment for hypertension, or readings at the examination exceeding the limit . Diabetes was coded present if an individual was treated for diabetes at the time of examination or if the fasting blood glucose level at the examination exceeded 126 mg / dl . History of coronary heart disease was determined according to the rose questionnaire, atrial fibrillation was diagnosed based on electrocardiogram findings, and peripheral artery disease was diagnosed based on history . Study participants were asked whether they were previous and/or current smokers or habitual daily drinkers as well as about their physical activity at leisure time; the amount of activity was graded into none (no physical activity), mild (daily walk), and moderate to intensive (daily sports). A lipid status was determined with standardized measurements for each study participant after a 12-h fasting . High - sensitivity c - reactive protein (hs - crp) was measured with a particle - enhanced immunoturbimetric assay, with a detection limit of 0.1 mg / l . We defined a high level of inflammation as hs - crp levels above the median of hs - crp distribution (> 1.9 mg / l) in our cohort . Metabolic syndrome was defined according to national cholesterol education program adult treatment panel iii criteria and revised american heart association / national heart, lung, and blood institute criteria (1). Participants were considered to have metabolic syndrome if they met three or more of the following criteria: 1) waist circumference> 102 cm in men and> 88 cm in women, 2) fasting serum triglycerides 150 mg / dl or treatment for hypertriglyceridemia, 3) serum hdl cholesterol <40 mg / dl in men and <50 mg / dl in women or treatment for low hdl cholesterol, 4) blood pressure 130/85 mmhg or use of antihypertensive drugs, 5) fasting blood glucose levels 110 mg / dl or use of antidiabetes drugs . A test battery assessing memory and learning abilities, psychomotor skills, and executive functions was administrated as described previously (13,14). To assess learning ability and intermediate memory recall, executive functions were assessed by three tests: the wisconsin card sorting test, part b of the trail making test, and digit span backwards, which is part of the wechsler adult intelligence scale, revised . We used summary measures of cognitive function by converting individual test results to z - scores and computing the average of the scores within each cognitive domain . Z - scores were not adjusted for age, sex, and education . For assessment of depressive mood we applied the eigenschaftswrterliste of w. janke, g. debus (1978) die eigenschaftswrterliste (ewl); handanweisung, gttingen: hogrefe, a validated multidimensional tool consisting of a list of given adjectives describing the emotional status of a test person at the time of the interview . All scans were obtained on a 1.5-tesla scanner (philips medical systems, eindhoven, the netherlands) with protocols described previously (13). White matter lesion (wml) volume, lacunes, thromboembolic infarcts, and brain volume were determined in each study participant . The scans were analyzed by two experienced investigators (c.e . And r.s . ). They first graded wmls according to our scheme (15), then marked and outlined each wml on a transparency that was overlaid on the proton density scans . They were blinded to clinical data of study participants . Independently from this visual analysis, lesion load measurements were done on proton density weighted images on an ultrasparc workstation (sun microsystems, santa clara, ca) by a trained operator using dispimage (16). The trained operator used a hardcopy overlaid by the transparency, with all lesions outlined by the experienced readers as reference, and segmented all lesions on the computer image . The total lesion volume (cm) was calculated by multiplying the total lesion area by slice thickness . Lacunes were defined as focal lesions that involved the basal ganglia, internal capsule, thalamus, or brain stem, not exceeding a maximum diameter of 15 mm . Brain volume was calculated from the t2-weighted spin echo sequence using the fully automated structural image evaluation of atrophy (sienax, part of the fmrib software library; http://www.fmrib.ox.ac.uk/fsl). Brain parenchyma percentage was estimated from the ratio of parenchymal volume to the total volume given by the outer surface of the brain . To determine the measurement errors in our dataset, significant differences in means of normally distributed continuous variables were tested by anova and significant differences of nonnormally distributed variables by the mann - whitney u test . Linear regression analysis was performed to estimate the association between metabolic syndrome and the z - scores of the specific cognitive domains . Model 1 included age and sex plus subjects' characteristics that were different across the metabolic syndrome, including years of education, leisure physical activity, and coronary heart disease . Although significantly more common in patients with metabolic syndrome, hypertension and diabetes were not used as covariates because all subjects with hypertension and diabetes were included in our definition of metabolic syndrome . Depressive mood was considered as a covariate based on its significant relationship to cognitive functioning . Model 2 investigated to what extent the associations between metabolic syndrome and cognition were mediated by mri findings and thus included all covariates of model 1 plus presence of lacunes, silent infarcts, wml volume, and brain parenchymal fraction . The relationship between metabolic syndrome and vascular lesions and brain atrophy was determined by multivariate regression analyses using model 1 covariates . Multicollinearity between mri variables was assessed using the variance inflation factor (vif). Usually, vif values> 10 are suggestive of multicollinearity and merit further investigation . The mean vif for mri variables in our study was 1.04 (range 1.011.08). Because data from the literature suggest possible sex differences in metabolic syndrome (6,17), we also stratified our analyses by sex . Hs - crp measurements were available in only a subset of our cohort, thus we did not include this factor in the primary model but performed a subanalysis in 541 subjects with hs - crp measurement to assess whether inflammation plays a causal role in the association between metabolic syndrome and cognition . This subanalysis was done with and without additional adjustment for nonsteroidal anti - inflammatory drugs or acetylsalicyclic acid use . Interactions of hs - crp and metabolic syndrome with cognitive functioning were tested by including the respective interaction term in the multivariate linear regression models . All analyses were done using stata (stata statistical software, release 6; statacorp, college station, tx). Risk factors were determined based on history and measurements at the examination, as previously described (13). Hypertension was considered a history of hypertension with repeated blood pressure readings 140/90 mmhg, treatment for hypertension, or readings at the examination exceeding the limit . Diabetes was coded present if an individual was treated for diabetes at the time of examination or if the fasting blood glucose level at the examination exceeded 126 mg / dl . History of coronary heart disease was determined according to the rose questionnaire, atrial fibrillation was diagnosed based on electrocardiogram findings, and peripheral artery disease was diagnosed based on history . Study participants were asked whether they were previous and/or current smokers or habitual daily drinkers as well as about their physical activity at leisure time; the amount of activity was graded into none (no physical activity), mild (daily walk), and moderate to intensive (daily sports). A lipid status was determined with standardized measurements for each study participant after a 12-h fasting . High - sensitivity c - reactive protein (hs - crp) was measured with a particle - enhanced immunoturbimetric assay, with a detection limit of 0.1 mg / l . We defined a high level of inflammation as hs - crp levels above the median of hs - crp distribution (> 1.9 mg / l) in our cohort . Metabolic syndrome was defined according to national cholesterol education program adult treatment panel iii criteria and revised american heart association / national heart, lung, and blood institute criteria (1). Participants were considered to have metabolic syndrome if they met three or more of the following criteria: 1) waist circumference> 102 cm in men and> 88 cm in women, 2) fasting serum triglycerides 150 mg / dl or treatment for hypertriglyceridemia, 3) serum hdl cholesterol <40 mg / dl in men and <50 mg / dl in women or treatment for low hdl cholesterol, 4) blood pressure 130/85 mmhg or use of antihypertensive drugs, 5) fasting blood glucose levels 110 mg / dl or use of antidiabetes drugs . A test battery assessing memory and learning abilities, psychomotor skills, and executive functions was administrated as described previously (13,14). To assess learning ability and intermediate memory recall, executive functions were assessed by three tests: the wisconsin card sorting test, part b of the trail making test, and digit span backwards, which is part of the wechsler adult intelligence scale, revised . We used summary measures of cognitive function by converting individual test results to z - scores and computing the average of the scores within each cognitive domain . Z - scores were not adjusted for age, sex, and education . For assessment of depressive mood we applied the eigenschaftswrterliste of w. janke, g. debus (1978) die eigenschaftswrterliste (ewl); handanweisung, gttingen: hogrefe, a validated multidimensional tool consisting of a list of given adjectives describing the emotional status of a test person at the time of the interview . All scans were obtained on a 1.5-tesla scanner (philips medical systems, eindhoven, the netherlands) with protocols described previously (13). White matter lesion (wml) volume, lacunes, thromboembolic infarcts, and brain volume were determined in each study participant . The scans were analyzed by two experienced investigators (c.e . And r.s . ). They first graded wmls according to our scheme (15), then marked and outlined each wml on a transparency that was overlaid on the proton density scans . They were blinded to clinical data of study participants . Independently from this visual analysis, lesion load measurements were done on proton density weighted images on an ultrasparc workstation (sun microsystems, santa clara, ca) by a trained operator using dispimage (16). The trained operator used a hardcopy overlaid by the transparency, with all lesions outlined by the experienced readers as reference, and segmented all lesions on the computer image . The total lesion volume (cm) was calculated by multiplying the total lesion area by slice thickness . Lacunes were defined as focal lesions that involved the basal ganglia, internal capsule, thalamus, or brain stem, not exceeding a maximum diameter of 15 mm . Brain volume was calculated from the t2-weighted spin echo sequence using the fully automated structural image evaluation of atrophy (sienax, part of the fmrib software library; http://www.fmrib.ox.ac.uk/fsl). Brain parenchyma percentage was estimated from the ratio of parenchymal volume to the total volume given by the outer surface of the brain . To determine the measurement errors in our dataset, significant differences in means of normally distributed continuous variables were tested by anova and significant differences of nonnormally distributed variables by the mann - whitney u test . Linear regression analysis was performed to estimate the association between metabolic syndrome and the z - scores of the specific cognitive domains . Model 1 included age and sex plus subjects' characteristics that were different across the metabolic syndrome, including years of education, leisure physical activity, and coronary heart disease . Although significantly more common in patients with metabolic syndrome, hypertension and diabetes were not used as covariates because all subjects with hypertension and diabetes were included in our definition of metabolic syndrome . Depressive mood was considered as a covariate based on its significant relationship to cognitive functioning . Model 2 investigated to what extent the associations between metabolic syndrome and cognition were mediated by mri findings and thus included all covariates of model 1 plus presence of lacunes, silent infarcts, wml volume, and brain parenchymal fraction . The relationship between metabolic syndrome and vascular lesions and brain atrophy was determined by multivariate regression analyses using model 1 covariates . The mean vif for mri variables in our study was 1.04 (range 1.011.08). Because data from the literature suggest possible sex differences in metabolic syndrome (6,17), we also stratified our analyses by sex . Hs - crp measurements were available in only a subset of our cohort, thus we did not include this factor in the primary model but performed a subanalysis in 541 subjects with hs - crp measurement to assess whether inflammation plays a causal role in the association between metabolic syndrome and cognition . This subanalysis was done with and without additional adjustment for nonsteroidal anti - inflammatory drugs or acetylsalicyclic acid use . Interactions of hs - crp and metabolic syndrome with cognitive functioning were tested by including the respective interaction term in the multivariate linear regression models . All analyses were done using stata (stata statistical software, release 6; statacorp, college station, tx). A total of 232 (28.3%) study participants had metabolic syndrome . There were 157 (67.6%) subjects with three, 55 (23.7%) with four, and 20 (8.6%) with five components of metabolic syndrome . Subjects with metabolic syndrome had lower educational status, less leisure physical activity, more coronary heart disease, higher frequencies of each component of metabolic syndrome, and higher hs - crp . Characteristics of the study cohort (n = 819) by the absence or presence of metabolic syndrome data are means sd, n (%), or median (interquartile range). Table 2 shows that subjects with metabolic syndrome performed worse on all cognitive tests, with significant differences in memory and executive functions, but there existed no significant differences in mri findings between subjects with and without metabolic syndrome . Metabolic syndrome and mri findings cognitive performance and brain mri findings in the sample (n = 819) by the absence or presence of metabolic syndrome data are means sd (range), n (%), or median (interquartile range). Mann - whitney u test . When using linear regression analysis with adjustment for age, sex, years of education, depressive mood, coronary heart disease, and physical activity at leisure time (table 3, model 1), the inverse associations between metabolic syndrome and memory, as well as executive functions, remained significant . The results were virtually unchanged following adjustment for mri findings (table 3, model 2). Stratification by sex demonstrated that the significant relationship between metabolic syndrome and impaired memory and executive functioning existed only in men but not in women (table 3). Relationship between metabolic syndrome and cognition: multivariate regression analysis * model 1: adjusted for age, years of education, depressive mood, coronary heart disease, and physical activity at leisure time . Model 2: model 1 plus adjustment for wml volume, presence of lacunes, silent infarcts, and brain parenchymal fraction . Figure 1 displays the relationship between the number of components of metabolic syndrome and performance on tests of memory and executive functioning . In men, with more components of metabolic syndrome present, memory (one component: = 0.38; two components: = 0.52; three components: = 0.64; more than three components: = 0.71; p for trend <0.001) and executive functions (one component: = 0.99; two components: = 1.02; three components: = 1.55; more than three components: = 1.90; p for trend = 0.010) were more impaired . Relationship between memory performance (upper panel), executive functions (lower panel), and number of metabolic syndrome components (indicated on the y - axis in the middle). Multivariate regression analysis stratified by sex and adjusted for age, years of education, depressive mood, physical activity at leisure time, and coronary heart disease . The squares on the x - axis indicate -coefficients, and the bars indicate 95% cis . An increasing number of metabolic syndrome components was associated with worse function in these domains in men only . A multivariate regression analysis adjusted for model 1 and 2 covariates showed that men, but not women, with metabolic syndrome who had high hs - crp levels (i.e.,> 1.9 mg / l) performed significantly worse on tests of memory (= 0.44; 95% ci [0.94 to 0.04]; p = 0.074) and executive functions (= 2.21; 95% ci [3.81 to 0.62]; p = 0.007) than their male counterparts with low hs - crp (memory: = 0.05; 95% ci [0.45 to 0.33]; p = 0.774; executive functions: = 0.48; 95% ci [0.74 to 1.7]; p = 0.437). After adjustment for nonsteroidal anti - inflammatory drugs or aspirin usage in the model, the results remained almost identical (data not shown). The interaction between hs - crp and metabolic syndrome was significant for executive functions (= 2.79; 95% ci [4.67 to 0.92]; p = 0.004) and showed a nonsignificant trend for memory (= 0.56; 95% ci [1.17 to 0.04]; p = 0.068). Middle - aged and elderly community - dwelling subjects with metabolic syndrome performed worse on tasks assessing memory and executive functions than their counterparts without metabolic syndrome . This was seen in men but not in women, and the more components of metabolic syndrome were present, the more impaired was cognitive functioning . Focal and diffuse brain lesions failed to explain the association between metabolic syndrome and cognition . The longitudinal aging study amsterdam also applied comprehensive neuropsychological testing and, similar to us, also found memory and processing speed deficits in metabolic syndrome patients (3). This pattern favors both cortical and subcortical brain damage as the underlying cause (18). So far, only few studies stratified their analyses by sex, and they yielded conflicting results . Vanhanen et al (4) found more alzheimer's disease in metabolic syndrome patients, and, after stratification by sex, this association existed predominantly in women . By contrast, laudisio et al . (6) reported that female patients performed cognitively better than male patients . So far, sex - specific differences in risk factor related biological mechanisms leading to cognitive decline remained widely unexplored . We can only speculate on the causes for more pronounced cognitive deficits in men than in women with metabolic syndrome . It is assumed to be a common denominator of metabolic disorders and cognitive dysfunctions by exerting detrimental effects on cholinergic and glutamatergic pathways which establish neuronal plasticity (19). Women have less insulin resistance than men and may thus be less amenable to metabolic syndrome related cognitive dysfunction . Several mechanisms have been reported, leading to a more insulin - sensitive environment in women . These include sex differences in adipose tissue distribution, estrogen effects on insulin and glucose homeostasis and proinflammatory markers, as well as higher levels of the insulin - sensitizing hormone adiponectin in women (20). We saw no significant differences in ischemic brain lesions or brain atrophy between subjects with and without metabolic syndrome . Our mri results were unexpected, as previous investigations described associations of metabolic syndrome with increasing grades of white matter abnormalities (11) and brain infarctions (10), and several studies indicated more pronounced brain atrophy in individuals with single components of metabolic syndrome such as diabetes (21) and hypertension (22). We probably recruited healthier participants than previous hospital- or population - based cohorts not excluding clinically overt cerebral disease (10,11). It remains to be determined whether ultrastructural tissue changes in subcortical or cortical tissue compartments not seen in conventional but new mri techniques, such as diffusion tensor imaging (12), occur with metabolic syndrome and whether they correlate with cognitive decline . Our analyses on the associations between metabolic syndrome, hs - crp levels, and cognitive functioning suggest that inflammation interacts in the relationship between metabolic syndrome and cognitive impairment . (3) and yaffe et al . (2,23) that described the detrimental effect of metabolic syndrome on cognition mainly in metabolic syndrome subjects with high levels of proinflammatory markers . The association between hs - crp and cognitive impairment in metabolic syndrome patients may at first seem to indicate that subjects with ongoing generalized atherosclerosis are at greater risk of concurrent and subsequent cerebrovascular damage and neurodegeneration, both of which cause cognitive decline . This, however, is unlikely based on our findings that the frequency of focal ischemic lesions and brain atrophy was similar in subjects with and without metabolic syndrome, and mri abnormalities were not related to cognitive test performance . These findings rather favor inflammatory processes being directly related to both metabolic syndrome and cognitive functioning irrespectively of their possible role in the etiology of vascular and primary degeneration . Increased hs - crp levels may well reflect lifetime overreactivity of the innate immune system to various exogenous and endogenous stimuli . This overreactivity may be genetically determined and confer the increased risk for developing both metabolic syndrome and cognitive impairment in later life . Consequently, the interactive association observed here of metabolic syndrome and hs - crp with cognitive functions is of particular interest . In this context it is also noteworthy that elevated levels of inflammatory markers have been repeatedly linked to an increased risk of dementia (23). Intriguingly, the honolulu asia aging study reported increased hs - crp levels 25 years prior to the occurrence of dementia (24). A strength of our study is the simultaneous assessment of cognitive function and brain mri in a large cohort of community - dwelling subjects who were prospectively examined using a standardized protocol . An important limitation of this study is its cross - sectional design, which does not permit to deduce a cause - and - effect relationship . Other limitations include missing measurements of hippocampal volume due to lack of coronal mri sequences and missing information on duration of metabolic syndrome . Sex - specific effects associated with proinflammatory markers and the lack of significant associations with mri abnormalities ought to be considered in future studies on mechanisms of cognitive impairment in metabolic syndrome.
Fourier transform infrared (ftir) and gas chromatography - mass spectrometry (gcms) are two essential techniques applied for analysis of edible oils [1, 2]. Fundamentally, ftir spectra illustrate absorption bands with characteristic frequency attributed to different functional groups whilst gcms reveals the compounds eluted at different retention times with mass spectra corresponding to compounds present, indicative of the fatty acid compositions . Conventionally the resultant signals from both instruments are analysed with the software equipped at the workstations for peak integration . The signal processing tool exclusively designed for ftir is not applicable to gcms chromatograms due to differences in data nature and characteristics . Therefore to analyse the output from both ftir and gcms, an analyst has to juggle between both instruments . When a large volume of sample is involved, the signal processing process can be exhaustive and time consuming . With the advances in computer technology, various alternatives have been made available reducing the dependence on the default signal processing tool; for instance, the digital data in csv format is readable on microsoft excel . Numerous algorithms have been developed for analysis of signal from various instrumentation techniques, that is, fourier transform infrared (ftir) [1, 3], gas chrnomatography - differential mobility spectrometry (gc - dms), gas chromatography - mass spectrometry (gc - ms) [57], high performance liquid chromatography (hplc), nuclear magnetic resonance (nmr), two - dimensional gas chromatography (gc - gc), and liquid chromatography - mass spectrometry (lc - ms) to allow mathematical integration of the signals including baseline correction, smoothing, and peak deconvolution on a personal computer . These algorithms however are designed to cater a specific source of signal; hence, when multiple sources of signals are involved more than one algorithm is required . In edible oil industry, continuous monitoring and extensive cross comparisons between products involving multiple instrumental techniques such as ftir and gcms are common and this often results in enormous amount of data that requires a more efficient way for data interpretation . This has motivated the development of a signal processing approach that aids in analysis of signals from multiple sources . In this paper, we report a synchronised signal processing algorithm that caters both ftir and gcms signal for evaluation of thermally degraded vegetable oils . Seven types of edible oils were studied including (1) palm oil, (2) canola and palm oil, (3) corn oil, (4) canola oil, (5) soybean oil, (6) blended palm, sesame and peanut oil, and (7) canola and sunflower oil . They were obtained from the local market and heated at 100c and 150c, respectively, for 15 min using a digital heating block . A total of 63 ftir spectra and gcms chromatograms, respectively, were produced (7 types 3 treatments 3 replicates = 63). All spectra were obtained using an atr - ftir of thermoscientific (thermo nicolet analytical instruments, madison, wi). The spectra were collected at a resolution of 4 cm in the range of 4000650 cm . Each spectrum was rationed against a fresh background spectrum recorded from the bare atr crystal . Prior to collection of each background spectrum, the atr crystal was cleaned with absolute ethanol to remove any residual . For gcms analysis, 100 l of oil was dissolved in 2 ml of dichloromethane (dcm). The sample was analysed on a shimadzu gc - ms system model qp500 with a medium polarity capillary column (bpx-5 column (29.4 m 0.25 mm), with film thickness of 0.25 m) with helium as the carrier gas . One microlitre of the sample was injected using splitless injection with injector temperature 300c according to the following scheme: 50c for 2 min with 10c / min up to 300c . The total runtime for each sample was 37 min . For ms detection, electron ionization with 70 ev was applied and mass fragments were detected between 40 and 500 m / z . The ftir spectra and gcms chromatograms were analysed using the synchronised algorithm developed in matlab r2012a . The synchronised strategy is an extension of the peak detection and matching algorithm designed for ftir, published in . First, all data files (csv format for ftir spectra and netcdf for gcms chromatograms) were converted into mat files . The algorithm requires input of the data nature whether it is one - dimensional or two - dimensional . If the data is two dimensional, the algorithm would obtain the total ion chromatogram (summing the intensities of all mass spectral of the same scan) compressing them into one - dimensional data . The data is subsequently baseline corrected according to asymmetric least squares, smoothed using soft heuristic thresholding (sym8 wavelet), and transformed into first derivative signal where the peak start, peak, and peak end are identified . Briefly, the algorithm would evaluate each data point of the derivative signal in succession; a peak start is labelled when the derivative signal is above zero and x times greater than the peak noise (average absolute change of derivative); when the signal crosses x - axis attaining negative values, the peak is located . As the derivative signal crosses x - axis again where the values become positive, peak end has arrived . Upon detection of a peak, the corresponding peak area is calculated as the sum of the detector output between the start and the end . The algorithm would evaluate the spectrum in turn to identify the peaks present; they will then be matched across samples for similar functional groups according to a predefined window size . For example, if a window of z1 scans is set and a peak at 1720 cm is targeted for matching, the algorithm would search through all samples for possible matching peaks ranging between 1720 z1 cm . The matching peaks are subsequently arranged in the same column with rows corresponding to samples; as a result, a table representative of peaks detected and matched is produced . It is important to evaluate the resultant table to confirm that parameters employed such as window size and peak threshold are adequate . If a potential matching peak is mismatched or unmatched, it is an indication that the window size would require fine - tuning . In terms of peak detection, it is possible that the algorithm suffers to identify poorly resolved bands or shoulder bands; sometime the peak noise threshold may be unsuitable, too small that noise is misidentified as signals or too large that signals are overlooked . Therefore, it is essential to manually verify the information on the peak table overlaying the spectra for visualization . For gcms data, peaks are detected similarly based on the total ion chromatograms; however, in the matching process, the candidate matching peaks are identified according to a predefined window size, z2 (in this paper, z2 = 50 scans (15 s)), and further confirmed with the mass spectra according to the similarity index . Spectra with correlation coefficient> 90% are considered corresponding to the same compound in which matching peaks are organised into a peak table as described above . For each compound the spectra were prealigned to some common peaks according to the strategy of retention time alignment in . In oil analysis, prealignment, and setting, an optimum window size is crucial to minimize erroneous matching as compounds eluted closely may exhibit mass spectra with high similarity, sometime more than 98% for example, 2,4-dodecadienal and 2,4-decadienal eluting at 11.98 min and 12.35 min, respectively . Two peak tables were produced resulting from the analysis of ftir and gcms spectra / chromatograms . The algorithm undoubtedly experiences some inherited shortcomings; nevertheless, it enables multiple output signals from analytical instruments to be processed efficiently and systematically . The peak table was preprocessed (square rooted, scaled to one, and standardised) and subjected to principal component analysis (pca) for further analysis . The algorithm yields two peak tables corresponding to the analysis of a total of 63 ftir and gcms spectra / chromatograms, respectively . Figure 2 shows the ftir spectra of seven edible oils treated under different temperatures: unheated, 100c, and 150c . Essentially the superimposed spectra of heated and unheated palm oil, corn oil, and blended palm oil exhibit slight variation suggesting little changes upon treatments . The scores plot of the ftir peak table in figure 3 shows that canola - based oils are differentiable from palm - based oil . After heating at 150c, various vegetable oils regardless of the origin are observed to cluster implying sharing of common features where palm oil encountered comparatively less alteration, inferring better thermal stability . Typically, heated cooking oils are challenged with the loss of unsaturation due to the attack of oxygen via radical reaction . The degree of unsaturation is often monitored based on several characteristic bands at 1650 cm (c = c stretching vibration of cis - olefins), 1417 cm (rocking vibrations of ch bonds of cis - disubstituted), and 3001 cm (ch stretching vibration of the cis - double bond). If only a limited number of spectra are involved, there is no issue related to identification of changes due to thermal treatment; however, the process can be exhaustive when numerous spectra are concerned . Figure 4 illustrates the relative abundance of some bands commonly reported for discrimination of thermally degraded oil . Evidently, the band corresponding to olefinic attribute at 14301330 cm reduces steadily / disappears upon heating confirming the loss of double bonds where palm oils are characterised by relatively less polyunsaturated compounds, which is not unexpected . Other lines of evidence of diminished unsaturation are observed at 3010 cm and 1650 cm . As oxidation continues, the degree of unsaturation is correspondingly reduced closing the gap of difference between polyunsaturated and polysaturated oils, leading to clustering of the thermally oxidised edible oils as demonstrated in figure 3 . In addition to loss of unsaturation, elevated temperature simultaneously triggers hydrolysis of triglycerides yielding fatty acids and glycerols, evidenced with increased band intensity at 1157 cm (c o ester groups). The band at around 1680 cm, indicative of the c = o stretching of conjugated unsaturated aldehydes, is profoundly detected in heated oils of which palm oil demonstrated greater abundance . Formation of trans - fatty acid, a common issue in thermally oxidised oil, is associated with the band near 966 cm; it is often identified in a small amount in fresh oil as a consequence of isomerisation of cis - unsaturated fatty acids upon bleaching, refining, and deodorization [1820]. The undesirable conversion is witnessed to be encouraged under increasing temperature confirmed by soaring of the band at 966 cm in which palm olein oil and blended canola - sunflower oil demonstrate higher concentration after heating . As suggested elsewhere, palm oil and sunflower oil are characterised by better efficiency of heat transfer, thus leading to higher conversion of trans - fatty acid [21, 22]. The information on the peak table is converted into bar charts that are verified by overlaying the spectral at a designated region as illustrated in figure 4; this approach enables rapid validation of the algorithm . Figure 5 illustrates the peaks precisely identified in a chromatogram and the inset shows several dienaldehydes commonly present in all chromatograms . Most cooking oils are enriched with vitamin e that exists in various forms mainly tocopherols and tocotrienols . Their presence essentially improves the antioxidation property of cooking oil as the compounds, during oxidation process, compete with unsaturated fats for lipid peroxy radical . According to al - saqer et al ., these compounds are prominently found in soybean, canola, sunflower, and corn oils with relatively lower amount in palm oil . In this study, only - and -tocopherols are identified; they are relatively more prominent in blended and pure palm oil as opposed to the findings of al - saqer et al . . Nevertheless the nutritional label on the products, when compared, indicates that blended and pure palm oils possess higher vitamin e corresponding to the relative abundance of the total tocopherols revealed by gcms: palm, peanut, and sesame (75 mg/100 ml); palm oil (60 mg/100 ml); corn (63 mg/100 ml); canola and palm (50 mg/100 ml); canola and sunflower (30.8 mg/100 ml); canola (20.3 mg/100 ml); soya (15 mg/100 ml). The higher concentration of vitamin e in palm based oil provides an explanation to the better oxidative stability suggested from the analysis of ftir spectra . The various forms of tocopherols are in addition convertible; several studies have reported the conversion of - and -tocopherols into - and -tocopherols [25, 26]. As observed in this study, the -form of tocopherol is distinctively found in unheated palm - based oil, corn oil, and canola - sunflower oils; upon heating, this compound is completely missing and replaced with -tocopherol that appears to degrade with increasing temperature . Squalene is also a compound commonly found in vegetable oils, particularly high in olive oils, with antioxidant property . As illustrated in figure 6, squalene is detected profoundly in blended and pure palm oil as well as corn oil corroborating the hypothesis drawn from ftir spectra that these varieties of oils are more resistant to oxidation . Plant sterols including sitosterol, campesterol, and stigmasterol are cholesterol - like molecules; the saturated analogues are suggested as effective cholesterol - lowering agents whilst those with ethylidene contain side chain that may behave as antioxidants [27, 28]. In this study, - and -sitosterols are identified with the former distinctively detected in canola and corn oils where the finding is in agreement with ratnayake et al . . During oxidation, fatty acids are typically converted into hydroperoxides and further broken down into secondary products such as aldehydes and ketones [3032]. It is found that dienaldehydes such as 2,4-nonadienal and 2,4-decadienal are consistently detected in all oil samples where the concentrations appear to increase appreciably after heating at 150c with no significant different concluded statistically (p> 0.05). Interestingly at 100c, a decline in the amount of dienaldehydes is consistently experienced in all oil varieties, possibly due to the chain reaction of transformation to ultimate by - products . The study demonstrates that output from ftir and gcms can be simultaneously analysed using a common signal processing approach for evaluation of thermally degraded vegetable oils . This would save the time for an analyst to juggle between instruments and applies some common guidelines to process the signals systematically . Undoubtedly, the approach is not a perfect and flawless method as there are possibilities that peaks are overlooked or mismatched; nevertheless, it offers a systematic strategy to process relatively large datasets of edible oils from gcms and ftir simultaneously with reasonable reliability.
Premature beats arising from foci other than pulmonary veins have been related to its pathogenesis . A 64-year - old female underwent superior vena cava (svc) isolation after triggers were identified originating from the svc following pulmonary vein isolation; immediately after svc isolation, she developed junctional rhythm with symptomatic hypotension requiring emergent management . Apical motion abnormalities were noticed in the echocardiography suggesting stress - induced cardiomyopathy which resolved 48 hours later . Although received a dual chamber pacemaker, intact sinus node function returned 2 weeks later . Superior vena cava isolation in those with trigger mediated atrial fibrillation following pulmonary vein isolation (pvi) is performed to enhance long - term outcomes . We present the first case of time course of recovery of sinus node function, injured during svc isolation . Atrial fibrillation (af) is the most common sustained arrhythmia; its incidence increases with aging . This condition could increase morbidity and mortality due to its high potential to cause heart failure, thromboembolic events and the related conditions associated to its treatment such as bleeding . The cornerstones for the management of atrial fibrillation are anticoagulation to prevent embolic events, control symptoms and avoid deterioration of heart function . Af arising from the pulmonary veins (pv's) was recognized by haissaguerre et al ., in a study that showed 94% of the time pvs are implicated . However, there is some evidence showing that non - pulmonary veins (npv's) premature beats could trigger af in up to 28% of cases . The most common npv foci include the left atrial posterior wall (38%) and superior vena cava svc (37%). Other areas acting as npv include crista terminalis, coronary sinus, ligament of marshall and interatrial septum . Catheter techniques looking for the electrical isolation of pv antrum and other atrial tissue originating premature beats has emerged as therapeutic option for symptomatic patients with atrial fibrillation, providing symptomatic benefit . We present a clinical case of a patient who developed a transient sinus node block as a complication during pvi and superior vena cava isolation (svci)., although patient did receive a dual chamber pacemaker, two weeks later, sinus node function returned back to baseline, identifying the time course of recovery of sinus node function . A 64 year - old female with drug refractory af and atrial flutter underwent pvi and cavotricuspid isthmus(cti) ablation . Basal measurements and conduction study were considered normal . During the isoproterenol testing the patient developed recurrent episodes of recurrent atrial tachycardia (at), left bundle branch block (lbb) and rapid ventricular response af (cycle length up to 410 msec). Pvi was carried out with irrigated radiofrequency (rf) ablation and lesions were tagged on geometry created using carto 3d sound system (biosense webster, diamond bar, ca, usa); we did a wide area circumferential ablation on all four pv's including carinal ablations, with evidence of entrance block . However, triggered af was inducible with isoproterenol, and triggers in vein of marshall was successfully ablated . Further testing revealed svc origination of npv triggers and svci was performed (see fig . 1, fig . 2). During immediate observation, junctional rhythm at 36 bpm (fig . 3) with hypotension requiring emergency administration of epinephrine and temporary transvenous pacemaker insertion was needed . An echocardiogram showed apical motion abnormalities, and coronary angiogram was negative for coronary artery disease . Patient was implanted with a permanent pacemaker after failure of recovery of sinus node function at 48 hours . On weekly observations in clinic, recovery of sinus node function yamaguchi et al . Showed that in those patients with npv foci pvi alone is not enough to reach long term responses suggesting that they should always be addressed during the index ablation procedure . Controversies still exist about the npv foci management especially with svc triggers and conflicting evidence exists based on randomized control trials (rct). . Showed in 2003 that there were no difference between patients randomly assigned to pvi versus pvi + svci regarding af recurrence after ablation . However in a more recent clinical trial evaluating 2 randomly assigned groups to pvi compared to pvi + svci, the authors found that those patients who underwent both procedures were significantly less prone to paroxysmal af after 12 months of follow - up . The most common are svc stenosis, phrenic nerve damage with diaphragmatic paralysis and sinus node injury (sni). Described 6 patients who developed sni after svci, in this clinical study 5 patients had transient junctional rhythm that resolved in the first 48 hours postprocedure and just one patient required permanent pacemaker implantation . Probably the sni is due to the damage to the atrial muscle sleeve that extends into the svc and contains sinus node pacemaker activity . To the best of our knowledge ours is the first report of sni associated with pvi + svci where time course of recovery of sinus node function was noted . Identification of npv triggers and subsequent successful ablation is performed in order to get higher responses in the long term follow up after the index procedure . Premature beats arising from svc triggering af is infrequent but when present has to be approached cautiously due to proximity of phrenic nerve and inadvertent sinus node injury.
Skin wounds are caused by the loss of connection in the skin and recovery requires cellular and biochemical reactions . Chronic wounds arise from the lack of physiological processes and the disease due to complications (e.g. Infection and amputation) has devastating consequences on societies . According to the principles of iranian traditional medicine (itm) this is a systematic review study that involved gathering data from three traditional medical textbooks, namely canon of medicine, tib - e - akbari and exir - e - azam with the keyword chronic wound or few tables were developed for nutrition measures as well as edible and topical medicinal herbs . Electronic databases such as pubmed, scopus, web of science, and cochrane library were searched for relevant articles and those related to effective medicinal herbs in the treatment of wounds were obtained . Depending on appearance and secretions, wounds are divided into two categories, namely simple and compound wounds . The prognostic factors are based on the age, weight, accompanied disease, as well as the quality and quantity of the wound secretions . Patient s diet is very important and oral medications have a major role in the whole body detoxification . Topical medications are used together with the above - mentioned treatments; noting that without detoxification, these medications are not effective entirely . Body detoxification is the first medical step, after which topical medications could lead to a better wound healing result.
We report the case of a patient who underwent a laparoscopic cholecystectomy with gallstone spillage 34 months before presenting to the thoracic surgery service . The patient first complained of streaks of hemoptysis at 6 months from the time of the original procedure . A lower lobe infiltrate was noted and treated successfully with oral antibiotics . Over the next 2 years, the patient's symptoms waxed and waned . Due to the chronic infiltrate in his lung, a thoracotomy was performed that revealed erosion of the stone through the right diaphragm with formation of a lung abscess . A high index of suspicion for a gallstone - related problem should be entertained by the practitioner when presented with a patient who has a right lung infiltrate and a history of open or laparoscopic cholecystectomy . Gallbladder perforation during laparoscopic cholecystectomy occurs in 10% to 32% of patients, with gallstone spillage in 0.2% to 20% of cases . There have been very few reported thoracic complications from this common problem . Before laparoscopic cholecystectomy, cholelithoptysis and intrathoracic complications from gallstones were rare because the operative field was packed off to facilitate the open surgery . Introduction of a pneumoperitoneum and intraoperative irrigation facilitates distant migration of gallstones, specifically to the subphrenic space . A review of the literature reveals 11 reported cases of gallstones being found in the thoracic cavity following cholecystectomy . Eight cases presented as cholelithoptysis and the latest one as massive hemoptysis from a gallstone associated abscess . We believe these findings reiterate the need for fastidious stone removal at the time of initial cholecystectomy . Furthermore, while most patients with this problem present with cholelithoptysis, a high index of suspicion should be entertained by the practitioner when encountering a patient who has a right lung infiltrate and a history of open or laparoscopic cholecystectomy . A 73-year - old man with a past medical history of hyper - tension and chronic obstructive lung disease presented with a 4-month history of biliary colic and mild pancreatitis . After 3 days of conservative management with nothing by mouth and intravenous antibiotics, his pancreatitis resolved, and he underwent laparoscopic cholecystectomy . He did well postoperatively and was asymptomatic for 6 months until he developed low - grade fevers and streaks of hemoptysis . Computed tomography of the chest revealed a 57-cm pulmonary infiltrate in the posterior basilar segment of the right lower lobe associated with a 59-mm oval calcification at the level of the right diaphragm (figure 1). Shows eccentric calcification of a soft tissue mass in the lower lobe of the right lung . A repeat ct scan 2 months later demonstrated marked improvement of the pulmonary infiltrate, but the persistence of a calcified nodule on the surface of the diaphragm . Over the next 2 years, the patient developed episodic bouts of scant hemoptysis every 2 to 3 months that resolved spontaneously . Thirty - four months after his laparoscopic cholecystectomy, after an episode of self - limited hemoptysis, he underwent a surveillance ct scan that revealed a new 53-cm opacity in the posterobasal segment of his right lower lobe . Further evaluation with a mediastinoscopy showed no evidence of malignancy . At the time of exploration, the right lower lobe was densely adherent to the diaphragm . To ensure an oncologic resection, the final pathology was returned as chronic pulmonary abscess and foreign body giant cell reaction to a gallstone . Transection of the gross lung specimen shows a gallstone encapsulated by surrounding fibrous reactive tissue . Gallstone spillage is estimated to occur in 0.2% to 20% of cases with very few causing complications . A case review of 1130 consecutive laparoscopic cholecystectomies performed at 2 different institutions demonstrated a 0.3% complication rate from intraoperative stone spillage at 13-year follow - up . Complications from gallstone spillage vary from trocar - site wound infections to overt intraabdominal abscess as well as gynecological and intrathoracic complications . Thoracic complications of retained gallstones with pleurobiliary and bronchobiliary fistulas were reported as early as 1955 . The mean time to onset of complications is 6 months, but a case report exists of a gallstone - associated subphrenic abscess presenting 10 years after the cholecystectomy . Our case represents a complication of a spilled gallstone at the time of laparoscopic cholecystectomy that eroded through the right diaphragm and caused pneumonitis presenting as intermittent hemoptysis . Although our patient never had an overt intraabdominal abscess, clinically and by imaging, he had a prolonged right subdiaphragmatic inflammatory process that allowed the intrathoracic migration of the gallstone . If stone spillage occurs, a serious attempt to retrieve as many stones as possible with a larger forceps should be made followed by copious amounts of irrigation with a large bore suction catheter . This should be performed laparoscopically without conversion to an open procedure, as overall complication rate of stone spillage is 0.3% . Before the advent of laparoscopic cholecystectomy, cholelithoptysis and intrathoracic complications from gallstones coughing up stones, was classically due to calcified peribronchial lymph nodes that eroded through the bronchial wall . There are 11 case reports in the literature of gallstones in the thoracic cavity following cholecystectomy (table 1). Nine cases presented as cholelithoptysis, and the latest one as massive hemoptysis from a gallstone - associated abscess . Complications of intrathoracic gallstones three possible routes of intrathoracic migration of gallstones exist: the lymphatic channels of ranvier, congenital diaphragmatic defects, and transdiaphragmatic tracts that form from local infection / inflammation . As seen in our case, prolonged chest symptoms, especially with findings in the rll, following a laparoscopic cholecystectomy should raise the suspicion of intrathoracic gallstones . One must search for an intraabdominal collection and consider antibiotic treatment, percutaneous drainage, or laparotomy . Thoracotomy is rarely required and only indicated for obstructing pneumonia, refractive hemoptysis, or nodules of uncertain cause . If stone spillage occurs during a laparoscopic cholecystectomy, documentation in the chart would be valuable to clinicians following the patient, allowing them to have a high clinical suspicion when abdominal or thoracic symptoms occur.
Spinal fusion and pedicle screw fixation techniques are usually used in cases of vertebral fractures, dislocation, scoliosis, kyphosis, spinal tumor and for severe back pain that does not respond to other therapies.123 the pedicular screw fixation offers a stable and safe possibility for stabilization during correction of malalignment.123 the pedicle is surrounded by many sensitive structures such as nerve root, dura, cord which are not visible during pedicle screw insertion . Screw malposition pedicle wall perforation, nerve roots and cord impingement and very rarely, damage to vascular structures.456 therefore, the exact location of entry points and screw orientation is of great importance . In case of conventional transpedicular fixation especially the minimally invasive pedicle screws insertion, the surgeon is only provided with intraoperative two dimensional x - ray images for the alignment and positioning of the pedicle screws and in up to 40% of the cases a perforation of the pedicle occurrs, depending on both the surgeon's performance and the definition of error.456 there was also much more radiation exposure to the surgeons during minimally invasive pedicle screws placement.78 in order to ease the surgeon's hand - eye coordination and to reduce the iatrogenic radiation injury to the surgeons, a robot assisted surgery is expected to increase the quality of percutaneous pedicle screw placement . This study to feasibility and clinical value of robot - assisted navigated drilling in percutaneous pedicle screw placement . Additionally, the results would form the basis for the development of a new robot for pedicle screw fixation surgery . The spine robot system [figure 1] was domestically developed by and the department of science and department of orthopedics at our university . The robot includes three main parts: robot arm, base of the robot arm and console [figure 2]. The robot, composed of six revolute joints, is a serial manipulator with 6 of freedom . The motion patterns of the robot consist of manual traction mode, longitudinal shift mode, angular deflection mode and horizontal shift mode . Therefore, the 6 of freedom robot can provide the surgeon with the appropriate entry point and insertion angle for the drill . The end of the robot arm equipped with bone drill holder which can hold the pneumatic drill . The pneumatic drill can be conveniently sterilized by separation from the robot and used for drilling the pedicle screw trajectory during operation . The bone drill holder integrated six dimensional force / torque sensor, surgeons can feel the stress changes of the drill through handle the operating lever during the drilling process . (1) robot arm, (2) base of the robot arm, (3) controller of the drill, (4) console operation interface of the console . (1) power button (2) touch screen operator interface (3) operating lever longitudinal shift (4) operating lever angular deflection (5) operating lever horizontal shift preoperative computed tomography (ct) of eight bovine lumbar spines (l1l5) in axial plane was captured for each vertebra, the entry points and trajectories of the screws were preoperatively planned designed specifically for percutaneous pedicle screw placement . During preoperative planning this process needs to be done for each of the vertebrae involved in the procedure . L. distance between the posterior median line of the spinous process and the entry point; (a) angle between the posterior median line of the spinous process and the insertion line bovine is a tetrapod, its anatomical characteristics and common fracture site is different from the human and bovine spine segments are presumed to have higher bone mineral density than human spines and the pedicles of the bovine spine were much more thin than human spines, all pedicle screws will make cortical perforation, so that we did nt insert pedicle screw into the pedicle to avoid the misjudgement about cortical perforation . The purpose of the preliminary study is to gain first insights into the feasibility and clinical value of robot - assisted navigated drilling for pedicle screw placemen . In each screw insertion, full procedures from preoperative tasks to postoperative tasks were tested and evaluated to determine whether they are proper to apply to clinical fields . We checked preoperative planning, robot movement and surgical procedure in every pedicle screw insertion case . Engineers and orthopedic surgeons participated in these experiments and they agreed that this system had proper roles for percutaneous pedicle screw insertion procedures and that those results were applicable to clinical applications . We positioned the relative positions of the drill and the bovine lumbar spines, drilled the bovine lumbar spines according to preoperative plans and then placed k - wires in the holes [figure 4]. The bovine spine used in our study were devoid of skin - soft tissue and muscles, it saved a lot of time . We noted surgical time and intraoperative fluoroscopy times and then we assessed the position of the k - wires through postoperative ct [figure 5]. Eight bovine lumbar spines (l1l5) were inserted 80 k - wires using the spine robot system . The most important characteristic of the spine robot system is that the angle of the drill can be deflected to keep drill tip in centre . This function is helpful for us to deflect the angle of the drill according to preoperative plan after the tip of the drill touch the entry point of the bony surface and then insert the drill to the bone; the whole operation process is smooth . (a) adjustment the pneumatic drill parallel to the upper vertebral body end plate, (b) adjustment the distance between the tip of the drill and the posterior median line according to preoperative planned distance, (c) longitudinal shift of the drill to the entry point on the bone surface, (d) adjusting the entry angle of the drill according to preoperative planned angle preoperative and postoperative computed tomography (ct) scan of the bovine lumbar spine . (a) preoperative plan through the preoperative ct scan, (b) evaluating the position of k - wire through the postoperative ct scan the spine robot system [figure 1] was domestically developed by and the department of science and department of orthopedics at our university . The robot includes three main parts: robot arm, base of the robot arm and console [figure 2]. The robot, composed of six revolute joints, is a serial manipulator with 6 of freedom . The motion patterns of the robot consist of manual traction mode, longitudinal shift mode, angular deflection mode and horizontal shift mode . Therefore, the 6 of freedom robot can provide the surgeon with the appropriate entry point and insertion angle for the drill . The end of the robot arm equipped with bone drill holder which can hold the pneumatic drill . The pneumatic drill can be conveniently sterilized by separation from the robot and used for drilling the pedicle screw trajectory during operation . The bone drill holder integrated six dimensional force / torque sensor, surgeons can feel the stress changes of the drill through handle the operating lever during the drilling process . (1) robot arm, (2) base of the robot arm, (3) controller of the drill, (4) console operation interface of the console . (1) power button (2) touch screen operator interface (3) operating lever longitudinal shift (4) operating lever angular deflection (5) operating lever horizontal shift preoperative computed tomography (ct) of eight bovine lumbar spines (l1l5) in axial plane was captured for each vertebra, the entry points and trajectories of the screws were preoperatively planned designed specifically for percutaneous pedicle screw placement . During preoperative planning, we measured angle a and distance l [figure 3]. This process needs to be done for each of the vertebrae involved in the procedure . L. distance between the posterior median line of the spinous process and the entry point; (a) angle between the posterior median line of the spinous process and the insertion line bovine is a tetrapod, its anatomical characteristics and common fracture site is different from the human and bovine spine segments are presumed to have higher bone mineral density than human spines and the pedicles of the bovine spine were much more thin than human spines, all pedicle screws will make cortical perforation, so that we did nt insert pedicle screw into the pedicle to avoid the misjudgement about cortical perforation . The purpose of the preliminary study is to gain first insights into the feasibility and clinical value of robot - assisted navigated drilling for pedicle screw placemen . In each screw insertion, full procedures from preoperative tasks to postoperative tasks were tested and evaluated to determine whether they are proper to apply to clinical fields . We checked preoperative planning, robot movement and surgical procedure in every pedicle screw insertion case . Engineers and orthopedic surgeons participated in these experiments and they agreed that this system had proper roles for percutaneous pedicle screw insertion procedures and that those results were applicable to clinical applications . We positioned the relative positions of the drill and the bovine lumbar spines, drilled the bovine lumbar spines according to preoperative plans and then placed k - wires in the holes [figure 4]. The bovine spine used in our study were devoid of skin - soft tissue and muscles, it saved a lot of time . We noted surgical time and intraoperative fluoroscopy times and then we assessed the position of the k - wires through postoperative ct [figure 5]. Eight bovine lumbar spines (l1l5) were inserted 80 k - wires using the spine robot system . The most important characteristic of the spine robot system is that the angle of the drill can be deflected to keep drill tip in centre . This function is helpful for us to deflect the angle of the drill according to preoperative plan after the tip of the drill touch the entry point of the bony surface and then insert the drill to the bone; the whole operation process is smooth . (a) adjustment the pneumatic drill parallel to the upper vertebral body end plate, (b) adjustment the distance between the tip of the drill and the posterior median line according to preoperative planned distance, (c) longitudinal shift of the drill to the entry point on the bone surface, (d) adjusting the entry angle of the drill according to preoperative planned angle preoperative and postoperative computed tomography (ct) scan of the bovine lumbar spine . (a) preoperative plan through the preoperative ct scan, (b) evaluating the position of k - wire through the postoperative ct scan preoperative ct of eight bovine lumbar spines (l1l5) in axial plane was taken for each vertebra, the entry points and trajectories of the screws were preoperatively planned designed specifically for percutaneous pedicle screw placement [table 1]. Assisted by spine robot system, the average time for system registration was (343.4 18.4) s, the time for procedure of drilling one k - wire was (89.5 6.1) s, times of fluoroscopy for procedure of drilling one k - wire were (2.9 0.8) s. overall, 12 (15.0%) of the 80 k - wires violated the pedicle wall . Four screws (5.0%) were medial to the pedicle, and 8 (10.5%) were lateral . The rate of the k - wire wholly within the pedicle was 85% [table 2]. Preoperative measurement index of the experimental group according to different vertebrae surgical results of the spine robot system for predrilled pedicle screw trajectory percutaneous pedicle screw placements with conventional and image guidance techniques have demonstrated acceptable results,9101112 but there were so much radiation exposure to the surgeons during minimally invasive pedicle screws placement.78 with the development of computer assisted surgery, spine robot system had been developed for pedicle screws insertion and even some spine robot system has already been used in clinic.131415161718 a biplane fluoroscopy guided robot system (bfrs) was developed by kim et al.15 for surgical robotic systems, minimally invasive surgeries and cooperative robotic systems, as well as enhanced surgical planning and navigation with preoperative and intraoperative image data . They pointed out that the bfrs might be helpful in improving the accuracy of percutaneous pedicular screw insertion procedures . In the future, they will attempt to improve the accuracy and reliability of the bfrs and to determine new clinical applications for the bfrs . The spine robot system in our study has motion patterns of the robot consist of manual traction mode, longitudinal shift mode, angular deflection mode and horizontal shift mode . Therefore, the 6of freedom robot can provide the surgeon with the appropriate entry point and insertion angle for the drill . The pneumatic drill can be conveniently sterilized by separation from the robot to ensure that sterility is maintained throughout the entire operation procedure . The bone drill holder integrated six - dimensional force / torque sensor, surgeons can feel the stress changes of the drill through handle the operating lever during the drilling process to ensure more safety during the whole drilling process . The spine robot system, which has already been used in the clinic, is spineassist.161718 kantelhardt et al.17 reported a retrospective cohort analysis comparing conventional open to open robotic - guided and percutaneous robotic - guided pedicle screw placement . Use of robotic guidance significantly increased the accuracy of screw position while reducing the x - ray exposure . Lieberman et al.18 pointed out that the robotic guidance group had fewer screw placement deviations, less surgeon radiation exposure, lower fluoroscopy time per screw and shorter procedure time compared to the no robotic guidance group . To our knowledge, the spineassist robot system only provided the optimized trajectory, the pedicle screws insertion was performed only by the surgeon but not the spineassist system itself . In the current study, the surgeons can perform the pedicle screws insertion technique behind the radiation protection screen using the tele - manipulation function of the spine robot system so that the radiation exposure to the surgeons can be decreased and the spine robot system can insert the pedicle screws itself . Bovine lumbar spine were used for robot - assisted navigated drilling because the bovine lumbar spines can be more easily available than human cadaver specimens . Due to traditional concept in china, a very few people accept body donation, so human cadaver specimens were hard to get . The purpose of the preliminary study is to gain first insights into the feasibility and clinical value of robot - assisted navigated drilling for pedicle screw placement, so we think that the bovine spine were acceptable for the study.19 the function of the spine robot system is to pre - drill pedicle screw trajectory, the system ca nt offer help for rod placement, when we have inserted the pedicle screws, we can insert the rod using some special instrument such as instrument in sextant system to place the rod through minimally invasive technique . The rate of the k - wire wholly within the pedicle in the current study was 85% . The reasons can be divided into the following two points: firstly, the pedicle of the bovine lumbar spine was too thin, little deviation of the insertion angle can cause the k - wires violated the pedicle wall . Secondly, we ca nt accurately determine the relative position of the drill and the bovine lumbar spine . So, the accuracy and reliability of spine robot system should be improved . In order to improve the accuracy and reliability of the spine robot system for clinical use, further research such as building the virtual surgery system and intraoperative electrophysiological monitoring system will be performed . In recent years, many researchers developed simulators for pedicle screw insertion; the simulators offer many helpful features to the surgeon with respect to complex cases and to the surgical trainee learning the basic technique of pedicle screw insertion.202122 this technology has also begun to be used in preoperative planning for selected cases, the surgeons can make the surgical plan, practice, and visualize pedicle screw surgery on a particular patient before operation through the simulator.2324 however, when the screws are being inserted, there is no projection fluoroscopy image provided to the surgeon . Next, we will develop a ct based patient specific pedicle screw insertion simulator to better prepare surgeons to perform pedicle screw insertion using free - hand technique under the projection fluoroscopy and help reduce the risk of pedicle screw misplacement.25 the second main research direction is to build intraoperative electrophysiological monitoring system . Based on strong evidence that multimodality intraoperative neuromonitoring (miom) is sensitive and specific for detecting intraoperative neurologic injury during spine surgery, it is recommended that the use of miom be considered in spine surgery where the spinal cord or nerve roots are deemed to be at risk, including procedures involving deformity correction and procedures that require the placement of instrumentation.2627 the basic function of the spine robot system can satisfy spine surgeons for percutaneous pedicle screw placement . Using the spine robot system, the operation time and intraoperative fluoroscopy times per pedicle screw was less, but we should improve the accuracy and reliability of spine robot system such as building the preoperative planning simulator and intraoperative electromyography monitoring system for clinical use . We think the spine robot system will be used in clinical practice with the development of preoperative planning simulator and intraoperative electromyography monitoring system in the near future.
Damage to the thoracic duct during spinal operations is a rare but potentially serious complication that manifests as a cervical chylous fistula, chylothorax, or chyloretroperitoneum . Serious insult to the structure can result in significant nutritional deficiency, respiratory dysfunction, and considerable immunosuppression . Conservative therapy initially consists of diet modification, electrolyte monitoring, and appropriate drain placement . If needed, definitive treatment requires ligation of the thoracic duct, a procedure that can result in significant morbidity . Current data regarding the occurrence of thoracic duct injury during spine surgery is primarily found as isolated case reports . This study is the first to report the overall incidence of thoracic duct injury during cervical spine surgery based on a multicenter retrospective review . We have conducted a retrospective multicenter case series study involving 21 high - volume surgical centers from the aospine north america clinical research network, selected for their excellence in spine care and clinical research infrastructure and experience . Medical records for 17 625 patients who received cervical spine surgery (levels from c2 to c7) between january 1, 2005, and december 31, 2011, inclusive, were reviewed to identify occurrence of 21 predefined treatment complications . The complications included reintubation for the purpose of a hematoma evacuation, esophageal perforation, epidural hematoma, c5 palsy, recurrent laryngeal nerve palsy, superior laryngeal nerve palsy, hypoglossal or glossopharyngeal nerve palsy, dural tear, brachial plexopathy, blindness, graft extrusion, misplaced screws requiring reoperation, anterior cervical infection, carotid artery injury or cerebrovascular accident, vertebral artery injuries, horner s syndrome, thoracic duct injury, quadriplegia, intraoperative death, revision of arthroplasty, and pseudomeningocele . Trained research staff at each site abstracted the data from medical records, surgical charts, radiology imaging, narratives, and other source documents for the patients who experienced one or more of the complications from the list . Copies of case report forms were transferred to the aospine north america clinical research network methodological core for processing, cleaning, and data entry . Of the 17 625 total patients reviewed, 9591 individuals underwent surgery of the cervical spine using an anterior approach only . The highest incidence for any single institution was 0.079%, while 19 of 21 institutions reported zero instances of thoracic duct injury . The first occurrence of thoracic duct injury was in a previously healthy male that was involved in a motor vehicle accident . As a consequence, the patient developed radicular pain for which physical therapy did not provide adequate symptom relief . Therefore, the patient opted to undergo anterior cervical discectomy and fusion (acdf) at the level of c5-c6 . Intraoperatively, a chyle leak was noted and the otolaryngology team was consulted to address the disruption of the thoracic duct . No residual effects were noted from the intraoperative leak during the follow - up period . This patient underwent acdf to address a herniated nucleus pulposus with associated radiculopathy at the level of c5-c6 . The patient was female, had a body mass index of 21.9, and had no comorbid conditions at the time of the index surgery . Approximately 2 months after surgery, the patient presented for outpatient follow - up during which a 2.5 2.5 cm nontender, mobile mass was noted superior to the incision . There were no accompanying symptoms of fever or dysphagia; needle aspiration of the lesion removed 3.5 ml of milky white fluid identified as chyle . The patient recovered uneventfully and no additional problems were noted during further follow - up . In all, the patient had 8 postoperative visits with the most recent occurring 3.5 years following the index operation . The first occurrence of thoracic duct injury was in a previously healthy male that was involved in a motor vehicle accident . As a consequence, the patient developed radicular pain for which physical therapy did not provide adequate symptom relief . Therefore, the patient opted to undergo anterior cervical discectomy and fusion (acdf) at the level of c5-c6 . Intraoperatively, a chyle leak was noted and the otolaryngology team was consulted to address the disruption of the thoracic duct . No residual effects were noted from the intraoperative leak during the follow - up period . This patient underwent acdf to address a herniated nucleus pulposus with associated radiculopathy at the level of c5-c6 . The patient was female, had a body mass index of 21.9, and had no comorbid conditions at the time of the index surgery . Approximately 2 months after surgery, the patient presented for outpatient follow - up during which a 2.5 2.5 cm nontender, mobile mass was noted superior to the incision . There were no accompanying symptoms of fever or dysphagia; no signs of infection were noted around the incision . Needle aspiration of the lesion removed 3.5 ml of milky white fluid identified as chyle . The patient recovered uneventfully and no additional problems were noted during further follow - up . In all, the patient had 8 postoperative visits with the most recent occurring 3.5 years following the index operation . The thoracic duct serves as the primary conduit for the return of lymph to the bloodstream from all lymphatic vessels except those found on the right side of the head, neck, thorax, and arm (figure 1). As such, it delivers three quarters of the lymph produced in the body to the venous circulation . Its origin is in the retroperitoneum at the cisterna chyli, located on the anterior surface of the first or second lumbar vertebra . After reaching the fifth thoracic vertebra, its course veers left and continues its ascent posterior to the aortic arch and the thoracic portion of the left subclavian artery . Traveling between the left side of the esophagus and pleura, it reaches the root of the neck and forms an arch rising approximately 3 to 4 cm above the clavicle . At the superior border of the clavicle, the thoracic duct is bordered by the left carotid sheath anteriorly, the omohyoid muscle laterally, the anterior scalene fascia posteriorly, and the esophagus medially . It then crosses anterior to the subclavian artery, vertebral artery and vein, and the thyrocervical trunk and terminates by opening into the junction of the left subclavian and left internal jugular veins . Drawing depicting the origin of the thoracic duct, its usual course of ascension through the thorax, and its termination in the root of the neck . Important variations in the course of the thoracic duct have been reported as a result of various cadaver studies . Gottlieb and greenfield found one cadaver out of 75 with a thoracic duct that remained on the right side during its ascent and emptied into the right internal jugular vein . The height of the arch of the thoracic duct in the root of the neck also varies, as it can be inferior to, at, or superior to the clavicle . Hart et al described a case in which the arch was situated 7 to 8 cm above the clavicle . Importantly, there is significant deviation in the termination pattern of the thoracic duct as it joins the systemic circulation . It may end as single or multiple outlets into the left internal jugular vein, the left subclavian vein, the left external jugular vein, the left brachiocephalic vein, the left transverse cervical vein, or the right internal jugular vein . Greenfield and gottlieb s study of 75 cadavers found 89.4% of thoracic ducts to have 1 termination, 6.6% to have 2 terminations, and 4% to have 3 terminations . The majority of reported cases of thoracic duct injury during spine operations have occurred during surgery of the thoracolumbar spine . Colletta and mayer describe a case report in which an intraoperative chyle leak is noted . Despite attempted intraoperative repair, the patient developed a postoperative chylothorax, which was subsequently managed with a chest tube and low - fat diet until resolution was achieved . Nakai and zielke reported 6 instances of chylothorax in an estimated 2000 cases of thoracolumbar spinal surgery; all were treated conservatively with a combination of diet modification, needle aspiration, and chest tube placement . Propst - proctor et al surveyed a total of 10 orthopedic spine surgeons who had combined to perform an estimated 1000 anterior thoracolumbar operations . They reported a total of 3 cases of chylothorax that were managed conservatively with closed chest tube drainage and a restricted fat diet . Similarly, bhat and lowery reported 3 operations complicated by chyloretroperitoneum and/or chylothorax that resolved with a combination of diet modification, chest tube drainage, and/or continued maintenance of a retroperitoneal drain . Su and chen also described a patient that experienced retroperitoneal chyle leakage after undergoing lumbar fusion; this patient had spontaneous cessation of drainage upon temporary clamping of his drain tube . Hussain et al described a series of 4 patients that developed chyloretroperitoneum following anterior lumbar surgery . Three patients symptoms resolved with fluoroscopic - guided percutaneous aspiration as well as drainage catheterization . The fourth patient had continued drainage of her abdominal lymphocele despite placement of a catheter and required reoperation 6 weeks after the initial surgery to create a peritoneal window to allow drainage of the lymphocele into the peritoneal space . Hart et al described a case of a patient undergoing a left - sided acdf in which an intraoperative chyle leak was identified secondary to disruption of the thoracic duct in the supraclavicular region . In this case, the arch of the thoracic duct extended 7 to 8 cm above the clavicle, making it significantly more superior than expected . Intraoperative suture ligation of the duct was conducted and the leakage was confirmed to have stopped; there were no postoperative complications . Warren et al reported a case of cervical lymphocele formation following right - sided acdf during which the right lymphatic duct was penetrated . Surgical closure of the lymphocele attempted 2 weeks postoperatively was unsuccessful; subsequent percutaneous drainage and sclerotherapy revealed a large branch of the thoracic duct communicating with the lymphocele . This branch was embolized via a percutaneous transcervical approach, resulting in immediate resolution of the patient s symptoms . The rate of thoracic duct injury during anterior cervical spine surgery was 0% at the majority of institutions reviewed (19 of 21). Estimates by nakai and zielke as well as propst - proctor et al suggest that the rate of thoracic duct injury during thoracolumbar surgery is approximately 0.3%: nakai and zielke reported 6 cases of injury out of an estimated 2000 cases, and propst - proctor et al found 3 instances out of an estimated 1000 patients . The present study is the first to report an incidence of thoracic duct injury following cervical spine surgery, revealing a substantially lower incidence of this complication in cervical surgery versus thoracolumbar . This discrepancy is possibly due to the longer anatomic course of the thoracic duct in the thoracolumbar area as compared to the cervical spine . The thoracic duct is subject to injury during any operation that involves the root of the neck . Such procedures are commonly conducted by thoracic surgeons and otolaryngologists in addition to spine surgeons . For instance, marthaller et al describe a series of patients that incurred damage to the thoracic duct following esophagectomy; this manifested through the formation of a chylothorax and/or a high - output chylous fistula . For such cases, the authors advocated for percutaneous embolization of the thoracic duct prior to open re - exploration . Similarly, patel et al describe a case of thoracic duct injury following neck dissection that was treated by percutaneous embolization . Thoracic duct damage leading to chylous leakage occurs in 1% to 2.5% of radical neck dissections . In a retrospective review of 221 patients that underwent neck dissection, de gier dietary modifications alone were sufficient to stop the leak in 5 of these patients . The other 6 cases necessitated initiation of total parenteral nutrition . Two of these individuals required surgical re - intervention via a pectoralis major muscle flap transfer . If left untreated, injury to the thoracic duct can lead to chronic chyle loss and eventually result in significant weakness, dehydration, and emaciation . Unfortunately, intraoperative diagnosis of lymphatic vessel injury can be difficult due to the usual fasting state of patients prior to surgery, which reduces lymphatic production and transport . Positive pressure application with the patient in trendelenburg position may facilitate identification of a suspected leak . If a leak is observed intraoperatively, suture ligation of the corresponding lymphatic vessel should be carried out . Postoperative presentation of a thoracic duct injury is dependent on the location of the injured vessel, although the presence of a variably cloudy and white fluid (ie, chyle) from an operative drain or upon aspiration is often seen . Conservative management of a chylothorax in these circumstances consists of a low - fat diet or total parenteral nutrition, supportive care (eg, correction of electrolytes, rehydration), and, in severe cases, adequate drainage via insertion of chest tube . Treatment with somatostatin or octreotide has also been shown to be effective in treating thoracic duct injury, as it decreases drainage output and results in earlier fistula closure . Definitive management in refractory cases involves ligation of the thoracic duct; however, conservative therapy for at least 1 to 2 weeks is recommended prior to taking this course . Limitations to consider when interpreting the results of this study include the consideration of biases (eg, selection bias, information bias) inherent to retrospective reviews . The multicenter, retrospective nature of the study prevented a standard methodology for identifying, documenting, and monitoring iatrogenic injury postoperatively between various institutions . This variation in follow - up protocols may have resulted in an underreporting of cases of thoracic duct injury occurring during the postoperative period . Additionally, there were likely other cases of thoracic duct injury that would have increased our understanding of the natural history and treatment of this complication that we did not encounter as they occurred on other services (eg, otolaryngology, thoracic surgery). The thoracic duct serves as the primary conduit for the return of lymph to the bloodstream from all lymphatic vessels except those found on the right side of the head, neck, thorax, and arm (figure 1). As such, it delivers three quarters of the lymph produced in the body to the venous circulation . Its origin is in the retroperitoneum at the cisterna chyli, located on the anterior surface of the first or second lumbar vertebra . After reaching the fifth thoracic vertebra, its course veers left and continues its ascent posterior to the aortic arch and the thoracic portion of the left subclavian artery . Traveling between the left side of the esophagus and pleura, it reaches the root of the neck and forms an arch rising approximately 3 to 4 cm above the clavicle . At the superior border of the clavicle, the thoracic duct is bordered by the left carotid sheath anteriorly, the omohyoid muscle laterally, the anterior scalene fascia posteriorly, and the esophagus medially . It then crosses anterior to the subclavian artery, vertebral artery and vein, and the thyrocervical trunk and terminates by opening into the junction of the left subclavian and left internal jugular veins . Drawing depicting the origin of the thoracic duct, its usual course of ascension through the thorax, and its termination in the root of the neck . Important variations in the course of the thoracic duct have been reported as a result of various cadaver studies . Gottlieb and greenfield found one cadaver out of 75 with a thoracic duct that remained on the right side during its ascent and emptied into the right internal jugular vein . The height of the arch of the thoracic duct in the root of the neck also varies, as it can be inferior to, at, or superior to the clavicle . Hart et al described a case in which the arch was situated 7 to 8 cm above the clavicle . Importantly, there is significant deviation in the termination pattern of the thoracic duct as it joins the systemic circulation . It may end as single or multiple outlets into the left internal jugular vein, the left subclavian vein, the left external jugular vein, the left brachiocephalic vein, the left transverse cervical vein, or the right internal jugular vein . Greenfield and gottlieb s study of 75 cadavers found 89.4% of thoracic ducts to have 1 termination, 6.6% to have 2 terminations, and 4% to have 3 terminations . The majority of reported cases of thoracic duct injury during spine operations have occurred during surgery of the thoracolumbar spine . Colletta and mayer describe a case report in which an intraoperative chyle leak is noted . Despite attempted intraoperative repair, the patient developed a postoperative chylothorax, which was subsequently managed with a chest tube and low - fat diet until resolution was achieved . Nakai and zielke reported 6 instances of chylothorax in an estimated 2000 cases of thoracolumbar spinal surgery; all were treated conservatively with a combination of diet modification, needle aspiration, and chest tube placement . Propst - proctor et al surveyed a total of 10 orthopedic spine surgeons who had combined to perform an estimated 1000 anterior thoracolumbar operations . They reported a total of 3 cases of chylothorax that were managed conservatively with closed chest tube drainage and a restricted fat diet . Similarly, bhat and lowery reported 3 operations complicated by chyloretroperitoneum and/or chylothorax that resolved with a combination of diet modification, chest tube drainage, and/or continued maintenance of a retroperitoneal drain . Su and chen also described a patient that experienced retroperitoneal chyle leakage after undergoing lumbar fusion; this patient had spontaneous cessation of drainage upon temporary clamping of his drain tube . Hussain et al described a series of 4 patients that developed chyloretroperitoneum following anterior lumbar surgery . Three patients symptoms resolved with fluoroscopic - guided percutaneous aspiration as well as drainage catheterization . The fourth patient had continued drainage of her abdominal lymphocele despite placement of a catheter and required reoperation 6 weeks after the initial surgery to create a peritoneal window to allow drainage of the lymphocele into the peritoneal space . Hart et al described a case of a patient undergoing a left - sided acdf in which an intraoperative chyle leak was identified secondary to disruption of the thoracic duct in the supraclavicular region . In this case, the arch of the thoracic duct extended 7 to 8 cm above the clavicle, making it significantly more superior than expected . Intraoperative suture ligation of the duct was conducted and the leakage was confirmed to have stopped; there were no postoperative complications . Warren et al reported a case of cervical lymphocele formation following right - sided acdf during which the right lymphatic duct was penetrated . Surgical closure of the lymphocele attempted 2 weeks postoperatively was unsuccessful; subsequent percutaneous drainage and sclerotherapy revealed a large branch of the thoracic duct communicating with the lymphocele . This branch was embolized via a percutaneous transcervical approach, resulting in immediate resolution of the patient s symptoms . The rate of thoracic duct injury during anterior cervical spine surgery was 0% at the majority of institutions reviewed (19 of 21). Estimates by nakai and zielke as well as propst - proctor et al suggest that the rate of thoracic duct injury during thoracolumbar surgery is approximately 0.3%: nakai and zielke reported 6 cases of injury out of an estimated 2000 cases, and propst - proctor et al found 3 instances out of an estimated 1000 patients . The present study is the first to report an incidence of thoracic duct injury following cervical spine surgery, revealing a substantially lower incidence of this complication in cervical surgery versus thoracolumbar . This discrepancy is possibly due to the longer anatomic course of the thoracic duct in the thoracolumbar area as compared to the cervical spine . The thoracic duct is subject to injury during any operation that involves the root of the neck . Such procedures are commonly conducted by thoracic surgeons and otolaryngologists in addition to spine surgeons . For instance, marthaller et al describe a series of patients that incurred damage to the thoracic duct following esophagectomy; this manifested through the formation of a chylothorax and/or a high - output chylous fistula . For such cases, the authors advocated for percutaneous embolization of the thoracic duct prior to open re - exploration . Similarly, patel et al describe a case of thoracic duct injury following neck dissection that was treated by percutaneous embolization . Thoracic duct damage leading to chylous leakage occurs in 1% to 2.5% of radical neck dissections . In a retrospective review of 221 patients that underwent neck dissection, de gier et al identified 11 individuals that suffered a chylous fistula . Two of these individuals required surgical re - intervention via a pectoralis major muscle flap transfer . If left untreated, injury to the thoracic duct can lead to chronic chyle loss and eventually result in significant weakness, dehydration, and emaciation . Unfortunately, intraoperative diagnosis of lymphatic vessel injury can be difficult due to the usual fasting state of patients prior to surgery, which reduces lymphatic production and transport . Positive pressure application with the patient in trendelenburg position may facilitate identification of a suspected leak . If a leak is observed intraoperatively, suture ligation of the corresponding lymphatic vessel should be carried out . Postoperative presentation of a thoracic duct injury is dependent on the location of the injured vessel, although the presence of a variably cloudy and white fluid (ie, chyle) from an operative drain or upon aspiration is often seen . Conservative management of a chylothorax in these circumstances consists of a low - fat diet or total parenteral nutrition, supportive care (eg, correction of electrolytes, rehydration), and, in severe cases, adequate drainage via insertion of chest tube . Treatment with somatostatin or octreotide has also been shown to be effective in treating thoracic duct injury, as it decreases drainage output and results in earlier fistula closure . Definitive management in refractory cases involves ligation of the thoracic duct; however, conservative therapy for at least 1 to 2 weeks is recommended prior to taking this course . Limitations to consider when interpreting the results of this study include the consideration of biases (eg, selection bias, information bias) inherent to retrospective reviews . The multicenter, retrospective nature of the study prevented a standard methodology for identifying, documenting, and monitoring iatrogenic injury postoperatively between various institutions . This variation in follow - up protocols may have resulted in an underreporting of cases of thoracic duct injury occurring during the postoperative period . Additionally, there were likely other cases of thoracic duct injury that would have increased our understanding of the natural history and treatment of this complication that we did not encounter as they occurred on other services (eg, otolaryngology, thoracic surgery). Nevertheless, surgeons operating in this area need to remain cognizant of the presence of this critical lymphatic structure and should be cautious of anatomic variants that may increase the risk of injury . The manifestations resulting from thoracic duct disruption are dependent on the degree of injury as well as its location . In many cases, prompt identification of damage to the duct will allow for conservative management and resolution of symptoms without significant long - term effects.
Neuromodulation has been established as an effective treatment for patients with overactive bladder (oab) where first - line therapies, such as the muscarinic antagonists, do not provide sufficient efficacy.1 currently, the two most common approaches to neuromodulation are sacral spinal nerve (sn) stimulation using the interstim device and percutaneous tibial nerve (tn) stimulation using the urgent pc. Sacral neuromodulation is included in the oab treatment guidelines of american urological association (aua), european association of urology, and international continence society.2,3 percutaneous tn stimulation is listed as optional therapy in the aua treatment guidelines.2 using a rat model in which reflex bladder rhythmic contraction (brc) is induced by filling with saline, we have characterized the relationship between inhibition of reflex bladder contraction and the frequency and intensity of sn stimulation.4 we have also compared the effects of stimulation of different bladder nerves, including spinal, tibial, and genital.5 dorsal genital nerve stimulation has not been approved clinically for oab treatment and is not included in this study . We now report on a further characterization of the response of the isovolumetric rat bladder to tn and sn stimulation, including a comparison of bilateral and unilateral stimulation, evaluation of desensitization to continuous and repeated stimulation, and the effects of simultaneous stimulation of spinal and tns . Results in the rat brc model appear to correlate with data from other animal models and with clinical observations . The characterization of the bladder effects of tn stimulation, and comparison of these effects to those of sn stimulation, as well as the interaction of stimulation at these two sites, could identify mechanistic differences and provide information useful in the use of nerve stimulation for the treatment of oab . Female sprague dawley rats (200300 g) were anesthetized with urethane (i.p ., 1.2 g / kg, 200 mg / ml in saline, sigma anesthetized rats were maintained at 37c with a heating pad and were euthanized by co2 asphyxia upon completion of experiments . The experimental protocols were approved by the institutional animal care and use committee of medtronic and non - clinical research board of medtronic (minneapolis, mn). To deliver unipolar tn stimulation, a bared portion of a teflon - coated, 40-guage, stainless steel wire (cooner wire co., chatsworth, ca) was placed bilaterally under each tn, which was exposed on the medial side of both hindlimbs above the ankle (72 rats, fig . Electrodes were also placed under both left tn and left l6 sn ipsilaterally (21 rats), and right tn and left sn contralaterally (26 rats). The wire electrode(s) were positioned, secured with silicone adhesive, and connected to a grass s88 stimulator (grass medical instruments, warwick, ri), through stimulus isolation unit(s) (siu - bi, grass medical instruments). Experimental model for tibial (tn) and spinal nerve (sn) stimulation (s, stim) unilateral stimulation was delivered on the left (l) side of the tn or sn and bilateral stimulation was applied simultaneously on both left and right (r) side of the nerve . Stimulation (10 hz, pulse width 0.1 msec) intensities varied from 0.8 times motor threshold intensity (tmot) up to six times tmot . D, e: histogram of motor threshold (tmot) distribution to tn and sn stimulation . In each rat, the threshold current (tmot) for biphasic pulses (pulse width = 0.1 msec, 10 hz for 26 sec) stimulation was defined as the lowest current required to evoke the first, barely observable, muscle contraction (hind - toe twitches and/or pelvic floor muscle contraction, 4, 5). For bilateral stimulation, the tmot was measured on each side separately, to allow differentiation of muscle responses to left or right nerve stimulation . A cannula (pe50) was inserted into the bladder via the urethra, and secured with a suture tie for intravesical pressure recording and saline infusion . The urethral cannula was connected via a t - type connector to a pressure transducer of the data acquisition system (ad instruments mlt0380d, colorado springs, co) and the signal of intravesical pressure was put through a dc amplifier (ad instruments, ml119). The other end of the t connector was attached to a syringe pump . To induce brc, saline was infused into the bladder via the syringe pump at a rate of 50 l / min to induce a micturition reflex (here defined as bladder contraction of a magnitude> 10 mmhg). The infusion rate was then lowered to 10 l / min and continued until three to five consecutive contractions were established . At this time each trial of recording lasted for up to 50 min including 15 min control, 515 min nerve stimulation, and 20 min post - stimulation . Two trials of the testing were performed with a random stimulation parameter in some rats . The bladder was emptied after finishing the first trial and brc was re - established by saline infusion . Electrical stimulation at a fixed frequency of 10 hz, which has been shown to be optimal for inhibition of bladder contractions by both low and high intensity stimulation,4,5 was tested unilaterally or bilaterally for 15 min (fig . 1a - a, b and b - a, b). The repeated 5 min stimulations were also assessed from variable length of 520 min between stimulations to allow recovery of the bladder inhibitory responses to tn stimulation from desensitization (fig . Finally, the combination of unilateral stimulation of sn and tn was examined using a 5-min stimulation protocol . The stimulation period of 5 min was chosen based on the results of a sustained 15-min tn stimulation which showed that bladder contractions were shut down only during the first 5 min of stimulation (see results section). With unilateral sn stimulation on the left side (0.82 times tmot), the stimulation of either the ipsilateral and contralateral tn (23 times tmot) was tested for a total of 10 possible combinations (fig . The stimulation intensity ranges were chosen based on the intensity dependent bladder inhibitory response curve to stimulations on either sn or tn alone . Either lower current intensities below which no response occurs or can be measured, or higher intensities above those producing a maximum response, were not tested . Sn or tn stimulation did not reduce the amplitude of bladder contractions,4,5 therefore only effects on frequency / interval of brc were studied . Data were calculated in 5 min bins, each having three control periods, two periods during stimulation, and four periods after stimulation . All data were normalized to the mean response during the 5 min immediately prior to stimulation . Time course for the brc response to stimulation was analyzed using repeated measures anova (prism 5, graphpadsoftware, inc . Bonferroni post - test was used to determine the statistical significance between individual time points . Sn or tn stimulation did not reduce the amplitude of bladder contractions,4,5 therefore only effects on frequency / interval of brc were studied . Data were calculated in 5 min bins, each having three control periods, two periods during stimulation, and four periods after stimulation . All data were normalized to the mean response during the 5 min immediately prior to stimulation . Time course for the brc response to stimulation was analyzed using repeated measures anova (prism 5, graphpadsoftware, inc . Bonferroni post - test was used to determine the statistical significance between individual time points . The threshold current (tmot) at which first visible motor contraction occurred to tn stimulation was 0.17 0.01 ma (n = 253; range: 0.010.6 ma; 95% confidence interval [ci]: 0.150.19 ma, fig . Tmot for sn stimulation was 0.17 0.01 ma (n = 121; range: 0.010.7 ma; 95% ci: 0.140.20 ma, fig . The tmot is distributed approximately normally, irrespective of tn and sn or left side and right side of the nerve roots . Figure 2 shows typical results of bilateral, electrical stimulation of the tn or sn on brc (3 times tmot, 10 hz, 15 min). There was no functional delay between application of the stimulus and effect on the bladder, that is, the next contraction expected to occur was abolished when the current was applied . However, the contractions re - appeared before tn stimulation was terminated (desensitization). In contrast, inhibition of brc using the same parameters (3 times tmot at 10 hz) of sn stimulation was sustained for 2 min post - stimulation . Typical experimental records showing the bladder rhythmic contraction (mmhg) to sustained 15-min bilateral tibial nerve stimulation (a) and spinal nerve (b) at three times motor threshold intensity (10 hz, pulse width 0.1 msec). If stimulation on the tn was applied continuously for 15 min, the frequency of spontaneous bladder contractions returned to pre - stimulation values before the end of the stimulation period (fig . Significant inhibition to tn stimulation was produced by the first 5 min stimulation at intensities equal or greater than three times tmot (vs. without stimulation, n = 29, p <0.05, unpaired student's t - test), and by the second or third 5 min stimulation at intensities five and six times tmot (vs. same time control without stimulation, n = 29, p <0.05, unpaired student's t - test, fig . 3b). The first 5-min bilateral tn stimulation at 10 hz, three times motor threshold decreased the frequency of contractions to 69.49 8% of control (n = 6). Effects of tibial nerve (a, b) and spinal nerve stimulation (c, d: 10 hz, pulse width 0.1 msec) on the frequency of the bladder rhythmic contraction . A, c: time course response of frequency of the bladder rhythmic contraction to bilateral tibial nerve (a) and spinal nerve (c) stimulation at motor threshold (tmot) and threefold of tmot (3 times tmot). Shaded areas are responses during electrical stimulation . +, p <0.05, versus control without stimulation; repeated measures anova, bonferroni post - test . B, d: intensity dependent effects of unilateral (uni) and bilateral (bi) tibial nerve (b) and spinal nerve (d) stimulations on frequency of bladder contractions during electrical stimulation . X - axis denoted increasing current intensity relative to multiples of motor threshold (tmot) stimulation . The mean contraction frequency during stimulation is expressed as a percentage of the control response prior to stimulation (% control). , p <0.05, versus values without stimulation, unpaired student's t - test; #, p <0.05, first 5-min stimulation versus second and third 5-min stimulation, , p <0.05, unilateral versus bilateral, unpaired student's t - test . The number of animals is indicated in each symbol . The decrease in bladder contraction frequency produced by the first 5-min stimulation at three and four times tmot was significantly greater than that produced by the second or third 5 min stimulation (p <0.05, paired student's t - test). These data again show decreased response to sustained tn stimulation . The inhibitory response was equal, regardless of whether unilateral or bilateral stimulation was applied (fig . 3b). Figure 3c and d shows the effect of sn stimulation at 10 hz on brc at different stimulation intensities . The inhibition of the contraction frequency to sn stimulation was greater than that of tn stimulation . The lowest intensity of sn stimulation that produced a statistically significant bladder inhibition (p <0.05, vs. without stimulation, n = 29, unpaired student's t - test) was 0.8 times tmot for bilateral stimulation and two times tmot for unilateral stimulation, to 67.46 15% (n = 9), and 32.52 16% (n = 6) of controls, respectively . The first 5-min bilateral sn stimulation at three times motor threshold decreased the frequency of contractions to 9.42 9% of control (n = 7). The inhibitory effect to bilateral stimulation at tmot is significantly greater than that produced by unilateral sn stimulation (p <0.05, unpaired student's t - test, fig . A comparison of the bladder inhibitory response to tn and sn stimulation shows that with either of continuous (15 min) or repeated 5 min stimulation, there is a clear difference between the responses to stimulation at the two sites . With only a 5-min interval between two 5 min stimulations, the second sn stimulation (tmot intensity, bilateral) produced a response (38.33 22% control) equal to the first (46.56 27% control, n = 4, p> 0.05, paired student's t - test). In contrast, the second tibial stimulation (three times tmot intensity) produced no inhibition (105.98 12% control, n = 16, combined data from 9 bilateral with 7 unilateral stimulations) while the first stimulation reduced bladder contraction to 65.44 11% control (p <0.05, paired student's t - test, fig . Effects of bladder inhibitory response to repeated 5-min stimulations (10 hz, pulse width 0.1 msec). A, b: typical experimental records showing the bladder rhythmic contraction (mmhg) to two 5 min tibial nerve (tn) stimulations at three times motor threshold intensity (10 hz, pulse width 0.1 msec). The contraction traces in (a) illustrate that with an interval of 10 min or less between tn stimulations, the response to a second stimulation was completely blunted; with 15 min between stimulations, the first and second stimulations were equieffective (b). C: inhibition of the bladder rhythmic contraction to first and second tn stimulation at three times motor threshold intensity and spinal nerve (sn) at motor threshold intensity with increasing interval between stimulations (x - axis). The number of animals is indicated in each symbol . *, p <0.05, first 5-min stimulation versus second 5-min stimulation, paired student's t - test . The effect of combined stimulation of spinal and tns is shown in figure 5 . With unilateral sn stimulation, the stimulation of either the ipsilateral or contralateral tn was tested . With 10 possible combinations tested, there was no significant additive effect of stimulation at the two sites . Only in the case of low intensity spinal (tmot) and high intensity tibial (3 times tmot) stimulation was there any additive effect, but the difference between spinal + tibial stimulation and tibial stimulation alone was not statistically significant . Intensity - dependent effects of combination of spinal nerve (sn) and tibial nerve (tn) stimulation on the frequency of bladder contractions . Unilateral tibial nerve stimulation was tested at intensity of two times motor threshold (tmot, a) and three times tmot (b). The responses are represented as a percentage of pretreatment values (% control), where the baseline response before stimulation is defined as 100% . The rat brc model has been used for prediction of the effect of nerve stimulation on bladder function.4,5 since the urethra is ligated, actual voiding is not measured . Furthermore normal rats are used, so bladder muscle and its innervations are normal . Nevertheless, considering the limitation of this animal model, the ability of nerve stimulation to inhibit the frequency of reflex bladder contractions is in good agreement with the ability to normalize micturition parameters in other animal models, such as cystometry in bladder irritated rats6 and rhythmic contraction and cystometry in anesthetized cats,7 as well as with clinical observations in oab patients.8 we have reported that both tn and sn stimulation was effective for inhibition of bladder reflex contractions.4,5 in those experiments, a single wire electrode was positioned under both sides of the nerve and current intensities were not controlled separately, and would depend on the functional impedance at each implantation site . In the present study, we specifically tested and controlled the delivery to both sides independently allowing a more complete characterization of stimulation thresholds for unilateral and bilateral stimulation of the sn and tn . Even though stimulation pulses were delivered differently in these three studies, it was consistently observed that the brc had a different sensitivity to stimulation of the tn and sn and that the response had a different time course . Sn stimulation produces bladder inhibition with a longer duration and greater efficacy than tn stimulation . The rat sn is composed of nerve fibers emerging from the tn and along pelvic nerve, and other somatic nerve bundles; the tn originates from the sns l4l6.9,10 it is possible that tn neuromodulation activates some, but not all of the fibers involved in sn neuromodulation . Tn stimulation triggers only toe twitches but sn stimulation evokes additional pelvic floor contraction and urethral sphincter contractions, which may produce further reflex detrusor inhibition, although effects on the urethra are unlikely since the urethra was expanded with a catheter in this preparation.5 in addition, sn has higher fiber density than tn,10 which may be responsible for the lower stimulation currents required for activation of a sufficient number of fibers for bladder control and may also contribute to the higher effectiveness of sn mediated bladder neuromodulation . High intensity stimulation of the tn or sn induces a strong skeletal muscle contraction . Previously we found that the marked inhibition of brc frequency by sn stimulation occurs in rats pretreated with pancuronium to block skeletal muscle contractions.4 therefore, such unwanted contraction does not seem to contribute to the bladder inhibition observed in response to sn or tn stimulation . There were some differences in the relationship between stimulation intensity and bladder inhibition; in particular, tn stimulation was effective over a narrow range (34 times tmot) and the duration of the inhibition was less than that from sn stimulation . These results may be a consequence of the more rapid diminution of the response to tn vis - - vis sn stimulation which we observed in the current study (see fig . 3). Compared with stronger bladder inhibition to bilateral vis - - vis unilateral sn stimulation, bilateral stimulation of the tn failed to produce more effective attenuation of bladder contractions . Bilateral symmetry of nerve responsiveness to electrical stimulation was demonstrated by equal motor threshold distribution between the left and right nerve roots . Though there is a difference in the results of bilateral stimulation on the sn and tn, the lack of a clear additive effect between spinal and tn stimulation would suggest that stimulation at either of these two sites inhibits the bladder via a similar effect on the micturition reflex arc . However, it is still possible that tn and sn stimulation target different points on the reflex arc and that the impulses from the different stimulation sites may travel through different neuronal pathways before reaching the brainstem . Both tn stimulation and low intensity sn stimulations do not alter the brc amplitude, suggesting their common mechanism of action through afferent limb of the micturition reflex arc.5,11 higher intensities of sn stimulation may directly depress the contractility of detrusor smooth muscle through the efferent limb.4 both tibial and spinal neuromodulation have been found to be effective in relieving symptoms in patients with oab . No side - by - side comparison of the two therapies in patients has been reported . It is difficult to compare reported results from individual patients since spinal stimulation is applied continuously12 and tibial stimulation is used intermittently (one 30-min stimulation once per week13). Tibial stimulation is applied intermittently since it is done trans - cutaneously as an office procedure rather than by a permanently implanted device . It is possible however, that stimulation at this site would not be effective if applied continuously, due to the potential for desensitization . A significant additive effect between spinal and tn stimulation was not observed (fig . However, there was a suggestion that the higher intensity tibial stimulation (3 times tmot) could be additive with a low intensity spinal stimulation (fig . Further experiments could determine whether this interaction is a consistent effect and if it could be enhanced by using different stimulation parameters . Based on this rat model, it would appear that neuromodulation of bladder activity by sn stimulation is preferable to tn stimulation . The data generated in this study does not suggest that stimulation at both sites would offer a therapeutic advantage over spinal stimulation alone.
Some forms, such as declarative memory, are explicit memories of objects, places, or events . Others forms, such as non - declarative memory, are implicit, like habits, skills, or priming, and do not require conscious awareness (gilbert et al ., 2001; squire, 2004). While this has been an established taxonomy of learning, the boundaries are often vague and difficult to demarcate . For instance, in associative learning there is a fine - grained distinction between declarative associative learning, which is dependent on the conscious association of events, and non - declarative unconscious associative learning, which occurs without awareness of the link between the related meaningful events (shanks, 1995). In an associative learning task, when two stimuli are systematically presented in a temporal sequence, a new relationship between these two items is learned . In classical trace learning, a neutral conditioning stimulus (cs) precedes and, therefore, causes the subsequent prediction of an emotionally charged or noxious stimulus (unconditioned stimulus, us; figure 1). During a differential eye - blink trace conditioning task, a specific tone (cs+) warns of a puff of air to the eye, whereas another tone (cs) does not . The presence of an anticipatory eye - blink response, which is a conditioned response (cr) to the cs+ tone, is highly correlated with participants verbal report of the relationship between the stimuli presented; this is known as the contingency (dawson and reardon, 1973; clark and squire, 1998). In contrast, during a delay conditioning task, for which there is no gap between the neutral stimulus (cs) and the puff of air to the eye, blink responses (cr) are elicited despite the lack of awareness of the contingency (clark and squire, 1998, 1999; figure 1). Furthermore, trace, but not delay, conditioning is strongly influenced by an expectancy of the us (clark et al ., 2001). Differential trace (upper graph) and delay conditioning (lower graph) for the eye - blink task . In trace conditioning there is a silent period between a tone and a puff of air to the eye . The cs+ tone is presented for 250 ms, followed by a 500-ms silent gap, before a 100-ms puff of air to the eye (aversive stimulus). A second neutral stimulus tone, cs, is presented alone . In delay conditioning, the tone lasts for 850 ms, covering the silent gap and co - terminating with the puff of air . Consequently, delay conditioning has been considered a hallmark of non - declarative learning and is systematically used as an associative learning task in vertebrates and invertebrates (lavond et al ., 1993). In contrast, human trace learning is dependent on conscious awareness of the contingency between stimuli (christian and thompson, 2003). Moreover it is this fact that has made human trace learning a potential turing - test of consciousness (koch, 2004). Though this turing - test potential has been influential in behavioral neuroscience, three arguments could pose a serious challenge to this notion: (1) trace conditioning can be learnt by almost every animal, even invertebrate sea slugs (glanzman, 1995); (2) trace conditioning can be elicited using subliminal stimuli (esteves et al ., 1994); and (3) clinically defined unconscious patients might learn trace conditioning (bekinschtein et al . It is in addressing these three arguments that we will characterize the theoretical boundaries of consciousness of learning . The evolution of wings can be used as an analogy for the evolution of conditioning . During the evolution of animals on this planet, wings appeared at least three times from three different ancestors . Primitive wingless insects known as bristletails used long antennae - like filaments at the ends of their bodies to glide down to tree trunks from forest canopies (yanoviak et al ., 2009). The second appearance, around 150 million years ago, involved the development of wings on dinosaurs . Some dinosaurs started to evolve lighter skeletons; their wrists changed and feathers grew to form wings (lewin, 1983; sullivan et al ., 2010). Though dinosaurs evolved wings, which gave birth to modern birds their capacity to fly has a different origin and ancestry than flying insects . The third appearance of wings developed in bats (simmons et al ., 2008). Though the three different wings serve a similar function, that is to fly, they were not derived from a common ancestor . Moreover, they are not controlled by the same machinery, and they do not obey the same rules . Is the same true of conditioning? Has a conditioning mechanism appeared only once in evolution? While the well preserved molecular machinery may suggest this, findings at the systems level are disparate and may suggest a different conclusion (barco et al ., molecular mechanisms underlying acquisition and consolidation of memory are in fact preserved in most species . However, learning relies on different network mechanisms in humans and sea slugs (takehara et al ., 2003). The plasticity of arrays of neurons and the ubiquitous evolutionary pressure of associative learning makes it extremely difficult to disentangle whether conditioning emerged only once during the course of evolution . It could be that conditioning emerged several times from several ancestral sources just as wings did . We are only left with the certainty that associative learning can be instantiated in simply a few neurons, as in sea slugs, or in millions of neurons, as in mammals . Humans show the highest degree of behavioral flexibility among animals, probably through a flexible network that relies on a frontoparietal hub . This flexibility, however, comes at a cost that results in slow, limited computational capacity . Trace conditioning does not seem to escape from this rule (zylberberg et al ., 2010). Arbitrary associations in humans are instantiated in this routing system that appears to be intrinsically related to consciousness (zylberberg et al ., 2011). On the contrary, in the sea slug these temporary associations may rely on direct connections (grol et al ., motor relations which bypass the central routing system (grol et al ., 2006). Trace conditioning in sea slugs, where consciousness is not required, does not necessarily imply that consciousness does not play a role in human trace conditioning . Alone, it does not imply more than, for example, a bacteria in need of oxygen breathing under water makes implications about human respiration . The sea slug aplysia californica only needs a few neurons to perform trace conditioning, while rabbits require hippocampal, frontal cortex, and cerebellar networks (christian and thompson, 2003). In contrast, rabbits seem to be more flexible than gastropods in their learning abilities, but not more than insects, i.e., fruit flies, (heisenberg et al ., 2001). In humans, as in rodents, trace conditioning is dependent of the hippocampus (james et al ., 1987; moyer et al ., 1990; clark and squire, 1998) while delay conditioning can be elicited without hippocampi (ivkovich and stanton, 2001; woodruff - pak and disterhoft, 2008). Delay conditioning seems to rely primarily on a functional cerebellum (mccormick and thompson, 1984; mauk and thompson, 1987; gerwig et al ., 2007). Functional imaging studies have shown that the hippocampus is activated by both trace and delay conditioning, but it is significantly more activated by trace (cheng et al ., 2008). These studies of neuroanatomy and the function of associative conditioning may indicate declarative memory s high dependence on the hippocampi and neocortex, as opposed to non - declarative memory s need for these structures, which is minimal (eichenbaum and cohen, 2000). Further evidence of a causal link between awareness of the contingency and trace conditioning comes from human and non - primate mammalian data . In humans the variability in trace conditioning responses and learning seems to be linked to attention (lovibond and shanks, 2002; carter et al ., 2003), suggesting a strong modulation by central cognitive processes . In healthy volunteers, increasing attentional load parametrically this elegant experiment used the classic n - back task to engage attentional resources and working memory . The engagement of these dramatically decreases the anticipatory responses to the us in trace conditioning . Interestingly, the interference paradigm used in humans to decrease conditioning has been replicated using mice (han et al ., 2003), demonstrating a similar functional frontotemporal network supporting successful trace conditioning in a non - primate mammalian species . In this study mice heard a tone and received a foot shock immediately after (delay conditioning) or, alternatively, the mice received a silent gap between tone and shock (trace conditioning). Two additional groups of mice experienced two lights flashing as interference for the tone shock conditions . Learning was impaired by light interference in trace but not in delay conditioning . In mice the light flashing acted as cognitive interference, just as working memory load might cause interference in trace conditioning with humans . In both populations, i.e., in humans and mice, the fact that attention is a key component of conscious learning and also that trace conditioning is affected by awareness may indicate that trace conditioning is a type of conscious learning . There is general agreement that exposure to a contingency between conditioned stimulus (cs) and us will create an associative process called conditioning (dickinson, 1980; rescorla, 1988). The difference between human and non - human animal models is that the former can easily produce a verbal or motor report of the relationship between the cs and us . If humans can form an internal representation of the contingency and verbalize it or make a voluntary response, then this behavior is taken as evidence of conscious awareness (lovibond and shanks, 2002). What if the cs is masked, or more generally, not accessible to verbal reports? This question is central to current theoretical discussions of the role of conscious processing in trace conditioning . The single - process model, asserts that a sole propositional learning process mediates expression cr and the expectancy of us (full network mapping; lovibond and shanks, 2002). The dual - process model, however, claims that these behavioral responses, both cr expression and us expectancy, are expressions of two independent learning processes (partial network mapping; perruchet, 1985; morris et al ., 1999; some studies find evidence in support of the single - process model (daum et al ., 1991, 1992; manns et al ., 2000a, b; weike et al ., 2007). While the series of experiments performed by perruchet, destrebecqz, cleeremans, and colleagues (destrebecqz and cleeremans, 2001; destrebecqz et al ., 2005, 2010; perruchet et al ., 2006) and experiments performed by others (ohman and soares, 1993, 1994, 1998; ohman et al ., 1995; weidemann et al ., the discussion is centered on the verbal reports of awareness of the contingencies (in questionnaires, motor evaluation or subjective ratings) and the measures of cr . Firstly, post - training questionnaires may elicit metacognitive process . Therefore the participant may verbally report the contingencies, not because they noticed them during the learning phase, but because they were forced to think about the contingencies after conditioning had finished . In this case it is difficult to know when conscious learning of the relationship between the stimuli occurred . It could have been during learning or it could have been when that participant was prompted about the relationship between stimuli . Secondly, if awareness is measured online during the experiment, as to avoid a post hoc metacognitive process, then participants may then take note of the contingencies of which they were previously unaware . The measurement of both verbal reports of contingencies and the cr is critical . In a series of studies, ohman et al . (1995) and ohman and soares (1993, 1994, 1998) argue that conditioning of electro - dermal responses to electric shocks can occur with masked, unconsciously perceived, stimuli . It has been found that unconsciously perceived stimuli only elicited a cr when fear - relevant stimuli, such as spiders and snakes (ohman and soares, 1993) or angry faces (esteves et al ., 1994), were presented as cs+ . In support of ohman s claims, one recent study showed that sensitivity to masking conditions was related to the cr of a masked cs but not an unmasked cs (cornwell et al ., 2007). In this study, sensitivity to the masked condition was a marker of unconscious processing, i.e., if participants were not aware of the masked item then this was taken to indicate that items were processed at an unconscious level . The depth of unconscious processing of the cs was linked to the intensity of the cr . Weak perception of the cs through masking may not elicit conscious recognition of it, but the cs may still be above an identification threshold . However, there are criticisms of these findings . One criticism is that the measure used to assess perceptual awareness of the cs may not be sufficiently sensitive to identify participants with residual awareness of stimulus features (pessoa, 2005; graziano and sigman, 2009). Additionally, some fear relevance effects in backward masking conditioning, as observed by ohman, could be due to selective sensitization rather than unconscious associative processes . Some methodological concerns have also been raised concerning the extent to which participants were truly unaware of the stimuli (lovibond and shanks, 2002). Firstly, not all verbal discriminative responses indicate awareness; discrimination of stimuli above chance levels does not necessarily imply conscious awareness (merikle and daneman, 2000; wiens and ohman, 2002). Another highly debated experiment was performed by nez and de vicente (2004), they have also showed that cr can be elicited when masked words are paired with a mild shock, and that this response is, as it was in the studies by ohman, related to the participants detection threshold . However, the results of this study are somewhat difficult to interpret as a higher proportion of participants in the unconscious masked condition produced a cr than in the conscious group . This study evaluated masked words paired with electric shocks; they used either a detection threshold, i.e., was the stimulus a word or a blank?, or an identification threshold, i.e., was it word1, word2 or not a word? When participants failed to detect a stimulus in the tachitoscope, half exhibited a cr above learning criterion (four out of eight). Yet, when participants were above detection threshold (conscious) only 11% (2 of 18) showed learning . There was a higher instance of conditioning, using a detection threshold, when stimuli were are presented unconsciously and when cr was measured using autonomic nervous system signals like skin conductance . On the contrary, when an identification threshold was applied, i.e., subjects had to differentiate between two words or two non - words, only 10% (1 of 10 participants) of participants in the unconscious condition exhibited cr, but 58% (7 out of 12) of participants in the conscious group exhibited cr . These contradictory results point to two different learning systems, the unconscious system, which bypasses central processes and consciousness - related workspaces . It also possibly directly links the early visual system with the autonomic nervous system and the conscious associative system, where the activation of the frontoparietal cortices may influence the autonomous nervous system giving rise to a different signal and a different type of learning . It is indeed the case that the variability, speed, and regularity of the cr was higher in the conscious identification group as compared to the unconscious detection learners (nez and de vicente, 2004). There are, however, caveats to this study s design that must be considered when discussing the conclusions . Firstly, in the identification threshold condition, words were repeated and could have therefore been deprived of their meaning . When a word is repeated it becomes easy to rely on low level features, such as the letter array, to determine the word s identity without the need to access its meaning . The participants decisions in this condition may not have been based on anything more than a surface features . Another methodological problem of this study is that the variability of the perceptual threshold was high and several subjects were therefore excluded leading to a small sample size . The final subgroups of learners were 2/18 and 4/8 for conscious and unconscious detection of a word / blank, respectively, and 7/12 and 1/10 subjects for conscious and unconscious identification of the word, respectively . The low number of participants complicates the statistical analysis and make conclusions difficult to extrapolate to other cases . When an electric shock is used as the aversive stimulus, as opposed to a puff of air to the eye or a loud tone, it may induce a general increase in arousal . This may, change detection thresholds and act as a confounding factor, leading to difficulty in interpreting the results . In short, there seems to be consistent evidence showing that trace conditioning can also be elicited by unconscious stimuli with a strong emotional content, which is not accessible for verbal report . There may be some specific residual forms of trace learning that can be mediated by the most likely candidates: unconscious processing and emotional stimuli . However, evidence of subliminal abstract, non - emotional stimuli eliciting trace conditioning is not conclusive . Careful and well - designed experiments are needed to robustly deliminate the boundaries of consciousness thresholds of stimuli in a given task . Their ability to elicit trace learning above and below this threshold should certainly be included in the agenda of highly relevant experiments in the next years . If trace conditioning is taken as an indirect test of awareness, then a clinically defined unconscious patient, i.e., those in a vegetative state (vs), that shows crs (learning) should be considered conscious . Conversely, if a clinically defined unconscious patient shows anticipatory responses in trace conditioning, then this learning may not necessarily be indicative of conscious awareness (bekinschtein et al ., 2009). They lack any behavior consistent with conscious awareness, such as, they do not follow someone with their eyes or head across the room, and they do not gesticulate or react to signs, words or commands . However, we used a classic differential trace conditioning eye - blink paradigm in 13 vs patients to investigate whether these unconscious patients might show learning (bekinschtein et al ., 2009). Moreover, we found a subset of these vs patients did show learning (see figure 2). Two vs could appropriately produce cr to cs+ and not to cs, and another four patients showed non - discriminatory anticipatory responses, i.e., producing the cr to both stimuli types . Patients that demonstrated learning eventually recovered by regaining awareness, as opposed to those patients that did not show learning . It is our belief that the patients that showed learning were partially conscious at the time of testing, but were unable to produce overt voluntary responses . In a recent trace conditioning study, using aversive noises and pleasant fanfares measuring skin conductance response, scott et al . (2011), showed that only participants attending to the stimuli and able to catch the rule of the experiment showed crs . If this paradigm is applied in the same disorders of consciousness patients group as the trace eye - blink conditioning, it may show convergence in conscious - dependent learning, and helping to better define the learning consciousness relationship . Anticipatory learning in normal and anesthetized participants, and in vegetative state patients during trace differential conditioning . Bars show mean muscle activity for baseline of the cs+(bs cs+), cs+ and baseline of the cs(bs cs), cs. Learning is in arbitrary units . Sedated subjects show no learning; vegetative state patients, as a group, show less muscle activity between cs+ and its baseline, and to cs. It is crucial to consider alternate hypotheses related to trace conditioning and the limited capacity of consciousness . Limited capacity implies that to learn trace conditioning, progressive gain of awareness of the contingency is required by maintaining a free global workspace clear of other contents (dehaene and naccache, 2001). The use of this limited space for other events or processes may weaken the link that establishes the association between the cs and the us . This in turn would prevent the associative relationship from being established . In our original study these sedated healthy participants showed no learning of the contingency, or cr, for eye - blink trace conditioning . The sedative (propofol) that anesthetized these participants ensured that they were indeed unconscious, and the levels of drug caused a significant decrease in absolute cerebral blood flow . In particular the propofol - related variations in the thalamic blood flow appeared to be linked to the midbrain reticular formation, thus suggests a close functional relationship between the two brain structures while unconscious (fiset et al ., 1999). These deeply sedated participants showed true unconsciousness through both diminished awareness and arousal (wakefulness). Moreover, the participants under low doses of propofol had a completely disorganized system for global integrated processing (davis et al ., 2007; stamatakis et al ., 2010). On the contrary, unconscious vs patients show dissociated wakefulness and awareness; they are regarded as being awake but not aware (jennett and plum, 1972). It could be that some vs patients retain partial functionality of the networks that support the acquisition of trace conditioning, but this is not enough to produce volitional movements . Sea slugs can learn trace conditioning, but they do not show a form of learning that reveals the flexibility typically displayed in conscious forms of learning (van den bussche et al ., 2008; heinemann et al ., 2009; see dehaene and changeux, 2011 for a review). Instead, these mollusks use the minimal numbers of systems necessary for successful associative learning . As far as we know sea slugs are not conscious, that is they are not conscious in the same way as humans are . Hence the mere observation of trace conditioning learning in this species does not provide evidence that trace conditioning is not an adequate signature of consciousness . Quite the opposite is true; humans are extremely sensitive to context and are continuously interpreting all incoming stimuli in multiple ways . This capacity for over - interpretation may help in social interaction, goal - oriented behavior and possibly changes the way that trace conditioning is encoded and processed . The expression of trace learning in humans through a display of non - stereotyped representations of the contingency relies on an entirely distinct neural architecture several studies have used masked or subliminally presented stimuli in order to make cs impossible to report (unconsciously perceived). This type of design has been used to explicitly determine whether conscious perception of the stimuli is necessary to achieve trace learning . Unfortunately, the results are inconclusive about whether trace learning, generally, is achievable through subliminal stimuli or under some specific circumstances . When it is not neutral, but of negative valence, the masked stimuli seems to be processed up to the point of forming an association with the us, despite not being reported, detected, or discriminated . The fact that clinically defined unconscious patients show trace learning suggest that they may have partial capacity for conscious awareness, as trace learning is dependant on some form of conscious awareness of the contingency . This result is further strengthened by data from sedated participants that show drug induced unconsciousness does not produce anticipatory responses above the baseline (bekinschtein et al ., 2009). Trace conditioning remains very much linked to awareness of the contingency between cs and us . We believe it is a combination of the timing between stimuli and the variations on the cs that will allow us to better frame, over the next few years, the boundaries between this basic form of learning and conscious awareness . The aforementioned analyses leads us to propose here three conditions which should be met in order for trace conditioning to be used as a test for conscious awareness: (1) a relatively stable, sustained attention to the stimuli, (2) a low central processing load to avoid interference and (3) a well defined stimuli, perceptually discernible and close in presentation time . If (1) is not met, then attention will deviate from the stimuli, and the creation of the association between cs and us will be disrupted . This would lead to sparse and inconsistent demonstration of the cr (armony and dolan, 2002). If (2) is not met then working memory capacity will be saturated by another task . Learning will then decrease, and as a result there will be low awareness of the contingencies . In this respect, trace conditioning seems to require the central resources of the attentional system in order for the association to be established, and this seems to be paired with awareness (carter et al ., 2003). If (3) is not met, then the stimuli will be ambiguous and the trace that is established will not be long - lasting or robust . This will lead to partial learning only and high variance in cr (lovibond and shanks, 2002; sehlmeyer et al ., we would like to raise one final important point that has not yet been emphasized . It is of paramount importance if trace conditioning is to be used in practical terms . Clark and squire (1998) established that in order to observe an anticipatory eye - blink response participants must be aware of the contingency at a level that is sufficient for a verbal report . The contrary, however, is not true (both in the original clark and squire data and in our data making more than 200 participants combined): some participants show verbal reports of the contingency, but do not show the cr of an anticipatory associative response . This simply may be because participants react in different ways to the anticipation of the air puff when they are aware that it is imminent . The most frequent reaction is a contraction of the muscle prior to the air puff, however, some participants may control this spontaneous response by relaxing and, inhibiting their blinks or by simply doing nothing . Hence, an improved statement is needed . While the absence of learning does not provide information concerning the participants degree of awareness the presence of trace learning may be taken as evidence in favor of conscious awareness . The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Latin america and the caribbean region is composed of 32 countries and puerto rico, an unincorporated territory of the united states . Twenty of these countries share a common history and culture resulting from the iberian colonization in the 16 century and are characterized by the use of the spanish and portuguese languages . The region covers an area of 21,069,501 km (14.1% of earth s land surface area) and is populated by 581.4 million people (8.3% of the world population), with approximately 79% of the people living in urban areas concentrated in the atlantic coastal regions, the central valley of mexico, the andean mountains, central america and the main caribbean islands; overall, this area has a per capita gross national income (gni) of us$8,981 . This territory constitutes one of the largest linguistic and culturally cohesive regions in the planet and represents the 4 largest economy in the world (latin america and the caribbean (lac) population database, 2005; almanaque mundial 2013, 2012; the world bank, 2013). The human development report 2013 of the united nations development program showed that the majority of the countries in latin america and the caribbean have reached a human development index (hdi) of 0.741, which is considered a high hdi; however, several countries in the area exhibit a medium hdi (human development report, 2013). This index suggests that the population in the region has reached an acceptable quality of social welfare and acceptable medical services, including prenatal and perinatal screening, fetal medicine, medical genetics services, and sophisticated molecular and genetics diagnostics laboratories; however, the region still suffers from important income inequalities and has the most unequal income distribution worldwide (human development report, 2013). Important sub - regional differences exist in every country of the region, even in countries with a very high hdi . Health services are concentrated in the primary urban centers, and there is a substantial lack of primary health - care centers in the rural areas to provide quality medical attention; additionally, almost no genetic services are available in several regions and countries (penchaszadeh, 2004). As with the health systems, the universities and research centers are also located in the main metropoles; however, the region stills suffers from minimal financial resources for research, low academic - entrepreneurial interactions and small inter - academic cooperation to develop research . Nevertheless, several governments are starting to support research activities, and several institutions with well - trained scientists and sufficient infrastructure are performing genomic research, primarily in countries such as brazil, mexico, argentina, and chile . The activities in the fields of human genetics and medical genetics in latin america and the caribbean began in the 195060s . Several original clinical descriptions of beguez - chdiak higashi syndrome (omim #214500), cant syndrome (omim #239850), congenital generalized hypertrichosis (omim #307150), grebe - quelce - salgado syndrome (omim #200700), acheiropodia (omim #200500), yunis - varon syndrome (omim #216340), ivic syndrome (omim #147750), odontotrichomelic syndrome (omim #273400), and several camptodactyly associated syndromes, among others [online mendelian inheritance in man (omim)], were first described by latin america geneticists; additionally, several regional hemoglobinopathies have been described and characterized . These scientists have also described notable genetic phenotypes clusters or large pedigrees for huntington s disease in maracaibo (negrette, 1963), laron syndrome in ecuador (berg et al ., 1992), early - onset alzheimer s disease in colombia (lopera et al ., 1997), jarcho - levin syndrome in puerto rico (cornier et al ., 2004), twin births in brazil (tagliani - ribeiro et al ., 2012), and actinic prurigo in several american - aboriginal communities, among others . Latin american geneticists also established remarkable birth defect registries, including the estudio colaborativo latino americano de malformaciones congnitas (eclamc) by dr . Eduardo castilla in south america and the registro y vigilancia epidemiolgica de malformaciones congnitas externas (rivemce) by dr . These registries are crucial for the understanding of several complex congenital diseases, such as orofacial clefts and neural tube defects, and several local studies have demonstrated that folic acid supplementation can help prevent neural tube defects . Several countries have national newborn screening programs, with very high coverage by comprehensive panels in costa rica, chile, and uruguay . The detection of congenital hypothyroidism has been effectively attempted by newborn screening programs in several latin american countries . Population geneticists have greatly contributed to the genetic studies of the aboriginal and modern latin american and caribbean populations . Finally, for the research of complex prevalent diseases, such as diabetes, cardiovascular disorders, psychiatric diseases, birth defects, and cancer, and for pharmacogenomic studies, the region offers unique conditions that are not present in the rest of the world, including environmental diversity, ethnic diversity, population structure, and a high rate of environmental exposures . This scenery favors the study of gene - environment interactions, and several admixture studies have analyzed the genetic factors involved in complex phenotypes . All of these developments and the promissing progress in the field of human and medical genetics inspired several of the founders and leaders of these disciplines in this region to create the red latinoamericana de gentica humana (relagh) in the early 1990s, with the aim of creating a network for human genetics researchers, students, and practitioners in latin america and the caribbean; the goal of this network was to contribute to the development of collaborative projects for education, research and assistance, and to represent the region before the international federation of human genetics societies (ifhgs). The idea of creating a latin american network of human genetics (relagh) arose during the ix international congress of human genetics in 1996 in rio de janeiro, brazil, at which the international federation of human genetic societies was created . Several latin american societies of human and medical genetics applied for membership to the new organization . After establishing that the executive committee of the ifhgs would include representatives of societies in europe, united states, canada, australia, and asia as full members, this committee suggested that latin america could form a regional representative group . In 1997, a meeting was held in san juan de el ro, queretaro, mexico; at this time, representatives of the human genetics field from a dozen latin american countries, and dr . Marcus pembrey, the ifhgs representative, organized relagh, stating that such a network would represent not only human genetics societies, but also persons in those countries that do not have associations or societies . A meeting in 1999 in buenos aires, argentina, primarily attended by eclamc members, resulted in the nomination of an organizing committee that consisted of representatives from brazil, argentina, and mexico . Several difficulties were overcome in the following years; therefore, in vienna, austria, during the 10 international congress of human genetics in may 2001, relagh was formally established through the participation of the presidents of five groups: the branch of human genetics of the sociedad argentina de gentica, the sociedade brasileira de gentica mdica, the asociacin colombiana de gentica humana, the asociacin mexicana de gentica humana, and the sociedad panamea de gentica humana . . Jos mara cant (mexico) was appointed the acting president for two years, and he agreed to create an assembly to evaluate the proposed governing statutes of relagh and chose the first board of directors . Dr . Roberto giugliani (brazil) was proposed as the relagh operator . During the ifhgs meeting at the same congress, relagh was accepted as a full member and part of its executive committee . In 2003, a new board of directors was appointed during the presidency of roberto giugliani (brazil). This board decided to form an executive committee to address the practical activities of the young network . This committee was based in porto alegre, brazil and was formed by roberto giugliani and ursula matte (chairpersons). The primary goals for this period were to draft and propose the bylaws of the network; to consolidate relagh as a representative of latin america in the ifhgs; to set up the network website and use it as the main communication tool between relagh members; to expand the relagh membership, and promote relagh as a hub for collaboration among human genetics groups in the latin american region; and to organize the latin american school of human and medical genetics (elag). Having achieved all of these goals, the relagh presidency was transferred to the new board of directors in 2006 (no business meeting was held in 2005). The executive committee decided to continue its activity and became the organizing committee of the elag, which has been a very successful course since 2005 . The bylaws of the network were initially established in march 2004 and modified in 2008 during the first latin american congress of human genetics in cartagena, colombia . Augusto rojas - martnez (mexico, 20112013); relagh is currently led by dr . Short courses in medical and human genetics are already offered to young students and professionals in north america (the bar harbor course on mammalian genetics) and europe (the european school of medical genetics). Young researchers and professionals in latin america should also be prepared to address the challenges of increased genetic applications in health sciences . The aim of elag is to promote a one - week state of the art human and medical genetics course that will expose a selected number of top - grade students to first rank researchers in the field . In addition to attending a strong teaching program, the students have the unique opportunity of discuss projects, techniques, clinical cases, and scientific issues with the faculty members . The participation of students and faculty members from several latin american countries stimulates further cooperation between the groups inside the continent . The target audience includes phd students, post - docs, and young physicians interested in the fields of human and medical genetics . The students are selected on the basis of a broad participation of latin american countries and on their individual performance according to academic records, curriculum vitae, and recommendation letters . The elag faculty consists of top rank scientists from latin america, north america and europe who are invited to lead lectures and courses . On - site accommodations are an important point, as informal contact between students and faculty is one of the major goals of this initiative . Regarding the financing of elag activities, the organizers apply to governmental organizations, research funding agencies, and companies to obtain the financial support for the faculty expenses, including transport, accommodation and meals, and to grant a limited number of competitive fellowships to students . The organizers also encourage the national agencies of latin american countries to grant fellowships to cover student expenses . The first elag course was held in 2005, and this course has been repeated every year . With the exception of 2007, when it was held in angra dos reis (200 km from rio de janeiro), all of the courses have been held at hotel samuara in rio grande do sul, brazil . This hotel is located inside a natural park, 10 km from the city of caxias do sul and 100 km from porto alegre, and provides a perfect environment for the development of this activity . Each course has 7075 students that come from 1015 different latin american countries and 2530 faculty members from latin america, north america, and europe . The first latin american congress of human genetics (clagh) was co - organized between relagh and the asociacin colombiana de gentica humana and was celebrated in cartagena, colombia in october 2008 . The meeting was attended by human and medical geneticists and many students from almost all of the region . A workshop on laboratory standards, organized by dr . Jean - jacques cassiman, in representation of the eurogentest, was held to contribute to the understanding of quality assurance norms and ethical standards that have been established by this european organization . The meeting was a very successful effort to launch research and academic activities in the region . During the business meeting, the bylaws of the network were modified, and the presidency was handed over to dr . The 2 clagh occurred in san jos costa rica in may, 2011 and was co - organized by relagh and the universidad de costa rica and the colegio de bilogos de costa rica, and was financed, in part, by the national ministry of science and technology . Francisco m. salzano (brazil) for his contributions in the development of the population genetics in latin america . The human variome project organization (hvp) held a meeting, which was presided over by dr . Richard g. cotton, the founder of the project, to encourage the participation of latin america in this project . During the business meeting, cotton, an inter - institutional mexican group created the first latin american node of the hvp in june 2012 . The node received financial support from the centro de investigacin y desarrollo en ciencias de la salud of the universidad autnoma de nuevo len in monterrey, in which the bioinformatics group coordinates the collection of relevant national data . A second hvp node was launched in march 2013 in venezuela, under the leadership of dr . The meeting was co - organized by the asociacin mexicana de gentica humana, which also celebrated the 38 mexican meeting of human genetics . The meeting was attended by more than 800 participants, including more than 70 lecturers by researchers from the usa, europe, australia, latin america and the caribbean and included the attendance of dr . In addition to six plenary lectures, a total of six workshops, 11 symposia, 80 oral, and 293 poster presentations were contributed by researchers from almost all of the latin america and caribbean countries . These events covered the topics of medical genetics, cytogenetics, complex diseases, population genetics, molecular biology, genetic epidemiology, and cancer . A 3-day workshop on public health genomics, financed by the consejo nacional de ciencia y tecnologa (mexico), was conducted as a preamble for the 3 clagh . This workshop introduced the concept of using genomic information for public health purposes to improve preventive care . Lecturers for this workshop included leading scientists from the fields of public health, medical genetics, human genomics, and bioinformatics from the usa, europe, argentina, brazil, and mexico . The hvp held a special symposium to promote the creation of more hvp country nodes in the region . During this meeting, the general assembly of relagh examined the status of the network, including the rules for membership, and elections were performed to form the new board . Lavnia schler - faccini (brazil) was elected as vice - president of the network for the period of 20142015 . This meeting showcased new technologies, including next generation sequencing and microarray platforms, that can facilitate genomic research studies of monogenic, chromosomal, and complex diseases, as well as made efforts to introduce these technologies into the clinic for diagnostic purposes . Despite the progress that has been made, the network is presently consists of national associations, societies, and sections of human and medical genetics from 13 countries (argentina, brazil, bolivia, chile, colombia, costa rica, cuba, ecuador, mexico, panama, peru, uruguay, and venezuela). Associations from nicaragua, and paraguay declared their interest in joining relagh during the last meeting held in the riviera maya, and these associations will most likely be included during the current presidency . This interest implies that relagh should increase its efforts to network with the associations of several countries in central america and the caribbean . Having decided since the very beginning to not collect fees from its members, relagh faces a continuous challenge in obtaining funding to support its activities . Obtaining funding has been accomplished with the help of the affiliated national societies, whose support has enabled relagh to conduct its meetings . However, more stable funding should be pursued to enable the development of projects that are aimed at establishing stronger connections between its members, such as regular newsletters, online educational projects, the maintenance of an updated database of research groups and services provided in the region, training / exchange programs, and other possibilities . Finally, the integration of available human resources and infrastructure in the region remains a challenge . Although this ability may compensate for the lack of professional and laboratory services in several countries and regions, ignorance about resources, the lack of communication and logistic, and administrative limitations, such as courier services and national customs, still restrict efficient international cooperation . The implementation of national directories of medical genetics services and diagnostic laboratories may be a step forward in accomplishing these tasks, and the current presidency of the relagh is working towards this goal.
Ocular melanoma (om), the second most common type of melanoma after cutaneous melanoma, accounts for 3.7% of all melanoma cases.1,2 the incidence rate of om is one per million in the united states (us), and 0.7 per 100,000 among caucasians alone (age - standardized to the world standard population).2,3 there are two major subtypes of om: those that arise from the iris, choroid, and ciliary body (uveal melanoma, um) and those from the conjunctiva (conjunctival melanoma, cm). Um is the most common primary intraocular malignancy among adults, and accounts for up to 85% of all cases of om.4,5 the incidence of um in the us is 4.9 per million and has remained stable over the last three decades.6 although um patients typically present with symptoms including blurred vision, visual field defect, metamorphopsia, or photopsia, 30% patients may be asymptomatic and upon routine eye examination, they are detected incidentally.7,8 diagnosis is accurately established by clinical examination in over 99% cases.9 modern diagnostic tools such as a and b ultrasonography, fluorescein angiography, and optical coherence tomography can significantly aid in diagnosis, making it possible to avoid biopsy in nearly all cases.10,11 historically, um has been treated with enucleation, radiation alone, or combination surgery and radiation; however since the late 1980s, a change in prevailing trends toward radiation has emerged.12 more recently, with growing understanding of the genetic and molecular basis of um, therapeutic trials utilizing adjuvant therapies such as crizotinib, sunitinib, valproic acid, interferon alpha, and dacarbazine have begun to emerge.11 regardless of the initial management method chosen, at least 30% of affected patients will develop metastatic spread to the liver, lung, bone, or skin within 10 years of successful local control of the primary neoplasm, with the liver being involved in up to 95% of cases.13,14 cm accounts for only 5% of all om patients.15 the cm incidence in the us is 0.4 per million and has been increasing progressively.10 among caucasian men alone, the incidence rate increased by 295% over the last 27 years.10 cm occurs most commonly in the bulbar conjunctiva, and rarely in the palpebral and forniceal conjunctiva, plica semilunaris, or caruncula.10,13 cm typically presents in patients over 60 years of age with raised pigmented lesions on the conjunctiva, often surrounded by prominent feeder blood vessels, and these lesions may even be amelanotic.10,13 cms are generally asymptomatic, causing only occasional pain and irritation.10 primary treatment of cm is surgical with wide local excision.1 additional therapies include adjuvant brachytherapy, cryotherapy, and topical chemotherapeutic agents such as mitomycin c.10 metastasis in cm typically involves the salivary lymph nodes, lungs, liver, skin, and brain, with the brain being the most common site of distant metastasis (involved in up to 25% of cases).10 at present, data detailing survival trends among om patients are based primarily on rare population - based studies involving small groups of patients from high volume melanoma centers.10,16 demographic and clinical factors influencing clinical outcomes in om patients, particularly cm, are not well understood . The current study examines a large cohort of om patients (both um and cm) to evaluate demographic, pathologic, and clinical factors that affect patient outcomes and alter therapeutic approaches . Data for the current study were extracted from the national cancer institute s surveillance, epidemiology, and end result (seer) database between 1973 and 2012 . Data from 17 seer registries (alaska native tumor registry, arizona indians, cherokee nation, connecticut, detroit, georgia center for cancer statistics, greater bay area cancer registry, greater california, hawaii, iowa, kentucky, los angeles, louisiana, new jersey, new mexico, seattle - puget sound, and utah) were extracted into seer stat software version 8.0.4 . There were 277,120 cases of histologically confirmed melanoma . A total of 8,165 cases with a primary diagnosis of om were identified using the seer international classification of disease for oncology (icd - o-3) codes, code c69.0 (cm) for the cm group, and c69.3 (choroid) and c69.4 (ciliary body and iris) for the um group . The demographic and clinical data that were extracted were age, sex, ethnicity, geographic location, prior malignancy status, tumor stage, laterality, and type of treatment received (surgery, radiation, both surgery and radiation, or no treatment / unknown). The endpoints and outcomes that were examined included overall survival, mortality, and 1-, 2-, and 5-year cancer - specific survival . Chi - square test was used to compare categorical data, while student s t - test and analysis of variance were used to compare continuous data . To determine independent factors that affect method was conducted, and odds ratios (or) were calculated . Long - term actuarial survival between conjunctival and unknown and missing data were not included in the multivariate analysis . A p - value of <0.05 was utilized to determine statistical significance . Ethics board approval to conduct the study was obtained from saint barnabas medical center who deemed patient consent was not required as the current study is a retrospective study utilizing data from the seer database and no specific patient identifiable information was utilized . The study cohort consisted of 8,165 om cases, which represented 2.9% of all melanomas in the seer database (19732012). Um accounted for 92.1% (n=7,516) and cm accounted for 7.9% (n=649) of all om cases (table 1), p<0.01 . Mean age - adjusted incidence of om was 5.5 per million, while mean age - adjusted cm incidence was 0.4 per million, p<0.001 . Cm and um patients had a similar mean age (61.718.6 years vs 61.415.0 years, p=0.63). Among all om patients, 21.0% (n=1,717) were <50 years of age, 67.7% (n=5,529) were between 5079 years of age, and 11.3% (n=919) were 80 years of age . Although most cm and um patients were between 5079 years old (59.4% vs 68.4%, p<0.01), more cm patients were under the age of 50 years (24.0% vs 20.8%, p=0.05) and 80 years (16.6% vs 10.8%, p<0.01) compared to um patients . The male - to - female ratio for om was 1.09:1 with 52.2% male (n=4,263) and 47.8% female (n=3,902), p<0.01 . Male - to - female ratio was similar for both cm and um (1.03:1.0 vs 1.1:1.0, p=0.47). Om, um, and cm were all more common among caucasians (93.9% in om, 94.7% in um, and 85.3% in cm, p<0.01). The highest incidence of om was among caucasians (93.9%; n=7,540), followed by hispanics (4.3%; n=342), african - americans (0.7%; n=54), and other ethnicities including asian, pacific islanders, and native americans (1.1%; n=91), p<0.01 . Cm had a significantly lower incidence among caucasians (85.3% vs 94.7%, p<0.01) and higher incidence in non - caucasian groups compared to um, including african americans (2.2% vs 0.5%, p<0.01), hispanics (8.7% vs 3.9%, p<0.01), and asian / pacific islanders / native americans (3.8% vs 0.9%, p<0.01). Among om patients, 50.1% (n=4,049) had right eye involvement, 49.8% (n=4,025) had left eye involvement, and 0.1% (n=3) had bilateral involvement (table 2). Cm had significantly less right eye involvement (45.5% vs 50.5%, p<0.01) and more left involvement (54.3% vs 49.4%, p<0.01), but comparable bilateral involvement (0.15% vs 0.03%, p=0.08) compared to um . In all, 90.1% (n=6,602) of all om patients presented with localized disease, 8.2% (n=601) with regional disease, and 1.7% (n=124) exhibited distant metastasis . Significantly fewer cm patients had localized disease compared to um patients (81.2% vs 90.9%, p<0.01). Conversely, cm patients had more regional (16.2% vs 7.5%, p<0.01) and distant diseases (2.6% vs 1.6%, p=0.09). In all, 47.1% (n=3,769) of om patients were treated surgically, while 39.9% of patients (n=3,192) were treated with primary radiotherapy (table 3). In all, 6.7% of patients (n=538) received both surgery and radiotherapy, while 6.3% of patients (n=504) had no treatment . Surgery was the primary treatment in cm (88.7%, n=565) and was used significantly more often than for um (42.8%, n=3,218), p<0.01 . Um patients underwent radiation therapy (43.0%, n=3,232) and radiation combined with surgery (7.0%, n=527) more frequently than cm patients in whom 0.9% (n=6) underwent radiation therapy and 2.4% (n=15) underwent combination therapy, p<0.01 . Mean overall survival for all om patients was 14.60.2 years (table 3), p<0.01 . Overall survival for cm patients was higher than for um patients (15.40.9 years vs 14.60.2 years), p<0.01 (figures 1 and 2). Overall and cancer - specific mortality was 45.5% and 21.5%, respectively, for om . Overall mortality was 46.1% among um patients and 38.8% among cm patients, while overall cancer - specific mortality was 24.9% among um patients and 20.0% among cm, p<0.01 . Mean cancer - specific relative survival at 1, 2, and 5 years for all om patients was 96%, 89%, and 71%, respectively . Similar mean cancer - specific survival at 1, 2, and 5 years was observed in um (96%, 89%, and 70%) and cm (95%, 88%, and 73%), p>0.05 . Multivariate analysis identified male sex (or 1.1, ci = 1.01.3), age over 50 years (or 4.0, ci = 3.44.6), and distant metastases (or 8.6, ci = 4.715) as independently associated with increased overall and cancer - specific mortality for om patients, p<0.005 . Surgical treatment alone was independently associated with increased mortality in um (or 2.6, ci = 2.03.3, p<0.005), while primary radiation treatment was independently associated with reduced mortality (or 0.5, ci = 0.40.7), p<0.005 . Mean age - adjusted incidence of om was 5.5 per million, while mean age - adjusted cm incidence was 0.4 per million, p<0.001 . Cm and um patients had a similar mean age (61.718.6 years vs 61.415.0 years, p=0.63). Among all om patients, 21.0% (n=1,717) were <50 years of age, 67.7% (n=5,529) were between 5079 years of age, and 11.3% (n=919) were 80 years of age . Although most cm and um patients were between 5079 years old (59.4% vs 68.4%, p<0.01), more cm patients were under the age of 50 years (24.0% vs 20.8%, p=0.05) and 80 years (16.6% vs 10.8%, p<0.01) compared to um patients . The male - to - female ratio for om was 1.09:1 with 52.2% male (n=4,263) and 47.8% female (n=3,902), p<0.01 . Male - to - female ratio was similar for both cm and um (1.03:1.0 vs 1.1:1.0, p=0.47). Om, um, and cm were all more common among caucasians (93.9% in om, 94.7% in um, and 85.3% in cm, p<0.01). The highest incidence of om was among caucasians (93.9%; n=7,540), followed by hispanics (4.3%; n=342), african - americans (0.7%; n=54), and other ethnicities including asian, pacific islanders, and native americans (1.1%; n=91), p<0.01 . Cm had a significantly lower incidence among caucasians (85.3% vs 94.7%, p<0.01) and higher incidence in non - caucasian groups compared to um, including african americans (2.2% vs 0.5%, p<0.01), hispanics (8.7% vs 3.9%, p<0.01), and asian / pacific islanders / native americans (3.8% vs 0.9%, p<0.01). Among om patients, 50.1% (n=4,049) had right eye involvement, 49.8% (n=4,025) had left eye involvement, and 0.1% (n=3) had bilateral involvement (table 2). Cm had significantly less right eye involvement (45.5% vs 50.5%, p<0.01) and more left involvement (54.3% vs 49.4%, p<0.01), but comparable bilateral involvement (0.15% vs 0.03%, p=0.08) compared to um . In all, 90.1% (n=6,602) of all om patients presented with localized disease, 8.2% (n=601) with regional disease, and 1.7% (n=124) exhibited distant metastasis . Significantly fewer cm patients had localized disease compared to um patients (81.2% vs 90.9%, p<0.01). Conversely, cm patients had more regional (16.2% vs 7.5%, p<0.01) and distant diseases (2.6% vs 1.6%, p=0.09). In all, 47.1% (n=3,769) of om patients were treated surgically, while 39.9% of patients (n=3,192) were treated with primary radiotherapy (table 3). In all, 6.7% of patients (n=538) received both surgery and radiotherapy, while 6.3% of patients (n=504) had no treatment . Surgery was the primary treatment in cm (88.7%, n=565) and was used significantly more often than for um (42.8%, n=3,218), p<0.01 . Um patients underwent radiation therapy (43.0%, n=3,232) and radiation combined with surgery (7.0%, n=527) more frequently than cm patients in whom 0.9% (n=6) underwent radiation therapy and 2.4% (n=15) underwent combination therapy, p<0.01 . Mean overall survival for all om patients was 14.60.2 years (table 3), p<0.01 . Overall survival for cm patients was higher than for um patients (15.40.9 years vs 14.60.2 years), p<0.01 (figures 1 and 2). Overall and cancer - specific mortality was 45.5% and 21.5%, respectively, for om . Overall mortality was 46.1% among um patients and 38.8% among cm patients, while overall cancer - specific mortality was 24.9% among um patients and 20.0% among cm, p<0.01 . Mean cancer - specific relative survival at 1, 2, and 5 years for all om patients was 96%, 89%, and 71%, respectively . Similar mean cancer - specific survival at 1, 2, and 5 years was observed in um (96%, 89%, and 70%) and cm (95%, 88%, and 73%), p>0.05 . Multivariate analysis identified male sex (or 1.1, ci = 1.01.3), age over 50 years (or 4.0, ci = 3.44.6), and distant metastases (or 8.6, ci = 4.715) as independently associated with increased overall and cancer - specific mortality for om patients, p<0.005 . Surgical treatment alone was independently associated with increased mortality in um (or 2.6, ci = 2.03.3, p<0.005), while primary radiation treatment was independently associated with reduced mortality (or 0.5, ci = 0.40.7), p<0.005 . The incidence of om is considerably lower than cutaneous melanoma; however, um is the most common primary intraocular malignancy in adults, with a mean age - adjusted incidence of 5.1 per million in the us population.1,4 um accounts for 85%90% of all oms in the published literature, while cm accounts for only 5%10% of cases.10,17 the current study reports a cm incidence of 0.4 per million over the study period from 1973 to 2012, which is relatively low compared to the um incidence of 5.1 per million . Mclaughlin et al and singh et al reported age - adjusted um incidence rates of 4.95.1 per million in the us, with the incidence remaining relatively unchanged over the last 35 years.2,4 yu et al and tuomaala et al reported cm incidence rates of 0.20.8 per million; however, unlike um, the incidence of cm has been rising over the last 40 years in tandem with a rising cutaneous melanoma incidence.16,18,19 yu et al reported a 295% increase in the incidence of cm among caucasian males between 1973 and 1999, while tuomaala et al reported a rising incidence from 0.4 to 0.8 per million between 1967 and 2000 in a danish population.18,19 the incidence of um and cm between sexes is approximately 1:1, which is consistent with most prior population - based studies.2,4,7 singh et al studied 4,070 um patients and noted that 51.8% of patients were males.4 in contrast, graell et al noted that 55.8% of their 303 um patients were females, while yu et al reported that 41% of their 206 cm patients were females.18,20 a higher incidence in females (55%) was also reported in a study of 194 dutch cm patients.14 lack of consensus implies there is no clear sex predominance for either cm or um, and both the sexes should be considered equally at risk . Om most commonly affects caucasians in the seventh decade of life, with cm patients being slightly older than um patients . These results are similar to large retrospective studies by graell et al and damato and damato who reported a mean age of 60.1 and 62.1 years for um patients, respectively, and anastassiou et al and kimura et al who reported a mean age of 60 and 62.3 years for cm patients, respectively.7,2022 mclaughlin et al conducted a large population - based retrospective study including 4,885 om patients and reported that the incidence of om was 810 times higher among caucasians compared to african americans.2 similarly, singh et al conducted a retrospective seer study and found that 97.8% of the 4,070 um patients were caucasians.4 caucasians in their seventh decade of life and older should be considered for fundoscopic screening examinations for om during regular health visits, with further workup warranted for patients presenting with visual symptoms.18 hispanics constituted the second largest ethnicity affected by om (4.2%), particularly cm (8.5%) in the current study . Similarly, hu et al conducted a retrospective seer study involving 168 cm patients and reported that 8.33% were hispanics, also comprising the second largest affected group.23 in terms of um, margo reported that hispanics were the most common ethnic group affected after caucasians, constituting 5.4% of the 873 um patients in his study.24 although caucasians are at the greatest risk, hispanic patients should also be considered a high - risk group, and periodic as well as symptomatic screening for om should be considered . Um is diagnosed far more often than cm and at earlier, localized stages, while cm has relatively higher rates of advanced disease stage at presentation . Mclaughlin et al reported that 67.2% of the 4,885 om (both um and cm) patients in their study had localized tumors at the time of diagnosis.2 missotten et al conducted a retrospective study involving 194 dutch cm patients and reported that 78.9% of patients had localized tumor, 4.64% had regional metastasis, and 16.5% had distant metastasis.14 regional metastasis of cm to preauricular and submandibular lymph nodes was very common and occurred in one - third of cm patients.15,21,25 um is routinely treated with primary radiation therapy, while cm is almost exclusively treated surgically . The majority of cm patients in the current study were treated with surgery only (87.1%), while radiation alone was used in 0.9% and combination therapy in 2.3% . Cm was previously believed to be one of the most malignant tumors and enucleation was historically the mainstay of therapy.26 currently, surgical en bloc excisional biopsy of the tumor, infrequently with adjuvant brachytherapy or chemotherapy like mitomycin c or cryotherapy, has become the preferred treatment for cm and preserves the eye.26 cm is far more surgically accessible given its external location, making it easier to resect than its uveal counterpart, resulting in excellent long - term prognosis and oncologic outcomes if resected early enough.26 enucleation was the mainstay of treatment for um until the results of the landmark collaborative ocular melanoma study (coms) involving 1,310 um patients was published and concluded that mortality rates following iodine (i)-125 brachytherapy did not differ significantly from enucleation (27% vs 28%, p=0.48).4,9,27 the coms centers also reported an increased trend toward diagnosing smaller ums with advances in diagnostic modalities . As a result, there has been a switch toward eye and vision sparing treatment for smaller tumors.27 in addition to the eye sparing and associated aesthetic benefits associated with avoiding enucleation, vision is preserved in up to 43% of patients receiving radiation therapy . Furthermore, patients with um who receive radiation therapy experience longer survival than patients who undergo surgical treatment.24,28 the coms study reported longer survival rates for medium sized melanomas when treated with i-125 brachytherapy compared to enucleation, with comparable 5-year all - cause mortality (19% vs 18%) and 5-year tumor - related mortality (11% vs 9%).24 despite this, radiotherapy as the primary treatment modality for um has been slow to take effect and, therefore, may be confounded by other factors such as earlier detection of um with improved diagnostic modalities allowing for earlier detection and treatment . Techniques such as proton beam radiotherapy and gamma knife radiosurgery have been increasingly investigated, with variable success.29 in a seer study involving 1,004 um patients, abrams et al reported similar 5-year overall survival (83.3% vs 82.5%, p=0.69) and 5-year cause - specific survival (88.3% vs 88.3%, p=0.92) with ebrt and brachytherapy.29 brachytherapy appeared more beneficial for early stage tumors, whereas ebrt was more favorable in late stage tumors, likely because it is more difficult to deliver prescriptive doses to tumors of advanced stages.29 in the current study, overall survival was longer for cm patients than um patients (15.40.9 years vs 14.60.2 years), and overall mortality for cm patients was lower than for um patients (38.8% vs 46.1%). In prior studies on um, the 5-year survival rates after enucleation, brachytherapy, and other methods have ranged from 25% to 66%.3034 isager et al studied 2,504 danish om patients and observed a 5-year mean cancer - specific mortality of 55%.10 the 5-year mean cancer - specific mortality for cm was noted to be 70%, which is comparable to the current study (73%).10 isager et al also noted that patients with iris melanomas had the highest observed and relative survival, patients with choroid and ciliary body melanoma had the lowest, and patients with cm had intermediate survival.10 these findings suggest that early management of cm is crucial to maximize survival as it has a relatively better prognosis than um . Given the difficulty in diagnosing um and the poor prognosis, periodic ophthalmologic screening of high - risk patients is required for earlier detection and treatment of um . More research and interest from the clinicians must take place in order to provide the best possible care as well as to improve the prognosis for um and cm . With the increasing knowledge of molecular and genetic biology on om, the optimal management for om is continually evolving.3538 to spare the need for surgery and radiation, transpupillary thermotherapy was introduced to conservatively treat small um tumors.39 alternative therapies including ipilimumab, kinase inhibitors, and histone deacetylase (hdac) inhibitors are beginning to be investigated . In a recent study with 746 um patients, moser et al reported improved survival with the use of ipilimumab (28 months vs 13 months, p=0.07), compared to those receiving only local therapy.40 similar improvements in survival were seen with bevacizumab (25 months vs 12 months, p=0.09).40 genetic counseling to identify bap1 mutations are also currently under investigation.41 a 15-panel genetic assay has also been developed, which helps to risk stratify tumors and can be used to recommend participation in therapeutic trials . Epigenetic drugs, including dna methyltransferase (dnmt) inhibitors and hdac inhibitors, have been shown to have anticancer properties and are beginning to be investigated for ocular tumors with hopes of restoring normal control of neoplastic genomes.42,43 vidaza (5-azacytidine), a dnmt inhibitor, has been shown to reduce ocular metastasis to the lung in murine xenograft models.42,44 tenovin-6, which inhibits the class 3 hdac sirtuin 1 and 2, has shown promise in eliminating um tumor cells and cancer stem cells.45 despite the results of this study, several limitations should be taken into consideration . Factors such as tumor size, tumor depth, and socioeconomic status were not included in the seer database . Furthermore, data on diagnostic imaging and long - term follow - up were not reported . Data on whether or not surgery and radiation were utilized were available in the seer database; however, specific details pertaining to the surgical procedure, such as surgical margins and the drugs and dosages utilized for chemotherapy, were not reported . Lastly, since seer registries are more likely to sample from urban than from rural areas, there may also be a degree of selection bias . However, despite these limitations, the seer database contains data obtained from 26% of the us population, and these findings can be generalized to the overall population . Om is a rare variant of melanoma . Although um and cm are variants of om, they have different clinical behaviors . Both um and cm are most common in the seventh decade of life and most often found in caucasians and hispanics . Both um and cm present most commonly as localized tumors, with cm more likely to have regional and distant metastasis at presentation . Um has higher overall mortality and cancer - specific mortality than cm . Surgery is the primary therapy for cm, while radiotherapy is the primary therapy for um and results in prolonged survival compared to surgery alone . Males, older age, and distant disease are all associated with an increased risk of mortality in om, with primary surgical treatment being an additional risk factor in um . Novel therapies such as transpupillary thermotherapy and targeted therapy were introduced to manage tumors conservatively without the need for invasive surgery . Although a variety of alternative modalities have been used to treat um with varying degrees of success, it remains uncertain how these therapies will impact future management and clinical outcomes . A deeper understanding of these rare tumors among clinicians is vital to guide therapeutic decision making in terms of the choice and timing of management.
There is growing evidence that drug combinations can be more effective than the sum of individual drug effects . One course of therapy that demonstrated this theory is the administration of paclitaxel (ptx) combined with 5-fluoroucacil (5-fu) to patients with gastric, breast, pancreatic, and other types of cancer, each drug using a different mechanism to kill tumor cells . Although the use of combined drugs appears to be promising in cancer therapy, the problem of bioavailability is still encountered because the drugs are often removed from blood circulation by macrophages or other molecular components associated with drug clearance from circulation . In a conventional anticancer drug combination regimen, the combined drugs that have synergistic actions in vitro are injected into the blood together but will distribute and be eliminated independently of each other . For example, ptx and 5-fu have half - lives of 1 h and 7 min, respectively, in mice . The different pharmacokinetic properties make it impossible to control the molar ratio of the two drugs taken up by the same diseased cells . Drug carrier systems have been proposed to address this problem based on the knowledge that the pharmacokinetic characteristics of individual drugs will depend on the carriers . Oil in water (o / w) nanoemulsions (nes), a nanocarrier system, can offer various advantages to therapeutics and targeting, for example, improvements of the chemical and/or enzymatic stability of carried therapeutic agents, long - term colloidal stability, and ease of manufacture and scale - up . However, o / w nes are designed to deliver hydrophobic drugs, which limit their application for the delivery of combined drugs, particularly for the combination of hydrophobic and hydrophilic anticancer drugs . In order to improve the compatibility between the cargo and the core of nes, in the present study, ptx and 5-fu were first conjugated with the functional vitamin e (ve) and tocopherol poly(ethylene glycol)succinate (tpgs), with ve and tpgs representing the oil core and pegylated emulsifying agent in the nes, respectively . The conjugated ptx and 5-fu were then loaded into the nes based on the core - matched technology (cmt) proposed by our lab (figure 1). The solubility and the stability of ptx and 5-fu in the core - matched nes is increased due to more favorable interactions with the core by the functional molecules anchored to the nes . Ptx and 5-fu modified by ve and tpgs, respectively, can easily come into contact with the matched ve core contributing to the prevention of rapid drug release and the observation of satisfactory pharmacokinetic properties . In addition, ptx is known to be a substrate of p - glycoprotein (p - gp), which has been involved in the multidrug resistance (mdr) of several cancers due to its overexpression . Tpgs has been extensively investigated as an inhibitor of p - gp, and some tpgs - based nanoparticles or copolymers with similar structures have been proven to overcome mdr . Our previous study has also reported that ve causes significant reversal of mdr due to the inhibition of atpase activity . Ve and 5-fu tpgs cannot only be encapsulated into a single nanocarrier to produce synergism by cmt but also reverse mdr in cancer treatment (figure 1). Schematic illustration of the core - matched nanoemulsions codelivering ptx and 5-fu to achieve the synergistic effects and reversal of mdr . Our aim was to design core - matched nes with the following functions: (1) ability to codeliver hydrophilic and hydrophobic anticancer drugs effectively; (2) improvement of the poor pharmacokinetics of ptx and 5-fu by promoting long circulation times and synergistic effects in vivo; (3) reversal of resistance to ptx by enhancing its antitumor activity . Ve and 5-fu tpgs were synthesized in our lab (figure 2). The synthesis and characterization of ptx ve and 5-fu tpgs is provided in the supporting information (figures s1s4). 5-fluorouracil injection was purchased from app pharmaceuticals, llc (schaumburg, il). Antibodies against p-53, -tubulin, p - gp, gapdh, and horseradish peroxidase (hrp)-conjugated anti - mouse or anti - rabbit whole igg were obtained from santa cruz biotechnology (san diego, ca). Deadend fluorometric tunel assay kits were obtained from promega (madison, wi). Other chemicals were purchased from sigma - aldrich (st . Chemical structures and synthetic design of ptx ve (a) and 5-fu tpgs (b). Kb-3 - 1 and kb-8 - 5 cells originally obtained from american type culture collection (atcc) (manassas, va) were maintained in rpmi 1640 and dmem medium (invitrogen, carlsbad, ca) containing 10% fetal bovine serum (invitrogen, carlsbad, ca), 100 unit / ml penicillin, and 100 g / ml streptomycin (invitrogen, carlsbad, ca). All experiments performed on animals were in accordance with and approved by the institutional animal care and use committee at the university of north carolina at chapel hill . Ve, 5-fu tpgs, tpgs, and ve were first dissolved in chloroform . Ve (10 mg / ml), tpgs (50 mg / ml), and ve (150 mg / ml) and 125 l of 5-fu tpgs (15 mg / ml) were then separately withdrawn on ice and added to an eppendorf tube with a screw cap . The chloroform was evaporated under nitrogen gas, and trace amounts of chloroform were further removed by keeping the mixture under vacuum in a desiccator for 1 h. following the addition of 1 ml of distilled water, the mixture was sonicated using a sonic dismembrator model 100 (fisher scientific, pittsburgh, pa) to produce the ne . In order to obtain a more homogeneous and fine particle size ne, the mixture was homogenized in a bullet blender (next advance, averill park, ny) using zirconium oxide beads (1:2; 1.0 mm diameter/0.5 mm diameter). The instrument was set at the eighth speed for 15 min . Particle size and zeta - potential of the nes were determined by dynamic light scattering measurements using a malvern zetasizer nano series instrument (westborough, ma). The loading efficiency of ptx ve was assessed, following high - speed centrifugation to separate the free drug from nes, using a liquid scintillation analyzer (tri - carb, packard bioscience company, waltham, ma). The shape and surface morphology of the nes were observed using a jeol 100cx transmission electron microscope (tokyo, japan). Prepared samples (5 l) were dropped onto a 200 mesh carbon - coated copper grid (ted pella, inc ., redding, ca) and then wicked off from the grid after 3 min . Grids were then stained with 1% uranyl acetate (5 l) for 1015 s and wicked for tem . Kb-3 - 1 and kb-8 - 5 cells were seeded into 96-well plates at a density of 1 10 cells per well and allowed to adhere overnight . Various concentrations of drug or formulation were added to the plates for 48 h. following incubation, 20 l of mtt reagent (5 mg / ml in pbs) was added to the culture medium and the cells were incubated for an additional 4 h at 37 c . The culture medium was then carefully removed, and 200 l of dmso was added to the wells to dissolve the formazan . Uv absorbance was measured at 570 nm using a bio - rad microplate imaging system (hercules, ca), and results were expressed as% cell viability (od of treated group / od of control group 100). Kb-8 - 5 cells growing exponentially were seeded at 1 10 cells / ml in 12-well plates . Tpgs ne, or combined ne for 48 h. the pbs solution served as the control . Ice - cold 70% ethanol was used to fix the cells at 4 c overnight . After centrifugation and removal of the supernatant, the cells were resuspended with 1 ml of staining buffer and washed once . After repeat resuspension, the cells were incubated with 10 l of rnase a (10 mg / ml) at 37 c for 30 min and then stained with 5 l of propidium iodide (1 mg / ml) at room temperature for 30 min . The cell cycle analysis was performed using a facscanto flow cytometry system (bd biosciences, san jose, ca). Adherent cells in culture dishes were washed with ice - cold pbs, scraped using a cold plastic cell scraper, and then gently transferred, in suspension, to a precooled microcentrifuge tube . Whole cell lysates were extracted with ripa buffer, and the protein concentration was measured using a pierce bca protein assay kit (thermo scientific, rockford, il). For each sample, approximately 50 g of protein was separated on a nupage 12% sds polyacrylamine gel and then transferred to a polyvinylidene difluoride (pvdf) membrane (bio - rad, hercules, ca). The membrane was blocked with 5% skimmed milk in pbs for 1 h. after incubation with primary antibody at 4 c overnight, it was washed with pbst (0.2% tween 80 in pbs) and then incubated with secondary antibody for 1 h. antibodies against p - gp, p-53, -tubulin, and gapdh were used at 1:2000, 1:200, 1:500, and 1:2000 dilutions, respectively . An anti - mouse antibody conjugated with hrp at a dilution of 1:10000 or an anti - rabbit igg at a dilution of 1:2000 served as the secondary antibodies in the experiment . The specific protein bands were visualized using a chemiluminescence kit (thermo scientific, rockford, il). Chemiluminiscent signals were detected using high - performance chemiluminescence film (ge healthcare bio - sciences, pittsburgh, pa). In vivo antitumor activity was evaluated in kb-3 - 1 and kb-8 - 5 bearing nude mice . Nude mice were inoculated with 5 10 kb-3 - 1 cells or kb-8 - 5 cells injected subcutaneously into their right or left flanks to establish the xenograft model . Once the tumor mass in the xenograft was established, mice were randomly divided into 5 groups (5 mice per group) and were injected, in the tail vein, with normal saline (the control group), taxol, ptx ve ne, 5-fu tpgs ne, or ptx ve combined 5-fu tpgs ne . Drug doses of 5 mg / kg ptx, 8 mg / kg ptx ve, and 30 mg / kg 5-fu tpgs were used for all treatments . Therapy was continued at days 3, 5, 7, and 9 (five doses in total). Tumor volumes were calculated as (length width)/2 from measurements taken every second day . Toxicity of the formulations was determined by monitoring mice behavior and weight loss and by he staining . Kb-3 - 1 and kb-8 - 5 tumor - bearing nude mice were given iv injections of the five formulations on days 1, 3, 5, 7, and 9 . Tumors were fixed in 10% formalin for at least 24 h before being embedded in paraffin and sectioned at a thickness of 5 m . In vivo tumor cell apoptosis the tunel staining was performed as recommended by the manufacturer (promega, madison, wi), and dapi mounting medium was dropped on the sections for nucleus staining . Images of tunel - stained tumor sections were taken with a fluorescence microscope (nikon corp ., the tunel - positive cells were counted using imagej software (national institutes of health, bethesda, md). Female cd-1 mice (1822 g) were intravenously injected with ptx ve and 5-fu tpgs combined ne, taxol, or 5-fu injection at a volume of 0.2 ml (8 mg / kg ptx - ve, 5 mg / kg ptx, and 2.22 mg / kg 5-fu). Blood samples were digested in tissue solubilizer (ge healthcare bio - sciences, pittsburgh, pa) at a ratio of 1 mg/10 l . Then, 200 l of hydrogen peroxide (30%) was added to 100 l of sample for decolorization . Following this, a scintillation cocktail (ultima gold xr, perkinelmer, waltham, ma) was added and the decolorized samples were stored in the dark for 40 min . The radioactivity was then quantified using a liquid scintillation analyzer (tri - carb, packard bioscience company, waltham, ma). The concentrations of drugs in the samples were determined, following subtraction of blank background radioactivity, by extrapolation from standard calibration curves . The statistical significance of group differences was analyzed using the student s t - test, and a value of p <0.05 was considered significant . In the study, the average particle size of the core - matched nes was 82 nm with a pdi of 0.165, which was considered effective for passive targeting of tumors . The morphology of the core - matched nes examined by tem (figure 3 and figure s5) exhibited a uniform and spherical shape . The loading efficiency of ptx ve in the core - matched nes was> 95%, 6-fold more than that of ptx alone, due to the solubility of ptx ve in the ve oil phase . The combined core - matched nes had a negative zeta - potential of 11.6 mv . The in vitro release of ptx ve in the core - matched nes was slow (figure s6), and no burst effect occurred . Therefore, the core - matched nes effectively avoid problems reported with other ptx formulations, including low entrapment efficiency, drug instability, and rapid drug leakage . Many therapeutic agents hitherto have not been successful due to their limited ability to reach the target tissue . Thus, a drug delivery strategy that selectively targets the malignant tumor is necessary . The core - matched nes have properties suited to synchronously delivering, in vitro and in vivo, both hydrophobic and hydrophilic anticancer drugs because of their nanoscale size and their stability within the matched core . Data in figure 4a, b show that ptx ve and 5-fu tpgs had a toxic effect in both ptx - sensitive and -resistant cells . In particular, the combined core - matched nes had the most significant cytotoxicity in both cell lines compared with the ne of the individual prodrug . Ve ne in the resistant kb-8 - 5 cells was 1.27 m, lower than that in the sensitive kb-3 - 1 cells (2.67 m). However, the ic50 of ptx in resistant cells was as much as 3 times that determined in sensitive cells (see table 1). It was shown that the codelivery of ptx ve and 5-fu tpgs in one single drug carrier could improve the sensitivity of ptx in resistant cells through the reversal of mdr via ve and tpgs inhibiting the atpase activity . The in vitro cytotoxicity assay revealed that the combined core - matched nes improved the ability of the individual prodrugs to cause decreased viability of both sensitive and resistant kb cells . Ve core - matched nes (see figure 4c) due to its inability to transport through the cell membrane following its precipitation from dmso after addition to the 96-well plate . The result also showed that the core - matched ne carrier plays a vital role in the delivery of water - insoluble ptx - ve in vivo and in vitro . Ve ne, 5-fu tpgs ne, and the combined ne against (a) kb-3 - 1 cells, (b) kb-8 - 5 cells, and (c) free ptx ve and ptx ve ne in kb-8 - 5 cells (mean sd, n = 6). The concentrations on the x - axis represent the concentrations of ptx ve . The synergism of ptx ve and 5-fu tpgs in cytotoxicity was assessed by the chou - talalay method . The combination index was calculated by the following equation: ci (combination index) = (d)1/(dx)1 + (d)2/(dx)2, where dx is the dose of one compound alone required to produce an effect (ic50) and (d)1 and (d)2 are the doses of compounds 1 and 2 necessary to produce the same effect in combination . The ci values calculated for kb-8 - 5 and kb-3 - 1 cells, respectively, were 0.9 and 1.0 . The ci theorem of chou - talalay offers quantitative definition for additive effect (ci = 1), synergism (ci <1), and antagonism (ci> 1) in drug combinations . The results indicated that the combined nes have a synergistic effect on resistant kb-8 - 5 cells and an additive effect on sensitive kb-3 - 1 cells . This could be ascribed to the reversal of mdr produced by tpgs and ve . Cell cycle checkpoints are used to monitor and regulate the progress of the cell cycle . It is known that ptx can arrest sensitive tumor cells at the g2/m phase by an unusual stabilization of the microtubule . When resistant kb-8 - 5 cells were treated with ptx ve ne, cells of the g2/m phase increased from 27.3% in the untreated group to 52.6% (figure 5). Ve was converted to the parent drug ptx, which blocked the g2/m phase in the cell cycle, even within the resistant cells, resulting in the reversal of mdr . Typical flow cytometric diagrams and g2/m phase distribution of the kb-8 - 5 cells treated with ptx ve nes, 5-fu tpgs nes, and the combined nes for 48 h (mean sd, n = 3). It has been reported that 5-fu exhibits three modes of cell growth modulation, namely, loss or accumulation of s phase cells, g2/m block, and g1/s arrest . In our study, the g2/m phase arrest was evident in kb-8 - 5 cells treated with 5-fu tpgs; 53.2% of cells were arrested probably because cells proficient in dna mismatch repair showed marked modulation in the g2/m phase . Furthermore, the combined cme showed the most significant effect on the cell cycle, with 61.4% of cells being arrested at the g2/m phase . Tubulin is the target for anticancer drugs such as taxol, tesetaxel, vinblastine, and vincristine . Previous studies have shown that ptx can enhance the expression of -tubulin in sensitive kb-3 - 1 cell lines but not in ptx - resistant cell lines . In the present study ve cme and the combined core - matched nes up - regulated the level of -tubulin in the resistant kb-8 - 5 cell line (figure 6). The result is consistent with the cell cycle assay above, which showed g2/m phase enhancement . P - gp, a marker for mdr, showed no change in both the untreated and treated groups . Previous studies have elucidated that ve and tpgs reverse mdr by inhibiting the atpase activity of p - gp rather than by regulating p - gp expression . Western blotting assays for p - gp, -tubulin, and p53 protein extracted from kb-8 - 5 cells treated with ptx ve nes, 5-fu tpgs nes, or the combined nes for 48 h. the p53 tumor suppressor is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad range of cellular damage 5-fu exerts its anticancer effects through incorporation of its metabolites into rna and dna interfering with their normal functions . Our previous studies have shown that both 5-fu and 5-fu pgs can increase the expression of p53 . Figure 6 shows that 5-fu pgs core - matched nes and the combined core - matched nes facilitate the up - regulation of p53 . This up - regulation is consistent with the reported ability of p53 to induce cell cycle arrest at the g2/m boundary of the cell cycle . As shown in figure 7a, the reductions in tumor volume observed in nude mice treated with ptx ve or 5-fu tpgs core - matched nes compared to control were not significant . A synergistic effect was observed in the combined core - matched nes, which exhibited significant inhibition of tumor growth compared to the control group . However, taxol showed a significantly reduced kb-3 - 1 tumor volume when compared to the other four groups . One was that kb-3 - 1 is a ptx - sensitive cell line, and the other is that the slow hydrolysis rate of ptx tumor growth inhibition in subcutaneous tumor models of (a) kb-3 - 1 cells and (b) kb-8 - 5 cells . Tissue he staining (c) after injection of the combined nes, via the tail vein, every second day for 10 days . All data are expressed as mean sd (n = 5); * p <0.05 for combined nes vs the individual nes and p <0.05 for combined nes vs the group treated with taxol . Advantageous properties of the core - matched nes loaded with both prodrugs were observed in the nude mice bearing resistant kb-8 - 5 tumors (figure 7b). The results showed that the core - matched nes were able to significantly inhibit the kb-8 - 5 tumor growth compared with the other four groups (tumor growth was reduced by 57.7% [t - test, p <0.05]). First, the core - matched technology prolonged the circulation of drug in vivo (data provided in pharmacokinetics study), facilitating greater drug accumulation in the tumor . Second, the codelivery of ptx and 5-fu in the prodrug form based on core - matched technology produced a synergistic effect . Third, ve and tpgs included in the prodrug and core - matched ne formulations help to reverse mdr . Side effects are one of the major problems of cancer chemotherapy . In this study, the toxicity of the combined core - matched nes on liver, spleen, and kidney was investigated using he staining after long - term treatment . Figure 7c showed no obvious damage observed in these tissues after treatment with the core - matched nes . Consistently, there was no decrease in body weight or noticeable change in activity . Ve and 5-fu tpgs core - matched nes caused the resistant tumor cell death . As illustrated in figure 8, ptx ve and 5-fu tpgs core - matched nes induced the most effective apoptosis of cells in kb-8 - 5 xenograft tumors compared with the control and individual prodrug nes . The percentage of apoptotic cells (32.1 7.6%) for the combined nes was 7-fold higher than for taxol (figure 8). The tunel assay for the sensitive kb-3 - 1 tumor cells indicated that taxol was able to induce significant cell apoptosis compared with other groups (figure s7). The results were consistent with the antitumor effect seen in the sensitive cell tumor (figure 7a). (a) tunel assay of the tumor sections from kb-8 - 5 tumor - bearing nude mice after a schedule of 5 doses . Tunel - positive cells are shown as green dots, and the nuclei stained by dapi are blue . (b) qualification of apoptosis; p <0.05 vs the control, p <0.05 vs the group treated with taxol, and p <0.05 vs the group treated with individual nes . The p53 is involved in chemosensitivity, and the loss of p53 function has been reported to enhance cellular resistance to a number of chemotherapeutic agents . The extraordinary enhancement of cell apoptosis, and hence reversal of mdr, observed in our study was ascribed to the up - regulation of p53 and the arrest of the g2/m phase in the cell cycle . Anticancer drugs gain access to solid tumors via the circulatory system and must penetrate through the extravascular space to reach cancer cells in sufficient concentration to cause lethal toxicity . These considerations will most likely result in limited distribution of chemotherapy drug in solid tumors and potentially give rise to clinical resistance . It is therefore necessary to improve delivery of the drug to the tumor and to prolong the blood circulation of the drug so that, aided by the epr effect, lethal concentrations in the tumor cells can be achieved . It is known that peg is the most widely used moiety for surface modification of nanoparticles, and pegylation can effectively retard the rapid uptake of nanoparticles by the mononuclear phagocyte systems . For this reason, 5-fu in our study was conjugated with tpgs and tpgs was also included in the core - matched nes . As figure 9a, b and table 2 show, the auc(0) of both ptx and 5-fu following the administration of the core - matched nes was much higher than after administration of taxol or 5-fu injection . The cmax of ptx in nes was found to be 64.9 mg / l, whereas the cmax following administration of taxol was 1.9 the mrt(0-t) of ptx from the core - matched nes was 29.6 h, significantly higher than that following taxol administration (1.3 h). This showed that ptx from core - matched nes existed in vivo much longer than ptx from taxol . Data in table 2 show that the elimination of 5-fu after administration of core - matched nes was also slower than after 5-fu injection . The half - life of 5-fu from the nes was nearly 3.5 times longer than that of 5-fu following administration of the 5-fu injection . The mice were injected intravenously with taxol, 5-fu injection, or core - matched nes (equal dose of 5 mg ptx / kg and 2.2 mg 5-fu / kg) containing 1 ci of tritium - labeled ptx and 5-fu . The results indicated that the core - matched nes facilitate the long circulation of ptx and 5-fu, which is closely associated with the high accumulation of chemotherapeutic agents within tumors and the improvement of antitumor efficacy . Although the elimination of 5-fu in the core - matched nes was more rapid than the elimination of ptx, it was still much improved when compared with the 5-fu injection (figure 9b). The antitumor activity observed has also shown the benefit of the combinational delivery with respect to the pharmacokinetics . In order to prolong the circulation of 5-fu even more, in future studies, it is planned that the 5-fu be conjugated with ve in order to avoid its loss through shedding of peg derivative from the interface of nes . Based on the core - matched technology proposed by our lab, ptx and 5-fu were coencapsulated into a single nanoemulsion in the form of prodrugs with high entrapment efficiency (> 95%), good stability, and fine particle size (<90 nm). The combined core - matched nes facilitate the long circulation of anticancer drugs in vivo, prevent drug loss through rapid clearance and metabolism, and render more drug accumulation in the tumor . Moreover, the codelivery of ptx and 5-fu in the single core - matched nes contributes to reversal of mdr and the production of profound synergism . All of these advantages result in the significant inhibition of resistant tumor growth with no obvious toxicity.

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