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Tuberculosis, one of the oldest diseases known to affect human being, is caused by bacteria belonging to the mycobacterium tuberculosis complex . Transmission usually takes place through airborne spread of droplet nuclei produced by patients with infectious pulmonary tuberculosis . Oral mucosa is a rare location for tubercular infection, and it may either be primary or more often secondary infection . Different areas of oral cavity like floor of mouth, soft palate, gingiva, lips, hard palate can be involved; however, hard palate and tongue are the commonest sites of involvement for oral tuberculosis . A 40-year - old male patient presented to our outpatient department because of difficulty in swallowing solid food for two months . This patient also complained of a painless ulceration in his soft palate, malaise, and weight loss since last two months . There is no history of rise of temperature, cough, hemoptysis, hoarseness of voice, regurgitation of food . Clinical examination revealed an irregular area of 3 2 centimeters over the soft palate and uvula . Hard palate, tonsil, tongue, pharynx were within normal limit [figure 1]. The regional cervical group of lymph nodes was not palpable when the patient presented to us . Palatal ulcer before treatment erythrocyte sedimentation rate was raised (77 mm / hour). Nasal endoscopy, fiberoptic laryngoscopy, barium swallow x - ray of esophagus, and x - ray paranasal sinus (water's view) were within normal limit . Punch biopsy was taken from the ulcerated lesions over the soft palate under topical anesthesia; bleeding controlled by application of local pressure . Histopathological examination of the tissue revealed presence of epithelioid cells, mononuclear inflammatory cells, langhans type of giant cells forming granulomas with focal caseous necrosis [figure 2]. Staining for acid - fast bacillus granulomatous inflammation pattern on histology (h and e, 400) in accordance with the existing guidelines, the patient was administered anti - tubercular medication (cat 1 four drugs for two months followed by two drugs for four months). His difficulty in swallowing reduced rapidly; his ulceration regressed within one and half months of chemotherapy . Although tuberculosis has definite affinity for lungs, it can affect any part of body including oral cavity . Oral manifestation of tb tuberculous involvement of the oral cavity is extremely rare, with incidence ranging from . 05 - 5% . An intact and healthy mucosa seems to provide a sufficient barrier to mycobacteria, with saliva also helping to control the organisms . Although oral tuberculosis has been well - documented, tuberculous lesions of the upper aerodigestive tract have become rare . Oral tuberculosis most commonly results from contact of infected sputum with oral mucosa or hematogenous dissemination in an older individual with pulmonary disease . In contrast, cases of primary infection arising through direct mucosal invasion by mycobacteria are uncommon and typically are seen in young patients, who often present with cervical lymphadenopathy with or without cutaneous sinus formation . The sites demonstrating the most frequent involvement with primary tuberculosis are the gingivae, vestibular mucosa, and extraction sockets . Mucosal lacerations and dental extractions have been implicated as predisposing an individual to the development of oral tuberculosis . Traditionally, the diagnosis of tuberculosis has been made on the basis of clinical, radiographic findings, and sputum examination . The diagnosis of oro - facial tuberculosis can be quite challenging, mainly because of a lack of definite signs and symptoms . According to pandit et al ., (1995), when considering the overall prevalence of tuberculosis of indian population, the presence of epithelioid cell granuloma is indicative of disease unless proven otherwise . 1991) reported two cases of primary tb of the oral cavity where smears and culture for afb, from the oral lesion and the sputum, were negative . They confirmed the diagnosis solely on the basis of history and histopathological examination, which only revealed giant cells and epithelioid cells . Oral ulceration can be a manifestation of primary syphilis and fungal diseases, and non - infectious processes such as chronic trauma and squamous cell carcinoma . Multinucleated giant cells of langhans type are frequently seen in various granulomatous lesions such as tuberculosis, leprosy, syphilis, sarcoidosis, crohn's disease, eosinophilic granuloma, and certain fungal diseases . Lepromatous leprosy lesions are associated with the involvement of superficial nerves leading to anesthesia and paresthesia, which may cause unrealized trauma leading to ulcers and secondary infection . Crohn's disease manifests itself as granulomatous nodules and ulcers in the oral cavity along with associated gastrointestinal symptoms . Fungal lesions such as histoplasmosis, blastomyosis, and coccidiodomycosis should also be considered during diagnosis of an oral lesion . Microscopically, organisms can be identified with stains such as hematoxylin and eosin (h and e), periodic acid - schiff (pas), or methenamine silver . Sporangia may be found free within necrotic tissue or within the epithelioid cells and giant cells of the granuloma . Fungal cultures can be an aid in identification of specific fungal species . In our patient, we got epithelioid cell granuloma, also the giant cells and caseous necrosis in histopathologic examination; his mantoux test elicited a strongly positive reaction with an induration of 14 18 mm after 72 hours, and patient was managed solely with anti - tubercular chemotherapy.
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Medical and paramedical staff, particularly in service in emergency planning, are frequently exposed to situations of great physical and psychological stress . An increasing number of articles in the literature have focused on the development of psychic and somatic symptoms in the health workforce . Emergency departments, in fact, may be challenging because of frequent unpredictability of daily work cases, coping with the acute phase of most disorders, and frequent facing of patient and their families expectations in unexpected critical cases / situations . This has induced an increasing number of researchers to investigate work - related psychological disorders, such as burnout syndrome (bos) and, more recently, posttraumatic stress disorder (ptsd).15 burnout denotes an occupational health concept of emotional and physical exhaustion, depersonalization, and decreased personal accomplishment . Bos has been defined as a state of physical, emotional and mental exhaustion caused by long - term involvement in emotional demanding situations experienced in the workplace.2 this definition highlights bos as a psychological state resulting from prolonged exposure to job stressors . Prevalence rates range from 10% to 50%, depending on profession, assessment tools, and population.2,3,69 literature has increasingly investigated ptsd prevalence rates, both in its full and subthreshold forms, as well as posttraumatic stress symptoms, among these same populations, upon the acknowledgment of the stressful characteristics of this kind of employment.2,7,9 emergency workers, in fact, must cope with a variety of duty - related stressors, including traumatic incident exposure . Accordingly, the diagnostic and statistical manual of mental disorders fifth edition (dsm-5) itself explicates, among the changes introduced in ptsd diagnostic criteria, that repeated or extreme indirect exposure to aversive details of the event(s), usually in the course of professional duties (eg, first responders, collecting body parts; professionals repeatedly exposed to details of child abuse)10 can be considered as a stressor (criterion a4). Studies aimed at assessing ptsd prevalence rates among emergency unit operators reported values between 10% and 17%.9,1114 a study on emergency rescuers serving in london15 reported work stress to be the most important morbidity factor, with a ptsd diagnosis reported in 15% of the individuals exposed to traumatic events while on duty . Clohessy and ehlers,13 in a study on a sample of 56 ambulance service workers of the oxfordshire ambulance nhs trust, found a ptsd prevalence rate of 21% according to dsm - iv16 criteria . The most reported symptoms were intrusive memories (49%), irritability (51%), and insomnia (47%). More recently, mealer et al2 reported a ptsd diagnosis in 18% of 332 university hospital nurses in the usa, while ptsd symptoms were reported in 22% of the same sample . Accordingly, an extensive systematic review17 of all empirical articles regarding emergency medical responders and conceptual literature on the constructs of interest in other related populations reported exposure to traumatic events to be between 80% and 100%, and rates of ptsd higher than 20% . In this same review, a modification of the stress process model is suggested to explain the relationships among occupational stress exposure, ptsd, and high risk of alcohol and other drug use . Most recently, fjeldheim et al5 reported 94% of 131 south african university paramedic trainees to have directly experienced trauma, with 16% meeting ptsd criteria and 7% chronic perceived stress, suggesting the need for efficient, ongoing screening of ptsd symptomatology in trauma - exposed high - risk groups . Recently, some authors have explored ptsd prevalence rates among emergency workers not only in its full - blown manifestation but also in partial and subthreshold forms.13,1820 clohessy and ehlers,13 in a study on 56 ambulance service workers in the united kingdom, found that 21% of responders met dsm - iii - text revised criteria for ptsd, while 22% met screening criteria for psychiatric symptoms . Mealer et al2 reported ptsd symptoms in 22% of the nurses operating in a university hospital, while genest et al22 evidenced the onset of intrusive symptoms in emergency operators who attempted basic life support . In the framework of an international italian spectrum - project, between researchers of the universities of pisa (italy), pittsburgh (usa), columbia (new york, usa), and california at san diego (usa), established to develop and test assessment instruments for assessment of the spectrum of clinical features associated with dsm psychiatric disorders, some researchers developed a multidimensional spectrum approach to ptsd . The spectrum model proposed highlights the significance of isolated symptoms and subthreshold symptom clusters that accompany each disorder classified in the dsm, and may follow, or be manifested, in concurrence with the main disorder.23 accordingly, the trauma and loss spectrum self - report (tals - sr)24,25 explores the spectrum related to ptsd upon a multidimensional approach across three major dimensions: that of the traumatic event, including the so - called low - magnitude events; that of the acute reaction; and that of the symptomatological clusters including maladaptive behaviors . Most recently, data on the ptsd symptomatological prevalence data according to dsm-5 criteria among survivors to natural and human - made disasters have been reported by some of researchers.26,27 little data are yet available on dsm-5 ptsd prevalence rates among operators of the emergency units in europe, particularly in italy . The aim of this study was to assess dsm-5 ptsd, as well as posttraumatic stress spectrum symptomatology, among the staff working in the emergency units of a major university hospital in central italy . A further aim of this study was to examine the impact of ptsd and posttraumatic stress symptoms on the work and social functioning of the same persons . A total sample of 110 persons employed, at the time of enrollment, at the emergency units (emergency medicine and emergency room) of the azienda ospedaliero - universitaria pisana (aoup) was included in this study . The ethics committee of the university of pisa approved all the procedures of evaluation and recruitment . Suitable candidates provided written informed consent after receiving an exhaustive description of the study and after having the opportunity to ask any questions in reference to the study . All persons were assessed by means of the tals - sr25 for posttraumatic stress spectrum symptoms and the work and social adjustment scale (wsas28) for work and social functioning . The tals - sr is a questionnaire developed for assessing posttraumatic stress spectrum symptoms.24,25 it includes 116 items exploring the lifetime experience of a range of losses and/or traumatic events and lifetime symptoms, behaviors, and personal characteristics that might represent manifestations and/or risk factors for the development of a stress response syndrome . The instrument is organized into nine domains including loss events (i), grief reactions (ii), potentially traumatic events (iii), reactions to losses or upsetting events (iv), re - experiencing (v), avoidance and numbing (vi), maladaptive coping (vii), arousal (viii), and personal characteristics / risk factors (ix). The responses to the items are coded in a dichotomous way (yes / no), and domain scores are obtained by counting the number of positive answers . In accordance with the aims of the study, all participants were asked to report symptoms related to work - related trauma exposure . According to previous studies29,30 a dsm-5 diagnosis of ptsd was assessed by using the following matching between dsm-5 symptoms criteria and tals - sr items: criterion b (b1 = 80; b2 = 77; b3 = 79; b4 = 78; b5 = 81);criterion c (c1 = 86; c2 = 87 and/or 88 and/or 89);criterion d (d1 = 90; d2 = 95; d3 = 85; d4 = 96; d5 = 91; d6 = 93; d7 = 92); andcriterion e (e1 = 108; e2 = 99 and/or 100 and/or 102; and/or 103 and/or 104; e3 = 106; e4 = 107; e5 = 105; e6 = 109). Criterion b (b1 = 80; b2 = 77; b3 = 79; b4 = 78; b5 = 81); criterion c (c1 = 86; c2 = 87 and/or 88 and/or 89); criterion d (d1 = 90; d2 = 95; d3 = 85; d4 = 96; d5 = 91; d6 = 93; d7 = 92); and criterion e (e1 = 108; e2 = 99 and/or 100 and/or 102; and/or 103 and/or 104; e3 = 106; e4 = 107; e5 = 105; e6 = 109). Due to the sample characteristics, the wsas is a test used to evaluate and measure the work and social adjustment . It includes five items that assess the individual s ability to perform the activities of everyday life and how these are affected in the week prior to the assessment . The second item assesses the ability to cope with household chores such as cleaning the house, looking after the children, and doing the shopping . The third item assesses private recreational activities carried out by the patient, such as going to the cinema, visiting museums, and reading . The fourth and fifth items investigate the family interaction and relationship: in particular, the fourth item investigates the social activities carried out exclusively with people who are not part of the family and includes activities such as parties, tours of pleasure, going clubbing, or going on romantic dates . The fifth item analyzes only the relations with family members with whom the person lives, and whether any problems of the subject under examination have interfered with this type of relationship . Each of the five items is rated on a nine - point scale ranging from 0 (not at all) to 8 (severe interference), so that the total scores are between 0 and 40 . To compare the groups, we used the chi - square test (or fisher when necessary) in the case of categorical variables, student s t - test for quantitative variables with gaussian distribution, and kruskal wallis and mann whitney tests for the ones with no gaussian distribution (see the total score wsas and the total numbers of ptsd criteria satisfied). To study the relation between the total wsas scores and the total numbers of ptsd criteria satisfied, we calculated spearman s correlation coefficient . All the processing statistics were conducted using the statistical package for social science (spss inc ., a total sample of 110 persons employed, at the time of enrollment, at the emergency units (emergency medicine and emergency room) of the azienda ospedaliero - universitaria pisana (aoup) was included in this study . The ethics committee of the university of pisa approved all the procedures of evaluation and recruitment . Suitable candidates provided written informed consent after receiving an exhaustive description of the study and after having the opportunity to ask any questions in reference to the study . All persons were assessed by means of the tals - sr25 for posttraumatic stress spectrum symptoms and the work and social adjustment scale (wsas28) for work and social functioning . The tals - sr is a questionnaire developed for assessing posttraumatic stress spectrum symptoms.24,25 it includes 116 items exploring the lifetime experience of a range of losses and/or traumatic events and lifetime symptoms, behaviors, and personal characteristics that might represent manifestations and/or risk factors for the development of a stress response syndrome . The instrument is organized into nine domains including loss events (i), grief reactions (ii), potentially traumatic events (iii), reactions to losses or upsetting events (iv), re - experiencing (v), avoidance and numbing (vi), maladaptive coping (vii), arousal (viii), and personal characteristics / risk factors (ix). The responses to the items are coded in a dichotomous way (yes / no), and domain scores are obtained by counting the number of positive answers . In accordance with the aims of the study, all participants were asked to report symptoms related to work - related trauma exposure . According to previous studies29,30 a dsm-5 diagnosis of ptsd was assessed by using the following matching between dsm-5 symptoms criteria and tals - sr items: criterion b (b1 = 80; b2 = 77; b3 = 79; b4 = 78; b5 = 81);criterion c (c1 = 86; c2 = 87 and/or 88 and/or 89);criterion d (d1 = 90; d2 = 95; d3 = 85; d4 = 96; d5 = 91; d6 = 93; d7 = 92); andcriterion e (e1 = 108; e2 = 99 and/or 100 and/or 102; and/or 103 and/or 104; e3 = 106; e4 = 107; e5 = 105; e6 = 109). Criterion b (b1 = 80; b2 = 77; b3 = 79; b4 = 78; b5 = 81); criterion c (c1 = 86; c2 = 87 and/or 88 and/or 89); criterion d (d1 = 90; d2 = 95; d3 = 85; d4 = 96; d5 = 91; d6 = 93; d7 = 92); and criterion e (e1 = 108; e2 = 99 and/or 100 and/or 102; and/or 103 and/or 104; e3 = 106; e4 = 107; e5 = 105; e6 = 109). Due to the sample characteristics, the wsas is a test used to evaluate and measure the work and social adjustment . It includes five items that assess the individual s ability to perform the activities of everyday life and how these are affected in the week prior to the assessment . The second item assesses the ability to cope with household chores such as cleaning the house, looking after the children, and doing the shopping . The third item assesses private recreational activities carried out by the patient, such as going to the cinema, visiting museums, and reading . The fourth and fifth items investigate the family interaction and relationship: in particular, the fourth item investigates the social activities carried out exclusively with people who are not part of the family and includes activities such as parties, tours of pleasure, going clubbing, or going on romantic dates . The fifth item analyzes only the relations with family members with whom the person lives, and whether any problems of the subject under examination have interfered with this type of relationship . Each of the five items is rated on a nine - point scale ranging from 0 (not at all) to 8 (severe interference), so that the total scores are between 0 and 40 . To compare the groups, we used the chi - square test (or fisher when necessary) in the case of categorical variables, student s t - test for quantitative variables with gaussian distribution, and kruskal wallis and mann whitney tests for the ones with no gaussian distribution (see the total score wsas and the total numbers of ptsd criteria satisfied). To study the relation between the total wsas scores and the total numbers of ptsd criteria satisfied, we calculated spearman s correlation coefficient . All the processing statistics were conducted using the statistical package for social science (spss inc ., chicago, il, usa), version 22 . Complete data were available for 83 (75.5%) persons because the remaining persons gave partial response or no response at all . Of the 83 persons included, 30 (36.1%) were males and 53 (63.9%) were females, with a mean age (sd) of 40.78.5 years . In particular, among all the responders, 15 (18.1%) were doctors, 51 (61.4%) were nurses, and 17 (20.5%) were health - care assistants . Further, 59 (71.1%) had a bachelor s degree or an advanced degree (these included not only doctors but also nurses, particularly younger ones who hold a university degree), while the remaining 24 (28.9%) were not graduates (including both some nurses and all of the health - care assistants). The department in which persons were employed was evaluated for the health - care assistants and the nursing staff only . All the doctors, in fact, were subject to rotation and served alternately in both departments . Among the health - care assistants and nurses, 26 (38.2%) were serving in the department of emergency medicine, while 42 (61.8%) were working in the emergency room . Clinical and demographic characteristics of the study sample are reported in table 1 . With regard to the prevalence of ptsd, 13 persons (15.7%, confidence interval [ci] 99%: [5.4%26.0%]) met dsm-5 criteria for the diagnosis of the disorder: one doctor (6.7%), four nurses (23.5%), and eight health - care assistants (15.7%). Of these, 3 were males (10.0%) and 10 were females (18.9%); 5 were below the age of 40 years (12.2%) and 8 over the age of 40 years (19.0). Concerning the education level, seven persons with ptsd had a bachelor s degree (11.9%), while six had a lower level of education (25.0%). As far as the comparisons of the mean ranks of the total wsas scores are concerned (mean sd, mean rank), females reported significantly higher scores than males (7.748.16 vs 4.375.79, p=0.022) (table 2). When correlating the total number of ptsd criterion symptoms satisfied (corresponding to the total number of tals - sr items corresponding to dsm-5 diagnostic criteria) and the total wsas scores, we found a moderate / strong significant correlation among health - care assistants (r=0.565, p=0.018) and nongraduates (r=0.428, p=0.037). Further, a moderate significant correlation was found among women (r=0.310, p=0.024) (table 3). Finally, nongraduates had significantly higher tals - sr domain iv (reaction to potentially traumatic events) mean scores compared to those of graduates (6.463.37 vs 4.813.04, p=0.033). Further, a significantly higher proportion of the former category reported at least one maladaptive item on the tals - sr compared to the latter category (7 [29.2%] vs 6 [10.2%], p=0.045). To the best of our knowledge, this is the first study exploring dsm-5 ptsd prevalence rates, as well as posttraumatic stress spectrum symptoms, in a sample of italian emergency service workers of a major university hospital . Our results show a ptsd prevalence of 15.7%, with higher, despite not significant, percentages among nurses and health - care assistants, women, older persons and nongraduates, as well as in those operating in the emergency room . Nongraduates also reported significantly higher mean scores on the tals - sr domain iv, exploring the acute reaction to potentially traumatic events, as well as a significantly higher proportion of persons presenting at least one maladaptive behavior, with respect to operators with graduate degrees . Among women, significantly higher scores on the wsas also emerged compared to men . When correlating the total number of ptsd criterion symptoms satisfied and the total wsas scores, a strong significant correlation emerged in health - care assistants and nongraduates, while a moderate significant correlation was found in women . The ptsd rate reported in the study sample is remarkable if compared to the recently published ptsd prevalence in the italian general population of 2.3%, diagnosed in accordance with dsm - iv - text revised criteria31 in the framework of the european study of the epidemiology of mental disorders (esemed).32,33 although caution has to be adopted because the two studies utilized different methods in the sampling and the diagnostic criteria adopted, and thus data cannot be directly compared, a possible higher vulnerability to the disorder can be seen in this professional category in italy . If we consider our sample, which enrolled all the staff employed at the aoup, as being possibly representative of italian emergency operators, it is important to highlight that we reported a ci 99% far from the 2.3% of de girolamo et al,32 and thus the ptsd rates could be considered potentially significantly different (p<0.01). In previous reports, some researchers explored symptomatological ptsd prevalence rates assessed by means of the tals - sr in accordance with either dsm - iv - tr or dsm-5 criteria in the general population exposed to a massive earthquake, reporting substantial correspondence.29,30 this may indirectly corroborate a substantial increase in ptsd rates among emergency workers with respect to the general population in italy . Noticeably, our results are also in line with previous reports conducted on rescuers and emergency workers across europe where ptsd prevalence rates ranging between 10% and 21% have been reported.9,1113,34 clohessy and ehlers,13 exploring a sample of paramedics and emergency medical technicians in the uk, showed that 21% of enrolled persons were affected by ptsd as diagnosed using both dsm - iii and dsm - iv . It is also important to highlight that despite the fact that most of the studies involved nurses and paramedics, only scant data are available on the medical staff . Maia and ribeiro,35 exploring 59 among nurses and medical staff from the national institute of medical emergency in the north of portugal, reported high exposure to events evaluated as traumatic and low prevalence of ptsd . It is important to note that most of the studies reporting higher ptsd prevalence rates are those conducted on paramedics and nursing staff, with our results showing lower, albeit not significant, rates in doctors compared to the other workers who are in line with these findings . However, caution should be adopted when interpreting our data, as a potential explanation for our findings would be that undergraduate emergency workers typically begin to work with trauma at younger ages than nurses and doctors do . There is evidence, in fact, that younger individuals are at higher risk of developing ptsd from the same trauma as opposed to older peers36 and that the more one is exposed to trauma, the higher is his / her risk of developing ptsd; thus, we may argue that having started working at an earlier age makes undergraduate workers more globally exposed to trauma than their colleagues . A third potential bias that should be taken into account in larger samples is related to the reasons for choosing emergency care professions; it is in fact more likely that these may be chosen by individuals with traumatic backgrounds, thus affecting the rates of possible complex ptsd . There is agreement on the fact that ptsd affects females about twice as much as males, also in italian samples, with more severe symptomatology.26,33,3741 however, studies exploring the possible confounding role of other risk factors, including work - related training and education, reported conflicting results.42,43 some authors, in fact, highlighted that differences between the sexes may be compensated for by adequate professional training, as shown in rescue workers, police agents, or firefighters, particularly in the united states.4345 our results seem to be in line with these reports despite needing to be corroborated in larger samples as they might be affected by the fact that a greater percentage of women reported lower education . Similarly, our data are in line with studies reporting a relationship between education level and ptsd . In this regard, epidemiological data from the esemed study in italy reported significantly higher ptsd rates in persons with lower instruction levels.33 as previously mentioned, our results also highlighted significantly higher scores on the tals - sr domain iv, exploring symptoms of acute reaction to trauma, and a significantly higher proportion of persons presenting at least one maladaptive behavior, with respect to operators with a graduate degree . Increasing literature has documented the relationship between acute distress reaction and ptsd, highlighting ptsd as the ideal candidate for secondary prevention programs.46,47 in this regard, maia and ribeiro35 found peritraumatic dissociation and distress symptoms to be the only predictors of ptsd symptoms among 59 nurses and medical staff from the national institute of medical emergency in the north of portugal . Maladaptive behaviors, including suicidal behaviors and substance use, fully entered the dsm-5 diagnositc criteria for ptsd and were repeatedly associated with higher severity of the disorder.4852 fjeldheim et al5 reported a rate of 23% of alcohol abuse in south african university paramedic trainees who directly experienced trauma . Our data as a whole seem to thus suggest an increased severity of posttraumatic stress reaction in emergency workers with lower educational levels . Consistently, we also found a strong significant correlation between the total number of ptsd symptoms and impairment in work and social adjustment in health - care assistants, nongraduates, and women . Some important limitations of the study should be borne in mind when interpreting our results . The most important is related to the limited sample size that may impact on the generalizability of our results . However, we underline the novelty of this study, which, to the best of our knowledge, is the first in the literature to report on ptsd and posttraumatic stress spectrum in emergency workers in italy . Second is the need of a standardized interview such as the structured clinical interview for dsm disorders to demonstrate the prevalence of ptsd suggested from our symptomatological data . Third, we may argue the fact that most severely avoidant ptsd cases may have not been enrolled because this same symptomatology prevented them from fulfilling the instruments . Fourth, we used a self - report instrument, instead of the rating of the clinician, in order to detect ptsd symptoms and also make the diagnosis and be influenced by co - occurring events . Nevertheless, the use of tals - sr allowed us to explore dsm-5 criterion symptoms . Fifth, as already mentioned in our first study, there is the lack of information on the presence of axis i psychiatric comorbidities . Despite the limitations mentioned above, this report suggests emergency health professionals to be at high risk for posttraumatic stress spectrum and related impairments in work and social adjustment . The increased vulnerability reported in specific subgroups, particularly women and operators with lower levels of education, should be further investigated in larger samples, with the aim to help identify specific education and training interventions for prevention programs.
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Disturbances in fatty acid metabolism may cause several key features of the insulin resistance syndrome, including impaired glucose regulation, dyslipidemia, and obesity . Thus systemic free fatty acid (fa) oversupply can decrease insulin - stimulated glucose uptake in skeletal muscle [1, 2], reduce insulin suppression of hepatic glucose production, and alter glucose - stimulated insulin secretion . Furthermore, an oversupply of fa to liver may cause dyslipidemia, including hypertriglyceridemia and the atherogenic lipoprotein profile . With regard to fa utilization, reductions in mitochondrial content and diminished fatty acid oxidation capacity in skeletal muscle and adipose tissue have been linked with obesity and insulin resistance [614]. Additionally, in conditions of impaired insulin action there is also a reduced ability to appropriately switch between glucose and lipid fuels (i.e., metabolic inflexibility), postulated to play an important role in the development of obesity . The obese zucker rat is an animal model possessing major metabolic features seen in conditions of human insulin resistance, including glucose metabolic insulin resistance, hypertriglyceridemia, and elevated nonadipose tissue lipid levels [16, 17], which have been implicated in both the development of insulin resistance and lipoapoptosis in tissues including the pancreas and heart . Inappropriate deposition of triglycerides and other bioactive fatty acid metabolites in a tissue may result from a systemic oversupply of fatty acid or from a local defect in fatty acid oxidation . The obese zucker rat has a loss - of - function mutation in the leptin receptor, and although leptin has been shown to enhance local rates of lipid oxidation [19, 20], the relative contribution of reduced fa utilization to the lipotoxic state observed in tissues of these animals in vivo is still not well established . We were interested in elucidating the mechanisms of lipid accumulation in non - adipose tissues of obese zucker rats: oversupply or underutilization? The aim of this study was therefore to determine how the fluxes and metabolic fate of fa are altered in obese versus lean zucker rats . Series 1 quantified fa uptake and metabolic fate in vivo at the individual tissue level based on the simultaneous combined use of [9,10-h]-(r)-2-bromopalmitate (h - r - brp) and [u - c]-palmitate (c - p). The h - r - brp tracer is used to estimate local fa (oxidative + nonoxidative) utilization while c - p is used to assess non - oxidative fa disposal into lipid stores . Series 2 examined fa oxidation and its dependence on fa levels, using nicotinic acid as an antilipolytic agent . Finally, series 3 was used to assess fa oxidation capacity in a comprehensive range of metabolically important tissues ex vivo . Our results give strong support for the hypothesis that the major factor responsible for tissue lipid accumulation in obese zucker rats is increased plasma fa availability . Experimental procedures were approved by the local ethics review committee on animal experiments (gteborg region). Male 8-week old lean (fa / fa) and obese (fa / fa) zucker rats (charles river wiga gmbh, suffield, frg) were maintained in a temperature controlled (2022c) room with a 12 h light - dark cycle (lights on at 06:00) and free access to rodent chow (r3 laktamin ab, stockholm, sweden) and tap water . At 07:00 on the morning of the study food was withdrawn . Then at 09:00 the rats were anaesthetized with na - thiobutabarbital (inactin, rbi, natick, ma), with the lean and obese rats receiving 120 and 180 mg kg (i.p . ), respectively . Body temperature was monitored using a rectal probe and maintained at 37.5c throughout the experiment . Animals were tracheotomized and catheters were placed in the right jugular vein for tracer administration and left carotid artery for continuous monitoring of arterial blood pressure and heart rate and for blood sampling via a device allowing minimum sample volumes . Arterial catheter patency was maintained by continuous infusion (10 l min) of a sterile saline solution containing sodium citrate (20.6 mmol l). Immediately following catheterization (described above), the sciatic nerves were exposed and cut bilaterally at the gluteal level . Unilateral electrical sciatic nerve stimulation was applied with ring electrodes at 0.5 hz to induce sustainable twitch contractions in muscles of one hind leg as described previously . Tracer infusates were prepared freshly each day . For each rat ~5 10 dpm h - r - brp and ~2.5 10 dpm c - p (amersham, solna, sweden), as well as 152 nmol na - palmitate (sigma, st . Louis, mo), were complexed to essentially fatty acid - free bovine serum albumin (bsa) (sigma) as detailed in . Unilateral sciatic nerve stimulation was commenced 70 min after completion of surgical preparation and 20 min prior to commencing tracer administration . All blood samples were collected via the carotid catheter into k - edta containing tubes (microvette cb300, sarstedt, nmbrecht, germany) via a device designed to reduce sample volume . Immediately before tracer infusion a 200 l basal blood sample was collected for determination of plasma insulin and substrate levels . Briefly, the albumin - palmitate - tracer complex was infused through the jugular catheter at 230 l min for 4 min . Blood samples were centrifuged immediately at 4c and a 25 l plasma aliquot placed directly into lipid extraction mixture (described in) for determination of plasma h - r - brp and c - p concentrations . After collection of the final blood sample, 16 min after commencing the tracer infusion, rats were killed with an overdose of thiobutabarbitol (120 mg kg). Tissues were collected and samples (~100 mg) were combusted for determination of total h and c content . Calculations . The clearance rate of h - r - brp by an individual tissue (kf *), an index of the ability of the tissue to utilize fas, was calculated as previously described, as (1)kf= mb0tcb(t)dt, where t is the time of tissue collection (16 min), mb is the total tissue h content (at t = t), and cb is the arterial plasma concentration of h - r - brp . An index of fa utilization rate (rf *) was calculated as (2)rf=cpkf, where cp is the arterial plasma fa concentration . An index of the clearance of c - p into storage products (kfs) was calculated as (3)kfs= mp0tcp(t)dt, where mp is the total tissue c content (at t = t) and cp is the arterial plasma c - p concentration . This assumes that all of the c label originating from locally activated c - p directed into oxidative metabolism would be lost from the tissue (largely as co2) by the time of tissue sampling . An index of the rate of fa incorporation into storage (rfs) was calculated as (4)rfs = cpkfs . Assuming certain conditions are met, rf * is proportional to the genuine rate of fa utilization (rf), that is, (5)rf=lcrf with a constant of proportionality (lumped constant) lc*. Since rf is the sum of oxidative disposal (rfox) and non - oxidative disposal (rfs), (6)rfox = lc1rfrfs . Note that when rfox = 0, lc * = rfs / rf*. We have previously suppressed fatty acid oxidation using pharmacological -oxidation inhibition to obtain crude estimates of lc * for different tissues in the rat . Lc * and the reliability of the rfox values derived vary in a tissue - specific manner with the following values assumed for the present work: skeletal muscle 0.27 and heart 0.19 . Estimates of whole - body plasma c - p clearance (kp) were calculated according to . Plasma h - r - brp and c - p were resolved using an acid lipid extraction procedure . Total tissue h and c levels were determined by combusting tissue samples using a packard system 387 automated sample preparation unit (packard instruments co., inc . Four groups (n = 2 - 3 per group) of both lean and obese zucker rats were studied in order to generate a range of plasma fa levels: a vehicle control group receiving a normal saline infusion and three groups receiving intravenous nicotinic acid infusion at the doses of 10, 100 and 1000 nmol kg min, respectively . Tracer infusates, ~2 10 dpm per rat [9,10 h] palmitic acid (h - p, amersham, solna, sweden) and 305 nmol na - palmitate (sigma, st . The tracer and na - palmitate were prepared in 150 l ethanol and added dropwise to 0.6 ml of continuously stirred 4% (w / v) essentially fatty acid - free bovine serum albumin (bsa, sigma, st . The infusate was made up to a final volume of ~3 ml per rat by addition of normal saline . After a 2 h postsurgery recovery period, two basal blood samples (~150 l) were collected 15 min apart for analysis of plasma fa, tg, glucose, and insulin . Immediately following collection of the second blood sample, intravenous infusions of nicotinic acid (or vehicle) and tracer the albumin - palmitate - h - p complex was infused at a constant rate (~1 10 dpm min, 17 l min). Arterial blood samples (~75 l) were collected 10, 20, 40, 60, 80, 100, and 120 min after the start of tracer infusion . For each sample, plasma was separated as quickly as possible in a refrigerated centrifuge . One 25 l aliquot was placed into 2 ml lipid extraction mixture, for determination of h - p and h2o; the remainder was used for analysis of fa level . After collection of the final blood sample, rats were killed with an overdose of thiobutabarbitol (120 mg kg). To discriminate h - p from total plasma h activity, a lipid extraction and separation procedure this involved an initial acid lipid extraction using a mixture of isopropanol - heptane-1 mol / l acetic acid (40: 10: 1 vol) followed by solid phase separation of free fatty acids (including h - p) from neutral lipids . H2o was estimated as the h - activity in the lower (isopropanol - water) phase of the lipid extraction procedure . Plasma fa mobilization was assessed using a constant infusion of h - palmitate (h - p). After attainment of isotopic steady states (<40 min after the start of tracer infusion), the plasma clearance rate of h - p (k) was calculated as (7)k = ipcp(), where ip is the tracer infusion rate (dpm min) and cp() is the steady state arterial concentration of h - p (dpm ml). The rate of appearance of plasma fas (ra) was calculated as (8)ra = cpk, where cp is the arterial plasma fa concentration (mol ml). Appearance of h2o in the plasma was linear from the earliest time point throughout the study consistent with rapid attainment of steady state of the labeled oxidation precursor pool . The fraction of plasma fa undergoing oxidation (fox) was estimated using the relationship (9)fox = vwdcw / dtip, where vw is the total water space of the rat (estimated from body weight, bw in g, using separate regression equations: for obese animals% water = 59.3 0.027 bw and for lean animals% water = 73.4 0.030 bw obtained from in - house water content analyses of obese and lean zucker rats, resp . ), cw is the plasma concentration of h2o, and t is the time from commencement of tracer infusion . The derivative above was estimated from the slope obtained from linear regression analysis of the h2o plasma versus time data for the period t = 10 to t = 120 min . An estimate of the whole - body rate of fa clearance into oxidation (kox) was calculated as (10)kox = foxk . The rate of whole - body fa oxidation (rox) was calculated as (11)rox = foxra . Fatty acid oxidation was measured in tissue homogenates using a modified version of a previously published method . Briefly, tissues were homogenized in either 9 volumes (epididymal white adipose tissue; wat), 19 volumes (cerebellum), or 39 volumes (heart, brown adipose tissue (bat), liver, red gastrocnemius, and white quadriceps) of ice - cold 250 mmol l sucrose, 10 mmol l tris - hcl, 1 mmol l edta . For assessment of palmitate oxidation, 50 l of tissue homogenate final concentrations of the reaction mixture were (in mmol l) 100 sucrose, 10 tris - hcl, 5 potassium phosphate, 80 potassium chloride, 1 magnesium chloride, 2 malate, 2 atp, 1 dithiothreitol, 0.2 edta, 2 l - carnitine, 0.05 coenzyme a (coa), 0.2 palmitate [+ 0.5 ci 1-c - palmitate], and 0.3% (w / v) fatty acid - free bsa . After 90 min of incubation at 25c, the reaction was stopped by the addition of 100 l of ice - cold 1 mol l perchloric acid . Co2 produced during the 90 min incubation was collected in 100 l of 1 mol l sodium hydroxide . C counts present in the acid - soluble fraction were also measured and combined with the co2 values to give the total palmitate oxidation rate . Insulin concentrations were determined using radioimmunoassay (rat insulin ria kit; linco research, st . Colorimetric kit methods were used for the measurement of plasma fa (nefa c; wako, richmond, va), triglycerides (triglycerides / gb; boehringer mannheim, indianapolis, in), and glucose (glucose hk; roche, stockholm). Differences between lean and obese groups were assessed using student's t - tests, assuming equal group variance . Systematic between - group differences in muscle parameters were assessed using 2-way analysis of variance (anova) using the program spss (spss, chicago, il). Linear regression analysis was performed using graphpad prism (graphpad software inc ., la jolla, ca). Body weights and general plasma factors for series 1 animals are summarized in table 1 . Estimates of body composition, lean and fat mass, have also been made as previously described . As expected, obese zucker rats weighed approximately 50% more than age - matched lean zucker rats, due to increased fat mass, and displayed hyperinsulinemia, hypertriglyceridemia, and a mild hyperglycemia . Plasma fa level and rate of appearance of fa (ra), calculated from the c - palmitate kinetics, are presented in figure 1 . Obese animals had substantially elevated systemic fa availability, compared to lean zuckers, due to an elevated rate of entry of fa into the plasma as shown by the ra data . Metabolic clearance rates of c - palmitate (kp, expressed per rat) were similar in lean and obese animals, despite the much greater total tissue mass of the obese animals (data not shown). The rate of h - r - brp clearance from plasma into a tissue (kf *) provides an index of the local ability to utilize fa for both oxidative and non - oxidative metabolism (storage), independent of the direct influence of plasma fa level . Tissue - specific clearance of c - palmitate into storage (kfs) indexes the local ability to store plasma fa . Independent of group, kf * and kfs had a range of 2 orders of magnitude across the different tissues sampled, with cerebellum having the lowest and liver the highest values (table 2). Also independent of group is the large difference between adipose tissue types with bat having a much greater ability to take up and store fa than wat . Comparing results for obese and lean animals, there were no differences in kf * or kfs values for liver, cerebellum, or wat . In bat and heart kf * was lower in the obese compared to the lean animals, while kfs was similar in the two groups . This indicates a reduced ability to metabolically sequester available fa and a preferential diversion towards non - oxidative disposal in these tissues of obese compared to lean zuckers . Results for five corresponding muscles from both hind legs are presented: one leg subject to repetitive efferent electrical stimulation of the sciatic nerve (stim - leg) versus the unstimulated control leg (con - leg). Examining first the results in the quiescent control leg muscles, it is apparent that independent of group, kf * and kfs roughly rank according to expected oxidative capacity with glycolytic muscle (wq and wg) <intermediate mixed fiber type muscle (edl) <highly oxidative muscle (rg and rq). There was no systematic difference in kf * in the quiescent muscles between lean and obese groups . Kfs did however tend to be modestly higher (by 15%, p <0.01, anova) in the quiescent muscles of the obese compared to the lean animals . These results suggest a similar ability to metabolically sequester available fa in the two groups but that in the obese animals there was a slight preference for disposal of fa into non - oxidative metabolism compared to the lean animals . Electrical stimulation of the sciatic nerve at the gluteal level induced twitch contractions in the lower leg muscles (wg, edl, and rg) but not in the thigh muscles (wq and rq). Correspondingly, for each lower leg muscle the kf * value was significantly higher in the stim - leg versus con - leg, while there were no differences in kf * between the stim - leg and con - leg for the 2 noncontracting thigh muscles (table 3). The average contraction - induced increase in lower leg muscle kf * was similar in both lean and obese groups (group effect, p> 0.05, anova). Sciatic nerve stimulation tended to induce a small increase in kfs (3 out of 3 muscles in lean and 1 out of 3 muscles in obese, table 3) which when averaged over all muscles was not significantly different in lean versus obese groups (p> 0.05, anova). Altogether, the relatively much larger contraction induced increase in kf * than kfs (in both groups) indicates that the contraction - induced increase in fa clearance is almost exclusively diverted into oxidation . Parameters reflecting in vivo metabolic fluxes of plasma fa in individual tissues are given in tables 4 and 5 . Rf * indexes the total rate of plasma fa utilization into both oxidative and non - oxidative metabolism . Rfs is an estimate of the rate of plasma fa incorporation into storage (non - oxidative metabolism) only . Obese animals had substantially higher rf * and rfs values compared with lean in the majority of tissues including cerebellum, liver, and wat (table 4), as well as all skeletal muscles examined (table 5). This was caused by the higher fa levels in the obese compared to the lean animals resulting from the higher rate of entry of fa into plasma described above . Only in heart and bat, was rf * similar in both lean and obese groups (table 4). Contraction increased rf * and rfs, in the lower leg muscles, an expected consequence of the increases in kf * and kfs, respectively (referred to above). The extent of the increase in rf * was greater in the obese compared to lean group: (p <0.05, group effect, anova). Figure 2 shows the relationship between rfs for wq and rq (con - leg only) and plasma fa level in individual animals . First it is apparent that in these quiescent muscles there is a simple linear dependence of plasma fa flux into storage on plasma fa concentration and that the same relationship seems to hold for both obese and lean groups, suggesting that the increased flux of fa into non - oxidative disposal in resting muscle in obese zuckers is a direct result of the higher plasma fa levels . Second, the red oxidative muscle (rq) has a much greater ability to store available fa than the glycolytic muscle (wq) as evidenced by the greater slope in rq compared to wq (p <0.0001). Tissue specific rates of fa oxidation cannot be calculated directly as the difference between rf * and rfs because of the slower kinetics of h - r - brp compared with native fa . Plasma fa oxidation (rfox) can however be estimated indirectly from rfs and rf * in some tissues including muscles (see table 6) as described in the materials and methods section . Rfox in quiescent hindlimb muscles were low in comparison to the flux of fa into non - oxidative disposal (rfs) and generally similar in obese compared to lean zuckers . The exception was in the rq muscle where the levels were higher in obese versus lean . Contraction (only occurring in wg, edl and rg) induced substantial increases in rfox to levels similar in magnitude to the levels of rfs in the corresponding muscles . Averaged across the three contracting muscles, the contraction induced increase in fa oxidation was similar in obese versus lean rats, 2.0 0.2 versus 1.6 0.3 mol/100 g / min (p> 0.05) respectively . Obese rats apparently also had similar rates of plasma fa oxidation compared to lean rats in two other contracting muscle tissues, diaphragm and heart . Thus diaphragm rfox for lean was 4.3 0.7 versus obese 5.9 1.8 mol 100 g min while rfox for the heart was for lean 43.4 6.1 versus obese 42.2 4.5 mol 100 g min . In series 1, obese zucker rats were observed to have a general elevation in plasma fa level and utilization at the whole body, as well as in muscle, fat, and liver compared to lean controls . To examine the dependence of fa oxidation on plasma fa availability, the antilipolytic agent nicotinic acid was used in several doses to suppress ra in order to generate a range of fa levels that overlapped in the lean and obese animals . Figure 3 shows the relationships between ra and rate of fa oxidation (rox) and plasma fa level, respectively . The nicotinic acid infusions applied succeeded in dose dependently reducing fa availability in both lean and obese zucker rats . In both groups there was a tight linear dependence of rox on ra (figure 3(a)), with regression line intercepts virtually coinciding with the origin . The slope of this relationship in the obese zucker was however 44% less than that in the lean animals (p <0.01), indicating that under conditions of an equal rate of systemic fa supply the obese zuckers would exhibit a reduced rate of fa oxidation compared to the lean zuckers . Strong linear relationships were also apparent between rox and plasma fa level in both groups of zucker rats (figure 3(b)) with the regression line for the obese zuckers having a lower intercept (p <0.05) and tending to have a lower slope than the line for the lean zuckers . Thus, under conditions of comparable fa levels, the obese zuckers would manifest a reduced fa oxidation rate compared to lean zuckers . In addition to assessing tissue specific fa metabolism in vivo, we also examined the tissues capacity to oxidize fa by measuring palmitate oxidation rates in tissue homogenates . Independent of obesity status, the results demonstrate a range of 2 orders of magnitude in the capacity to oxidize fa across different tissues of the body, lowest in wat and highest in the heart (figure 4). In the obese animals there was only one tissue, bat, where the fatty acid oxidation capacity was actually lower (67%) than that observed in the lean animals, again consistent with the previously mentioned phenotypic difference . All other tissues of the obese animals examined showed either a similar capacity (heart, liver, cerebellum, and wat) or a moderately enhanced (+ 35%) capacity (skeletal muscles) to oxidize fa compared with the tissues of the lean animals . To gain insight into the metabolic functionality of individual tissues, in vivo fa flux data are compared with the ex vivo estimates of fa oxidation capacity for individual tissues in figure 5 . Genuine rates of fa uptake (y - axis) were estimated from rf * values (table 3) using procedures described in the materials and methods section . In association with marked insulin resistance, nondiabetic obese zucker rats exhibit substantial accumulation of triglycerides and lipid intermediates in non - adipose tissues, including liver and muscle [2628]. This lipid accumulation could result from disturbances in tissue fatty acid uptake or metabolic fate including systemic oversupply, a locally enhanced ability to take up plasma fa or impairment of their oxidation . Which of these factors predominates in obese animals in vivo has not been resolved . In this study in vivo fa metabolism, both at the whole - body and individual tissue levels, completely novel information concerning the flux of fa from plasma and its metabolic fate was obtained by applying a method based on the combined use of the non--oxidizable fa analogue tracer, h - r - brp and c - palmitate . The results demonstrate that oversupply of plasma fa is a major factor in the fatty acid overload of non - adipose tissues of the obese zucker rat . We have shown in the current study and previously that the rate at which fa enters the plasma (ra) in the fasting state in the obese animals is more than twice the rate of lean animals of the same age . The increased flux of fatty acid into the plasma has to be matched by a corresponding increase in flux of fatty acids into the tissues . Even after subtracting the fraction of ra that disappears into the body fat, which is much greater in the obese animals, the remainder that supplies the non - adipose tissues of the body is still more than doubled in the obese compared to the lean animals, 14 versus 6 mol min, respectively, based on an average adipose tissue disposal equal to rfs for epididymal fat (table 4) and a body fat mass of 38% of body weight in obese versus 14% in lean animals (table 1). The consequence of systemic fa oversupply is a general elevation of fa flux into non - oxidative metabolism in the tissues . Thus, in the obese animals the flux of plasma fa into storage metabolism was substantially increased in liver, skeletal muscle, wat, and the heart compared to the corresponding fluxes in the lean animals (tables 4 and 5). The group independent, linear relation between rfs in quiescent muscles and plasma fa concentration (figure 2) shows the importance of systemic fa in generating lipid overload in this metabolically important tissue, consistent with the old idea that fa metabolism is supply driven . Our in vivo observations agree well with the results of previous ex vivo studies of skeletal muscle fa metabolism . Thus in both perfused hindlimbs and incubated muscles from normal rats, tg synthesis was found to be a linear function of the perfusate / media albumin - bound fa concentration, and synthesis rates were substantially higher in oxidative than glycolytic muscles . Tissue fa uptake is determined both by plasma and extracellular fa availability and the ability of the tissue to take up fa . An enhanced ability of skeletal muscle to take up fa has been documented to occur in vivo in skeletal muscle of high fat fed rats, which like the obese zucker rat, exhibit insulin resistance associated with lipid accumulation . There is also evidence suggesting that this could be the case in the obese zucker rat . Ex vivo studies in perfused hind limbs from lean and obese zucker rats where perfusate fa concentration had been equalized showed that in noncontracting muscle in the absence of insulin, fa uptake is augmented in obese compared to lean zuckers [34, 35]. However this difference, which appears to be causally associated with the degree of translocation of putative fa transporter proteins, is condition dependent and can be abolished by insulin stimulation or contraction . Moreover we found no evidence that the in vivo ability of muscle tissue to take up fa, assessed by the parameter kf *, is enhanced in the obese zucker rats . While this is in contrast to the increased fa uptake reported in muscle of high fat fed rats, it should be noted that a comparable situation apparently exists in humans where fractional extraction of fa and perfusion of the leg were both determined to be similar in obese and lean volunteers in a classic study of kelley and colleagues . To summarize, our data suggests that at least under the conditions of this study, skeletal muscle fa oversupply in obese zucker rats can be exclusively attributed to increased fa availability . However, our data provide no evidence that absolute rates of muscle fa oxidation in vivo were reduced in the obese compared with the lean zucker rats . Information about the rate at which plasma fa enters the tissue and is immediately oxidized, rather than stored, was obtained by combining the tissue data derived from h - r - brp, which reflects oxidative and non - oxidative fate, with that derived from c - palmitate incorporation into storage, reflecting non - oxidative metabolism only . Using this approach in the anesthetized preparation employed here, substantial levels of direct plasma fa oxidation were only in evidence in contracting muscles, including the beating heart and contracting diaphragm, as well as in contracting hindlimb muscles (table 6). Most significantly, similar rates of direct oxidation of plasma fa were apparent in the working muscles of obese and lean animals . While the absolute rate of muscle fa oxidation is not reduced in the obese animals, this does not preclude an abnormality in the control of fa oxidation . One potential abnormality relates to the primary genetic defect in the obese zucker, the loss - of - function mutation in the leptin receptor . Leptin has been shown to acutely increase fa oxidation in skeletal muscle and in the isolated heart . On the basis of these effects, one would expect an inappropriately low rate of fa oxidation in these animals, relative to the prevailing plasma fa concentration . Indirect support for this mechanism in the liver has been provided in diabetic obese zucker rats where transgenic overexpression of functional leptin receptor resulted in a remarkable reduction of the hepatic lipid content . A confounding factor preventing direct comparison of oxidation rates in the obese compared with the lean animals is the higher prevailing plasma fa levels in the obese rats, which on its own would tend to drive higher rates of fa oxidation by simple mass action . To circumvent this issue we therefore studied the relationship between whole - body fa oxidation and fa availability by infusing the antilipolytic agent nicotinic acid in various doses to separate subgroups of animals (figure 3). This revealed that indeed at comparable levels of plasma fa availability there was a lower rate of fa oxidation in the obese animals . Our ex vivo studies of palmitate oxidation seem to exclude the possibility that this results from a limitation in the capacity of tissues to oxidize fa . A much more likely explanation relates to the known elevation of tissue malonyl - coa in the obese zucker rats which may be responsible by suppressing cpt i activity and diverting fa into storage as reviewed by . In support of this, studies in our laboratory using pharmacological acc inhibition in obese zucker rats have shown normalization, relative to lean zuckers, of both hepatic malonyl - coa levels as well as ability to oxidize fa at the whole - body level (oakes et al ., unpublished observation). To investigate whether a defect in fatty acid oxidation was apparent at the subcellular level we assessed the capacity of a comprehensive range of tissues to oxidize palmitate . Skeletal muscle homogenates from the obese animals had a modest enhancement in the rate of palmitate oxidation (figure 4) compared with the lean animal, in qualitative agreement with the recently reported increases in the activities of several important enzymes involved in mitochondrial oxidation in the muscle of obese compared with lean zucker rats [36, 42]. All other tissues of the obese zucker rat, with the exception of bat, possessed a similar capacity to oxidize fa compared to the lean rats . Our data are consistent with the findings of noland et al . Obtained using similar methods in liver, muscle, and heart of lean and obese zucker rats . Overall the present data provide evidence that the excess lipid accumulation in non - adipose tissues, including skeletal muscle, of the obese zucker rat is primarily due to an increased fa availability rather than a major intrinsic impairment in the ability to oxidize fatty acids . This deduction fits well with an in vivo study indicating equivalent mitochondrial oxidation capacity in skeletal muscle of diabetic zucker rats compared to control rats . Holloway et al . Also recently concluded, based on ex vivo studies, that intramyocellular lipid accumulation results from increased delivery of fa to the muscle cytosol rather than a defect in fatty acid oxidation . However, our in vivo findings diverge from those of holloway et al . In the major cause of the increased delivery (supply of fa to the myocyte versus enhanced plasma membrane transport) and these discrepancies likely relate to methodological differences in assessing fatty acid metabolism in the in vivo setting versus isolated ex vivo assessments . Comparing the estimates of in vivo fa uptake versus ex vivo fatty acid oxidation capacity provides information about the metabolic functionality of the individual tissues (figure 5). Thus, the unique storage function of wat is made apparent as it is the only tissue where fa uptake substantially exceeds oxidization capacity . By contrast, fa uptake in bat lies well below its high capacity to oxidize, probably reflecting a rather low level of sympathetic activation under the physiological conditions of this study . Skeletal muscle also takes up much less fa than it is capable of oxidizing in the quiescent state, but sustainable twitch contractions causes uptake to approach oxidation capacity . In the beating heart, uptake and oxidation capacity were approximately matched, indicating that cardiac fatty acid oxidation is virtually maximal under the physiological conditions of this study . It is indeed remarkable that the intact heart seems to be able to extract fa from the plasma and oxidize it at approximately the maximal rate attainable in vitro where membrane barriers are absent and concentration gradients are presumably negligible . The points for the liver are also located on the line of equality of uptake and oxidation . However, unlike the heart, the liver exports a substantial fraction of the fatty acid taken up as plasma fa in the form of vldl triglyceride and it also has a much greater capacity to transiently store fa as endogenous tg . Based on ethical and practical considerations, whenever possible we use anesthetized preparations rather than conscious chronically catheterized animals for acute experiments . However one limitation is the fact that the skeletal musculature, of major importance for whole - body metabolism, in this situation is essentially inactive . To circumvent this problem we performed unilateral nerve stimulation to induce repetitive twitch contractions in a defined set of muscles in the lower hindlimb . This allows a comparison of substrate metabolism in specific contracting oxidative and glycolytic muscles of the stimulated leg with the corresponding contralateral resting muscles in the same animal . Our data show that contraction increases muscle fa uptake and oxidation without lowering the flux of fa into non - oxidative disposal . This implies that the contraction - induced increase in fa uptake is not simply the result of an elevated pull of fatty acids into mitochondrial -oxidation . The latter is an important mechanism in the heart where increased respiration results in a fall in mitochondrial acetyl - coa, in turn leading to a fall in cytosolic malonyl - coa, relieving inhibition of carnitine palmitoyl transferase 1 (cpt i) and thereby increasing transport of fatty acyl - coa into the mitochondria for oxidation . A contraction - associated increase in skeletal muscle fa uptake without a reduction in fa storage is consistent with the involvement of a push mechanism and may be the result of enhanced membrane transport due to translocation of the fatty acid translocase (fat / cd36) [46, 47]. While the methodology employed in this study offers a unique opportunity to study in vivo metabolism of plasma fa in a diverse range of tissues, an important limitation should be acknowledged: the method does not provide a complete assessment of overall tissue specific fa metabolism . Information is generated about the direct contribution of circulating fa to tissue specific fa metabolism but there are two significant alternative sources of fa: circulating tg and endogenous tg . The contribution of circulating tg to tissue specific fa metabolism is potentially large . Thus, using results from a previous study, rates of vldl esterified fa secretion in the fasting state corresponded to 44% and 71% of the fa ra for the lean and obese zucker rats respectively . Since tg secretion must be matched by tissue tg uptake, the substantially elevated (~4-fold higher) vldl tg secretion in the obese zucker compared to lean age - matched zuckers is almost certainly also a major contributor to tissue lipid overload . However, little is known about the true in vivo tissue fate of this tg - fa and methods that can provide quantitative information about the contribution of both plasma tg - fa and fa are needed . A perhaps even more challenging issue is experimentally determining the contribution of the intracellular tg pool . While pulse labeling wash - out studies might be achievable for highly oxidatively active tissues like the heart, assessing tg turnover in tissues with modest rates of turnover is likely to be difficult . In summary, our assessments of in vivo fa fluxes demonstrate that the major factor responsible for non - adipose tissue lipid overload in the insulin resistant obese zucker rat is systemic fa oversupply . There was no evidence for a widespread impairment in the capacity of tissues from the obese animals to oxidize fa . At the whole - body level the absolute rate of fa oxidation was not reduced in obese zucker rats under physiological conditions, but the marked increase in fa availability was not being matched by an equivalent elevation in fa oxidation, indicating some disturbances in fatty acid utilization . When conditions of equivalent fa availability were achieved in vivo, the obese animals exhibited a mild defect in fa oxidation compared to lean zucker rats . We showed that in skeletal muscle, fatty acid uptake associated with contraction was channeled into oxidation . By contrast, non - oxidative disposal of fa in skeletal muscle was heavily influenced by availability and remarkably did not appear to be diminished by a contraction mediated increase in local oxidative metabolism.
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Renal cell carcinoma (rcc) is the most common malignancy of the kidney and a cancer of increasing incidence . In korea, rcc accounted for 1.85% of the total cancer occurrence in 2012, and the incidence rate was 8.2 per 100,000 people . The incidence of this cancer is relatively low among genitourinary malignancy, leading to the management of a lower volume of rcc cases in most hospitals . Therefore, it is difficult to make generalizations based on studies of rcc performed by single center due to the lower number of cases and the possibility of selection bias . Therefore, it is necessary to establish a well - qualified multi - institutional database (db) system comprising a large cohort to conduct clinical rcc researches due to the lower incidence of this neoplasm . And this database should include more variable data than the cancer statistics of national cancer center or health insurance review & assessment service . Examples include the renal mass study of croes (clinical research office of the endourological society), corona (collaborative research on renal neoplasms association) project [5 6789], saturn (surveillance and treatment update renal neoplasms) projects and japanese multicenter studies, which have reported high - quality research results . Some multi - institutional retrospective studies on rcc have also been conducted in korea, but there have been no large db systems to collect data on korean rcc . Here, we report the establishment of the 1st web - based rcc db system in korea (korean renal cell carcinoma, korcc) and summarize the basic characteristics of korean patients with rcc who underwent surgical management . The new web - based db system was established to collect basic demographic and clinicopathological characteristics of a large cohort of patients with rcc in korea . This project was approved by local ethics committee at seoul national university bundang hospital (irb number: b1202/145 - 102). The data included basic demographic (such as age, gender, height, and weight) and clinicopathological characteristics (such as clinical stage, perioperative parameters, pathological stage, fuhrman nuclear grade and survival data) (tables 1, 2, 3, 4, 5). Private information (such as resident registration number and hospital i d number) was excluded to protect the patients' personal information . Data from a total of 6,849 patients were collected from 8 tertiary care hospitals that agreed to participate in organizing korcc study group as of 1 july 2015 . These hospitals were chonnam national university hwasun hospital (308 cases), chungbuk national university hospital (178 cases), korea university anam hospital (105 cases), kyungpook university medical center (890 cases), national cancer center (618 cases), seoul st . Mary's hospital (883 cases), seoul national university bundang hospital (1,440 cases) and seoul national university hospital (2,427 cases). Data were collected for the management of renal masses of all tumor stages (pt1 - 4, n0 - 2, m0 - 1) at the department of urology at each hospital from 1990 to present (time frame varies among hospitals due to differences in their own db collections). We established the 1st web - based db of korcc, a database comprising renal mass management cases from multiple centers in korea (www.mebica.net). A total of 6,849 patients were collected nationwide from 8 tertiary care hospitals in korea . We analyzed data of 5,281 patients with surgical management (mean follow - up [fu], 32 months) (tables 1, 2, 3, 4, 5). The patients' mean age was 55.712.7 years, and 71.2% of the patients were male (fig . 1). Hypertension, diabetes mellitus and chronic kidney disease (defined as grade 3 or more) were identified in 38.5%, 14.9%, and 19.4% of the patients, respectively . The most common symptom was incidentally detected renal mass (4,048 cases, 76.9%), and the 2nd most common was gross hematuria, which accounted for 10.4% . Preoperative characteristics - such as clinical stage, tumor size, laterality, and location - were described in table 2 . Clinical t1a was the most common (54.3%) stage, and the mean tumor size was 4.84.2 cm . Radical nephrectomy accounted for 62.7% of the total cases, and an open approach was used in 50.7% and 52.2% of radical and partial nephrectomies, respectively (table 3). Warm ischemia was the most common type of ischemia (83.0%) and mean ischemic time was 25.211.3 minutes . Intraoperative and postoperative transfusions were done in 7.2% and 3.6% of the patients, respectively (table 4). A total of 2.9% of severe postoperative complications were of clavien - dindo grade 3 . The most common nuclear grade was fuhrman grade 2 (51.8%) and the most common histological type was clear cell (85.2%). The 5-year overall, cancer - specific and recurrence - free survival rates were 88.1%, 92.2%, and 88.0%, respectively (table 6). Kaplan - meier survival curves according to stage, nuclear grade and histologic type are shown in figs . 1, 2 . We report the 1st establishment of a web - based db system to collect clinicopathological characteristics of rcc in korea . The korcc web - based db system was launched in 2013, finally leading to the participation of 8 centers nationwide in 2014 . It is necessary to collect data from a large cohort of rcc patients to provide better analysis of rcc data in korea . Data from approximately 7,000 rcc cases were collected, and this project remains ongoing to collect more cases . We believe that this db can represent korean rcc due to its large cohort size and nationwide distribution . Therefore, we invited 8 nationwide tertiary care centers to participate in this project, and they all agreed to join . This led to the creation of a db that represents rcc characteristics of korean patients . Second, because this system is web - based, there is possibility that patients' data could be leaked due to security problems . Therefore, we chose an experienced company that has built many web - based clinical trial db systems (mebixon co., seoul, korea). Personal information - such as resident registration number and hospital identification number - was not included in our db system . This led to the establishment of a secure web - based db system of rcc in korea . For the good quality of this database, we limited the number of hospital as above . In the future, if the database works well, we will invite other hospitals to participate in korcc and provide chances to investigate with this database . There have been many multicenter based studies on rcc, and croes was the 1st group to establish an international electronic data management system . The global renal mass study driven by croes was started in 2008 to provide a prospective international study on the indications, treatment modalities and outcomes of the management of renal masses . Each center participating in this project included all of the patients with the diagnosis of renal mass during a one - year period . Other examples of international multi - center collections were the corona and saturn projects, which were not web - based . Nevertheless, this type of multicenter collection is necessary to obtain better knowledge on rcc through a db with a large cohort . This rate was much higher than the rate of approximately 50% in a previous report . Recently, the more common applications of imaging modalities for the evaluation of a variety of nonspecific symptoms has led to higher rates of incidental mass detection and an increased proportion of early stage tumors . More than half of the patients (54.3%) presented with clinical t1a stage tumors and metastatic tumors were identified only in 6.3% of patients . It is probable that this rate overestimated the real pattern of presentation because patients with advanced stage disease who were managed by hematooncologic department were not included in our db system . This relatively higher proportion of low clinical stage contributed to excellent overall and cancer - specific survival rates . We will likely find more incidental tumors with lower stages with the increasing use of imaging studies in the future . An open approach was used in 50.7% and 52.2% of radical and partial nephrectomies, respectively . Minimally invasive approaches - such as laparoscopic and robotic surgeries - were used for the remaining cases . Interestingly, robotic approach was used in 3.1% and 23.8% of radical and partial nephrectomies, respectively . It is well known that the application of robotic surgery in partial nephrectomy can provide good surgical quality including a reduced ischemic time, due to its enhanced 3-dimensional vision and excellent degree of freedom . These factors contributed to nearly a quarter of the patients undergoing partial nephrectomy being managed by a robotic approach . Although it represented a relatively lower (3.1%) proportion, robotic technology was also used in radical nephrectomy . Pure laparoscopic surgery is a challenging procedure, and some surgeons find laparoscopic radical nephrectomy difficult to perform . Robotic radical nephrectomy can bridge the gap between open and pure laparoscopic approaches for some urologists . As the number of people with private insurance has increased and the cost barrier is not an issue for these patients, the application of robotic technology will continuously increase in the future . Another notable f inding was that proportion of clear cell type was higher (85.2%) in our db compared to that in previous reports (60%-80%). Whether this phenomenon is korean - specific is not known . The proportion of sarcomatoid differentiation has ranged from 2% to 5% in past reports; our db revealed a proportion of 2.5%, which was equivalent to other reports . In addition to this error, some patients' data were lacking, causing incomplete data collections for some individual patients . Second, the length of fu length was rather short (mean, 32 months) but this weak point will be solved with long - term fu in the future . Some patients with advanced stage disease could visit the department of internal medicine and they could be managed without surgical intervention . Then, the database will be able to represent the whole cohorts of korean rcc . In conclusion, the 1st web - based db system to collect rcc data was established in korea . We report the 1st establishment of a web - based db system to collect rcc data in korea.
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Mitochondria play a key role in apoptosis triggered by a wide variety of stimuli since they release important proapoptotic factors from their intermembrane space . Normally, it acts as mobile electron carrier shuttling electrons between ubiquinol cytochrome c oxidoreductase (complex iii) and cytochrome c oxidase (complex iv) of the respiratory chain allowing cell life . On the other hand, upon apoptotic induction, cytochrome c is released from mitochondria in the cytosol where it carries out a proapoptotic function by binding the adapter protein apoptosis protease - activating factor-1 (apaf1). Consequently, it promotes, in presence of atp / datp, the assembly of the multiproteic complex apoptosome, which binds and activates the caspase-9, thereby initiating the activation of a caspase cascade which leads to apoptotic cell death [2, 3]. Mitochondrial outer membrane permeabilization (momp) and cytochrome c release from mitochondria during apoptosis are tightly regulated by the proteins of the bcl-2 family . This family of proteins includes both antiapoptotic members (e.g., bcl-2 and bcl - xl) repressing momp and release of apoptogenic factors from mitochondria and pro - apoptotic members promoting momp (e.g., bax and bak, bid) [4, 5]. The phospholipid cardiolipin (cl) seems to have a key role in the process of the cytochrome c release . Only 15% of cytochrome c is free in the intermembrane space, while most of it is attached to the outer leaflet of the mitochondrial inner membrane with the mitochondrion - specific anionic phospholipid cl . It has been recently found that posttranslational modifications or interaction with hydrophobic anions such as cl causes the activation of cytochrome c into a peroxidase with selective catalytic competence toward cl [7, 8]. Cytochrome c tightly bound to cl was proposed to possess this peroxidase activity and to catalyze cl peroxidation, most likely by utilizing the high production of reactive oxygen species (ros) generated in the mitochondria during the first stages of apoptosis . Cytochrome c has a lower affinity for peroxidized cl, and peroxidation of cl enables the dissociation of cytochrome c from mitochondrial inner membrane allowing the release of cytochrome c from mitochondria . However, the final release of cytochrome c requires additional steps such as the permeabilization of the outer membrane . Cl is also involved in mitochondrial outer membrane permeabilization since it enables docking and activation of some pro - apoptotic bcl-2 proteins [911]. These findings strongly suggest an active role of the membrane in modulating momp that has been underestimated so far . Cytochrome c is part of the respiratory chain whose complexes are located across the mitochondrial inner membrane, and membrane integrity greatly influences the respiratory activity of the cells . Based on this considerations, we analyzed the respiratory changes during apoptosis and their temporal relationship with the release of cytochrome c in order to get information useful to unravel the mechanisms of release of this hemoprotein from mitochondria . We therefore analyzed the respiratory activity of a rat cell line of neuronal derivation (pheochromocytoma-12, pc12 cells) by polarographic measurement of oxygen consumption in intact cells . This cell line manifests many features of sympathicoblasts, the cells that give rise to postmitotic sympathetic neurons . Indeed, they respond to nerve growth factor (ngf) by shifting to a nonproliferating neurite - bearing phenotype and acquiring many of the properties characteristic of sympathetic neurons among which electrical excitability . For this reason the clonal pc12 cell line is widely used for various studies on neuronal cell differentiation and function . During staurosporine- (sts-) induced apoptosis in nave pc12 cells, we observed an uncoupling event preceding the reduction of cytochrome c oxidase- (cox-) respiratory activity . Our investigation has also revealed different kinetics of decrease in 2,4-dinitrophenol- (dnp-) uncoupled and cox - dependent respiration with the former showing, at very early stage, a faster kinetics of decrease compared with the latter . This suggests an effect of sts on the respiratory activity, which is independent of cytochrome c release . This hypothesis is confirmed by our finding that overexpression of bcl-2 protects from release of cytochrome c and massive decrease in respiration, while it has no effect on the early decrease in dnp - uncoupled respiration induced by sts . Since the cytochrome c is part of the respiratory chain as the electron donor for complex iv, it is expected that its release from mitochondria during apoptosis would cause changes in the respiratory activity of the cells . We analyzed the nature and the extent of these changes by measuring the rate of oxygen consumption in naive pc12 cells and in naive pc12 cells undergoing apoptosis by treatment with the protein kinase inhibitor sts [13, 14]. We measured the oxygen consumption of the cells (endogenous respiration), the oxygen consumption in presence of the uncoupler dnp (dnp - uncoupled respiration, which represents the highest endogenous respiration rate that cells can reach being free of control by the proton gradient) and the rate of respiration measured in presence of the complex iii inhibitor antimycin a, the artificial membrane - permeant electron donor tmpd (n, n, n,n-tetramthyl-1,4-phenylendiamine), and the reducing agent ascorbate that maintains tmpd in a reduced state (tmpd - dependent respiration). Since antimycin a inhibits complex iii and blocks the electron flux upstream of cox, the tmpd - dependent respiration provides a measure of the cox and cytochrome c - dependent oxygen consumption independently of the upstream segment of the respiratory chain . The respiration of both untreated and sts - treated pc12 cells resulted being 100% antimycin a - sensitive (data not shown). Upon treatment with sts we observed a time - dependent decrease in the endogenous, dnp - uncoupled and tmpd - dependent respiration rates . After 5 hr of treatment with sts, the dnp - uncoupled respiration rate decreased to ~45% of the dnp - uncoupled respiration rate of untreated cell and to ~28,8% after 9 hr of treatment (figures 1(a) and 2(b)). The oxygen consumption relative to prolonged period of sts treatment (9 h and 24 h) is overestimated since, being very light, it is difficult to collect all dying cells with the usual centrifugation speed; and it is not advisable to use higher speed since it would affect the respiration of alive cells . The kinetics of decrease in tmpd - dependent cox respiration rate during sts - treatment was similar to the kinetics of decrease in endogenous and dnp - uncoupled respiration rates . However, very interestingly, by focusing on the first few hours of treatment, we observed that, the decrease in dnp - uncoupled respiration rate was significantly faster compared with the cox - dependent respiration rate decrease . In fact, after 1 hr of sts treatment, the tmpd - dependent respiration rate was decreased only by 4% relative to the rate of untreated cells, while the dnp - uncoupled respiration rate was decreased by 15% (figure 1(b)). Since the cox - dependent respiration depends directly on cytochrome c and decreases less than the overall uncoupled respiration, this means that the uncoupled respiration is affected by some other modifications than the absence of cytochrome c. this finding suggests therefore that there is an impairment of the mitochondrial respiration soon after the apoptotic induction that is not due to the release of cytochrome c. to better understand the temporal correlation between decrease in respiration and cytochrome c release in sts - induced apoptosis we analyzed cytochrome c localization by confocal immunofluorescence microscopy . 2 um sts - treatment of pc12 cells promoted nuclear fragmentation (figure 2(a), sts 3 h; white arrow) and cytochrome c release from mitochondria as clearly resulted from the different immunolocalization of the mitochondrial marker hsp60 and cytochrome c (figure 2(a), sts 3 h; yellow arrows). Figure 2(a) (white arrow) shows an example of cell considered apoptotic; it is shrunk and has a fragmented nucleus . By analyzing several fields, we calculated the percentage of cells showing cytochrome c release and the percentage of cells showing nuclear fragmentation (cell death) at each time point in order to compare these kinetics with the changes in respiration . Since cytochrome c release from mitochondria during apoptosis precedes nuclear fragmentation, we detected cells in which the cytochrome c had been released from mitochondria while the nucleus was not yet fragmented (figure 2(a), sts 3 h; yellow arrowheads) but never the opposite . For this reason, we obtained a kinetics of cytochrome c release slightly more rapid than the kinetics of nuclear fragmentation (figure 2(b)). Notably, once released from mitochondria, cytochrome c localizes throughout the cell and, as sometimes reported [16, 17], into the nucleus (figure 2(a), sts 3 h; yellow arrows). During the immunofluorescence staining, a high number of late stage apoptotic cells detached from the dish and were lost which means that we run the risk to underestimate their number . To solve this problem we calculated the real percentage of apoptotic cells by collecting all of them, also from the supernatant, through high speed centrifugation and by staining all nuclei with dapi . The difference between the percentage of cell death calculated by immunofluorescence on a dish and the one obtained by high speed centrifugation was used to correct the underestimated percentage of cytochrome c releasing cells evaluated by means of the immunofluorescence staining; dead cells detaching during the experiment are at the very final stages of apoptosis and have already released cytochrome c . We compared these kinetics with the changes in respiration reported in figure 1, and for this purpose we expressed data shown in figure 2(b) as percentage of cells with mitochondrially localized cytochrome c and percentage of alive cells (figure 2(c)). We observed that the progressively lower rate of respiration upon sts treatment highly correlates with the decreased number of alive cells due to apoptotic death . Interestingly, the decrease of cells with mitochondrial cytochrome c localization highly correlates with the kinetics of cox - respiration decrease, but such a decrease is slower than that observed in dnp - respiration (figure 2(c)). Such an analysis strengthens the conclusion that the decrease in tmpd - dependent respiration is mainly due to cytochrome c release, while the decrease in dnp - uncoupled respiration precedes cytochrome c release and must be due to another mechanism of impairment of the respiratory chain system . To confirm that the early changes in respiration observed during apoptosis were really independent of cytochrome c release, we decided to study the respiration of pc12 cells stably overexpressing the apoptotic inhibitor bcl-2 (pc12-bcl-2 cells), which is known to block the release of cytochrome c from mitochondria [19, 20]. In fact, pc12-bcl-2 cells treated with 2 um sts do not detach from the dish and do not undergo apoptosis as shown by dapi staining (figures 3(a) and 3(b), nuclei). Also after prolonged exposure to sts, cytochrome c and hsp60 maintain the same localization in pc12-bcl-2 cells (figures 3(a) and 3(b)). The analysis of the respiration of these cells (figure 4(a)) shows that bcl-2 strongly protects against long - term respiratory decrease . In fact, after 24 h of treatment the uncoupled endogenous respiration decreases to ~44.5% of the rate of untreated cells while in absence of bcl-2, under sts treatment, respiration decreased to ~16% of the rate of untreated cells (or even less considered the overestimation cited) (figure 1(a)). The difference observed for the cox - dependent respiration is even greater; indeed, after 24 h of treatment, pc12-bcl-2 cell respiration decreases only to ~70.7% of the untreated cell rate, while the cox - dependent oxygen consumption of naive pc12 cells falls to ~19.8% of the control . The protection of the respiratory activity by bcl-2 is mainly due to the fact that bcl-2 blocks the release of the cytochrome c from mitochondria (figures 3(a) and 3(b)). However, the early decrease in dnp - dependent respiration that we have found during the first hours of apoptosis occurs also in bcl-2-overexpressing cells, although cytochrome c is not released . Indeed, after 1 h of sts treatment, both in pc12-bcl-2 and naive pc12 cells, dnp - uncoupled respiration decreases to ~84.6% of the respiration rate of untreated cells, and the cox - dependent respiration decreases only to ~96% of untreated cell respiration . After two hours of sts treatment, the uncoupled endogenous respiration falls to ~72% and ~74% of the control in pc12-bcl-2 and naive pc12 cells, respectively, while the tmpd - dependent respiration reaches the 86% of the control in naive pc12 cells and remains almost unchanged in pc12-bcl-2 cells (92%) (figures 1 and 4). Since in pc12-bcl-2 cells cytochrome c is not released from mitochondria, this experiment confirms that, in the first phases of apoptosis, the maximum possible respiration rate achievable in the cell decreases and that the respiratory chain is impaired independently of the release of cytochrome c. moreover, the comparison between the respiration of naive pc12 and pc12 cells overexpressing bcl-2 also shows that the ratio between the absolute values of dnp - uncoupled and endogenous respiration (dnp / end) decreases in pc12 cells immediately after sts treatment, while, in cells overexpressing bcl-2 remains unchanged (figure 5). This suggests an early uncoupling respiration event induced by sts that is prevented by bcl-2 . The dnp - dependent respiration is uncoupled from the synthesis of atp since it occurs when there is a dissipation of the proton gradient which cannot be used to provide the energy for the synthesis of atp . In this condition, the consumption of oxygen is the highest possible since it is free of control by the proton gradient and by the synthesis of atp . Dnp - dependent respiration rate is therefore higher than normal endogenous respiration since it is the maximum rate achievable by the respiratory chain . For this reason the ratio beween dnp - dependent respiration and endogenous respiration (dnp / end) is higher than 1 . If the ratio between dnp and end decreases, this means that endogenous respiration of the cell tends to become uncoupled . In the case of pc12-bcl2 cells the ratio remains constant which means that there is no uncoupling, while in pc12 cells this ratio decreases and this indicates an early uncoupling event (figure 5). In order to define whether the decrease of respiration, under sts treatment, was caspase dependent we pretreated pc12 cells with the broad - spectrum caspase inhibitor z - val - ala - asp (ome)-ch2f (z - vadfmk) before sts incubation . While z - vadfmk blocks apoptosis also after 24 h of sts treatment (figure 2(a), zvad - sts 6 h, nuclear morphology), it did not affect the extent of respiration rate reduction (figures 6(a) and 6(b)). In fact, the decrease in endogenous, dnp - uncoupled and cox - dependent respiration upon sts treatment was perfectly comparable with the respiration rates measured in pc12 naive cells undergoing apoptosis (figures 1(b)). Active caspases are indeed not required for the mitochondrial release of cytochrome c in sts - induced cell death . In fact, the analysis of the localization of cytochrome c by confocal immunofluorescence over several hours after apoptotic induction shows a gradual release of cytochrome c not affected by zvadfmk pretreatment (figure 2(a), zvad - sts 6 h, arrows). To exclude the possibility that the early reduction of respiratory activity we detected during the first phase of apoptosis was due to the mere protein kinase inhibition activity of sts, without any relationship with its apoptotic induction, we tested bisindolylmaleimide - i (bis - i), an sts - analog which is a protein kinase inhibitor without apoptotic functions [22, 23]. The treatment of pc12 cells with bis - i shows that this drug does not induce apoptosis (data not shown) and does not inhibit the respiration of cells (figure 6(c)). The lack of respiratory changes in cells treated with bis - i confirms that the decrease in respiration and the uncoupling of respiration induced by sts correlate with its apoptotic role . As stated before, sts - treated pc12-bcl-2 do not die and do not show nuclear fragmentation typical of apoptotic cells also after long treatment (figures 3(a) and 3(b)). However, despite the absence of fragmentation, we can clearly observe a strong condensation of chromatin (figure 3(a)), which is very similar to the first stage of condensation observed in sts - treated pc12 cells and preceding fragmentation (figure 2(a), sts 3 h, nuclei yellow arrowheads). Sts - treated pc12-bcl-2 cells bearing this strong nuclear condensation (figure 3(a)) are alive since they respire (figure 4) but do not replicate (figure 3(c)); they do not die but stay in a quiescent state . In fact, when sts is removed by changing the medium, all nuclei regain a normal appearance and cells started replicating again (figure 3(d)). We conclude that the first stage of nuclear modification in sts - induced apoptosis is not dependent on cytochrome c release and is not a point of no return of the apoptotic cascade . The overall decrease in respiration we detected during sts treatment of pc12 cells can obviously be related to the release of cytochrome c. however, we also found an impairment of the uncoupled respiration occurring in the first phase of sts - induced apoptosis and preceding the release of cytochrome c. this phenomenon was also reported in 143b cells, while in anti - fas antibody - treated jurkat cells, the loss of tmpd - dependent and of endogenous respiration occurred nearly simultaneously and followed cytochrome c release from mitochondria . This difference in the sequence of events might rely on different pathways taking place in fas- and sts - induced apoptosis . Regarding the cause of this early respiration impairment independent of cytochrome c release, we hypothesize a modification of the mitochondrial inner membrane integrity likely to be present at the level of the phospholipid cl . Indeed, cl is intimately linked to the mitochondrial bioenergetic machinery and is also actively involved in the release of cytochrome c. it has also been demonstrated that the modifications of the binding between cl and cytochrome c and also the binding between cl and some bcl-2 proteins precede and prime mitochondria for momp and for cytochrome c release . Cl is crucial for maintaining the integrity and the function of the mitochondrial inner membrane and is required for the optimal activity of most of the respiratory chain complexes . For these reasons, cl modifications (e.g., peroxidation by ros and cytochrome c peroxidase activity or tbid binding or redistribution within and between membranes) immediately result in structural changes of the mitochondrial inner membrane which destabilize mitochondria and affect the activity of membrane embedded respiratory chain complexes peroxidation of cl does occur following a wide variety of apoptotic stimuli also including sts . For all these reasons we hypothesize that the early changes in respirations observed might be due to perturbation of the mitochondrial membrane at the level of cl . Our analysis also suggests that the first stage of apoptosis is characterized by an early uncoupling event . Indeed, in the first hours of sts treatment of pc12 cells, the ratio between the absolute dnp - uncoupled and the endogenous oxygen consumption decreases (figure 5), indicating a difference between endogenous and dnp - uncoupled respiration decrease rate (figure 1). This uncoupling event occurs after 1 h of sts treatment when tmpd respiration is only slightly affected, and it is therefore presumably independent of cytochrome c release . However, the uncoupling is blocked by the overexpression of bcl-2 (figures 4 and 5). Therefore, bcl-2 does not prevent the early decrease in respiration induced by sts (figure 4) but prevents the uncoupling of respiration, which might be the step following the decrease of the maximum rate of respiration and ultimately leading to cytochrome c release . We suggest that the uncoupling of respiration might correlate with the step of the cytochrome c release process involving the bcl-2 family of proteins . Interestingly, the uncoupling of respiration is known to be also caused by cl modifications, supporting our hypothesis of cl as early target for apoptotic changes in mitochondria . Our experiments, in addition, show a strong degree of nuclear condensation (never reaching fragmentation) occurring in absence of cytochrome c release (figure 3(a)). This partial chromatin condensation might be the first step of nuclear modifications leading ultimately to fragmentation, and it has been reported to involve dna double - strand breaks [25, 26]. We assessed that, in nave pc12 cells, this early nuclear condensation occurring in bcl2-overexpressing cells is reversible and that it is not a point of no return in the apoptotic pathway (figures 3(c) and 3(d)). This confirms the finding that momp inhibitors allow cells with preapoptotic chromatin condensation to repair their dna and to return to a normal nuclear morphology . Finally, we observed that cytochrome c redistributed within the nuclei once lost its mitochondrial localization after induction of apoptosis, and, after long sts treatment, as previously reported, it became finally degraded (figure 2(a), zvad - sts 6 h, white arrow). This means that the nuclear localization of cytochrome c is quick, and indeed it occurs not only when the nuclei are fragmented but also when the chromatin is not yet fragmented or at a very early stage of condensation (figure 2(a), yellow arrows). This nuclear cytochrome c accumulation was zvad - independent (figure 2(a), zvad - sts 6 h). In conclusion, we found that mitochondrial bioenergetics is perturbed previously and independently of the release of cytochrome c during apoptosis . This finding may shed new light on the mechanisms leading to the release of cytochrome c. indeed, considering the interdependencies existing between bioenergetics and apoptosis, their coinvestigation in a holistic approach is strongly needed . Pc12 cells were cultured at 37c in a 5% co2 atmosphere in f-12 k (gibco) medium supplemented with 2 mm l - glutammine, 1,5 g / l sodium bicarbonate, 15% horse serum, 2.5% fetal bovine serum (fbs), and penicillin - streptomycin . For apoptotic induction, cells were seeded at 210/ml into tissue culture dishes coated with 20 g / ml poly - l - lysine (sigma - aldrich). After 2 days the medium was replaced, and after 2 hours 2 m sts (sigma - aldrich) in dmso was added . 100um z - vadfmk (kamiya biochemicals) was added 30 min before sts . Apoptosis was assessed by nuclear morphology . At different time points upon sts treatment, both cell floating in the medium and cells adherent to the dish were collected by centrifugation, washed with pbs, fixed in 4% paraformaldehyde (pfa), and stained with 1 g / ml of the fluorescent double - strand dna - binding dye 4,6-diamidino-2 - 2phenylindol (dapi, sigma - aldrich) for 8 min . Cells with large nuclei containing uniformly stained chromatin were counted as alive cells, while cells containing fragmented nuclei and/or pyknotic nuclei were counted as dead cells . Values were obtained from 4050 fields of 1 - 2 slides for a total of around 1000 cells / experiments . A human bcl-2 cdna was subcloned from sffv - bcl-2 n1 into the neomycin resistance marker - containing plasmid pcdef3 under the control of the ef-1a promoter and then used for transfection into pc12 cells by electroporation . Exponentially growing pc12 cells (10) were resuspended in 0.8 ml of pbs, mixed with 18 mg of bcl-2-pcdef3 and 5 mg of pgl1, and incubated on ice for 10 min . Electroporation was performed with a biorad gene pulser by using a setting of 960 mf and 300 v. cells then were incubated on ice for 10 min and plated in f12-k medium supplemented with 15% horse serum and 2.5% fbs on collagen coated dishes (biocoat). After 48 h stable transfectant overexpressing bcl-2 (pc12-bcl-2 cells) was selected by using 0.3 mg ml g418 (geneticin; invitrogen) for 2 weeks . Pc12 cells were suspended in td buffer (137 mm nacl, 5 mm kcl, 0.7 mm na2hpo4, 25 mm tris - hcl ph 7.4 at 25c) at 3 10 cells / ml, and the respiration rate was measured in an oxygraph (yellow spring instruments, model 5300). The same measurement was assessed after addition of 40 m dnp or dnp and 20 nm antimycin a (sigma - aldrich) or dnp, antimycin a, 400 m tmpd (fluka) and 10 mm ascorbate (sigma - aldrich) as described . Oxygen consumption rate was expressed in nanomoles of oxygen consumed per minute and mg of cellular proteins (nmolo2/min / mg) as determined by the bradford procedure (biorad) and as percentage of the untreated pc12 cell respiration . The oxygen consumption rate measured in presence of cells, dnp, antimycin a, ascorbate, and tmpd was corrected by subtraction of the oxygen consumption rate due to the autooxidation of ascorbate / tmpd in absence of cells . Cells were cultured on collagen - coated dishes at a concentration of 5 10 cells / ml . After 2 days cells were treated with sts, then washed with pbs, fixed in 4% pfa in pbs for 10 min at rt, washed, permeabilized with pbs containing 0,4% triton x-100 for 5 min, and washed again with pbs . After blocking the cells with pbs containing 2% horse serum (hs - pbs) for 30 min, they were incubated with mouse anticytochrome c monoclonal antibody 6h2.b4 (bd pharmingen) diluted 1: 300 in hs - pbs and rabbit anti - hsp60 antiserum (stressgene) diluted 1: 200 for 1 h at 37c in a humidified chamber . Following 3 washes in hs - pbs (5 min each), the cells were incubated with fitc conjugated goat anti - mouse igg (jackson immunoresearch laboratories) diluted 1: 200 and lissamine - rhodamine conjugated goat anti - rabbit igg (jackson immunoresearch laboratories) diluted 1: 200 for 1 h at room temperature . The specimens were washed 3 min in hs - pbs and incubated with sytox green (molecular probes) 25 nm for 15 min at room temperature . Cells were washed again 3 times (5 min each) in pbs, mounted in fluoroguard antifade reagent (biorad), and analyzed on a zeiss 510 laser scanning microscope . Proteins were then transferred to a polyvinylidene difluoride (pvdf) membrane (biorad), and the membrane was treated for 1 h with blocking solution (0.02% tween 20, 0.02% nan3, 5% nonfat milk in pbs) at room temperature . The membrane was then incubated for 4 h at 4c with mouse anti - bcl-2 monoclonal antibody (sc-7382) (santa cruz biotechnologies) diluted 1: 1000 in blocking solution . It was then washed 3 times in pbs (10 min each) and 1 time with washing solution (150 mm nacl, 50 mm tris - hcl ph 7.5) and then incubated for 1 h at rt with sheep anti - mouse igg peroxidase - linked (amersham) diluted 1: 2000 in washing solution containing 5% nonfat dried milk . Finally the membrane was washed 5 times in washing solution (10 min each), and specific protein complexes were identified using super signal west pico chemiluminescence reagent (pierce biotechnology) by autoradiography.
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, more than 9000 articles related to sids were published and over 100 sids explanations appeared in medical hypotheses . Sids occurs when an infant dies suddenly, unexpected by history, and without a cause found at forensic autopsy or thorough death - scene investigation . In the past four decades several multifactor models three inter - related causal spheres of influence model in which any two of these three could cause sids: (1) subclinical tissue damage (2) deficiency in postnatal development of reflexes and responses, and (3) environmental factors . Filiano and kinney also proposed a triple risk model but required three risk factors, similar to emery's, to act simultaneously and described them as (1) vulnerable infant; (2) critical development period; and (3) environmental stressors . These and other models do not, however, address the mathematical character of sids . This paper addresses each characteristic factor of sids that must be explained: the gender distribution; the age distribution; the effect of prone and supine sleep positions; the seasonal variation; risk factors of anemic apnea, respiratory infection, and neurological prematurity, and links them together with other causes of respiratory deaths as a proposed unifying theory . This paper uses data on infant live births and deaths as reported by the u.s . The sids data are given by international classification of diseases (icd) for 19791998 as 9icd and for 19992005 as 10icd . Although technically a sids diagnosis requires an autopsy without causal findings, not all these sids were autopsied and the percentage of sids without autopsy in the us has decreased monotonically from 1979 to the present day . However, cdc lists sids for all autopsied and nonautopsied cases without distinction . In the case of an interracial parentage consequently the cause of death and race of sids will have measurement error involved which will increase the chi - square 1 d.f . Test statistics (1) of comparisons between predicted and observed sids numbers and races, above those for tabulated p - values that assume the variance is from sampling error only . Therefore although p - values are presented they may give incorrect implications for rejection of hypotheses if they fall <p = .05 . We therefore rely upon the overall consistency of age and gender data to support our mathematical construct of sids . A characteristic male fraction is associated with sids . Because of difficulty in detecting congenital anomalies and other subtle causes of death in <28-day neonates, postneonatal sids the cdc reports there were 62,933 male and 40,952 female postneonatal sids during 1979 to 2005 for a male fraction of 0.606 . Figure 1 shows the male fraction of us postneonatal sids of all races over this period fluctuating slightly about the mean value of 0.606 as the sids rate decreased markedly from the discovery that the prone sleep position was a major sids risk factor which was followed by a back - to - sleep campaign in 1992 . Naeye et al . First hypothesized that the male excess in infant mortality could be x - linked . Mage and donner [810] showed that this excessive male fraction could be related to an unknown x - linked gene locus with a dominant allele (a) protective against sids, perhaps by providing enzymatic activity to allow anaerobic oxidation to take place in respiratory control neurons of the brainstem during transient periods of cerebral anoxia . The corresponding recessive allele (a), with frequency q, would not provide this protection and could allow critical cerebral neurons to die of anoxia, and sids to occur when the dominant allele a is not present . For xy males and xx females the recessive allele frequency (q) can be determined from (1a) and (1b), as the ratios of susceptible infants (1a)fmmm = q2fbq mb, (1b)q = fm / fbmm / mb, where fm and mm are postneonatal female and male sids and fb and mb are female and male live birth rates, respectively, so q represents the female postneonatal sids rate divided by the male postneonatal sids rate per 1000 live births . The global average male fraction of 0.612 for autopsied postneonatal sids is higher than the 0.606 us male fraction for total autopsied and nonautopsied sids, perhaps due to false positive sids that have a lower male fraction . With a 5% average male excess live birth rate, when stratified by race, we obtain from the cdc us 19792005 total postneonatal sids and birth data there is a white male fraction of 0.622, black male fraction of 0.570, and other races combined = 0.594 . Using (1b), q white = 0.639, q black = 0.779, and for other races combined q = 0.724 . Such variation in allele fraction between races along with the establishment of hardy - weinberg equilibrium is expected for each racial grouping from genetic drift over a long period of time . By necessity we accept here the cdc racial designations and neglect the presence of interracial infants . In our genetic analysis we assume that all sids infants have only the recessive allele (a) and require their probability of genetic susceptibility pg to equal 1 . To support this genetic mechanism, we note in table 1 that other causes of respiratory deaths in infancy have a statistically similar male fraction to 0.606 for postneonatal us sids when all races are combined . Not all of these cdc reported cases were autopsied, and false positive sids occur from infanticide by gentle suffocation that is virtually indistinguishable from sids at autopsy, so statistical testing assuming no autopsy error may not be cause for rejection at p = 0.05 . Rds, also known as hyaline membrane disease, had a male fraction of 0.610 . Bronchopulmonary dysplasia had a male fraction of 0.613 . With the discovery that the prone position is a risk factor, there is a trend to parse postneonatal sids into true sids and subcategories . Two such alternatives to sids are accidental suffocation and strangulation in bed and unknown ill - defined unspecified - causes which had male fractions of 0.593 and 0.597, respectively . . Suffocations by inhalation of food or other foreign object (siffo) in infancy had a male fraction of 0.600 very close to 0.606 (p = 0.52) for all postneonatal sids . The risk factors for infant inhalation of food or other object are morsel size, rounded shape, and slippery surface, like a grape . However, types of infant food, and mode and manner of preparation are identical for males and females, so these risk factors are independent of gender . We hold that all this tabulated male fraction similarity of order 0.61 is strong evidence of a common x - linked recessive susceptibility to the same terminal mechanism of cerebral anoxia . Furthermore, the virtually identical male fraction of 0.6053 compared to 0.6057 for sids occurs for these same siffo icd codes combined for all children ages 1 to 14 years in the us from 1979 to 2005, with 2,324 male and 1,515 female (p = 0.98). When broken into ages 14, 59, and 1014 years none of these groups are rejected . The implications of this consistent male fraction from infancy through adolescence is emphasized in the later discussion section . The siffo + igc data for the next cdc age group of 1519 years with a higher male fraction is not shown here because higher teenage male alcohol consumption is a new positive bias factor (496 male, 277 female: male fraction = 0.642). The male fractions in 19791998 of all us infant deaths by all icd 9 chapters and for 19992005 in their icd 10 equivalents are shown in table 2 . These data show the well - known male excess in virtually all icd classes of infant death, with only the neoplasms showing no male or female excess as expected from a purely random initiation process as the 5% us male live birth excess corresponds to a male fraction of 105/205 = 0.5122 . (1) the differing male fractions for most of these disease classes are essentially similar between the two periods 19791998 and 19992005 . This suggests that there is something physiological involved that provides the apparent characteristic excess male risk for each such class of cause of death . For example, certain conditions arising in the perinatal period with some 350 000 deaths covered by icd9 and 100 000 covered by icd10 have male fractions of 0.566 and 0.567, respectively . (2) the approximately 0.61 male fractions of table 1 for respiratory causes shown are found as expected for the congenital anomalies of the respiratory system (0.602 icd9 and 0.579 icd10) and diseases of the respiratory system (0.587 icd9 and 0.581 icd10). What is surprising is that the male fraction of mortality from diseases of the digestive system in infancy is similar to that from respiratory causes, 0.588 in 9icd and 0.601 in 10icd . In the uk from 1979 to 2006 other diseases of the digestive system (not ulcer - appendicitis - hernia - obstruction - chronic liver disease - cirrhosis) were 458 male and 329 female for a male fraction of 0.581 similar to the us data . We speculate that a linkage between the mechanism for the similar male fraction from digestive disease as sids may be from digestive causes such as malabsorption of iron and glucose in celiac disease and insufficient vascularization that would limit uptake and transport of glucose, respectively . This could lead to hypoglycemia that is a known risk factor for sids and sudden death [15, 16]. In the older infant, the resistance to hypoxia is much less than for the neonate, reflecting the diminished stores of glycogen and therefore limited substrate for anaerobic metabolism . An enzyme, such as glucose-6-phosphate dehydrogenase (g6pd) could play a role as its x - linked gene locus is at xq28 and it has a great multiplicity of alleles that are associated in their deficiency with nonspherocytic hemolytic anemia, and anemia is a likely risk factor for sids . G6pd catalyzes initiation of glucose oxidation via the hexose - monophosphate pathway that may be a critical requirement for neuronal survival during cerebral anoxia . There could be more complicated x - linked processes such as requiring two (or more) independent x - linked alleles with probabilities q1 and q2, with probability of simultaneous presence (q = q1q2) that would equal the qvalues listed above for a single x - linked allele . Alternatively, a gene locus such as g6pd could have many recessive alleles (q1, q2, q3,) that are nonprotective of sids that could sum up to the q values listed above for the same risk of sids (q = q1 + q2 + q3 +). We have chosen a single - gene x - linkage process for simplicity of discussion, and note that any genome - wide association study required to test our model can test for all possibilities . The age distribution of sids is unique: any viable hypothesis for the cause of sids must account for its characteristic age distribution . . Raring first noted that the unique and characteristic age distribution of sids appeared to follow a 2-parameter lognormal model . Mage reviewed the sids age literature and in a meta - analysis of 15 global sids age data sets obtained the distribution of some 20 000 ages of sids shown in figure 2 . In construction of figure 2, 1-month is <28 days of life . Age data in weeks of life were divided by 4.33 to convert to months and the althoff data from cologne reported as age within midmonth intervals (e.g., 1.52.5 month) were plotted to estimate the corresponding integer month intervals (e.g., 1 - 2 months and 2 - 3 months) for pooling with the other monthly sids data . The total of 19,949 sids includes 194 sids deaths that are predicted to occur after 1-year in these 15 cohorts by use of an exponential fit to monthly data intervals 5 to 12 that was then extrapolated and summed from 13 to 41 months . These data in figure 2 were fit by a 4-parameter lognormal distribution, also known as the johnson sb distribution, shown as (2). Here dp(m) is the probability of sids occurring between ages m and m + dm in months, median = 3.1 months and standard deviation = 0.6617, as fit by maximum likelihood (2)dp(m)dm=(22)1[(m+0.31)1 + (41.2m)1] exp [log e2([(m+.31)(41.2)]/[(41.2m)(+.31)])/22]. Equation (2) can be interpreted as a sum of products of three age dependent terms, denoted as pn, pi, and pa . Let pn = 1/(m + 0.31) represent a risk factor of neurological prematurity leading to delays in development of respiratory reflexes and responses, that decreases with increasing age . Neurological prematurity is a risk factor that is maximal at birth and decreases as the infant physically matures . Kinney has found that an important subset of sids appears to have a deficiency in serotonin receptors that is hypothesized as a causal factor of those sids . Let pi = 1/(41.2 m) represent an infection risk factor that increases with increasing age . Secondary sids, that have findings of low - grade respiratory infection at autopsy that of itself is insufficient to cause death . Risk of such infection increases with age as infants lose passively acquired maternal immunoglobulin (igg) and they have increased exposure to pathogens as they have more contacts both within and without their immediate family . Us dhhs linked birth and death certificate data for 19952004 show, in table 3, that the rate of sids increases monotonically with live birth order (lbo). It has been suggested that older school - age siblings may be an important respiratory infection vector . We assume here that the infant lives with two parents, all older siblings survived to the time of sids death, and no adoption of the sids infant or older siblings took place . For lbo 6 we assume only 5 siblings have contact with the infant . Let the probability of a family member not carrying a respiratory infection communicable to the infant at any time = p. for infants with family size = 2 parents + (lbo 1) siblings the probability of not having an infection vector present is equal p. the probability of an exposure to at least one carrier is then 1 p. by least squares analysis we found p = 0.9 with a scaling factor of 2.5, so we model the rate of sids per 1000 = 2.5 (1 . Infant anemia has not been considered directly as a risk factor for sids per se, because accurate hemoglobin [hb] levels cannot be determined after death due to rapid hb breakdown resulting in the mottled and reddened areas known as livor mortis . A study in mice shows how hb is already significantly decreased in the first postmortem hour . Because the exact time of sids during sleep is not known it would be impossible to correct for the variable amount of hb lost between the instant of sids death and autopsy . There is, however, indirect evidence suggesting a relationship between anemia and sids: the peak incidence of sids coincides with the nadir [of hb] in the physiological anemia of infancy . Anemia does contribute to apnea and apparent life - threatening events (altes) from causing longer cyanotic breath - holding spells [2932] that are risk factors for sids, leading to the apnea hypothesis . [3, 32]. Therefore anemia is treated by us as a risk factor for sids . Let pa = exp [log e ([(m + 0.31)/(+ 0.31)]/[(41.2 m)/(41.2)])/(2)], as found in the johnson sb model as (2), represent an anemia - cum - apnea risk factor rising from 0 at birth, reaching a peak at the median (= 3.1 months) and decreasing to zero at 41.2 months . Anemia in infancy may be defined relatively as any value for the hemoglobin [hb] less than two standard deviations (<2) below the mean for age, or absolutely as less than a fixed value, such as 13.5 g / dl which is the 2 level below mean cord blood hb and mean hb at 1 week . Infant physiological anemia is a risk factor that is virtually zero at birth due to placental transfusion during labor and at birth hb concentration in the blood can reach + 2 of 23.7 g / dl . We propose that this high at birth hb phenomenon accounts for the relative protection from sids during the first week of life . In the following weeks, total hb decreases rapidly as fetal hemoglobin (hbf) is removed faster than it can be replaced by adult hemoglobin (hba). A nadir in total hb occurs at or about 2 months of age for a term infant that corresponds to the 63rd day mode of the sids sb age distribution [18, 21]. Table 4 shows the 2 hb g / dl level (lowest 2.5% of all infants). By definition, approximately 25 in 1000 term infants have a hb value below the 2 value shown, and preterm infants will fall under this value with a higher frequency, perhaps related to their increased risk of sids . Of those 25 in 1000, the one with the lowest hb would be at the highest risk of apnea and therefore sids . If physiological anemia is considered as a hb deficit from a fixed level of 13.5 g / dl, that could, combined with apnea, cause transient hypoxia and inability to meet neuronal oxygen demand in the brainstem of sids susceptible infants . If so, it could correspond, as shown in table 4, to the rise - and - fall factor pa modeled from the johnsonsbdistribution as (2) fit to figure 2 . The presence of respiratory infection as a risk factor fits the characteristic of sids of a seasonal dependency, maximizing in the winter and minimizing in the summer, that has been associated with wide seasonal temperature changes . Mage showed that in hawaii, a semitropical us state with only narrow seasonal change in mild temperatures, that 384 sids varied seasonally with calendar day (t) between 1979 and 2002 as a cosine function shown as (3) where the maximum sids rate is predicted to occur on january 30th (t = 30): (3)mortality on day t=0.810 + 0.241 [1+cosine2(t30)365.25],0<t<365.25 . This equation may be interpreted to show that in hawaii, 23% of sids have a seasonal infection component (0.241/1.051) and that 77% of sids occur at a constant rate due to other physiological factors such as anemia and neurological prematurity that have no known seasonality . The winter flux of infection vector would come via visitors from the us mainland and japan, so the authors expect that the proportion of seasonal infections and sids may be larger in temperate zones of the us with colder winter temperatures than found in hawaii . There has been a tendency for the winter peak to be reduced since the start of the back - to - sleep campaign that may be due to the lessening of the hypoxia caused by low - grade seasonal respiratory infection when sleeping supine [38, 39]. The pre-1992 lognormal form of the age distribution of sids [40, 41] remained the same during the change of preferred sleep position from prone to supine . Pollack found the age distributions of us sids between 1989 and 1999 were virtually unchanged in the two cohorts . He reported that the stability of this distribution is remarkable when one considers the large decline in sids incidenceas shown in figure 1 . Malloy and freeman also found little change in age distribution for us sids between 1992 and 1999 (p = 0.025). The derivation and its explanation for this consistency is aided by a venn diagram shown as figure 3 . Let a prone sleeping infant be susceptible to sids in both the pg pa pn and pg pa pi areas of figure 3 even if missing the pi or pn risk factors, respectively . Note that the sum of pg pa pn + pg pa pi represents two overlapping areas on the venn diagram because the central segment (pg pa pn pi) is counted twice . Let the probability of a prone sleeping child = pp and that of a supine sleeping child = ps . For simplicity we include the side sleeping position with the prone, and we require pp + ps = 1 . Then the probability of dying of sids at age m while prone (ppsids) is written as, (4)ppsids = pp pg pa ([pn+pi]pnpi). One can then write the probability of supine sids (pssids) as, (5)pssids = ps pg pa pn pi . Combining (4) and (5) we get the total probability of sids (psids) as (6)psids = pp pg pa (pn+pi)+(pspp) pg pa pn pi . We then note that the sum of pn + pi has a similar mathematical form as pn pi as follows: (7a)pn+pi=1(m + 0.31)+1(41.2m)=[(41.2m)+(m+0.31)][(m+0.31) (41.2m)], (7b)pn+pi=41.5[(m+0.31)(41.2m)], (7c)pn pi=1[(m+0.31)(41.2m)]. Thus the mathematical form for the age distribution of both supine sids and prone sids can be represented by the same relationship of c pa/[(m + 0.31) (41.2 m)], where c is a constant, which implies that, in terms of relative probability at different values of m, (7b) and (7c) are the same . This is consistent with the report that there were similar frequencies of pathological findings in both supine and prone sids confirming that the mode and cause of sids death is apparently the same for both sleep positions . This derivation shows how the venn diagram and johnson sbage distribution predict that supine and prone sids have the same age distribution, with lower rates for the supine sids . This corresponds to the supine requirement to have all 4 risk factors (pg pa pn pi) as opposed to only 3 risk factors (pa pg pi or pa pg pn) that can allow a prone sids to happen more readily . Factors that make the prone sleep position a risk factor for sids are rebreathing of exhaled breath with reduced oxygen and increased carbon dioxide and the finding that presence of a fan in the infants sleep environment, that disperses exhaled breath, decreases the sids rate . Other hypotheses than the x - linkage hypothesis of naeye et al . For the male excess in sids and other causes of infant respiratory mortality have appeared in the literature [4547]. The mechanism behind the excess perimortality rate in male infants is not known . A genetic factor leading to reduced tolerance analyzed amniotic fluid and showed the male fetus developed pulmonary surfactants slower than the female fetus and suggested that this deficit at birth may cause the male excess in infant respiratory distress syndrome (rds) that matches that of sids . This is not likely because the measured deficit should decrease with maturity as the infant ages, but cdc reports that the male fraction of rds between 28 and 364 days [0.617] is greater than the male fraction [0.604] on the first day of life when the deficit is maximal . Patterson et al . Found in their sids cases that males had a larger deficiency in serotonin receptors in the brainstem than females and suggested that this may be related to the male excess in sids . As for the male surfactant deficit cited above, a greater male serotonin - receptor deficit at birth should decrease with infant maturity, but the 0.606 male fraction of sids between 28 and 364 days is also greater than the 0.548 male fraction for 06 days (which may partially be related to false positive sids from undiscovered infanticide or subtle congenital anomalies). L'hoir et al . Found in their study in the netherlands that male infants were placed to sleep in the prone position more often than females, and were more likely to turn prone from a side sleeping position than females, and suggested that this may be related to the male sids excess . However, as shown in figure 1, the sids male fraction remained essentially the same as the recommended sleep position in the us changed from prone (pre-1992) to supine (post-1992), even though the sids rate dropped by a factor of three from 1979 to 2005 . Furthermore, any other hypothesized cause for sids that suggests that the sids male excess in mortality is related to a male underdevelopment relative to the female cannot explain the fact that virtually exactly the same male fraction of 0.605 occurs for siffo between 1 and 14 years as the 0.600 in the first year of life shown in table 1 . The risk factors for siffo in children are independent of gender because food in the us is not chosen or prepared differently for males and females . Types of food that are most often recovered from the upper airway at infant autopsy are raw carrot and apple, round and slippery items such as hotdog pieces without skin removed, candy, nuts, and grapes [4951]. Foreign objects swallowed by children over 1 year of age are often balloons and small coins such as pennies . Although the rates of siffo decrease with age, as dentition and swallowing control develop, and the types of food items eaten by children change as they go from infancy to 14 years of age (e.g., chewing gum is often inhaled), the male excess remains the same up to 14 years . As opposed to sids that predominantly occurs during sleep, siffo predominantly occurs while the infant is awake or being fed, and immediate first aid is attempted that is successful in approximately 99% of all cases [52, 53]. Yet, assuming equal siffo risks for males and females, more males than females cannot be resuscitated in exactly the same proportion as dying in sids . Virtually all other risk factors posited for sids are either independent of gender (e.g., parental smoking or autosomal genetic conditions) or are inoperative for siffo between 1 and 14 years of age except the possible x - linkage . An obvious potential cause of an infant male excess for any icd class may be due to an androgen excess in the male . We discussed this previously and showed that during the first year of life, the sids male fraction remains relatively constant while the male serum testosterone is slightly higher than the female's at birth, peaks for three months during the first six months to aid testicular descent, and falls back towards zero for the second six months of life, so an androgen interaction is not a likely factor . As in any epidemiology study, there is always a finite probability that these data are the result of happenstance and coincidence, known as sampling error, and that next year's data may be cause for rejection of the developments presented . We have shown how all characteristic properties of sids, its gender, age, and seasonal distributions, along with the observed risk factors of apnea, respiratory infection, and neurological prematurity, can be tied to each other mathematically . These relationships presented here explain how the supine position reduces the rate of sids and why it does not change the gender distribution or the form of the age distribution from those of sids occurring predominantly in the prone position . Because all sids risk factors except the hypothesized x - linkage are independent of gender, we propose that equal numbers of males and females, per equal numbers of live births, are at risk of having potentially fatal risk factors that we previously defined here as pa, pi, and pn . Approximately 2/3 of all males and 4/9 of all females have a genetic risk factor pg that is necessary to cause sids but not sufficient by itself infants with the protective allele and the three other risk factors (see figure 3) may be among the cohort of those presenting with apparent life - threatening episodes (altes) that do not then or later progress to sids . It is proposed that sids may occur for those genetically susceptible infants when repeated transient coincidences of factors reduce the oxygen supply (apnea, anemia, rebreathing exhaled breath, etc .) During a period of increased oxygen demand (low grade respiratory infection raising body temperature). If the infant has a residual neurological prematurity, auto resuscitation by the gasp reflex may be delayed causing acute cerebral anoxia that may cause some respiratory - drive neurons in the brainstem to die (emery's subclinical tissue damage). When a sufficient number of such neurons die, the next sleep with identical risk factors causing anoxia may reduce the number of functioning neurons below a minimum critical requirement so auto resuscitation is impossible . The protected infant with an x - linked dominant allele (a) could switch over from aerobic oxidation to anaerobic oxidation to keep those critical neurons alive during the same transient anoxic conditions so that autoresuscitation could occur . In summary, the quadruple risk model presented here, with factors developed from pre-1994 gender data and from pre-1992 sids age data, predicts the age and gender distributions for post-1992 data as shown . The factors determining the age distribution mesh with the medical literature's findings of the risk factors for sids . Should the genetically susceptible infant pass through infancy unscathed, the genetic susceptibility to cerebral anoxia can still penetrate in childhood if anoxic circumstances arise as shown by the identical us postneonatal sids male fraction of 0.606 occurring in us children aged 1 to 14-years suffocating from inhalation of food or other foreign objects . So, in the absence of any other plausible explanation in the medical literature for the same siffo male excess from birth to 14 years of age as sids, a common x - linkage remains as the only possibility . Furthermore there was a 45% excess adult male completion rate of suicide attempts by coal - gas inhalation in paris between 1949 and 1962 (completions of 58% male versus 40% female). In conclusion, although modern thought is now that sids is a composite of independent and different causes of death, they all appear to have the same male fraction . We reason that all those different causes of death lead to the same cerebral anoxia that may result in respiratory failure from the absence of an x - linked dominant allele that supports anaerobic oxidation in respiratory control neurons of the brainstem . Proof of this unifying mechanism must await genetic testing to identify, if correct, the unknown recessive x - linked allele that is exclusively present in all these icd codes with the statistically similar male excess of sids.
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This study demonstrates the application of an iterative two - stage multi - method strategy to improve quality of service (ie, design - based research). It represents a continuous and systematic appraisal of an operational process with the objective of improving the quality of health service . There was a notable difference between the sizes of the first and second survey samples (n=282 vs n=121). The ratio of first time to follow - up patients, however, was similar in both of the survey samples . The patient flow analysis (pfa) used in our study was adapted and did not cover all the stages from the entry point to the exit point within the health care process . Finally, our study corroborates previous research that the perception of waiting considerably influences patient satisfaction . However, providing information about wait time as well as distractions from waiting can improve the perception of quality of service . Health care delivery is currently at the midpoint of a period of transition; a period characterized by the appraisal and continuous improvement of services that health care providers are offering to patients . Steered by increased consumer demands for easily accessible, cost - effective, and efficient health services, many health systems have been forced to redesign and standardize their operational processes . In 2001, the institute of health care published a report that defined six aims to help improve the quality of health care, and these were outlined in a framework of guiding principles that would help health institutions stay abreast of an increasingly competitive health care market.1 timeliness was one of these six aims and involved providing timely care to patients that would help reduce harmful delays . Better recognized as wait time in the literature, this important but understudied aspect of health care has been described as an essential determinant of patient satisfaction in health care practice,2 particularly because long wait times have been found to result in negative perceptions of the quality of services provided in outpatient departments3,4 and the resultant decrease in patient satisfaction in turn may influence the return rate to outpatient departments, an essential element in the treatment of complex and chronic conditions.5 the atrium medical center is a large non - university teaching hospital in the southern part of the netherlands . The hospital houses about 1,300 hospital beds spread over three locations, ie, heerlen (main hospital site), and kerkrade and brunssum (satellite hospital sites). In addition to the outpatient consultations provided at the three sites, the site in heerlen provides clinical services, ie, long - term hospital admission and the delivery of acute, intensive, and complex care . Elective (non) surgical procedures are provided in brunssum, while medical diagnostic services are provided in kerkrade . Approximately 200 medical specialists, 150 specialist registrars, and 3,600 other hospital staff are responsible for the health care services to the 300,000 inhabitants of the region . As a teaching hospital, the atrium medical center also caters for the professional training of undergraduate (90 medical students) and postgraduate (135 specialist registrars) physicians, as well as nursing staff (200 trainee nurses and midwives) and in - house employees . Each year, the hospital records about 30,000 long - term and 35,000 short - term (<24 hours) clinical admissions . Hospital admission records show that our pediatric department notes approximately 1,900 long - term and 770 short - term clinical admissions, while the pediatric ambulatory clinic (spread over the three locations) notes yearly 4,900 first consultations and 7,800 follow - up consultations.6 the atrium medical center is a large non - university teaching hospital in the southern part of the netherlands . The hospital houses about 1,300 hospital beds spread over three locations, ie, heerlen (main hospital site), and kerkrade and brunssum (satellite hospital sites). In addition to the outpatient consultations provided at the three sites, the site in heerlen provides clinical services, ie, long - term hospital admission and the delivery of acute, intensive, and complex care . Elective (non) surgical procedures are provided in brunssum, while medical diagnostic services are provided in kerkrade . Approximately 200 medical specialists, 150 specialist registrars, and 3,600 other hospital staff are responsible for the health care services to the 300,000 inhabitants of the region . As a teaching hospital, the atrium medical center also caters for the professional training of undergraduate (90 medical students) and postgraduate (135 specialist registrars) physicians, as well as nursing staff (200 trainee nurses and midwives) and in - house employees . Each year, the hospital records about 30,000 long - term and 35,000 short - term (<24 hours) clinical admissions . Hospital admission records show that our pediatric department notes approximately 1,900 long - term and 770 short - term clinical admissions, while the pediatric ambulatory clinic (spread over the three locations) notes yearly 4,900 first consultations and 7,800 follow - up consultations.6 in 2011, we observed a decline in the positive patient satisfaction ratings of the services delivered in our pediatric ambulatory clinic . We also found out that there was growing dissatisfaction from our hospital management board with respect to the utility of resources for our operational processes, and patients were unhappy with some of our services as evidenced by the outcome of a focus group interview on the quality of our care pathway for pediatric asthma care . One of the major complaints on further analysis was about patients ease of access to our services and, in particular, wait time . As it has been found in the literature that teaching hospitals tend to have longer wait times than non - teaching hospitals,2,5 and because our institution (specifically our department) had a teaching setting, the need arose to perform a formal evaluation of patients perceptions of our services, ie, operational processes, and also to investigate for methods to help 1) reduce patient wait time; 2) overcome barriers to effective patient flow; and 3) improve the efficiency of care . Based on the identified problem, we decided to investigate wait time in our pediatric ambulatory clinic and investigate patients perception of the services provided . The goal of the survey was to improve the ultimate customer experience of patients and their parents . More specifically, we aimed to obtain more insight into the operational processes of our services, including identifying elements that influence the wait time, the specific expectations of patients regarding our services, and recommendations on how to serve them better . Not only was it essential for us to understand the impact of wait times and their effect on our patients experience of care, but it was equally important for us to understand the managerial implications of these findings and what is required to restore and/or improve patient satisfaction . As the project was a service improvement initiative, we decided to design a two - stage process improvement study made up of a process evaluation, an intervention, and a re - evaluation at a later stage (see figure 1). We decided to conduct an explorative survey to investigate the operational process within our ambulatory care setting . We chose to investigate our wait time using a pfa, and with the help of a standardized questionnaire investigated patients perceptions of the services provided in our clinic . The study was conducted at the three locations of the outpatient department of the atrium medical center . As the atrium medical center is a teaching hospital, both pediatricians and pediatric residents work in the ambulatory clinics, which serve patients aged 018 years with a wide range of socioeconomic backgrounds . All patients who took part in the survey were insured, as this is obligatory in the netherlands . An explanation of the objective of the survey and the request for informed consent from all respondents was achieved through the questionnaire prior to participation in the survey . All the physicians involved in the delivery of care during the survey were blinded to who and when ratings were taking place . As the survey was service improvement oriented and did not involve research on human, ethical review board approval was not sought . We designed an 18-item questionnaire that included a simple written survey to assess the wait time . Some of the items included in the questionnaire focused on investigating patients satisfaction with the wait time, the quality of the facilities provided in the waiting area, whether patients were provided with information about the wait time, whether they were willing to see other health care providers in case of long wait times, and finally, for general recommendations on how to serve them better . In addition to answering to specific short answer questions, respondents could add qualitative comments to the items (figure s1). The first stage of the study (process evaluation) was conducted in the months of june, august, and september of 2013 . Based on the findings of the process evaluation, a three - stage intervention was designed to address the identified problems . Stage 1 involved reporting the outcome of the survey back to the health workers at different points of the service chain, ie, the attendants at the reception desks, the medical team (pediatrician and residents), and the back - office administrative staff . Stage 2 on the other hand involved analyzing the various recommendations obtained from the different stakeholders, while stage 3 involved synthesizing the various recommendations into concrete improvement plans that resulted in structural improvements to the waiting rooms, consultation rooms, and back offices . The most important improvement plan was where the changes were performed to wait time, ie, the length was limited to a maximum of 10 minutes and to consultation time, which was increased to 20 minutes for residents, and a 15-minute buffer was added to the allocated consultation time for the pediatricians . The second survey, ie, re - evaluation study, was performed between january and march 2014 at the three locations of the pediatric ambulatory clinic . The objective of this second survey was to evaluate the effect of the process improvement intervention we performed in 2013 . We analyzed the data by means of descriptive statistics and chi - square analysis using spss 22.0 (ibm corporation released 2013, ibm spss statistics for windows, version 22.0; ibm corporation, armonk, ny, usa). Qualitative data were clustered into an excel spreadsheet (2003, microsoft corporation, redmond, wa, usa). Based on the identified problem, we decided to investigate wait time in our pediatric ambulatory clinic and investigate patients perception of the services provided . The goal of the survey was to improve the ultimate customer experience of patients and their parents . More specifically, we aimed to obtain more insight into the operational processes of our services, including identifying elements that influence the wait time, the specific expectations of patients regarding our services, and recommendations on how to serve them better . Not only was it essential for us to understand the impact of wait times and their effect on our patients experience of care, but it was equally important for us to understand the managerial implications of these findings and what is required to restore and/or improve patient satisfaction . As the project was a service improvement initiative, we decided to design a two - stage process improvement study made up of a process evaluation, an intervention, and a re - evaluation at a later stage (see figure 1). We decided to conduct an explorative survey to investigate the operational process within our ambulatory care setting . We chose to investigate our wait time using a pfa, and with the help of a standardized questionnaire investigated patients perceptions of the services provided in our clinic . The study was conducted at the three locations of the outpatient department of the atrium medical center . As the atrium medical center is a teaching hospital, both pediatricians and pediatric residents work in the ambulatory clinics, which serve patients aged 018 years with a wide range of socioeconomic backgrounds . All patients who took part in the survey were insured, as this is obligatory in the netherlands . An explanation of the objective of the survey and the request for informed consent from all respondents was achieved through the questionnaire prior to participation in the survey . All the physicians involved in the delivery of care during the survey were blinded to who and when ratings were taking place . As the survey was service improvement oriented and did not involve research on human, ethical review board approval was not sought . We designed an 18-item questionnaire that included a simple written survey to assess the wait time . Some of the items included in the questionnaire focused on investigating patients satisfaction with the wait time, the quality of the facilities provided in the waiting area, whether patients were provided with information about the wait time, whether they were willing to see other health care providers in case of long wait times, and finally, for general recommendations on how to serve them better . In addition to answering to specific short answer questions, respondents could add qualitative comments to the items (figure s1). The first stage of the study (process evaluation) was conducted in the months of june, august, and september of 2013 . Based on the findings of the process evaluation, a three - stage intervention was designed to address the identified problems . Stage 1 involved reporting the outcome of the survey back to the health workers at different points of the service chain, ie, the attendants at the reception desks, the medical team (pediatrician and residents), and the back - office administrative staff . Stage 2 on the other hand involved analyzing the various recommendations obtained from the different stakeholders, while stage 3 involved synthesizing the various recommendations into concrete improvement plans that resulted in structural improvements to the waiting rooms, consultation rooms, and back offices . The most important improvement plan was where the changes were performed to wait time, ie, the length was limited to a maximum of 10 minutes and to consultation time, which was increased to 20 minutes for residents, and a 15-minute buffer was added to the allocated consultation time for the pediatricians . The second survey, ie, re - evaluation study, was performed between january and march 2014 at the three locations of the pediatric ambulatory clinic . The objective of this second survey was to evaluate the effect of the process improvement intervention we performed in 2013 . We analyzed the data by means of descriptive statistics and chi - square analysis using spss 22.0 (ibm corporation released 2013, ibm spss statistics for windows, version 22.0; ibm corporation, armonk, ny, usa). Qualitative data were clustered into an excel spreadsheet (2003, microsoft corporation, redmond, wa, usa). Two hundred and eighty - two respondents, of which 71% were follow - up patients, completed the first survey . The majority of patients arrived on time at the outpatient clinic; only 8% arrived> 10 minutes after the appointment . Seventy - nine percent of the patients were called in within 20 minutes after their appointment time . At baseline, the majority of the respondents were satisfied with the wait time to see the doctor (89%) and with the available amenities provided (96%97%), eg, waiting room, consultation rooms, and the atmosphere of the clinics setting (see table 1). The satisfaction with length of wait time, atmosphere, waiting area, and provided amenities is displayed in table 2 . Only 50% of the follow - up patients were aware that the consultations with the doctor lasted 15 minutes . Eighty - nine percent of the respondents thought that the length of the consultation was sufficient, while 11% preferred that the consultation time be lengthened . A large number (99%) of the participants responded that there was enough time to ask questions to the doctor . The majority (90%) of the respondents were of the opinion that the wait time did not influence the quality of the consultation . Of the respondents who were dissatisfied with the wait time, 20% thought that this influenced the quality of the consultation, whereas 80% thought that it did not influence the quality of the consultation . Thirty - two percent of the respondents were informed about the wait time, whereas 25% were not informed . Forty - two percent of patients responded that this was not applicable as they were attended to promptly, ie, no wait time . One hundred and twenty respondents, 75% of whom were follow - up patients, completed the second survey, performed 2 months after implementation of the intervention . After intervention, 79% was called in within 20 minutes after the appointment (see tables 1 and 3). The patients satisfaction with the wait time was 91% (see table 1). For the majority of the patients (55%), 1020 minutes the satisfaction with the length of time of the consultation has risen to 96% (p=0.05) (see table 1). The majority (98%) the satisfaction with the waiting environment was high (97%99%), although some parents responded that the waiting area was inconvenient to oversee playing children (see tables 1 and 2). A minority (24%) was informed about the wait time, 32% was not informed, and 42% of patients responded that this was not applicable due to no wait time . In both surveys, half of the patients were willing to see a different pediatrician if this would lead to a shorter wait time . The main argument to see the same pediatrician was that the child was familiar with the pediatrician and that the pediatrician had all the inside information about the child and its background . Other parents were neutral about seeing the same pediatrician, as long as the pediatrician had access to the medical history of the child, or preferred a shorter wait time instead of waiting to see the same pediatrician (see tables 4 and 5). It should be noted that for some questions where respondents could add qualitative comments, only a few did make use of this by providing their comments . Waiting with infants or children with known behavioral problems was the most reported reason for why parents experienced wait time as a problem . Also, having a scheduled appointment after the consult was a reason for dissatisfaction with waiting (see tables 4 and 5). Parents reported that they would like to be informed about the expected wait time, preferably as soon as they checked in at the front desk . Two hundred and eighty - two respondents, of which 71% were follow - up patients, completed the first survey . The majority of patients arrived on time at the outpatient clinic; only 8% arrived> 10 minutes after the appointment . Seventy - nine percent of the patients were called in within 20 minutes after their appointment time . At baseline, the majority of the respondents were satisfied with the wait time to see the doctor (89%) and with the available amenities provided (96%97%), eg, waiting room, consultation rooms, and the atmosphere of the clinics setting (see table 1). The satisfaction with length of wait time, atmosphere, waiting area, and provided amenities is displayed in table 2 . Only 50% of the follow - up patients were aware that the consultations with the doctor lasted 15 minutes . Eighty - nine percent of the respondents thought that the length of the consultation was sufficient, while 11% preferred that the consultation time be lengthened . A large number (99%) of the participants responded that there was enough time to ask questions to the doctor . The majority (90%) of the respondents were of the opinion that the wait time did not influence the quality of the consultation . Of the respondents who were dissatisfied with the wait time, 20% thought that this influenced the quality of the consultation, whereas 80% thought that it did not influence the quality of the consultation . Thirty - two percent of the respondents were informed about the wait time, whereas 25% were not informed . Forty - two percent of patients responded that this was not applicable as they were attended to promptly, ie, no wait time . One hundred and twenty respondents, 75% of whom were follow - up patients, completed the second survey, performed 2 months after implementation of the intervention . After intervention, 79% was called in within 20 minutes after the appointment (see tables 1 and 3). The patients satisfaction with the wait time was 91% (see table 1). For the majority of the patients (55%), 1020 minutes the satisfaction with the length of time of the consultation has risen to 96% (p=0.05) (see table 1). The majority (98%) the satisfaction with the waiting environment was high (97%99%), although some parents responded that the waiting area was inconvenient to oversee playing children (see tables 1 and 2). A minority (24%) was informed about the wait time, 32% was not informed, and 42% of patients responded that this was not applicable due to no wait time . In both surveys, half of the patients were willing to see a different pediatrician if this would lead to a shorter wait time . The main argument to see the same pediatrician was that the child was familiar with the pediatrician and that the pediatrician had all the inside information about the child and its background . Other parents were neutral about seeing the same pediatrician, as long as the pediatrician had access to the medical history of the child, or preferred a shorter wait time instead of waiting to see the same pediatrician (see tables 4 and 5). It should be noted that for some questions where respondents could add qualitative comments, only a few did make use of this by providing their comments . Waiting with infants or children with known behavioral problems was the most reported reason for why parents experienced wait time as a problem . Also, having a scheduled appointment after the consult was a reason for dissatisfaction with waiting (see tables 4 and 5). Parents reported that they would like to be informed about the expected wait time, preferably as soon as they checked in at the front desk . In this paper, we describe the process of how a pfa was used to gain a better insight of the wait time in our pediatric ambulatory clinic . A questionnaire was designed to identify elements that influenced wait time, specific customer expectations, and perceptions of the quality of our services . Our aim was to improve the ultimate customer experience of our patients (and their parents) and, in particular, increase their satisfaction with our wait times and waiting environment in our clinic . In addition, an intervention based on the outcomes of the first survey was designed, which included increasing consultation time and carrying out structural improvements in the waiting rooms . In general, our findings showed that there was high satisfaction with the wait time, both prior to and after the intervention . Satisfaction with the available amenities and the waiting area and atmosphere was also high among the respondents . We also saw an increasing trend of higher satisfaction with the increase in duration of the consultations from 15 minutes to 20 minutes for residents and addition of a 15-minute buffer to the total allocated time for pediatricians consultations . Unfortunately, the intervention did not result in a decrease in wait time or a significant increase in patient satisfaction with wait time . It has been well established that patient satisfaction is significantly influenced by wait time, with longer wait times decreasing patients satisfaction of the overall quality of the services.2,4,7,8 moreover, increased wait time negatively influences the perceived quality of care received from the clinician, including various quality elements, such as reliability, assurance, and empathy, as well as the likelihood of return visits.4,8 so minimizing wait time is of essence in establishing good quality of care . The findings of our survey suggest that for the majority of the patients, a wait time of no more than 20 minutes was acceptable . These findings are consistent with hill and joonas who reported 1630 minutes to be an acceptable wait time for the majority of patients . If a patient would have an unacceptable wait time, this could lead to negative overt action by the patient, including switching to a different medical service provider.4 for the majority of patients, unacceptable experiences would have to occur fairly frequently before they would lead to some type of response . However, for some patients, an infrequently occurring unacceptable wait time would be the reason to undertake action and, in the worst case, switch doctors.4 another thing we observed in our study was the ambivalence among our respondents toward seeing a different pediatrician even if this would lead to a shorter wait time . The physician s familiarity with the patient s problem and their expertise were the most reported reasons for why respondents preferred the same specialist . As was discussed by hill and joonas, patients are able to separate their assessment of how competent the doctor is from how they feel about him / her as a person and their ultimate satisfaction with the total experience.4 in our study, 90% of the respondents found that the wait time did not influence the quality of the consult . Of the respondents who were dissatisfied with the wait time, the majority (80%) of them felt that this did not influence the quality of the consult . Wait time did not appear to influence patients to like the doctor less.4 however, it was discussed that at some point, regardless of how good the doctor is perceived to be, patients become dissatisfied with the provider as they experience unacceptable wait time.4 these abovementioned points further buttress the importance of minimizing wait time . It has been shown in the literature that the use of a time - flow study can be a useful and effective technique to identify inefficiencies in the patient visits, target interventions, and measure effectiveness of interventions.5 furthermore, the ease of performing a pfa in an ambulatory setting negates the need to hire external consultants, thus avoiding additional expenditures . In addition, it allows clinical staff members who are familiar with patient flow designs, develop a sense of ownership in the process.5 in contrast to other studies using pfa,3,5,9 the patients and their parents in our survey were able to provide qualitative comments . As mentioned earlier, the perception of waiting time has been shown to be a better indicator of patient satisfaction than actual waiting time.8,10 therefore by assessing our patients opinions, we gained additional insights into the patients perception of our operational process . This enabled us to identify specific expectations regarding our services and obtain patients recommendations on how to improve these services . In this way, we were able to design an intervention based on patients perception of care and assess the patients satisfaction after the intervention . There are several factors that have been found to influence the perception of wait time . For example, anxiety makes waiting seem longer, unexplained waits are longer than explained waits, and unoccupied wait time feels longer than occupied wait time.11 additionally, some of the comments provided in our study showed that waits with infants or children with behavioral problems could be of particular inconvenience and lead to a negative perception of wait time . For example, the provision of specific activities or toys to engage children with in waiting rooms, distracts their attention from the length of waiting and minimizes the chances of unwanted tantrums . Therefore, it could be an important contributor to improve the waiting experience for children in hospitals by improving environmental attractiveness.10 providing information about wait time can be an important factor in establishing patients satisfaction . In our study, respondents preferred to be informed about the wait time as soon as they checked in at the front office . As was previously discussed, dissatisfaction with wait time was of less influence on the overall satisfaction of emergency room patients when appropriate information about delays was provided.2 there are a number of limitations in our study that should be mentioned, including the difference between the sample sizes (n=282 vs n=121). It is our assumption that this difference may have been as a result of redundancy, ie, patients refusing to participate again having taken part in the first survey . However, as the ratio of the new to follow - up patients was approximately equal in both surveys despite the different sample sizes, it is unlikely that this is the explanation for the smaller group in the second study . It is our assumption that the difference was probably due to a loss of motivation among the reception desk staff who were responsible for handing out the questionnaires . Nonetheless, since this observational study was performed to obtain information about the patients view of accessibility to caregivers and the quality of care received, we believe the sample sizes were large enough to achieve this goal . A second limitation was the fact that we used a pfa to identify the wait time from entrance to the doctor s room . Ideally, a pfa should include the time the patient is seen by the nurse (for obtaining physical measurements) to identify inefficiencies in the whole care process . Considering minimizing wait time, it would be better to perform a pfa from entrance to exit, including the time spent with the nurses to identify elements that could lead to delay . On the other hand, our study aimed to gain insight into the patients perception of our operational process and identify areas for improving care . As perceived wait time is more important than actual wait time, we chose to focus on qualitative comments and included measuring wait time by means of pfa . In summary, our study showed that the satisfaction with wait time was high and equal, both prior to and after the intervention . Satisfaction with the available amenities and the waiting area and atmosphere was also high among the respondents . We also saw an increasing trend of higher satisfaction with the increase in duration of the consultations from 15 minutes to 20 minutes for residents and addition of a 15-minute buffer to the total allocated time for pediatricians consultations . Unfortunately, the intervention did not result in a decrease in wait time or a significant increase in patient satisfaction with wait time . On further analysis, we think that this finding may have been biased by the already highly perceived satisfaction with our services . Another possible explanation could be because of the preferential lengthening of the consultation time for residents alone . It is theoretically possible that due to their additional expertise, increasing the consultation time for the specialists may have resulted in a significant improvement in the patient satisfaction scores and reduced wait time . As our study represents the continuous appraisal of operational processes, the search for ways to reduce wait time and improve patients satisfaction with the provided care remains an area for further investigation.
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Tlrs are transmembrane proteins which contain extra - cellular domains composed of leucine - rich regions (lrr) that interact with specific pathogen associated molecular patterns (pamp) ligands . The cytoplasmic domains of tlrs, which are homologues to the interleukin-1 receptor (il-1r) signaling domain, are called toll / interleukin-1 receptor (tir) domains . All tlrs recruit certain adaptor proteins that contain a tir domain and a death domain . 5 adaptor proteins are linked to the tir domain - myd88, mal (myd88 adaptor - like)/tirap (tir domain - containing adaptor protein), trif (tir domain - containing adaptor inducing interferon - beta), tram (trif related adaptor molecule), and sarm (sam and arm containing protein). Mammalian tlrs comprise of at least 11 members, which are categorically divided into 2 membrane receptor groups . The tlrs associated with the cell surface are tlr1, tlr2, tlr4, tlr5, tlr6, tlr10, tlr11, tlr12, and tlr13, whereas the tlrs associated primarily with endosomal membranes are tlr3, tlr7, tlr8, and tlr9 . Phylogenetically, tlrs are generally divided into 6 families - tlr1, tlr3, tlr4, tlr5, tlr7, and tlr11 . Different tlrs recognize different microbial products i.e. Tlrs are differentially activated by various types of pamps such as bacterial dna, lps, peptidoglycan, teichoic acids, flagellin, pillin, viral dsrna, and fungi zymosan . Activated tlrs differentially trigger the expression of cytokines like the interferons, the interleukins - il2, il6, il8, il12, il16, and tnf- . Binding of adaptor proteins to the tir domain of tlrs trigger the downstream signaling cascade, leading to the activation of different transcription factors . Pamp - specific activation of tlrs converges at the level of tir domain signaling to induce activation of nf- and inflammatory gene expression to clear infectious agents . Tlr1 and tlr6 cooperate with tlr2 to discriminate certain differences between triacyl and diacyl lipopeptides, respectively . Cellular tlr4 is present in 2 different sizes, the larger is a 130 kda heavily glycosylated, md-2-dependent, surface translocated form, and a smaller 110 kda partially glycosylated form is found in the golgi . Dna recognition and tlr3 is associated with the recognition of viral dsrna, whereas tlr7 and tlr8 are implicated in viral - derived ssrna recognition . Tlrs are the key initiators of innate and adaptive immune response due to high production of proinflammatory cytokines and chemokines, upregulation of co - stimulatory molecules, and activation of antigen presentation . Tlr recognition of pathogens leads to expression of genes such as inflammatory cytokines and co - stimulatory molecules . Antigen - specific acquired immunity developed due to phagocytosis - mediated antigen presentation together with tlr - dependent gene expression of inflammatory cytokines and co - stimulatory molecules . In human nave b cells, most of the tlrs are expressed at low to undetectable levels, but tlr9 and tlr10 expression is highly increased following b - cell receptors (bcr) triggering where as memory b cells express several tlrs at constitutively high levels . This difference in tlr9 expression correlates with responsiveness to its agonist, cpg dna . In response to cpg, the human memory b cells proliferate and differentiate to immunoglobulin secreting cells . While nave b cells do the same only if simultaneously triggered through bcr . The expression of tlrs induced by bcr in human nave b cells prevents polyclonal activation in a primary response as it restricts stimulation of antigen - specific b cells . Thus, in human b cells, tlrs are downstream of bcr and plays a role both in primary response and in the memory phase . Autoreactive b cells are present in the lymphoid tissues of healthy individuals, but since they are subject to self - tolerance mechanisms, they remain silent . However, when tolerance to self - antigens fails, a complex of self - reactive antibodies against self- or cross - reactive dna co - engages the antigen receptor and the tlrs, leading to a continuous activation of these auto - reactive b cells and the development of autoimmune diseases . The maturation of antigen - presenting cells (apcs), in response to signals received by innate immune system, may lead to the breakdown of tolerance . This process is mainly activated by tlrs that have been triggered by self - antigens . High proliferation of autoreactive immune cells leads to development of autoimmunity, which is mainly due to defects in the maintenance of tolerance . Mechanisms of clonal deletion along with the regulation of expression of co - stimulatory molecules on apcs and t regulatory cells (t regs) are known to maintain the tolerance . Clonal deletion maintains the central tolerance, whereas a co - stimulatory molecule expression maintains the peripheral tolerance . Several factors play a role in the breakdown of tolerance such as genetic predisposition, self- antigenic load, immature self t - cells, and co - expression of co - stimulatory molecules during apc maturation and absence of treg functions . In autoimmunity, self antigens cannot be easily eliminated because of its excess number or is ubiquitous . Tlr ligand binds to their specific microbial products and to the dendritic cells (dcs) which further activate them and thus induce signals for maturation towards tolerogenic type . These developments of autoreactive t cells and the suppression of tregs cells result in the breakdown of tolerance . Thus, a set of events that follow activation through tlr results in the production of autoantibodies and cytokine response leads to autoimmune diseases . Immunoglobulin g containing immune complexes (ics) bound to mammalian chromatin effectively through the process which involves bcr recognition of the ics, and the dna is delivered to tlr9 in an endosomal / lysosomal compartment . Tlr9 breaks the tolerance of autoreactive b cells by hypomethylated cpg dna present in dna . However, low affinity b cells do not proliferate in response to protein containing ics . Plasmacytoid dcs (pdcs) and myeloid dcs (mdcs) get stimulated and secrete cytokines through a link of fc gamma receptors (fcrs) and tlr9 . Mammalian chromatin can also directly stimulate dna - reactive cells through tlr9-dependent process but under appropriate conditions . Rna and rna - associated autoantigens stimulate the bcr / tlr7 and thus leads to the activation of autoreactive b cells . Tlr 3, 9 and 7 induces the production of ifn - a, viral dsrna binds to tlr 3 and activates nf-b which helps in the induction for formation of apoptotic bodies that further promote pdcs to produce ifn-. Despite of many potential mechanisms for triggering of autoantibody production by microorganisms, it is also possible that certain autoantigens, independent of infection, are endogenous ligands for pattern recognition receptors (prrs) and have a proactive role in loss of immune tolerance . Importantly, ifn- markedly upregulates expression of tlr7 and tlr adaptor protein myeloid differentiation primary - response gene 88 (myd88) and can also increase the response of b cells to tlr9 ligands . Aberrant expression or regulation of relevant tlrs might render b cells hyper - responsive to endogenous ligands, and therefore, predispose an individual to the development of systemic autoimmune disease . Responses to both rna- and dna - containing ics are blocked by inhibitors of tlr7 or tlr9, and b cells that are deficient in tlr7 or tlr9 respond poorly to rna and dna - containing immune complexes, respectively . The age of onset is between 16 and 55 years, and there is a higher frequency of sle in women typically during their child bearing years . Increased serum levels of ifn- and chronically activated pdcs are characteristic of sle patients . It has been reported that pdcs selectively express tlr7 and tlr9 . The cellular activation by ics by dcs, neutorphils, and monocytes play an important role in pathogenesis of sle . Tlr9 is thought to play a role in the production of autoantibodies in sle. [1214] chronic stimulation of tlr9 by sle dna - ics leads to overproduction of ifn- that exacerbates sle pathogenesis . Both chloroquine and cpg oligonucleotides, known inhibitors of tlr9 signaling, blocked sle dna - ic induced ifn- production in pdcs . The interaction of tlr9 with sle dna - ics in lysosomes was found to be rapid, but transient . It is also possible that an exacerbated response to pathogens initiated by tlr2 and tlr4 may potentiate sle attacks . Tlr9 was originally identified as a receptor that could distinguish between bacterial (or viral) dna and mammalian (host) dna, on the basis of high frequency of hypomethylated cpg motifs in non - mammalian dna . Tlr7 was identified as a receptor for viral single - stranded rna (ssrna). The link between dna - ics, fcrs, and tlr9 has been formally established by using tlr9-deficient primary cell populations and tlr9-transfected cell lines . Dna - ics from sle patients were shown to stimulate production of cytokine mrna by hek293 (human embryonic kidney 293) cells that had been transfected with tlr9 and fcriia, this complex co - localize with tlr9 and fcriia in acidic lysosomes . Sle or sjgren's syndrome (ss) patients often have high titers of antibodies specific for small nuclear ribonucleoproteins (snrnps), which are macromolecular complexes consisting small nuclear rna (snrna) and associated proteins . It has been found that igg purified from the sera of sle patients induce ifn - a production by pdcs when igg is mixed with necrotic - cell debris or purified snrnps . This ifn--inducing activity is inhibited by chloroquine or bafilomycin, agents that interfere with the acidification of endosomes and block activation of tlr7 and tlr9 . Cytokine production is also blocked by oligodeoxynucleotide (odn) sequences that are known to inhibit the activation of tlr7 and tlr9 . Data originating predominantly from experimental animal models of autoimmune disease suggest that inappropriate activation of tlr pathways by endogenous or exogenous ligands may lead to the initiation and/or perpetuation of autoimmune responses and tissue injury . It is reasonable to assume that the association between infection and autoimmunity is often caused by tlr - mediated induction of proinflammatory cytokine and chemokine expression and upregulation of co - stimulatory molecule expression by apcs . The ability of microbial tlr ligands to trigger disease onset has been documented in experimental models of arthritis, multiple sclerosis (ms), experimental allergic encephalomyelitis (eae), myocarditis, diabetes, and atherosclerosis . Adoptive - transfer studies in mouse models of arthritis and in mice with eae indicate that endogenous tlr ligands might contribute to the pathogenesis of related autoimmune diseases . Transfer of serum to tlr4-deficient mice induces joint swelling that resolves more quickly than in tlr4-sufficient (control) mice, indicating that endogenous tlr4 ligands have a role in the perpetuation of disease . The transfer of t cells to myd88-deficient recipients lead to only minimal disease, and tlr9-deficient recipients had a much attenuated clinical score compared with tlr - sufficient (control) mice . Tlr2-deficient mice subjected to occlusion of renal arteries produce significantly less pro - inflammatory cytokines and chemokines, showed less leukocyte infiltration, and developed less severe renal injury than tlr - sufficient (control) groups of mice . To evaluate the overall impact of tlr activity on production of autoantibodies and on development of systemic autoimmune disease, in vivo verification of the in vitro studies is needed . Tlr7 and tlr9 have key roles in production of pathogenic autoantibodies and/or in development of clinical features of autoimmunity in experimental animals . Lpr / lpr mice deficient in the tlr adaptor protein, myd88 do not produce anti - nuclear antibodies (anas), and these mice develop marked lymphoproliferative disease . Tlr9-deficient lpr / lpr mice show strong cytoplasmic reactivity, but this reactivity is relatively rare among lpr / lpr mice that are sufficient in tlr9 . Remarkably, renal disease in tlr9-deficient autoimmune - prone mice was significantly worse than in tlr9-sufficient mice . It remains to be determined whether this reflects a role for tlr9 in the clearance of cell debris, an increase in the pathogenicity of rna - containing immune complexes, differential expression of tlr7 or tlr9 by a treg population or any another mechanism . B - cell expression of tlr9 has an important role in promoting antibody response to dna and dna - binding proteins, such as histones . The absence of functional tlr9 has a marked effect on disease outcome, and tlr7 deficiency can also influence autoantibody production . Ehlers et al . Reported that tlr9 is a myd88-dependent inducer of igg2a and igg2b autoantibodies, which are implicated in the progression of lupus - prone mice . Wu and peng reported that genetic ablation of tlr9 has a protective role in the onset of sle - like syndrome in mrl / lpr mice because tlr9-deficient animals have low suppressive activity of treg and demonstrated that tlr9-deficient mice possess a higher titer of anti - dsdna antibody than control c57bl/6 mice . This suggests that diminished expression of tlr9 could be involved in enhanced production of autoantibodies . It will be important to evaluate whether aged tlr9-deficient mice lacking other autoimmune - related genes develop autoimmune diseases . Viral double - stranded rna (dsrna) activates dcs to secrete type i interferons and cytokines, which are associated with disease activity in sle or with autoimmunity in general . Systemic exposure to unmethylated cpg - dna (ligand of tlr9) can induce eae and aggravate the ic glomerulonephritis in mrl lpr / lpr mice . Tlr3 is the only known tlr that depends on signaling through the adaptor molecule trif (toll - il-1 receptor domain - containing adaptor inducing ifn-) and rna helicase rig-1, which is followed by a robust induction of ifn responsive genes . These findings may point toward the recognition of viral dsrna via tlr3 on dcs not only as an important component of virus - induced immunity but also as hypothetical link to viral infection induced aggravation of preexisting autoimmunity . Immunotherapeutic role of tlrs is emerging in treating autoimmune conditions, suggesting that the selective targeting of tlrs might be useful . Initially, extracellular tlr agonists were designed to compete with natural microbial ligands for binding to tlrs . More recently, basic research to identify new targets for drug development has begun to explore modulation of tlr intracellular signaling pathways, in addition to tlr ligand binding . The common signaling pathways used by all members of tlr superfamily are being targeted, with drugs that block nf-b and p38 mitogen - activated protein kinase (mapk) in clinical development for diseases such as ra and psoriasis . Decoy peptides and mimetics, plant polyphenols, and chemically - modified anti - sense oligonucleotides that inhibit different molecular events in tlr signaling pathways to modulate the inflammatory response have been tried . The molecular mechanisms of these inhibitors range from interference with protein - protein interactions between signaling proteins, inhibition of transcription factor activity, to perturbation of the plasma membrane . These inhibitors are derived from host, pathogen, and plant sources and by rational design . Taken together, these studies represent promising avenues for the development of novel - tailored immune therapeutics that can relieve by inflammatory and autoimmune diseases on human health and quality of life . At present anti - malarial agents have been used to treat sle for more than a century, and hydroxychloroquine (plaquenil) is still considered to be an effective treatment for cutaneous sle and for sle - associated polyarthralgia, pleuritis, and pericarditis . The potential for therapeutic development of tlr antagonists several biotechnology and pharmaceutical companies have programs to develop new drugs like agonists of tlrs to enhance immune responses against tumors and infectious agents or to correct allergic responses; or antagonists designed to reduce inflammation due to infection or autoimmune disease . The potent immunogenicity of lipopeptides is due to their ability to activate dcs by targeting and signaling through tlr-2 . In addition, the simplicity and flexibility in their design makes tlr antagonists highly attractive vaccine candidates for humans and animals . Tlrs represent attractive drug targets for modulation of immune response and hold promising applications for the treatment of infection and inflammation . Two main strategies for targeting tlrs seem to be promising for drug development such as targeting tlrs with synthetic agonists or antagonists and targeting the protein - protein interaction involved in the tlr - activated signaling cascade . Specifically, blockade of tir / tir interaction between tlrs and adapter proteins by low molecular weight is particularly encouraging for the development of orally - available agents . To address the need for additional and potentially superior vaccine adjuvants, a number of tlr agonists are currently being tested . While lps is fairly toxic in humans, monophosphoryl lipid (mpl), another tlr4 ligand with a greatly improved safety profile, finally, with the rapid progress in the development of therapeutic agonists for the tlrs, there is accompanying attention to, the theoretical possibility that such therapy may induce autoimmunity or autoimmune diseases . Tlr3 ligands have been identified in the synovial fluid of patients with ra, and tlr9 ligands have been found in the immune complexes of sle patients . These considerations have also prompted interest in the potential of tlr antagonists, as therapeutic agents for autoimmune diseases in near future.
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Accurate and automatic segmentation of medical images is an essential component of a computer - aided diagnosis (cadx) system . However, medical image segmentation is typically a difficult task due to noise resulting from the image acquisition process, irregular shape and variable size of anatomical objects, as well as the characteristics of the object's neighborhood . For example, a lung nodule or a colon polyp is usually embedded in a complex surrounding region . In ct imaging, the intensities of such lesions (e.g., juxtavascular nodules, juxtapleural nodules, or colonic polyps) are usually very similar to their adjacent tissues . In this case, traditional intensity - based or morphological methods [15] may fail to accurately segment the lesion . In particular, graph cut methods [612], which construct an image - based graph and achieve a global minimum of energy functions are often found in image segmentation . Two key issues in the graph cut technique are as follows: (1) how to define an energy function so its minimization leads to the desired result; (2) how to represent the energy function in terms of the graph construction . In most graph cut methods, the graph vertices are constructed at the image pixels, and the segmentation energy is composed of intensity terms only . Proposed a framework to simultaneously segment and register the lungs and nodules in ct data . For segmentation, a 2d pixel - based graph cut algorithm was applied to 3d lung and nodule datasets . It is noted that, when a graph vertex is placed at each image pixel, the number of nodes in the graph increases exponentially with the image size, dramatically increasing the computation time . To improve the efficiency, li et al . It is known that pixel intensity can be locally erroneous due to noise and other image acquisition issues, such as partial volume effect (pve). Xu et al . Presented a graph cuts - based active contour approach to object segmentation, which slabaugh and unal extended by incorporating a shape prior . These methods show promising results, but require manual initialization and quantitative evaluation on different types of lesions which are not available . Liu et al . Applied ordering constraints into an energy smoothness term based on an initial labeling . Zheng et al . Constructed a graph laplacian matrix and solved linear equations for the estimation of ground glass opacity (ggo) nodules in ct, with assistance from some manually drawn scribbles for which the opacity values are easy to determine manually . More general - purpose graph cut methods show much utility in image segmentation, however, are ideally suited to nonmedical images . Other medical imaging approaches apply to nodule segmentation but are evaluated only on very small ct datasets . Further, most available methods require user interaction to identify initial foreground and background seeds . The goal of this paper is to develop an automatic and robust superpixel - based graph cut method for accurate segmentation of different types of lesions in ct imaging including solid and ggo nodules, as well as colonic polyps . One of the original contributions of this paper is that our graph is built on mean - shift superpixels . In this paper, we obtain superpixels by using five - dimensional mean shift clustering that incorporates joint spatial, intensity, and shape (jsis) features . The superpixels express the local structure of the data in the five - dimensional feature space . Mean shift clustering reduces the data variation, thereby helping to produce accurate segmentations . By connecting superpixels instead of pixels, a second original contribution is our energy function, which incorporates the image intensity and the shape feature into a markov random field (mrf) minimized with graph cuts . In particular, the intensity and shape information appear in both our unary and pairwise terms . Compared to our previous work in, we have improved our unary term in the energy function by considering both image intensity and shape features and investigated the method on a relatively large dataset that contains different types of nodules, including juxtavascular, juxtapleural, as well as ggo . Moreover, the experimental results, evaluated both visually and quantitatively, demonstrate high performance for ct lung nodule segmentation . Initial results on colon polyps demonstrate the generalization of the method to other anatomies . The paper is organized as follows: section 2 describes the proposed algorithm, which covers five - dimensional mean shift clustering based on jsis features (section 2.1) and automatic graph cut segmentation of the mean shift jsis superpixels (section 2.2). Section 3 presents our experimental results, followed by a discussion and future work in section 4 and conclusion in section 5 . Our approach is a combination of five - dimensional mean shift clustering followed by energy minimization based on graph cuts . The method is also guided by prior knowledge about the lesion (e.g., nodule / polyp). The flow chart of our method is illustrated in figure 1 . In the following sections, the method first computes the jsis features, which are then clustered in a five - dimensional space using mean shift . In this section, we review the volumetric shape index feature and our five - dimensional mean shift approach . The volumetric shape index (si) at voxel p can be defined as follows [14, 15]: (1)si(p)=121arctank1(p)+k2(p)k1(p)k2(p), where k1(p) and k2(p) are the principal curvatures, defined as (2)k1(p)=h(p)+h2(p)k(p),k2(p)=h(p)h2(p)k(p), where k(p) and h(p) are the gaussian and mean curvatures, respectively . The calculation of the gaussian and mean curvatures is based on the first and second fundamental forms of differential geometry . A practical approach for computing these forms is to use the smoothed first and second partial derivatives of the image as suggested in . In this paper, prior to shape index calculation, a single - scale 3d gaussian filter, with standard deviation of 1.5, is applied to obtain a smoothed image . Every distinct shape, except for the plane, corresponds to a unique shape index . For example, a shape index value of 1.0 indicates a sphere - like shape (e.g., lung nodule or colonic polyp), while 0.75 indicates a cylinder - like shape (e.g., vessel or colonic fold). Based on the definition, the volumetric shape index directly characterizes the topological shape of an isosurface in the vicinity of each voxel without explicitly calculating the isosurface . This feature provides rich information that complements the image intensity and is useful for automated segmentation . In particular, lesions in a ct image may appear within an area of complicated anatomy (such as a lung nodule neighboring a blood vessel or colonic polyp attached to the colon wall) where adjacent structures have similar image intensities but different shapes . Note that we use the term spherical concentration throughout the paper to describe a spatially connected set of voxels that have a high shape index value . For each voxel, the 3d spatial location, intensity, and volumetric shape index features are concatenated in the joint spatial - intensity - shape domain of dimension d = 5 . Given n data points pi, i = 1,,n in a five - dimensional space, the multivariate kernel is defined as the product of three radially symmetric kernels (3)khshrhsi(p)=ck,5k(||pshs||2)k(||prhr||2)k(||psihsi||2), where ck,5 is a normalization constant which assures that k(p) integrates to 1 . P, p, and p are the spatial location, intensity, and shape index features, associated with each voxel p; hs, hr, and hsi are the kernel bandwidths for spatial, intensity, and shape index kernel functions . The normal kernel is used in this paper, where k(p) = (2)exp (1/2||p||). In the mean shift framework, the shape index feature can be combined with the intensity and spatial features for clustering . The mean shift vector with three kernel bandwidths (spatial, intensity, and shape index) is defined as (4)mhshrhsi(p) = i=1npig(||pis / hs||2) g(||pir / hr||2)g(||pisi / hsi||2)i=1ng(||pis / hs||2) g(||pir / hr||2)g(||pisi / hsi||2)p, where g(p) = k(p). The mean shift vector always points in the direction of the maximum increase in the density function . It is noted that the mean shift algorithm estimates the modes (the densest regions) of the multivariate distribution underlying the feature space . The set of points that converge to the same mode is defined as the attraction basin . Mean shift maps all the data samples to the local maxima of their corresponding attraction basin based on five - dimensional features . Each superpixel has a constant shape index and intensity, represented with mode maps, namely, an intensity mode map (mi) and a shape index mode map (msi). Additionally, there is a spatial mode map (ms) that is not incorporated directly in our energy function; figure 2 shows a nodule attached to a vessel and its corresponding intensity and shape index mode maps . It can be noted that the mode maps ((c) and (d)) from jsis mean shift clustering can be seen as filtered images and are less contaminated by outliers . In the following section, graph cut - based segmentation is applied to these superpixels . The mode map obtained by the above jsis mean shift technique expresses the local structure of the data in a given region of the feature space . The number of resulting superpixels depends on the kernel bandwidth and the data itself; a key advantage of the mean shift clustering is its ability to locally group regions of similar intensity and shape, reducing the data variance in superpixel . The aim of this section is to group superpixels into two classes: foreground (lesion) and background (nonlesion). It is known that the image labeling problem can be formulated using an energy function in a bayesian framework in the context of maximization a posteriori (map) and mrf theory and can be solved by energy minimization . The energy function includes both unary and pairwise terms, the latter providing smoothing by modeling the interaction between neighboring superpixels . In this section, a novel energy formulation that incorporates shape and intensity in both unary and pairwise terms is defined on superpixels and minimized with graph cuts using the maxflow / mincut method . Two key issues are addressed in the following subsections: (1) how to initialize the segmentation and (3) how to define the energy function for minimization . In this paper, we assume that a nodule (or polyp) is generally either spherical or has a local spherical concentration, while a blood vessel (or colonic fold) is usually oblong . The initial seeds for the graph cut are computed automatically based on a spherical concentration . A spherical concentration s is defined, using hysteresis thresholding, as a 3d connected region that satisfies the following criteria . All voxels in the region have a shape index greater than or equal to a low threshold tl . At least one voxel in the region has a shape index greater than or equal to a high threshold th . Typical thresholds are in the range tl [0.8,0.9) and th [0.9,1]. The background seeds are determined using adaptive enlargement of the spherical concentration . A distance transform of s is computed and then adaptively enlarged (e.g., factorfdistancemax, where factor is the enlargement factor and the enlargement of the foreground region is designed so that the background region does not cover the foreground object . Figure 3 shows an example of the initialization for a juxtavascular nodule using the above method, where tl and th were set to 0.82 and 0.92, respectively . Figures 3(b) and 3(c) are the initial foreground and background regions, respectively, where factor is set to be 10 . The thresholds of tl and th are fixed throughout the paper and are based on the assumption that a nodule which may not be entirely spherical has at least spherical elements concentrated within the nodule . In fact, using shape index to extract the spherical concentration is the first step in our automatic lung nodule detection algorithm . Very high detection sensitivity can be achieved at this stage . In most cases (e.g., high contrast solid nodule), the sphericity concentration covers the core part of the nodule . However, in the case of some part - solid or ggo nodules, the initial foreground region might spread in the nodule object . Figures 4 and 5 show examples of two different segmented nodules using automatic calculation of foreground and background regions, where, in figure 4(b), the initial foreground contains the core part of the solid nodule object, while in figure 5(b), the foreground region scatters in the part - solid nodule (it is noted, the foreground region is a 3d connected region but it may appear as several small regions in each 2d slice as shown in figure 5(b)). Figures 4(d) and 5(d) show the segmentation results by using the proposed graph cut - based method based on the initial foreground (b) and background seeds (c). However, occasionally the above initialization may fail to give good foreground and background seeds, as the spherical concentration may contain part of the nodule as well as lung tissue . More discussion for the initial foreground seeds will be given in section 4 . In this section, we employ a graph g = (v, e), defined with vertices v v representing superpixels determined from five - dimensional mean shift clustering, and edges e connecting adjacent superpixels . A key difference from the usual graph construction is that we connect superpixels instead of the original pixels . As a result, the number of vertices in g is greatly reduced compared to the original number of pixels in the image . The lesion segmentation problem is formulated as a binary labeling problem, so the goal is to assign a unique label li {0,1} (where 0 is background and 1 represents foreground (lesion)) to each superpixel i by minimizing the following energy function e(l): (5)e(l)=ive1(li)+(i, j)e2(li, lj), where e1(li) is the unary data term representing the cost to assign the label li to the superpixel i, e2(li, lj) is the pairwise smoothness term representing the cost of assigning the labels li and lj to adjacent superpixels i and j, and is a weighting factor . The details of energy minimization via the graph cut algorithm for binary labeling can be found in . Below we unary data termwe are given the initial foreground {mm} and background regions{mt}, automatically calculated from the previous section, where m and t are the superpixel indices for initial foreground and background, respectively . Figure 6 shows the schematic diagram for different types of superpixels, for example, foreground, background, and uncertain superpixels . For each superpixel i, the unary term is defined as (6)e1(li=1)=cifcif+cib, e1(li=0)=cibcif+cib, where ci and ciare the costs of a superpixel i to the foreground superpixels and background superpixels, determined from the initialization . The foreground cost (ci) is calculated as (7)cif = min m{1exp ((si(i)mmf)2i2)} (1exp ((ssi(i)1.0)2si2)), where si(i) and ssi(i) are the intensity value and shape index value at superpixel i, determined from mean shift clustering . It is noted that the intensity and shape features are normalized to the same scale . Also, in this paper, the parameters i and si are determined experimentally (e.g., it can be seen that ci encourages a superpixel to have the same label as the initial foreground superpixels if the superpixel has similar intensity to the foreground superpixels and also its shape feature is closer to one . Similarly, the background cost ci is defined as (8)cib = min t{1exp ((si(i)mtb)2i2)} exp ((ssi(i)1.0)2si2). We are given the initial foreground {mm} and background regions{mt}, automatically calculated from the previous section, where m and t are the superpixel indices for initial foreground and background, respectively . Figure 6 shows the schematic diagram for different types of superpixels, for example, foreground, background, and uncertain superpixels . For each superpixel i, the unary term is defined as (6)e1(li=1)=cifcif+cib, e1(li=0)=cibcif+cib, where ci and ciare the costs of a superpixel i to the foreground superpixels and background superpixels, determined from the initialization . The foreground cost (ci) is calculated as (7)cif = min m{1exp ((si(i)mmf)2i2)} (1exp ((ssi(i)1.0)2si2)), where si(i) and ssi(i) are the intensity value and shape index value at superpixel i, determined from mean shift clustering . It is noted that the intensity and shape features are normalized to the same scale . Also, in this paper, the parameters i and si are determined experimentally (e.g., it can be seen that ci encourages a superpixel to have the same label as the initial foreground superpixels if the superpixel has similar intensity to the foreground superpixels and also its shape feature is closer to one . Similarly, the background cost ci is defined as (8)cib = min t{1exp ((si(i)mtb)2i2)} exp ((ssi(i)1.0)2si2). Pairwise smoothing term the second term e2(li, lj) in (5) is defined as (9)e2(li, lj)=|lilj|(wsi(ei+esi)+(1wsi)eiesi), where ei is an intensity energy term denoting the intensity difference between two adjacent superpixels i and j, defined as ei(li, lj) = 1/(||si(i) si(j)|| + 1). This means that superpixels with similar intensities have a larger ei, which leads to assigning them the same labels . E si is the shape energy term denoting the shape difference between two adjacent superpixels, defined as esi(li, lj) = 1/(||ssi(i) ssi(j)|| + 1). Similar to the intensity term, the shape energy term captures the shape features for the two adjacent superpixels . If adjacent superpixels have similar shape, esi is larger, with high probability, both superpixels have the same label.as can be seen from (9), the intensity term and shape term are combined through a weighting function wsi which is defined as (10)wsi(ssi(i),ssi(j))=exp ((ssi(i)ssi(j)si)2).it is noted that when two adjacent superpixels have the same shape, namely, wsi = 1, the energy depends on the first term of (9). However, when two adjacent superpixels have very different shapes, wsi is small, so the (9) depends on the second term . Figure 7 shows a juxtavascular nodule segmentation that uses different pairwise smoothing terms . Due to pve in ct imaging, part of the nodule's pixels (e.g., figure 7(a3)) has relatively low intensities, compared to those on other slices . With just the first term of (9), the pixels with low intensity (but similar shape) can still be correctly identified as being part of the nodule object, as seen in figure 7(b3). However, some small amounts of vessels (which has similar intensity but a different shape feature value) are also included in the segmentation, as seen in figures 7(b1b3). This is because the first term equally considers the similarity for both of the intensity and shape features . Figures 7(c1c3) are the results using the second term only in (9). It can be seen that the shape feature is only used as a weighting to the intensity feature . Superpixels with different shapes result in a lower weighting; this is why part of the nodule can be properly separately from the adjoining vessel despite the similar intensity, as shown in figures 7(c1c2). However, the superpixels with lower intensity due to pve are wrongly identified as background due to the different intensities, compared to that on the other slices, as shown in figure 7 (c3). Figures 7(d1d3) are the results by combining both of terms as in (9), in which the nodule boundary can be properly delineated despite the pve and the presence of vessels with similar intensities . Figure 8 shows a step - by - step example of using different cost functions for the energy minimization . Figures 8(a1) and 8(b1) are two contiguous slices for one 3d vascular nodule . Figures 8(a2) and 8(b2) are the unary cost (7) for each slice, respectively . The dark area in the image indicates lower cost for being foreground, while the bright area means high cost . Figures 8(a3) and 8(b3) are the pairwise smoothing term (9) for each slice, respectively . It can be seen that there are low costs for the boundary, while high costs are usually within the regions; figures 8(a4) and 8(b4) are the minimization results in which the energy function uses both of unary term (6) and pairwise smoothing term (9). The second term e2(li, lj) in (5) is defined as (9)e2(li, lj)=|lilj|(wsi(ei+esi)+(1wsi)eiesi), where ei is an intensity energy term denoting the intensity difference between two adjacent superpixels i and j, defined as ei(li, lj) = 1/(||si(i) si(j)|| + 1). This means that superpixels with similar intensities have a larger ei, which leads to assigning them the same labels . E si is the shape energy term denoting the shape difference between two adjacent superpixels, defined as esi(li, lj) = 1/(||ssi(i) ssi(j)|| + 1). Similar to the intensity term, the shape energy term captures the shape features for the two adjacent superpixels . If adjacent superpixels have similar shape, esi is larger, with high probability, both superpixels have the same label . As can be seen from (9), the intensity term and shape term are combined through a weighting function wsi which is defined as (10)wsi(ssi(i),ssi(j))=exp ((ssi(i)ssi(j)si)2). It is noted that when two adjacent superpixels have the same shape, namely, wsi = 1, the energy depends on the first term of (9). However, when two adjacent superpixels have very different shapes, wsi is small, so the (9) depends on the second term . Figure 7 shows a juxtavascular nodule segmentation that uses different pairwise smoothing terms . Due to pve in ct imaging, part of the nodule's pixels (e.g., figure 7(a3)) has relatively low intensities, compared to those on other slices . With just the first term of (9), the pixels with low intensity (but similar shape) can still be correctly identified as being part of the nodule object, as seen in figure 7(b3). However, some small amounts of vessels (which has similar intensity but a different shape feature value) are also included in the segmentation, as seen in figures 7(b1b3). This is because the first term equally considers the similarity for both of the intensity and shape features . Figures 7(c1c3) are the results using the second term only in (9). It can be seen that the shape feature is only used as a weighting to the intensity feature . Superpixels with different shapes result in a lower weighting; this is why part of the nodule can be properly separately from the adjoining vessel despite the similar intensity, as shown in figures 7(c1c2). However, the superpixels with lower intensity due to pve are wrongly identified as background due to the different intensities, compared to that on the other slices, as shown in figure 7 (c3). Figures 7(d1d3) are the results by combining both of terms as in (9), in which the nodule boundary can be properly delineated despite the pve and the presence of vessels with similar intensities . Figure 8 shows a step - by - step example of using different cost functions for the energy minimization . Figures 8(a1) and 8(b1) are two contiguous slices for one 3d vascular nodule . Figures 8(a2) and 8(b2) are the unary cost (7) for each slice, respectively . The dark area in the image indicates lower cost for being foreground, while the bright area means high cost . Figures 8(a3) and 8(b3) are the pairwise smoothing term (9) for each slice, respectively . It can be seen that there are low costs for the boundary, while high costs are usually within the regions; figures 8(a4) and 8(b4) are the minimization results in which the energy function uses both of unary term (6) and pairwise smoothing term (9). In this section, we present results demonstrating the effectiveness of the proposed algorithm applied to clinical ct scans . The first, visual analysis is performed on several different types of ct pulmonary nodules and colonic polyps to provide insight into the method's performance for any visual gross missegmentation, such as a failure in separating nodules from vasculature . The second, quantitative experiments evaluate the segmentation results on large nodule datasets, containing 101 solid nodules and 80 ggo nodules . It is noted that, all the nodules in our database are confirmed by three experienced thoracic radiologists . However, each nodule boundary was manually delineated by one qualified radiologist . In all experiments, the three kernel bandwidths (spatial hs, intensity hr, and shape index hsi in (4)) in the five - dimensional jsis mean shift clustering were set to 3.0, 6.5, and 3.0, respectively . The proposed graph cut algorithm was applied to the mean - shift superpixels using the energy formulation based on (5)(10), where the weighting factor was set to be 1 . The mrf parameters were selected manually based to maximize the overlap ratios with the ground truth data . Attached nodules figure 9 demonstrates a detailed example of the segmentation of a solid nodule attached to a small vessel . The figure shows (a) the original ct lung nodule on five contiguous slices, (b), and (c) corresponding intensity mode and shape index mode values on two of the slices of the superpixels resulting from the five - dimensional jsis mean shift clustering . Since each superpixel is an attraction basin in the 5d feature space, each superpixel has a different pair of intensity and shape index mode values . In figure 9(b), after five - dimensional jsis mean shift clustering, most of voxels within the nodule on the same slice have the same mode (such as the value of in intensity mode map of the first slice and 5 of the third slice). It can be seen that, although the intensity modes within nodule are very different, their shape modes are quite similar . The nodule superpixels can be merged using the proposed graph cut method . Figure 9(d) shows the segmented nodule resulting from a graph cut without the shape feature, for which the intensity energy term ei was only used in the pairwise term (9). The algorithm correctly segments most of the nodule (specifically, the part of nodule in the middle three slices). However, the other parts of the nodule with lower intensity (e.g., pixels on the first and the last slice) are wrongly identified as background due to the different intensities . It can be seen that, by combining both the intensity energy ei and shape energy esi into the pairwise energy term (9), the parts of the nodule with low intensity can be successfully segmented due to the similar shapes on the first and last slices . The intensity of the nodule is very similar to that of the adjoining vessel and heart wall . Figure 10(a) shows the nodule on three continuous ct slices; figures 10(b), and 10(c) are two intensity mode maps corresponding to the same nodule slice produced by four - dimensional mean shift clustering (without the shape index feature) and five - dimensional mean shift clustering (with the shape index feature), respectively . It can be seen that, without considering the shape feature, a large amount of nonnodule voxels (e.g., heart) have a same superpixel as that of nodule voxels (e.g., however, using the proposed five - dimensional jsis feature, most of the nonnodule object is separated from the nodule . This is due to the different local shapes of the nodule and the surrounding anatomy (such as the adjoining vessel and heart wall). The proposed five - dimensional mean shift clustering not only considers spatial and intensity information, but also shape features . Figure 10(d) shows the final segmented nodule using graph cut on the above four - dimensional mean shift superpixels, for which only the intensity term is used in the energy function . It is noted that the superpixel with intensity mode value of 24 in figure 10(b) produced from four - dimensional mean shift without taking into account the shape feature contains part of the nodule but also nonnodule objects (such as heart). By representing each superpixel as a node in the graph construction and minimizing the energy function defined in (5), this superpixel is identified as nonnodule object . That is why most of the nodule voxels within this super pixel were wrongly identified as background (nonnodule object). For comparison, it is noted that, by using the proposed method, the misclassified nodule part in figure 10(c) can be successfully identified as being part of nodule object . Figure 9 demonstrates a detailed example of the segmentation of a solid nodule attached to a small vessel . The figure shows (a) the original ct lung nodule on five contiguous slices, (b), and (c) corresponding intensity mode and shape index mode values on two of the slices of the superpixels resulting from the five - dimensional jsis mean shift clustering . Since each superpixel is an attraction basin in the 5d feature space, each superpixel has a different pair of intensity and shape index mode values . In figure 9(b), after five - dimensional jsis mean shift clustering, most of voxels within the nodule on the same slice have the same mode (such as the value of 570 in intensity mode map of the first slice and 5 of the third slice). It can be seen that, although the intensity modes within nodule are very different, their shape modes are quite similar . The nodule superpixels can be merged using the proposed graph cut method . Figure 9(d) shows the segmented nodule resulting from a graph cut without the shape feature, for which the intensity energy term ei was only used in the pairwise term (9). The algorithm correctly segments most of the nodule (specifically, the part of nodule in the middle three slices). However, the other parts of the nodule with lower intensity (e.g., pixels on the first and the last slice) are wrongly identified as background due to the different intensities . It can be seen that, by combining both the intensity energy ei and shape energy esi into the pairwise energy term (9), the parts of the nodule with low intensity can be successfully segmented due to the similar shapes on the first and last slices . The intensity of the nodule is very similar to that of the adjoining vessel and heart wall . Figure 10(a) shows the nodule on three continuous ct slices; figures 10(b), and 10(c) are two intensity mode maps corresponding to the same nodule slice produced by four - dimensional mean shift clustering (without the shape index feature) and five - dimensional mean shift clustering (with the shape index feature), respectively . It can be seen that, without considering the shape feature, a large amount of nonnodule voxels (e.g., heart) have a same superpixel as that of nodule voxels (e.g., 24 in figure 10(b)). However, using the proposed five - dimensional jsis feature, most of the nonnodule object is separated from the nodule . This is due to the different local shapes of the nodule and the surrounding anatomy (such as the adjoining vessel and heart wall). The proposed five - dimensional mean shift clustering not only considers spatial and intensity information, but also shape features . Figure 10(d) shows the final segmented nodule using graph cut on the above four - dimensional mean shift superpixels, for which only the intensity term is used in the energy function . It is noted that the superpixel with intensity mode value of 24 in figure 10(b) produced from four - dimensional mean shift without taking into account the shape feature contains part of the nodule but also nonnodule objects (such as heart). By representing each superpixel as a node in the graph construction and minimizing the energy function defined in (5), this superpixel is identified as nonnodule object . That is why most of the nodule voxels within this super pixel were wrongly identified as background (nonnodule object). For comparison, it is noted that, by using the proposed method, the misclassified nodule part in figure 10(c) can be successfully identified as being part of nodule object . Pleural nodulea pleural nodule is defined as a nodule attached to the pleural wall . Similar to the above two examples, figures 11(b) and 11(c) compare results based on the 4d approach (no shape feature) to the 5d approach (using the shape feature). It can be seen that, without considering the shape feature, the algorithm is unable to segment the nodule on the last slice, while this missing part can be identified as part of the nodule by using our method . Figure 11(a) is a 3d nodule in four contiguous ct slices . Similar to the above two examples, figures 11(b) and 11(c) compare results based on the 4d approach (no shape feature) to the 5d approach (using the shape feature). It can be seen that, without considering the shape feature, the algorithm is unable to segment the nodule on the last slice, while this missing part can be identified as part of the nodule by using our method . It is challenging to properly segment ggo boundaries due to the faint contrast, irregular shape, and fuzzy margins of ggo nodules . Figure 12 shows an example of the five - dimensional method on a large ggo nodule (containing four slices as shown in figure 12(a)), attached to small vessels with similar intensity . The segmentation results shown in figure 12(c) demonstrate good performance of the method . For comparison, it can be seen that, without considering the shape feature, a large amount of small adjoining vessels are wrongly identified as part of the nodule object . However, using the shape index feature in both the mean shift clustering and the graph cut energy function, the nodule boundary can be properly delineated from the background despite the low intensity of the nodule and the presence of other nontarget structures with similar intensity.quantitative evaluation of ggo nodule segmentation and further discussion will be given in next subsection . It is challenging to properly segment ggo boundaries due to the faint contrast, irregular shape, and fuzzy margins of ggo nodules . Figure 12 shows an example of the five - dimensional method on a large ggo nodule (containing four slices as shown in figure 12(a)), attached to small vessels with similar intensity . The segmentation results shown in figure 12(c) demonstrate good performance of the method . For comparison, it can be seen that, without considering the shape feature, a large amount of small adjoining vessels are wrongly identified as part of the nodule object . However, using the shape index feature in both the mean shift clustering and the graph cut energy function, the nodule boundary can be properly delineated from the background despite the low intensity of the nodule and the presence of other nontarget structures with similar intensity . Quantitative evaluation of ggo nodule segmentation and further discussion will be given in next subsection . The segmentation of colonic polyps in ct images is a complex task due to the irregularity of polyp morphology and the complexity of surrounding regions . Results showing the proposed method applied to colonic polyp segmentation appear in figures 13 and 14 . Similar to the nodule segmentation mentioned above, for comparison, figures 13(b) and 14(b) show results without the shape feature, while figures 13(c) and 14(c) are the results based on the proposed method . It can be seen that, by considering the shape index feature in both the mean shift clustering and the graph cut energy, both polyp boundaries can be properly delineated and separated from nonpolyp tissues . The performance of our superpixel - based graph cut algorithm was also compared with that of a standard pixel - based method, where the graph was constructed on each pixel and only the intensity was considered in the energy function . Figure 15 shows segmentation results based on the two methods on three different nodules: (a) one large vascular ggo nodule, (b) one part - solid nodule, and (c) one solid nodule . Testing was performed on a system with a 2.39ghz cpu and 2 gb memory . Table 1 shows the total number of vertices used in the graph cut algorithm and the computation time for both of pixel - based and superpixel - based methods . It is noted that the computation time in table 1 includes construction of the graph and energy minimization . The majority of the computation time is in the graph construction, which includes the calculation of the both energy terms for each vertex . For the superpixel - based method,, it can be seen that, on the superpixel - based graph, the fewer vertices result in a much faster run - time . Furthermore, from figure 15, it is noted that on the pixel - based graph, only the core part of the nodule object is segmented while the outside part is wrongly labeled as a nonnodule object as shown in the middle row for each nodule figures 15(a)15(c). However, the proposed method shown in the third row for each nodule can correctly identify the nodules, separating nodules from the adjoining vessels . Since the superpixels from the five - dimensional jsis mean shift algorithm express the local structure of the data, it produces better results and improves the speed significantly . In this section, the proposed algorithm has been evaluated on a large set of 130 thoracic ct scans with a slice thickness ranging from 0.5 mm to 2.0 mm . Each scan was read individually by three experienced thoracic radiologists to produce a gold standard of 181 nodules (101 solid nodules and 80 ggo nodules). Among those solid nodules, 28 nodules are isolated nodule (in this paper, isolated nodule is a nodule not located near distracting structures of similar intensity, like large blood vessels, the pleural wall, etc . ), 53 are vascular nodules, and another 20 are pleural nodules . The size of the nodules ranged between 5 mm to 20 mm in diameter . To produce the ground truth, dice's coefficient (r) between each segmented nodule and the ground truth nodule is calculated as follows: (11)r=2||vsvg||||vs||+||vg||, where vs and vg are the segmented nodule object and the ground truth object . The notation |||| gives the total number of voxels in the region, and the operator is an intersection . Figure 17 shows the overall distribution of the coefficients (r) calculated based on (11) for all solid nodules (101 nodules) using the proposed method and the method without the shape feature . It is noted that, without the shape feature, the mean dice coefficient for the whole dataset is 0.71 with standard deviation () of 0.1 . However, using the proposed method, the mean dice coefficient has been increased to 0.79 with the decreasing to 0.06 . For comparison, we split all the solid nodules into three different types (isolated, vascular and pleural nodules) and evaluated the segmentation performance for each type separately . Table 2 is the summary of dice coefficients for each of the types of nodules, where the mean and standard deviation are calculated for the two different methods . For the isolated nodules, both methods give good results . For these nodules, the mean dice coefficient for the 4d method is 0.80, and 0.81 for the 5d method . As can be seen in figure 16(a), in general, isolated nodules have high contrast and well - defined boundaries, so the image intensity is the predominant feature, hence both methods obtain similarly good results . However, the proposed 5d method provides better results in the presence of pve . In such cases, by considering the shape feature, the parts of the nodule with low intensity but similar shape can be successfully segmented . For both vascular nodules and pleural nodules, the proposed method gives a much better mean dice coefficient . Especially for vascular nodules, the mean dice coefficient increased from 0.68 to 0.8 with decreasing from 0.1 to 0.06 using the proposed method . In general, vascular nodule is embedded in a complex surrounding anatomy with similar intensities, and considering the shape feature provides rich information for the accurate segmentation . For pleural nodules, the mean dice coefficient is 0.73 for the proposed method and 0.67 without the shape feature . Among these 20 pleural nodules, however, figure 18 shows an example of a small pleural nodule segmentation based on the proposed method, where the segmented nodule missed a small part of the nodule on the last slice, and figure 19 shows a case where the segmented nodule boundary includes a small amount of lung wall tissue . The proposed method has also been evaluated on 80 ggo nodules (with pure and part opacified components). Most ggo nodules exhibit low contrast, irregular shapes and are often attached to vessels with very similar intensities . Figure 20 shows the overall distribution of the dice coefficients computed between each segmented ggo nodule and the ground truth, based on the proposed method . The proposed method provides a robust approach that combines both of local shape features and intensity . Experimental results presented in the previous section demonstrate the promise and generalization of the proposed method to different types of lung nodules in ct . Most of nodules can be properly delineated from the lung parenchyma despite the presence of other nontarget structures such as vessels or the lung wall . Based on the quantitative evaluation shown in table 2, the mean dice coefficient of the proposed method for both isolated nodules (0.81) and vascular nodules (0.8) is on average higher than that of pleural nodules . Moreover, for the vascular and pleural nodules, the mean dice coefficient has been greatly improved when considering the shape feature, compared to without shape feature . Figures 18 and 19 show two small pleural nodules for which the proposed method either missed a small amount of nodule or included a small amount of adjoining tissues . We further investigated these cases for probable causes . In the mean shift clustering step, different fixed bandwidths are used in (3) for both the intensity and shape features, respectively . However, a fixed bandwidth might not be ideal, as a bandwidth that is too large might create superpixels that are too large, grouping small differences in intensity or shape that should otherwise be ungrouped . This can lead extraneous parts included in the final segmentation . To improve the performance, variable bandwidths for mean figures 12 and 15(a) show our visual results on two ggo nodules, and figure 20 provides a quantitative measure on 80 ggo nodules . The mean dice coefficient of ggo nodules is, as expected, relatively lower compared to that of solid nodules . However, it is more difficult to segment ggo nodules due to the large variations in the intensities and vague boundaries . As an initial study, both in terms of a visual analysis and a quantitative evaluation on a relatively large dataset in general, ggo nodule has shown certain opacity pattern as shown in figures 12 or 15(a); texture features such as grey level cooccurrence (glcm) can be incorporated into mean shift clustering for the further improvement of the ggo segmentation . Also, in this paper, the initial foreground seeds for graph cut segmentation are automatically obtained based on spherical concentration . For our data, the initialization, described in section 3, never failed completely, that is, the foreground initialization always returned some seeds within the nodule object for subsequent processing . In most cases, the initialization provided good foreground and background seeds for the graph construction and cut . The concentration of the high shape index values can either cover the core part of the object (e.g., solid nodules) or scatter within the nodule (e.g., some part - solid or ggo nodules). However, in the case of some part - solid or ggo nodules, the spherical concentration might be on the nodule boundary, which contains part of the nodule as well as the lung tissue . An example can be seen in figure 21, where the initial foreground is on the top of the nodule border that contains some nonnodule parts, while the automatically obtained initial background also contains part of the nodule object . That can lead to the final segmentation missing part of the lesion . To resolve this issue, we are investigating a robust method which not only considers the shape feature but also intensity for the automatic generation initial seeds . The polyp boundaries can be accurately delineated from the adjoining nonpolyp tissues with very similar intensity . However, further analysis on a large dataset with different types of colonic polyps is required to test the generalization of the proposed method on this anatomy . A five - dimensional jsis mean shift clustering is firstly used to produce superpixels comprised of intensity and shape index mode maps . A graph cut algorithm is then applied using a novel energy formulation that considers not only image intensity but also shape . The jsis features provide rich information for lesion segmentation . Both by visual inspection on different types of lesions (lung nodules and colonic polyps), as well as using a quantitative evaluation on 101 solid nodules and 80 ggo nodules, demonstrate the potential of the proposed method . The method cannot only successfully segment lesions adjacent to structures of similar intensity but different shape, but also can correctly identify some part of lesions with different intensity (due to pve in ct imaging) but similar shape.
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A 67-year - old man, 164.5 cm in height and 64.7 kg in weight, was admitted to the hospital for investigation of abnormal shadows on chest x - ray in both lungs . Approximately 3 months before admission, he was diagnosed with pancreatic ductal adenocarcinoma and underwent surgery, which consisted of a distal pancreatectomy, splenectomy, and left adrenalectomy . The patient did not have hypertension, diabetes mellitus, cardiovascular disease or other associated disorder . No abnormal findings were detected in the preoperative physical examination, electrocardiography, pulmonary function test or echocardiography . His chest x - ray and computed tomography scan revealed multiple pulmonary small nodules in both lungs . Hematogenous pulmonary metastasis of pancreatic ductal carcinoma was highly suspected, and pulmonary wedge resection of the left upper lobe by vats was scheduled for tissue confirmation . Surgery was performed under general anesthesia using a dlt (bronchocath left 37f; mallinckrodt medical, athlone, ireland). We confirmed indirectly that the endotracheal and endobronchial cuffs were not overinflated by palpation of the pilot balloon . After the patient's position was changed to right lateral decubitus, the inflation of the endotracheal and endobronchial cuffs were reconfirmed as above, and the placement of the tube was again confirmed by fob . We also confirmed that the cuffs were not overinflated before and after one - lung ventilation . Anesthesia was maintained with 2 - 3% isoflurane with o2 and air, 1 l / min each during two - lung ventilation . One - lung ventilation was provided in volume control mode with 400 ml tidal volume . The fio2 was 1.0, respiratory rate was 12 breaths / min and peak airway pressure was maintained at less than 18 cmh2o . The surgeon confirmed that the left recurrent laryngeal nerve was left untouched and remained preserved . The operation time was approximately 1 hour . At the end of the surgery, glycopyrrolate 0.4 mg and pyridostigmine 15 mg were given after confirming 4 twitches in train - of - four (tof) stimulation of the ulnar nerve . The tidal volume was 150 - 250 ml, and the patient was able to open his eyes following a verbal order, so extubation was performed . Thinking this was due to upper airway spasm following the stress of extubation or vocal cord edema, we applied a jaw thrust maneuver with 10 l / min of oxygen via facial mask . Both lung sounds were decreased, but the peripheral arterial oxygen saturation by pulse oximetry (spo2) was 100% . Five minutes later, the stridor had not abated, but the patient seemed to breathe well without the jaw thrust maneuver, so he was transferred to the postanesthesia care unit (pacu) without reintubation . In the pacu, an electrocardiogram, non - invasive blood pressure, and spo2 were monitored, and 10 l / min o2 was applied to the patient via facial mask . We began to suspect that the patient might have other reasons besides laryngeal spasm or vocal cord edema for his stridor . An otolaryngologist examined the patient with a fiberoptic nasopharyngolaryngoscopy and diagnosed him with bilateral vocal cord paralysis of median type with a 1 mm gap . We planned a tracheostomy, but an arterial blood gas analysis showed mild hypercapnia and normal arterial oxygen pressure (paco2 54 mmhg, pao2 223 mmhg), and the surgeon persistently opposed any invasive procedure . We decided to monitor him in the intensive care unit (icu), keeping in mind the possibility of an emergency tracheostomy . In the icu, 1 mg of midazolam two hours later, the patient became fully awake and was able to say " ah " . Bilateral vocal cord paralysis was present, however, and identical to the patient's last examination . The next day, inspection of the vocal cords was again performed and revealed fully recovered vocal cord movement . Thereafter, the patient was transferred to the general ward and discharged six days after surgery ., many reports have been published about unilateral or bilateral vocal cord paralysis after general anesthesia, but it is uncommon for paralysis to occur in patients who have undergone lung surgery with a dlt . In this case, we could suspect two possible causes for the paralysis: first, recurrent laryngeal nerve injury during the operation, and second, recurrent laryngeal nerve injury occurring due to intubation with a dlt . We can rule out the surgery - related injury, because the surgeon did not perform a sternotomy and manipulate the nearby origin of the internal thoracic artery, which is where the left vagus nerve passes . Ellis and pallister proposed that compression injury by an overinflated cuff within the larynx could be inflicted on the anterior branch of the recurrent laryngeal nerve passing the medial side of the thyroid lamina . We fixed a dlt at 32 cm from the incisor and reconfirmed the location of the cuff by using fob . For this reason, the probability of cuff inflation within the larynx can be ruled out . Analyzed the risk factors of vocal cord paralysis and found that vocal cord paralysis occurred more often in conditions that included older patients, patients with hypertension or diabetes mellitus, and increased intubation time . They suggested that aged patients have fragile tissues in the laryngeal system that are more susceptible to mechanical damage, and these tissues could be easily injured by cuff pressure and the tracheal tube itself . The size of tube has also been correlated with the incidence of postoperative laryngeal injury [6 - 8]. Published data of stout et al . Imply that the incidence of postoperative hoarseness and vocal cord injury might be directly correlated with the size of the endotracheal tube used . Knoll et al . Proposed that a dlt could harm a patient due to its curved endobronchial lumen during intubation as well as extubation . In their study about the usefulness of prophylactic dexamethasone for sore throat and hoarseness after tracheal extubation with a dlt, park et al . Explained that one of the reasons for their finding of increased severity of sore throat according to time was structural injuries in the vocal cords from the dlt . The outer diameter of a 37f dlt is a little larger than that of the 8 mm single - lumen tube, which corresponds approximately with that of a 35f dlt . Therefore, we can hypothesize that the tissues of aged patients are susceptible to microcirculatory insufficiency, and compression injury by a large - sized and curved dlt could occur more easily . Furthermore, because the extubation was done while the patient's respiratory drive was recovering and because we could not observe the vocal cords during extubation, we can predict that the vocal cord injury could have occurred more easily during extubation . In this case, the recovery time was shorter than that of other reports . It is known that the usual recovery time of vocal cord paralysis is three months but can vary between six weeks to one year . There has been no report of such a short recovery time as in this case, when vocal cord paralysis developed after endotracheal intubation . There have been several reports, however, about transient bilateral vocal cord paralysis associated with laryngeal mask airway; in those reports, the paralysis was diagnosed as recurrent laryngeal nerve neuropraxia, a transient episode of motor paralysis with little or no sensory or autonomic dysfunction . The quick recovery in this case suggests a neuropraxia of the recurrent laryngeal nerve as the most likely diagnosis . Laryngospasm is a reflex closure of the upper airway from spasm of the glottic musculature . During laryngospasm, there is no airflow or vocal sound, and the true vocal cords cannot be seen . We confirmed the fixation of the true vocal cords with a laryngoscope, so laryngospasm could be ruled out . Despite the possibility of quick recovery, if bilateral vocal cord paralysis is diagnosed by a laryngoscope, tracheostomy or transient endotracheal intubation would be the correct treatments . In this case, midazolam 1 mg injected by a pulmonologist was based on the experience that low dose midazolam had diminished dyspnea in patients who had undergone examination with a rigid bronchoscope . Midazolam administration can cause unexpected respiratory failure, so it should have been administered more carefully . In addition, we did not apply continuous positive airway pressure, because it was not available at the time . In this report, a case of bilateral vocal cord paralysis after wedge resection of the lung is described . The increased susceptibility of tissue to damage in older patients and the use of a dlt have been identified as possible causes of the paralysis . When faced with stridor in a patient who has undergone lung surgery using a dlt, bilateral vocal cord paralysis should be considered as an etiology, and a prompt fiberoptic laryngoscopic examination should be done.
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Maternal moderate and severe anemia have been considered as conditions that affect adversely maternal and perinatal outcomes . For such reason the identification of these cases is important since appropriate treatment may reduce the risk for adverse outcomes . Iron is the most common treatment currently used to control iron deficiency and anemia particularly in low - income countries, where anemia is more frequent . Physiologically, hemoglobin concentration drops during gestation, and for such reason the hemoglobin cutoff to define anemia was set at 11 g / dl of hb . Maternal hb levels are higher at high altitudes in all trimesters of pregnancy than at sea level . However, the reduction of hemoglobin levels during pregnancy occurs in populations at low and at high altitudes . This is due to plasma volume expansion as a mechanism to improve arterial uterine flow to the placentae . Thus, a normal hb value at first booking in the first trimester does not preclude the presence of anemia in a second measurement of hemoglobin as pregnancy advances . A recent study showed a higher risk for anemia in pregnant women with low body mass index (bmi)., anemia, particularly moderate and severe anemia is associated with high risk for low birthweight [3, 9, 10], it is important to monitor hemoglobin values during pregnancy . The present study has been designed to determine changes in hemoglobin concentration at second measurements after a normal hemoglobin concentration was detected at first booking during pregnancy at low and at high altitudes . We will determine rates of anemia, normal hemoglobin values and erythrocytosis (hb> 14.5 g / dl). In addition, risk factors for moderate / severe anemia will be assessed in a multivariable analysis . This is a secondary analysis of a large data base obtained from the perinatal information system in peru which includes 379,816 pregnant women and their babies obtained from 43 maternity units in peru . Data were obtained at the three geographical regions in peru: coast, highlands, and amazon . The study was authorized by the institutional review board at the universidad peruana cayetano heredia . As the study used secondary analysis of data, the study was exempt from formal review . From the total sample size, a selection was made of all women diagnosed as nonanemic and without erythrocytosis at first booking (hb ranging from 11 to 14.5 g / dl) and having a second measurement of hemoglobin during the same pregnancy ., 57,231 pregnant women were living at low altitude (<2000 m), 11,349 women at moderate altitude (20003000 m), and 20,714 women were living at high altitudes (> 3000 m). Data on gestational age at first and second hemoglobin measurement were recorded . With a second hemoglobin value, rates of pregnant women with anemia (hb <11 g / dl), normal hemoglobin values (1114.5 g / dl), and erythrocytosis (hb>14.5 g / dl) were determined . As demonstrated previously, no correction of hemoglobin cutoff to define anemia at high altitude is needed; hence, the present study used the definition of anemia as suggested who for populations at sea level [1, 11]. The degrees of anemia were classified as mild (hb 9.010.9 g / dl), moderate (hb 7.0 <9 g / dl), and severe (hb <7 g / dl). Body mass index was calculated from prepregnancy body weight and height . Low bmi was defined as <19.9 kg / m and high bmi as> 25 altitude was defined as low (01999 m), moderate (20002999 m), and high (3000 m). The following perinatal outcomes were assessed: stillbirths, preterm deliveries and small for gestational age . Stillbirths were diagnosed when fetal deaths occurred after 20 weeks of gestation or with a weight higher than 500 g. preterm births were defined as deliveries before 37 weeks of gestational age . Small for gestational age was defined if birthweight was below the 10th percentile of the williams reference chart . The adverse maternal outcomes assessed were preeclampsia, premature rupture of membranes (prom), and postpartum hemorrhage (pph). Preeclampsia was defined as the presence of pregnancy - induced hypertension (systolic pressure of 140 mm hg and/or diastolic pressure of 90 mm hg) and proteinuria (300 mg/24 h) after 20 weeks of gestation . Pph was defined as women after parturition with blood losses of 500 ml or more . Data are assessed according level of altitude (low, moderate, and high) and according to degree of anemia (moderate / severe and mild), and erythrocytosis . If data were normally distributed, then a one - way analysis of variance were used (anova). Data expressed in proportions were assessed by the chi - square test . The confidence interval (ci) the prevalences of women with moderate / severe anemia (hb <9 g / dl), mild anemia (hb 910.9 g / dl), normal hemoglobin (hb 1114.5 g / dl), and erythrocytosis (hb> 14.5 g / dl) were calculated for the total sample, as well as for groups at different altitudes (low, moderate, and high altitude). Estimates of crudes and adjusted odds ratio (or) with 95% ci were computed as measures of association between the variables . A logistic regression analysis to determine the risk for moderate / severe anemia at second hemoglobin measured was assessed . The estimates were adjusted for gestational age at second hemoglobin, hemoglobin value at first measurement, altitude, maternal age, body mass index, maternal education, marital status, prenatal care visits, parity, the presence of preeclampsia, cardiopathy, and diabetes . Concentration of hemoglobin at first booking in nonanemic women increased as altitude increased (p <0.05), whereas birthweight decreased as altitude increased . No differences were observed in gestational age at birth at the three altitude groups studied . Concentration of hemoglobin at second measurement also was increased as altitude increases, but values were lower than those at first measurement, particularly at low altitude, in which values were reduced to 94%, whereas at moderate altitude . They were reduced to 98.4% and at high altitude to 99.2% . Gestational age at first and second hemoglobin was higher as altitude of residence increased (table 1). Most of the studied sample remained with normal hemoglobin values at second hemoglobin measurement (75.1). However, 21.4% of women became anemic at the second hb measurement . In all, 2.8% resulted with moderate / severe anemia and 3.5% with erythrocytosis (figure 1). Women with anemia in the second hb measurement showed lower hb measurements in the first hb . Conversely, women with erythrocytosis at the second hb measurement showed higher hb levels in the first measurement (table 2). In all cases hb was higher as altitude increased . The rate of stillbirths was higher with moderate / severe anemia and with erythrocytosis (p <0.05), which remained higher after controlling for confounders (table 3). The rate of preterm births was increased in women with severe anemia (9.01%; or = 1.81). After adjusting for confounders, moderate / severe anemia (or = 1.73) and mild anemia (or = 1.28) were risks for preterm deliveries (table 4). The rate of sga was significantly higher in mothers with erythrocytosis, almost twice, than in women with normal hemoglobin . Lowest rate of sga was observed with a maternal hb level of 910.9 g / dl (mild anemia) (table 5). The rate of preeclampsia was significantly higher in moderate / severe and mild anemia and in mothers with erythrocytosis, which remained after controlling for confounders (table 6). The highest risk for pph was observed with moderate / severe anemia (twenty times higher than in woman with normal hb levels) (table 8). An increased risk for moderate / severe anemia was associated with higher gestational age at second measurement of hemoglobin, bmi <19.9 kg / m, living without partner, prenatal visits care <5, first parity, multiparity, and preeclampsia . Lower risk for moderate / severe anemia were observed with normal high hb level at first booking, living at moderate and high altitude, and with high bmi (table 9). The present study has been designed to determine changes in hb levels after a first hb measurement in 89,294 pregnant women detected normality (hb 1114.5 g / dl). According to the study results an increase in the rate of anemia according to pregnancy progress has been previously published in other settlings . Moderate / severe anemia has been characterized as the conditions more associated with maternal and perinatal adverse outcomes . Our results demonstrated that 2.8% of women classified as nonanemic in the first hb measurement showed moderate / severe anemia in the second measurement . Our data showed that moderate / severe anemia were associated with stillbirths, preterm births, preeclampsia, and pph . The strength of this study is that it was done thanks to a large sample of women recorded through a perinatal information system that included 379,816 pregnant women and their newborn babies from different maternity units throughout peru . This database is an important tool for monitoring conditions and trends related to pregnancies and newborns, including service characteristics and women's risk factors . Data revealed that a percentage of women became anemic during the progression of pregnancy despite having a first normal hb measurement . This is an important finding since moderate and severe anemia are harmful for mothers and neonates . Risks for moderate / severe anemia are preventable, or can be managed through appropriate management, such as with prenatal care, parity, preeclampsia, and low bmi . Increasing the number of prenatal visits will result in better monitoring of hemoglobin values, and can control or reduce the effect of preeclampsia . Low bmi is a characteristic of low - income countries and is associated with increased risk of maternal anemia, low birthweight, and preterm births . The present results are in accordance with other studies where low prevalence of anemia was observed in women with highest bmi, and high prevalence of moderate / severe anemia were associated with malnutrition [14, 15]. Our data suggest that in situation in which a normal hemoglobin value was first observed in a woman with low body mass index, supplementation with iron should be recommended . It is interesting to note that populations living at moderate and high altitudes had lower risk for moderate / severe anemia in a second measurement of hemoglobin during pregnancy . This suggests that women living in these altitudes bearing normal hemoglobin values at first booking should not be treated with iron supplementation . In recent years, it has been demonstrated that anemia defined as less than 11 g / dl of hb is lower at moderate and at high altitudes, since populations at these altitudes are characterized by increased hemoglobin values to compensate for the effect of altitudinal hypoxia [3, 10]. However, we assumed that most cases of anemia were related to iron deficiency, which was supported by the finding that low bmi was associated with moderate / severe anemia . However, results of our analysis were similar to those obtained in a study in which bmi was obtained at first prenatal care . It is possible that erythrocytosis found in the second measurement of hb after a normal value of hb at first booking could be due to the effect of iron supplementation, since in peru a ministry of health regulation orders pregnant women be supplemented with iron . However, low adherence may explain the low prevalence of erythrocytosis in a second hb measurement during pregnancy . The results of this study confirm that prevalence of anemia increases as pregnancy progresses and that a normal value at first booking should not be considered sufficient, as further hb values should be indicated and observed during the course of pregnancy.
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In the bioinformatics and computational biology (bcb) domain, biological sequence alignment is a quite common task . It includes comparison of dna (nucleotide), rna (nucleotide), and protein (amino acid) sequences . In such comparison, a query sequence is aligned to subject sequences from a large database to find similarities . Scientists are used to adopting sequence alignment to infer biological information of a newly discovered sequence from a set of previously known sequences . For instance, if a recently discovered sequence is similar to a known disease gene, then the biological information about the functionality of the new sequence can be inferred . In addition, biological sequence alignment plays an important role in the study of evolutionary development and the history of species . Being one of the most important tools applied in finding biological sequence alignment, blast (basic local alignment search tool) has been widely implemented on commodity pc clusters . But with the exponential growth of the biosequence databases (see figure 1), meeting the computational requirements using current platforms is becoming a difficult task . Compared to general - purpose pc microprocessors, field programmable gate arrays (fpgas) are able to provide a much higher degree of bit - level data parallelism which leads to higher computational speed . Scientists have been investigating parallel architecture design for the blast algorithm on fpgas to achieve higher computational efficiency . This paper proposes a design and implementation of a parallel architecture on fpga for blast, namely, systolic array - based parallel architecture for the blast algorithm . The design is implemented with very high speed integrated circuit hardware description language (vhdl), making it compatible across a number of fpga architectures (e.g., xilinx, altera). We first present the overview of the blast algorithm as well as the related work . After that, architecture performance including storage resource requirements, fpga resource consumption, and estimated speedups against other architectures will be analyzed based on synthesizing, functional, and timing simulation . Although heuristic algorithms produce local alignments that are not always optimal, they are normally much quicker than exhaustive dynamic programming algorithms . The execution time of biological sequence alignment is crucial to bioinformatics scientists, especially due to the fact that the size of the biosequence databases has grown exponentially over years . The three main steps of the blast algorithm are finding hits, ungapped extension, and gapped extension . (1) finding hitsin this step, we use a protein sequence which consists of eight residues (amino acids) as an example shown below: pqgefgvy . In this step, we use a protein sequence which consists of eight residues (amino acids) as an example shown below: pqgefgvy . The query sequence is divided into overlapping k - words as illustrated below with k = 3: word 0: pqg word 1: qge word 2: gef word 3: efg word 4: fgv word 5: gvy.six words are extracted from this sequence . Generally speaking, (m k) + 1 words would be extracted when the size of the sequence is m. after dividing the query sequence, these k - words plus a score matrix are used to find the possible matching words which have a score with at least the threshold value of t. finally, subject sequences in the database are scanned one by one to find exact matches to those possible matching words . Previous work shows that this step could be finished by high - level application software running on a pc . By designing an architecture which can implement every step of blast on a fpga, the interface issue for software and hardware connection can be avoided, saving execution time . Instead of finding exact matches to those possible matching words, in this paper, the implementation searches for the exact matches of those k - words directly on the fpga . (2) ungapped extensionin this step, each hit recorded in the last step is extended by aligning with a subject sequence in both directions without any gap . A score matrix, for example, blosum50, is used to compute residue pair alignment scores . The extension process is terminated when the total score of the alignment is below a threshold value . These results from ungapped extension are called high - scoring segment pairs (hsps). In this step, each hit recorded in the last step is extended by aligning with a subject sequence in both directions without any gap . A score matrix, for example, blosum50, is used to compute residue pair alignment scores . The extension process is terminated when the total score of the alignment is below a threshold value . These results from ungapped extension are called high - scoring segment pairs (hsps). (3) gapped extension in this stage, the smith - waterman or needleman - wunsh algorithm is applied to perform the gapped alignment in both directions for those collected hsps . After this step,, the smith - waterman or needleman - wunsh algorithm is applied to perform the gapped alignment in both directions for those collected hsps . After this step, we choose the needleman - wunsch algorithm for gapped extension implementation because of its ability to find optimal global gapped alignment between the query sequence and the subject sequence . Suppose that there are two sequences, q and s, whose lengths are m and n, respectively . Q[1: i] denotes the first i characters in sequence q and s[1: j] denotes the first j characters in sequence s. the similarity score between q[1: i] and s[1: j] can be obtained from the similarity scores computed for q[1: i 1] and s[1: j 1]. A dynamic programming score matrix d is built, where each element of the matrix denotes the best alignment score between the q[1: i] segment of q and the s[1: j] segment of s. the first step of needleman - wunsch algorithm is to create the score matrix d with m + 1 columns and n + 1 rows . M and n are lengths of sequences q and s. the first row and the first column are assigned the value zero . The computation complexity of the initialization step is o(m + n). The second step is comprised of filling the matrix . Di, j is used to express the cell (i, j) of the scoring matrix d. di, j is defined as the maximum of the similarity scores computed in positions (i 1, j 1), (j, j 1), and (i 1, j) associated with gap penalty or si, j . The following equation is used to compute the value of the cell (i, j): (1)d(i, j)=max{d(i1,j1)+si, j, d(i1,i)+d, d(i, j1)+d . In this equation, si si, j = 1 if the ith character in q is equal to the jth character in s (match score); otherwise si, j = 0 (mismatch penalty). D(i 1, j) + 1 is the score of an alignment between xi and a gap in y. d(i, j 1) + d is the score of an alignment between a gap in x and yj . Trace back can form the best global alignment between q and s. it starts from the bottom - right value in the matrix and ends at the up - left value . For each step, the next alignment pair which is added to the front of the alignment segment accords to 3 rules: (1) if the cell (i, j) was derived from cell (i 1, j 1), the pair of alignment xi and yj is added to the front of the alignment segment . (2) if cell (i, j) was derived from cell (i 1, j), xi and a gap in y are added to the front of the alignment segment . (3) if cell (i, j) was derived from cell (i, j 1), a gap in x and yj are added to the front of the alignment segment . In some cases it's hard to decide which cell cell (i, j) comes from . The diagonal cell is chosen under this situation to shorten the length from the bottom - right cell to the upper - left cell . It corresponds to the following alignment: t_g g c c a g t c c g and t t g_c_a_t_g . Fpgas not only provide high speedups and high bit - level data parallelism compared to regular processors, but also include the convenience of reprogrammability . More and more bioinformatics scientists have designed parallel fpga architectures for the blast algorithm in order to solve the biological sequence alignment issue . Many architectures have been proposed already such as mercury blastn [1214], tree - blast, mercury blastp, rc - blast, fpga / flash accelerator, multiengine blastn accelerator [18, 19], and many industry applied systems like bee2 and mitrion . Most of them adopt the word position record - based search (wprbs) method, which constructs all storage tables to record the position of each word in query sequence, then sends those words to the accelerator one by one where the storage table is searched to find the hit . Although this approach is widely used, it still suffers from time and storage limitations . Using wprbs, at most one hit can be found in one - clock cycle because only one word can be searched per clock cycle . Fpga memory port number also limits wprbs searching capacity no matter where the storage tables are stored: internal chip memory or external chip memory . Take storing tables in an external memory for instance, mercury blastn and mitrion built a hash table from the query . During the searching process, an accelerator checks words in the subject sequence database against the hash table one by one . An external sram attached to the fpga is used to store the hash table since the internal block rams do not have enough storage space to hold tables for long query sequences . This situation obviously causes a timing issue: accessing delay to external sram leads to long pipeline cycle time . The database is formatted as an index structure in which every word is associated with its position in the sequence and its adjacent environment so that short, ungapped alignments could be computed simultaneously, avoiding many random accesses to the database . But the size of the database index is very large, a 150 gb index for the human genome is needed when storing a 40 amino acid substring environment . This requires 50 times more storage than the raw data . In the future, with the exponential growth of the database, the storage resource requirement will become a serious issue . To get better searching efficiency, multiengines blastn adopted 64 identical computing units in a single chip to compare the query with 64 subject sequence in database at the same time . Mercury blastp proposes a two - hit generator for accelerating the first stage of blastp . One is used as a subject sequence database and the other is used as a results container which receives hsps lists from the basic layer . This layer scans all subject sequences from database for the query sequence in order to get a hsps list . After that, the list is sent to the host computer software that assigns statistical significance specified by bioinformatics scientists . In the top layer of this system, a dell precision t1500 desktop computer with an intel core i7 microprocessor and 4 gb memory runs the host software . In the middle layer, two 1 gb the basic layer is the systolic array - based parallel architecture that implements the blast algorithm . This layer is mapped to one fpga chip (xilinx virtex-5 xc5vlx110 t) which is embedded on the xilinx ml509 evaluation platform . The initialization set and data control block is used to initialize the status of registers for the hit finder array and to forward subject sequence as well as query sequence to the hit finder array . For example, it can find adjoined overlapping hits tkel and kelp then combine them into tkelp . Ungapped extension blocks extends ungapped hits in both directions until the score meets a thread value t. gapped extension block extension gapped extension on hsps through exhaustive algorithm like needleman - wunsch . Multiple hits detection module is used to detect 3-word hits and record the hits' address in the query and the subject sequence . Compared to wprbs method which could detect at most one hit in one clock cycle, as the figure illustrated, there is a systolic array with 32 processing units, every 3 units are connected to one 3-input and gates . The value of each register is sent to the corresponding hits information extraction units for recording the hits address in the query and the subject sequence . The systolic array and the hits information extraction unit clock_en (array_clk in figure 4) drives hits information extraction units; clock_out is the clock source of the systolic array . These two clocks work together driving a pipelined architecture without any pause . At each clock_out rising edge, query or subject sequence moves forward for one processing unit; at each clock_en rising edge, a bit value at a certain position in each register is used to detect the location of hits . The whole architecture works as follows: first, a query sequence with 32 characters is forwarded into the systolic array so that each processing unit holds a character from the query sequence . Then the subject sequence is driven in to the systolic array by each internal clock rising edge . Meanwhile, the incoming subject character and the query character which are held by the unit are compared, if they are identity, the logic 1 would be generated; otherwise, the logic 0 would be generated . So, the systolic array with 3-input and gates can detect multiple hits at one internal clock rising edge . The architecture of the processing unit in the systolic array is illustrated in figure 6 . In the implementation, 20 3-bit long binary numbers (00001 to 10100) are defined to present 20 amino acids in a protein . Shift registers are able to shift characters to the adjacent processing unit so that the query and the subject sequence can go through the systolic array . When a new character from the query or the subject sequence is shifted into the unit, the corresponding i d register would increase its value by 1 . Each processing unit of the systolic array will compare two characters in it at each internal clock rising edge . As shown in figure 4, outputs of 32 3-input and gates goes into 2 16-bit registers . The systolic array with 32 processing units cooperates with 32 3-inputs and gates to detect hits in both sequences . If there is high similarity between query and subject sequence, the multiple hits detection module may output a large amount of hits per clock cycle . Ungapped extension block is relatively slower than multiple hits detection module and the systolic array in this situation . As a result previous research [23, 24] suggested that in many cases there is a high similarity between the query sequence and the subject sequence . Processing those hits not only wastes the execution cycle for multiple hits detection module, but also consumes additional extension time for ungapped extension block because one hit is extended twice . For instance, two adjacent hits atk and tkp are found but they are actually one hit if they are not combined into one hit, they would have been recorded twice in multiple hits detection module and extended twice in ungapped extension block . In this section, we implement hits combination block to address this issue . It can detect the overlapping hits and merge them to reduce verbose hits and maintain the sensitivity of blast . This block contains a hits (first in first out) fifo buffer which is used to store hits location addresses from both query and subject sequence . The algorithm implementation engine executes hits combination algorithm then forward the hit addresses and hit length to the ungapped extension block . The order of the hit addresses in the hits fifo buffer follows the direction of the stream flow (figure 5). For example, the address of akl should be stored in the fifo buffers first, then the klp . The combination algorithm is triggered when there is more than one record in the hits fifo buffer . Here, the hit addresses in subject sequence are recorded and forwarded to the following operations . First, the algorithm implementation engine calculates the ending address of the ith and (i + 1)th hit in subject sequence . In this paper, the starting address is the recorded address minus 1, because the recorded address is the address of the middle word in a 3-word hit . The ending address is the address of the last character in the sequence, so (ending address = starting address + (seqence length 1)). Here, the sequence length is equal to 3 . Then, a comparison between ith hit's ending address and (i + 1)th hit's starting address is performed . If ith hit's ending address is greater than or equal to the (i + 1)th hit's starting address, there would be an overlap between two hits . The algorithm can determine new addresses in both subject sequence and query sequence of the merged hit and calculate its length . In the next step, the algorithm records the new address and hit length at the ith place in hit's fifo buffer . If ith hit's ending address is less than the (i + 1)th hit's starting address, the (i + 1)th hit address will be chosen to be compared with (i + 1)th hit address . This operation continues until the hits fifo buffer is empty . Because multiple hits detection module can detect multiple hits in one internal clock cycle, multiple modules working together in parallel speeds - up hit detection . In order to construct a longer array, as figure 8 shows, the large systolic array contains many multiple hits detection modules . Every module matches one hits combination block, each of which records and combines hits detected by its mapped module . Then fifo combination modules combine hits fifos and deliver hits information to larger hits fifos . In this architecture, multiple hits detection modules are connected in a serial way to extend the array comparison size . Each hits combination block maps to a multiple hits detection module so that each block can detect overlapping hits and merge them simultaneously . The score matrix blosum50 [10, 25] is applied to find the alignment score for a pair of words . If this block detects a hit in a fifo, the address of the hit word in query sequence, and subject sequence and the hit length would be extracted from the fifo to compute both extension points . After this step, the ungapped extension algorithm implementation engine can finish the ungapped extension step . The data flow in the ungapped extension block is shown in figure 9 . The extension procedure in algorithm implementation engine after computing the addresses of characters k and p, the starting addresses of the extension, the algorithm drives the extension in both directions . Each pair of the residues own one alignment score which can be found in the blosum50 score matrix . In figure 13, the alignment score l and d is 3, and the score of the pair then endpoints of this high - scoring segment in both directions on both query and subject sequences are recorded and forwarded to the gapped extension block for gapped alignment . In this step, hits which are sent from hits fifo of the hits combination block are extended to either side to identify a hsp . Mercury blastn prefilters the hit's characters because only one hit can be searched at one clock cycle . Fpga / flash could get the subsequence directly because it constructs the index for each word . Compared to methods applied in mercury blastn and fpga / flash, multiple hits detection module is more efficient since it could detect multiple hits in one clock cycle . However, the serial ungapped extension process is slower than multiple hits detection module . To speedup this step, we proposed two methods in the above sections: adding hits combination block between multiple hits detection module and ungapped extension block; dividing the long components array into several parallel components array groups . As shown in figure 11, we propose one more parallel architecture for ungapped extension block . In this architecture, each hits fifo of hits combination block connects to an ungapped extension block . Each block connects to the query sequence memory as well as initialization set & data control block because the context of the hits is necessary for extension . System may slow down if hits from all hits fifos are queued in only one ungapped extension block . In this architecture, hits information could be distributed into different paths and save processing time . In this block, hsps from the fifo buffer are used to perform gapped extension . The gapped algorithm implementation engine will be triggered once the block detects that there is a pair of segments in fifo . This paper focuses on the first two steps of blast algorithm including finding hits and ungapped extension because they consume most execution time in blast algorithm . In order to implement the needleman - wunsch algorithm on the fpga we have mapped multiple hits detection module, hits combination block, and ungapped extension block architecture on the xilinx xc5vlx110 t fpga which is embedded on ml509 education board . The architecture was designed under xilinx ise design suite 12.1 in very high - speed integrated circuit hardware description language (vhdl). In our system, the xilinx ml509 education board has a pci express(x1) interface which could provide 500 mb / s throughput . The board is then connected to the host computer: dell precision t1500 pc with a 2.80 ghz intel core i7 microprocessor and 4 gb memory . Ncbi blast software runs on the host . In our experiment, a systolic array - based parallel architecture with 2048 processing units was implemented on the xilinx ml509 . Except for this implementation, we also mapped systolic array based architectures with 1204, 2048, and 3072 processing units to different fpga devices under xilinx ise design suite . The clock values came from the timing analyze tool called postplace and route which integrated in the ise . We will analyze the architecture performance from two aspects: one is comparing our architecture with previous architectures on hardware, the other is comparing our architecture against blast software versions . For hardware comparison, we focus on 3 parts including synthesis performance, storage requirement, and word scanning speed . For software versions comparison, a hardware performance comparison is made between ours and other fpga architectures from 3 aspects including synthesis performance, storage requirement, and word scanning speed . Tree - blast and mercury blastn are wprbs architectures, families of fpga - based accelerators is a systolic array - based architecture . All data which reflects the capability of architectures in the comparison from previous publication . Because every four components need a bram to index the scoring matrix, the input array size the system could process is restricted by bram . As a result, the query size is limited up to 600 on xc2vp70 and 1024 on xc4vlx160 . Xia et al . Implemented a fpga - based accelerator which is also a systolic array - based one for blast algorithm called families of fpga - based accelerators . In addition, the data stream in the array needs to be shut down to record hits addresses . As a result, it needs more registers to store the systolic array status in order to resume it . In our architecture, wprbs is replaced by multiple hits detection module to find hits so that the bram is not the bottleneck of array size anymore but luts are . We can build 2048 components on xc5vlx110 t which is two times the array size of tree - blast . Based on synthesis report our design has longer array size and consumes less resource, comparing with tree - blast built on xc4vlx160 with 78% of the slices . For synthesis performance comparison, we also implement our architecture with 2048 components on fpga xc2vp70 and fpga xc4vlx160 . We constructed two systolic arrays with 1024 and 3072 processing units to make a comparison in synthesis performance with the families of fpga - based accelerators . The memory usage is 3408 kbits in total which is 20.3% of the memory capacity in xc5vlx110 t . Since many wprbs architectures need to record the position of each word in query sequence, they generally consume much more storage . For instance, rc - blast spends 64 k x 64bits memory to store the query index in xilinx 4085xla . Mercury and mitrion have external sram to store the hash table because internal memory is not enough for the record . Compared to these methods, our architecture saves storage, avoids the complexity of memory access, and simplifies the fpga layout and routing . The speed of multiengine blastn accelerator which applies 64 identical parallel engines reaches 6400 m matches / s . The word scanning speed of our multiple hits detection module approach achieves 14450 m matches / s which is over 2 times than related work . Our design is mapped to the xilinx xc5vlx110 t fpga to accelerate the first two steps of ncbi blast family programs . As table 3 illustrated, swiss - prot from ebi and drosoph.net from ncbi blast are two databases in experiments . Swiss - prot is a protein sequence database while drosoph.net is a database for dna sequences . The swiss - prot in our experiments is uniprotkb / swiss - prot protein knowledgebase release 2012_11 . Queries which are picked from drosoph.nt translated into six - frame protein sequence before searching against swiss - prot . Tblastx is sued for searching six - frame translation of dna sequence against six - frame translation of dna database . For each algorithm, we built 6 arrays with different sizes (128 - 3k) to carry different queries with different lengths . For each length, 15 queries with the same length are picked up randomly from the relevant database . The final execution time for a certain array is the average of running time of 15 queries . The execution time of the software versions in this table comes from the previous work . The hardware execution time (ht) is less than software execution time (st) for all cases . Blastp software version only spends 1901 ms in searching database with a 128 long query while 25132 ms is taken when it is searching database with a 3-k long query . Index construction not only needs more time but also increases the size of searching object . In our architecture, however, the execution time does not increase so rapidly . This is because the searching time is equal to the sum of the time of database stream flowing through the array and the extension time of valid hits . The time of database stream flowing is determined by the size of database; the extension time of valid hits is influenced by the architecture design . In this experiment, the hits combination block and the parallel design reduce the number of valid hits and their extension time . For the same reason, the execution time of tblastn, blastx, and tblastx which are implemented on the fpga does not increase sharply . From table 3, it can be seen that tblastn software version is slower than blastp software version for the same query length . The reason is that all dna sequences are translated into the 6 possible potential proteins in tblastn software version before the searching . The translation time is not counted for the hardware version in our experiments because the translation is a one - time job, and results can be used for many other applications . For tblastx software version, both queries and the database are translated into the six possible potential proteins before searching . The translation time is not counted for the same reason as in tblastn . Our architecture could implement 3 steps of blast algorithm simultaneously because the fpga is a good platform to parallel the blast algorithm . Paralleling blast algorithm on the fpga patternhunter has 48 m/4s word scan speed at most while ours could get 14450 m matches / s . In this paper, we presented a systolic array - based fpga parallel architecture for blast algorithm . The architecture contains four different kinds of blocks which are multiple hits detection module, hits combination block, ungapped extension block, and gapped extension block . We designed the multiple hits detection module to improve the efficiency of the bottleneck step . The multiple hits detection module based on the systolic array could find more than one hit in only one clock cycle . The performance is faster than wprbs searching approach used in other proposed architectures cited in this paper . In order to speedup hits searching, hits combination block between multiple hits detection module and ungapped extension block merges adjacent overlapping hits into one hit in order to speedup the extension step . Lastly, we proposed a parallel architecture for ungapped extension block as figure 12 illustrated . We have implemented multiple hits detection module, hits combination block as well as ungapped extension block . Based on the ise synthesis report, we compared our architecture performance to the performance of other related work . First, compared to tree - blast, a twice longer array can be built in our architecture with less fpga resource . Thirdly, the word scanning speed of our architecture faster than mercury blastn's word scanning speed . Last, compared to blastp software, our architecture is more suitable for dealing with longer sequences . In the future, we hope to design an efficient architecture for gapped extension block . To satisfy more users' requirement the multiple hits detection module needs to be changed, for instance, 3-input and gate should be changed to 11-input and gate . The clock_division block needs to be redesigned to guarantee the pipeline works correctly, more details will be addressed in the further design . Our architecture is only available for users who use xilinx xc5vlx110 t fpga because we only test it on that fpga to guarantee the correctness of its behavior and timing . After enough tests on different devices,
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The identification of effective strategies to address the prevention and treatment of childhood obesity is critical to improving the health of the us population . National data from 2009 and 2010 show that nearly one in three children in america is either overweight or obese, and the numbers are even higher among certain demographic groups . In the short term, obesity poses significant risks for children's physical health and psychosocial well - being [2, 3]. In the long term, many of today's children will age into adulthood with obesity that began in childhood and will experience the negative health consequences associated with obesity as adults, such as type ii diabetes . Addressing the high prevalence of childhood obesity will require coordinated and collective efforts in multiple sectors and settings government, health care, school, workplace, and community that influence the food and physical activity environments in which children live [2, 5]. Primary care providers (pcps), defined for purposes of this paper as physicians, physician's assistants, nurse practitioners, registered nurses working in a primary care setting (e.g., community health center), or clinicians working in a school - based health center setting, have important roles in meeting obesity prevention goals . Primary care providers have traditionally measured patients' heights and weights to assess growth, development, and body mass index (bmi) and treated obesity and health - related conditions, but there is a recognized need to expand these roles to include advocacy, modeling healthful behaviors in the community, and counseling individuals and families about obesity prevention [5, 6]. A number of scientific organizations have published recommendations or guidelines for primary care providers to address childhood obesity prevention and treatment (see table 1). The most recent, by the institute of medicine (iom) in its 2012 report accelerating progress in obesity prevention, includes the goal to expand the role of health care providers, insurers, and employers in obesity prevention . Health care providers have a role in each of the four strategies recommended by the iom to achieve this goal: strategy 4 - 1: provide standardized care and advocate for healthy community environments;strategy 4 - 2: ensure coverage of, access to, and incentives for routine obesity prevention, screening, diagnosis, and treatment;strategy 4 - 3: encourage active living and healthy eating at work; andstrategy 4 - 4: encourage healthy weight gain during pregnancy and breastfeeding and promote breastfeeding - friendly environments . Strategy 4 - 1: provide standardized care and advocate for healthy community environments; strategy 4 - 2: ensure coverage of, access to, and incentives for routine obesity prevention, screening, diagnosis, and treatment; strategy 4 - 3: encourage active living and healthy eating at work; and strategy 4 - 4: encourage healthy weight gain during pregnancy and breastfeeding and promote breastfeeding - friendly environments . Recommendations by the white house task force on childhood obesity, the american academy of pediatrics [8, 9], the american heart association, and other health organizations have focused primarily on the health care provider's role of assessment and monitoring of bmi, encouraging and supporting recommendations for physical activity and healthy eating, and serving as positive role models for obesity prevention . The guide to community preventive services recommended behavioral interventions to reduce screen time but noted insufficient evidence for provider - oriented interventions (e.g., provider education, feedback, or reminders) for obesity prevention and treatment . Despite these recommendations, pcps data from a 2008 national survey of pcps found that fewer than half of all pcps assessed bmi percentiles regularly in children, and only 18% reported referring children for further evaluation or management . Most (58%) reported never, rarely, or only sometimes tracking patients over time concerning weight or weight - related behaviors . National survey data from 2007 found that 12% of physician office visits of all child or adult patients included counseling about nutrition or diet . Several studies identified a lack of office time to gather background information from families as a major impediment to addressing healthy weight [1417]. Other obstacles include lack of awareness of the issue, lack of comfort or skill counseling families on the issue, need for organizational prompts, and lack of familiarity with available community resources for lifestyle counseling or obesity prevention programs [5, 11, 1821]. Evidence suggests that with the right interventions and activities, pcps can effectively play an expanded role in preventing and treating obesity among children and adolescents . Obstetricians and gynecologists also play an important role in prenatal care, monitoring maternal weight gain, and encouraging and supporting breastfeeding . The purpose of this review is to identify effective or promising practices in the expanded roles that are now recommended for pcps (see table 1). These roles include the following: weight status assessment and monitoring: assessment and monitoring of bmi, nutritional intake, physical activity level, and other indicators of weight status in children and adolescents;healthy lifestyle promotion: dissemination of healthy lifestyle recommendations and materials as part of primary prevention efforts in the primary care setting, excluding healthy lifestyle promotion that is part of patient treatment (item no . 3);patient treatment: use of evidence - based techniques, such as behavioral and motivational counseling, within the primary care setting to treat patients identified as overweight or obese (treatment may include healthy lifestyle promotion);clinician skill development: education and training on evidence - based assessment and counseling techniques;clinical infrastructure development: implementation of capacity building within the primary care setting, such as improvements to organizational systems or care models used by providers;community program referrals: referral of patients to community - based obesity treatment programs outside of the primary care setting;community health education: dissemination of healthy lifestyle recommendations and materials as part of prevention efforts in the community setting;multisector community initiatives: participation in multisector obesity prevention and treatment initiatives to achieve policy and systems goals; andpolicy advocacy . Support of and advocacy for policy changes in the broader community setting . Weight status assessment and monitoring: assessment and monitoring of bmi, nutritional intake, physical activity level, and other indicators of weight status in children and adolescents; healthy lifestyle promotion: dissemination of healthy lifestyle recommendations and materials as part of primary prevention efforts in the primary care setting, excluding healthy lifestyle promotion that is part of patient treatment (item no . 3); patient treatment: use of evidence - based techniques, such as behavioral and motivational counseling, within the primary care setting to treat patients identified as overweight or obese (treatment may include healthy lifestyle promotion); clinician skill development: education and training on evidence - based assessment and counseling techniques; clinical infrastructure development: implementation of capacity building within the primary care setting, such as improvements to organizational systems or care models used by providers; community program referrals: referral of patients to community - based obesity treatment programs outside of the primary care setting; community health education: dissemination of healthy lifestyle recommendations and materials as part of prevention efforts in the community setting; multisector community initiatives: participation in multisector obesity prevention and treatment initiatives to achieve policy and systems goals; and policy advocacy . This review, guided by a socioecological framework and a systems approach [5, 23], focuses on the intersection between pcps (including community health centers) and public health in the community . Studies of child or family interventions in primary care settings or community interventions with a direct link to primary care (e.g., a community intervention with active referral to pcps) were the focus of the review . Although other reviews on childhood obesity and health care have been published since iom's 2005 report on preventing childhood obesity, the extent to which they summarize the specific roles of primary care providers in implementing the intervention varies [2426]. This review updates the most recent reviews of literature on the primary care role in obesity prevention and treatment published from 2005 to 2012, addresses 2012 iom recommendations that emphasize both a clinical and community advocacy role for pcps, and incorporates multisector interventions and community advocacy - specific interventions involving pcps . It also recognizes the new imperatives or opportunities to do more based on the affordable care act changes requiring preventive care as an essential health benefit and eliminating cost sharing for preventive services . We conducted a review of clinic- and community - based obesity interventions with a primary care component to identify evidence of effective roles of primary care in addressing the epidemic . The first phase of the scan was conducted in march 2011 (figure 1). We searched for literature published from 2005 to 2011 using search terms listed in table 2 . Our search included pubmed and other databases (esco academic search premier, cochrane central register of controlled trials, eric, and health technology assessments) and resulted in 669 articles . For these articles, we reviewed abstracts for relevance and to ensure that the intervention took place in the united states, which refined the list to 147 articles . We retained the articles that described an obesity intervention for children and/or families that took place in a primary care setting, a school health center, or a community setting (community health center; pediatrician; tribal health center; special supplemental nutrition program for women, infants, and children (wic) clinic; and so on) with some link to primary care . The elimination of unrelated school - based, policy, environmental change, and workplace interventions that did not include a link to the primary care setting, as well as articles that focused primarily on primary care recommendations (rather than primary care interventions), further refined the scan to 112 articles . We reviewed the remaining 112 full - text articles to ensure that each article fits the original criteria and to document the findings . We updated the review in january 2013 for the literature published in 2011 and 2012 . The additional review resulted in 36 articles that met our criteria, for a total of 96 articles considered in this paper . These sources included 63 articles describing specific interventions; 14 that reviewed existing interventions; 13 summarizing recommendations for the treatment and prevention of childhood obesity; and 6 that summarized the results of topic - related focus groups with parents, children, or clinicians . The full list of citations for articles considered in this review is available upon request from the authors . The 96 articles that met the criteria for this review provide examples of how pediatricians, pcps, and communities have implemented clinic- and community - based programs and initiatives targeting the prevention, screening, diagnosis, and treatment of obesity among children and adolescents . The interventions reviewed typically took place in a primary care clinic, pediatrician's office, community health center, school - based health clinic, university research program, wic clinic, or other community setting . In this section, we summarize the paper findings regarding the efficacy of these efforts, when available, and describe how these interventions align with current recommendations for obesity prevention and treatment in the nine areas identified in table 1 . Because an article could address multiple primary care physician roles (e.g., weight assessment as well as treatment of obesity), articles can be cited in more than one category . Methods used to assess changes in children's weight status include change in bmi; bmi z - score (i.e., the number of standard deviations of the value for an individual away from the mean value of the reference population); bmi percentile for age and gender; bmi velocity; kilograms or pounds lost; percent healthy weight, overweight, and/or obese; and waist and hip girth . The majority of interventions lasted between four and 12 weeks, and most follow - up efforts occurred over less than 12 months . Annual assessment of weight status through the use of bmi compared with age - sex bmi percentiles in growth charts in children and adolescents is widely recognized as a standard of care in the primary care setting [2, 9, 28]. Although a healthy weight assessment routinely involves some form of measuring body weight, evidence suggests that a complete assessment should also include indicators of healthy diet, active living, and child and family health history [8, 9]. Most interventions reviewed included an evaluation of the patient's overall health through patient and/or parent discussions or questionnaires in addition to assessment of weight status by use of bmi or growth charts . Methods used to assess or monitor weight included bmi, bmi z - scores, and comparisons to reference growth charts and standards for overweight and obesity . Integrated parent - completed questionnaires into routine primary care visits to assess family history of diabetes and cardiovascular disease, parents' height and weight, child's television and play habits, and child's intake of meals in front of the television; information collected from parents was used to inform weight assessment and guide the content of counseling and goal setting for overweight patients . Recommendations state that pcps should track annual bmi assessments over time to assist clinicians in recognizing major changes in weight relative to height [8, 9]. Two articles reviewed suggested that integration of bmi assessment into frequently used electronic medical record (emr) systems or hand - held personal digital assistants could facilitate increased use of bmi as a screening tool and as a method of effectively tracking bmi over time for the purposes of monitoring [18, 20]. However, only one article reviewed specifically discussed integration of bmi collection into emr systems . Found that customized emr templates designed to facilitate assessment of bmi and screening and counseling for overweight patients increased the frequency of children screened for bmi, as well as the diagnostic rate for overweight and obesity . Articles reviewed suggest that multiple barriers might limit the assessment and monitoring of bmi in the clinic setting, including lack of familiarity with the use of bmi; lack of agreement about the utility of bmi as a screening and intervention tool; lack of office time to gather background information from families; and lack of practice - level resources conducive to simple, frequent use of bmi [11, 1821]. Several articles noted the importance of familiarizing clinicians with weight assessment tools, including bmi assessment calculators, centers for disease control and prevention guidelines for bmi interpretation, and educational materials to increase uniformity of screening and improve clinician self - efficacy [20, 3032]. Perrin et al . [20, 31] suggested that age - specific office - based tools may assist practitioners in communicating results of bmi assessment to families and in evaluating the patient's readiness to change . Another study that promoted use of bmi tools found a significant decrease in bmi at 5 months, but not 12 months, among children participating in a primary care - based program that combined clinician training in weight assessment with an eight - week, family - based behavioral intervention . Multiple recommendations suggested that promotion of healthy lifestyle behaviors such as adherence to recommended dietary guidelines, increased participation in physical activity, and limiting screen time and sedentary behavior should be incorporated into standard clinical practices for clinicians who serve children and adolescents [79, 12, 3436]. These recommendations apply to both prevention and treatment of obesity in the primary care setting . Healthy lifestyle promotion as used in clinic - based treatment interventions is discussed in more detail in the following section; here, we describe notable examples of healthy lifestyle promotion as it pertains to the prevention of overweight and obesity in children and adolescents . The iom suggests that providers utilize a multifaceted approach to patient education, recognizing that patients may have different learning styles, needs, and preferences . Incorporation of healthy lifestyle promotion in the primary care setting may involve distribution or display of educational materials on nutrition, physical activity, and screen time in conjunction with verbal counseling of patients . Kubik et al . Described a prevention intervention that incorporates educational brochures on behavior - regulated activities, a kid's goal board, and a parent tip board in the waiting room area . Perrin et al . Suggested that pcps should incorporate messages about healthy weight management, such as limiting screen time and sugar - sweetened beverages and increasing physical activity, into conversations with patients and parents during regular office visits . These conversations might be particularly important for children who are more likely to be overweight . Materials including healthy weight messages should be made available in multiple languages representative of the populations served by the clinic . It is notable that recommendations by health organizations regarding reduced screen time have become more common in recent years [7, 12]. This is expected as screen time is a more recently accepted measure of inactivity (i.e., as one measure of physical activity) compared to more established measures such as healthy eating . It is also notable that, although several recommendations stated the need for pcps to promote healthy weight gain during pregnancy, provide information and resources on breastfeeding, and promote guidelines for weaning children at the appropriate age [7, 9, 35, 36], few articles reviewed specifically addressed healthy lifestyle promotion among pregnant or breastfeeding mothers . Those that did [20, 21, 39] summarized recommendations for incorporating healthy lifestyle messages into prenatal or postnatal visits; however, none described specific health promotion interventions for pregnant or breastfeeding women in the primary care setting . Although few health organization recommendations specifically addressed the role of the pcp in the treatment of overweight and obesity in children and adolescents [2, 12, 28], most of the articles reviewed that occurred in or were intended for the primary care setting involved interventions that were designed to treat the children who were identified as overweight or obese through bmi assessment . The format of the treatment and the intensity, frequency, and length of engagement with clinicians varied across studies . Many of the health organization recommendations on promotion of healthy lifestyle discussed in the previous section also apply to obesity treatment interventions; most of the interventions shown to be successful included promotion of improved nutrition and exercise habits and reduced screen time . For children with a bmi above a specified percentile, treatment interventions that incorporated individual case management or patient - centered counseling as a means for achieving examples of individual case management include private, age - appropriate conversations with clinicians regarding achieving healthy weight; goal setting; motivational interviewing; and conversations with registered dieticians about patient readiness, diet, and exercise . Of the seven studies in this category, six measured positive results including weight loss, improved lifestyle habits, or increased parent confidence using provider recommendations after patients participated in multiple individual sessions with the providers [15, 4045]. Successful studies emphasized the need for providers to engage the patient in a dialogue about lasting lifestyle changes and the benefits of training clinicians on how to address ambivalence about making behavioral changes . The tone and language used to communicate messages regarding obesity and being overweight is important . Two articles discussed strategies that pcps could use to deliver diagnosis and treatment options . In focus groups, parents expressed preferences for health care providers to communicate using clinical terms to explain the rationale for their concern and to provide specific treatment recommendations [46, 47]. When a treatment plan is established for an individual, many primary care practices sponsor or refer patients to interventions that provide group classes or activities to support individuals and families . Content of primary - care - based group interventions was diverse, including in - person physical activities, educational grocery store visits, interactive nutrition and exercise sessions, family cooking courses, and group discussions . Though the pcp might make the initial referral to interventions of this type, his or her role in the actual group treatment intervention was less clear from the articles reviewed . Described a group - based intervention for parents using the national institute of health's we can! Curriculum, which is facilitated by pcps; mcclaskey described a community - health center intervention involving group nutrition and physical sessions led by physicians . However, dieticians, interventionists, or nurses carried out the majority of primary care - based group interventions . Multiple recommendations from health organizations cited the role that pcps can play in educating parents about healthy eating, physical activity, and reduced screen time [79, 35, 36]. Kwapiszewski and lee wallace found it critical to have full support from all intervention partners (providers, parents, and children) in commitment to lifestyle changes to treat obesity . Most interventions in both the patient - based or group format involved some form of parent involvement, with parents present during individual or group counseling sessions with a pcp or in attendance at parent - only meetings with a focus on goal setting, modeling healthy behaviors, or nutrition and/or physical activity decision making . Recommendations suggest that in order to effectively prevent, diagnose, and treat obesity and overweight in children and adolescents, clinicians must be adequately trained in standardized, evidence - based assessment and counseling techniques . Moreover, clinicians must be comfortable communicating results of weight assessment and monitoring to patients and their families . . Suggested that trainings that include the full spectrum of care, rather than weight assessment alone, might be more effective in improving efficacy, as providers could be more likely to diagnose a child as overweight or obese when they have the tools and comfort level to provide counseling and treatment . Multiple articles reviewed suggested the need for physician training and decision support for use of techniques and tools for counseling on behavioral treatment approaches for childhood obesity [11, 24, 50, 51]. Seven of the articles reviewed involved training for physicians, nurses, and/or registered dieticians on the use of motivational interviewing techniques, goal setting for parents and children, and/or evidence - based tools for facilitating discussions on obesity . In most cases, training took place in person in a group format, though stahl et al . Clinicians trained on the use of a brief, structured intervention for school - age children involving the use of flash - cards and take - home games reported increased physician comfort and competence discussing obesity issues . Pediatricians and registered dieticians who received training on motivational interviewing techniques reported the need for more role - playing activities and experience asking open - ended questions . Web - based or in - person primary care clinician trainings for assessment of bmi, healthful eating, and active living habits among children and adolescents were found to be effective in increasing provider confidence in weight assessment [31, 40], rates of bmi assessment, and use of behavioral screening tools after at least a year [32, 38]. Trainings included reference charts for bmi and laboratory values, guidelines for discussion about healthy behaviors, and decision support charts . At least one training included guidance on assessment of parental readiness for change . In 2004, the american dietetic association stated the need for pcps to take into account regional and cultural differences when promoting healthy diet patterns among diverse populations and ethnic groups . Examples of ways that interventions took the cultural, linguistic, or literacy needs of their subjects into account included providing materials and activities in multiple languages, offering recipes to groups that incorporate cultural food preferences, or tailoring materials to families who might have low literacy levels . Of the three studies that explicitly described their efforts to adapt obesity interventions to their population(s), these studies used diverse means to adapt to the needs of their populations, which included latino families (bilingual and bicultural project staff), ethnically diverse youth (traditional recipes), and families with low literacy levels (adapted educational materials). Results from a focus group with latino parents suggest that, among this population, health messages can be especially well received coming from a trusted health care provider . However, a culturally competent health educator might help to extend the benefits of an obesity treatment program beyond a brief encounter with a provider . Few recommendations cited the need for pcps to advocate for systemic changes in clinical practices to promote screening, diagnosis, and treatment in the primary care setting . However, five of the articles reviewed focused specifically on an intervention that implemented some form of capacity building within the clinic setting, such as improvements to organizational systems or care models used by providers . Each identified structural gap in primary care services and implemented systemic solutions focused on reorganizing clinical care delivery . One such study evaluated the implementation of a patient - centered medical home system in a community health center and found positive outcomes in lifestyle changes, reduced bmi, and increased physical activity in the patients one year after implementation . Another study evaluated the adoption of principles of continuous quality improvement and adult learning theory in the office environment; physicians found these tools helpful, which resulted in increased bmi screening documentation . A third study assessed the effectiveness of integrating practice - based pediatric obesity prevention and treatment clinics within existing primary care settings; these clinics were shown to be helpful in improving nutrition status among obese children . Several studies reviewed involved systems changes targeted at multiple primary care clinics or health care organizations . Described the steps to health king county (steps) initiative, which promoted clinic staff training and integrated clinic systems changes across three local health care organizations . The maine youth overweight collaborative sought to improve clinical decision support in 12 primary care sites; findings showed increased assessment of bmi and use of behavioral screening tools, as well as increased parental satisfaction with services . An activity frequently cited in journal articles is the physician's role in the identification and recruitment of children and families into obesity prevention or treatment interventions . Of the 38 articles reviewed that described community - based interventions, 14 reported physicians referring their patients to research studies (8 articles) or other community - based programs (6 articles). However, physician recommendations rarely called out this role, with one exception . In 2010, the white house recommended that pcps and insurance companies connect pregnant women and new mothers to breastfeeding support programs . The obesity interventions described in these articles were mostly family - based counseling and treatment programs, lasting from eight weeks to six months, including group education sessions for parents and children, home visits, follow - up telephone calls, automated messages, and/or other family - oriented activities . Some were branded with program names and set curricula, including kids on the geaux, kids n fitness, healthy kids healthy weight, energize!, smart choices for healthy families, and family insulin resistance management firm . Of the 14 articles reviewed, nine reported positive outcomes, including weight loss or reductions in bmi scores [6265, 6771]. However, some study limitations included a small sample size and a low participant retention rate [62, 65]. Factors that reportedly contributed to the success of the programs included the dosage of the intervention, the use of healthy eating strategies and behavior modification techniques, and family participation in physical activities . Three of the articles assessed the value of the pcps' referral role in the interventions . Reported that, in the smart choices for healthy families program, physician involvement was seen as a valuable partnership . While physicians recognized the importance of referring patients to community - based programs that they did not have time to offer, program lay leaders saw the benefit of physician referrals, including improved behavior change among the provider's patients . Reported that parents perceived the obesity treatment program as an extension of their pediatrician's care because of the close partnership between the pediatrician and program trainers, the pediatrician's recommendation of the program, and followup with patients who were in the program . However, a third article compared physician referrals less favorably with other community program recruitment strategies . Because the number of physician referrals was not as high as expected, the program deployed additional recruitment methods, including the use of radio ads and posting of program flyers . Outside of their usual clinical role, pcps have a unique opportunity to serve as role models, educators, and promoters of healthy lifestyle practices to their patients and other community residents . In 2005, the american academy of pediatrics encouraged physicians to make use of community - based resources outside of their traditional hospital and outpatient office settings to instruct residents on the effects of individual and community factors on child health status and to promote the well - being of all children in the community . This might seem a natural role for physicians, extending their health promotion efforts with their patients to the community . Unfortunately, although many health care providers are aware of the childhood obesity epidemic, are concerned about its health impacts, and want to work on its prevention, they continue to see themselves primarily as clinical practitioners and not as health educators or advocates in the broader community . Pcps can fill several roles in such community health education efforts by serving on leadership teams; providing advice on community messages; volunteering as institutional partners in the funding, planning, and evaluation of community awareness campaigns; and collaborating with community partners on marketing healthy food choices and physical activity . Of the articles reviewed, four focused on the physician's role in community - level obesity prevention initiatives . Health care providers served on the leadership team of the switch program, which included a community awareness campaign to modify key health behaviors, increasing physical activity, improving nutrition, and reducing screen time . Pcps also served on the community task force that led the tioga county fit for life initiative, a comprehensive primary prevention program that used school - based health education classes, a virtual wellness club, and community health fairs to promote healthy nutrition and physical activity . Two other studies reported that health care providers were involved in community initiatives that implemented the national we can! Program developed by the national heart, lung, and blood institute (although their roles were not specified) [76, 77]. Although it is important to note the participation of health care providers in these initiatives, outcomes were not reported in three of the four articles [74, 76, 77]. The fourth study, a five - year longitudinal analysis of grade - specific rates of overweight and obesity of participating children, showed that overweight and obesity rates increased in all cohorts . Factors cited for the program's failure included inadequate reach of key health messages and lag time between the messages' dissemination and uptake . It is fair to assume that the involvement of pcps in these initiatives was not responsible for their lack of reported success . In contrast, health care providers have played important roles in numerous more effective community interventions targeting both obesity prevention and treatment (see the next section). Over the past decade, pcps have been encouraged to build partnerships across disciplines to work collaboratively with public health departments and other colleagues, to identify and decrease barriers to the health and well - being of the children in their communities, and to coordinate and focus new and existing services for the benefit for all local children [34, 35]. In the articles we reviewed, health care providers participated in six multisector obesity prevention and treatment initiatives that achieved intermediate policy and systems goals [7880]; changes in children's food and physical activity environments [80, 81]; and population - level health outcomes, including reduced bmi scores [82, 83] and changes in overweight and obesity prevalence trends [78, 79, 83]. Two projects used a multisector intervention model that started as a community - based research study at tufts university . In shape up somerville, 50 medical professionals were trained on childhood obesity guidelines and current bmi screening practices as part of a community - wide effort in somerville, massachusetts, to increase daily physical activity and healthy eating through programming, physical infrastructure improvements, and policy work . North carolina's health department patterned its childhood obesity prevention demonstration projects after shape up somerville . The state offered grants, training, technical assistance, and state - level partnerships and other resources to support local obesity prevention and treatment efforts in five counties . Pcps were also involved in bmi assessment and treatment in community initiatives in delaware and california . Delaware's 5 - 2 - 1-almost none initiative targeted multiple sectors, including schools, child care providers, and primary care settings, to implement policy and practice changes, in addition to implementing a media - based social marketing campaign . Pcps promoted universal bmi assessment, preventive health messages, and early intervention and treatment of childhood obesity . The california endowment's healthy eating active communities program worked in six communities to prevent childhood obesity in five childhood environments schools, after - school programs, neighborhoods, health care, and advertising . As part of the initiative, pcps were trained on the importance of tracking bmi scores, delivering obesity prevention messages, linking families to community programs, and improving local nutrition and physical activity environments . Two other communities included community - based bmi assessments in their multisector initiatives . In the healthy living cambridge kids program in cambridge, massachusetts, schools conducted bmi assessments and then referred students with high bmi scores to pediatricians for followup . The initiative included changes in city policies, implementation of a 5 - 2 - 1 community awareness messaging campaign, physical education enhancements in schools, food service reforms, family outreach, and farm - to - school - to - home programs . In the karanja research study, american indian / alaska native tribes were randomly assigned to either a community - wide intervention that used five strategies raising community awareness; providing health education; supporting behavior change; enhancing public health practice; and modifying local breastfeeding environments or policies to increase breastfeeding, limit consumption of sugar - sweetened drinks, and promote water consumption or to an intervention that combined these community - wide activities with family - level interventions, including bmi assessment, counseling, and treatment . Health care providers conducted the bmi assessments in wic clinics and maternal child health practices as part of routine visits . Another promising initiative is the healthy weight collaborative (hwc), a national quality improvement effort to share and spread promising and evidence - based practices to prevent and treat obesity among children . In this learning collaborative, the national initiative for children's healthcare quality is working with about 50 community teams of primary care, public health, and community - based organizations to implement and test an integrated change package of strategies . These include (1) building a community coalition; (2) implementing a healthy weight messaging campaign; (3) conducting weight status assessments and follow - up plans; (4) integrating activities across community sectors; and (5) advocating for food and physical activity policy change . The hwc evaluation will be completed in 2013 . Seven other studies in the review featured school - primary care partnerships or primary care interventions in school - based health centers . In four projects, nurses, nurse practitioners, and physicians in a school - based health center or wic clinic offered counseling and treatment services to students identified with high bmi scores . The results of these programs were either not evaluated, minimal [14, 86], or mixed . The other articles described school bmi assessment projects and a student walking project, whose outcomes were not evaluated . Several recommendations encourage health care professionals to support and advocate publicly for a number of policy changes, including increasing funding for childhood obesity prevention research; prioritizing capital improvement projects and school and community sports programs to increase opportunities for physical activity among students; and social marketing to promote healthful food choices, breastfeeding, and other healthy behaviors [2, 8, 9]. Although multisector community initiatives have used policy advocacy successfully to alter obesogenic community environments, one article reported on an initiative to increase public advocacy activity among pcps . Funded by the robert wood johnson foundation, the project sought to recruit, train, and reinforce 160 pcps to become change agents and leaders in community advocacy to prevent childhood obesity . Posttraining surveys showed that the training had increased participants' comfort and motivation advocating publicly for healthy behaviors, including active living (26%), healthy eating (25%), breastfeeding (24%), and school and worksite policies (15%). Identifying successful models that integrate primary care, public health, and community - based efforts is important to accelerating progress in preventing childhood obesity . This review aimed to identify the roles that pcps play in childhood obesity prevention and treatment initiatives in the united states and, in doing so, to determine effective or promising strategies for primary care and community settings . The review, based on 96 peer - reviewed articles published from 2005 to 2012 that met study criteria, demonstrates that pcps are increasingly being included in childhood obesity interventions, consistent with current recommendations from scientific and professional organizations . The review indicated an average of about 10 relevant articles published yearly during the period from 2005 to 2011 and nearly twice that number in 2012, supporting the increased attention to health care providers in the prevention of childhood obesity . The rise in obesity among children indicates the need for new strategies that encompass more than individual - level behavior change or postassessment treatment . The prenatal and early childhood periods are critical times for growth and healthy lifestyle development . In the first two years of life, primary care pediatricians, wic clinics, and community health centers have several opportunities during well - child visits to counsel parents about healthy lifestyles, to model healthful behaviors, and to refer families to community resources . Outside of their clinical role, primary care physicians can also serve as role models, educators, and promoters of healthy lifestyle practices and serve as leaders in community obesity treatment and prevention initiatives . However, national survey data on health practitioners and research studies suggest that pcps continue to see themselves primarily as clinic - based practitioners and not as health educators or advocates in the broader community . Although this review identified nearly 100 articles addressing the topic, the ability to draw conclusions about the effectiveness of pcps' roles in childhood obesity initiatives based on the review is limited by the lack of consistent reporting across studies about (1) specific pcp role(s) beyond referral and bmi assessment, (2) the level and duration of pcp involvement, and (3) child clinical outcomes or process outcomes . Interventions ranged from four to 12 weeks in duration, depending on the study and intervention methods . In addition, there was a general lack of long - term followup results; of the 20 interventions reviewed that had a significant impact on weight status, nine included followup over more than six months [33, 43, 54, 67, 70, 71, 79, 82, 83], and only three followed participants for more than one year [71, 79, 83]. In many cases, evaluations of the initiative interventions that did include an evaluation component used a range of outcome measures, including improved weight status, increased provider or parent knowledge, or increased rates of provider assessment of weight status or use of counseling tools, which made it difficult to compare the efficacy of results across articles reviewed . While change in weight or weight status was a frequently used outcome, the methods of weight assessment varied between interventions . Moreover, very few of the interventions reviewed utilized a randomized control study design, further limiting the ability to draw meaningful conclusions about the effectiveness of the interventions reviewed . For these reasons, the results of this review are primarily descriptive . While it is difficult to draw conclusions about the efficacy of the interventions considered due to the limitations mentioned previously, multisector community childhood obesity initiatives with primary care involvement were more likely to report positive outcomes than obesity initiatives in a single setting (school or clinic based). Multisector obesity prevention and treatment initiatives that achieved intermediate policy and systems goals included partnerships across disciplines, including pcps, addressed children at all points along the prevention continuum, and used an ecological approach targeting individual, organization, system, and policy change . Positive outcomes included improvements in children's food and physical activity environments, reduced bmi scores, and changes in overweight and obesity prevalence . Successful models that integrated primary care, public health, and community - based efforts also shared several similarities: multisector messaging within a community;weight assessment training for clinicians;modeling of healthy behaviors for children (to reinforce their understanding of the concept);promotion of culturally competent approaches;parental involvement . Multisector messaging within a community; weight assessment training for clinicians; modeling of healthy behaviors for children (to reinforce their understanding of the concept); promotion of culturally competent approaches; parental involvement . Because interventions of this type inherently involve multiple components, it is difficult to disentangle the roles to ascertain which individual components were especially successful or effective . Additionally, very few studies documented long - term effectiveness of interventions of this type, demonstrating a need for studies that measure the impact of multisector obesity initiatives over multiple years . Despite these limitations, this review provides a useful resource for pcps, community organizers, researchers, and policymakers planning childhood obesity initiatives in their communities or primary care settings . Future research on community - based childhood obesity interventions should collect and report information on the specific roles that pcps played in the initiative, including the level of training and counseling skills, presence of role modeling, referrals to community resources, number and type of community partnerships, and public advocacy activity . Reporting on the process or implementation of the initiative as well as child - level and population - level outcomes will contribute to the evidence base for effective strategies by pcps in the prevention and treatment of childhood obesity.
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Presence of associated anomalies suggests a developmental origin of the arachnoid cysts . We present a case of a 6-year - old boy who presented with paraparesis and a brief review of the etiology and pathogenesis in relevance to clinical decision making is also presented . A 6-year - old boy presented to us with complaints of recent onset difficulty in walking and dribbling of urine . On examination he had grade 3/5 power in both lower limbs and absent reflexes . An mri spine was performed which revealed a clearly defined intradural cystic lesion extending from l2 to s2 which was hypointense on t1-weighted images [figure 1] and hyperintense on t2-weighted images [figure 2] with tethering of cord . A tentative diagnosis of lumbosacral arachnoid cyst was made and the patient was taken up for surgery . Multiple level laminectomies were performed and on opening the dura a tense cyst was found [figure 4] along with short and thickened filum terminale . There were no other associated lesions . Postoperatively the child's motor power began to improve though his bladder was kept catheterized . Postoperative mri was done which showed complete resolution of cyst [figure 5]. At follow - up of 2 months patient t1-weighted mri showing hypodense intradural cystic lesion extending from l2 to s2 t2-weighted image showing hyperintense lesion syrinx in the cervicodorsal spine intra operative photograph showing tense cyst on opening the dura postoperative mri showing complete decompression of cyst arachnoid cysts of the spine are collections of cerebrospinal fluid (csf) within protrusions of arachnoid that can occur in a perineural, extradural, intradural, or intra - extradural site . These lesions are most often located posterior to the spinal cord but have also been identified anteriorly . The thoracic spine is the most common location followed by lumbosacral and cervical spine . Spinal arachnoid cysts are equally prevalent in men and women usually presenting in the fourth and fifth decades of life . Rarely they may present in children with other anomalies thus lending support to congenital etiology of spinal arachnoid cysts . Idiopathic arachnoid cysts in children are associated with neural tube defects and in adults with spinal deformities . Type 1 included extradural cysts, type 2 included cysts involving nerve root also known as tarlov perineural cyst and type 3 consisted of intradural cysts . Intradural cysts are outpouchings of arachnoid that, regardless of size, lie entirely within the dural space . Lumbosacral arachnoid cysts are believed to occur as a disorder of secondary neurulation . While patients with arachnoid cysts / meningoceles are usually neurologically normal, 90% of patients with meningocele have associated occult spinal lesions such as tight filum terminale, split cord malformation, and epidermoids . Alterations in the arachnoid trabeculae that overlie the spinal cord are probably the root cause . These alterations may be a congenital or may be caused by inflammation from previous trauma, surgery, or subarachnoid hemorrhage . In some cases no cause can be established, it has been hypothesized that these lesions develop from the septum posticum of schwalbe which is an arachnoid membrane dividing the midline posterior cervical and thoracic subarachnoid space . But such a hypothesis does not explain cysts occurring anterior to the cord . According to the hydrodynamic theory normal pulsations of the csf dilate weak areas of arachnoid which then progressively enlarge and form a cyst . A ball valve effect at the neck of the diverticulum is probably responsible for the progressive enlargement of the cyst . These pockets of fluid lead to further disruption of normal pulsatile csf flow and are capable of expanding and developing into cysts . Most spinal arachnoid cysts are asymptomatic and are discovered incidentally on magnetic resonance (mri). Mri is the most sensitive and specific study for detecting a spinal arachnoid cyst and for assessing the extent of the cyst wall . The signal intensity of arachnoid cyst fluid is the same as csf on t1 and t2 images . Sometimes higher signal intensity is seen on t2 which maybe related to increased protein content of sequestered fluid or absence of motion effects . Diffusion - weighted mri not only helps differentiate the arachnoid cyst from epidermoid cyst, abscess, or tumors but also evaluates spinal cord atrophy and inflammatory changes . Although ct myelography has been used to establish or refute communication between the intraspinal subarachnoid space and the arachnoid cyst, it is more sensitive in determining whether a communication exists rather than locating the communication . 12 kinematic mri (cine - mri) helps locate the communication which appear as pulsating turbulent flow voids . The presentation of sacral arachnoid cysts varies from long standing low back pain to neurological deficits such absent deep tendon reflexes or bowel bladder abnormalities . Radicular pain if present is relieved by lying down which pushes fluid out of the cyst . The treatment decision needs consideration of the extent of the cyst, the point at which the spinal cord is maximally compressed and presence or absence of communication between cyst and subarachnoid space . Aspiration under mri guidance is advised for small cysts which have no communication with subarachnoid space . For moderate - sized cysts complete excision is advised and multiseptated cysts extending over several segments, cyst fenestration can be done to avoid extended laminectomy . Percutaneous cyst drainage can be tried as a temporary measure to decrease symptoms or as a test of success of operative management . The success of the treatment modality employed ultimately depends on the degree of correlation of the mri findings with the patient's symptoms . On histopathology, the walls of arachnoid cysts are usually seen as fibrous and lined by meningothelial cells . These cells are usually negative for gfap, s-100, transthyretin (prealbumin), and cea staining thus differentiating them from epithelial cysts . Surgery typically results in excellent outcomes in terms of resolution of symptoms, and is effective across a large range of cyst sizes . Arachnoid cysts of the spine may occasionally be encountered in the work up of neurological symptoms or incidentally . It is important to be aware of the possibilities of other congenital anomalies of the spine and investigate for the same . For good surgical outcome
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Caudal block is the most popular and commonly used regional anaesthetic technique in children with a high success rate, for surgeries below the level of the umbilicus . It reduces the requirement of inhaled and intravenous (iv) anaesthetic agents, attenuates the stress response to surgery, facilitates a rapid and smooth recovery and provides satisfactory post - operative analgesia but with the limitation of relatively short duration of analgesia with single shot technique . Use of caudal catheter for continuous infusion is usually not preferred due to high risk of catheter contamination from faecal soiling . To overcome this limitation, opioids, alpha 2 agonists and ketamine have been studied with local anaesthetics to increase the efficacy of caudal analgesia but are associated with adverse effects such as nausea, vomiting, pruritus, urinary retention and respiratory depression (in case of caudal opioids), hypotension, bradycardia (with caudal alpha 2 agonists) and more sedation with ketamine . Among local anaesthetics, ropivacaine provides a greater margin of safety, less motor blockade, less neurological and cardiac toxicity and similar duration of analgesia in comparison to bupivacaine . Corticosteroids have a strong anti - inflammatory action and have been used via epidural, intrathecal, caudal and perineural routes in adults for prolonging post - operative analgesia . This prospective double - blind study was designed to investigate whether dexamethasone as an adjuvant to 0.2% ropivacaine enhances the analgesic potency in paediatric herniotomies performed under caudal block, . This randomised controlled double - blind interventional study was initiated with due permission from the institutional ethical committee . A written informed parental consent, of 128 patients of 15 years age group, belonging to american society of anaesthesiologists (asa) physical status i and ii undergoing elective inguinal herniotomy was taken . Patients having bleeding / coagulation disorder, local infection, sepsis, bacteraemia, vertebral deformity, pre - existing neurological diseases, allergy to ropivacaine or any other drug to be used were excluded from this study . All parents were explained the about the anaesthetic technique and perioperative course during a pre - operative visit on the day before surgery . Patients were randomly allocated to two groups (64 patients in each group), and randomisation was done by pulling chits out of a partially sealed box . Blinding was done by preparation of the medication according to assigned group by one investigator and block was performed by the second investigator who was blinded to group assignment . A received a bolus of 1 ml / kg of 0.2% ropivacaine with 1 ml saline . Group b received a bolus of 1 ml / kg of 0.2% ropivacaine with dexamethasone 0.1 mg / kg in saline to make a total volume 1 ml . On receiving the patient in the operation theatre, monitoring was attached in the form of electrocardiogram (ecg), pulse oximetric oxygen saturation (spo2), non - invasive blood pressure (nibp) and parameters recorded . Pre - medication was done with injection glycopyrrolate 0.008 mg / kg and injection midazolam 0.05 anaesthesia was induced with injection ketamine 2 mg / kg and as a protocol of our hospital, thereafter caudal block was performed with 5 cm short bevelled 22-gauge needle in lateral decubitus position taking aseptic precautions . Once the needle was in epidural space, study drug was injected slowly, according to the group assigned in the chit after negative aspiration for blood and cerebrospinal fluid . Anaesthesia was maintained with halothane and oxygen 40% and n2o 60% with patient breathing spontaneously via jackson rees breathing circuit as the procedure was of short duration . All patients were monitored by a standard protocol in a uniform pattern during anaesthesia and surgery . Continuous monitoring of vital parameters- heart rate, ecg, respiratory rate, nibp, spo2 were done, and values were recorded before and after pre - medication, induction, caudal block, after incision and thereafter every 10 min until the surgery was over . At the end of surgery, all anaesthetics were discontinued . Any side effects such as breath holding / apnoea, hypotension, involuntary movements, and nausea / vomiting were also noted . After surgery patients were shifted to the post - anaesthesia care unit for further observation and monitoring . Sedation by 5-point sedation score, post - operative pain by faces, legs, activity, cry and consolability tool (flacc) score were determined on emergence from anaesthesia and 1,2,4,6, 12, 24 h until the first dose of rescue analgesia was given in form of paracetamol suppository 15 mg / kg when the flacc score was 4 . Other adverse events such as nausea, vomiting, respiratory depression, bradycardia, hypotension, urinary retention, facial flushing and pruritus were also observed . The duration of analgesia was defined as the time period between administration of block until the time flacc score reached 4 . The sample size was calculated based on anobservation made by jo et al ., they had taken three groups in their study as compared to two groups in this study . The sample size was calculated as 64 subjects for each of two groups at alpha error 0.05 and power 80% assuming a difference of means to be detected in time of first rescue analgesic requirement in ropivacaine plain group and ropivacaine with dexamethasone of 10 min with expected standard deviation 20 min . The two groups were comparable with respect to age, weight and duration of surgery . Intraoperative haemodynamic parameters were maintained within 20% of base value in both the groups having no significant variation . The mean duration of analgesia in group b was significantly more than in group a, i.e., 478.046 104.57 min and 248.4 54.1 p <0.001 respectively . When pain score was compared within two groups, it was observed that at 4, 6, 12 h, it was significantly higher in group a as compared to group b and in first 2 h there was no significant difference in pain scores . Our study demonstrated a significant prolongation in the post - operative analgesia by adding dexamethasone to caudal ropivacaine . There was a decrease in pain score and demand for rescue analgesic requirement during 24 h post - operative period and time for first analgesic administration was significantly longer with dexamethasone . The effect of iv dexamethasone in decreasing post - operative pain has been studied in children mainly in otolaryngological procedures with a wide range (0.41.0 mg / kg) of dexamethasone . Variation in dosage of dexamethasone, type of surgery and anaesthetic techniques, use of opioids intraoperatively and different methods for assessing post - operative pain may be responsible for conflicting results obtained in these studies . In one study, iv dexamethasone in combination with caudal ropivacaine in paediatric orchiopexy was used and reduced severity of post - operative pain and prolonged analgesic duration were observed . Other studies where dexamethasone was used as an adjuvant to local anaesthetic via perineural epidural intrathecal and caudal and routes for various surgeries supported its role in potentiating and prolonging the analgesic effect . We used dexamethasone with a preservative in the form of methyl paraben and propyl paraben . Epidural corticosteroids have been used safely since long in the treatment of low back and radicular pain . The safety of methyl paraben and propyl paraben has been proven for intrathecal injection in human and animal models. [20 the exact mechanism of epidural or perineural dexamethasone is not known, but it is believed to have local anaesthetic effect by direct membrane stabilising action on nerves . Use of dexamethasone is more common because it is a clear liquid (non - particulate) steroid . The effect of dexamethasone on the spinal cord is due to the presence of transcription factor nuclear factor - kappa b (nf-b), present throughout the nervous system . Dexamethasone by regulating nf-b inhibits central sensitisation after surgery and potentiates analgesia of the caudal block . Systemic administration of dexamethasone attenuates the level of bradykinin and by inhibiting phospholipase a29 and the expression of cyclo - oxygenase-2 in peripheral tissues and in the central nerves system resulting in decreased production of prostaglandins, contributing to analgesia . Until date, no significant adverse effects have been reported for epidural dexamethasone and caudal dexamethasone . We used 0.1 mg / kg of dexamethasone as an adjuvant to 0.2% caudal ropivacaine . The addition of 0.1 mg / kg dexamethasone to caudal ropivacaine for paediatric herniotomies as a single shot injection resulted in a significantly longer duration of analgesia as compared to the use of ropivacaine alone, and the quality of analgesia was better after the first 2 post - operative hours, without any side effects.
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Since it first appeared as a clinical syndrome in central africa in the late 1970s, ebola hemorrhagic fever, now termed ebola virus disease, has intrigued infectious disease physicians, virologists, and epidemiologists because of the striking clinical presentation associated with the end stage of the disease, its high case fatality rate, and the ease with which it is transmitted among close contacts, including caregivers . Isolation and identification of the zaire ebola virus soon after the first outbreak gave clinicians, scientists, and public health representatives tangible evidence of the pathogen responsible for severe illness in 318 people which resulted in 280 deaths and fueled four decades of research on the biology of the highly lethal pathogen . This virus, currently referred to as ebola virus, is rapidly disseminated to lymph nodes by monocytes, macrophages, and dendritic cells where it quickly spreads to the liver and the spleen . This process is extremely efficient, making development of a therapeutic regimen to treat ebola virus disease a race against the clock due to the narrow window between the time when viremia and/or the onset of fever and other clinical symptoms can be detected and death . Several experimental candidates have shown promise for treating ebola virus disease in animal models of infection, however, there are currently no therapeutic or preventative agents approved for human use . Basic supportive care (fluid and electrolyte replacement; administration of antibiotics and antimalarials for concurrent infections and antiemetics for gastrointestinal symptoms) continues to be the cornerstone of therapy for ebola virus disease and can notably improve outcomes when administered early in the course of the disease . The current outbreak in west africa not only emphasizes the important relationship between early detection of infection and supportive treatment, it also highlights a critical need for a well tolerated, highly effective ebola vaccine that can rapidly elicit protection with a single dose . According to the world health organization (who) at the time of this writing (october 30, 2014) there have been 13,703 cases of ebola infection, 4,922 of which have resulted in death . It is estimated that more than 521 of the reported cases have been healthcare workers, the majority of which (99%) reside in countries with widespread and intense transmission, and that more than half (52%) of these people did not recover from infection . Even though this might be an underestimate of the actual situation due to delays in reporting of data and the rapid evolution of the outbreak, this represents a significant loss to an already understaffed and resource poor healthcare system in a region of extreme poverty and civil unrest . Considering that the who and other modeling experts have predicted that more than 20,000 new cases of ebola infection will occur by the end of november 2014 and that in the worst case scenario 1.4 million cases will be seen before the current outbreak ends, an effective needle - free vaccine would bolster the medical response and health care infrastructure of affected nations by allowing a large number of medical personnel to provide aid and immunizations to those under outbreak conditions without concern for their personal health . The overall goal of these studies was to identify an immunization platform that is easy to administer and capable of eliciting long - term protection from ebola infection . Using results generated in mouse and guinea pig models, two studies were designed to evaluate the clinical profile of a recombinant adenovirus serotype 5-based vaccine given as a single dose by respiratory or sublingual (sl) administration to non - human primates (nhp). The first study, conducted with 9 male cynomolgus macaques, was designed to identify a subtherapeutic dose for each route of administration to assist in identifying formulations that improved vaccine performance . The second study, conducted with 11 macaques, was designed to evaluate a novel formulation that improved the immunological profile of the vaccine after intranasal administration in rodents and to evaluate the longevity of the immune response conferred by each platform . A comprehensive assessment of the immune response elicited by each vaccine platform after exposure to ebola virus was performed in this study . Full toxicological profiles were generated for each animal after immunization in both studies . To our knowledge, this is the first evaluation of a needle - free ebola vaccine in primates and one of the first to demonstrate long - term protection from lethal infection . The e1/e3 deleted recombinant adenovirus serotype 5 vector expressing a codon optimized full - length ebola glycoprotein sequence under the control of the chicken -actin promoter (ad - cagoptzgp) and a host range mutant adenovirus serotype 5 (ad5mut) that can replicate in non - human primates were amplified in hek 293 cells and purified as described . Concentration of each virus preparation was determined by uv spectrophotometric analysis at 260 nm and with the adeno - x rapid titer kit (clontech, mountain view, ca) according to the manufacturer s instructions . Preparations with infectious to physical particle ratios of 1:37 were used in these studies . Buffers and reagents used in the production and purification of each virus preparation were of the highest quality available and were tested for the presence of endotoxin using a qcl-1000 chromogenic lal end point assay (cambrex bioscience, walkersville, md). All reagents contained less than 0.1 e.u./ml, and each virus preparation contained less than 0.2 e.u./ml . Sterility of each preparation was confirmed employing the methods outlined in the united states pharmacopeia for parenteral products . A two cell line bioassay was performed on each preparation to determine the presence of rca as described . Less than one rca was detected for every 3 10 virus particles tested . Non - human primate studies were conducted under a contract at bioqual inc ., the animal management program of this institution is accredited by the american association for the accreditation of laboratory animal care and meets nih standards as outlined in the guide for the care and use of laboratory animals . This institution also accepts as mandatory phs policy on humane care of vertebrate animals used in testing, research, and training . Twenty male cynomolgus macaques (macaca fascicularis) of chinese origin were allowed to acclimate for 30 days in quarantine prior to immunization . Animals received standard monkey chow, treats, vegetables, and fruits throughout the study . Husbandry enrichment consisted of commercial toys and visual stimulation . Specific details about the primates used in each of these studies are summarized in tables 1 and 2 . Animals were screened for signs of prior exposure to adenovirus (anti - ad5 nab, ad5 dna, t cell responses) 4 days prior to immunization . Samples were taken for evaluation of blood chemistry and adenovirus shedding (nasal and oral swabs, urine, feces) 6 h after immunization and on days 1, 2, and 7 . On day 20, serum and bal were collected for assessment of shedding and anti - ad5 nab and anti - ebola gp antibody levels . Bal, pbmcs, and ilns were also screened for ebola gp - specific cd8 and cd4 t cells at this time point . On day 38, additional samples were taken for assessment of anti - ebola gp and anti - ad5 antibodies and antigen - specific t cell proliferation (ebola gp and ad5). 42 days after immunization, nhps were shipped to the national microbiology laboratory in winnipeg, canada, for challenge . After an acclimation period, primates were challenged with 1,000 pfu of ebola (1995, kikwit) by intramuscular (i m) injection . Two animals were given the vaccine by intramuscular injection in a total volume of 1 ml of potassium phosphate buffered saline (kpbs) divided equally between the left and right deltoid muscles . Three animals were given the vaccine by the sublingual route by placing 50 l of the preparation under each side of the tongue and waiting for 15 min between doses to allow for absorption . This was achieved by slowly dispensing two 250 l volumes of the preparation into each nostril and waiting for 15 min between doses to allow for absorption . The remaining dose of the vaccine (5 ml volume) was instilled into the lungs via an endotracheal tube . This route of administration will be referred to as respiratory immunization or as intranasal / intratracheal (in / it) throughout the manuscript to illustrate that the vaccine was administered to the respiratory mucosa by two different routes . One primate was given 1 ml of kpbs divided equally between the left and right deltoid muscles . Blood was collected 6 h after immunization and on days 1, 2, and 7 . Full blood chemistry panels and complete blood counts were performed by idexx bioresearch (west sacramento, ca). Two animals (negative controls) were given 1 ml each of kpbs divided between the left and right deltoid muscles . The respiratory formulation was prepared at five times the working concentration [10 mg / ml poly(maleic anhydride - alt-1-octadecene) substituted with 3-(dimethylamino)propylamine (anatrace, maumee, oh)], sterilized by filtration, and diluted with freshly purified virus in kpbs (ph 7.4) prior to use . Three animals were given the vaccine in this formulation in the respiratory tract as described for study 1 . Three animals were given an adenovirus serotype 5 host range mutant virus to establish pre - existing immunity (pei) by i m injection 28 days prior to immunization with the vaccine by the sublingual route as described above . Three animals with no prior exposure to adenovirus were given the vaccine by the sublingual route for comparison . Animals were transported to the national microbiology laboratory in winnipeg and, after an acclimation period, transferred to the biosafety level 4 (bsl-4) laboratory there for challenge . Challenge studies were approved by the canadian science centre for human and animal health (cschah) animal care committee following the guidelines of the canadian council on animal care . For challenge, animals were infected by intramuscular injection at two sites with a total volume of 1 ml of freshly prepared ebola virus (strain kikwit 95, passage 3 on veroe6 cells) of an inoculum containing 1,000 times the 50% tissue culture infectious dose (tcid50) in diluent (minimal essential medium containing 0.3% bovine serum albumin). Ebola virus titers were confirmed (1.21 10 tcid50/ml) by back - titration of the challenge preparation following administration of the virus . Animals were monitored daily and scored for disease progression using an internal filovirus scoring protocol approved by the cschah animal care committee . The scoring system graded changes from normal in the subject's posture, attitude, activity level, feces / urine output, food / water intake, weight, temperature, and respiration and ranked disease manifestations such as a visible rash, hemorrhage, cyanosis, or flushed skin . Samples were taken for assessment of anti - ebola gp antibodies and full blood panels on days 3, 7, 14, 21, and 28 postchallenge and upon death . Hematological analysis of samples was performed in the bsl-4 lab with a horiba abx scil abc vet animal blood counter, and blood chemistries were analyzed with a vetscan vs1 (abraxis). Ifn- elispot assays were performed in triplicate according to the manufacturer s protocol (bd biosciences, san diego, ca) with 5 10 peripheral blood mononuclear cells (pbmcs) per well in crpmi media (rpmi 1640, 1 mm l - glutamine, 50 m -mercaptoethanol, 10% fbs and 1% penicillin / streptomycin). Cells were stimulated with three peptide pools for the ebola glycoprotein (2.5 g / ml) for 18 h. spots were visualized with the aec substrate (bd biosciences) and quantified with the elispot plate reader (aid cell technology, strassberg, germany). The frequency of cd8 and cd4 t cells producing ifn-, il-2, il-4, and cd107a were assessed by flow cytometry with the following antibodies: cd3 alexa fluor 700 (clone sp34 - 2) and cd4 peridinin chlorophyll protein (percp)-cy5.5 (clone l200) from bd biosciences (san jose, ca); cd8 phycoerythrin (pe)-cy7 (clone rpa - t8), cd107a brilliant violet 421 (clone h4a3), il-2 alexa fluor 488 (clone mq1 - 17h12), il-4 pe (clone 8d4 - 8), and ifn- allophycocyanin (apc, clone b27) from biolegend (san diego, ca). One million pbmcs were stimulated overnight with peptides (5 g / ml) using golgiplug (0.5 l / ml) and golgistop (0.6 l / ml) in the presence of the anti - cd107a antibody . After surface staining for cd3, cd4, and cd8, samples were incubated two times (30 min each) in cytofix / cytoperm (bd biosciences) for permeabilization . Intracellular staining was performed, and the samples were kept overnight in pbs/1% paraformaldehyde . Approximately 250,000500,000 events were captured on a bd lsr ii flow cytometer and data analyzed with flowjo vx0.6 software (tree star, ashland, or). Blood was collected from each primate in edta tubes, shipped same day and pbmcs isolated as described previously . Cells were resuspended in r10 medium (rpmi 1640, 2 mm l - glutamine, 50 m -mercaptoethanol, 10% fbs, and 100 iu / ml penicillin and streptomycin) and stimulated using either an ebola glycoprotein - specific peptide library (2.5 g / ml), a first generation adenovirus that is genetically identical to the vaccine but does not contain a transgene cassette (adnull, 1,000 moi), or 5 g / ml cona (sigma, st . After 3 days, cells were fed by removing 50 l of spent medium and replacing it with 100 l of fresh r10 medium . On day 5, cells were washed with phosphate buffered saline (pbs) for subsequent immunostaining for cell surface markers and for ki-67, an intracellular marker for proliferation as described . Proliferation was calculated by subtraction of values obtained from cells cultured in medium alone . Flat bottom, immulon 2hb plates (fisher scientific, pittsburgh, pa) were coated with purified ebola virus gp33637tm - ha (3 g / well) in pbs (ph 7.4) overnight at 4 c . One hundred microliters of each dilution were added to antigen - coated plates for 2 h at room temperature . Plates were washed 4 times and incubated with a hrp - conjugated goat anti - monkey igg antibody (1:2,000, kpl, inc ., gaithersburg, md) for 1 h at room temperature . Optical densities were read at 450 nm on a microplate reader (tecan usa, research triangle park, nc). Primate sera were heat inactivated at 56 c for 45 min and then serially diluted in 2-fold increments in dulbecco s modified eagle s medium (dmem) in triplicate prior to incubation at 37 c for 1 h with an equal volume of medium containing ebov - egfp (100 pfu per well) as described . Serum mixtures were then added to vero e6 cells and placed at 37 c for 2 days and then fixed in 10% phosphate buffered formalin . Gfp levels were quantified by a fluorescent plate reader (aid cell technology). These assays were performed under bsl-4 conditions at the national microbiology laboratory in winnipeg . Primate sera were heat inactivated and serially diluted as described for the ebola virus assay . Samples were incubated with a first generation adenovirus serotype 5 expressing beta - galactosidase for 1 h before they were added to hela cell monolayers . An equal volume of medium containing 20% fbs was then added to each well, and infections continued for 24 h. cells were then histochemically stained for beta - galactosidase expression as described . Positive cells were quantified by visual inspection with a lecia dm lb microscope (leica microsystems inc ., bannockburn, il). For both assays, the serum dilution that corresponded to a 50% reduction in transgene expression was calculated by the method of reed and muench and reported as the reciprocal of this dilution . Total rna was extracted from whole blood using a qiamp viral rna mini kit (qiagen). Ebola virus rna was detected by a qrt - pcr assay targeting the rna polymerase (nucleotides 16472 to 16538, af086833) and lightcycler 480 rna master hydrolysis probes (roche diagnostics gmbh, mannheim, germany). The reaction conditions were as follows: 63 c for 3 min, 95 c for 30 s, and cycling of 95 c for 15 s, 60 c for 30 s for 45 cycles with a lightcycler 480 ii (roche). Primer sequences for this assay were as follows: ebovlf2 cagccagcaatttcttccat, ebovlr2 tttcggttgctgtttctgtg, and ebovlp2fam fam - atcattggcgtactggaggagcag - bhq1 . Urine and bal fluid were concentrated using amicon ultra 100k centrifugal filter devices (millipore, billerica, ma). Dna was isolated from blood, concentrated bal, and oral and nasal swabs using a qiamp dna mini kit according to the manufacturer s instructions (qiagen, valencia, ca). Dna was isolated from rectal swabs using a modified protocol and the qiamp dna mini kit . Dna was extracted from the urine concentrate using a qiaamp viral rna mini kit (qiagen) according to the manufacturer s instructions . Dna was isolated from stool samples using a qiaamp fast dna stool mini kit (qiagen). Quantification of viral dna was determined by real time pcr according to a published protocol . Dna amplifications were carried out using a viia 7 real - time pcr system (life technologies, carlsbad, ca) with the following cycling conditions: 50 c for 2 min, 95 c for 10 min, 95 c for 15 s, and 62 c for 1 min for a total of 41 cycles . Primer sequences, used to amplify a region of the adenovirus serotype 5 hexon protein, were 5-act ata tgg aca acg tca acc cat t-3 (forward) and 5-acc ttc tga ggc acc tgg atg t-3 (reverse). The internal probe sequence, tagged with 6fam fluorescence dye at the 5 end and tamra quencher at the 3 end, was 5-acc acc gca atg ctg gcc tgc-3. Each sample was run in triplicate in a given pcr assay . Two primate studies are summarized here . The first, referred to as study 1, involved 9 male cynomolgus macaques and served to identify suitable doses of vaccine that were semiprotective for further evaluation of test formulations to improve survival in the nhp model . The second study, referred to as study 2, evaluated a novel formulation for the respiratory platform and involved refinement of the sublingual platform in naive animals and those with prior exposure to adenovirus . The workflow and treatment schedules for each study are depicted in figures 1 and 7 . Administration of the vaccine at a dose of 1.4 10 infectious virus particles (ivp)/kg to the respiratory and the sublingual mucosa was well tolerated with no adverse reactions noted . Of particular note is that all animals experienced a transient increase in serum phosphate levels 6 h after immunization with a primate from each treatment group falling outside normal values (22473, i m, 1.4 times normal, 50459, in / it, 1.2 times normal, 62125, sl, 1.3 times normal, figure 2a). Phosphate levels for all animals reached their nadir at the 24 h time point and were within the normal range for the remainder of the study . Blood urea nitrogen (bun) levels peaked for all animals 24 h after immunization . Two of these animals, one given the vaccine by i m injection (40347, 29 mg / dl) and another given the vaccine by the in / it route (52945, 33 mg / dl), had levels that were notably outside of the normal range (figure 2b). These values returned to normal by 48 h and remained so throughout the course of the study . Serum aspartate aminotransferase (ast), a standard indicator of adenovirus toxicity, was significantly elevated above normal values in all animals 24 h after immunization except for one animal given the vaccine by the sl route (62125) and another given the vaccine by the i m route (40347). Ast levels fell 48 h after immunization with only a few animals remaining above normal limits (figure 2c). Ast values for all animals were within normal limits by the 7 day time point . Serum alkaline phosphatase (alp) of two animals fell outside the normal range during the study . Samples from one animal given the vaccine by i m injection were only mildly over the normal acceptable limit (22473, figure 2d) while those of an animal immunized by the in / it route (52945) were 2 times the normal acceptable limit . In both cases, this parameter was high throughout the study and this elevation was not in response to the vaccine . Other serological parameters evaluated during the first week after immunization (calcium, creatinine, albumin, globulin, total protein, total bilirubin, alanine aminotransferase (alt), glucose, sodium, potassium, chloride, and cholesterol) all fell within normal limits during the course of the study . Study 1: clinical parameters evaluated over time in non - human primates immunized by various routes . Cynomolgus macaques were given a single dose of vaccine by i m injection or by the respiratory or the sl route . Each line represents alterations for each parameter during the course of therapy for one primate . In each panel: red lines / squares: saline control . Adenovirus shedding was also evaluated using a standard real time pcr assay to detect adenovirus genomes in serum, nasal swabs, bal fluid, oral swabs, urine, and feces (figure 3). A significant number of adenovirus genomes were found in the serum of one animal immunized by the respiratory route 2 days after immunization (50459, 1,452 genomes / ml serum, figure 3a) and another immunized by i m injection 7 days after treatment (22473, 7,296 genomes / ml serum). As expected, substantial amounts of adenovirus serotype 5 genomes were found in nasal swabs obtained from primates immunized by the in / it route (50459, 4.2 10, 52483, 1.4 10, 59245, 7.5 10) 24 h after immunization (figure 3b). Swabs from one primate immunized by the sl route also contained a notable amount of ad5 genomes (62361, 8,090) at the 24 h time point . Swabs from one animal immunized by the in / it route contained a significant amount of adenovirus genomes 2 days after immunization (52945, 6,333). Samples taken at days 7 and 20 fell below detection limits of the assay . Very low amounts of ad5 genomes were found in the bal fluid of animals immunized by the in / it route 20 days after immunization (figure 3c). Oral swabs taken 24 h after treatment from one nhp immunized by the in / it route (52483, 4.5 10, figure 3d) and two animals immunized by the sl route (62125, 4.9 10, and 62361, 9.3 10) contained significant numbers of adenovirus genomes . Swabs collected from animals at the 2 day time point did not contain any adenovirus genomes . A significant number of virus genomes were detected in the urine of 2 animals within 6 h after treatment (52945, 621 copies / ml, and 62361, 1,228 copies / ml). Adenovirus dna was also found 24 h after treatment in the urine of 3 animals (50459, 1,163, 62361, 801, and 62125, 116 copies / ml, figure 3e). Samples from all other animals throughout the time course of this study fell below detection limits of the assay . Interestingly, adenovirus genomes were only detected in the feces of animals immunized by the in / it route (figure 3f). As early as 6 h after immunization, 2,362 and 7,302 adenovirus genomes were found in fecal samples from animals 52483 and 52945 respectively . Feces collected from animal 50459 24 h after vaccination contained 5,919 adenovirus genomes . This increased to 7,405 in samples taken from the same animal at the 48 h time point . Samples from animal 52945 also taken 48 h after treatment contained 2,772 virus genomes . Study 1: adenovirus genomes are released predominantly in the nasal mucosa and feces after respiratory immunization and in the oral and nasal mucosa after sublingual immunization . Male cynomolgus macaques were given either 1 10 ivp by i m injection or 1 10 ivp by the respiratory or the sl route . Dna was isolated from each sample, and viral genomes were determined by real time pcr . Twenty days after immunization, pbmcs were isolated from whole blood and incubated with peptides specific for ebola glycoprotein (gp). Cells were then subjected to intracellular cytokine staining for cd8 and cd4 surface antigens and ifn- and sorted by flow cytometry . At this time point, few cells responsive to ebola glycoprotein could be detected in pbmcs obtained from any of the animals (data not shown). A similar trend was observed in samples taken from iliac lymph nodes (ilns) of animals . Profound responses were seen in samples obtained from the bal fluid of animals given the vaccine by the in / it route . The strongest response was seen in cd4 cells with 12.5% of the population obtained from primate 52945 and 3.03% of the population from primate 50459 responding (figure 4a). Although the response from the third primate in this treatment group (52483) was small in comparison (0.71%), it was significantly higher than that observed in animals given the vaccine by i m injection . Study 1: respiratory immunization induces strong antigen - specific t cell responses after administration of a single dose of a formulated adenovirus - based ebola vaccine . (a) quantitative analysis of ebola glycoprotein - specific cd4 t cells in bal fluid . Cells were isolated from whole blood 20 days after immunization and stimulated with a peptide library for ebola glycoprotein or peptides specific for the mhc class ii associated invariant chain peptide that binds the mhc class ii groove of cells (h - clip, negative control). Each cell population was stimulated for 5 h, stained for phenotypic markers, and analyzed by flow cytometry . (b) quantitative analysis of ebola glycoprotein - specific cd8 t cells in bal fluid . Cells were treated as described for panel a. (c) magnitude of the antigen - specific response of mononuclear cells isolated from whole blood of macaques . Pbmcs were isolated 20 days after immunization from whole blood and evaluated for ifn- secretion after stimulation with an ebola gp - specific peptide library by elispot . (d) magnitude of the antigen - specific response in mononuclear cells isolated from iliac lymph nodes (ilns) of primates . Mncs were isolated 20 days after immunization from ilns and evaluated for ifn- secretion after stimulation with an ebola gp - specific peptide library by elispot . (e) proliferative capacity of ebola gp - specific t cells collected 38 days after immunization of naive primates by various routes . The proliferative capacity of cd4 (white bars) and cd8 (black bars) t cells isolated from whole blood was evaluated for each animal by stimulation for 5 days with an ebola gp - specific peptide library and subsequent staining for ki-67, an intracellular marker for proliferation . (f) proliferative capacity of adenovirus serotype 5-specific t cells after immunization by various routes . Cells were isolated from whole blood 38 days after immunization and stimulated for 5 days with a first generation adenovirus that does not contain a transgene cassette (adnull, moi 1:1,000). The proliferative capacity of cd4 (white bars) and cd8 (black bars) t cells was determined by intracellular staining for ki-67 . Animal numbers displayed in each panel and their corresponding treatments are summarized in table 1 . Pbmc and iln populations were further analyzed for ifn- production in response to ebola gp by elispot . Samples from animals immunized by the i m route (22473 and 40347) both had significant numbers of ifn- producing cells (255 and 642 spot forming cells (sfcs)/million mononuclear cells (mncs) respectively, figure 4c). Pbmc samples from two nhps immunized by the sl route (52165, 62361) also had measurable numbers of ifn- producing cells (257 and 98 sfcs / million mncs). Samples from nhps immunized by the in / it route contained the highest numbers of ifn- producing cells (1,100, 607, and 2,055 sfcs / million mncs). Samples from the ilns of 2 nhps given the vaccine by the in / it route (50459 and 52945) contained approximately 7 and 18 times the number of ifn- producing cells found in the saline control (animal 22457) respectively (figure 4d). 38 days after immunization, the proliferative capacity of cd4 and cd8 cells in response to ebola gp and adenovirus serotype 5 was assessed by a ki-67 staining assay . Two samples, each obtained from animals immunized by the respiratory route, contained significant numbers of proliferative ebola gp - specific cd4 t cells (50459, 11.9%, and 52945, 6.5%, white bars, figure 4e). The sample obtained from nhp 50459 also contained the most ebola gp - specific cd8 t cells (8.8%, black bars, figure 4e). The sample from nhp 62125 immunized by the sl route contained the second highest amount of cd8 t cells (4.9%). All remaining samples contained approximately 34% cd8 t cells that could proliferate in response to ebola gp except for that from animal 52483 (1.1%). Only one sample obtained from a primate immunized by the in / it route, 52165, contained a significant population of proliferative adenovirus 5-specific cd4 t cells (8.1%, white bars, figure 4f). One sample from a primate in the in / it group (50459) and another from the sl group (62125) contained notable populations of cd8 cells that proliferated in response to ad5 (9.4 and 9.3% respectively, black bars, figure 4f). All remaining samples contained approximately 4% cd8 t cells that could proliferate in response to adenovirus except for animal 40347 (2.2%). Anti - ebola gp and anti - adenovirus antibody levels were assessed in serum and bal fluid 20 and 38 days after immunization (figure 5). Marked levels of anti - ebola gp igg antibodies were found in serum from animals immunized by the i m and the in / it routes 20 days after treatment (figure 5a). Anti - ebola gp antibodies were found in the serum of only one of the animals immunized by the sl route (52165). This animal also had ebola gp - specific igg antibodies in bal fluid 20 days after treatment (figure 5b) that were similar to those found in samples from animals immunized by the respiratory route . Bal from animals immunized by the i m route did not contain any detectable levels of anti - ebola gp antibodies . One sample from a primate immunized by the i m route (40347) contained a significant amount of circulating anti - adenovirus neutralizing antibodies (nabs, 1,007 reciprocal dilution, figure 5c). The sample from the remaining animal in the i m group and 2 others from the in / it group contained anti - adenovirus nab titers of 200 reciprocal dilution . Serum from animals immunized by the sl route did not contain measurable levels of anti - adenovirus 5 nabs . Study 1: respiratory immunization induces strong anti - ebola gp and minimal anti - adenovirus antibody responses in serum and bal fluid . These samples were screened for the presence of anti - ebola gp antibodies by elisa . Serum collected on day 20 was also screened for anti - adenovirus 5 nabs using an infectious titer assay (panel c). Data in panel c is reported as the dilution at which the infectious titer of a first generation adenovirus expressing the beta - galactosidase transgene was reduced by 50% . In each panel, error bars represent the standard error of samples assayed in triplicate from each primate for each time point . 62 days after immunization, nhps were challenged with 1,000 pfu of ebola virus (1995, kikwit). One primate immunized by i m injection (40347) and one animal immunized by the sl route (62125) succumbed to infection 6 days after challenge (figure 6a). At this time animal 62125 had a clinical score of 23, and substantial petechiae were noted upon necropsy . Primate 40347 had a temperature of 40.3 c and a clinical score of 25 and experienced notable bleeding . One primate immunized by the in / it route (52483) and one primate immunized by the sl route (62361) died the following day . Each of these animals had clinical scores above 25 and significantly decreased food intake the previous day . The remaining primate immunized by the sl route (52165) expired 8 days after challenge . One of the primates vaccinated by i m injection (22473) and two of the animals immunized by the in / it route (50459, 52945) survived challenge (50 and 67% survival i m and in / it respectively, figure 6a). A slight increase in weight of one animal immunized by the in / it route (50459) was noted during the study period . Changes in body temperature (figure 6c) and clinical scores (figure 6d) for each primate were in line with survival results . The most striking changes in hematology and blood chemistry values were observed around day 5 postchallenge in the animals that did not survive . These include significantly elevated liver enzymes with alt (figure 6e) and alp (figure 6f) values rising to levels 27 and 16 times baseline respectively and blood urea nitrogen levels rising to 7.5 times normal values before the animals expired (figure 6 g). Platelet counts, however, dropped to half the baseline values in these animals (figure 6h). In contrast, a sharp increase in platelets was noted in samples obtained from animals that survived challenge . Other hematology and blood chemistry values in these animals remained largely unchanged (data not shown). Naive male cynomolgus macaques (see table 1 for characteristics) were challenged 62 days after immunization with a lethal dose of 1,000 pfu (1,000 tcid50) of ebola virus (1995, kikwit). (d) daily clinical scores for each primate using a standard, approved scoring methodology throughout the challenge . Variations in serum (e) alanine aminotransferase (alt), (f) alkaline phosphatase (alp), (g) blood urea nitrogen (bun), and (h) platelets (plt) were noted in animals that did not survive challenge . The most exciting finding extracted from study 1 was that the combined in / it administration of the vaccine was able to confer long - term immunity to ebola . Since it was not known if immunity induced by adenovirus - based vaccines for ebola is persistent over time, we decided to extend the length of time between respiratory administration of a formulated version of the vaccine and challenge . A secondary goal was to increase the dose of vaccine given by the sublingual route and to evaluate the ability of the sublingual vaccine to confer protection in animals with prior exposure to adenovirus since improved responses in this population were observed in studies with rodents . The long - term immune response of surviving animals postchallenge was also a major point of interest in this study especially in animals receiving vaccine containing a novel formulation and in those given the sublingual vaccine to identify parameters to target during additional refinement of each immunization platform . Three male cynomolgus macaques were given the vaccine in a potassium phosphate buffer (ph 7.4) containing an amphiphilic polymer (formula weight (fw) 39,000) formulation that improved the antigen - specific immune response in rodent models of infection . The goal was to immunize this group as early in the study as possible so that there would be a significant amount of time between immunization and challenge (figure 7). 42 days after these animals were immunized, 3 macaques were given 1 10 particles of a host range mutant adenovirus serotype 5 that can replicate in non - human primates by intramuscular injection to establish pre - existing immunity . 42 days later, animals were then given the vaccine by the sublingual route . At this time the animals had an average circulating anti - adenovirus antibody titer of 320 160 reciprocal dilution . Three naive animals were also given the same dose of vaccine by the sublingual route at the same time for comparison . Eleven male cynomolgus macaques of chinese origin were immunized according to the schedule depicted in the figure . Animals were shipped to the national microbiology laboratory (nml) in winnipeg 126 days after immunization for challenge on day 150 of the study . Animals were screened for signs of prior exposure to adenovirus and ebola (anti - ad5 nab, ad5 dna, anti - ebola gp antibodies) 1 week prior to the initiation of the study . Samples were taken for evaluation of blood chemistry and adenovirus shedding (nasal, oral, rectal swabs, urine, feces) 6 h after immunization as well as on days 1, 2, and 7 . On day 20, serum and bal were collected for assessment of shedding, anti - ad5 nabs, and anti - ebola gp antibodies . On day 42, additional samples were taken for assessment of anti - ebola gp and anti - ad5 antibodies and antigen - specific t cell proliferation (ebola gp and ad5). 150 days after immunization, nhps were shipped to the national microbiology laboratory in winnipeg for challenge . In / it: intranasal / intratracheal . In contrast to the first study, a notable spike in creatine phosphokinase (cpk) was detected in the serum of all animals 24 h after immunization (figure 8a). This enzyme increased to 8 times baseline in one animal immunized by the in / it route (810003, 8,209 iu / l) and to 10 times baseline in a primate with pre - existing immunity to adenovirus immunized by the sublingual route (804819, 4,483). A notable spike in serum lactate dehydrogenase (ldh) was also noted at the 24 h time point . This was not as sharp as that seen with cpk with the highest elevations found to be approximately 3 times baseline (804317, 849 as seen in the first study, serum ast increased in all primates after immunization . This occurred at the 24 h time point for animals immunized by the respiratory and sublingual routes but was not observed in primates with pre - existing immunity to adenovirus until 48 h (figure 8c). As in the first study, serum alkaline phosphatase (alp) levels varied between primates, however, in this trial a distinct drop in this parameter was noted in samples collected from most animals between the 6 and 24 h time points, after which values remained constant (figure 8d). Other serological parameters evaluated during the first week after immunization (calcium, creatinine, albumin, globulin, total protein, gamma glutamyl transferase (ggt), total bilirubin, alanine aminotransferase (alt), bun, glucose, sodium, potassium, phosphate, chloride, and cholesterol) all fell within normal limits throughout the course of the study (data not shown). Study 2: clinical parameters demonstrating transient changes after immunization of naive non - human primates and those with pre - existing immunity to adenovirus immunized by various routes . Naive cynomolgus macaques were given a single dose of vaccine by the respiratory (in / it) or the sl routes . A separate group of animals first received a dose of an adenovirus serotype 5 host range mutant virus 42 days prior to immunization . Black lines / squares: sl immunization (primates with pre - existing immunity to adenovirus). Orange lines / diamonds: sl immunization (naive primates). Adenovirus genomes were only found in serum samples collected from animals immunized by the respiratory route (figure 9a). The most significant numbers of virus genomes detected in any of the biological samples collected throughout the second study were found in nasal swabs collected from primates 6 h after in / it immunization [810003 (8.18 10 genome copies (gc)), 809077 (1.44 10 gc), and 802197 (1.36 10 gc, figure 9b)] and in oral swabs collected from primates 6 h after sublingual immunization: [804317 (9.06 10 gc), 805257 (1.74 10 gc), and 808233 (7.92 10 gc, figure 9d)]. As seen in the first study, adenovirus genomes were only found in the bal fluid of animals immunized by the in / it route (figure 9c). Urine collected from one naive animal immunized by the sl route and another with pre - existing immunity also immunized by the sl route 6 h after treatment contained notable amounts of adenovirus (808233, 9,821 gc; 807243, 2,363 gc, figure 9e). Adenovirus genomes were found in feces collected from one primate with pre - existing immunity to adenovirus 24 h after immunization by the sl route (804819, 2.71 10 gc) and in another primate 2 days after it was immunized by the in / it route (802197, 6.51 10 gc, figure 9f). Virus continued to be shed in feces of this animal 1 week after immunization (802197, 2.52 10 gc). Adenovirus dna was found on rectal swabs collected from each animal throughout the course of the study (table 3). Study 2: adenovirus genomes are released in the serum and nasal mucosa after in / it administration of formulated vaccine and in the oral mucosa after sublingual immunization . Male cynomolgus macaques were given either 1.6 10 ivp / kg of vaccine in a formulation of 10 mg / ml poly(maleic anhydride - alt-1-octadecene) substituted with 3-(dimethylamino)propylamine by the respiratory route or 2 10 ivp / kg of vaccine in potassium phosphate buffered saline by the sl route . Dna was isolated from each sample, and viral genomes were determined by real time pcr . Animal numbers and corresponding treatments are outlined in table 2 data were obtained by real - time taqman pcr on dna isolated from samples as described . None detected . Sample fell below the detection limit of the assay (10 viral genomes/100 ng of dna). The ebola virus glycoprotein - specific t cell response was examined in pbmcs isolated from whole blood immediately prior to challenge, 150 days postimmunization . Multiparameter flow cytometry provided a comprehensive analysis of the types of antigen - specific t cells elicited by each treatment (figure 10). The cd4 t cell population present in animals immunized by the in / it route was much more diverse than the cd8 t cell population (figure 10a, b). Six specific cd4 t cell subpopulations were found in animal 802197 with the most predominate phenotype being cd4 cd107a il-2 (39% of the cd4 population, figure 10a). This animal also had the most diverse antigen - specific cd8 t cell population with 4 different subpopulations detected by intracellular staining (figure 10b). Cells that were cd4 il-2 were most prevalent (45%) in this primate . The cd8 population in this animal was composed of 3 specific subtypes with relatively equal distribution (cd8 cd107a il-2, cd8 ifn-, and cd8 il-2). The cd4 t cell population was less diverse in primate 810003 with the majority of antigen - specific cells also having the cd4 il-2 phenotype (85%). The cd8 il-2 subpopulation was the most prominent of two types of antigen - specific cd8 t cells found in this primate . Study 2: mucosal immunization elicits diverse populations of t cells capable of responding to ebola glycoprotein 150 days after treatment . Quantitative analysis of cd4 t cell populations secreting individual and combinations of cytokines in response to antigen stimulation after in / it administration (panel a), sl administration to naive animals (panel c), and sl administration to those with pre - existing immunity to adenovirus (panel d). Panel b reflects the quantitative analysis of cd8 t cell populations after immunization by the in / it route . Each positively responding cell was assigned to one of 8 possible categories reflecting the production of ifn-, il-2, and il-4 alone or in combination . Pie charts depict the variety of t cell populations found in each individual animal . Cd4 t cells were not found in samples obtained from primate 808233 (sl immunization). A single cd8 il-2 population was detected in samples from primate 804819 (pei - sl) and is not illustrated as a pie chart . Cd4 and cd8 t cell populations were noticeably less diverse in animals immunized by the sl route (figure 10c). Antigen - specific cd4 t cells were not detected in samples collected from primate 808233 . Cd4 ifn- il-2 cells were present to a lesser degree than cd4 ifn- cells in samples collected from animal 805257 (25% and 75% of the population respectively). The most diverse cd4 population elicited by sl immunization was found in primate 804317 with cd4 il-2 cells being the most prominent of 5 different subtypes identified in this population . Antigen - specific cd8 t cells were only found in samples collected from this animal with the majority being of the cd8 cd107a phenotype (92.6%) and the remaining cells of the cd8 il-2 phenotype (7.4%, data not shown). Pre - existing immunity to adenovirus did not noticeably alter the diversity of t cells elicited by sublingual immunization (figure 10d). Five distinct subpopulations of cd4 t cells were found in primate 809227 with those of the cd4 il-2 being the most prominent (63.1%). A single population of cd8 cd107a cells was also found in samples collected from this animal (data not shown). Cd4 il-4 cells were the most prominent of the two antigen - specific cd4 t cell populations found in samples collected from primate 807243 . Antigen - specific cd8 t cells were not detected in samples collected from this animal . Sl immunization induced a single population of cd4 il-2 cells and a single population of cd8 cd107a cells in primate 804819 . Anti - ebola gp and anti - adenovirus antibody levels were assessed in serum and bal fluid at various time points after immunization (figure 11). Antigen - specific antibody levels mildly increased between day 20 and day 104 in serum collected from two animals immunized by the in / it route (0810003, 1.5-fold increase, 0809077, 1.3-fold increase, 0802197, no change, figure 11a). Antibody levels remained high at the 142 day time point and were comparable to those found in animals immunized by the respiratory route in the first primate study . Significant anti - ebola gp antibody levels were detected in the bal fluid of only one primate immunized by the in / it route (0802197, figure 11b). Samples obtained from one of the animals immunized by the sublingual route (0808233) contained the highest level of anti - ebola gp antibodies than any of the other animals given a single dose of vaccine (figure 11c). It is also important to note that a significant change in anti - ebola gp antibody levels between day 20 and day 57 postimmunization was detected in samples obtained from only one animal in this treatment group (0805257, 2.4-fold increase). Samples from only one of the animals with prior exposure to adenovirus immunized by the sublingual route contained anti - ebola gp antibodies above the detection limit of the assay (809227, figure 11d). While a notable amount of anti - adenovirus neutralizing antibody (nab) was detected in the serum of one primate 20 days after immunization by the in / it route (802197, 1:640 reciprocal dilution), circulating anti - adenovirus nabs were low in samples obtained from other primates immunized in the same manner (figure 11e). Anti - adenovirus nabs were not found in the bal of any of the primates immunized by the in / it route during the course of the study (data not shown). While anti - adenovirus nabs were quite high in the serum of one animal with pre - existing immunity 20 days after immunization by the sl route (809227, 1:2,560 reciprocal dilution), they were not detected in samples collected from two naive primates immunized in the same manner (805257, 804317, figure 11f). Study 2: respiratory immunization induces production of antigen - specific antibodies that are sustained over time . Serum was collected from cynomolgus macaques immunized by the in route (panel a) on days 20, 104, and 142 after immunization and analyzed for anti - ebola gp igg by elisa as described . Serum was also collected from naive primates (panel c) and those with pre - existing immunity to adenovirus (panel d) on days 20 and 57 after immunization . These samples along with bal fluid (panel b) collected from all primates were screened for anti - ebola gp antibodies in the same manner . Serum from animals immunized by the in / it route (panel e) and from animals immunized by the sl route (panel f) was also screened for anti - adenovirus neutralizing antibodies . In each panel, error bars represent the standard error of samples assayed in triplicate from each primate for each time point . 150 days after immunization, animals were challenged with 1,000 pfu of ebola virus (1995, kikwit). Six days after challenge, both primates given saline, two animals immunized by the sl route (804317, 808233), and one animal with pre - existing immunity to adenovirus immunized by the sl route (809227) expired from infection (figure 12a). The remaining primates with pre - existing immunity succumbed to infection on days 7 (804819) and 8 (807243) respectively . The remaining animal given the vaccine by the sl route (805257) expired on day 9 . These animals experienced minimal changes in body weight (figure 12b) and temperature (figure 12c) during the course of infection with their clinical scores peaking at about 47 days after challenge (figure 12d). Study 2: a single dose of a formulated adenovirus - based vaccine protects from lethal challenge 150 days after immunization . Cynomolgus macaques given a single dose of 1.4 10 ivp of ad - cagoptzgp formulated with 10 mg / ml poly(maleic anhydride - alt-1-octadecene) substituted with 3-(dimethylamino)propylamine in phosphate buffered saline survived lethal challenge . Animals succumbing to infection experienced a change of 10% of body weight during the active infection period . Body temperature declined in each animal during challenge with the most dramatic drops observed in animals that were not protected from infection . Clinical scores were recorded for each primate by a blinded technician using a standard, approved scoring methodology . Lymphocytes of surviving animals recovered from an initial drop 3 days after challenge and remained stable throughout the remainder of the study . (f) elispot analysis of the cellular immune response in surviving animals 14 days after challenge . Pbmcs were isolated from whole blood and stimulated with a peptide pool spanning the ebola glycoprotein . Samples were collected from animals on day 3 and day 14 and at the time of death . (i) blood urea nitrogen (bun) profile of immunized animals during challenge . Blue lines / triangles: in / it immunization . Orange lines / diamonds: sl immunization . Black lines / squares: animals with pre - existing immunity to adenovirus immunized by the sl route . A notable drop in lymphocyte levels of all animals lymphocytes abruptly spiked in one animal immunized by the sl route (808233) and another with pre - existing immunity to adenovirus (804819) 6 days after challenge . Lymphocyte levels of primates immunized by the in / it route slowly increased to day 14 where they remained constant . Elispot analysis revealed that a significant amount of mncs capable of producing ifn- in response to stimulation with ebola gp peptides were present in pbmcs isolated from whole blood of surviving animals 14 days after challenge (figure 12f). A sharp drop in platelet counts was noted in all animals that did not survive challenge (figure 12 g). Mild drops in platelet counts were observed in animals immunized by the in / it route 3 days after challenge . Alt (figure 12h) and bun (figure 12i) sharply rose to values as high as 24 and 6 times baseline respectively in animals that succumbed to ebola infection . Assessment of sera taken during challenge revealed that primates immunized by the in / it route had very high levels of circulating anti - ebola gp antibodies (figure 13a). These were neutralizing since very low levels of infectious ebola were found in samples taken from two primates 3 days postchallenge (figure 13b). Infectious ebola virus was not detected in any samples collected from the third animal in this treatment group (809077). Ebola virus genomes were also not detected in samples taken from any of the animals immunized by the respiratory route (table 4). Although samples from two animals immunized by the sublingual route also contained high levels of anti - ebola neutralizing antibody (804317, 808233, 1,280 reciprocal dilution, figure 13c), they were only partially neutralizing since a concentration of 316 tcid50/ml was found in samples collected from both primates at the 3 day time point that escalated to 1.47 10 and 6.81 10 tcid50/ml respectively by the 6 day time point (figure 13d). The number of circulating virus genomes in these animals followed a similar trend (table 4). One animal that was exposed to the adenovirus serotype 5 host range mutant prior to immunization by the sl route (804819) also had high levels of anti - ebola gp circulating antibodies (1,280 reciprocal dilution, figure 13e), however, ebola virus rna was detected in samples collected from this animal at a concentration of 8.19 10 genome copies / ml (table 4). This animal expired before any infectious virus could be detected in its serum (figure 13f). Anti - ebola gp antibodies generated by a formulated adenovirus - based respiratory vaccine are neutralizing while those produced by an unformulated sublingual vaccine are partially neutralizing . The neutralizing capacity of antibodies in serum collected from each primate was assessed using a fluorescence neutralization assay (panels a, c, and e). The amount of ebola virus present in the serum of animals during challenge was determined using a standard infectious titer assay (panels b, d, and f). In each panel, data obtained from animals given saline prior to challenge with ebola are included as red symbols and lines for reference . Tcid50 = median tissue culture infectious dose 50 or the amount of virus that will produce pathological change in 50% of cells that are infected in culture . These assays were performed under bsl-4 conditions at the national microbiology laboratory in winnipeg . Data were obtained by quantitative rt - pcr on rna isolated from whole blood as described . Sample fell below the detection limit of the assay (86 viral genomes / ml). Units are genome copies per milliliter of whole blood (gc / ml). The ongoing epidemic in west africa is the largest ebola outbreak ever recorded and is rapidly crossing borders . In response to this public health crisis, the who has supported a movement to initiate small phase i clinical trials of vaccine candidates that have successfully prevented non - human primates from developing ebola virus disease after exposure to a lethal dose of the virus . One candidate, an attenuated vesicular stomatitis virus (vsv) that expresses the ebola glycoprotein in place of its own envelope protein, has shown efficacy in inducing both prophylactic and postexposure protection from infection . While this fast - acting platform clearly holds promise for people in high - risk settings who may have already been exposed to ebola, the longevity of the immune response elicited by the vaccine has only just been evaluated in rodent models of infection . A second vaccine candidate, a bivalent recombinant chimpanzee adenovirus serotype 3 virus expressing the glycoproteins of both ebola and sudan species, the most lethal of the known ebola viruses, is also undergoing clinical testing . This platform, which does not generate a protective immune response as rapidly as the vsv - based vaccine, requires supplementation with a modified vaccinia ankara virus also expressing the ebola and sudan glycoproteins to generate long - term protective immunity . Both of these vaccine candidates have been developed and are entering the clinic as injectable products . The ideal characteristics for an effective ebola vaccine should be greatly influenced by the population where infections are endemic . Long - lasting protection from ebola is necessary for at - risk populations (medical personnel) and for rural villagers where repeated prime - boost regimens are not feasible . Development of easy to administer, noninvasive immunization platforms eliminates the potential for transmission and spread of other blood - borne pathogens like hepatitis and hiv due to needle stick injuries that occur from unsafe practices during large immunization campaigns and from improper handling of biomedical waste . Establishment of mucosal as well as systemic immunity to ebola is also important since transmission of the virus occurs through direct contact of mucosal areas with body fluids of infected individuals . While the manner by which we delivered the vaccine to the respiratory mucosa may seem complex by traditional standards, the in / it dual route method was previously found to stimulate a significant igg response as early as 14 days after immunization, which is much earlier than that observed after intramuscular injection . Thus, we envision this approach to be quite attractive when rapid immune protection is desired especially in the case of first responders to an outbreak . Another important consideration is that of the prevalence of pre - existing immunity (pei) to adenovirus serotype 5 in the global population and the negative impact it might have on the potency of our vaccine . We have previously found that the performance of the unformulated vaccine when given by in / it administration to primates at a dose equivalent to that used in the studies outlined in this manuscript was not affected by pre - existing immunity to adenovirus 5 . However, survival was not complete as 25% of the naive animals and those with pre - existing immunity to adenovirus succumbed to infection . It is also important to realize that, in this case, the vaccine was not given alone but in combination with another adenovirus vector expressing interferon alpha, which may play a significant role in development of the antigen - specific immune response necessary for survival from ebola infection in animals with prior exposure to adenovirus . In contrast, respiratory administration of our formulated vaccine alone afforded full protection to all animals challenged at a much later time after immunization (figure 12). While this improvement in the absence of an immunostimulatory cytokine may suggest that our formulated vaccine may improve the potency of the vaccine in those with prior exposure to adenovirus, additional dose ranging studies in animals with pei to adenovirus are greatly warranted . Both the unformulated and the formulated vaccines given by the respiratory route were well tolerated by each primate with only mild, transient changes in a few blood chemistry parameters noted (figures 2 and 8). Shedding of the vaccine was also minimal as the adenovirus was fully cleared from the nasal passages, urine, and feces within 48 h after immunization (figures 3 and 9). Administration of the unformulated vaccine by the respiratory route induced notable systemic and mucosal ebola gp - specific t cell responses (figure 4) as well as anti - ebola gp - specific antibodies in the circulation and bal fluid (figure 5). To date, a considerable body of evidence indicates that, regardless of vaccine platform, a robust antibody response to ebola glycoprotein is essential for protection from lethal infection . Additional studies have shown that a strong antigen - specific t cell response is required to prevent the dysregulation of host protective immune responses during ebola infection and is supportive when the antibody - mediated response is suboptimal . These principles were illustrated by the primate given the respiratory vaccine that did not survive challenge in study 1 as the t cell response and the antibody mediated response were significantly lower in samples collected from this animal than in samples collected from other primates immunized in the same manner . For the past 20 years, there has been solid evidence in the scientific literature that heterologous prime - boost regimens are required for induction of long - lived protective cd8 t cells against a variety of microbial infections . As a result, there are very little if any data delineating the durability of the immune response generated by a single dose of a recombinant adenovirus - based vaccine since most of these platforms involve a priming dose of a recombinant dna plasmid containing an antigenic sequence similar to that encoded in a recombinant adenovirus used as a boost or the use of two different adenovirus serotypes expressing the same antigen . Detailed study of prime - boost regimens revealed that this approach elicited a diverse cd8 t cell population with a variety of immunological functions as defined by the production of two or more cytokines at one time and cell surface mobilization of the degranulation marker cd107a . While the presence of polyfunctional t cells has been shown to be important for protection in some infectious disease models, the role of polyfunctional t cells is not yet clear in the context of ebola infection . Previous studies in rodent models of ebola infection revealed that pre - existing immunity to adenovirus compromised the production of polyfunctional cd8 t cells, which was indicative of poor survival upon challenge . In the non - human primate model, it has recently been found that the presence of cd8 ifn-tnf- t cells correlated with survival while other studies suggest that polyfunctional cd4 t cells, especially those producing ifn- and il-2 in response to stimulation with ebola gp peptides, are important for survival . Data generated in our study best correlate with these latter studies as samples obtained from two of the primates receiving the respiratory vaccine that survived challenge contained highly diverse cd4 t cell populations and the most diverse cd8 t cell populations of all immunized animals (figure 10). However, one common cell surface marker / cytokine profile could not be detected among the surviving animals . To our knowledge, this is the first time that durable protection from a single dose respiratory recombinant adenovirus - based ebola vaccine has been demonstrated in non - human primates . The step and hvtn studies, which utilized an adenovirus serotype 5-based hiv vaccine, and some non - human primate data generated with recombinant adenoviruses have raised concerns that induced antigen - specific, vector - specific, or total cd4 lymphocytes at mucosal surfaces may lead to enhanced hiv-1 infection . Here, we show that respiratory administration of a recombinant adenovirus 5-based vaccine does induce strong cd4 t cell responses in bal fluid and in the periphery that are long lasting . In contrast, sl immunization did not foster production of antigen - specific cd4 t cells in bal but did support antigen - specific t cell responses in the periphery, which were not hampered by prior exposure to adenovirus . While it is not clear if these observations would have adverse impact in populations where there is currently a heightened need for an ebola vaccine and where hiv is quite prevalent, the fact that adenovirus genomes were detected from rectal swabs taken from animals at 7 and 20 days should be further evaluated . Because these samples were contaminated with residual fecal matter, screening of rectal biopsies taken 104 days after immunization for signs of inflammation and cellular activation one very important point illustrated in the second primate study is that the antibody response generated by a given vaccine platform may not be fully neutralizing . This was somewhat evident in the first primate study as two animals with what appeared to be strong ebola gp - specific antibody responses as determined by an elisa assay did not survive challenge (figure 5 and 6). Tcid50 and quantitative rt - pcr assays performed in the second study provided solid evidence that antibodies generated after immunization by the sl route could not effectively neutralize the virus (figure 13). The highly diverse ebola gp - specific cd4 t cell populations found in one naive animal immunized by the sl route and another with pre - existing immunity could not compensate for the poor antibody - mediated immune response generated by this immunization strategy . While these results were disappointing, it is important to realize that the primary goal of this project was to develop an adenovirus - based vaccine that could be given by either the respiratory or the oral route . During development of the oral platform, we discovered that sublingual administration of the vaccine could elicit protective responses to rodent - adapted ebola . Recently, studies evaluating this route of immunization have gained presence in the scientific literature with promising results for a variety of pathogens . The respiratory platform is attractive for use in areas where there is a dire need for ebola vaccines and therapeutics because it allows for self - immunization in a needle free capacity . However, devices required for instillation into the nose and lung can be bulky, require some skill for proper use, and may require refrigeration for storage . Many of the dosage forms used for sublingual delivery are compact and can stabilize compounds at ambient temperature . Thus, refinement of this platform for adenovirus - based vaccines and other ebola vaccine candidates is warranted.
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A 17-year - old male was referred to emergency department immediately after a gsi . On arrival, he was conscious, the vital signs were within normal limits, and no neurological deficit was noted . The right globe was ruptured and light perception was negative on the right eye . On physical examination, a single bullet entry hole on the right posterior scapular area was detected [fig . (a) particular appearance of skin wound including small contusion, skin introflection, and simple ecchymosis with frayed margins in the right posterior scapular area . (b and c) the preoperative appearances of the globe in emergency and operation room the only detectable exit wound was the right orbit . The route of the bullet was identified by computed tomography (ct) scans obtained in several projections and signs of the damage along the path of the bullet entering from the right scapular region and leaving the body from the right orbit were confirmed [fig . Identified route was; entrance from the right posterior scapular region, passing neighboring to right lung, moving upward to the cranium by side of the carotid artery and the vein, fracturing lateral and posterior wall of the maxillar sinus, entering the orbit fracturing the orbital floor, and leaving the body through the orbit perforating the right eye [fig . Orbital computed tomography, sagittal section; along the path of the bullet, there were signs of emphysema and hematoma in the soft tissue and at the right parapharyngeal, the masticator and the inferiotemporal muscles due to penetrating injury . Furthermore, bone fracture fragments were observed along this path, especially in the lateral and the posterior wall of the maxillar sinus primary reparation under general anesthesia was performed . However, as the double perforation was so severe no postoperative visual function was preserved . Unusual presentations of bullet trajectory in gsi can create surgical and/or medico - legal diagnostic problems . Since the face and neck region is packed with the vital structures in a relatively small volume of space, even the smallest of movements by a penetrating missile may injure a major vein, artery, and main nerve trunk simultaneously . Moreover, especially the injuries to the neck and maxillofacial region could end with high morbidity and mortality . Gsi to the orbit, especially the ones penetrating the globe could have devastating effects on all intra- and peri - orbital structures . As the bullet has both forward and rotatory movements, it possesses much higher amounts of kinetic energy to cause more damage in the eye . The energy is dissipated as the bullet slows within the soft tissues or the orbit . High - velocity injuries also cause secondary damage due to the fragmentation of bone, which is shattered by the missile on impact and enhance the injury . Nature and severity of the damage and preservation of visual function depends on the direction of impact and the part of globe involved . In case of perforating injury, loss of vision is common as in our case or even loss of eyeball and late enophthalmos in many cases . Primary evisceration may be needed in cases with severely ruptured globe if reconstruction is not possible . On the other hand in closed injuries, globe concussion, retinal detachment, optic nerve avulsion, or chorioretinal lacerations some unusual routes of bullet in gsi are reported in the literature . In these awkward injuries, the prediction of the trajectory is very difficult without additional radiological investigations . Especially in case of any high velocity projectile wounding, the physician must be aware of the fact that the bullet's course will not be a linear but most probably a complicated one . The entry wound and the exit wound should be both carefully explored . Over - concern with the entry wound ct is the procedure of choice to detect any hemorrhage, air, bullet, bone fragments, hemothorax, nerve lesion, musculoskeletal lesions, and vessels injuries . Ct imaging is also useful for assessing the missile path and the anatomical structures at risk . Prognosis of the injury depends on the course of the bullet or shrapnel fragment and the multidisciplinary team approach . Moreover, even the crime investigation might be enlightened by the demonstration of the bullet's route . Herein, a very unusual route of a bullet entering from the scapular area, passing through the neck and ending with eye perforation is reported . The accurate detection of entrance and exit wounds, path and extent of tissue damage were difficult . Although the area pierced by the bullet was rich in neurovascular structures - many of which are extremely important - by chance the patient did not suffer any life - threatening injury . As the dynamics of the shot was investigated, he was thought to be injured with a trajectory from below to above while he was running away from the gunshot . The knowledge of the path of the missile, an open - minded approach, interdisciplinary care, and close clinical observation of the patient are critical for the assessment of management in atypical gunshot wounds.
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A 26-year - old man was admitted to the emergency department with a 24-hour history of diffuse abdominal pain that had started in the epigastric area, then localized at the right lower quadrant (rlq), followed by nausea and vomiting . Physical examination revealed a mcburney incision scar . Tenderness and rebound tenderness were noted in the rlq during palpation . White blood cell (wbc) count was 17400 cells / mm with a neutrophil percentage of 78%, whereas c - reactive protein (crp) was in normal reference ranges . Contrast - enhanced computed tomography (cect) scan of abdomen and pelvis showed pericecal free pelvic fluid, cecal inflammation and inflammatory changes in the rlq with a dilated tubular structure extending from the base of the cecum (figure 1). Yellow arrow: periceceal free pelvic fluid, cecal inflammation; red arrow: right lower quadrant with a dilated tubular structure (stump appendicitis). After adhesiolysis, a remnant suppurative appendiceal stump 5 cm . In size was noted . The postoperative period was uneventful and the patient was discharged on the seventh postoperative day . Histopathological examination confirmed stump appendix 5 cm in size with features of local peritonitis (figure 2). On surface epithelium ulceration, all the layers of appendix wall leukocyte infiltration (hematoxylin and eosin 100). Most patients diagnosed with stump appendicitis present with typical symptoms and findings of acute appendicitis, including pain that starts periumbilically and migrates to the rlq with anorexia, nausea and vomiting . Leukocyte count and crp levels tomography scan is more useful than ultrasound to diagnose stump appendicitis, as ultrasonographic findings are not characteristic [2, 4]. Cect scan can reveal findings that support the diagnosis of stump appendicitis, such as inflammatory changes in pericecal region, thickening of cecal wall, abscess formation, presence of fluid in right paracolic area, and air - filled tubular structure [5, 6]. Clinical diagnosis of stump appendicitis may be difficult due to underlying conditions like mental retardation, pregnancy, immune suppression and steroid use . Medical history of appendectomy can also lead to delay or even missed diagnosis of stump appendicitis . Therefore, stump appendicitis should be considered in differential diagnosis of patients with acute abdomen indication, appendectomy or mcburney s incision scar . Cecal diverticulitis should also be considered in differential diagnosis of stump appendicitis since cecal diverticulitis is clinically indistinguishable from acute appendicitis . It has been reported that almost 70% of patients with cecal diverticulitis underwent surgery based on preoperative diagnosis of acute appendicitis, and correct preoperative diagnosis was made in only 5.3% of 318 patients . It is also reported that time interval from initial appendectomy to stump appendectomy may vary from 2 months to 50 years [1, 9]. In the present case, rate of perforation for stump appendicitis (detected during surgery) approaches 68% and length of hospital stay increases due to delayed diagnosis . Appropriate stump length to be left after appendectomy is 35 mm to prevent stump appendicitis . Stump appendicitis has been reported after both open and laparoscopic appendectomy; there is very little difference between various surgical techniques in terms of increase in incidence of stump appendicitis . In both open and laparoscopic appendectomy, a longer stump can be obstructed with fecalith, which may lead to chronic inflammation causing ischemia of appendiceal wall and eventually perforate and/or suppurate . While some authors do not support this idea, incidence of stump appendicitis has increased relative to the increase of laparoscopic appendectomy . In an open or laparoscopic approach, careful appendix artery dissection of taenia coli appendiceal cecal junction identification is very important, especially for subserous appendix . In the present case, retrospective examination of patient s medical records of first appendectomy revealed multiple abscesses and adhesions in right iliac fossa (rif). It is important to understand that a history of appendectomy is, by itself, insufficient to exclude diagnosis of appendicitis . Presence of mcburney scar may be a warning for surgeons to consider stump appendicitis during an emergency examination . Identification of appendiceal base by tracing taenia coli to appendix is very important to prevent stump appendicitis . Appendiceal stump of less than 5 mm in length can minimize incidence of stump appendicitis . Careful evaluation of clinical and computed tomography (ct) scan findings may prevent delay in diagnosis, and decrease morbidity and length of hospital stay.
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As in other countries worldwide, the number of traffic accidents in turkey is increasing each year, from 500,664 in 2000 to 1,034,435 in 2010 . The high number of traffic accidents yielding injuries and fatalities makes them of great importance to emergency departments (ed). It is thought that by determining common injuries resulting from accidents and by taking the necessary precautions, morbidity and mortality could be reduced . The material damage resulting from accidents has a strong adverse effect on a country s economy . Physicians who are aware of the costs of traffic accidents will be able to take a more cost - effective approach to trauma . By retrospectively evaluating the files of patients who presented at hacettepe university medical faculty adult ed because of traffic accidents between 2000 and 2010, this study aimed to determine the epidemiology of traffic accidents and to analyze the costs . It was determined that between 1 january 2000 and 31 december 2009, 3712 patients presented at hacettepe university adult ed following traffic accidents . For all the patients included in the study, a record was made of various demographic and epidemiological characteristics such as age, gender, arrival time, arrival condition, time to arrival at hospital, presence of any life - threatening condition, glasgow coma score, revised trauma score (rts), findings and results, outcomes (admittance to hospital, self - discharge, death), and length of stay (los) in the ed . Injuries were evaluated separately as head, thorax, abdomen, extremities and other injuries . The details of costs pertaining to that date with the patient file number were recorded . Mean costs per capita were calculated separately for each year, and then the mean cost for each year was converted to american dollars according to the mean exchange rate for that year . Differences between groups were evaluated by kruskall - wallis, mann - whitney, and wilcoxon tests according to variable type and parametric test assumptions . The study comprised an examination of 2003 patient files that could be accessed from a total of 3712 patients who presented at hacettepe university medical faculty ed following traffic accidents between 1 january 2000 and 31 december 2009 . The patients included in the study were 901 females (45%) and 1102 males (55%) with a mean age of 39.6816.15 years (range 1593 years). When the years were examined, the most presentations following traffic accidents were in 2004 with 307 (15.4%), followed by 2005 with 287 (14.3%). The highest incidence was seen in the months of may, july, and june, with 217 (10.8%) in may and the lowest number of presentations was in february and march at 120 (6%) for each month . When the distribution of cases was calculated according to season, it was determined that the most of 28.4% of presentations occurred in the summer months . In respect of the time of presentation, it was determined that the most presentations (n=671, 33.5%) were between 6 pm and 12 am (midnight) (table 1). In the evaluation of the time taken to reach the emergency department, it was determined that 51.5% presented within the first 30 minutes after the accident, 76.5% within the first 60 minutes, and 88.7% within the first 2 hours (table 2). Most patients (1675=83.6%) arrived at the ed by ambulance . According to the manner of injury in the patient records, 1907 (95.2%) were from motor vehicle accidents (table 3). The most frequently seen trauma was head trauma (18.3%), followed by extremities (16.7%), thorax (7.3%), and abdominal trauma (3.5%). Distribution of the outcomes according to the age, glascow coma scale(gcs) and revised trauma scores(rts) were given in table 6 . The results as to whether or not there were life - threatening conditions according to the forensic reports written in the ed are given in table 8 . Mortality occurred in 7 patients (0.4%) who had been given a non - life - threatening report . The costs of 1998 patients in this study were evaluated and the costs of 5 patients were not evaluated . The mean cost was found to be 983.54364.4 tl; minimum 3 tl, maximum 84,941 tl . The costs for each year were converted to american dollars using the mean exchange rate for that year . The study examined 2003 records that could be accessed from a total of 3712 patients who presented at hacettepe university faculty of medicine emergency department following traffic accidents between 1 january 2000 and 31 december 2009 . The patients were 901 females (45%) and 1102 males (55%), which is similar to the sex distribution found in the literature . The age of patients in the study ranged from 15 to 93 years, with a mean age of 39.6816.15 years . Reported a mean age of 35.814.3 years, marmor et al . Reported 27 years, and etinolu et al . Reported 35 years [36] this mean age of traffic accidents affects the young and productive population; therefore, in addition to financial loss, there is thought to be a loss to the workforce and daily functioning . When the number of traffic accidents was examined according to year, the lowest number was 83 (4.1%) in 2000 . This result may be due to a lack of records in the past and it is thought that positive steps have been taken over the years in improving recording systems . Highest numbers of presentations occurred in may, july, and june . In a study by varol et al ., the most accidents occurred in august (17.9%). The observation that most traffic accidents occur in the summer months agrees with data in the literature . This may be due to evening rush - hour traffic congestion, limited visual fields at night, and human error due to tiredness, lack of attention, and alcohol intake . The hours of most presentations were found to be 6 pm to 12 am (midnight) in a study by aygencel et al ., 12 pm (noon) to 6 pm by beyazta et al ., and 3 pm to 7 pm by mishra et al . . In the current study, 1607 patients (83.62%) were brought to the ed by ambulance . As 52.5% in a similar study . In a previous epidemiological study of trauma in turkey, patient transfer by ambulance the increase in this rate is thought to be due to use of the 112 ambulance system having become effective and public awareness of first aid and emergency procedures . The ed was reached within the first 30 minutes after the accident by 51.5% of the patients in the current study . In a study by wong et al ., the mean period before reaching hospital was 28 minutes . The period before reaching hospital is known to be significant for mortality and morbidity . Of the patients in the current study, the mortality rate associated with the motor vehicle accidents was 46 (2.4%) and 1 (1.6%) with motorcycle accidents . Eken et al . Showed a mortality rate in motorcycle accidents of 5.7% . In a study by mon et al ., the injury and mortality rate of motorcycle accidents was 3.5 times higher than that of other motor vehicle accidents . The result of the current study is at the expected level as there is widespread motorcycle use in ankara . The most frequently determined pathology was head trauma (18.3%), followed by extremity trauma (16.7%), and these results agree with the literature . Taking these results into consideration, precautions giving protection in the event of an accident should be considered a priority in motor vehicle safety equipment . In a study by weninger et al ., head trauma occurred in 66% of patients, thoracic trauma in 55%, abdominal trauma in 55%, spinal trauma in 27%, at least 1 long bone fracture in 70%, and multiple fractures in 51% . A reason that these rates are higher than those of the current study could be that only high velocity motor vehicle accidents were included in that study . In the current study, 1639 patients (81.8%) were discharged from the ed, 248 (12.4%) were hospitalized, and 48 (2.4%) died . Reported that 89.7% of patients were discharged from the ed, 8.4% were hospitalized, and 1.1% died . It is necessary to have monitoring in different departments of patients who have been stabilized by initial interventions, both in terms of patient morbidity and ed turnover . In the current study, the average los in the ed was 4031416 minutes, with a median of 180 minutes . In a study by mishra et al ., 10% of cases spent less than 1 hour at the hospital, 41.6% spent 16 hours, and 48.3% spent more than 6 hours . In the current study, only los in the ed was calculated and the total los in the hospital was not included . This excessive period and the low rate of hospitalization may be associated with a bed shortage in our hospital . In the current study, this is an expected outcome due to factors such as comorbidities and weaker compensation mechanisms in the elderly . Traffic accidents constitute the great majority of criminal cases that present at the ed . Of criminal cases presenting at the emergency department, in the current study, 7 patients (0.4%) died who had been given an emergency department report that there was no life - threatening condition . In the light of this result, it is thought that it is necessary to be very thorough in the preparation of forensic reports, and physician training on the writing of such reports should be reinforced . Annual losses to the turkish economy of 350450 million dollars are caused by traffic accidents . In the current study, the mean cost per capita was found to be 9834364 tl (7553357 dollars). If labor force and functional losses are considered, this amount could be much higher . 1910 pounds sterling by morris et al ., and 3600 euros by jacobs et al . . When compared with these studies, the mean cost of the current study was lower, which may be due to differences in economic status and costs of healthcare in various countries . Removing unnecessary tests would reduce costs, but the basic solution is to increase preventive measures against traffic accidents and driver education must be improved.
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Copper is present in a large number of enzymes, which are involved in electron transfer, activation of oxygen, and other small molecules as well as superoxide dismutation [1, 2]. It serves as an essential cofactor for a variety of proteins involved in neurotransmitter synthesis as well as in neuroprotection via the cu / zn superoxide dismutase . Copper in excess is toxic while cu deficiency can lead to serious disease [35]. Copper ions have also showed antimicrobial activity against a wide range of microorganisms [68]. The determination of trace amounts of copper, because of its importance in health, medical, and industrial processes, is of great interest to analytical chemists . The most common techniques are icp - ms [9, 10], atomic absorption [11, 12], capillary electrophoresis, and uv / vis spectrophotometry [1416]. In addition, some methods use organic solvents such as chloroform or acetonitrile which are for the issue of environmental concern or worldwide storage crisis . Among these techniques, visible absorption spectrophotometry represents the most convenient technique because of the availability of the instrumentation, simplicity, speed, precision, accuracy, and low cost . As it was conducted in our previous studies, 6-naphthyl and anthracenyl substituted, 1,2,4-triazine-3-thione form colored complexes with cu(ii) in basic media [1720]. In this investigation the synthesis of 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thione has been synthesized as a chromogenic reagent for the determination of cu(ii) and validation of the developed method is reported . All spectra recordings and absorbance measurements were carried out on a shimadzu, 160a uv / vis spectrophotometer . Atomic absorption (nmr spectra were recorded on a bruker ft-500 spectrometer (bruker, rheinstetten, germany) with tetramethyl silane (tms) as internal standard . 2-methoxynaphthalene, thiosemicarbazide, amylnitrite, aluminium chloride, and acetyl chloride were used for ligand synthesis and were purchased from fluka (switzerland) or merck chemical companies . Solvents (acetone, acetonitrile, ethyl alcohol, and methanol) were of hplc grade and prepared from merck (germany). Copper nitrate solution . A stock standard solution of 1 mg / ml cu(ii) was prepared by dissolving 0.5 g pure elemental copper in hot concentrated hno3, cooling, and adjusting the volume to 500 ml by addition of the distilled water . Acetyl chloride (0.4 g) and alcl3 (0.67 g) were mixed in a mortar in dry condition and then 2-methoxynaphthalen (i) (0.5 g) was added and mixed in oxygen protected condition . The product was re - crystallized from ethanol - water to yield 0.454 g (72%), mp . C nmr (125 mhz, cdcl3) was 32.68 (ch3co), 55.36 (och3), 112.71, 123.58, 124.05, 128.65, 128.13, 128.78, 130.37, 131.44, 133.31, 153.91, and 205.22 . Was then refluxed with amyl nitrite in ethanol in presence of sodium ethoxide for 48 h in dry condition . The product (iii), 2-methoxynaphthylglyoxal aldoxime, was then extracted with diethyl ether, dried, and crystallized from water - ethanol . The reaction product (iii) and thiosemicarbazide were refluxed for 3 h in dilute hydrochloric acid . The progress of the reaction was monitored by tlc using a mixture of chloroform and methanol as a mobile phase . The reaction mixture c nmr (125 mhz, dmso) was 57.04 (och3), 114.25, 123.56, 124.45, 128.03, 128.56, 131.21, 131.26, 132.48, 154.85, 158.53, 173.76, and 188.74 . H nmr (500 mhz, dmso) was 3.87(s, 3h, ch3), 7.387.56 (m, 3h), 7.577.62 (m, 2h), 810 (s, 1h), and 833 (bs, 1h, nh). Mass m / z (%) was 269 (7), 241 (5), 239 (20), 165 (10), 153 (20), 151 (100), and 138 (4). General procedure for determination of cu(ii). In a series of 5 ml volumetric flask, 1 ml (2.520 g / ml) of cu(ii) and 2 ml of mndtt (0.002 m) were taken and the volume was adjusted to 5 ml with methanol . The absorbance of the solutions was recorded against reagent blank at 475 nm in a 1 cm quartz cell . 40 mg of an amalgam (110-plus) containing ag(i) (45%), sn(ii) (30%), and cu(ii)(25%) was taken in a 50 ml beaker and 10 ml hno3: hcl (1: 1, v / v) solution was added . The mixture was heated at 120c for 30 min until the dissolution is completed and the resulting solution reaches minimum volume . The solution cooled and transferred to a 100 ml volumetric flask and diluted to mark with double distilled water . The reagent 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thione (mndtt) was synthesized, due to our recent studies for preparing more sensitive chromogenic reagents, for determination of trace amount of some cations such as cu(ii), ni(ii), and hg(ii). To achieve more sensitivity, complexing moiety, 1,2,4-triazine-3-thione ring, different solvent systems (acetonitrile, water, 0.1 m naoh, chloroform, ethanol, and methanol) were examined to find out more suitable solubility, better absorption spectra, and greener solvent . The results showed that the reagent, mndtt, was not soluble in chloroform and water . Both the reagent and cu - mndtt complex were soluble in 0.1 m naoh, acetonitrile, ethanol, and methanol . Acetonitrile not only had lower sensitivity but also was not considered as an environmental and health friendly solvent [25, 26]. Eventually, comparison of the absorption value of cu - mndtt complex in methanolic and ethanolic media showed that the most suitable solvent for both reagent and complex was methanol (table 1). Therefore, methanol which had greater absorbance value and was also considered as one of the greener solvents was selected for subsequent experiments . 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thion, which is synthesized as a new reagent, reacts with cu(ii) forming a red - colored complex in methanol . The absorption spectra of cu - mndtt complex versus the blank and the ligand (mndtt) in methanol was recorded in the wavelength region of 200800 nm (figure 2). The ligand shows a maximum wavelength at 346 nm while the spectrum of cu - mndtt reveals a maximum at 475 nm which increased as a function of cu / mndtt molar ratio according to the curve reported in figure 3 . The influence of ph on the complex formation using britton - robinson buffer (ph = 512) and 1 m naoh was studied by measuring the real absorbance of the solution containing cu(ii) (20 g / ml) in the presence of the reagent mndtt against the reagent blank . The results illustrated in figure 4 reveal that maximum absorbance of the colored complex was obtained at ph = 12 . Comparing the results obtained in the absence of buffer (table 1) with the above values reveals that the absorbance of the produced complex decreased in the presence of aqua buffer solution . The effect of cationic (cetrimide), anionic (sodium lauryl sulfate), and nonionic (tween 80) surfactants was studied and results showed that surfactants caused turbidity in solution in different surfactant concentrations ., the absorbance of a 25 g / ml solution of cu(ii) at the optimum condition was recorded over a period of 3 h with an interval of 30 min, after 24 and 48 hours . The results showed that the complex was completely stable for at least 3 hours and there were no significant changes (<3%) in the absorbance of the complex after 24 hours . . The chemical structure of cu - mndtt complex was determined by limiting logarithmic method . As it is shown in figure 5 the logarithm of absorbance intensities of cu - mndtt complex versus log [cu] 1.57 104.68 10 m) at fixed concentration of mndtt (5.58 10 m) and log [mndtt] (5.75 101.72 10 m) at fixed concentration of cu(ii) (1.57 10 m) the values of the slopes of these lines were 0.8463 and 1.586, confirming the 1: 2 ratios for the complex formation reaction . The opposed method was calibrated using 6 series in the range of 2.520 g / ml . The analytical parameters of the proposed method are given in table 2 . The detection limit (3 sd / k) and quantification limit (10 sd / k) (where sd is the standard deviation of the y - intercept and k represents the slope of the straight line), as defined by iupac, were found to be 0.33 and 1.10 g / ml, respectively . The accuracy and precision of the proposed method were determined at three different concentrations within the same day (n = 3) and over three different days (n = 9). Percentage relative standard deviation (rsd%) as precision and percentage relative error (er%), which shows accuracy of the suggested method, was less than 3.68% and 2.40%, respectively . The effect of diverse ions was determined using a standard solution containing 20 g / ml of cu(ii) and 20 g / ml of the studied ions . The method was completed according to the general procedure and the absorbance value was obtained against the reagent blank at 475 nm . Similar to the results obtained for the naphthyl derivative of 1,2,4-triasine-3-thione (6-(2-naphthyl)-2,3-dihydro-1,2,4-triasine-3-thione) obtained in previous study, the new reagent had no interference with fe(ii), ba(ii), mg(ii), ca(ii), cd(ii), co(ii), mn(ii), and sr(ii). Ni(ii), hg(ii), and pd(ii) ions form complexes with mndtt which have maximum absorbance between 400 and 500 nm . As there was no suitable masking agent for these ions, derivative spectrophotometric method has been recommended for analyzing cu(ii) in the presence of ni, hg, or pd ions . Application of the method to real sample . In order to evaluate the analytical applicability of the proposed method, it was applied for the determination of cu(ii) in a dental amalgam 110-plus . The results and recoveries presented in table 4 indicate the percentage of recovery 97.5 and 96.5 for spectrophotometry and atomic absorption method, respectively . Using the two - tailed t - test and f - test methods, it was revealed that there was no significant difference between the results obtained from these two methods (p value> 0.05). Relative recovery . The relative recovery was determined using the standard addition method (n = 3). The percent relative recovery of 101.66 1.24 indicates that no interference with other components in amalgam has been observed . A simple, rapid, sensitive, and accurate method for determination of cu(ii) using the newly synthesized reagent, 6-(2-methoxynaphthyl)-2,3-dihydro-1,2,4-triazine-3-thion, was developed . Comparison of characteristic features of some spectrophotometric methods reported earlier for the determination of copper reveals the suitability of the present work in terms of molar absorptivity, linear range, interferences, lod, and so forth (table 5). The use of mndtt as a complexing reagent was utilized for nonextractive determination of cu(ii) in dental amalgam . Additionally, the method was much safer, since only a small amount of methanol was used which is considered as green chemistry for determination process . Therefore, the proposed method can be recommended for pharmaceutical and industrial samples.
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Tracheobronchopathia osteochondroplastica (to) is a rare benign airway disease typically characterized by the presence of multiple rock - garden - like nodules in the lower trachea and upper main bronchi (1). Because of the absence of cartilage in this region of the airway, these nodules involve the anterior and lateral walls of the trachea and the bronchus, sparing the posterior membranous wall (2). Several reported cases have demonstrated successful surgical intervention and bronchoscopic laser therapy for advanced symptomatic patients (2,3). We herein report the successful bronchoscopic resection of a symptomatic localized polyp due to to using a high - frequency snare . An 80-year - old japanese man was admitted to our hospital for the evaluation and management of multiple tracheobronchial polyposis and right middle lobe atelectasis . He had a history of polyarteritis nodosa and had been treated with corticosteroids (prednisolone 6 mg / day). Chest computed tomography (ct) revealed diffuse calcified lesions throughout the cartilaginous regions of the trachea and bronchi, right middle atelectasis, and airway polyps (4 - 9 mm) in the left trachea and the left main bronchus (fig . 1). The bronchoscopic findings showed diffuse edematous mucosal lesions with polyposis on the left side of the trachea, the right middle bronchus and the left main bronchus (fig . 2). A spirometric analysis demonstrated an obstructive impairment, and the forced expiratory volume in one second (fev1) was 1.36 l, and fev1% was 43% . A transbronchial biopsy to make a diagnosis of the airway polyp was performed, and endoscopic mucosal resection was also carried out using a high - frequency snare to improve ventilatory insufficiency . To was pathologically confirmed in the resected submucosal cartilaginous tissue, and mature ossifications were also observed in the tissue (fig . After resecting the airway polyp, the spirometric data of the fev1 and fev1% improved from 1.36 l to 1.69 l and from 43% to 93%, respectively . A: a coronal view of the chest mediastinal window shows diffuse calcified lesions throughout the cartilaginous regions of the trachea and bilateral bronchi . Noncalcified endobronchial airway polyps are also seen on the left side of the trachea and the upper side of the left main bronchus (white arrows). B: a transverse view of the chest mediastinal window demonstrates right middle lobe atelectasis and an endobronchial airway polyp with small calcified lesions (white arrow) in the right middle lobe bronchus . There are no remarkable abnormal findings in the trachea (a) and carina (b), however, bronchoscopy showed a diffuse edematous mucosa with polyposis in the trachea (a), carina (b), right middle bronchus (c) and left main bronchus (d). A: an endobronchial polyp lesion obtained from the left main bronchus demonstrated submucosal calcification, ossification and cartilage formation surrounded by chronic airway inflammatory cells . B: an enlarged view shows the polyp lesion to consist of submucosal ossification and inflammatory cells . The comprehensive etiology of to remains to be elucidated, however, chronic airway infections, irritant exposure, several metabolic disorders and genetic factors have been proposed to be causative factors of to (3,4). This patient showed typical chest ct findings (fig . 1) and unusual bronchoscopic features (fig . Long - term corticosteroid administration might be a potential explanation for the atypical bronchoscopic findings . Tajima et al . Reported that bone morphogenetic protein-2 (bmp-2) played an important role in nodule formation and might synergistically act with transforming growth factor 1 (tgf-1) to promote an inductive cascade of to nodules (5). The airway polyp in our patient did not include mature ossifications in contrast to the previously reported cases (2,4), and the long - term corticosteroid administration in this patient might be related to these pathological atypical findings, such as the suppression of calcified lesion formation . However, there has so far been no report describing the effects of corticosteroids on initiating and enlarging airway polyp formation; thus, further studies are necessary to clarify the mechanism of airway polyp formation and effective treatment . Reported that chronic airway inflammation might be an important factor in the formation of to, and they discussed the potential clinical effects of inhaled corticosteroids to improve the symptoms in patients in the early stage of this disease (3). No guidelines have yet been established for the management of to, and systemic or inhaled corticosteroid treatment might be one of treatment choices for to without any problematic clinical symptoms, as seen in the present patient . In conclusion, we herein reported a rare case of to accompanied by unusual bronchoscopic features, such as multiple tracheobronchial polyposis, which was successfully treated using a high - frequency snare . To is a benign disorder, however, to may cause various clinical symptoms and spirometric impairments that necessitate the resection of airway polyps . Physicians should therefore be aware of this disease and its clinical symptoms and include it in the differential diagnosis.
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Traditionally, the emotional needs of dementia family caregivers are conceptualized with the transactional model of stress and coping (lazarus and folkman, 1984; pearlin et al ., 1990). In this model, the focus is specifically on the emotion of stress, with emphasis on improving coping skills and lowering stress level . This has translated into caregiver services like disease education, skills training, stress management and respite care . However, stress is only one of the emotions experienced by caregivers, and there are a complex range of other emotions, such as guilt, denial, sadness and anger . From the recent evidence, this whole array of emotions can be better understood using the concept of predeath grief (blandin and pepin, 2015). Predeath grief is the emotional response as dementia family caregivers mourn for the psychologically absent patient and anticipate impending losses (blandin and pepin, 2015). It is prevalent among dementia caregivers (chan et al ., 2013) and can be more overwhelming than handson care issues (frank, 2007). Caregivers who do not recognize the presence of predeath grief tend to deny the losses they experienced and attempt to control the uncontrollable progression of dementia . They become more paternalistic in their communication with the patients and more authoritarian in making decisions related to the patients . Such behaviours create inequity in the dyadic patient caregiver relationship, with the caregivers feeling more helpless and the patients losing the sense of autonomy (piiparinen and whitlatch, 2011). With this understanding, it is not surprising that predeath grief has been linked to negative consequences like caregiver burden (holley and mast, 2009), caregiver depression (sanders and adams, 2005; chan et al ., 2013) and caregivers' desire to institutionalize the patient prematurely (walker et al ., 1995). However, the published literature on predeath grief is derived primarily from the caucasian population (chan et al ., 2013). Because the expression of grief varies with culture (eisenbruch, 1984) this study investigates the existence (primary aim) and the characteristics (exploratory aim) of predeath grief in a multiethnic asian population using a wellestablished predeath grief scale the marwit meuser caregiver grief inventory (mmcgi). Ethical approval was obtained from the domain specific review board of national healthcare group, singapore . The following inclusion criteria were used: (i) spouses or children of patient with dementia; (ii) caring for a patient with dementia not residing in nursing home; (iii) able to read in english; and (iv) aged at least 21 years . The other scales of related construct, such as prolonged grief scale (pg12), zarit burden interview (zbi) and centre for epidemiologic studies depression scale (cesd), were also included to contrast with mmcgi . Meuser caregiver grief inventory is a predeath grief scale developed empirically from extensive qualitative interview of dementia caregivers (marwit and meuser, 2002). Its 50 items are assessed through 5point scales and summed to generate a total score and three subscale scores corresponding to the different dimensions of loss personal sacrifice burden, heartfelt sadness and longing, and worry and felt isolation . In the original us study (marwit and meuser, 2002), mmcgi showed high internal consistency reliability, with cronbach's alpha ranging from 0.90 to 0.96 . It also had good construct validity, with strong correlation with anticipatory grief scale (pearson's correlation coefficient, r = 0.798). Meanwhile, personal sacrifice burden subscale correlated with caregiver strain index (r = 0.730), heartfelt sadness and longing subscale with anticipatory grief scale (r = 0.666) and worry and felt isolation subscale with wellbeing scale (r = 0.718). The authors of mmcgi proposed using one standard deviation above the normative mean of a population to indicate high predeath grief and a need for further interventions . In the us population, this translates to cutoff scores of> 175 for mmcgi,> 68 for personal sacrifice burden subscale,> 59 for heartfelt sadness and longing subscale and> 52 for worry and felt isolation subscale . Such normative values were not available for the singapore population at the time of this study . Prolonged grief scale is a 12item predeath grief scale for caregivers modified from a postdeath grief scale, the inventory of complicated grief . It is based on the diagnostic criteria of prolonged grief disorder proposed for icd11 (prigerson et al ., 2009). Its first 11 items are rated on 5point scales and summed to produce a quantifiable total score, while item 12 is scored dichotomously for the absence or presence of sociooccupational dysfunction . It assesses the perceived burden experienced by caregivers of older persons (zarit et al ., 1980). Zbi has five domains burden in the relationship, emotional wellbeing, social and family life, finances and loss of control over one's life (rankin et al ., 1994). Zbi has been validated in singapore (seng et al ., 2010), with good reliability and construct validity . Centre for epidemiologic studies depression scale is a 20item depression scale, assessed through 4point scales . Its four domains are depressed affect, positive affect, somatic symptoms and interpersonal problems (radloff, 1977). Cesd was previously validated in singapore (stahl et al ., 2008), with cronbach's alpha of 0.700.79 and diagnostic performance of 6482% . The three dementia severities in the revised third edition of diagnostic and statistical manual of mental disorders (american psychiatric association, 1987) were used as a brief measure of dementia staging . From the three options, participants chose the description that best described the patient with dementia still capable of independent living (mild stage), needs some assistance with daily living (moderate stage) or needs roundtheclock supervision (severe stage). This brief measure was previously shown to have reasonable agreement with clinical dementia rating scale (= 0.560.6) (forsell et al ., 1992; juva et al ., if predeath grief exists in the asian context (primary aim), it should be measurable on mmcgi with high internal consistency reliability and with convergent and discriminant validities consistent with the original caucasian constructs (marwit and meuser, 2002). We had four hypotheses regarding convergent validity: mmcgi measures the experience of predeath grief . Therefore, it should show stronger correlation with another predeath grief scale, pg12, than with zbi or cesd.personal sacrifice burden subscale captures the experience of individual losses due to caregiving . It resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd.worry and felt isolation subscale measures the feelings of losing connection with others . It is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd.heartfelt sadness and longing subscale resembles the traditional concepts of grief, that is, one's intrapersonal reactions to lost relationship . Therefore, it should show stronger correlation with another predeath grief scale, pg12, than with zbi or cesd . It resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd . It is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd . Heartfelt sadness and longing subscale resembles the traditional concepts of grief, that is, one's intrapersonal reactions to lost relationship . Hence, it should correlate stronger with pg12 than with zbi or cesd . We made two hypotheses regarding discriminant validity: heartfelt sadness and longing subscale should correlate poorer with depression scale cesd, consistent with ample evidence demonstrating the difference between grief and depression (prigerson et al ., 2009).mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd, because they are distinctly different constructs . Heartfelt sadness and longing subscale should correlate poorer with depression scale cesd, consistent with ample evidence demonstrating the difference between grief and depression (prigerson et al . Mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd, because they are distinctly different constructs . First, linear regression was performed, with mmcgi as dependent variable, to identify factors associated with predeath grief . All variables with p 0.075 in univariate regression were entered into multivariate regression, and variables with p> 0.05 in multivariate regression were removed through stepwise backward selections . Second, the mean scores of mmcgi and its subscales were compared with those from the original us study (marwit and meuser, 2002) using onesample ttest . We calculated the sample size using bonett and wright's formulas (bonett and wright, 2000, 2015), setting the 95% confidence interval width at 0.2 . Referencing to the original study (marwit and meuser, 2002), we expected the cronbach's alpha of mmcgi to approximate 0.90, corresponding to a minimum sample size of 11; spearman's correlation coefficient between mmcgi and pg12 should approximate 0.8, corresponding to a minimum sample size of 72 . Ethical approval was obtained from the domain specific review board of national healthcare group, singapore . The following inclusion criteria were used: (i) spouses or children of patient with dementia; (ii) caring for a patient with dementia not residing in nursing home; (iii) able to read in english; and (iv) aged at least 21 years . The other scales of related construct, such as prolonged grief scale (pg12), zarit burden interview (zbi) and centre for epidemiologic studies depression scale (cesd), were also included to contrast with mmcgi . Marwit meuser caregiver grief inventory is a predeath grief scale developed empirically from extensive qualitative interview of dementia caregivers (marwit and meuser, 2002). Its 50 items are assessed through 5point scales and summed to generate a total score and three subscale scores corresponding to the different dimensions of loss personal sacrifice burden, heartfelt sadness and longing, and worry and felt isolation . In the original us study (marwit and meuser, 2002), mmcgi showed high internal consistency reliability, with cronbach's alpha ranging from 0.90 to 0.96 . It also had good construct validity, with strong correlation with anticipatory grief scale (pearson's correlation coefficient, r = 0.798). Meanwhile, personal sacrifice burden subscale correlated with caregiver strain index (r = 0.730), heartfelt sadness and longing subscale with anticipatory grief scale (r = 0.666) and worry and felt isolation subscale with wellbeing scale (r = 0.718). The authors of mmcgi proposed using one standard deviation above the normative mean of a population to indicate high predeath grief and a need for further interventions . In the us population, this translates to cutoff scores of> 175 for mmcgi,> 68 for personal sacrifice burden subscale,> 59 for heartfelt sadness and longing subscale and> 52 for worry and felt isolation subscale . Such normative values were not available for the singapore population at the time of this study . Prolonged grief scale is a 12item predeath grief scale for caregivers modified from a postdeath grief scale, the inventory of complicated grief . It is based on the diagnostic criteria of prolonged grief disorder proposed for icd11 (prigerson et al ., 2009). Its first 11 items are rated on 5point scales and summed to produce a quantifiable total score, while item 12 is scored dichotomously for the absence or presence of sociooccupational dysfunction . It assesses the perceived burden experienced by caregivers of older persons (zarit et al ., 1980). Zbi has five domains burden in the relationship, emotional wellbeing, social and family life, finances and loss of control over one's life (rankin et al ., 1994). Zbi has been validated in singapore (seng et al ., 2010), with good reliability and centre for epidemiologic studies depression scale is a 20item depression scale, assessed through 4point scales . Its four domains are depressed affect, positive affect, somatic symptoms and interpersonal problems (radloff, 1977). Cesd was previously validated in singapore (stahl et al ., 2008), with cronbach's alpha of 0.700.79 and diagnostic performance of 6482% . The three dementia severities in the revised third edition of diagnostic and statistical manual of mental disorders (american psychiatric association, 1987) were used as a brief measure of dementia staging . From the three options, participants chose the description that best described the patient with dementia still capable of independent living (mild stage), needs some assistance with daily living (moderate stage) or needs roundtheclock supervision (severe stage). This brief measure was previously shown to have reasonable agreement with clinical dementia rating scale (= 0.560.6) (forsell et al ., 1992; juva et al ., if predeath grief exists in the asian context (primary aim), it should be measurable on mmcgi with high internal consistency reliability and with convergent and discriminant validities consistent with the original caucasian constructs (marwit and meuser, 2002). We had four hypotheses regarding convergent validity: mmcgi measures the experience of predeath grief . Therefore, it should show stronger correlation with another predeath grief scale, pg12, than with zbi or cesd.personal sacrifice burden subscale captures the experience of individual losses due to caregiving . It resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd.worry and felt isolation subscale measures the feelings of losing connection with others . It is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd.heartfelt sadness and longing subscale resembles the traditional concepts of grief, that is, one's intrapersonal reactions to lost relationship . Therefore, it should show stronger correlation with another predeath grief scale, pg12, than with zbi or cesd . It resembles caregiver burden measured by zbi and should correlate stronger with zbi than with pg12 or cesd . It is a feature of grief reaction and should correlate stronger with pg12 than with zbi or cesd . Heartfelt sadness and longing subscale resembles the traditional concepts of grief, that is, one's intrapersonal reactions to lost relationship . We made two hypotheses regarding discriminant validity: heartfelt sadness and longing subscale should correlate poorer with depression scale cesd, consistent with ample evidence demonstrating the difference between grief and depression (prigerson et al ., 2009).mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd, because they are distinctly different constructs . Heartfelt sadness and longing subscale should correlate poorer with depression scale cesd, consistent with ample evidence demonstrating the difference between grief and depression (prigerson et al ., 2009). Mmcgi should correlate poorly with finances subscale of zbi and positive affect subscale of cesd, because they are distinctly different constructs . First, linear regression was performed, with mmcgi as dependent variable, to identify factors associated with predeath grief . All variables with p 0.075 in univariate regression were entered into multivariate regression, and variables with p> 0.05 in multivariate regression were removed through stepwise backward selections . Second, the mean scores of mmcgi and its subscales were compared with those from the original us study (marwit and meuser, 2002) using onesample ttest . We calculated the sample size using bonett and wright's formulas (bonett and wright, 2000, 2015), setting the 95% confidence interval width at 0.2 . Referencing to the original study (marwit and meuser, 2002), we expected the cronbach's alpha of mmcgi to approximate 0.90, corresponding to a minimum sample size of 11; spearman's correlation coefficient between mmcgi and pg12 should approximate 0.8, corresponding to a minimum sample size of 72 . Table 1 shows the basic demographics of the participants and the patients with dementia they cared for . Basic demographics of the caregivers and the patients with dementia they cared for (n = 72) sd, standard deviation; mmcgi, marwit meuser caregiver grief inventory . P value derived from univariate linear regression, with mmcgi score as the dependent variable . Cronbach's alpha, which measured the internal consistency of responses, was 0.97 for mmcgi . Meanwhile, the cronbach's alpha was 0.94 for personal sacrifice burden subscale, 0.92 for heartfelt sadness and longing subscale and 0.89 for worry and felt isolation subscale . Contrasting among the three scales of pg12, zbi and cesd (table 2), mmcgi expectedly correlated stronger with pg12 (spearman's = 0.79, p <0.001) than with zbi or cesd, while personal sacrifice burden subscale correlated stronger with zbi (= 0.74, p <0.001), heartfelt sadness and longing subscale with pg12 (= 0.76, p <0.001) and worry and felt isolation subscale with pg12 (= 0.79, p <0.001). Spearman's correlation coefficient between the mmcgi (total and subscale scores) and the other scales of related constructa mmcgi, marwit meuser caregiver grief inventory; pg12, prolonged grief scale; zbi, zarit burden interview; cesd, centre for epidemiologic studies depression scale . P <0.001 for all the values in the table, after bonferroni adjustment . As hypothesized, heartfelt sadness and longing subscale correlated poorer with cesd (= 0.58, p <0.001). Mmcgi also correlated poorly with finances subscale of zbi (= 0.37, p = 0.162) and positive affect subscale of cesd (= 0.33, p = 0.530). Table 1 includes the p values derived from univariate regression analysis of factors related to mmcgi . In multivariate linear regression (table 3), factors associated with higher mmcgi score included caring for patients with severe dementia, spousal relationship, secondary or below education and malay ethnicity . Employment and primary caregiver status both had p> 0.05 and were removed from the final model . The final model in multivariate linear regression, with mmcgi score as the dependent variable . Mmcgi scores for the respective variables were also shown mmcgi, marwit meuser caregiver grief inventory; sd, standard deviation; ci, confidence interval . Table 4 compares the mmcgi total and subscale scores between the current study and the original us study (marwit and meuser, 2002). The worry and felt isolation subscale score was 13.8% higher in this study (p <0.001), while the rest of the scores were comparable with those of the usa (marginal difference of 4.4% to 3.7%). Comparison of mmcgi scores between the original us study (n = 166) and the current singapore study (n = 72) mmcgi, marwit meuser caregiver grief inventory; sd, standard deviation; ci, confidence interval . Prior to this study, there were uncertainties about the applicability of predeath grief concept to the asian population . In many ways, this study supported the existence of predeath grief in asia and attests to the universality of human emotions . We showed that predeath grief was measurable in a reliable and valid manner, using an established predeath grief scale of mmcgi . The risk factors of spousal caregivers and advanced dementia reported here were similarly highlighted in a systematic review of the caucasian studies (chan et al ., 2013). Even the mmcgi total and subscale scores were largely comparable with those of the usa (marwit and meuser, 2002). These results suggest a need for clinicians to be attuned to the predeath grief experience of dementia caregivers even in the noncaucasian populations . This is particularly important because predeath grief in dementia caregivers is commonly disenfranchised (frank, 2007), and its expression is not generally acceptable in society . Unless the clinicians inquire about its presence, dementia caregivers are unlikely to volunteer such feelings and will suffer in silence until they experienced the irrevocable adverse effects of predeath grief . By such time, it may be too late to intervene . Asian caregivers showed more worries and felt isolation, and certain ethnicity experienced more predeath grief . To attune to the predeath grief experience of caregivers, clinicians need to be mindful of the disparate presentations of predeath grief in different cultures . In the example of asian caregivers, strong expression of worries and felt isolation may be an indicator of underlying predeath grief . Such cultural sensitivity is also necessary to tailor personcentred interventions and to better support the emotional needs of caregivers in multiethnic populations . The influence of culture on predeath grief is an area worth further exploration in future researches . The caregiver risk factors in this study (spouse, lower education and selected ethnicity) paralleled the predictors of complicated postdeath grief from a systematic review (chan et al ., 2013) previous studies showed that greater predeath grief is a key predictor of postdeath complicated grief (blandin and pepin, 2015). Identifying shared risk factors between predeath and postdeath grief opens a window of opportunity to target efforts of case finding and early intervention in the predeath setting . Such efforts can prevent the development of complicated grief following bereavement (schulz et al . One of the strengths of this study lies with the location where it was conducted . Singapore is a cosmopolitan city in asia and holds a number of major asian cultures, including that of the chinese, indian, malay muslim and eurasian . This environment provided an opportune test bed to answer our research question on the applicability of predeath grief in asia . They alert readers on the likely presence of predeath grief even in the other parts of asia, and serve as an impetus for further researches on predeath grief in asia . In this study, limited proportion of spousal caregivers participated because a number of them could only read in chinese and not in english . We also restricted the number of variables entering multivariate regression so that the analyses remain valid given the sample size . Even with these two limitations, we have had reasonable evidence from this study to show that predeath grief do exist among dementia family caregivers in asia . We hope to better represent spousal caregivers and identify even more risk factors in our ongoing study involving chinese mmcgi and larger sample size . In summary, predeath grief is applicable even to the noncaucasian population, and it is detectable with an objective scale like mmcgi . Clinicians working with noncaucasian populations need to be attuned to its presence and be sensitive to the influence of culture on its expression key points predeath grief is also applicable to dementia caregivers in a noncaucasian population, and it is detectable with an objective scale like mmcgi.to prevent the adverse outcomes of predeath grief, clinicians working with noncaucasian populations need to be sensitive to its presence and to the influence of culture on its expression.identifying shared risk factors between predeath and postdeath grief allows targeted efforts of case finding and early intervention in the predeath setting, thus preventing complicated grief following bereavement . Predeath grief is also applicable to dementia caregivers in a noncaucasian population, and it is detectable with an objective scale like mmcgi.to prevent the adverse outcomes of predeath grief, clinicians working with noncaucasian populations need to be sensitive to its presence and to the influence of culture on its expression.identifying shared risk factors between predeath and postdeath grief allows targeted efforts of case finding and early intervention in the predeath setting, thus preventing complicated grief following bereavement . Predeath grief is also applicable to dementia caregivers in a noncaucasian population, and it is detectable with an objective scale like mmcgi . To prevent the adverse outcomes of predeath grief, clinicians working with noncaucasian populations need to be sensitive to its presence and to the influence of culture on its expression . Identifying shared risk factors between predeath and postdeath grief allows targeted efforts of case finding and early intervention in the predeath setting, thus preventing complicated grief following bereavement.
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Use of computers by students has reportedly increased due the current information society and rapid economic growth1 . Students lifestyle choices including computer overuse, along with a lack of health education and exercise lead to changes in physical posture and increases in pain2 . Trunk stability is related to the body s ability to move and involves increased activity of the core muscles and erector spinae3, 4 . Muscles that comprise trunk stability stabilize the body and the spine irrespective of limb movements since they act like a corset . Studies have reported that once trunk stabilization is secured, activities of the abdominal muscles, pelvis, waist, and hips harmonize and movements of the limbs occur more smoothly6 . In addition, trunk stabilization exercises are important to preventing functional movement impairment of the abdominal muscles7 . Researchers have also reported that trunk stabilization exercises increase weight bearing toward the immobile side and that they are effective for postural control and activities of daily living8, 9 . Trunk stabilization exercises maximize spinal movement and stability through repeated reinforcement10 . A sling, which is used to enhance stability and mobility during trunk exercises, has the following advantages: the participant s own weight serves as the resistance and the exercises can be performed in small areas, are simple, and include a variety of motions . Thus, the use of a sling is popular11 . A previous study reported on how one can maintain balance in the prone position as well as advantages of trunk stabilization exercises12 . When trunk movement is parallel to the provided external resistor, transversus abdominis (tra) contractions are activated to maintain a neutral trunk position13 . When the shoulder joints are bent to a 90 horizontal alignment of the pelvis posture, abdominal internal oblique (io) and tra activities are high11 . Oh et al . Compared abdominal external oblique (eo) and abdominal io by controlling the elbow joint angle during push - ups at 0, 45, and 90. as a result, activity was higher when the angle during sling exercises was 0. those authors concluded that trunk stabilization exercises effectively promote stability of the trunk as well as the muscles around the proximal joint14 . As shown above, although trunk stabilization exercises have clinical therapeutic effects, there have been few formal studies on the effect of different shoulder joint angles on trunk stabilization exercises . The purpose of this study was to identify whether bending the shoulder joints with a sling strapped at an angle in the prone position effectively thickens the core muscles and increases static and dynamic balance abilities in a standing position . In this study, 91 students of both genders, all in their 20s, and who were enrolled at sun moon university located in asan city were randomly selected as subjects . The study was approved by the sun moon university institutional bioethics committee (smu-14 - 07 - 03). All participants provided written informed consent and were informed of the study s purpose and procedures . The inclusion criteria involved 1) a full understanding of the researchers instructions and purpose of study; 2) good health; and 3) written informed consent . Exclusion criteria consisted of 1) structural abnormalities of the spine during the three months prior to the study; 2) pain in the spine, such as back pain; 3) drug use; 4) regularly performing weight - training activities that may affect the mechanical structure of the abdominal muscles . Subjects were randomized into three group: the shoulder joints 60 flexed trunk stabilization exercises group (group 1), shoulder joints 90 flexed trunk stabilization exercises group (group 2), and shoulder joints 120 flexed trunk stabilization exercises group (group 3). The experiment was conducted one by one in a separate time in order to blind subjects to the information about the angle . In group 1, the exercises were performed with a sling strapped on both ankles at the shoulder joints bending 60 in the push - up position . In group 2, the same condition at the shoulder joints bending 90 was used, while in group 3, the same condition at the shoulder joints bending 120 was used . The intervention period lasted a total of 8 weeks, after which time the thickness, static balance, and dynamic balance of the core muscles were measured to identify the pre- versus post - intervention changes . When the measurer says start in a state when a sling has been strapped, the participant must move both knees away from the floor11 . Before both knees fall back to the floor, the shoulder joints bending angles (60, 90, and 120) are set using an electronic goniometer . After both knees are away from the ground, the hip joint angle must be maintained at 0. the measurer should always observe whether the participant maintains the posture and angle in the prone state . According to each shoulder joint angles, trunk stabilization exercises were performed for 30 minutes three times a week for a total of 8 weeks (warm - up, 5 minutes; exercises, 20 minutes; cool - down, 5 minutes). After the exercises were conducted for 40 seconds, each subjected rested for 20 seconds . A 3.5-mhz convex array transducer with an ultrasonic measurement device was used to measure trunk muscle thickness . On the ultrasound images, one physical therapist skillful at measuring ultrasound images applied ultrasound gel between the transducer and the skin and placed the b - mode scanner so that the center of the transducer contacted the 2.5 cm site frontward at the midpoint between the 12th rib and the iliac spine ridge centered around the armpit15, 16 . To measure muscle thickness, a vertical line was then drawn at the left and right ends of the image and the muscle thickness was measured in the order of tra, abdominal io, and abdominal eo17 . For a measurement posture the participant was asked to comfortably bend the hip and knee joints in the supine position to minimize lumbar lordosis and pull the belly upward . When the participant performed an abdominal throw - operation, the therapist gave visual feedback for the abdominal muscle to contract while looking at the monitor . A dynamic balance functional reaching test was used to measure dynamic stability18 . During this inspection, the subject is asked to extend his / her legs as far as possible with heels not falling . At this time, arm movement is measured from end to end using a ruler pasted on the wall . In other words, this procedure aims to test a subject s ability to extend their legs as far as possible while stepping forward without losing their balance . The ability to stretch farther indicates a wider stability limit, suggesting better dynamic balance16 . Balance measuring equipment was used to evaluate static balance . A stability index (st) and a weight distribution index (wdi) an st base on each of the 4 force - plates for the toes and heels of the right and left feet showed the balance stability by measuring the posture fluctuation based on changes in weight, while the wdi showed a weight - loaded value using percentages19 . The subject was asked to place one bare heel on the force place where the sole - shaped heel portion was drawn . Measured postures were divided into eyes open, eyes closed, standing on a soft supportive surface, and eyes closed while standing on a soft supportive surface . For each posture, the subject was instructed to maintain it for 32 seconds . For the eyes after the operation, the st and the wdi were comprehensively calculated and displayed on a computer screen connected to the equipment . In this study, a comprehensive st value and a wdi value were used, for which lower values indicate good balance19 . We performed the shapiro - wilk test to determine the distribution of the subjects . To compare pre- versus post - experimental data in each group, the paired samples t - test was used . To compare pre- versus post - experimental data of the three groups, one - way analysis of variance (anova) was performed . When a significant difference was found, post hoc comparisons were performed using a bonferroni correction . The measurement data of each item are presented as means and standard deviations . The significance level for statistical verification the mean age height, weight, and bmi were not significantly different among the groups (table 1table 1.general characteristicsgroup 1(n=14)group 2(n=15)group 3(n=14)age (years)20.6 0.920.5 1.120.2 0.4height (cm)16,930 8.1167.3 7.8171.8 7.0weight (kg)60.7 12.163.0 8.367.6 11.7bmi (kg / m)22.1 2.119.6 1.221.2 2.2 meansd, group 1: the shoulder joints 60 bending trunk stabilization exercises group, group 2: shoulder joints 90 bending trunk stabilization exercises group, group 3: shoulder joints 120 bending trunk stabilization exercises group, bmi: body mass index). Meansd, group 1: the shoulder joints 60 bending trunk stabilization exercises group, group 2: shoulder joints 90 bending trunk stabilization exercises group, group 3: shoulder joints 120 bending trunk stabilization exercises group, bmi: body mass index group 1 and group 2 showed significantly increased tra thickness after the experiment (p <0.05). Group 3 showed a significant post - experiment increase in eo thickness (p <0.05) (table 2table 2.comparison of core muscle thickness pre- vs. post - interventions(mm)group 1(n=14)group 2(n=15)group 3(n=14)eopre0.99 0.301.00 0.170.96 0.18post0.87 0.180.86 0.160.94 0.13iopre1.03 0.120.96 0.141.06 0.22post1.05 0.101.08 0.201.03 0.23tra pre1.20 0.161.21 0.131.28 0.24post1.43 0.261.58 0.311.49 0.46meansd, group 1: the shoulder joints 60 bending trunk stabilization exercises group, group 2: shoulder joints 90 bending trunk stabilization exercises group, group 3: shoulder joints 120 bending trunk stabilization exercises group, eo: external oblique, io: internal oblique, tra: transverse abdominal, comparison of the three groups pre- vs. post - interventions using one - way anova, comparison of differences before and after interventions in each group using a paired t - test, * p<0.05, * * * p<0.001). Meansd, group 1: the shoulder joints 60 bending trunk stabilization exercises group, group 2: shoulder joints 90 bending trunk stabilization exercises group, group 3: shoulder joints 120 bending trunk stabilization exercises group, eo: external oblique, io: internal oblique, tra: transverse abdominal, comparison of the three groups pre- vs. post - interventions using one - way anova, comparison of differences before and after interventions in each group using a paired t - test, * p<0.05, * * * p<0.001 changes in dynamic balance increased from 18.00 4.51 to 21.98 3.86 in group 2 . The post - test results revealed no statistical significance in the differences before and after dynamic balance at 60, 90, and 120 of bending . Changes in static balance showed statistical differences in eyes closed, pillow with eyes closed, and stability index values in group 2, in which the shoulder joints were bent at 90 using a sling (p <0.05). The post - test results found no statistical significance in each difference in st, wdi and in 60, 90, and 120. this study found that groups in which the shoulder joints were bent to 60 and 90 showed improved tra strength . This finding suggests that the tra muscle activity was increased . In previous studies, when the shoulder joints are bent at 90 or less, the closer the trunk, the more significant the activation of the tra11, 19 . Tra fibers run parallel around the abdominal wall, playing a role in allowing a force the same as the rim following contraction . In addition, tra stabilizes the lumbar spine and is first activated by the weight of the torso due to the limb movement18 . This study s experimental method requires a force to align the trunk against gravity with the shoulder bent at various joints ., it is reported that shoulder joints can securely move since their bending angle is related to the activation of the follicle through the significant difference from 30 to 110 in the shoulder joint bending angle19 . Reported that the movement of the far limbs may affect muscle activity and that the force of contracting muscles can be transferred to other connected muscles and tendons through the site of origin . In addition, myers et al . Reported that the deltoid was involved when the shoulder joint connected to the trapezius is bent20 . Based on previous research, this study also found that trunk stabilization exercises depending on the shoulder joint angle impact the contraction of the muscle belly through the myofascial meridians of the dorsal arm line, where the arm muscles deeply contract and the chest waist fascia to increasing the activity21 . In the group in which the shoulder joint was bent to 120, the eo strength was significantly improved compared to that in the other groups . For stevens et al ., the muscle activity of the eo in the leg lifted in a posture of lifting the arms and legs was higher because the eo on the lifted side of the torso turning direction in maintaining the spine against gravity contracts more by adjusting the body turning occurring in the lifting process22 . Additionally, it may be because the eccentric contraction of the eo resulted in increasing muscle activity to maintain trunk stability as the trunk and arms go farther . For static balance, group 2 showed a statistically significant difference in ec, pc, and st values . For st, a significant difference was seen only in the eyes - closed posture . Visual and proprioceptive senses perform an important function in reflective postures and movement control23 . Static balance on a soft supportive surface is significantly reduced when vision is blocked24 . Reported that trunk stability is divided into three sub - systems: a passive system in charge of the stability provided to the non - contractile tissue in a passive tense form; the transmission of power, an active sub - system made by shrinking organizations, reducing the stress exerted on the vertebral body and spinal joint, adjusting pain, and reinforcing the joint with active stabilization; and an adjustable sub - system consisting of proprioceptive senses and the central nervous system25 . Ec and pc better demonstrate the effects of balance, and an improved balance ability helps with neurological stabilization, which consists of an active sub - system of trunk muscles, proprioceptive senses, and the central nervous system . However, for wdi, no significant change was seen in eo, ec, or po . Periyasamy et al . Reported that foot pressure distribution is affected by a number of factors such as foot anatomy, body mass, gender, and joint range of motion26 . In this study, since measurements were performed three times in each standing posture and foot position, no statistically significant difference was created in wdi dynamic balance was improved overall in all three groups, but statistically significant differences were found only in group 2 . Muscle stability and strength increase as the motor control for harmonious muscle mobilization between the large trunk muscles and the small intrinsic muscles is emphasized27 . In addition, the automatic activity of the tra is thought to be the protective mechanism of the lumbar spine13 and has been reported as part of the deep muscle that provides the lumbar stability during functional movements . The tra is reportedly involved in proactive attitude adjustments regardless of upper and lower limb movement direction28 . From these results, trunk stabilization exercises of the shoulder joint bent at 90 were the most effective since they provide trunk stability, which then improves one s dynamic balance sense . Such increases are considered to contribute to the dynamic stability required by functional movements . First, subjects were limited to healthy adults in their 20s . As such, the subject cohort was small, making it difficult to generalize the results obtained . Second, compared to the 8-week intervention period, the pre- and post - intervention comparison was made and no subsequent evaluation was conducted . Thus, it was impossible to determine the long - term effect of the intervention . Third, the subjects private lives, sex, habits, and personal athletic career were not considered . Therefore, further studies of a larger number of subjects with follow - up studies are required to evaluate the retention of the improved effects . The program applied in this study thus, it is necessary to normalize the program as a more specific and structured intervention program through design improvements and modifications . Therefore, trunk stabilization exercises according to shoulder joint angle using a sling have positive effects on core muscle strength and balance . The entire muscle was strengthened under trunk stabilization exercises applied at the shoulder joints at angles of 120 or higher . In trunk stabilization exercises using a sling according to various shoulder joints angles, the shorter distance between the arm and the trunk allows the tra to act, whereas a longer distance contributes to trunk stability due to the contraction of the eccentricity of abdominal eo . To improve one s balance capacity for functional activities, the shoulder joints should be bent at 90 before training . This study is significant in that it has presented the orientation for muscle strengthening training involved in trunk stabilization for use in future clinical experiments . Related studies to demonstrate and systemize the effects on the core muscle are required.
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We sequenced 12 bat brains positive for lyssavirus antigen detected by immunofluorescence and reverse transcription the 400-bp 5 variable extreme of the nucleoprotein gene of these eblv-1 strains was amplified by specific eblv-1 nested rt - pcr and sequenced by using the following primers: seqvar1f 5-1acgcttaacaaccagatcaaag22 - 3, seqvar2f 5-51aaaaatgtaacacyyctaca70 - 3, eblvseqvar1r 5-596cagtctcaaagatctgttccat575 - 3, and eblvseqvar2r 5-552tagttcccagtattctgtcc533 - 3. all rabies - positive serotine bats came from southern spain (huelva, seville, murcia, and badajoz) and were molecularly identified as e. isabellinus (8). An alignment was performed by using clustalx (www.clustal.org) to combine the obtained sequences and other available eblv-1 sequences from genbank, including a duvenhage virus used as the outgroup (table a1). Before conducting further analyses, we used jmodeltest (http://darwin.uvigo.es/software/jmodeltest.html) to select the best fitting substitution model for our sequences according to the corrected akaike information criterion . Maximum - likelihood phylogenies were reconstructed by using phyml (http://atgc.lirmm.fr/phyml) software and by using a generalized time - reversible model and the parameter estimated in the analyses . Maximum - parsimony analyses were conducted by using paup * 4.0b10 (http://paup.csit.fsu.edu/) weighting transversions 15 according to the transitions / transversion ratio estimated in the jmodeltest analyses . Confidence in the topologies for the maximum - likelihood and the maximum - parsimony analyses was established with 1,000 bootstrap replicates . A bayesian phylogenetic inference was obtained by using mrbayes version 3.1 (http://mrbayes.csit/fsu.edu/) with random starting trees without constraints . Two simultaneous runs of 10 generations were conducted, each with 4 markov chains, and the trees were sampled every 100 generations . Net p - distances between groups were calculated by using mega4 (www.megasoftware.net) (figure 1). European bat lyssavirus 1 (eblv-1) phylogenetic reconstruction based on the first 400 bp of the nucleoprotein gene . The tree was obtained by bayesian inference run for 10 generations; trees were sampled every 100 generations . Bootstrap supports after 1,000 replicates for each node are also shown for maximum - parsimony (green numbers) and maximum - likelihood (blue numbers) analyses . Net p - distance values (as percentages) between groups are indicated by arrows . A parsimony - based network is presented for each major lineage; sizes of yellow circles are proportional to the number of individuals sharing a given haplotype, and reconstructed haplotypes (median vectors) are shown in red . Duvv, duvenhage virus . The genetic structure and relationships between haplotypes were examined within the main lineages through a parsimony - based network built with a median - joining algorithm implemented in the network 4.5.1 program (11). To evaluate and compare genetic variability and polymorphism among lineages, we estimated the number of haplotypes, mutations, and segregating sites as well as haplotype diversity and nucleotide diversity by using dnasp version 4.5 (12) for the major clades (table). Finally, to investigate population dynamics across lineages, the fu fs and tajima d statistics were calculated (table). These 2 statistics are considered to be the most powerful tests for detecting expansion events (13). * eblv, european bat lyssavirus; n, no . Sequences; s, no . Segregating sites; eta, no . Mutations; hap, no . Haplotypes; hd, haplotype diversity; varhd, haplotype variance; pi, nucleotide diversity; thetanuc, estimated population mutation rate per site; k, average no . All phylogenetic analyses, regardless of the reconstruction criterion used, formed a monophyletic cluster of the eblv-1 strains from spain (only the bayesian inference reconstruction is shown). The bayesian inference, maximum - likelihood, and maximum - parsimony analyses identified the cluster from spain and eblv-1a and eblv-1b as being monophyletic (figure 1), although only maximum - likelihood and maximum - parsimony analyses suggested a closer relationship between eblv-1a and the cluster from spain . The genetic differentiation of the eblv-1 strains from the iberian peninsula matches their association with another bat species (figure 2), which suggests that the host bat s evolutionary history plays a major role in eblv-1 molecular epidemiology, as has been proposed for rabies virus in bats in north america (14). Geographic distribution of eptesicus serotinus bats (red), e. isabellinus bats (blue), and cases of rabies in bats (green dots), europe, 19902009 . Obtained from rabies bulletin europe (www.who-rabies-bulletin.org/). The low genetic diversity and the fu fs and tajima d statistics (table) all suggest rapid population expansion of eblv-1a, which is consistent with the star - like structure of the network for this lineage (figure 1). Conversely, haplotype and nucleotide diversity descriptors (table) have the highest values for eblv-1b and a complex network structure with differentiated subnetworks . The lineage from spain also has low diversity and a star - shaped network, but neutral evolution cannot be rejected on the basis of the fs and d statistics . Net distances are similar within and between lineages, except for eblv-1a, which is slightly more differentiated (figure 1). Consequently, the suggested eblv-1 expansion from spain into europe (15) is not supported by our results, which record the highest variability and most complex phylogenetic structure for france and the netherlands (figure 1). This complex structure suggests either a longer evolutionary history in these areas or a recent contact of distinct bat lineages in this zone . The results of this study show that the strains from spain do not belong to subtype 1b because of their association with a different reservoir (e. isabellinus bats). Moreover, what is currently considered to be eblv-1b seems to include at least 4 lineages that are more genetically diverse and have a complex history . Eblv-1a, however, has low genetic diversity despite its extensive geographic distribution, suggesting a relatively recent and successful expansion of this lineage . These results call into question the current classification of eblv-1 into 2 single subtypes . To provide a better understanding of eblv-1 molecular epidemiology in europe, additional studies that consider different genes should be conducted and the current classification should be revised accordingly.
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The potential energy landscape of a protein is a high - dimensional object, where locating the global potential energy minimum, the protein folding problem, is often still considered a since calculations using models where all degrees of freedom of all atoms in the protein are included are computationally demanding, coarse - grained models with simple interaction potentials are often used . The bln model, in which protein residues are represented by hydrophobic (b), hydrophilic (l), and neutral (n) beads, is a widely studied representation . In the present work we employ a version of the bln potential with stiff harmonic terms to restrain the bond lengths and bond angles:(6)where rij is the distance between two beads i and j. the first term is a harmonic bond restraint with kr = 231.2 and re =, for consistency with previous work . The main features of the landscape are independent of the precise value chosen for kr over quite a wide range of values . The second term is an angle restraint with k = 20 rad and e = 1.8326 rad . If two or more of these beads are n, then a = 0 and b = 0.2 . For all other sequences, a = b = 1.2 . Thus, the main chains of the -barrel prefer all - trans conformations, but the turn regions are more flexible . If both residues are b, then c = d = 1 . If one residue is l and the other is l or b, then c = 2/3 and d = 1 . If either of the residues is n, then c = 1 and d = 0 . The 46-residue sequence (bln-46) b9n3(lb)4n3b9n3(lb)5l was designed to exhibit a frustrated energy landscape, with a four - strand -barrel as the global minimum . This protein has several alternative -barrel structures with energies close to the global minimum, but separated from it by large barriers, compared to experimentally relevant thermal energies. (9) when all non - native attractive interactions are removed to make a go potential,(24) most of the frustration is removed and the potential energy landscape forms a single folding funnel. (9) in the go model all of the attractive interactions that are not present in the native state are set to zero . This potential is identical to the original bln potential except that dbb = 0 for any pair of residues where rij> 1.167 in the global minimum of the unperturbed model . Placing salt bridges in key locations can also reduce the frustration . The 69-residue sequence (bln-69) b9n3(lb)4n3b9n3(lb)4n3b9n3(lb)5l folds into a six - strand -barrel . Studies using automated histogram filtering,(25) replica exchange monte carlo,(26) statistical temperature molecular dynamics,(27) and conformational space annealing(28) indicate that bln-69 has a frustrated potential energy surface . The energy landscape of the bln-69 protein is less well understood than that of its 46-residue counterpart . With more degrees of freedom, it has a much larger conformational space, but how frustrated is it? To answer this question, we analyze the energy landscape of bln-69 and the corresponding go model using disconnectivity graphs, where minima are connected by nodes representing the highest transition state on the lowest pathway between them . Finding the global minima of frustrated systems, such as the bln proteins, we assess the performance of basin - hopping and genetic algorithms on the bln model proteins . The disconnectivity graphs for the 69-residue bln and go model proteins were constructed from databases of stationary points generated using the pathsample program,(31) which organizes independent pathway searches using the optim program. (32) the databases of stationary points include 298 856 minima and 258 477 transition states for the bln potential, and 53 901 minima and 75 389 transition states for the go potential . All the transition state searches in optim were conducted in cartesian coordinates(33) using a quasi - continuous interpolation scheme to avoid chain crossings, with local maxima accurately refined to transition states by hybrid eigenvector - following . Successive pairs of local minima were selected for connection attempts within optim using the missing connection algorithm. (37) performance of a local energy minimization after structural perturbations, in combination with an accept / reject test, transforms the potential energy surface into the basins of attraction of local minima,(38) and removes downhill barriers. (39) the resulting basin - hopping (bh) algorithm has proved very effective for locating the global minimum in a wide range of systems . In the present work all bh searches were performed using the gmin program. (42) to improve the efficiency, a restart procedure was used with the newrestart keyword . If the lowest minimum did not change for a fixed number of steps (30 000 in the present work), a new search was initiated from a random starting structure . A cyclic taboo list(43) of 10 structures was also employed to prevent revisits to configuration space that had already been explored, using the avoid keyword with a distance tolerance of 2.0 for reseeding, in reduced units . Genetic algorithms (ga) are another popular type of global optimization algorithm . If all structures are subjected to energy minimization before fitness evaluation, the resulting lamarckian genetic algorithms have also been very effective in optimizing the structures of clusters(46) and proteins . In our ga, each structure is represented by a genome comprising the sequence of torsional angles describing the protein backbone conformation . Offspring structures are generated by one - point crossover where the genomes from both parents are cut at the same random point and the offspring s genome comprises one section from each parent . Mutants were generated by making copies of the parent and offspring structures and replacing one randomly selected torsion angle . We use an elitist genetic algorithm, where parent structures are retained in the population . After each generation, all structures (parents, offspring, and mutants) are sorted in order of fitness and the fittest are taken forward to the next generation . Thus, the energy of the least fit member of the population is always less than or equal to that of the least fit in the previous generation . If no better structures are found in a generation, a new epoch is initiated and all members of the population are replaced with random structures . Optionally, a small number of the fittest structures can survive from one epoch to the next . To maintain population diversity, a duplicate predator operator is used. (49) if two members of the population have the same energy (within 0.1) and the same torsional angles in the backbone (within 10), the least stable one is removed from the population . The global minimum structure for the 69-bead bln protein has the three b9 chains forming a hydrophobic core, which is surrounded by the three (lb)4 chains (figure 1), as characterized in previous studies . The bln-69 disconnectivity graph is frustrated (figure 2), with a number of deep funnels, each of which leads to a structure close in energy to the global minimum . There are at least 33 other structures within of the global minimum . Of the 10 lowest - energy structures, four lie in the same basin as the global minimum and only differ by changes in the turn regions . The other six low - lying minima lie at the bottom of distinct funnels and have different arrangements of the -strands (figure 1). Although the overall landscape is frustrated and multifunnel in character, local relaxation within individual funnels to the alternative low - lying minima is expected to be quite rapid . This structure is associated with multiple relaxation time scales for the global minimum and features in the heat capacity . The five most stable structures of the 69-residue bln protein, ordered in increasing energy with the global minimum on the left . Residues are colored red (b), blue (l), and green (n). The third structure is in the same basin as the global minimum and differs only by the position of the turn residues . Disconnectivity graph of the 69-residue bln protein showing the 11 343 minima accessible from the global minimum by transition states lower than 8. the five lowest minima are illustrated close to the bottom of the branches to which they correspond . These representations were generated using the vmd program (ref (53)) with a coloring scheme for the beads that varies from red to blue (n - terminus to c - terminus). The 69-residue go model (go-69) has a funneled energy landscape, where most of the local minima are linked to the global minimum by barriers less than 4 (figure 3). There are only three other structures within of the global minimum, and they are all connected by low - energy transition states . These structures only differ from the global minimum by small rearrangements of the turn regions . It is interesting to note that the disconnectivity graphs for both the 69-residue go and bln proteins are similar for structures accessible by transition states lower than 6 from the global minimum (figure 4). This structure implies that the energy landscape for the bln protein in this region is described by the making and breaking of native contacts, with little influence from non - native contacts . Disconnectivity graph of the 69-residue go protein including the 1061 minima accessible from the global minimum by transition states lower than 8 relative to the global minimum . The structure of the global minimum is illustrated using the vmd program (ref (53)) with a coloring scheme for the beads that varies from red to blue (n - terminus to c - terminus). Disconnectivity graphs of the 69-residue go (left) and bln (right) proteins including the minima accessible by transition states lower than 6 relative to the global minimum . The performance of the search algorithms was assessed for both the 46- and 69-residue bln proteins . For each system, 100 searches were performed from random starting points and the mean time to the first encounter of the global minimum structure was recorded . For ga, the first - encounter time was taken at the end of the generation where the global minimum was found . We present the mean first - encounter time in terms of the number of energy evaluations and the number of minimization steps . We consider the number of minimization steps to be the fairest test of the performance of the search algorithm because the number of energy evaluations depends on the convergence criteria used in the local energy minimization . Both of the search algorithms have a number of parameters that can be optimized to improve performance, and the best sets of parameters for the bh and ga searches are listed in table 1 . Searches were also performed for the go model proteins using the same search parameters . The most effective optimization algorithm for bln-46 reported in previous work is bh, which required an average of 6000 energy minimizations to find the global minimum. (17) the introduction of a taboo list and a restart operator reduce this figure to 4400 minimizations in the present work (table 2), which required an average of 79 s of cpu time on an intel xeon e5405 cpu (single core, clock speed 2.0 ghz). The 46 beads were randomly placed in a sphere of radius 3.0 to generate the initial configurations . The other searches prematurely converge to other minima, and after 75 000 generations 10% have not found the global minimum . Introducing the epoch operator improves the performance of the ga, with all searches finding the global minimum within three epochs (210 generations). The best performance is obtained when no structures are carried over from one epoch to the next . For the bln-69 protein the bh algorithm requires an average of 26 000 energy minimizations to locate the global minimum (table 3), which corresponds to an average of 1100 s cpu time on an intel xeon e5405 cpu (single core, clock speed 2.0 ghz). Our ga requires a similar number of minimizations to find the global minimum (table 3). The performance of the new epoch operator is improved if the fittest structure from each epoch is transferred to the next . The conformational space annealing algorithm has the best published performance for this system, requiring an average of 9.4 10 energy evaluations to find the global minimum. (28) however, the number of minimizations is not given, and therefore it is difficult to separate the performance of the conformational space annealing algorithm from the choice of minimization algorithm . Values in parentheses are the standard deviations . The 69 beads were randomly placed in a sphere of radius 3.0 to generate the initial configurations . As expected, the global minima of the go proteins are significantly easier to find than for the bln proteins . With bh, there is a 10-fold reduction in the number of energy minimizations required when moving from the bln potential to the go potential for both chain lengths . With ga, the number of minimizations required is a factor of 3 smaller for the go proteins . We have shown that the bln-69 protein has a frustrated potential energy landscape, with multiple low - energy minima lying at the bottom of funnels separated by high barriers . Nevertheless, both the ga and bh algorithms locate the global minimum quite efficiently for both bln-46 and bln-69, with significant improvements over previous work . Searches for bln-69 require between four and six times more energy minimizations than for bln-46 . In future work we plan to examine this scaling in terms of metric disconnectivity graphs and measures of landscape complexity. (19) another area of interest is to investigate the energy landscapes and ease of global optimization for potentials that are intermediate in character between the bln and go limits, in order to identify the transition from a poor to a good folder . This analysis would also allow us to understand the effect of non - native interactions with different strengths on the thermodynamics and kinetics of protein folding.
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Considered as an organ, the collective mass of hematopoietic bone marrow in a healthy adult is greater than that of the liver . The major proportion of this hematopoietic activity is directed to producing neutrophil leukocytes . Each day a healthy human adult releases about a hundred billion neutrophils into the circulating blood . This baseline production is greatly expanded in inflammatory states and by treatment with a granulocyte - colony stimulating factor (g - csf) such as filgrastim . In addition to stimulating neutrophil hyperplasia, that is, increased production, g - csf treatment also results in hypertrophic changes, that is, larger neutrophils with severalfold greater azurophilic granule content and proportionally increased myeloperoxidase (mpo). The neutrophil serves as the principal leukocyte of the acute inflammatory response and is the primary microbicidal phagocyte of the innate and acquired immune defense systems . Accomplishing such when stimulated by microbial products, complement activation peptides, cytokines, et cetera, neutrophils fuse their specific (a.k.a ., secondary) granules with their cytoplasmic membrane (i.e., specific degranulation), thus providing the increased surface - to - volume ratio necessary for locomotion and exposing the cytokine receptors and opsonin receptors required for close endothelial contact and diapedesis (i.e., transit) from the vascular space through the endothelial lining into the tissue interstitial space . Chemotactic locomotion to the site of infection is directed by concentration gradients of bacterial products, anaphylatoxin, cytokines, et cetera . Contact and receptor - mediated recognition of an opsonified (e.g., complement-, immunoglobulin - labeled) microbe results in phagocytosis, formation of a phagosome, and fusion with azurophilic (a.k.a . The morphologic changes of phagocytosis are associated with magnitudinal increase in hexose monophosphate shunt metabolism of glucose and with proportionally increased molecular oxygen (o2) consumption, that is, the respiratory burst . This mitochondria - independent metabolic activity is required for effective microbicidal action [5, 6]. The resulting microbicidal oxygenation reactions have exergonicities sufficient to produce electronic excited products yielding light emission in the visible spectrum, that is, chemiluminescence or luminescence . Two reducing equivalents (i.e., two electrons (e) plus two protons (h)) are transferred from nadph to the oxidase where they distribute in a manner allowing for univalent (one equivalent) reduction of o2, yielding hydrodioxylic acid (ho2; a.k.a . Ho2 is an acid with a pka of 4.9 and, as such, dissociates yielding a proton and its conjugate base, the superoxide anion (o2). Production of ho2 within the neutrophil phagosome or phagolysosome dynamically acidifies the confined space . As the ph of the space approaches the pka (i.e., 4.9), the ratio of ho2 to o2 approaches unity, removing the anionic barrier to direct radical - radical disproportionation [10, 11]. In such acidic milieu, ho2 reacts directly with o2 to produce hydrogen peroxide (h2o2) and singlet molecular oxygen (o2) [1012]. These activities provide the optimal milieu and h2o2 substrate for myeloperoxidase (mpo) oxidation of chloride (cl) to the chloronium (cl) state yielding hypochlorous acid (hocl). Spontaneous reaction of hocl with an additional h2o2 yields cl, h2o, and o2 . The order of electronegativity values for fluorine (f), oxygen (o), and chorine (cl) is 4.0, 3.5, and 3.0, respectively . The energy difference separating o2 from water (h2o) is large, that is, a difference of 1.23 volts (v) or 96.5 kilocalories per mole (kcal mol), and provides the driving force of life on earth . The merriam - webster dictionary defines combustion as a chemical reaction that occurs when oxygen combines with other substances to produce heat and usually light . As such the heat of reaction calculations for the reaction of o2 with ethylene liberates 93 kcal mol (i.e., 389 kilojoules per mole (kj mol)) or the energy equivalent to an einstein (i.e., one mole) of ultraviolet photons . The electronegativity of oxygen predicts the high exergonicity of oxygenation reactions, but such reactions are not spontaneous . Placed together in a chamber, o2 does not react with ethylene . For reaction to occur, a photon or spark with energy sufficient to initiate ethylene bond homolytic cleavage must be applied . This presentation addresses the questions: why does not oxygen spontaneously react with organic material? Why is oxygen reduction, a process that decreases its thermodynamic potential, required as a first step for neutrophil combustive microbicidal action? And what is the best viewpoint for considering oxygen reactivity? The review provides the background physics and chemistry for a fundamental perspective with regard to oxygen reactivity in particular, neutrophil combustive microbicidal action with its associated luminescence, and reaction chemistry in general . Hopefully, it will encourage interested researchers out of their comfort zone and broaden their scientific outlook . Particles are of two symmetry types, each with unique statistical mechanical properties [17, 18]. They are either bose - einstein particles (bosons) or fermi - dirac particles (fermions). According to the exchange principle, a pair of particles, that is, a and b, can be described by a wave function, (a, b), representing the space and spin coordinates of the particles . Even if the particles are indistinguishable (e.g., electrons), the particle sites are distinguishable . Each site is unique with regard to its spin - state, that is, spin - up () or spin - down (). Exchange can be symmetric: (a, b) = (b, a); or exchange can be antisymmetric: (a, b) = (b, a). The wave functions of bosons are symmetric to exchange of a pair of particles; that is, (a, b) = (b, a). Bosons obey ordinary commutation; a b = b a. rotating a boson through 360 degrees, 360, returns it to its original state . . Photons, the force carrier particles of electromagnetic energy, are bosons and are described by the planck equation: e = h, where e is energy, h is planck's constant, and is frequency . Such products include large bosons with mass, such as alpha () particles . The character of atomic and molecular orbitals can likewise be considered as bosonic or fermionic . As will be developed subsequently, the frontier orbital of an atom or molecule is bosonic if composed of paired antisymmetric electrons . As such, the wave functions of fermions are antisymmetric to exchange of particles; that is, (a, b) = (b, a). Fermions are characterized as having half - integer spin and appear as multiples of the basic unit (1/2), where (h - bar) equals planck's constant (h) divided by 2. fermions anticommute; that is, a b b a. rotating a fermion through 360 degrees, 360, changes the phase but does not return the fermion to its original state . An additional 360 degrees' rotation, 360, is required to return the antisymmetric particle to its original state . The frontier orbitals of atoms, such as hydrogen (h), nitrogen (n), and oxygen (o), and molecules, such as o2 and nitrous oxide (no), have singly occupied atomic or molecular orbitals (so(a)mo). Atoms and molecules with fermionic orbitals are radical and paramagnetic . Atomic hydrogen (h) is composed of a positively charged nuclear proton (h) and a negatively charged electron (e). H is thousandfold more massive than e, and, as such, the kinetic and potential energies of e are described as its orbit relative to the massive h. the orbital wave function describes the energy possibilities . Using polar coordinates, the position of e can be described as its distance, r, from its nucleus and two angles, and . The total wave function is isolated into 3 separate contributions, (r,,) = r(r)()(), where r(r) is the radial component and () and () are the angular components . The radial component yields the principal quantum number, n. the angular components yield the azimuthal quantum number, l, and the magnetic quantum number, ml . The degree of orbital degeneracy is the square of the principal quantum number, n. when n = 1, the degeneracy is 1 = 1, yielding the 1s orbital . When n = 2, the degeneracy is 2 = 4, yielding the 2s, 2px, 2py, and 2pz orbitals . The azimuthal quantum number, l, describes the shape of the orbit and the orbital angular momentum of e. the magnetic quantum number, ml, describes the number of orbitals with a given value of l. the value of the total orbital angular momentum, l, is l = [l(l + 1)]. This intrinsic quantum mechanical property, that is, spin, is described by the spin quantum number, s. the magnitude of s is restricted to a value of 1/2 . The total spin angular momentum, s, of a system is expressed by the equation s = [s(s + 1)]. The intrinsic spin with its value of (1/2) (abbreviated to 1/2) is a quality of fermions without analogy in classical physics . Just as l gives rise to ml, s gives rise to the spin quantum number ms . Only two values are allowed for ms . When ms = 1/2, e is described as spin - up (); when ms = 1/2, e is described as spin - down (). Each e of an atom or molecule is defined by its five quantum numbers: n, l, ml, s, and ms . The pauli exclusion principle states that no two electrons of a given atom can have identical quantum numbers; that is, the total wave function for a system must be antisymmetric to the exchange of any pair of electrons . For an orbit to accommodate two electrons, the electrons must have opposite spins . If one orbital e has ms = 1/2 (), the other orbital e must have ms = 1/2 (). As such, the total spin quantum number, s, for an orbital electron - couple is 1/2 + 1/2 = 0 (). Orbital coupling of the fermionic electrons results in spin - neutralization . In effect, the antisymmetric fermions combine into a symmetric boson . This concept is illustrated in figure 2 by the reaction of two h atoms to generate molecular h2 . Multiplicity is a spectroscopic term and indicates the number of wave functions possible for the system; that is, singlet indicates 1, doublet indicates 2, triplet indicates 3, et cetera . In its ground (lowest energy) electronic state, atomic h has a single e in the 1s orbital and has an s value of 1/2 or 1/2; thus |2(1/2 or 1/2)| + 1 = 2; the multiplicity is doublet . For molecular h2 the value of s is 0; thus, |2(0)| + 1 = 1; the multiplicity is singlet . In figure 2, the spin quantum number of each e is represented as spin - up (ms = 1/2 =) or spin - down (ms = 1/2 =). The circle surrounding the orbital electron - couple of the homo of h2 symbolizes the bosonic character of the filled orbital . When the electrons are antisymmetric, that is, and, overlap is constructive resulting in chemical bonding; that is, there is an increased probability of finding electrons in the internuclear region between the two h nuclei . Combining the two atomic 1s orbitals yields the bonding sigma molecular orbital, . Note that bonding lowers the energy of the system . When the electrons are and or and, the overlap is destructive and no bonding occurs . Frontier orbital theory focuses on the initial orbital conditions of the reactants and on reactive transition with special emphasis on the highest occupied and lowest unoccupied orbitals [21, 22]. The frontier orbitals, that is, the highest occupied atomic or molecular orbital (ho(a)mo) and the lowest unoccupied molecular orbital (lumo), define the reactive possibilities . As depicted in figure 2, the electrons of the singly occupied atomic orbital (soao) of the two h atoms constructively overlap in a radical - radical (i.e., doublet - doublet) annihilation producing the bonding homo orbital of singlet multiplicity, diamagnetic molecular hydrogen (h2). For the h atoms, the electrons of the 1s orbitals have identical energies, but with covalent bonding to form h2, the wave functions overlap and split producing two molecular orbitals, one with lower and one with higher energy than the original 1s atomic orbitals . The bonding orbital () is lower in energy and stable, thus promoting bonding . The antibonding orbital () is of higher energy . Populating the antibonding orbital promotes bond breaking . Consequently, if a photon of sufficient frequency is captured by a electron, its electronic excitation to the orbital results in h2 bond cleavage . The wigner - witmer spin conservation rules describe reaction symmetry possibilities in terms of reactant and product multiplicities [24, 25]. The spin conservation rules state that the overall spin angular momentum of a system must be conserved . Reactant and product state possibilities can also be considered in terms of fermionic and bosonic frontier orbital character . In table 1 reactants and products are presented in terms of multiplicity and also in terms of bosonic or fermionic orbital character . As depicted in figure 2, combining two doublet multiplicity h atoms produces singlet multiplicity h2 . With regard to orbital spin character, the two antisymmetric fermionic electrons of the atomic orbitals are coupled (condensed or combined) resulting in the bosonic electron pair of the orbital of h2 . Since the pauli principle requires the total wave function for any system of electrons to be antisymmetric to exchange an orbital pair of electrons, only phase - opposite electrons can occupy a given orbital state . Hund's maximum multiplicity rule states that the electronic configuration with highest multiplicity has the lowest energy . The greater the number of wave functions possible for a system, the lower the energy . Figure 3 depicts the combination of two nitrogen atoms (n) to yield molecular nitrogen (n2). Ground state atomic n is a paramagnetic triradical with one electron in each of its three 2p orbitals . Note that each electron has the same ms value resulting in quartet multiplicity; that is, |2(3(1/2 or 1/2))|+1 = 4 . Combining the atomic orbitals of the two quartet multiplicity nitrogen atoms (n) generates the filled (bosonic) (1s and 2s) and orbitals; constructive overlap of the phase - opposite electrons of the three 2p orbitals of the two n atoms generates one and two bonds of triple - bonded ground state singlet multiplicity n2 . The triradical n atoms combine to produce nonradical n2 . From the fermion - boson frontier orbital perspective, each n presents a complex of trifermionic soaos, that is, (i.e., s = 3(1/2)) or (i.e., s = 3(1/2)). Consistent with table 1, antisymmetric coupling produces triple - bonded bosonic (i.e., s = 0) n2 . Frontier orbital overlap of two oxygen atoms with antisymmetric somo is constructive producing o2 as depicted in figure 4 . As per table 1, combining two ground state triplet multiplicity paramagnetic, diradical oxygen atoms (o) produces an electronically excited singlet multiplicity, diamagnetic, nonradical o2 . Note the product o2 obeys the spin symmetry rules but violates hund's maximum multiplicity rule . As such, o2 is electronically excited (indicated by) with an energy of 22.5 kcal mol greater than that of triplet multiplicity ground state o2 . The spin conservation rules predict that the change in spin multiplicity, that is, a singlet - to - triplet transition, is of low probability . As such, electronically excited o2 is metastable with a relatively long half - life . The estimated four - microsecond lifetime of o2 is sufficient to allow its participation as an electrophilic reactant in dioxygenation reactions . However, such reactions are restricted to within a radius of about 0.2 microns (m) from its point of generation [27, 28]. As depicted in figure 5, unreacted o2 relaxes to its triplet multiplicity ground state (o2) by emitting a 1270 nm (near infrared) photon . As per the maximum multiplicity rule, ground state molecular oxygen is a triplet multiplicity, paramagnetic diradical with one e occupying each of its two somos; it is bifermionic . Ground state o2 can be in either the (i.e., s = 1/2 + 1/2 = 2(1/2) = 1) or the (i.e., s = 1/2 1/2 = 2(1/2) = 1) state; thus, the multiplicity is |2(1) + 1| = 3, that is, triplet . The vast majority of biological molecules are singlet multiplicity that is, s = 0and, as such, present bosonic frontier orbitals . For such molecules, chemistry is confined to bosonic homo - lumo exchange of a composite orbital electron - couple . Antisymmetric fermionic frontier orbital overlap is constructive resulting in covalent bonding of the bosonic singlet product . As described in table 1, constructive somo - somo overlap of a doublet reactant and a triplet reactant produces a doublet product; that is, the fermionic character is preserved . Direct reaction of ground state triplet multiplicity o2 with singlet multiplicity organic molecules violates the spin conservation rules, and, as such, combustion is not spontaneous . Overlap of the bifermionic (the 2 somo) frontier orbitals of triplet multiplicity o2 with the empty lumo or bosonic homo of singlet multiplicity organic molecules is not constructive . Such reactions are symmetry - restricted, and the only product possible would also be a bifermionic triplet . O2 with its triplet multiplicity ground state the relaxation of such excited triplet multiplicity molecules to their singlet ground state, that is, triplet - to - singlet transition, violates the conservation rules and is of low probability . The delayed relaxation of an excited triplet to its singlet ground state by photon emission is responsible for the phenomenon of phosphorescence . Considering the complexity and variety of potentially pathogenic organisms, neutrophil microbicidal action must be simple, potent, and focused so as to maximize microbicidal action and minimize collateral damage . Microbicidal combustion is realized by changing the frontier orbitals of oxygen from bifermionic to bosonic . Oxygenation reactions yield electronically excited products that relax to ground state by emission of light in the visible spectral range . Respiratory burst metabolism mobilizes the reducing equivalents required for changing the frontier orbitals of o2 . When it does occur, the semiquinone of a riboflavin prosthetic group is typically involved . Such flavoenzymes mark the departure from two equivalent cytoplasmic transfers to one equivalent cytochrome transfer . One equivalent reduction of o2 changes its multiplicity from triplet to doublet, thus reducing its overall fermionic orbital character . The flavocytochrome enzyme nadph oxidase catalyzes the reduction of bifermionic triplet multiplicity o2 to fermionic doublet multiplicity ho2 [9, 10]. Acidic dissociation of ho2 yields its conjugate base, doublet multiplicity superoxide anion (o2). As per figure 1 and table 1, direct somo - somo orbital overlap of ho2 with o2 produces singlet multiplicity ground state h2o2 plus o2 [10, 12]. The h2o2 produced serves as substrate for myeloperoxidase (mpo) oxidation of chloride producing hypochlorous acid; that is, h2o2 + cl h2o + hocl . This reaction involves the bosonic homo of h2o2 and the bosonic lumo of hocl producing the intermediate singlet multiplicity chloroperoxy acid (hoocl) and singlet multiplicity h2o . Disintegration of the chloroperoxy intermediate yields ground state singlet multiplicity chloride and electronically excited singlet multiplicity oxygen . The net reaction is h2o2 + hocl o2 + h2o + cl + h. the bosonic orbital symmetry of o2 allows direct constructive spin - allowed overlap with the bosonic frontier orbitals of biological substrates producing singlet multiplicity dioxygenated products of bosonic symmetry . In conventional combustive burning, sufficient energy must be applied to a nonradical (singlet) substrate to produce homolytic bond cleavage yielding two radical (doublet) products . The fermionic somo of these doublet multiplicity radical products can now constructively overlap with one of the fermionic somos of triplet multiplicity ground state o2 . As described in table 1, the product of doublet - triplet reaction will have doublet multiplicity . Such radical - diradical reactions yield heat plus additional radical products that can further participate in the radical propagation process of burning . Neutrophil microbicidal action is combustion by a different pathway . Instead of radicalizing a substrate to facilitate reaction with diradical o2, the bifermionic orbitals of ground state triplet o2 are converted to the bosonic orbitals of o2 . In its electronically excited singlet multiplicity state, the bosonic frontier orbitals of oxygen can participate in spin - allowed electrophilic oxygenation reactions with a broad spectrum of singlet multiplicity biological molecules . As per spin conservation, such products are nonradical, diamagnetic, and unable to participate in the radical propagation reactions that are characteristic of burning . The microsecond lifetime range of o2 confines reactions to within about a 0.2 m radius from its point of origin [27, 28]. Thus, phagolysosomal generation of o2 in close proximity to the target microbe guarantees that combustive action is focused on the microbe with minimal collateral damage to the host . By changing the spin quantum number of oxygen, the neutrophil not only realizes its potent electrophilic microbicidal action, but also limits such combustive action to the target microbe . One carbonyl is in the ground singlet multiplicity state, and the other carbonyl is in the electronically excited n singlet multiplicity state . As depicted in figure 6, the n description indicates that an electron from the ground state nonbonding (n) orbital of the atomic oxygen (o) component occupies the orbital of the electronically excited carbonyl function . When the spin of the excited electron is antisymmetric to that of the ground state n electron of o, the electronically excited carbonyl is singlet multiplicity . Thus, -to - n relaxation to ground state is spin - allowed and yields a photon with energy proportional to the difference between the two orbital states . In bioluminescence and chemiluminescence processes, dioxygenated products disintegrate by oxygen - oxygen bond cleavage producing the singlet multiplicity n electronically excited carbonyl ., electronic excitation to the n state can occur when a photon of proper energy is captured by an electron in the nonbonding (n) orbital of the o component of the carbonyl . As depicted in figure 6, relaxation of the electron from of the singlet excited carbonyl back to the n orbital of o results in photon (h) emission and returns the carbonyl function to its ground singlet state . The frequency of the emitted photon is the energy difference that separates the n orbital from the orbital . Photons are symmetric bosons; their absorbance or emission does not affect the electron spin . In fluorescence, photon capture promotes an electron of an orbital to an appropriately higher energy orbital . Such photon excitation results in transient antibonding and fermionic character, but the overall spin symmetry is retained; that is, singlet character is retained . Relaxation of an n electronically excited singlet multiplicity carbonyl to its singlet ground state by fluorescence or chemiluminescence is spin - allowed, and, as such, the lifetime of the excited state is very short - lived, typically in the nanosecond range . The chemiluminescence produced by neutrophils is an energy byproduct of microbicidal combustion and varies with the molecular composition of the microbe . Different microbes present substrates that vary with regard to dioxygenation susceptibility and combustion quantum efficiencies, that is, the photon yield per dioxygenation reaction . Native luminescence is easily measured using a luminometer, but the photon yield is insufficient for measurement of less than a hundred thousand neutrophils . In order to increase photon yield and impose reaction specificity, chemiluminigenic substrates are susceptible to dioxygenation producing endoperoxides or dioxetanes ultimately yielding n electronically excited carbonyl products . In addition to increasing sensitivity, chemiluminigenic substrates can be selected for specificity with regard to the type of oxygenation activity measured . The sensitivity and specificity obtained using selective chemiluminigenic probe in combination with phagocytic and chemical stimuli allow simultaneous quantification of multiple neutrophil metrics using a microliter or less of unseparated whole blood . When subjected to discriminant function analysis, such neutrophil luminescence measurements allow assessment of host inflammatory state and hematopoietic marrow stress [3235]. Neutrophil leukocytes respond to cytokines and other agents by fusing specific granules with the cytoplasmic membrane, that is, specific degranulation . Such fusion effectively increases the surface - to - volume ratio of the neutrophil, as is required for locomotion and phagocytosis, and for upregulated membrane expression of cytokine and opsonin receptors, as is required for effective recognition and phagocytosis . Microbicidal effectiveness is optimized by coordinating phagocytosis with activation of nadph oxidase, resulting in dynamic acidification and h2o2 production . Fusion of the lysosomal azurophilic granules with the microbe - containing phagosome produces a phagolysosome with a relatively high mpo concentration in an acidic milieu replete with h2o2 . The neutrophils of patients with chronic granulomatous disease (cgd) have defective nadph oxidase function and consequently have defective microbicidal combustion and do not chemiluminesce . These cgd neutrophils phagocytose microbes and release mpo into the phagolysosomal space, but the neutrophil respiratory burst is defective and microbicidal action is poor . Consequently, cgd patients have an increased susceptibility to infection . Interestingly, the incidence of pneumococcal and other streptococcal infections is not increased in cgd patients; that is, infection rates for these microbes are about equivalent to those for healthy subjects . Pneumococci and streptococci are cytochrome - deficient lactic acid bacteria (lab) that produce lactic acid and h2o2 as metabolic products [38, 39]. Phagocytosis of live lab by cgd neutrophils does result in microbicidal action . In the case of cgd neutrophils, the phagocytosed lab provides the acid and h2o2 necessary for effective mpo - mediated microbicidal combustion and chemiluminescence . Human neutrophils contain a relatively high concentration of mpo (about 5% of the dry weight of the neutrophil) in their azurophilic granules . Following departure of the neutrophil leukocyte from the hematopoietic marrow and a relatively short transit time in the circulating blood, the neutrophil leaves the circulating and enters the tissue pool . However, even in the absence of inflammation and infection, healthy humans have significant concentrations of neutrophils present in tissue fluids . The oral and vaginal cavities have neutrophil concentrations in proportion to blood neutrophil concentrations [41, 42]. When neutrophils transit from the blood to tissue and body spaces, they carry mpo into these spaces . The oral and vaginal cavities have acidic ph . The normal florae of these spaces are cytochrome - negative lab that generate lactic acid and h2o2 as metabolic products . The transit of neutrophils into such spaces delivers mpo into milieux that are rich in lab . However, lab show relatively poor mpo binding . When small concentrations of neutrophil - free mpo are added to bacterial suspensions containing viridans streptococci with other competing bacteria, a potent synergistic microbicidal action is directed against mpo - binding gram - positive, for example, staphylococcus aureus, and gram - negative, for example, pseudomonas aeruginosa and escherichia coli, microbes . This lab - mpo synergistic action may explain the dominance of streptococci and lactobacilli in healthy oral and vaginal cavities . Selective binding of mpo to a microbe guarantees that o2 is generated in close proximity to the target microbe, resulting in selective, focused combustive microbicidal action . The o2 lifetime of a few microseconds limits damage to within about a 0.2 m radius of its point of generation [27, 28]. Thus, combustion is focused on potential pathogens with minimal collateral damage to the host cells and the normal lab flora . These rules forbid the direct reaction of triplet oxygen with singlet organic molecules, and, as such, combustion is not spontaneous . Chemical reactions involve frontier orbitals . Changing the multiplicity of oxygen changes its frontier orbitals, eliminating the symmetry barrier to its electrophilic reactive potential . The redox reactions of neutrophil respiratory burst metabolism change triplet ground state (diradical) o2 to singlet electronically excited (nonradical) o2 . In addition, other singlet multiplicity (nonradical) microbicidal reactants, for example, h2o2 and hocl, are generated . O2 has one empty (lumo) orbital and one full (homo) orbital with bosonic character . This highly electrophilic lumo can participate in spin - allowed bosonic lumo - homo reactions with biological molecules . The dioxygenated products of such combustions yield the electronically excited carbonyl functions that relax by photon emission, that is, luminescence or chemiluminescence . Considering frontier orbital reactivity in the bosonic - fermionic terms of particle physics broadens the perspective for explaining oxygen chemistry in particular and for appreciating reaction chemistry in general . Radical reactions involving singly occupied atomic or molecular orbitals (so(a)mo) are uncommon in biology under normal conditions, that is, in the absence of high energy radiation or heat producing homolytic bond cleavage . Biological reactions involving one electron transfer are typically restricted to flavoenzymes and to highly ordered cytochrome electron transport systems such as those found in mitochondria . Chemical reactions can be generalized as constructive bosonic transfers (i.e., homo - lumo) or constructive fermionic annihilations . Radical - radical (i.e., so(a)mo - so(a)mo) orbital overlap is typically constructive; that is, fermionic radicals react with fermionic radicals . Overlap of a bosonic homo with a fermionic so(a)mo is not constructive . As illustrated in figure 1, the first steps of the hexose monophosphate metabolic pathway involve dehydrogenation of glucose-6-phosphate and reduction of nadp yielding nadph; these redox reactions involve balanced two equivalent bosonic transfers . The bosonic nature of the antisymmetric orbital electron - couple may be essential for such biologic redox transfer . There are advantages to transferring electrons as a bosonic orbital - couple . As stated by dirac, if a state has zero total angular momentum, the dynamical system is equally likely to have any orientation, and hence spherical symmetry occurs . This has significance with regard to the heisenberg uncertainty principle which states that the uncertainty of momentum (p) multiplied by the uncertainty of position (x) is always equal to or greater than (1/2); that is, px (1/2). The spin momentum of the bosonic orbital electron - couple is known, that is, zero . Thus, the intermolecular redox transfer of a bosonic orbital electron - couple may proceed by a quantum tunneling mechanism analogous to the emission of a bosonic alpha particle from an atomic nucleus in alpha radiation decay . Such a quantum tunneling mechanism explains the facility for electron - couple transfer in biologic redox reactions.
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When cells migrate, they extend dynamic membrane - bound actin - rich tubular protrusions known as filopodia . Formins are a family of large multi - domain proteins that nucleate and polymerize actin to form linear actin filaments like those found within filopodia . In this review formins function as dimers and nucleate actin by means of a formin homology 2 (fh2) domain that binds globular actin monomers . Interaction of the adjacent formin homology 1 (fh1) domain with profilin effectively recruits actin monomers to the formin dimer, facilitating the polymerization process . A subset of formins, known as diaphanous - related formins (drfs), bind to and drfs are rendered inactive by interaction of a c - terminal diaphanous autoregulatory domain (dad) with an n - terminal diaphanous inhibitory domain (did) (fig . 1). The binding of a rhogtpase to the n - terminal gtpase binding domain (gbd) contributes to disruption of this autoinhibitory interaction, which results in the activation of the drf . In the case of the drf fhod1, phosphorylation of c - terminal serine and threonine residues by rock other domains found in drfs include a a dimerization domain (dd) and a coiled coil (cc) region, and some groups believe that the dd and did together constitute a loosely defined formin homology 3 (fh3) domain . An n - terminal phospholipid - binding basic domain (bd) mdia1 has three stretches of basic amino acids in this domain, which allows it to localize to the plasma membrane, while mdia2 has two . In contrast, the drf daam1 has only one stretch of basic amino acids in its n - terminal, and the distribution of constitutively active daam1 is restricted to the cytoplasm . This might explain why only mdia1 and mdia2 have been implicated in the formation of membrane - bound cell surface protrusions like filopodia and lamellipodia, while mdia3lacking this bd has only been reported to generate cytoplasmic actin structures . Several rhogtpases and other proteins have been reported to bind to mdia1 - 3 . These are summarized in figure 1 . The shortest fragment(s) known to bind the respective mdia isoforms is shown for each interacting protein . Excluded from this diagram are ywk - ii, which binds a 223 aa fragment of hdia1 that shares 96% aa sequence identity with mdia1 (903 - 1125 aa), and inf2, which binds to aa 1181 - 1262 and aa 1051 - 1193 of what might be longer splice variants of mdia1 and mdia3 respectively . Mdia1 - 3 form several types of actin - based cellular structures . Within the cytoplasm, mdia1 gives rise to stress fibers, and mdia2 drives the actin dynamics that power vesicle movement and creates the actin scaffold for constriction of the contractile ring during cytokinesis . At the plasma membrane, both mdia1 and mdia2 a role for formins in lamellipodial protrusion is not surprising once thought to be structures comprised of dendritic networks of branched microfilaments, lamellipodia have recently been reported to contain linear actin filaments as well . We have found mdia3 to be capable of inducing filopodia in n1e115 neuroblastoma cells, despite not being able to detect the presence of endogenous mdia3 protein in this particular cell line . All three mdia isoforms have also been linked to invadopodia protrusion, while mdia2 alone plays a role in forming the filopodial precursors of dendritic spines . Actin dynamics leading to the formation of the phagocytic cup in macrophages are believed to involve mdia1 and mdia2 too . Previous studies have pointed to a role for mdia2 in mammalian filopodia . A decrease in filopodial protrusion was seen in mouse fibroblasts overexpressing constitutively active cdc42 and microinjected with anti - mdia2 antibodies, as well as cells cotransfected with activated cdc42 and a non - functional mdia2 mutant . In nih3t3 fibroblasts, constitutively active mdia2, when overexpressed alone in b16f1 melanoma cells, also accumulated at filopodial tips . Furthermore, knockdown of mdia2 protein in mouse hippocampal neurons reduced the formation of the filopodial precursors of dendritic spines . In more recent work, mdia1 - 3 have been shown to induce filopodia in neuronal cells when overexpressed on their own . However, only mdia1 was seen within filopodia, as observed by time lapse imaging of live cells . The lack of mdia2 and mdia3 in neuronal filopodia implies that these drfs might be involved in the initiation of filopodium formation but not the elongation of the structures . One possibility is that mdia2 and mdia3 generate short microfilaments that are subsequently elongated by mdia1 to form mature filopodia . This would tie in with the suggestion that mdia2 is a relatively strong nucleator but poor elongator of microfilaments, based on observations that it elongates microfilaments at a slower rate than mdia1 . The filopodia induced by full - length wildtype mdia2 appeared cylindrical and of even thickness along their length, and so did the filopodia formed by fh1fh2-mdia2, a fragment of mdia2 that consists of only the fh1 and fh2 domains . This is unlike the club - shaped filopodia obtained by transfecting b16f1 cells with constitutively active mdia2, where the structures are packed with shorter microfilaments at their distal ends but contain relatively few long microfilaments that extend toward the base of the protrusions . The high overexpression of constitutively active mdia2 could have resulted in endogenous mdia1 protein becoming a limiting factor on the process of filopodial microfilament elongation, giving rise to the club - shaped morphology of the filopodia . In addition, both full - length mdia2 and fh1fh2-mdia2 localized mostly to the cytoplasm in these experiments it is likely that less of the protein was present in filopodia, and there was enough endogenous mdia1 to elongate the smaller number of short microfilaments generated, thus resulting in filopodia of even thickness along their shafts . These interpretations of the findings would further implicate mdia1 as the key mdia isoform that elongates microfilaments to form mature filopodia . It would be interesting to see if knocking out mdia1 affects mdia2-driven filopodial protrusion will the resulting mdia2-induced filopodia be shorter in length? Mdia1 was seen throughout the shafts of filopodia when overexpressed alone or together with irsp53 or constitutively active rif in neuronal cells . This is in contrast to the tip nucleation model of filopodium formation, where formins are expected to be found only at the tips of the protrusions . One possible explanation is that mdia1 dimers play a dual role in filopodium formation and are involved in not just polymerising the actin filaments but bundling them together as well . Another possibility is that filopodia consist of short, discontinuous actin filaments that do not span the entire length of the filopodial shaft . This has been shown by cryo - electron tomography studies to be the nature of the actin filaments that constitute dictyostelium filopodia . Superresolution microscopy studies would be able to reveal detail at the nanometre scale and help elucidate the specific locations of mdia1 and mdia2, as well as other proteins associated with filopodial protrusion, within the structures with much greater accuracy . This would help in establishing a better understanding of the roles of these proteins in the various stages of filopodium formation . Cdc42 works through irsp53, which recruits to the plasma membrane the following proteins that modulate actin dynamics: n - wasp, mena, wave2 and eps8 . The rif pathway to filopodia does not require irsp53, n - wasp, mena or wave2 . As for the mdia proteins, mdia1 appears to be the only isoform common to both pathways . In the filopodia of neuronal cells overexpressing irsp53, mdia1 but not mdia2 was present, and was observed to interact with irsp53 within the structures . While both mdia1 and mdia2 were present in rif filopodia, only mdia1 interacted with the rhogtpase . In addition, knockdown of either of these two isoforms resulted in a decrease in rif - driven filopodium formation, while irsp53 filopodia were affected only by the silencing of mdia1 expression . Taken together, it appears that mdia2 is not required for irsp53 to form filopodia, and we have found that coexpressing mdia2 with irsp53 leads to a loss of filopodia instead . It remains to be seen as to why cells need two or even moreyetundiscovered pathways to form filopodia, and why irsp53 requires only mdia1 when rif appears to require both mdia1 and mdia2 . Also, rif has been shown to bind the gbd of mdia3, however the significance of this interaction has yet to be investigated . Mdia1 and possibly mdia2 are involved in stress fiber formation in addition to filopodial protrusion . These two types of actin - based cellular structures appear to be linked in fish fibroblasts, microfilaments in filopodia can become incorporated into stress fibers, and it has been suggested that the reverse might occur in rat embryonic fibroblasts, with the actin freed up by the dissolution of stress fibers facilitating the protrusion of filopodia . Rif interacts with both mdia1 and mdia2 and is able to trigger the formation of both filopodia and stress fibers . The dual role of these three proteins might point to a major role for them in controlling the balance between these two types of actin structures in cell migration . Mdia2 appears to be specific for the rif - mediated pathway whereas mdia1 is required for the pathways controlled by cdc42 and rif . It remains to be seen how exactly rif utilizes two different formins, mdia1 and mdia2, to form filopodia . How rif couples membrane deformation with actin dynamics to give rise to these structures has also yet to be resolved . Apart from mdia1 and mdia2, are there other proteins specific to the rif pathway to filopodium formation? What potential roles do irsp53 family proteins (irtks, mim, abba and pinkbar) play in filopodial protrusion? These are some of the important questions to address in future studies on mammalian filopodia.
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Patients who report having diarrhea for more than four weeks should be evaluated for chronic diarrheal illness, since most infectious enteritides and other causes of acute diarrhea generally resolve within this period . Flexible sigmoidoscopy or colonoscopy is often used by many physicians to find the organic cause of chronic diarrhea, and some gastroenterologists perform routine mucosal biopsies to exclude organic causes of chronic diarrhea that can only be diagnosed microscopically even when the endoscopic findings are normal . Diseases that cause diarrhea when the endoscopic findings are normal or nonspecific include collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis . It is also known that normal - appearing colorectal mucosa in patients with inflammatory bowel disease can show histological abnormalities and melanosis coli, a sign of laxative abuse, may be seen only microscopically . Finding these histological abnormalities in grossly normal colonic mucosa may alter the therapeutic decisions by implicating the use of immunosuppressive drugs, such as corticosteroid, or the need for close clinical follow - up . There are controversies surrounding routine mucosal biopsies taken during colonoscopy in patients with normal looking mucosa who have chronic gastrointestinal symptoms . Some suggest colonic biopsies should be taken routinely, while others argue about the cost - effeciveness of the procedure . But, it is difficult to conclude the cost - effectiveness of routine mucosal biopsies since these studies had different and obscure inclusion criteria of patients, and different protocols for each biopsy site . Recently, sporadic cases of these histological abnormalities, especially collagenous colitis, have been reported in korea . However, the incidence, of these histological abnormalities among patients with chronic diarrhea has not been reported in korea . The purpose of this study is to determine the incidence of clinically important histological abnormalities, prospectively in chronic diarrhea patients with strict inclusion criteria who have grossly normal or nonspecific colonoscopic findings, to ascertain the clinical significance of these findings to therapeutic decision - making and outcome . One hundred and eighteen consecutive patients suffering from diarrhea who met our inclusion criteria and visited the gastroenterolgy unit of samsung medical center during a fifteen months period (from april, 1995 to june, 1996) were evaluated prospectively in this study . The inclusion criteria were as follows; 1 . Patients suffering from chronic nonbloody diarrhea with consistency of watery to loose stool and a frequency of more than 2 times a day for more than a month, 2 . Exclusion of patients with specific colonoscopic findings which may explain the cause of diarrhea, such as chronic inflammatory bowel disease, colon cancer and large villous adenoma . Colonoscopic examinations were done with olympus cf-200i or cf-200l video - colonoscope, and two pieces of biopsies were taken from six different parts of the colon; cecum, ascending colon, mid - transverse colon, descending colon, sigmoid colon and rectum . Of the two biopsy specimens taken from the same part of the colon, one piece was stained with hematoxylin - eosin while the other was stained with masson - trichrome to determine the thickness of the subepithelial collagen band . Each biopsy specimen was reviewed by a pathologist who had no information on clinical diagnosis prior to interpretation of microscopic findings . Collagenous colitis was diagnosed when the subepithelial collagen band thickness, averaging over 10 intercryptal spaces, was measured to 10 m or greater using the masson - trichrome stain and signs of epithelial damage, such as epithelial detachments and flattenings, were present . If the subepithelial collagen band thickness was between 5 and 10m and other features were present, the biopsy was classified as lymphocytic colitis was diagnosed when diffuse presence of intraepithelial lymphocytes was present equal to or more than 10% of colonic epithelial cells and presence of epithelial flattening, increase in crypt lymphocytes, crypt distortion and increase in lamina propria chronic inflammation . If intraepithelial lymphocyte infiltration was less than 10% but other features of lymphocytic colitis were present, the biopsy was classified as having some features of lymphocytic colitis . Eosinophilic enterocolitis was diagnosed when inflammatory cell infiltrate was almost exclusively composed of eosinophils, eosinophilic infiltration outside the gi tract was not present and parasitic infestation was not present . Diagnosis of nonspecific colitis was made when increased lamina propria inflammatory cell infiltrate was present but did not meet the criteria of the above disorders . Concurrent microbial, biochemical and physiologic tests were done in a proportion of the patients . These included stool parasite ova, stool occult blood, stool white blood cells, stool fat, stool culture, fasting blood glucose level, serum calcium, phosphorous, thyroid function tests and breath hydrogen test . One hundred and eighteen consecutive patients suffering from diarrhea who met our inclusion criteria and visited the gastroenterolgy unit of samsung medical center during a fifteen months period (from april, 1995 to june, 1996) were evaluated prospectively in this study . The inclusion criteria were as follows; 1 . Patients suffering from chronic nonbloody diarrhea with consistency of watery to loose stool and a frequency of more than 2 times a day for more than a month, 2 . Exclusion of patients with specific colonoscopic findings which may explain the cause of diarrhea, such as chronic inflammatory bowel disease, colon cancer and large villous adenoma . Colonoscopic examinations were done with olympus cf-200i or cf-200l video - colonoscope, and two pieces of biopsies were taken from six different parts of the colon; cecum, ascending colon, mid - transverse colon, descending colon, sigmoid colon and rectum . Of the two biopsy specimens taken from the same part of the colon, one piece was stained with hematoxylin - eosin while the other was stained with masson - trichrome to determine the thickness of the subepithelial collagen band . Each biopsy specimen was reviewed by a pathologist who had no information on clinical diagnosis prior to interpretation of microscopic findings . Collagenous colitis was diagnosed when the subepithelial collagen band thickness, averaging over 10 intercryptal spaces, was measured to 10 m or greater using the masson - trichrome stain and signs of epithelial damage, such as epithelial detachments and flattenings, were present . If the subepithelial collagen band thickness was between 5 and 10m and other features were present, the biopsy was classified as lymphocytic colitis was diagnosed when diffuse presence of intraepithelial lymphocytes was present equal to or more than 10% of colonic epithelial cells and presence of epithelial flattening, increase in crypt lymphocytes, crypt distortion and increase in lamina propria chronic inflammation . If intraepithelial lymphocyte infiltration was less than 10% but other features of lymphocytic colitis were present, the biopsy was classified as having some features of lymphocytic colitis . Eosinophilic enterocolitis was diagnosed when inflammatory cell infiltrate was almost exclusively composed of eosinophils, eosinophilic infiltration outside the gi tract was not present and parasitic infestation was not present . Diagnosis of nonspecific colitis was made when increased lamina propria inflammatory cell infiltrate was present but did not meet the criteria of the above disorders . Concurrent microbial, biochemical and physiologic tests were done in a proportion of the patients . These included stool parasite ova, stool occult blood, stool white blood cells, stool fat, stool culture, fasting blood glucose level, serum calcium, phosphorous, thyroid function tests and breath hydrogen test . Of one hundred and eighteen patients included in this study, 79 were male and the average age was 43.0 years (1871 years). The average frequency of diarrhea was 3.51.7 bowel movements per day and the average duration of symptoms was 45.752.4 months (1240 months). The gross colonoscopic findings were normal in the majority of cases (71.1%), but some showed nonspecific findings (8.5%), such as mild mucosal edema and hyperemia . Incidental findings, such as small polyps (smaller than 0.7 cm, 19 cases), diverticula (3 cases), colonic cyst (1 case), submucosal tumor (1 case) and angiodysplasia (1 case) were discovered during the examinations (table 1). In the microscopic examinations, two cases of collagenous colitis (fig . 1) and one case of lymphocytic colitis (fig . The gross colonoscopic findings of the cases diagnosed as collagenous colitis and lymphocytic colitis were completely normal, but showed nonspecific edema and hyperemia in the case of eosinophilic enterocolitis . One specimen showed compatible features of ulcerative colitis, but the gross features showed only mild focal mucosal hyperemia at the rectum . Four cases of melanosis coli were discovered during the microscopic examination, gross findings of which were categorized as normal . Sixteen cases (13.5%) of borderline histological abnormalities were observed where 8 cases showed possibility of collagenous colitis and 8 cases showed some features of lymphocytic colitis . Results of biochemical, microbial and physiological tests, which were done to a portion of patients, are summarized in table 3 . In particular, fasting hyperglycemia (140mg / dl), 2.2% (2/92), hyperthyroidism, 3.8% (2/53) and hypocalcemia, 3.2% (3/93) were discovered . Positive rate of breath hydrogen test was 69.6% (32/46). In summary, among 118 patients included, 7.6% of patients showed clinically significant histological abnormalities (collagenous colitis, lymphocytic colitis, eosinophilic enterocolitis, ulcerative colitis and melanosis coli), and 13.5% of patients showed borderline histological abnormalities (fig . Many physicians and gastroenterologists frequently diagnose a patient with chronic diarrhea as irritable bowel syndrome when they get a normal result in colonoscopy, but we had a suspicion that many of those diagnosed as irritable bowel syndrome may have organic causes . Among the various causes of chronic diarrhea, two organic conditions that reportedly cause diarrhea, when colonoscopy and colonic radiography are normal, are collagenous colitis and lymphocytic colitis . Recently, much attention has been paid to those conditions which can be diagnosed by taking biopsies from normal - appearing colonic mucosa, which otherwise would be diagnosed as irritable bowel syndrome . First described in 1976 and 1980, collagenous colitis and lymphocytic colitis respectively have been recognized with increasing frequency in the past few years, as endoscopically normal mucosa has been biopsied more frequently in patients with chronic unexplained diarrhea . Recently, two cases of collagenous colitis have been reported in korea, suggesting the possibility that collagenous colitis and lymphocytic colitis might play some etiologic role in chronic diarrhea in korea . Macintosh et al performed sigmoidoscopic biopsies on 134 patients with nonspecific lower gastrointestinal symptoms and reported that they did not find any cases of collagenous colitis or lymphocytic colitis . Since the symptoms of the majority of patients with collagenous colitis and lymphocytic colitis manifest as chronic watery diarrhea, they might have included cases that are unlikely to be collagenous colitis or lymphocytic colitis . Only rectal biopsies were performed in their study, which suggest that the cases that are confined to the right side of the colon may have been missed . Marshall et al . Have performed mucosal biopsies from normal - looking mucosa obtained from 111 chronic diarrhea patients . They included all patients who experienced a change in their bowel habits, which is a too broad definition of diarrhea . Their data included 1 case of possible collagenous colitis and 13 cases that showed some features of lymphocytic colitis of which the clinical significance was denied by the authors . As there have been a few cases that reported development of collagenous colitis in sequential biopsy specimen, we thought that these cases should not be considered as trivial or nonspecific findings . Unlike the results by macintosh, et al . And marshall, et al ., we were able to find clinically significant histological abnormalities, findings that could alter therapeutic decisions in 7.6% of patients (9/118); 2 cases of collagenous colitis and 1 case for each of the following - lymphocytic colitis, eosinophilic enterocolitis, ulcerartive colitis and 4 cases of melanosis coli . The differences between our result and the previous reports can be accredited to more strict patient selection, more broadened biopsy field and possibly a racial difference between caucasians and asians . We also think that the borderline histological abnormality group (13.5%) observed in our study should be followed carefully for a possible development of collagnous colitis or lymphocytic colitis . Careful long - term clinical follow - up and, if necessary, follow - up colonoscopy with biopsies are mandatory . It should be noted that when collagenous colitis or lymphocytic colitis is suspected, biopsy sites should include the whole length of the colon, including the right side colon . The lesions can be discontinuous and thus absent in some biopy specimens, even in multiple specimens from a given area . It is difficult to determine the optimal number of biopsies that should be taken, and from what locations within the colon but, based on available information, we suggest that, at least six, biopsies should be taken approximately evenly spaced between the cecum and the rectum to exclude those conditions . Our results of the biochemical and microbial tests need to be mentioned . Although our results cannot be used to define the relative etiologic roles of each metabolic and biochemical cause of chronic diarrhea, since the tests were not performed in all of the patients included in the study, they provide valuable information that metabolic causes, such as diabetes melitus, hyperthyroidism, hypocalcemia and microbial causes, such as parasitic infestation, may play a role in a significant proportion of the patients . Our results show that 69.6% of patients (32/46) were positive for breath hydrogen test, but it was not possible to attribute the cause of the patients symptoms to lactose intolerance because therapeutic trials of lactose free diet were not performed on those who were positive for breath hydrogen test . It is peculiar to find that the positive rate of breath hydrogen breath test showed lower than the expected positive rate in general korean patients, which is approximately 85% . We speculate that this low rate of positive breath hydrogen could be due to false negative tests, which may reflect increased small bowel motility in patients with chronic diarrhea . In conclusion, clinically significant histological abnormalities can exist in significant percentages, in spite of normal or nonspecific colonoscopic findings, which can justify routine mucosal biopsy in the evaluation of chronic diarrhea patients . The clinical significance of borderline histological abnormalities needs to be determined by careful follow - up studies.
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Redox biological signaling has significantly developed over the last couple of decades with small molecules such as no, co, h2s, and h2o2 being identified as competent signaling agents . Nitroxyl (hno), the oneelectron reduced / protonated form of no, has been discussed as a signaling agent, but the lack of specific & selective detection methods hampers a better understanding of its biology . Significant advances in hno detection have occurred with the development of new copper and phosphinebased fluorescent probes and new electrochemical & mass spectrometric methods . With these mechanistically different ways to detect hno, questions regarding hno's biology and endogenous formation can be approached . The most significant result is that the azaylide intermediate generated from the reaction of these probes and hno rapidly and reliably undergo a staudinger ligation resulting in fluorescence and a stable amide byproduct . Reaction of these probes with rsno gives a similar azaylide, but in this case, the staudinger ligation does not occur or proceeds inefficiently . Flow cytometry experiments show that these reactions also occur in cells and that these probes only detects hno in cells . One of the challenges starting this project was to not let our previous ideas about the chemistry dictate what experiments to do . We knew that rsno reacts with phosphines to yield azaylides that should undergo ligations and yield fluorescence . Should we continue if we were going to see that our probes were not selective? Zhengrui miao noticed that no one had ever really directly compared the response of these probes to hno and rsno . He did this comparison and found fluorescence generation from hno treatment was greatly enhanced compared with rsno, and these findings initiated the study . The overall lesson is to keep an open mind and don't plan the outcome of your experiments!
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Tricyclic alkaloids represented by cylindricines, lepadiformine (3) and fasicularin are unique metabolites of ascidians . Cylindricines a and b isolated from clavelina cylindrical were the first members of the family based on the perhydropyrrolo[2,1-j]quinoline and the perhydropyrido[2,1-j]quinoline ring systems, respectively . The structures and relative stereochemistries were unambiguously established by x - ray crystallography of their corresponding picrates . Moreover, these two compounds were reported to interconvert through aziridinium intermediate forming 3:2 equilibrium mixture upon standing in a solution . Compound 3 exhibited cytotoxicity against several tumor cell lines and in vivo and in vitro cardiovascular activity . More recently, fasicularin, isolated from nephteis fasicularis, was reported as a cytotoxin to vero cells and was found to act as a dna - damaging agent in the assay using a dna repair - deficient yeast strain . As a part of collaborative investigation of biologically active compounds from indonesian marine organisms, we herein report the isolation and structures of two new tricyclic alkaloids polycitorols a (1) and b (2) together with known lepadiformine (3) from a marine ascidian . Specimens (65.0 g, wet) of the ascidian of the family polycitoridae were collected by scuba (25 m) off flores, indonesia in august 2001 and were stored in ethanol after its collection . The sample was exhaustively extracted with meoh and the resulting extract was chromatographed on a silica gel column followed by reversed - phase hplc to give polycitorol a (1), b (2), and lepadiformine (3). Polycitorol a (1), colorless oil, []d + 15 (c 0.2, chcl3), was shown to have a molecular formula of c17h31no by hreims (m / z 265.2410, m, + 0.4 mmu). Strong absorption bands at 3384 and 1671 cm in the ir spectrum suggested that 1 contain hydroxyl and amine functionalities . The h nmr spectrum contained a number of unresolved multiplets in the high field region (1.212.24), while no olefinic signals were observed . The c nmr spectrum revealed the presence of a quaternary carbon (77.9), two heteroatom bearing methines (67.0, 71.9), one methine (44.2), one ch2oh (65.0), one methyl, and eleven methylenes . Therefore, alkaloid 1 is tricyclic, and the nitrogen atom was suggested as a tertiary amine . The c nmr spectrum of 1 displayed 17 carbon signals indicating that 1 has two less carbons than 3 . A mass fragment ion at m / z 208 was formed by the cleavage of c4h9, thus, differing in the length of the alkyl side chain from 3 . Cosy correlations revealed that a methine proton at 2.97 (c 67.0) correlated with two methylene protons at 1.58 and 2.05 confirming the c2/c15 linkage . Further 2d nmr analysis (table 1) allowed us to elucidate the planar structure of 1 . Enhancement of h-2 at 2.97 and h-4ax at 1.57 upon irradiation of h-9ax at 2.15 suggested a cisfusion between rings a and b. the alkyl side chain at c-2 occupied equatorial position on the basis of mutual noe enhancements between h-2 and h-5 . Noe between one of h-12 protons and h-13 may indicate the primary hydroxyl group as shown . Polycitorol b (2), []d 10.3 (c 0.6, chcl3), was isolated as a colorless oil . The molecular formula c17h31no was determined by hreims, indicating that it was isomeric to 1 . Comparison of nmr data of 1 and 2 revealed that considerable higher field shifts were observed for a methine at 58.3 and a quaternary carbon at 66.5 in the c spectrum of 2 . Furthermore, additional methine signal at 4.06 (c 60.5) suggested that 2 had a secondary rather than a primary alcohol . The cosy spectrum indicated that this proton coupled to two nitrogenattached methylene protons (3.53, 3.36) and two other protons (1.78, 2.11). Thus, the aforementioned data indicated that the pyrrolidine ring was rearranged to a piperidine ring . The relative stereochemistry of a / b rings was shown to be identical to that in 1 with a c-2 butyl side chain based on similar noe data, particularly a set of mutual noe enhancements between h-2, h-4ax and h-9ax . A trans fusion between rings a and c was suggested by noe between h-13/h-2, h-14ax / h-2 and h-5/h-2 . Thus, rings a and c of 2 adopted a much more rigid trans 1-azadecalin system and the c-13 hydroxyl group occupied equatorial position . Two new tricyclic alkaloids, polycitorols a (1) and b (2), have been isolated along with the known lepadiformine (3) from a marine ascidian . The new compounds contain a butyl side chain instead of a hexyl group observed more commonly in known related compounds . Nmr spectra were taken on a jeol 500 ft nmr spectrometer and referenced to chcl3 solvent signal at 7.26 for h nmr and 77.0 for c nmr spectra in cdcl3 as a solvent and also referenced to tms signal at 0.00 for h nmr and 49.0 for c nmr spectra in cd3od as a solvent . Hplc separations were carried out on a hitachi l-6000 or shimadzu lc 9a pumps equipped with waters 486 or hitachi l-4000 uv detector and waters r401 differential refractometer . Columns used for hplc were reverse phase nacalai 5c18-ar ii (10 250 mm). Tlc was carried out on precoated silica 60f254 plates and visualized with vanillin - etoh-1% h2so4 . The specimens (65.0 g, wet wt .) Of the ascidian (family polycitoridae) were collected by hand using scuba (25 m) at misa is ., labuhanbajo, flores, indonesia in august 2001 and were stored in ethanol after its collection . Adrian gittenberger, national museum of natural history naturalis, leiden, the netherlands . A voucher specimen (0117j78) is deposited at department of chemistry, biology, and marine science, university of the ryukyus . A photo of the specimen can be viewed at http://www.ascidians.com/families/polycitoridae/polycitoridae_blue/polycitoridaeblue.htm a sample of tunicate (65.0 g wet weight) was cut into small pieces and soaked in acetone (300 ml) for 7 hr . After decantation, fresh solvent was added, and the procedure was repeated three times . The oil was chromatographed on silica gel by eluting with step gradient of hexane / ch2cl2/etoac / meoh . Fraction v was selected for further purification using hplc (rp-18, meoh / h2o / tfa) to afford compounds 1, 2 and 3 . Polycitorol a (1): colorless oil; []d + 15.3 (c 0.2, chcl3); ir (film) 3384, 2937, 2867, 1671, 1455, 1201, 1132 cmh and c nmr see table 1; eims m / z 265 (25, m), 234 (100), 222 (97), 208 (45), 194 (5), 178 (5); hreims found m / z 265.2410 (calcd for c17h31no, 265.2406). Polycitorol b (2): colorless oil; []d 10.3 (c 0.6, chcl3); ir (film) 3384, 2937, 2869, 1670, 1457, 1201, 1132 cmh and c nmr see table 1; eims m / z 265 (6, m), 222 (29), 208 (100), 193 (7), 150 (5); hreims found m / z 265.2406 (calcd for c17h31no, 265.2406). Nmr spectra were taken on a jeol 500 ft nmr spectrometer and referenced to chcl3 solvent signal at 7.26 for h nmr and 77.0 for c nmr spectra in cdcl3 as a solvent and also referenced to tms signal at 0.00 for h nmr and 49.0 for c nmr spectra in cd3od as a solvent . Hplc separations were carried out on a hitachi l-6000 or shimadzu lc 9a pumps equipped with waters 486 or hitachi l-4000 uv detector and waters r401 differential refractometer . Columns used for hplc were reverse phase nacalai 5c18-ar ii (10 250 mm). Tlc was carried out on precoated silica 60f254 plates and visualized with vanillin - etoh-1% h2so4 . The specimens (65.0 g, wet wt .) Of the ascidian (family polycitoridae) were collected by hand using scuba (25 m) at misa is ., labuhanbajo, flores, indonesia in august 2001 and were stored in ethanol after its collection . Adrian gittenberger, national museum of natural history naturalis, leiden, the netherlands . A voucher specimen (0117j78) is deposited at department of chemistry, biology, and marine science, university of the ryukyus . A sample of tunicate (65.0 g wet weight) was cut into small pieces and soaked in acetone (300 ml) for 7 hr . After decantation, fresh solvent was added, and the procedure was repeated three times . The oil was chromatographed on silica gel by eluting with step gradient of hexane / ch2cl2/etoac / meoh . Fraction v was selected for further purification using hplc (rp-18, meoh / h2o / tfa) to afford compounds 1, 2 and 3 . Polycitorol a (1): colorless oil; []d + 15.3 (c 0.2, chcl3); ir (film) 3384, 2937, 2867, 1671, 1455, 1201, 1132 cmh and c nmr see table 1; eims m / z 265 (25, m), 234 (100), 222 (97), 208 (45), 194 (5), 178 (5); hreims found m / z 265.2410 (calcd for c17h31no, 265.2406). Polycitorol b (2): colorless oil; []d 10.3 (c 0.6, chcl3); ir (film) 3384, 2937, 2869, 1670, 1457, 1201, 1132 cmh and c nmr see table 1; eims m / z 265 (6, m), 222 (29), 208 (100), 193 (7), 150 (5); hreims found m / z 265.2406 (calcd for c17h31no, 265.2406).
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The serous layer is also divided into two parts, the parietal pericardium which is intimately fused to the fibrous pericardium and also the visceral pericardium which is a part of the epicardium . In between the visceral and parietal pericardium is a thin potential space consisting of 2030 cc of serous fluid that fills a potential space known as the pericardial cavity . In the case of ccp, the fibrotic pericardium thickens and forms a noncompliant casing surrounding the heart . Pericardial involvement in end - stage renal disease (esrd) is manifested most commonly as acute uremic or dialysis pericarditis and infrequently as chronic constrictive pericarditis . The cause of 60% of cases of ccp is unknown and is labeled as idiopathic . Moreover, ccp is commonly confused with other diagnoses such as end - stage - liver failure, idiopathic cardiomyopathy, and restrictive cardiomyopathy . In our case presentation, we report an advanced case of constrictive pericarditis associated with end - stage - renal disease and follow with a literature review . A 27-year - old armenian man with a history of hypertension, end - stage - renal disease on hemodialysis, and recently diagnosed ccp presented to our hospital with massive ascites, dyspnea, and hypotension . He has had esrd over the past three years, the cause of which was most likely secondary to uncontrolled hypertension . His social history included a 1 pack - year of tobacco smoking and no alcohol or recreational drug use . On physical exam, his vitals were 8090 s/3040 s and he had decreased breath sounds on the left side, decreased heart sounds, some crackles diffusely, and 2 + pitting edema diffusely over the lower extremities . Over the past few months, he had developed massive ascites, and a through workup was undertaken to discover the underlying cause of his symptoms which included intrahepatic pressure measurements, cholecystectomy, appendectomy, liver biopsy, and a peritoneal biopsy . In the hospital, he was dialyzed aggressively and received multiple paracentesis (the first of which drained 6 liters of greenish fluid), but the ascites continued to return in 1 - 2 days . An echocardiogram showed normal left ventricular systolic function, ef 55%, dilated left and right atrium, and evidence of pressure and volume overload over the right ventricle (right ventricular hypertrophy). A ct scan showed pericardial thickening and bilateral pleural effusions (figure 1). A left and right heart cardiac catheterization demonstrated a thick peal around the heart, ejection fraction of 55%, pap of 30 mm hg, lved pressures of 3035 mm hg, and also equalization of pressures on both the right and left side of the heart (figure 2). Following the diagnosis of ccp, a partial pericardiectomy was performed; however, the patient did not improve and a salvage total pericardiectomy soon followed . At sternotomy, no specific etiology for ccp was found after numerous histopathological, serological, and bacteriological studies including cbc with differential, hepatocellular function tests, esr, crp, bnp, ana, rf, aso titer, anti - dna abs, ppd test, hiv elisa, thryroid studies, pericardial fluid analysis (ldh, total protein, cell count, glucose, gram stain, and cultures), and cardiac biomarkers (troponin i). His labs did not reveal any evidence of hyperparathyroidism; on presentation calcium was 9.5 meq / dl, phosphorus 3.8 mg / dl, pth was 45, and patient was not on any vitamin d analog medication . The patient developed complications from the total pericardiectomy including septic shock, and shortly thereafter expired following a terminal extubation . The most common cause of constrictive pericarditis (up to 4555% of cases) is idiopathic or viral in etiology [24]. The most common identifiable causes include postcardiac surgery (37%), pericarditis (1620%), and mediastinal radiation (9%) [2, 3, 5]. Other recognized but less common causes include connective tissue disorders (such as ra and sle), malignancy (especially breast cancer, lung cancer and lymphoma), and local trauma . Tuberculosis was once recognized as the most common cause of constrictive pericarditis and continues to be a major problem in the developing world . Other causes include purulent infections, histoplasmosis, and chronic renal failure treated by chronic dialysis . In the case of our patient, no specific etiology was found after numerous histopathological, serological and bacteriological exams . Though undetected viral cause and the large majority of constrictive pericarditis, because of his esrd and recent initiation of hd just a few months prior to his presentation, we believe the esrd is the underlying etiology of his constrictive pericarditis . The pericardium in esrd patients commonly will present as an acute uremic or dialysis pericarditis and less commonly as chronic constrictive pericarditis . Following a medline literature search, we were only able to find a few such association [5, 8, 9] between ccp and esrd when using the keywords: renal failure and constrictive pericarditis and esrd and pericarditis . Thus, end - stage - renal disease should be considered in the underlying etiology of constrictive pericarditis . Constrictive pericarditis is a rare disorder that manifests predominantly as right heart failure and, in severe cases, systemic hypotension and circulatory collapse . Some common clinical features include edema, ascites, raised jugular venous pressure, pleural effusion, and hepatomegaly, all commonly found with right - heart strain ., a plain chest radiography may show pericardial calcification and unexplained pleural effusions especially in severe stages of the disease . Echocardiography may validate the presence of small ventricular dimensions with preserved systolic function, and dilated atria, all findings present in our patient . Constrictive pericarditis may be differentiated from other forms of heart failure such as restrictive cardiomyopathy by the presentation of thickened pericardium on computed tomography and magnetic resonance imaging . Localized fibrosis and intrapericardial calcifications are some of the histopathological features often associated with constrictive pericarditis . During early diastole, ventricular filling is rapid due to high systemic venous pressure which results in the elevation and equilibration of the diastolic pressures of all four heart chambers . However, late diastolic filling is inhibited by the rigid pericardium; it is restricted as the intracardiac volume reaches the limit set by the noncompliant pericardium . Our case was an exception to this rule as cardiac output was preserved, at least in part, most likely due to the high end diastolic filling pressure or lvedp (measured to be equal on both the right and left ventricle at 30 mm hg). The definitive treatment of constrictive pericarditis is total pericardiectomy and aggressive treatment of any underlying disease process . Total rather than partial pericardiectomy yields better outcome and should be undertaken as early as possible which has been shown to yield a better clinical outcome . Also, survival is dependent on the underlying etiology, and patients with idiopathic constrictive pericarditis usually have the best outcome following pericardiectomy . Long - term survival ranged from 78% at 5 years, 57% at 10 years, and limited by severe preoperative functional disability, renal failure, and nonresectable myocardial calcifications . Our unfortunate patient had many of these risk factors and survived just 2 weeks following his total pericardiectomy . Thus, in patients with end - stage - renal disease, chronic indolent or subclinical dialysis associated with pericarditis may lead to constrictive pericarditis . Both the etiology and physiology of constrictive pericarditis are reviewed and it is of value for clinicians to consider esrd as an underlying cause in their workup of constrictive pericarditis after first ruling out other more common etiologies.
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Since 1965 when brnemark et al.1 introduced and established the osseointegration concept, dental implant has achieved enormous development and progress . Defined as direct connection between bone and implant surface, osseointegration is formed by the process of bone formation between bone and implant surface . Because the success and failure of implant is determined by osseointegration, it is a precondition for prosthetic repair through implant . The primary stability is obtained by mechanical fixation of the implant with bone, and this is one of the basic conditions for osseointegration.2 primary stability is related with implant surface area, geometry, length, contact area between implant and bone . Other factors include ratio of spongy bone vs. cortical bone, and implant technique.3 the secondary stability is generated secondarily by bone formation and bone remodeling in the process of osseointegration due to biological fixation in the interface between bone and implant.4 therefore, we can evaluate the degree of osseointegration through the measurement of changes in the implant stability.5 meredith et al . Reported on the use of the resonance frequency analyzer to evaluate the stability of implant, and demonstrated the ability of the device to evaluate the changes in stiffness of the interface in the early in vitro experiment.6,7 recently, histomorphologic studies suggested that the resonance frequency value has a high correlation with the level of contact between bone and implant.8 - 11 this discovery supports the use of resonance frequency analysis to evaluate the changes in the process of osseointegration and bone healing after placement of implant . The resonance frequency analyzer can measure clinically and noninvasively the stability of implant and estimate the degree of osseointegration . In this study, we used a recently developed magnetic resonance frequency analyzer to measure the stability of implant . For measurement unit, implant stability quotient (isq) is used which is recorded as a number between 1 and 100 with 100 representing the highest stability.12 this study intends to measure and analyze the changes in implant stability without load during an early healing period of six months after placement of three different types of implants by one stage implant . In this way, this study will provide useful information for prosthetic treatment planning through immediate and early loading after placement of implant as well as the evaluation of long - term prognosis for it . A total of 28 patients (25 males and 3 females, mean age: 58.6 9.23) were selected among the patients who visited between march and september 2004 with the main purpose of implant placement . Inclusion criteria were as follows adults aged 18 or olderpatients who understand and agree to this studyadequate oral hygiene (1 or lower mean modified sulcus bleeding index, 1 or lower mean modified plaque index)sufficient bone volume to place the planned implantone or more edentulous mandible parts which are six months or longer after dental extraction (however, adjacent teeth must be healthy and properly repaired)fertile women who received pregnancy test no later than one week before surgery and have been confirmed to be negative adults aged 18 or older patients who understand and agree to this study adequate oral hygiene (1 or lower mean modified sulcus bleeding index, 1 or lower mean modified plaque index) sufficient bone volume to place the planned implant one or more edentulous mandible parts which are six months or longer after dental extraction (however, adjacent teeth must be healthy and properly repaired) fertile women who received pregnancy test no later than one week before surgery and have been confirmed to be negative exclusion criteria were as follows smokers over 10 cigarettes / daya history of alcoholism or drug addition during the past 5 yearssevere teeth clenching or bruxismrisk of subacute bacterial endocarditisuncontrolled hypertension or diabetespatients with malignant tumor smokers over 10 cigarettes / day a history of alcoholism or drug addition during the past 5 years severe teeth clenching or bruxism risk of subacute bacterial endocarditis uncontrolled hypertension or diabetes patients with malignant tumor the three types of 45 implants were divided into 3 groups including osseospeed (group a, astra tech, sweden), camlog (group c, biotechnologies ag, switzerland), and replace (group r, nobelbiocare, sweden) in this study . One assigned operator placed implants by one stage technique in accordance with the surgical protocol suggested by the manufacturer . Bone quality classification followed the criteria proposed by lekholm & zarb, and type 1, 2, 3 or 4 was determined on the basis of the sense of resistance during bone drilling . After placement, isq was measured, a healing abutment was connected, and sutured . For magnetic resonance frequency analyzer, this study used osstell (gteborg, sweden). A special smart peg was connected to the implant body at 4 - 5 n / cm torque, and measurements were made at 2 - 3 mm away so that the probe tip of the analyzer would point to the small magnet above the smart peg (fig . If the probe measures two values simultaneously and the difference between these two values is 3 isq or higher, the values must be indicated simultaneously . Isq was measured immediately after placement, after 3 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks and 24 weeks . In addition, we examined the existence of discomfort at the time of healing abutment removal and the smart peg connection . The data were analyzed by implant type, bone type, healing time, and location . The data were recorded as implant isq over time, and were analyzed by implant type, bone type, healing time, and location . For the analysis by bone type, interaction between time and type with two - factor effect mixed model was used . Here, the comparison between the time points used contrast comparison in the two - factor effect mixed model . For comparison of bone types at different time points, the multi - comparisons of anova and tukey were used (p = .05). The analysis by mandible location used the time effect with two - factor effect mixed model and the location effect with two - factor effect mixed model . The data were recorded as implant isq over time, and were analyzed by implant type, bone type, healing time, and location . For the analysis by bone type, interaction between time and type with two - factor effect mixed model was used . Here, the comparison between the time points used contrast comparison in the two - factor effect mixed model . For comparison of bone types at different time points, the multi - comparisons of anova and tukey were used (p = .05). The analysis by mandible location used the time effect with two - factor effect mixed model and the location effect with two - factor effect mixed model . The surgeries produced no complications in all the patients and the isq numbers were obtained without causing inconvenience to the patients . Bone types 2 and 3 were grouped together, because recent papers demonstrated that it was difficult to reliably differentiate the drilling resistance in intermediate bone . The two - factor mixed model anova was used to determine the existence of interaction between bone type and time in groups a, c and r (p = .0022, p = .017, p = .0018). In other words, the change patterns of isq by time were different by bone type (fig . 2 - 4). Furthermore, the isq values of bone types were compared through multi - comparisons of anova and tukey . In the groups a, c and r, the measurements immediately after placement showed statistical differences in bone types 1, bone types 2 & 3, and bone type 4 (p <.05). The mean isq of implants immediately after placement was over 70 in the bone type 1, between 65 and 70 in the bone type 2 & 3, and between 48 and 50 in the bone type 4 (fig . A, between week 0 and 6, isq increased 4.78% in bone type 1, 8.73% in bone type 2 & 3, and 56.47% in bone type 4 . The bone type 1 did not show significant change (p = .052), while bone types 2, 3 and 4 showed significant changes (p = .044, p = .0326). In group c, between week 0 and 6, isq increased 0.52% in bone type 1, 6.38% in bone type 2 & 3, and 49.11% in bone type 4 . The bone type 1, 2 & 3 showed slow increase between six weeks and six months . The bone types 1, 2 & 3 showed significant changes (p = .0356, p <.0001), but type 4 did not show significant changes (p = .3715). In group r, between week 0 and 6, isq increased 7.37% in bone type 1, 11.87% in bone type 2 & 3, and 47.81% in bone type 4 . The bone types 1 did not show significant changes (p = .6411), but type 4 showed significant changes (p = .0005, p = .0462). According to the two - factor mixed model, groups a and c did not show significant differences in stability changes by healing time according to the anatomical location between maxilla and mandible . In group r, the change of stability by healing time showed significant difference between maxilla and mandible (p = .0238). However, the comparison between maxilla and mandible by the two - sample t - test did not find significant difference among 3 groups (fig . 9 - 12). According to the two - factor mixed model, groups a and c did not show significant differences in stability changes by healing time according to the anatomical location between maxilla and mandible . In group r, the change of stability by healing time showed significant difference between maxilla and mandible (p = .0238). However, the comparison between maxilla and mandible by the two - sample t - test did not find significant difference among 3 groups (fig . The need for clinical diagnosis tools with high accuracy to recognize the stability of implant and early healing changes is increasing along with the immediate and early load concept . The resonance frequency device invented by meredith4 has been used for clinical reference of the stability of implant placed in various bone types . There are several reports about implant stability influenced by healing time.8,10,11 this study also found through an analysis with the interaction between time and type with two - factor effect mixed model that the interaction of time and bone quality had significant influence on the isq values . The result of implant stability immediately after placement was bone type 4 <type 2 & 3 <type 1 in all implant groups . The implant stability measured immediately after placement is primary stability resulting from the mechanical press - fitting of implant with a greater diameter than the hole in the bone, and is influenced by the stiffness of adjacent bones.3,4 in other words, the higher the stiffness of adjacent bones, the higher the primary stability . Therefore, the stability immediately after placement of bone type i that has a high volume of cortical bone and a small volume of spongy bone is higher . On the other hand, bone types 2 & 3 and type 4 have a less volume of cortical bone and a more volume of spongy bone, which decreases the stiffness of bones and lowers the stability immediately after placement of implant.13 the three bone types experienced great changes in stability between week 0 and week 6 . In an experiment with rabbits, robert14 reasoned that human bone quality would undergo significant changes between week 0 and week 6 by the formation of woven bone and the deposition of lamellar bone . Furthermore, the size of changes between week 0 and week 6 varied by bone type . The ascending order of the size of changes was bone type 1> type 2 & 3 <type 4 . However, the cancellous healing showed was fast because in addition to trabecular remodeling, the bone is close to bone marrow that has a rich vascular system and mesenchymal progenitor cells which can be differentiated into osteoblasts . Therefore, it seems that the closer to bone type 4, the higher implant stability by the fast regeneration of woven bones.15 the slow increase of implant stability between week 6 and six months (plateau effect) has been reported by cochran et al.16 this phenomenon has a correlation with the strengthened bone formation concept around the implant . Robert14 believed that the later stage of the deposition of the lamellar bone into the grids in human woven bones and the interface remodeling begins at week 6 and continues until week 18, and the deposition of lamellar bone gives sufficient strength to withstand load . The changes of stability by healing time according to the mandible location were not significantly different between the upper and lower mandible groups . There are many existing studies that found that the lower mandible shows higher stability.17,18 the result of this study was different, which seems to be due to the small number of samples (there were five cases of bone type 4 in the 13 maxilla cases). The implant length may be an important factor that influences isq value.19 however, this study could not analyze the differences due to the insufficient number of samples by length . Within the limitation of this study, we found that the time / bone quality interaction had significant influence on isq values through an analysis among implant groups a, c, and r. in other words, in all the implant groups a, c and r, the change patterns of isq over time differed by bone type . Implant stability increased greatly between week 0 and week 6 (order of increase: type 1 <type 2 & 3 <type 4), and showed slow increase between week six and six months (plateau effect). However, no significant difference in stability changes by healing time according to locations was found in all the implant groups . More studies are required about the mechanotransduction effect during the early implant loading in various implant systems, and the effect of this on the magnetic resonance frequency analyzer during the early healing period.
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Dilated cardiomyopathy (dcm) is the most common form of primary cardiomyopathy and is a leading cause for heart transplantations approximately 30%50% of affected individuals have a familial form of dcm . To date, at least 40 disease genes have been reported to be associated with familial dcm, in which; mutations of scn5a gene which encodes the alpha subunit of the major sodium channel in the heart have been demonstrated to result in myocardial damage and dcm [4, 5]. Previously, scn5a has been implicated in conduction disturbances and several electrophysiological phenotypes [6, 7], including atrioventricular block (avb), long qt syndrome, sick sinus syndrome, sudden infant death syndrome, and atrial fibrillation [8, 9]. Meanwhile, more and more scn5a mutations were discovered to be related with dcm, such as a1180v, d1275n, w156x, r225w, and r222q . We first reported finding a1180v, a single nucleotide mutation, c.3539c> t, that occurred in exon 20 of the scn5a gene in a three - generation chinese family with age - related dcm and progressive avb in 2008 . In that study, a1180v was detected in all studied patients and in six younger unaffected members of the pedigree . Experimental data revealed that a1180v channels exhibited a negative transform of voltage - dependent inactivation of the cardiac sodium channels, which lead to a rate - dependent sodium current reduction, and a moderate increase in late sodium current . Correspondingly, our clinical study revealed qrs widening at high heart rates and qtc prolongation at rest in unaffected carriers . These findings suggest that a1180v is a functional mutation that can contribute to the development of dcm . However, data deposited in the dbsnp database show that a1180v has been found in two small sample groups of the asian population . In the first sample group, a1180v (dbsnp refsnp number, rs41310765) was present in a cohort of 47 han people (r31_hanla) who were part of the los angeles panel at a carrier frequency of 6.4% . In another sample group (her_asian - panel) of 16 individual asians selected from the coriell cell repository, genetic sequencing yielded a carrier frequency of 6.2% (http://www.ncbi.nlm.nih.gov/projects/snp/snp_ref.cgi?rs=41310765). To date, the data obtained from the dbsnp database show that a1180v is virtually absent in other ethnic groups . Thus, these unpublished data suggest that a1180v might be a common variant in asians, which is inconsistent with our previous observations . In order to further evaluate the pathogenic role of a1180v in familial dcm and make sure the frequency of a1180v among healthy han chinese, we followed up the three - generation chinese family and expanded the control size to screen for a1180v . Besides, we analyzed its frequency in other database as well . We followed up all the a1180v carriers and noncarriers of the three - generation chinese family for 6 years . All individuals were interviewed with the symptoms of cardiovascular disease, such as typical chest pain, palpitation, cyanosis, and syncope . 12-lead electrocardiogram (ecg) and echocardiography were performed on the family members to make a diagnosis . 460 unrelated healthy han chinese living in shanghai, ranging from 20 to 70 years old, were enrolled to the control group from a national high technology research and development program (the national 863 program) of china . Individuals with known cardiovascular risk factors other than age, gender, and smoking history were excluded . The enrolled participants did not show any abnormalities under comprehensive clinical evaluation, including review of medical history, physical examination, 12-lead ecg, submaximal exercise ecg, and blood biochemical examinations . Each of the participants and individuals whose data are included here signed an informed consent form after a complete and clear explanation of the nature and purpose of the study . The study was approved by the local ethics committee of zhongshan hospital, fudan university . Dna was extracted from 2 ml venous blood according to kit procedure (sigma, usa) and stored at 80c . A 20 ul mixture was prepared for each reaction and included 1x hotstartaq buffer, 2.0 mm mg, 0.2 mm dntp, 0.2 m of each primer, 1 u hotstartaq polymerase (qiagen inc .) And 1 l template dna . The dna was amplified by cycling at 95c for 15 min; 11 cycles of 94c for 15 s, 62c0.5c per cycle for 40 s, 72c for 1 mins; 24 cycles of 94c for 15 s, 57c for 30 s, 72c for 1 mins; 72c for 2 min . The oligonucleotide sequences of the primers were as follows: 5-acccccatcatcgtagctctt-3 and 5-tcctgctctggcctccatac-3. The pcr products were purified by 1 u sap and 6 u exo i, and they were added into 8 l pcr products . The mixture was incubated at 37c for 60 mins, followed by incubation at 70c for 10 mins . Reaction mixture included 2 l bigdye3.1 mix, 2 l sequencing primer (0.4 m, 5-acccccatcatcgtagctctt3) and 1 - 2 l purified pcr product . The cycling program was 96c for 1 min; 28 cycles of 96c for 10 s, 50c for 5 s, 60c for 4 mins . Besides, we have excluded some familial data to make sure that the frequency of a1180v from 1000 genomes project was calculated from unrelated individuals . Comparison of the a1180v carrier frequency between our studies and the results of dbsnp database was tested by using the fisher's exact test . Combined with our former study and the 6 years of clinical followup of the affected pedigree, we discovered that a1180v related dcm was preceded by progressive avb which mostly occurred in the third decade of life . During the follow - up period, the condition of an a1180v carrier (ii-8 in figure 1) who had been diagnosed with dcm before aggravated gradually and she now was diagnosed with severe congestive heart failure . Besides, iii-1 with a1180v, developed to dcm and complete atrioventricular block (avb) and another 3 carriers (iii-2, iii-3, iii-13) progressed to different degree of avb . Figure 2 displays changes of echocardiograms of iii-1 and ecg recordings of iii-2 in the follow - up period . None of the individuals in the pedigree showed any sign of long - qt or brugada syndrome and other diseases . After screening a1180v in 460 individuals, we found that a1180v was absent in this group (0/460, 0%). Figure 3(a) gives an example of the sequencing results, and figure 3(b) is a sequencing result of a a1180v carrier in 2007 . The combined carrier frequency, 0% (i.e., 0 out of 660 cases), of a1180v from our studies remarkably suggests that a1180v is not a common variant among healthy chinese, and the data significantly differs from that in the dbsnp database (p = 5.3 10). Data from the 1000 genomes project revealed a carrier frequency of 0.51% (i.e., 1 out of 197 cases) for a1180v among chinese participants . The data was obtained by analyzing the sequencing results of 97 northern and 100 southern chinese adults . Since the southern chinese group was familial related, the data from known offspring were excluded from the calculation of the carrier frequency to make sure the value was calculated from unrelated individuals (http://browser.1000genomes.org/homo_sapiens/variation/population?db=core;r=3:38616415-38617415;v=rs41310765;vdb=variation;vf=12532571). The data also dramatically differs from those deposited in the dbsnp database (p = 0.013). We have presented the followup and sequencing data identifying that a1180v of scn5a was a potential risk factor for dcm, and it is absent among healthy han chinese . Compared with the former results in 2008, 5 a1180v carriers exhibited age - related progressive avb and dcm, and the a1180v carrier frequency among han chinese in our study is 0% out of 660 control cases . Evidence is now being accumulated that scn5a mutations are associated with development of cardiac dilation [13, 1618]. Mcnair et al . Have reported that mutations in scn5a were detected in 1.7% of dcm families with a shared mechanism of disruption of the voltage - sensing mechanism of this channel leading to dcm . Moreover, some scn5a mutations which have been found to be a primary factor of leading dcm were characterized by an age - related phenotype of cardiac dilation with different types of arrhythmia [11, 20]. And the pathogenic characteristic of a1180v conformed to the pattern as well . Since the cardiac sodium channel is principally in charge of the fast depolarization of the myocardium, some suggested that the cardiac dilation may result from long - term arrhythmia [22, 23]. However, our experimental evidence indicated that a1180v could alter calcium homeostasis as a consequence of alterations in intracellular sodium concentrations, and the former condition might damage myocardium directly and subsequently lead to cardiac dilation . Our data have revealed that a1180v specifically appeared in the affected pedigree and was absent among healthy controls, however, why do such significant discrepancies exist for a1180v among different studies of the same ethnic population? (1) different asian ethnic subgroups may have different genetic backgrounds that cause differing prevalence of a genetic variant . For example, r1193q (another scn5a variant) is more prevalent in asians living in taiwan (frequency~12%) compared to asians living in japan (~2%). Most chinese - americans are descendants of people from southern china, so they may have a genetic background that differs slightly from the majority of the subjects enrolled in our studies . The population of the shanghai area consists mostly of people who have immigrated from other areas of china . (2) relatively small sample sizes may also account for high carrier frequency of those data deposited in the dbsnp database . (3) the sequencing depth of different sequencing technologies varies greatly within and between experiments, and it may introduce bias as well . At present, lots of gene mutations were found playing pathogenic roles in both familial and sporadic diseases [26, 27], then another question is that whether a1180v would be a risk factor among sporadic dcm patients as well . In our recent studies of the distributions of candidate mutations in dcm - related genes in sporadic dcm patients conducted using high - throughput sequencing technology, we identified a1180v in 1 out of 68 patients with dcm . The patient, a 41-year - old male, was diagnosed as having dcm with nyha class i, lvedd 78 mm, and lvef 24%, but no other diseases . However, the patient carrying a1180v also carries other mutations in several dcm - related genes, including g1117s, p1119r, y498h of lama4, w768s of rbm20, p398s of vcl, and r659w of scn5a . Although we found that a1180v might also be a risk factor in sporadic dcm patients, we cannot exclude the possibility that a1180v could work together with other gene mutations . In addition to genetic factors, our previous study of the dcm pedigree carrying a1180v demonstrated that the a1180v channel lost current only at high heart rates, which suggested that physical activity and lifestyle that increased average daily heart rate might enhance the phenotype for a1180v carriers . Therefore, environmental factors may also play a role in expression of the pathogenic mechanism of a1180v . Further studies of genetic and environmental factors and followup of a1180v carriers will provide more information about the relationship between the mutation and the risk of dcm . Studies of transgenic mice may also provide a step to test the hypothesis and to explore the underlying mechanism . In conclusion, herein we provide evidence that the a1180v is a potential cause for dcm rather than a common variant in han chinese, although additional genetic or environmental factors may potentiate the risk for dcm in a1180v carriers.
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Premature ovarian aging (poa) is defined by elevated age - specific basal follicle stimulating hormone (fsh) cut - off levels with menstruation . The age - specific cut - off level under the age of 33 years (our patient's age) is 7.0 miu / ml . There are four main causes for pof, namely, idiopathic, genetic, autoimmune, and viral causes . Can we extend causes of pof to poa? If causes are similar and we evaluate women for poa, can we delay the process of pof? With these thoughts, we evaluated this patient and we are reporting the case . A study by gleicher et al . Titled concluded that presumed underlying etiologies of poa follow a similar distribution pattern as reported for pof . They proved the hypotheses that poa is a precursor stage of pof and hence requires similar evaluation . She had an elevated basal fsh level of 28 miu / ml 3 months back . Her height was 1.68 m and she weighed 50 kg with a body mass index of 19 kg / m . A transvaginal ultrasound scan showed a normal - sized uterus but ovaries were not visualized . The repeat basal fsh level (after 6 weeks) was 27 miu / ml . Since the basal fsh level was above the age - specific cut - off level (7 miu / ml for 33 years of age), diagnosis of poa was considered and karyotype was requested . Jacobs et al . Described the first association of triple x syndrome with pof in 1959 . A total of 21 cases of pof with triple x syndrome have been reported in the literature, but to the best of our knowledge, this is the first case report of poa with triple x syndrome . Genetic causes comprised approximately 16% of the total in the study conducted by gleicher et al . Both autosomes and x chromosomal involvement they are turner mosaicism, partial x chromosome deletion, x chromosome mosaicism, x chromosome inactivation, and fmr 1 (fragile site mental retardation x gene). X chromosome partial deletions are more common, while balanced x chromosome to autosome translocation of xq13q26 is rare, but documented . Autosomes involved are at the following gene loci: 3q, 13q, 14q, 17q, 15q, and 11p . Genetic defects are proposed to cause poa and pof by increasing atresia of ovarian follicles due to apoptosis or failure of follicle maturation and thus decreasing the pool of primordial follicles . Triple x syndrome women also suffer from psychiatric disorders like schizophrenia, eeg abnormalities, scoliosis, and genitourinary malformations . Poa with triple x syndrome and primary infertility is treated with ovulation induction by gonadotrophins, because of elevated basal fsh values . Prenatal diagnosis for pregnant women with triple x syndrome is definitely required as there will be 25% chance of x chromosomal abnormalities in the offspring . Hence, we conclude that it is essential to consider karyotyping for all cases of poa, and age - specific basal fsh values will help us detect cases of poa.
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Hyperspectral imaging, as a tool for spectrochemical analysis, integrates the advantage of conventional imaging and spectroscopic technique, which can obtain both spatial and spectral information from a tested object and has been widely used in detecting quality of fruit products . Hypercubes, are made up of hundreds of contiguous wavebands for each spatial position of a target . Hypercubes are three - dimensional (x y) blocks of data, comprising two spatial dimensions (x and y direction) and one wavelength dimension (). Hyperspectral images often contain mass wavebands, which can result in modelling complicated . In addition, the existence of multicollinearity problem could reduce the accuracy of the calibration models . Feature transformation is a process that creates a new set of features and this method has been applied to extract feature in hyperspectral data . Used principal component analysis (pca) and independent component analysis (ica) to reduce the spectral dimension of the hyperspectral reflectance images of cucumber leaves and combined them with linear regression model to estimate chlorophyll concentration based on the extracted pcs and ics . Implemented minimum noise fraction (mnf) rotation on important wavebands to extract the defective feature of hyperspectral images of loquat fruits and finally obtained that the identification accuracy was 92.3% . However, those methods only were used to eliminate useless information in view of spectra and neglect the relationship between spectral values and chemical concentrations . Feature transformation aims at preserving the topological structure of the data whereas the variables selection aims at enhancing the predictive power . Recently, many studies proved that the selection of important variables can predigest calibration modelling and improve the results in terms of accuracy and robustness . Moreover, selection of important wavelengths instead of full spectra of hyperspectral images has an advantage to generate chemical spatial distribution and provide a reference for developing portable multispectral imager . Random frog (rf) methodology is a novel and efficient technique for variable selection, which borrows the framework like reversible jump markov chain monte carlo (rjmcmc) [7, 8]. It executes a search in the model space through both fixed - dimensional and transdimensional moves between different models, and then a pseudo - mcmc chain is computed and used to calculate selection probability (sp) for each variable . Important variables can be selected in terms of the ranking of all variables based on sp . Hu et al . Used a combination of rf selected reflectance and transmittance spectra from hyperspectral data to predict blueberry mechanical properties, and the results showed that prediction models based on rf had similar results with full spectral model . Li et al . Detected tea polyphenols (tp) of 14 cultivars of tea using infrared spectroscopy with important wavenumbers selected by interval partial least squares (ipls) combined with rf; finally, a linear formula with 18 wavenumbers provided satisfactory results for predicting tp measurement . Yu et al . Employed rf and partial least squares regression (plsr) to establish calibration model for predicting total nitrogen content of pepper plant on the basis of hyperspectral imaging in the region of 3801030 nm . The hypercube could provide visualization of biochemical constituents of a sample by calculating the chemical value of each pixel based on the spectral prediction model . There are many ways to develop calibration models, such as principal components regression (pcr), multiple linear regression (mlr), partial least squares regression (plsr), backpropagation neutral network (bpnn), and least - square support vector regression (ls - svr). The widespread uses of plsr make it possible to process visualization of hyperspectral images . Jin et al . Applied rcs - plsr model to shift the spectrum of each pixel into its mc value for visualizing the mc distribution map in peanut kernels . K value spatial distribution map in grass carp and silver carp fillets was generated by employing the successive projection algorithm- (spa-) plsr model of the hyperspectral images (4001000 nm). Simplified mlr model was used to visualize the thiobarbituric acid (tba) values distribution in fish fillets and obtained good results (of 0.8395 and rmsep of 0.1147 mg some authors attempted to realize chemical concentration visualization of hyperspectral images using nonlinear calibration model . For example, bpnn model considerably improved the performance of prediction set (rp of 0.938 and 0.965, rpd of 4.590 and 9.335) for detecting lycopene and total phenolic content in intact tomatoes and the bpnn model made it possible to predict the bioactive compounds in each pixel of the hyperspectral images . . Used hyperspectral imaging technique to detect different browning levels of lychee pericarp fruits that were affected by moisture contents . A few studies reported that spa - ls - svm was successfully applied to generate various indexes (freshness, total viable counts (tvc), and warner - bratzler shear force (wbsf)) distribution of meat for detecting quality of meat products . Other methods also could be used to map component distribution based on hyperspectral images; siripatrawan and makino detected fungal infection on brown rice grains at early stage by using unsupervised self - organizing map (som) and visualized data classification of different levels of fungal infection . Mulberry (fructus mori) fruits with bumpy surface and good taste were popular in food processing . Total soluble solids (tss), which include the carbohydrates, organic acids, proteins, fats, and minerals, can contribute to the quality of fruits . Consequently, there is a need in the main producing industries to determine tss rapidly and nondestructively to assure that fruits meet a minimum level of acceptance . Spectroscopic techniques have been proved to be an effective approach to detect the internal quality of fresh fruits . Unfortunately, the spectroscopic technology fails to provide the quality parameters spatial information, which is essential to detail the analysis of the products' features . What is more, based on the scanning mode, spectroscopic technology generally detects the small areas of fruits' surface and the information of the whole sample might be lost in the testing process . Visualization of chemical components using hyperspectral imaging has received an increasing attention in food - processing industry . The specific objectives of this study were to (1) investigate the potential of vis - nir hyperspectral imaging to detect tss of the mulberry fruits, (2) select the important wavelengths using rf algorithm, (3) evaluate the performance of the different calibration models with rf - plsr and rf - ls - svm based on linear and rbf kernel function, (4) compare the tss visualization results which were developed with plsr and ls - svm and select the optimal calibration model, and (5) predict tss value of each pixel in tested samples on the basis of the optimal calibration model and generate tss spatial distribution . Mulberries from local orchards (hangzhou, zhejiang, china) were selected for the research . Prior to measurement, all 310 fruits were mature with the absence of any green area . Each single fruit constituted a sample, and as a result, 310 samples were collected and stored in the refrigerator at a constant temperature at 3c in the laboratory before the hyperspectral images were acquired . Samples were removed from the refrigerator and placed under room condition (~20c) for more than 2 hours . Mulberry samples were scanned by a push - broom hyperspectral imaging apparatus with reflectance mode as shown in figure 1 . The hyperspectral imaging system mainly consisted of an imaging spectrograph (imspectorv10e, spectral imaging ltd ., finland) covering the spectral range of 3801,030 nm; a ccd camera (c8484 - 05, hamamatsu, japan) coupled with a zoom lens (oles23, specim, spectral imaging ltd ., oulu, finland); an assembled illumination source coupled with two 150-w quartz tungsten halogen lamps (fiber - lite dc950 illuminator, dolan jenner industries inc ., usa); a mobile platform operated by a stepper motor (ircp0076, isuzu optics corp ., taiwan); and a computer with the spectral imaging system v10e software (isuzu optics corp ., taiwan), which was used to set and adjust the parameters of the device, including exposure time, motor speed, imaging acquisition, wavelength range, and image correction . The spectral resolution is 2.8 nm; the resolution of ccd camera is 672 512 (spatial spectral) pixels . Some parameters of apparatus for acquiring hyperspectral images should be set and adjusted before acquiring hyperspectral images of mulberries . In this work, the moving speed of mobile platform is 1.6 mm / s, exposure time of the ccd camera is 0.008 s, and the distance from the lens to samples is 295 mm . The whole system (except the computer) was assembled in a dark chamber to minimize the effects of ambient light during the sample scanning [4, 24]. Due to the existence of dark current in ccd camera and the uneven intensity of the light source in different bands, several bands with weaker light intensity contained the biggest noises . Here, raw hyperspectral images were calibrated using the white and dark reference based on (1) for weakening the effect of dark current in the ccd camera and the uneven intensity of light in different bands:(1)i = irawidarkiwhiteidark, where i is the calibrated hyperspectral images, iraw is the raw hyperspectral images, iwhite is the white reference images (~99% reflectance), and idark is the dark reference images (~0% reflectance). Hyperspectral data were extracted from the calibrated hyperspectral images using envi software (version 4.6, itt visual information solutions, boulder, usa). Before acquiring accurate spectra of the samples, background information should be removed in batches and this process has shorter processing time than manually extracted roi of the individual sample . The results subtracted from waveband at 893 nm (a) and 569 nm (b) are shown in figure 2(c), in which it was found that there were big differences on gray value between tested samples and background images . Then threshold of 0.4 was used to remove the background and good masks (d) were obtained . By implementing the masks in the original hyperspectral images, the background information was removed . The separated region was identified as the region of interest (roi) of the sample . The average spectrum of spectra of all pixels in a mulberry was considered as the spectrum of a sample and 310 spectrums were collected . To avoid the low signal - noise ratio and diminish the problem of high dimensionality of feature spaces, the wavebands of 4201,000 nm were considered in the analysis . Ultimately, a spectral data matrix of 310 460 (samples wavebands) was obtained for further analysis . Total soluble solids (tss, brix%) in mulberry samples were measured using traditional destructive tests . After acquiring the hyperspectral data, each fruit unit was juiced and tss were measured by using way-2s digital refractometer (shanghai precision & scientific instrument co., ltd ., the instrument range covered from 0 to 95% with temperature correction and refractive index accuracy is 0.0002 . All measurements were averaged over the data from three replicates in a room at 20c . The key steps of rf are illustrated in figure 3 and the detailed algorithm of rf was described in literatures [7, 8]. Before running rf algorithm, five parameters (t, q,,, and) should be assigned to proper values . T was the number of iterations and needed to be sufficiently large to achieve convergence (t = 10,000); q was the number of variables in the initialized variables set (q = 50); controlled variance of a normal distribution (= 0.3); was a coefficient explained in step 2 (= 3); represented the upper bound of the probability (= 0.1). Partial least squares regression (plsr) is a multivariate data analysis technique which generalizes and combines features from principal component analysis (pca) and multiple linear regression (mlr). Plsr has been successfully used in developing multivariate calibration models, as it uses the concentration information (y) in determining how regression factors are computed from the spectral data matrix (x), thereby reducing the impact of irrelevant x - variations in the calibration model . There were three steps to develop plsr; the first step is to decompose the matrix and the model is(2)x = tp+e, y = uq+f, where t and u are the score matrices of x matrix and y matrix, p and q are the loading matrices of x matrix and y matrix, and e and f are the errors which come from the process of pls . It must build the following linear correlation:(3)u = bt+e, where b = (tt)ty . Ls - svm is an alternate formulation of svm regression proposed by suykens et al . . The main advantage is that it is computationally more efficient than the standard svm method . Optimization problem of ls - svm is formulated:(5)minjw, e=12wtw+12k=1nek2 subject to the constraints(6)yk = wtxk+b+ek, k=1,,n, where is the regularization parameter which balances the model's complexity and the training errors; ek is the random error; xk and yk are input and output variables; k is sample number . And then, lagrange function is applied to solve the optimization problem(7)lw, b, e,=jw, ek=1nkwtxk+b+ekyk, where k r is lagrange multipliers . The solution of the above equation can be obtained by partially differentiating with respect to each variable:(8)lw=0w=k=1nkxk,lb=0k=1nk=0,lek=0k=ek, k=1,,n,lk=0wtxk+b+ekyk=0, k=1,,n . When the variables w and e are removed, the equation can be rewritten as a linear function group(9)01t1+1iba=0y with(10)y = y1,,yn,1=1,,1,=1,,n,=kl,n, where k (xi, xj) is defined as the kernel function and must satisfy mercer's condition . Kernel function can map sample in original space to high - dimensional feature space to solve the linear inseparable problem . There are several typical examples of kernel function such as linear kernel, polynomial kernel, rbf, and sigmoid kernel . Each kernel has some parameters, while rbf kernel function is strongly recommended and widely used for its performance and complexity . Ls - svm with rbf kernel and linear function were selected in our work to compare the predictive performance with plsr . Grid - research and leave - one - out cross - validation were used to find out the optimal (gam) value and (sig2), which is the bandwidth in the case of the rbf kernel . Is the regularization parameter, determining the trade - off between structural risk minimization principle and empirical risk minimization, and is important to improve the generalization performance of ls - svm models, while controls the value of function regression error and influences the number of initial eigenvalues and is absent in linear kernel function . In this case, we use leave - one - out cv to determine the tuning parameters . The performances of models were evaluated using correlation coefficient (r) and root mean square error (rmse) in calibration set (rc, rmsec), cross - validation set (rcv, rmsecv), and prediction set (rp, rmsep). Generally, an optimal model should offer high r values and low rmse values; small difference existed between calibration and prediction set . The optimized model was employed to predict tss of mulberries . As shown in figure 4, in method (i), the hyperspectral image of a sample is a 3d data cube (x y) (a), and there are n pixels in x and y direction, respectively . First, unfold the three - dimensional data matrix into a two - dimensional (n n) matrix and the data matrix with n n pixels being defined as variable xi (b), and plsr model (c) was applied to predict the chemical value of each pixel, forming a prediction image (g). In method (ii), the pixels at the same position at important wavebands were extracted and arranged in a row, and all pixels were arranged at column; in all, i n data matrix (d) was formed . Ls - svm was used to calculate tss values of pixels, 1 n data (e) was figured out, and then (e) was folded into a n n matrix (f), namely, the image of the tested sample with predictive tss value . Pseudo - colour images were created with different colours representing different levels of tss that were predicted by the optimal simplified model . There are many kinds of noise in hyperspectral images, such as electrical noise from ccd detector; the noise was caused by transmitting procedure and others . The presence of the noise seriously affects the feature extraction and recognition accuracy of the tested objects . Median filtering, in which gray value of every pixel is set to be the average gray value in a certain neighbourhood window, is a nonlinear filtering technique that has been successfully applied to many signal and image processing tasks . Median filtering (5 5) was applied to denoise hyperspectral images in this study . Random frog (version 2.0) toolbox, ls - svm lab v1.8 toolbox, median filtering toolbox, and the visualization program were finished with matlab r2009a (version 7.8) software . Origin pro 8.0 sr0 (origin lab corporation, northampton, ma, usa) software was employed to design the curve figures . All the processes were run on a pc (cpu: g2020 @ 2.90 ghz, ram 4.00 gb) operating on windows 7 . In order to establish the calibration models of the tss in mulberries, all mulberry samples were divided into a calibration set and prediction set according the ratio about 3: 1 (235: 75) by spxy method, in which sample set partitioning is based on joint x - y distances . Table 1 summarized the measured tss of all samples, the maximum and minimum value of calibration set were 10.99brix and 3.21brix, and the range of prediction set was 9.863.86brix . Usually, calibration set with a wide range of the chemical component could establish a robust model . Spectra of all mulberries covering the range of 4201,000 nm are displayed in figure 5 . Lower reflectance (<10%) in the visible region of 420650 nm was attributed to the relatively homogeneous and dark purple or black colour of mature fruits and mainly caused by anthocyanin and chlorophyll [35, 36]. Reflectance for mulberry samples started to increase dramatically from 650 to 800 nm and reached a peak at 850 nm . An obvious valley located around 960980 nm, which was attributed to the combination effect of -oh groups from carbohydrate and water . Chemometrics methods were introduced to analyze the spectra and establish the relationship between spectra and measured tss to determine the internal quality of mulberries in the future study . Figure 6 displays sps of wavelengths, and a small number of wavelengths displayed had high sp (over 0.9); most of the wavelengths were with low sp, and these results showed that there existed a lot of useless information to make tss content be predicted using hyperspectral imaging . If the cutoff of sp was 0.7 (1) and 0.85 (2), there were 23 (960, 929, 814, 849, 432, 500, 877, 995, 903, 915, 831, 956, 967, 954, 923, 511, 454, 840, 835, 527, 509, 850, and 883 nm) and 11 (960, 929, 814, 849, 432, 500, 877, 995, 903, 915, and 831 nm) important wavelengths selected, respectively . These wavebands were ranked by descending order of sps . The concentration of anthocyanin and chlorophyll caused low reference in the visible region and contributed to the maturity of mulberry fruits . Wavelengths at 814 nm, 975989 nm, and 981 nm correspond to second and third overtones of nh, 877 nm and 900950 nm were assigned to ch third overtone, and 960 nm was assigned to oh second overtone . These bands are assigned to mono- and ploy - carbohydrates (fructose, glucose, and pectin) and water in mulberry . In order to establish calibration models with fewer features, two important wavelengths sets (23 versus 11) were employed to establish calibration models and these results were compared to obtain optimal visualization map of tss in mulberry . Multivariate analyses, developed with leave - one - out cv, were used to find accurate plsr and ls - svm models for the prediction of tss . The predictive models of tss in mulberry fruits were built using the two kinds of the selected wavelengths (23 versus 11) and the results of these models are enumerated in table 2 . Overall, ls - svm regression models had better performance for predicting tss than plsr models because ls - svm is a nonlinear regression model and it could transform the original data into a high dimension space to make linear solution . Ls - svm with rbf kernel function based on 23 wavelengths with rp of 0.956 and rmsep of 0.430 could provide the most effective tss estimation compared to other models, while ls - svm models with linear function had similar results with rf - plsr models (except with full wavelengths). This consequence caused by the rbf kernel function of ls - svm has an advantage in conducting samples in multidimensional space . In addition, linear kernel function in ls - svm was considered as a special form of rbf kernel function . Model (9) with 11 important wavelengths had an approving expression with rp of 0.925 and rmsep of 0.557, and these results were equal to the performance of the full - plsr model (rp of 0.959 and rmsep of 0.411). When the number of important wavelengths reduced to almost a half (11 versus 23), model (9) only had a little reduction of 3.24% (rp) and an increase of 29.5% (rmsep) compared to model (6). Although ls - svm had better performance, plsr with 23 important wavelengths offered acceptable results . Model (4) provided a reliable result of rp of 0.899 and rmsep of 0.675 . Compared with full - plsr model, rc, rcv, and rp in model (4) showed a slight reduction of 3.88%, 2.52%, and 5.47%; rmsec, rmsecv, and rmsep provided a change of 0.213, 0.101, and 0.228, respectively . However, 95.0% of the variables (23 versus 460) were removed in rf - plsr model . The accuracy of rf - plsr model was higher than spa - mlr model in the literature of for predicting tss of mulberries, because hyperspectral imaging could provide both spectra and image information about tested samples . In addition, spectra of whole fruit were averaged as the spectrum of the sample to avoid the loss of spectra . But beyond that, there were only two wavelengths in the spa - mlr model which meant that useful spectral information might be over - removed . However, rf - plsr model for predicting tss of mulberries was not as good as monte carlo - uninformative variable elimination- (mc - uve-) spa (mc - uve - spa) model for predicting tss of ya pear . This phenomenon could be explained that the mulberry fruit has a bumpy surface that has more influence on the spectra than does a smooth surface . Tss prediction of mulberries based on rf - plsr model was parallel to the prediction tss of blueberries that were acquired by interval partial least square- (ipls-) plsr model . In order to seek an optimal calibration model for realizing tss visualization in mulberry fruits, models (4), (8), and (9) the comparative maps of six samples developed by three models are exhibited in figure 7 . Six mulberry fruits were used to compare the reliability of tss distribution maps, which were predicted by three models . Plsr (figure 7(a)) and ls - svm with linear kernel function (figure 7(b)) could provide clearly tss distribution, while ls - svm with rbf kernel function (figure 7(c)) failed to display tss visualization of mulberry fruits . There are four possible reasons to explain this phenomenon: (1) the special modelling way of ls - svm with rbf kernel function, which needed to transfer the raw data into high - dimensional space, might change the original data form; (2) there were two parameters (,) to control the predicted results of ls - svm, which might add the complexity of ls - svm; (3) using calibration model with 235 variables to predict a map (such a map of a mulberry was about 100 200 pixels) might produce problem of overfitting; (4) the mulberry fruit with bumpy appearance would bring the variation of spectral reflectance, which might affect the accuracy of models . The linear kernel function is the special form of rbf kernel function, and ls - svm with linear kernel function (ls - svm - linear) (b) had better tss distribution (b) than that of ls - svm - rbf . However, in figure 7(b), it was hard to distinguish the tss level of mulberry and a mistake was made in sample (2). The measured tss value of sample (2) was 8.2brix and it was the highest tss value among these six samples, while in figure 7(b), it had the lightest colour . Plsr had the best performance compared to ls - svm for its simple linear combination . At the same time, the fitting effects of plsr and ls - svm with linear kernel function (ls - svm - linear) were compared and expressed in figure 7(d). The correlation coefficients between measured tss values and predicted tss values in plsr and ls - svm with linear kernel function (ls - svm - linear) were 0.857 and 0.500, respectively . Although ls - svm could offer satisfactory results in calibration model, plsr with simple algorithm was feasible to map tss distribution in mulberry fruits . When the simplified model was finally established, it was subsequently employed to predict tss in each pixel of the image resulting in new pseudo - colour images and this process was called prediction map . As the last step of analyzing hyperspectral image, rf - plsr model was used to predict tss of each pixel and transferred its hyperspectral image to the tss distribution map . The multilinear function for the tss prediction of the mulberries was obtained:(12)ytss=16.2070.19110.81821.71731.4814 + 0.0075 + 0.03761.29170.19381.11291.017101.606110.230120.168130.132140.92415 + 0.04216 + 0.023171.541181.57419 + 0.04720 + 0.040211.474221.25223,where i is the spectral reflectance value at the wavelength of i nm (i was 960, 929, 814, 849, 432, 500, 877, 995, 903, 915, 831, 956, 967, 954, 923, 511, 454, 840, 835, 527, 509, 850, and 883, resp .) And ytss is the predictive tss values of the mulberry fruits . New hyperspectral data with only 23 wavelengths could speed up the visualization process and make it easier to establish a multispectral imaging system . The function (12) was employed to predict the tss of each pixel within the mulberry fruits hyperspectral images . The predicted tss concentration of each pixel was mapped with a linear colour scale using different colours from red to blue to represent different tss concentrations from high to low . Mulberries with higher tss values have more pixels shown in red, such as in figure 8 (16) samples . The more the pixels coloured in green and blue, the lower the tss values . There were unsatisfying results: the blue pixels in samples (13) and (14) were saturated points and were not considered in the analysis . Different values in the maps of tss distribution were in quantitative proportion to the spectrum of the corresponding pixels . The ability to provide spatial information makes hyperspectral imaging available to focus on detecting both external and internal quality of fruits . To sum up, the successful mapping of tss distribution in mulberries suggested that the application of hyperspectral imaging to realize the visualization of mulberry fruits' internal quality is feasible and promising . The plsr and ls - svm model based on 23 and 11 wavelengths had a good performance to predict tss of mulberries, which indicated that rf algorithm was effective in reducing three - dimensional data . It could be revealed that plsr was feasible to map chemical component concentration (tss) distribution of mulberry fruits . This research provided a theoretical basis for developing the instrument for measuring the internal quality of fruits and made it possible to sort mulberries based on tss spatial distribution.
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The folding and assembly of proteins and protein complexes is essential for all aspects of cellular function, with profound consequences on the long - term health of the cell, as well as organismal lifespan [1 - 5]. The challenge to proteome stability over the course of a lifespan is substantial and is impacted by mutations, expressed polymorphisms and intrinsic errors in gene expression on the one hand [5 - 7], and by the acute effects of environmental and metabolic stresses on the other [8 - 12]. The rate of error accumulation determines the flux of destabilized, meta - stable proteins that tend to misfold and aggregate, thereby putting the health of the cell at risk . Proteostasis is achieved when the flux of metastable proteins associated with biosynthetic processes is balanced by the function of basal protein quality control networks, responsible for protecting the proteome [3, 7]. Because proteomic composition and stability are dynamic over the lifespan of an organism, the functional status of the proteome is also constantly monitored by stress signaling pathways so as to prevent protein misfolding and aggregation [5, 13 - 15]. Proteostasis is, therefore, closely associated with the regulation of stress response pathways, including the heat shock response, the unfolded protein responses [14, 15], the oxidative stress response, and the metabolic stress response [17, 18]. Over - expression of thecorresponding stress response transcription factors, such as hsf-1, atf-6 and ire-1, skn-1, and daf-16, or enhancing the activity of chaperones, the ubiquitin proteasome system or autophagy, all have significant beneficial effects on lifespan extension and suppression of age - related misfolding diseases [12, 19 - 24]. While cells possess elegant and efficient mechanisms to autonomously activate stress response pathways and deploy resources proportional to their needs, there are provocative examples in which proteostatic networks fail to adjust to cellular demands or when stress responses are poorly activated . These include a limited heat shock response in early mammalian development and the absence of stress response activation in somatic cells of caenorhabditis elegans mutants deficient in neuronal signaling . Other examples are, the reduced proteostasis capacity and stress response activation in different tissues of aged animals and in late - onset neurodegenerative diseases, including huntington s disease (hd), alzheimer s disease (ad), amyotrophic lateral sclerosis (als) and parkinson s disease (pd) [5, 25 - 34]. Recent studies mainly conducted in c. elegans uncovered several cell - nonautonomous pathways that modulate cellular quality control systems . This list includes cell - nonautonomous regulation of stress responses, such as the heat shock response and the er and mitochondrial unfolded protein responses [36, 37], as well as transcellular signaling of proteostasis deficiencies in which the expression of misfolded protein in one tissue can induce a systemic response [38, 39]. Moreover, the same signal can inversely regulate proteostasis maintenance and the response of somatic cells to heat shock, suggesting that the capacity of quality control systems can be regulated cell - nonautonomously by various signals that can differentially modulate organismal responses to proteins damage . We, therefore, asked why does proteostasis become imbalanced with age? Specifically, is the accumulation of damaged proteins an inherent property of proteostatic networks? If not, are such networks malleable at different stages over the lifespan of an organism? Several possible non - mutually exclusive mechanisms can explain the failure of proteostasis networks to maintain the proteome of aged animals . Protein damage and misfolding in aged individuals could result from a limited efficiency of cellular quality control networks in repairing or removing misfolded proteins throughout an organism s life, leading to a gradual accumulation of damaged proteins over time (fig . For example, declining translation fidelity may result in an increased load of damage proteins as the individual ages (fig . Finally, the ability of cellular quality control networks to maintain the proteome and rebalance itself may be differentially regulated during the lifespan of the organism, leading to a rapid remodeling of cellular folding capacity and stress tolerance (fig . One prediction that ensues from these proposed mechanisms is that the rate of damage accumulation should determine whether proteostasis is remodeled during adulthood . If reprogramming of proteostasis that affects the efficiency of cellular quality control networks does occur, then the rate of damage accumulation should be modified over the course of an organism s life, leading to differential deterioration of cellular proteome stability . Specifically, a rapid change in the regulation and activation of stress responses is expected . Studies in c. elegans have demonstrated that cellular quality control networks are modified as animals undergo transition to a reproductively mature state . The temporal requirement for both daf-16 and hsf-1 was found to change upon transition to adulthood such that knockdown of daf-16 during reproductive adulthood was sufficient to modulate lifespan, whereas knockdown of hsf-1 during development was mostly associated with lifespan modulation [40, 41]. Altered temporal regulation was also apparent for c. elegans c - jun n - terminal kinase (jnk) signaling . While the jnk homolog kgb-1 enhances daf-16 nuclear localization and transcriptional regulation during c. elegans development, this function is reversed upon transition to adulthood . Likewise, epidermal growth factor (egf) signaling up - regulated the expression of genes associated with the ubiquitin proteasome system yet down - regulated the expression of some chaperones at the time of transition to reproductive adulthood . A change in the activation of jnk signaling and expression of proteasome subunits was also observed in adult drosophila melanogaster, although expression modulation was not monitored early in adulthood [31, 44, 45]. Changes in expression of quality control machinery components are associated with changes in proteostasis function over time . For example, the rate of protein clearance is modulated early in adulthood in worms, flies and rats [43, 46 - 48]. Likewise, expression levels of autophagy - associated genes declined with age, concomitantly with reduced autophagic activity in adult drosophila and rats [31, 49]. These data demonstrate that major signaling pathways that modulate stress - resistance, proteostatic capacity and longevity demonstrate a switch - like change in behavior upon transition to adulthood (fig . Such regulatory changes are associated with suppressed activation of several stress response pathways, including the heat shock response and the er unfolded protein response (upr), at the point of transition to reproductive adulthood in worms and flies [37, 50, 51]. Stress responses enable the cell to adjust the expression of chaperones and other cyto - protective genes under proteotoxic conditions, thereby ensuring stress survival, recovery and adaptation . Thermo - resistance and induction of heat shock genes sharply declines 8 - 12 h following transition to adulthood . Likewise, activation of stress resistance and the upr in response to a range of er perturbations, such as exposure to tunicamycin or thapsigargin, declines early in adulthood [30, 37]. The induction of responses to other stress stimuli examined, such as the oxidative stress response and mitochondrial unfolded protein responses (upr), also declined sharply upon transition to adulthood (itay valenci, personal communication). These data suggest that stress - induced transcriptional activation is strongly dampened early in adulthood and exhibits switch - like behavior associated with reproduction onset . This supports the hypothesis that quality control networks are remodeled with age and highlights the transition to adulthood as being a critical window during which time regulation of proteostasis can be modified . However, proteostasis may also be remodeled at other stages during the lifespan of an organism, such as the modulation of the upr and heat shock response seen during development . If cellular quality control networks undergo remodeling upon transition to adulthood, one would expect proteostatic capacity to decline early in adulthood . As the activity of quality control networks reduces, interactions with a given protein could change the rate at which that protein misfolds . Indeed, in a model of huntington s disease in which animals express poly - glutamine (polyq) containing 35 repeats, aggregation and toxicity followed the onset of reproduction when animals were cultivated at 20c (~4 days post - embryo) or 25c (2.5 days post - embryo) [51, 53]. Thus, aggregation - prone protiens quickly became insoluble upon transition to adulthood . Monitoring the functions and folding of meta - stable, temperature - sensitive (ts) mutant proteins demonstrated that these proteins also become unstable early in adulthood . For example, expression of a ts myosin resulted in age - dependent mislocalization of myosin and uncoordinated movement in c. elegans . The phenotype was not muscle - specific, as it was also apparent in neuronal cells and coelomocytes when animals expressing a ts mutant version of dyn-1 were examined . Other temperature - sensitive mutant proteins assayed for changes in their folding capacity, such as ras, the acetylcholine receptor and perlecan (unc-52), all showed a rapid loss of function early in adulthood (days 2 - 6 of adulthood) [30, 51]. Finally, wild type proteins also show age - dependent misfolding and associated decline in function, albeit later in adulthood [30, 51, 54]. For example, wild type myosin also showed age - dependent mislocalization and misfolding, although the onset occurred later in adulthood [30, 51]. The age - dependent misfolding of wild type proteins is supported by an unbiased examination of age - related aggregation of the c. elegans proteome using a systematic proteomic approach and mass - spectrometry, where an age - dependent increase in insolubility of several hundred proteins was seen . It is interesting to note that some proteins displayed an early onset of misfolding, i.e., as early as day 3 of adulthood . The fact that many of the same proteins were identified in two independent studies [55, 56] emphasizes the vulnerability of some proteins to proteostasis capacity during adulthood . The wide - range remodeling of quality control networks upon transition to reproductive adulthood, therefore, results in age - dependent collapse of cellular proteostasis (fig . 2), the severity of which depending on the genetic polymorphism of the organism [6, 57 - 59]. The transition between development and adulthood represents a critical window during which time individuals must partition resources between parent and progeny so as to maximize reproductive fitness . Because changes in proteostatic composition and capacity coincide with the onset of oocyte biomass production, it was suggested that disrupting the function of the reproductive system would affect the observed decline in proteostatic maintenance seen during adulthood [24, 30, 43, 51]. Reproduction is linked to aging by endocrine signaling from germline stem cells (gscs) and the somatic gonad . Removal of gscs by laser ablation or through mutations inhibiting proliferation increases the lifespan of c. elegans and modulates several signaling pathways (reviewed in). In contrast, removal of the entire gonad did not lead to extended lifespan, suggesting that longevity triggered by germline elimination is not a simple consequence of sterility but that the somatic gonad likely emits signals that modulate lifespan [61, 62]. In fact, bile acid - like (dafachronic acids) steroid signals from the somatic gonad are modulated upon transition to adulthood and were found to extend c. elegans lifespan by activating the daf-12 steroid hormone receptor [63 - 65]. Thus, there is a hormone - dependent switch upon transition to adulthood that regulates reproduction and lifespan . The link between reproduction and lifespan is not specific to c. elegans but was also observed in other model animals . For instance, ablation of gscs of the nematode pristionchus pacificus [62, 66] or enhanced differentiation and loss of gscs in drosophila melanogaster also extended lifespan . Moreover, transplantation of young ovaries into old mice increased the lifespan of the recipients by promoting longevity [68, 69], supporting a role for signals from the reproductive system, and specifically from gscs, in modulating lifespan . The association between the timing of proteostatic remodeling and longevity further supports a possible link between proteostasis and reproduction . To establish whether reproductive status is linked to the altered expression of and requirement for quality control machineries, as well as to restriction of protein folding capacity in adulthood, the effects of inhibiting reproduction, and specifically gsc proliferation, on proteostasis networks and their capabilities were examined in c. elegans . Removal of gscs by laser ablation or mutations inhibiting gsc proliferation was shown to modulate the function of several transcription factors that mediate stress responses, such as daf-16 and hsf-1 [61, 62, 71 - 73]. Moreover, germline - less mutant animals were found to induce the expression of autophagy - associated genes, such as lgg-1, bec-1 and unc-51 . Likewise, inhibition of germline proliferation mildly reduced the expression of various proteasome subunits, while leading to a specific 3-fold induction in the expression of the rpn-6.1 subunit . The augmented level of rpn-6.1 was shown to affect expression of other proteasome subunits . When the transcriptional response of p. pacificus was monitored following germline laser ablation, whole genome microarray analysis identified over 3000 differentially expressed genes . This population was highly enriched for ribosomal and translation-, proteasome-, protein folding- and refolding - associated genes . Furthermore, the expression of tcer-1, a transcriptional elongation factor, and heat shock genes (following exposure to heat shock) was modulated in c. elegans [51, 72]. Thus, affecting gsc proliferation modulated the expression of different proteostatic components, including those belonging to the translational, chaperone, autophagic and proteasomal machineries [24, 51, 72, 74]. Given that the expression of specific sets of proteostatic components is determined by different signaling pathways [24, 51, 74], it would seem that gsc signaling activates several different regulatory programs that differentially modulate somatic functions . It is interesting to note that down - regulation of any of the gsc - dependent signaling pathways is required for increased lifespan, with these pathways differentially modulating proteostasis functions and stress response regulation [24, 51, 74]. This suggests that only the combined actions of all of these pathways are sufficient for lifespan enhancement, while proteostasis function can be modulated by partial activation of these pathways . This is in agreement with chemical and genetic manipulation of lifespan - extending pathways in which lifespan, proteostasis and stress resistance were mechanistically dissociated [75 - 78]. Modulated expression of proteostasis components was shown to be tightly associated with widespread effects on proteostatic functions, specifically of quality control systems . Induced expression of autophagy genes in germline - less animals was associated with increased autophagosome numbers . Gsc - dependent activation of autophagy is regulated by the foxa transcription factor pha-4, which was specifically required for the expression of autophagy genes and autophagosome formation . Likewise, increasing the expression lipl-4, which is regulated by gsc signaling, can also activate autophagy [74, 79]. The induced expression of rpn-6.1 in germline - less mutant animals resulted in increased levels of chymotrypsin - like proteasome activity and decreased levels of highly polyubiquitinated proteins in c. elegans . This activity is mediated mainly by daf-16 and kri-1 but not by hsf-1 . Over - expression of rpn-6.1 was sufficient to increase proteasome activity in wild type animals . Moreover, rpn-6.1 is essential not only for animals lacking gscs but also for other long - lived mutants, such as those with reduced iis (daf-2), mitochondrial electron transport chain (isp-1) or food intake (eat-2) levels . Thermo - resistance is also modulated by gscs [51, 80]. In this case, diverse mutations in the c. elegans reproductive system were examined to determine whether disrupting reproduction modulates stress resistance in adulthood . When decline in the ability to induce a heat shock response was monitored, it was noted that only mutants exhibiting defects in gsc proliferation but not other sterile mutants gained the ability to mount an effective stress response in different somatic tissues during adulthood . In agreement, inducing dna damage in germ cells or inhibiting checkpoint genes also gave rise to thermo - resistance in adulthood [81, 82]. Other repair and defense responses, such as the oxidative stress response [61, 81] or the innate immune response to pathogenic bacteria [83, 84], are also modulated in germline - less animals . Together, these observations demonstrate that signals from gscs can regulate different stress responses and improve c. elegans survival under stress conditions . Inhibition of gscs proliferation also modulated the ability of somatic cells to prevent protein aggregation . When a model for huntington s disease in c. elegans in which animals expressing polyq with 35 repeats in the body wall muscle was considered, germline - less animals displayed reduced aggregation, as well as reduced toxicity . Likewise, mimicking gsc - dependent activation of proteasomal function by over - expression of rpn-6.1 was sufficient to reduce aggregation and toxicity in animals expressing polyq with 67 repeats in c. elegans neurons, suggesting that proteostatic capacity is increased when germline proliferation is inhibited . Proteostatic capacity was also monitored by employing metastable proteins, such as temperature - sensitive unc-52, as probes over the lifespan of c. elegans . While the functions of disparate metastable proteins in wild type animals were compromised with age, unc-52(ts) function was rescued in germline - less animals . In a similar manner, while the myofilaments of wild type animals were mostly disrupted, myofilament integrity in germline - less animals was well maintained and was associated with improved motility . Furthermore, age - dependent mislocalization of the neuronal protein dyn-1 was modified by inhibition of gsc proliferation . Thus, inhibition of gsc proliferation abrogated the decline in protein quality control seen early in adulthood in different somatic tissues . For example, while daf-16/kri-1/tcer-1 and hsf-1 are required for the activation of the heat shock response in adulthood, other modifiers, such as daf-12, daf-9, daf-36, nhr-80, and pha-4, differentially modulate other aspects of somatic quality control function [24, 51, 74, 80]. Taken together, several different signaling pathways are modulated upon transition to adulthood by gsc inhibition, with these signaling pathways drastically remodeling somatic proteostasis . Thus, a switch - like mechanism links gsc status with the maintenance of somatic proteostasis via regulation of the expression and function of different quality control machineries and cellular stress responses that progressively leads to a decline in the maintenance of proteostasis in adulthood why would inhibition of germline proliferation lead to improved quality control in the soma? A restricted capacity of somatic maintenance has been suggested to result from the allocation of resources to reproduction at the cost of somatic tissue maintenance and lifespan . The link between gsc proliferation and somatic functions may, therefore, stem from gscs communicating their commitment to reproduction to the somatic gonad . Proliferation of gscs then activates signaling cascades that modulate metabolism and somatic functions that, in turn, would affect the rate of aging . If so, then not just inhibition of gsc proliferation but any damage to the germline would be expected to be reported to the soma and affect somatic maintenance, in general, and protein quality control networks, specifically . In agreement, different mutations in dna damage checkpoint genes or mutations that block programmed cell death affect somatic stress resistance and lifespan [82, 86]. These mutations likely only affect proliferating gscs, since c. elegans somatic cells are post - mitotic and resistant to dna damage - induced program cell death . In fact, somatic cells of intact and germline - less animals do not express dna damage response - signaling proteins or activate programmed cell death . Although it should be noted that use of the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (fudr), suggested to inhibit dna replication in gscs, was found to modulate proteostasis and stress resistance in germline- and gonadogenesis - defective mutants . Given that radiation, inactivation of checkpoint proteins and impairment of nucleotide excision repair in gscs all lead to improved stress survival and activation of proteasome expression and function, it would seem that dna damage in the germline, like inhibition of gsc proliferation, results in improved activation of stress responses and changes in the expression of proteostatic components [81, 82, 86]. Signals to the soma can be transmitted via induction of an innate immune response, while cell - nonautonomous signals from the soma, requiring kri-1, mediate germline cell death in response to dna damage . It is of note that kri-1 was also shown to affect protective signals from the gsc to the soma, suggesting the existence of crosstalk between the germline and the soma . A tradeoff between reproduction and somatic maintenance would also be expected to respond to environmental conditions that impact reproductive success . Given that reproduction is affected by environmental conditions, such as food availability and temperature [90, 91], crosstalk between gscs and the soma should result in the transmission of environmental cues to the gscs . Indeed, starvation conditions in adulthood often lead to death due to internal hatching of progeny and to reduced germline number and size [91, 92]. Animals starved from the early fourth larval stage (i.e, before the onset of oocyte biogenesis) were more likely to escape this fate by delaying the onset of oogenesis and embryo production and hence producing fewer viable progeny, suggesting that environmental cues uncouple gsc proliferation from stem cell maintenance [92, 93]. Not only food depravation but also changes in food sources can be buffered by somatic functions to maintain the germline and thus enhance reproductive robustness . The transition to adulthood may, therefore, correspond to a regulatory window during which time environmental conditions and gsc competence are weighed to determine reproductive potential and the mode of proteostasis required . However, current data have not uncovered the nature of the signals originating from the reproductive system, how and by whom they are received, or the mechanism of proteostatic remodeling . Answering these questions will further our understanding of how protein folding is regulated in multi - cellular organisms . This is particularly important given the large number of diseases associated with age and misfolding . Understanding the nature of the signal and how it is received may, therefore, offer novel directions for treatment of protein misfolding diseases; diseases with different etiologies but similar underlying biology.
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Numerous publications have clearly defined the role of pkc as transforming oncogene in fibroblasts and epithelial cells . Overexpression of pkc in nih3t3 fibroblasts and frc / tex cl d rat colonic epithelial cells was shown to increase cell proliferation, enhance anchorage - independent colony formation, and induce a highly tumorigenic in vivo phenotype with tumor incidence of 100% [1, 2]. In addition, nih3t3 fibroblasts with pkc overexpression were invasive and displayed a polarized morphology with extended long cellular membrane protrusions . Epidermis - specific pkc transgenic mice developed highly malignant and metastatic squamous cell carcinomas in response to 12-o - tetradecanoylphorbol-13-acetate stimulation . Pkc was demonstrated to transform androgen - dependent lncap prostate cancer cells into an androgen - independent variant . Moreover, transgenic mice with selective overexpression of pkc in the prostate epithelium developed prostate hyperplasia and prostate intraepithelial neoplasia . Our laboratory demonstrated that inhibition of pkc in mda - mb231 cells, a highly metastatic breast cancer cell line with elevated pkc levels, was sufficient to dramatically decrease in vivo tumor growth and reduce the incidence of lung metastasis . Subsequently, pkc was shown to promote an invasive and motile phenotype in hnscc through modulation of rhoa presumably through posttranslation phosphorylation . Rhoa, a member of the rho gtpase family, has been implicated to be involved in the development and/or progression of numerous cancers . A recent report showed that overexpression of rhoa is sufficient to immortalize human mammary epithelial cells . Moreover, mir-31 was reported to be inversely associated with metastasis through inhibition of rhoa in breast cancer patients . Multivariate analysis revealed that elevated rhoa is an independent prognostic biomarker of poorer overall survival in pancreatic adenocarcinoma . High levels of rhoa correlated with venous invasion, advanced ptnm stage, and prognosis in hepatocellular carcinoma [13, 14]. Increased rhoa is associated with tumor progression in ovarian carcinoma and lymph node metastasis and overall survival in bladder carcinoma [15, 16]. Similarly, rhoa was shown to be biomarker for lymph node metastasis and overall survival in esophageal squamous cell carcinoma . Rhoa, rac2, and cdc42 were found to be elevated in premalignant dysplastic and hnscc cell lines in comparison to normal keratinocytes . Furthermore, based on their immunohistochemistry analyses, rhoa was suggested to be a promising biomarker of malignancy and/or aggressiveness in head and neck squamous cell carcinoma (hnscc). Our previous work provided the initial evidence linking two proteins, pkc and rhoa, intimately involved in metastasis . Pkc was shown to signal through rhoa to modulate cell invasion and motility in hnscc . In this study interestingly, an active atp - docked pkc conformation is not required for pkc to bind to rhoa indicating that the pkc-rhoa complex is assembled independently of the transient substrate - kinase interaction at the catalytic site of pkc. Stoichiometric fret analysis with hek293 cells overexpressing mcherry - pkc and egfp - rhoa revealed that the pkc-rhoa complex is assembled in the cytoplasm and subsequently translocates to the cell membrane . Our work revealed that pkc phosphorylates rhoa but, intriguingly, also has a kinase - independent action to function as a chaperone to traffic rhoa to the cell membrane . Human pkc cdna was cloned into pentr / d - topo vector (invitrogen, carlsbad, ca) by pcr from a human cdna library (clontech, mountain view, ca). The n - mcherry - tagged pkc was made by inserting pkc open reading frame into bglii / xbai site of mcherry - c1 vector (clontech, mountain view, ca). Mcherry - pkc/k437r mutant was generated using the quikchange lightning kit (agilent technologies, inc ., the positive control plasmid mcherry - linker - egfp for stoichiometric fret analysis was made by inserting mcherry dna fragment into nhei / bglii sites and followed by eliminating the sequence between bamh1 and bglii sites within the multiple cloning site in vector egfp - c1 (clontech), resulting in a 10 amino acid long in - frame linker sglkdppvat . Hek293 cells were purchased from atcc (rockville, md) and cultured in dulbecco's modified eagle's medium supplemented with penicillin (100 units / ml), streptomycin (100 g / ml), and 10% fetal bovine serum . Recombinant pkc was incubated with recombinant rhoa in kinase buffer (24 mm tris (ph 7.4), 0.5 mm edta, 0.5 mm egta, 10 mm -mercaptoethanol, 1 g / ml leupeptin, 1 g / ml aprotinin, and 50 g / ml pmsf) containing pkc activators, phosphatidylserine and diacylglycerol, and [p]atp for 30 minutes at 25c . Subsequently, termination buffer consisting of 7.5 m guanidine - hcl was added to stop the reaction . Rhoa was phosphorylated by pkc in vitro and then subjected to digestion by trypsin, chymotrypsin, or glu - c . Following enzyme digestion the sample was acidified to 0.5% trifluoroacetic acid concentration and stored at 20c until further analyzed . The digested rhoa protein was analyzed by reverse - phase nanoscale lc - ms using a waters qtof premier mass spectrometry system . Prior to analysis edta and diammonium phosphate were added to sample for a final concentration of 25 mm each, and 1125 ng of digested protein was analyzed . Peptides were separated using acetonitrile / water mobile phases containing 0.1% formic acid on a waters nanoacquity uplc system employing a 300 m i d 20 mm c-18 5 m particle symmetry trap column and a 75 m i d 150 mm c-18 1.7 m beh analytical column . Peptides were trapped for 15 minutes at 3 l / min followed by gradient elution using 028% acetonitrile in 40 minutes through the analytical column at 300 nl / min . Esi was conducted at approximately 3.3 kv using in - house prepared spray emitters . Emitters were constructed by sleeving a 7 cm piece of 20 m i d 90 od fsc into a 3 cm piece of 100 m i d 360 od fsc and gluing the junction with epoxy . The polyimide coating on the terminal end of the emitter was burned off using a microtorch, and the emitter was used with a waters nanoease esi mount . The qtof premier mass spectrometer was programmed to collect alternate scan ms data as previously described [19, 20]. Briefly, ms data collection was performed by a low collision energy acquisition of 0.8 seconds followed by a high collision energy acquisition for 0.8 seconds without quadrupole mass filtering across a 501990 m / z mass range . This was performed in an alternating fashion during a 65-minute run and termed lc - ms analysis . Low collision energy acquisition records all peptide precursor mass data, while the high collision energy portion of the acquisition collected peptide fragmentation data . Following the low collision energy acquisition set at 10 volts, collision energy was ramped from 10 volts to 40 volts over the 0.8 second high collision energy acquisition to accommodate peptides requiring different collision energy for fragmentation . Glu - fibrinopeptide at a concentration of 200 fmol/l in 25% acetonitrile / water/0.1% formic acid was introduced as a lockspray calibrant through a second esi probe at 0.5 l / min using an auxiliary uplc pump . Lockspray data was collected for 1 second every 30 seconds over a 65-minute analysis . Following an lc - ms analysis, data processing was performed using waters plgs software version 2.3 build 23 using the following parameters: low - energy threshold 100 counts, high - energy threshold 10 counts, and an intensity threshold of 1000 counts . Data processing combined the signal intensity of all charge states generated from a given peptides into singly charged mh values and determined the peak apex for both low - energy precursors and all fragment ions within the vicinity of the precursor . First, the data was used to search human refseq database version 17 within plgs software using the following search parameters: peptide and fragment tolerance was automatic, the minimum fragment ion matches per peptide were 3, the minimum fragment ions per protein were 7, the minimum peptides matches per protein were 1, missed cleavages were 2, the false positive rate was 4%, and modifications allowed were acetyl n - term, carbamidomethyl - c, carbamyl n - term, and phosphorylation at sty . Second, low - energy precursor mh data was copied into excel and compared to mh values calculated for predicted rhoa trypsin, chymotrypsin, or glu - c protease peptides bearing up to 4 phosphate groups . Experimental mh masses that matched within 0.03 da of gpmaw calculated values were evaluated manually in plgs or masslynx protein / peptide editor software . Possible phosphopeptide assignment was made when the measured mass was within 0.03 da of the calculated phosphopeptide mass and greater than 3 accurate mass product ions could be assigned to a peptide sequence . Confident site - specific phosphorylation also used this criteria but further required fragment ions including phosphorylated serine or threonine amino acids ., san diego, ca) was incubated with recombinant rhoa (cytoskeleton, denver, co) in pkc kinase buffer for 30 minutes at 25c . The binding reaction was immunoprecipitated using agarose - conjugated anti - pkc antibody or igg (abcam, cambridge, ma) at 4c with gentle agitation overnight . The suspension was centrifuged at 1,000 g for 1 minute, and the agarose beads were washed three times with ice - cold pbs and resuspended in sds sample buffer . The same procedure was performed to immunoprecipitate the binding reaction containing his - tagged pkc-kinase domain (biobasic inc ., markham, canada), and rhoa except agarose - conjugated anti - his antibody (abcam) was used . The immunoprecipitated samples were boiled in sds sample buffer, resolved by sds - page, and transferred to immobilon membrane . The membranes were incubated with anti - rhoa (cytoskeleton) or anti - his (millipore, billerica, ma) antibodies and visualized by ecl using the fast western kit (pierce, rockford, il). Hek293 cells were seeded on 35 mm glass - bottomed dishes one day prior to transfection with mcherry - pkc and egfp - rhoa . Fluorescence microscopy experiments were performed in the center for live - cell imaging (clci) at the university of michigan medical school using an olympus ix70 inverted microscope (olympus, center valley, pa). Experiments involving live - cell imaging employed a heated stage (harvard apparatus, inc ., holliston, ma) in combination with hepes - buffered medium to maintain cell viability and activity for several hours of microscopic observation . Illumination was provided from a 100 w halogen lamp for phase - contrast microscopy and by an x - cite 120 metal halide light source (exfo, mississauga, canada) for fluorescent microscopy . The microscope was equipped with 100x (oil immersion; uplan fl, na = 1.30), 40x (lcplanfl, na = 0.6), and 10x (cplan, na = 0.25) objectives . Rockingham, vt) were used for fluorescent imaging; in particular set number 86014v2 includes filters used for gfp (excitation 492 nm / bp18, emission 535 nm / bp40) and mcherry (excitation 572 nm / bp23, emission 630 nm / bp60). The excitation and emission filters were mounted in a lambda 10 - 3 automatic filter wheels (sutter instrument co., novato, ca) allowing rapid filter switching . Images were collected using a coolsnap hq2 14-bit ccd camera (photometrics, tucson, az). All devices were controlled through metamorph premier v7.7 software (molecular devices, downingtown, pa). Quantitative analysis of the imaging data and the preparation of presentation quality images were performed using metamorph v7.7 software . Quantitative stoichiometric fret analysis of the data was performed with proprietary software created by the clci staff using matlab r2009a (the mathworks, natick, ma), and this fretcalc software can be obtained from the university of michigan tech transfer . The methods and algorithms used in fret stoichiometry have been previously described [21, 22]. Our laboratory reported that pkc modulates rhoa activity in hnscc presumably through posttranslation phosphorylation . In silico prediction of phosphorylation sites identified multiple serine and threonine residues that are putative pkc phosphorylation sites on rhoa suggesting that direct phosphorylation of rhoa through pkc may be a possibility . To determine if pkc can directly phosphorylate rhoa, we performed an in vitro kinase reaction and incubated recombinant pkc with rhoa in the presence of pkc activators, phosphatidylserine and diacylglycerol, and p - atp . Pro - q diamond, a phosphoprotein staining reagent, confirmed rhoa as a substrate for pkc (figure 1(b)). Next, we identified the phosphorylation sites on rhoa using phosphopeptide mapping with liquid chromatography - mass spectrometry / mass spectrometry (lc - ms / ms). Lc - ms analysis of peptides resulting from trypsin digested rhoa showed about 83% coverage, whereas the combined data from trypsin and glu - c digested rhoa showed 100% coverage of the serine and threonine residues on rhoa . Thus, it is reasonable to conclude that a comprehensive phosphopeptide map of rhoa was generated using trypsin and glu - c . Our results provide the first evidence that rhoa is a direct substrate for pkc phosphorylation . There is limited, although intriguing, literature demonstrating that a kinase preassembles with its substrate prior to a kinase phosphorylation event . The preassembled kinase - substrate complex not only increases specificity but also shortens the time between kinase activation and phosphorylation of the substrate [23, 24]. To determine if pkc preassembles with rhoa without an active atp - docked pkc conformation, recombinant pkc and rhoa were incubated in atp - free in vitro kinase buffer . As shown in figure 1(c), pkc was able to bind to rhoa under this condition . Moreover, the kinase domain of pkc was sufficient to bind to rhoa demonstrating that the rhoa docking site is within the pkc kinase domain (figure 1(d)). These results indicate that the binding between pkc and rhoa does not require an active atp - docked pkc kinase conformation, and thus, the interaction between these two proteins is more complex than a transient substrate - kinase intermediate state . There is evidence that a preassembled kinase - substrate complex not only enhances phosphorylation specificity and efficiency but also plays a role in cellular localization . To determine if pkc mediates the localization of rhoa, hek293 cells were transfected with mcherry - pkc or egfp - rhoa or cotransfected with mcherry - pkc and egfp - rhoa . Subcellular localization of pkc and rhoa was visualized in living cells using fluorescence microscopy in the presence or absence of phorbol 12-myristate 13-acetate (pma). Activation of pkcs with pma is associated with the translocation of pkcs to the cell membrane . As expected, pkc was translocated from the cytoplasm to the cell membrane following pma stimulation in hek293 cells overexpressing mcherry - pkc (figure 2(a)). In contrast, in hek293 cells overexpressing egfp - rhoa, rhoa remained at the cytoplasm following pma treatment . As shown in figure 2(b), pma treatment induced rhoa to colocalize with pkc at the cell membrane in hek293 cells transfected to overexpress mcherry - pkc and egfp - rhoa . Our data showed that pkc traffics rhoa to the cell membrane following a general pkc activation signal in hek293 cells . To better define the interaction between pkc and rhoa with spatiotemporal resolution, hek293 cells overexpressing mcherry - pkc and egfp - rhoa were stimulated with pma, and images collected over a 12.5 minute time course were subjected to quantitative stoichiometric fret analysis . Pma treatment induced an obvious reorganization of mcherry - pkc and egfp - rhoa in the cell from the cytoplasm to the cell membrane as evidenced by comparing the ia and i d images at 0 min and 12.5 min after pma stimulation, respectively (figure 3). Furthermore, pma - treatment resulted in an overall increase in ed, a measure of the fret efficiency of the interaction between mcherry - pkc and egfp - rhoa . The initial increase and peak in ed occurred in the cytoplasm followed by an elevation of the pkc-rhoa interaction at the cell membrane for the entire time course . Recruitment of rhoa and the increase in fret activity was especially robust in the actively ruffling regions of the cell (upper and bottom right corners of the cell). Taken together, fret analysis demonstrated that in response to pma stimulation, the pkc-rhoa complex is recruited to the cell membrane over time, and furthermore, the pkc-rhoa complex may be preassembled in the cytoplasm prior to translocation to the cell membrane . Our in vitro protein binding experiments indicate that an active atp - docked pkc confirmation is not required for pkc to bind to rhoa . The kinase - inactive pkc mutant (k437r) contains a point mutation in the atp binding pocket to prevent atp occupancy . Interestingly, pkc/k437r is localized to the cell membrane in unstimulated hek293 cells overexpressing mcherry - pkc/k437r (figure 4(a)). Hek293 cells cotransfected with mcherry - pkc/k437r and egfp - rhoa showed colocalization of these two proteins at the cell membrane without pma stimulation . In support of these observations, quantitative stoichiometric fret analysis confirmed the interaction between mcherry - pkc/k437r and egfp - rhoa at the cell membrane prior to pma stimulation (figure 4(b)). The fret signal at the cell membrane did not change significantly after pma treatment indicating that the pkc/k437r - rhoa molecules at that cell compartment were already in complex with each other . These results indicate that the pkc-rhoa interaction occurs in the absence of atp and thus do not require an active catalytic site on pkc. The k437r mutation appears to alter the conformation of pkc to expose the critical amino acids required to interact with rhoa . Our data confirm that cell membrane localization of rhoa is mediated independently of a pkc-rhoa phosphorylation event . Regulation of rhoa activity is tightly controlled in the gdp / gtp cycle through the coordinated interactions between gtpase activating proteins (rhogaps), guanine dissociation inhibitors (rhogdis), and guanine nucleotide exchange factors (rhogefs). Rhogaps deactivate rhoa by enhancing the intrinsic gtpase activity of rhoa to hydrolyze gtp to gdp . In addition to this well - described regulatory pathway, there is evidence that posttranslational phosphorylation is an alternate mechanism used to control rhoa . Protein kinase a (pka) was reported to phosphorylate s188 of rhoa resulting in relocalization of gtp - bound rhoa from the membrane to the cytoplasm, possibly through enhanced interaction with rhogdi . Protein kinase g (pkg) activation was demonstrated to increase rhoa stability resulting in an increase in total rhoa protein levels . Furthermore, phosphorylation of s188 on rhoa protected rhoa, particularly the gtp - bound active form, from ubiquitin / proteasome - mediated degradation . In the present study, it is interesting that three different kinases, pka, pkg, and pkc, are able to phosphorylate s188 suggesting that phosphorylation of s188 may be a nondiscriminatory mechanism to enhance rhoa stability . In addition to s188, t127 was identified as a novel rhoa phosphorylation site suggesting that pkc may sequentially phosphorylate rhoa to fine tune rhoa levels and/or activation . Ongoing efforts in our laboratory will delineate the physiological significance of t127 and s188 phosphorylation on rhoa function in hnscc . We made the novel observation that recombinant pkc associates with recombinant rhoa in the absence of atp indicating that the pkc-rhoa complex is assembled without an active atp - docked pkc conformation . Additionally, kinase - inactive pkc was sufficient to colocalize with rhoa at the cell membrane in live hek293 cells providing further evidence that the pkc-rhoa interaction is much more involved than as a transient kinase - substrate transition state . These observations is consistent with a published report demonstrating that the mitogen activated protein kinase (mapk) substrate complexes are often spatially separate from the kinase active site and the substrate phosphorylation site . The region of the kinase that binds to a substrate has only been identified in two cases, for c - jun aminoterminal kinase 2 (jnk2) and c - terminal src kinase (csk) [29, 30]. The substrate - docking sites for jnk2 and csk were identified to be within the kinase domain and in proximity, within 50 amino acids, to the catalytic loop of the kinase [29, 30]. Consistent with these results, the kinase domain of pkc is sufficient to bind to rhoa . Our work showed that pkc binds to rhoa within the kinase domain and without the requirement of atp . The accepted model of pma - mediated activation of pkcs is that pma changes pkcs from a closed to an open conformation resulting in translocation of pkcs to the cell membrane . Fret results showed that the initial response to pma is an increase in the molecular interaction between pkc and rhoa in the cytoplasm . This observation indicates that the active pkc conformation is required to expose the rhoa docking site and thus allow rhoa to complex with pkc. The interaction between pkc and rhoa showed an early peak at the cytoplasm and then decreased to basal levels for the remainder of the time course . In contrast, a gradual but prolonged increase in fret intensity was observed at the cell membrane, in particular the actively ruffling regions of the cell, over the entire time course . A plausible explanation is that, following a pkc activation signal, pkc and rhoa are assembled in the cytoplasm, and the resulting complex is subsequently trafficked to the cell membrane . It is important to point out that pma does not induce translocation of rhoa to the cell membrane without the presence of pkc. Therefore, the recruitment of the pkc-rhoa complex to the cell membrane is completely dependent on the cellular localization of pkc in response to a stimulus . Fluorescence microscopy showed that kinase - inactive pkc is predominantly localized to the cell membrane under basal conditions . This result suggests that the pkc/k437r is in an open conformation capable to interact with chaperone proteins involved in pkc translocation . The cellular localization of rhoa is concentrated at the cell membrane in cells cotransfected with mcherry - pkc/k437r and egfp - rhoa . Similarly, fret analysis showed that the interaction between pkc/k437r and rhoa is concentrated at the cell membrane rather than at the cytoplasm in unstimulated cells . Furthermore, pma did not alter the extent of the molecular interaction between pkc/k437r and rhoa at the cell membrane . Fret analysis with pkc/k437r confirmed our in vitro observations and showed that pkc is able to recruit rhoa to the cell membrane without a pkc-rhoa phosphorylation event . Taken together, these findings support a kinase independent role of pkc as a chaperone to traffic rhoa to the cell membrane . Work from our laboratory provided the initial evidence linking two proteins, pkc and rhoa, intimately involved in metastasis . The pkc-rhoa signaling module was shown to modulate cancer cell invasion and motility . However, the molecular mechanism of pkc regulation of rhoa remains to be elucidated . In this study, our results revealed that pkc has both kinase - dependent and kinase - independent functions to regulate rhoa; pkc directly phosphorylates rhoa and, moreover, serves as a chaperone to translocate rhoa to the cell membrane.
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Industrial areas are characterized by a high density of industries, sharing common infrastructures, such as transport networks, waste water treatment plants, and waste incineration plants . They have historically attracted, and may still attract, hundreds of employees who have settled in the vicinity of the plants . With extensive urbanization, industrial areas have been embedded in the urban landscape, increasing the nuisances and the exposure of the population . For instance, in the south of france, the industrial area of l'etang de berre hosts 430 industries classified for the protection of the environment and more than 60% of the seveso ii (referring to the european directive 96/82/ce) plants of the region . About 16 towns representing more than 300,000 inhabitants are exposed to the plumes produced by these plants . People living near major industrial areas are facing complex situations of exposure: occupational and environmental exposure, multiexposure to chemicals combined with exposure to noise, dusts, visual pollution, stress, and so forth the possible associated health risks are of the highest concern to the population . Quantitative health risk assessments, based on the comparison of a hypothetical exposure (assessed through measured or modeled concentrations in different matrices combined with scenarios of exposure) to toxicological reference values or to regulatory values, have been extensively used for regulatory purposes . For instance, several risk assessments around large french industrial areas found that the levels of some compounds, including benzene, particulate matter (pm), and so2, could be considered too high [2, 3]. They confirmed that the concerns of the population were legitimate and triggered actions to reduce those specific pollutions . Yet, quantitative health risk assessments can neither tell if and how many people are actually suffering because of the pollution, nor they can take into account the integrated burden of the multiexposure to the chemical, physical, and perceived industrial pollution . The answers to these questions belong to epidemiologists and raise several methodological issues: what kind of study should be used, which health outcomes should be investigated, how to assess exposure, and how to control for confounding factors? In this paper, we performed a literature review of the published studies investigating the health of population exposed to industrial air pollution around major industrial sites . The objectives were (1) to identify the reasons why studies were performed, (2) to list the health outcomes that have been investigated, (3) to describe the study designs that have been used, and (4) to describe and discuss the exposure assessments . The objectives were not to perform a systematic review but to collect a representative sample of the different practices that can be used in that field . The work focused on studies investigating the chronic effects of air pollution from large industrial areas and major complexes grouping several plans or multicenter studies involving similar types of industry that could be or not part of larger industrial complexes . Papers published between 1980 and 2012 were searched based on the scopus database that includes pubmed and other relevant literature database . As an initial research using key words referring to industry retrieved very few papers, we searched epidemiological studies on the impacts of air pollution around point sources . On a second step, the initial search equation was ((air pollutants [mesh] or air pollution [mesh]) and epidemiology [subheading]) or ((air pollution [title / abstract] or air pollutants [title / abstract]) and (epidemiology or epidemio * or case control study or cohort or cross sectional study or prevalence or incidence or surveillance or survey or health risk or risk assessment or health or health effects or exposure or health impact * or mortality or adverse effects)) and (industry or industrial) and (residents or residential or inhabitants or neighborhood * or vicinity or living area or living near or surrounding * or populations). Papers were analyzed focusing on the types of industries, the study design, the health indicators, and the exposure assessment . The objectives were to identify the methods but not to discuss the results reported by each paper . From the initial search 230 papers in english or french were selected based on their title . Based on the abstracts, 155 papers were excluded (58 environmental studies only, 35 looking at exposure through water, soil, or food and not air directly, 36 using industrial areas as one source among other air pollution sources, 10 description of the health of a population without links to exposure, 8 on nuclear installations, 4 toxicological studies, 3 studies focusing on acute exposure after an accident, and 1 literature review). Two reports from the grey literature were added, but no specific search was performed to identify such reports on a larger scale . 77 papers were finally included in the review, published between 1989 and 2011 . While papers were available from many different countries, a majority of papers came from 3 european countries: the united kingdom, italy, and spain (table 1). One paper may provide results for several studies, and 27 studies were focusing on cancer, 25 on morbidity, 9 on biomonitoring, 7 on mortality or birth outcome, and 3 on mental health . The reasons for doing an epidemiological study on cancer near a major industrial area were frequently concerns from the population, explicitly quoted by 7 studies [1, 7, 12, 15, 17, 25, 27]. Few studies gave details on the social background, showing that the health issues crystallized the concerns of the population . The controversy was such that the health concerns were central issues in a public inquiry, and received extensive media coverage . Our study was requested by the local authority, to help resolve this controversy . Study was undertaken in response to concerns of a local pressure group based [] about an alleged cluster of cancer, especially of the larynx, and leukaemia among children [] there was also concern about several deaths among teachers and pupils at the nearby comprehensive school . In 11 other studies, concern of the population is not mentioned, but the authors justified their study with references to an overincidence of cancers or mortality observed in the area by previous investigation [46, 8, 9, 14, 16, 19, 26, 28, 31]. By contrast, multicenter studies refer to the literature and possible etiology in relation to the emissions to justify their choices [2022, 24], although geographical variations of the incidence of the cancers investigated are also used as a justification for focusing in a specific region or on a specific cancer [10, 18, 23]. Study areas vary from very rural areas with about 2,000 inhabitants to highly populated areas with several hundred thousand people [1, 4, 12]. The industries involved in the studies are highly heterogeneous and usually have been operating since several decades before the study period, with areas sometimes industrialized since the 19th century . Six studies were on refineries [6, 16, 17, 26, 28], including one multicenter study in the united kingdom, and 3 on petrochemical plants [15, 27], including one multicenter study in louisiana . Larger sites gather a variety of different industries . For instance, teesside includes iron, steel industries, chemical, and heavy engineering industries . By 1945 in france, a site like etang de berre involves oil refining, oil storage, petrochemical and organic chemical activities, chlorine chemistry, steel and metal working, waste incineration plant, and the port for ore and oil tankers . Among the multicenter studies, industrial sites of different natures were involved in a study in italy [4, 5] and in spain . Wilkinson et al . Studied 11 petrol refineries corresponding to 7 industrial areas . In a study investigating the petrochemical industries in louisiana, simonsen et al . Used three different criteria to aggregate the industries: (1) all sites were considered as a whole, without regard to specific emissions; (2) sites were classified on the basis of their standard industrial classification code as either belonging to the petrochemical industry or not; (3) sites were classified on the basis of the international agency for research on cancer (iarc) carcinogen rating assigned to their specific chemical releases . European registries of polluting industries were extensively used in spain [10, 18, 2024, 32, 33] to perform the multicenter studies . In some cases [10, 23, 24], all sites were included . For instance, the study by cambra et al . Included 66 sites, aggregated into 6 categories: 4 energy production plants, 28 metalworking industries, 8 cement industries, 44 chemical industries, and 17 others . In other cases, only the industries corresponding to one activity, for example, metal production [20, 22] or paper, pulp, and board industries, were included . Most of the studies (20/27) used a geographical ecological design, based on standardized mortality or morbidity ratios, searching for a possible overincidence of the mortality or the morbidity . Poisson regression and similar statistical designs were used to assess a relationship between health indicators and exposure, taking into account confounding factors (mostly socioeconomic) (table 2). For instance, zambon et al . Included 172 cases of sarcoma and 405 controls in their study . The multicenter design was used either for case - control studies [4, 5, 7, 10] or for standardized incidence or mortality ratio studies [14, 18, 2024, 31]. Lung cancer was the most commonly studied [1, 5, 710, 15, 16, 18, 19, 21, 24, 25, 28, 34], based on registries, mortality data, or hospitalizations data . Other cancers investigated were leukemia [6, 15, 20, 2527], digestive cancers, non - hodgkin's lymphoma and sarcoma, either based on mortality or registry data . The latency of cancer was usually taken into account as the number of years of residence in the area before deaths . It varied from at least 1 year (e.g.,) to 10 years (e.g.,) and was sometime unspecified . Distance was used as the method to assess the exposure in 19 of the studies . The use of distance is seen as a way to overcome the lack of measurement data, but also to reduce the latency problem, as clearly stated by pless - mulloli et al . : areas closest to steel and chemical plants at the time of study were also close 40 years earlier, an important consideration given the long latency of lung cancer . However, this requires the assumption that people were also living in the same area 40 years earlier . Several options were used for the distance (table 2), for instance, exposed group (near) 5 km from a metal production plant, intermediate 5 km from any industrial installation other than metal production and processing, unexposed group (far), consisting of towns having no eper - registered industry within 5 km of their municipal centroid (reference level), distance: 02 km, 07.5 km, and eight bands around refinery perimeters with outer limits at 0.5, 1, 2, 3, 4.5, 5.6, 6.6, and 7.5 km, three concentric circles with radii of 3, 8, and 10 km for descriptive purposes and 10 concentric circles with a radius increasing from 1 to 10 km to define nine bands . Exposed group (near) 5 km from a metal production plant, intermediate 5 km from any industrial installation other than metal production and processing, unexposed group (far), consisting of towns having no eper - registered industry within 5 km of their municipal centroid (reference level), distance: 02 km, 07.5 km, and eight bands around refinery perimeters with outer limits at 0.5, 1, 2, 3, 4.5, 5.6, 6.6, and 7.5 km, three concentric circles with radii of 3, 8, and 10 km for descriptive purposes and 10 concentric circles with a radius increasing from 1 to 10 km to define nine bands . Additional refinement may be added, taking into account, for instance, the residential history . Bhopal et al . Made an original combination of different metrics to characterized exposure: perceived exposure areas (criteria not specified), modeled exposure (model not specified), and the 24-hour mean daily measures of so2 and smoke over 56 months . In finland, the exposure area was based on distance, but that distance was chosen based on measurements of sulfuric acid and the prevailing wind directions . Edwards et al . Also mentioned that their choice of the distance was guided by a validation study using data from historical records and measurements . Another example of a complex exposure assessment initially relying on distance is given by yu et al . : to account for the effects from monthly prevailing wind, they defined exposure wedges for each month by the monthly prevailing wind direction . Only addresses located within the exposure wedges were considered exposed during the particular month, and the exposure opportunity scores for these residences were assigned by the inverse of distance to the relevant petrochemical complexes . Although reference sites are usually defined as the farthest to the plant, some studies include a further subclassification taking into account proximity to traffic, urban, semiurban, and rural areas . For instance, the industrial area can be defined based on the distance between the subject's residence and an industrial installation (industrial distance), as the area defined by the first decile of industrial distance . Models were used by only 5 studies . The industrial source complex model in long - term model was used by zambon et al ., and atmospheric dispersion model system version 3-adms 3 was used in france [1, 31]. The other two models were not detailed [12, 15]. In the etang de berre study, results from the models were combined with measurements to obtain a map of the annual mean levels of so2, which were then grouped in three classes of exposure based on quartiles . Derived two indicators from the air pollution model, corresponding to different hypotheses about the mode of exposure: the concentrations alone represented exposure from inhalation only; the number of years the plant had operated and the degradation speed in soils provided a cumulative ground - level concentrations since the start of the activity . The lack of emission data is a key limitation to modeling, acknowledged by some authors . In france, viel et al . Used a complex process to recreate emissions based on exposure judgment in order to be able to complete the dispersion modeling . Measures alone were used by one study only, taking advantage of a relatively dense air quality monitoring network for so2 and pm10 . More frequently, measures were used to describe areas previously chosen based on distance or modeling, and measurements were not input in the statistical models . For instance, in the case of stenungsund in sweden, models (unspecified) of ethylene levels based on the emissions of year 2000 were used to classify a low and a high exposure area . Measurements were performed in the high exposure areas (ethylene, propylene, benzene, 1,3-butadiene, 1,2-dichloroethane (edc), and vinyl chloride) in 2001 - 2002 and 2006 - 2007 . They were used to perform ahealth risk assessment but not directly in the epidemiological study . In the area of teesside, abundant routinely available air quality data, reflecting long standing concerns about air pollution there, were used to check the validity of the selection of study areas based on residential proximity to industry as a proxy for exposure . Again, there is a great diversity of the industries involved in the studies, similar to those described for cancer . Concern was a major motivation quoted by 12 studies [1, 12, 35, 36, 38, 41, 46, 48, 51, 54, 57]. For instance, bhopal et al . Stated that one of the major concerns among the residents [] was an apparent increase in the incidence of asthma in the area . Reference to previous studies showing over - incidences of cancer, mortality or asthma are also quoted by 11 studies [37, 40, 42, 45, 48, 49, 52, 53, 56]., the prevalence of childhood asthma [] was approximately twice that of a control area []. One study mentioned that an acute episode had severe impacts, resulting in hospitalizations . A majority of the studies focused on the respiratory health of children (17 studies), using questionnaires specifically defined for the study or standardized questionnaires such as the isaac questionnaire from the international study of asthma and allergies in childhood [39, 45, 47, 54], or the questionnaire from the american thoracic society (ats) [40, 43]. Few studies used additional data from general practitioners (gps) [12, 13, 49, 59]. Studies involved from 200 to 500 children [41, 43, 47] to more than 3000 children . 6,399 adults were also interviewed in teesside, while in india the respiratory health of 2573 women was investigated . One study in thailand investigated short - term memory dysfunction in children through questionnaires (table 3). One study focused on odor annoyance, based on the observation that odors from industrial sources, such as the petrochemical plants in sarnia, have been shown to considerably impact general health and well - being by affecting both the physiological and psychosocial status of people . Compared gps information on the respiratory health of 874 children for two periods: when the industry was operating and after its closure . Stenlund et al . Investigated the influence of a measure taken to reduce air pollution (predominantly dust and soot) on perceived pollution, risk perception, annoyance, and health symptoms through interviews of 684 people . Five studies used an ecological approach to study standard rates ratio based on hospital admissions or disease incidence . Two studies quantified the relationship between symptoms and measurements through a time - series analysis and a case - cross over analysis . Participants of the cross - sectional surveys were selected based on their city of residence (or school), and distance was again the preferred method to define the exposed versus nonexposed cities . In most studies, a finer exposure assessment was performed for the participants, based on information collected through the questionnaires, modeling, or measurements . When measurements were available, they were not always used to assess exposure . For instance, moraes et al . Mentioned that concentrations were available for several pollutants (pm, nox; so2, o3, benzene, toluene, and xylenes) but used them for descriptive purposes only (in comparison to the world health organization air quality standards). White et al . Reported that they did not have the budget for a model and that concentration and emissions data were missing . Therefore, they add that they rely on a meteorologically estimated exposure index based on wind direction and speed . Aylin et al . Also explained that they had to use distance because input data for the dispersion modeling were missing . Selected the participating cities based on the annual averages of so2, no2, and o3 and mentioned that the reference area is polluted but considered proposed two indicators to characterize the long - term versus short - term exposure: short - term exposure was assessed through pm10 measurements, and long - term exposure was defined as living near an active site . Regarding short - term, acute exposure, dubnov et al . Developed a complex indicator for episodes when nox and so2 concentrations were high . For each episode, they computed an integrated concentration value (icv) as nox multiplied by so2 summarized the results over the entire study period (3 years). One study compared the associations between emergency department visits and so2 concentrations obtained from fixed monitors and from an air dispersion modeling and found some differences increasing with the distance . They were all geographical ecological studies, distance being used as the exposure indicator except in one study relying on so2 dispersion modeling . One study was multicentric, focusing on 10 coke works operating in england and listed in the coke oven managers association . Seven studies on birth outcomes were identified, with three focusing on the same petrochemical area in taiwan [28, 65, 66]. The main sites were those already investigated for other health issues, such as teesside . Again, concerns of the population were the main reason for investigation in the studies focusing on a single area [12, 13, 67], while results from the literature and etiology were the reasons for the three multicenter studies [6870]. In taiwan, studies were justified on observed excess cancer mortality among women [28, 71]. Distance was the method used by all the studies but one, although extensive measurements were available in some sites, like in israel, for instance . In that case, the measurements and the wind rose were used to validate the choice of the distance, resulting in a large exposed area, up to 20 km . By contrast, in the multicenter study in texas, proximity to industrial sites was defined at 1 mile or less . Three studies investigated mental health, psychological distress [72, 73], and one study investigated perceived pollution, perceived health and stigma . All relied on postal questionnaires that may be complemented by a smaller number of semistructured face - to - face interviews . Participants were located in three areas distant to the site (1.5, 7, and 8 km) (table 7). The local background and concerns of the population were not the main motivation in the two studies in the united states based on industrial registries [72, 73]. On the contrary, population concern was a major issue in the study on teesside, as stated by bush et al ., a place stigmatized not only for its heavy industry (technological stigma) but also on the basis of air pollution and poor health . Two studies investigated the psychological distress of the population in relation to their proximity to industries registered in the toxic release inventory through questionnaires . In these studies, the main assumption is not that an over - exposure to air pollutants can create adverse psychological effects, but that proximity to industrial activity is psychologically harmful because many individuals perceive industrial activity negatively, as a potential health threat or a sign of neighborhood disorder . Therefore, exposure was defined based on distance, taking into account the volumes of the emissions as a proxy for facility size and visibility . The authors made the assumption that industrial facilities are not likely to impact residents' mental health if residents are unaware of them . They propose a method to compute a potential visual exposure to industrial activity for each resident [72, 73]. Nine biomonitoring studies were reviewed . In none, even the one based in teesside, concern of the population participants were always recruited based on their residency in a city close to the industry . Additional data were usually collected to refine the exposure assessment of each participants for instance, near chlor alkali plant in sweden and italy, measurements of total gaseous mercury and a dispersion model (transport air pollution model (tapm)) were used to assess the exposure at residence (table 6). However, it was interesting to note that when studying cancer, very few results were statistically significant, although several studies concluded on a gradient of risk following the exposure gradient [4, 1921]. The risks estimated by the multicenter studies were also statistically nonsignificant, although significant risks may be found when a subanalysis of the study focuses on a single industry or a subgroup of industries [23, 24]. Morbidity, and especially less severe outcomes such as respiratory symptoms, eyes symptoms or consultations to the general practitioners tended to increase with exposure [35, 39, 40, 4245, 5356, 62]. Similar results were found for hospitalizations for respiratory and cardiovascular causes [1, 34, 36, 52]. In the studies of declared health, complaints about odors or dust all studies on mental health underlined the influence of living near major industrial sites on psychological distress [7274]. Epidemiological studies investigating the impacts of air pollution produced by major industrial sources are scarce, as only 77 papers were found in this review . However, our search is likely to be incomplete, and the limits of this search are probably the largest on the biomonitoring studies and the mental health studies, as we did not included these as explicit key words in the search . However, given that the papers we included in the review were written by different teams, in different areas and at different periods, we are still confident that it can give a good overview of the practices in the field . Yet, it has to be noted that several papers were produced by the same team and/or part larger initiatives on industrial pollution, which may limit the diversity of practices reported . We also included two reports from the grey literature in the review [1, 36], but there are probably many unpublished work on the health status around industrial areas . For instance, bentov et al . Performed a study on the congenital malformation of a large industrial estate in israel, explaining that their study was initiated by the israel ministry of health, following complaints of residents of surrounding localities who blame the ip emissions for the odor nuisance and suspect that possible long- or short - term health disorders could be attributed to this exposure . It is likely that other health outcomes have been investigated given the context, yet no paper was found on that area . Similarly, rusconi et al . Mentioned that an excess of respiratory symptoms in children was observed in the sarroch region, near a major petrochemical area, referring to unpublished data . Several reasons may explain the low number of publications; few epidemiological studies may be performed because of the complexity of collecting health and exposure data or because quantitative risk assessment is extensively used to study industrial pollution . There may also be a publication bias, with studies showing no link between exposure and health not being published . In many of the cases, the studies are justified by a concern from the population; that is, epidemiology is used to test the hypothesis made by the population that the industries impair their health . It is also used to investigate areas where an overincidence of a health outcome had been previously observed . There are few initiatives to identify the health effects of a given industry independently of the local context, and these initiatives are mostly multicenter studies based on industrial registries indeed, whatever the topic (cancer, mental health, etc). In summary, the multicenter studies based on industrial registries are not taking into account the local context to select the areas under investigation, while mostly all others studies do . Therefore, there is likely to be a bias in site selection where to perform epidemiological studies, based on the existence of a local social mobilization . It would be interesting to understand why in some areas industries raised high concerns and lead to epidemiological studies, while in others there is such social mobilization, and if these reasons may result in biases in the result of the studies . On the other hand, it is essential to answer the population concerns, and, as stated by ginns et al ., the kind of epidemiological study we have conducted regards local concerns and beliefs as a nuisance', the effect of an already sensitized population and an obstacle to scientific enquiry' that seeks to uncover real' health effects . A more socially informed epidemiology, however, would wish to give lay beliefs some prominence, to regard local concerns as data that are as valid as those derived from more formal questionnaires such as that used in the present study . A similar conclusion was reached by phillimore on teesside, showing that concern is an obstacle for epidemiology, especially when using questionnaires, as it introduces a bias in the population answer . But concern is also seen as an important issue by social scientists, including its possible health consequences [82, 83]. It is also interesting to note that several authors of the papers on mental health in these reviews are affiliated to social sciences department and that the papers were not published in epidemiological journals [7274]. This calls for a broadening of the competency when answering the populations concerns near major industrial sites, that is, including a social sciences dimension in the analysis and not underestimating the influences of the industry and of its designation as a possible danger on the stress and well - being of the population . Multicentric design is believed to be a solution to the local biases, as the influence of the confounding factors may decrease as the number of sites increases . However, it is difficult to identify relevant sites that could be included in the same studies . In the literature, the choices to aggregate industries based on large classes may hide differences linked to the industrial processes used, the size of the plant, its operating time, and so forth . Yet, multicenter studies may not fully answer the local concerns, and as ramis stated, each industrial source has its own characteristics, and subsequent studies will therefore have to address these on a case - by - case basis . Independently of the health outcome and the statistical design used, the lack of information on the environmental and industrial background of the sites is striking in many papers . As industrial sites emit a complex mixture of pollutants, with plumes varying in composition and over time and space, epidemiologists have to rely on measurements and modeling of a subset of pollutants to assess an integrated exposure . Modeling is seen as the most efficient tool to avoid exposure misclassification . In teesside, environmental data, land - use data, historical data, and data on the perception of air pollution and odors were analyzed to check that the distance to the site was an interesting proxy . Globally, measurements did not show large differences between exposed and nonexposed areas, but the dispersion models confirmed a gradient of pollution with distance . However, environmental data and modeling are not easily accessed, especially when investigating past exposures . Indeed, several authors mentioned that emissions data were not available to perform a dispersion modeling or that they could not afford the cost of such modeling . Some authors underline that some environmental data collected for regulatory purposes are not usable for epidemiological studies . It is striking to see that in many areas the population is highly concerned by the environmental pollution and its consequences, and that these concerns are answered through complex epidemiological studies, relying on poor environmental data . In short, there is a discrepancy between the expectancies of the population, the investment in collecting and analyzing health data, and the poor accessibility to key emissions and concentrations data . When distance is the only possible choice, hodgson et al . Advised to integrate knowledge of the factors that drive exposure, for example relative emissions, and wind direction . Interestingly, odors are mentioned by several authors as an issue, but data are used to define the exposure area (e.g.) And not to investigate a possible health impact . The bias in exposure assessment and the ecological bias are likely to limit the possibility of ecological studies to reveal low relative risks with statistically significant results, especially when studying cancer with a latency of several decades . Leukemia may be the only cancer for which the latency is a priori short enough to allow a good reconstruction of exposure based on present data . A combination of multicentric studies and local studies could be efficient ways to increase knowledge on the health effects of industrial areas and answer the concerns from the population . As stated below, multicenter studies would limit local biases, and sites would not be selected based on an a priori population concern or over incidence . However, criteria to decide that sites are similar enough to be included in a multicenter study need to be defined . A focus on sites where the population requests more information could then be performed, with the support of social scientists . An investigation of the mental health impacts would be highly relevant, as this issue seems to have been poorly taken into account by epidemiologists so far . For the multicenter and the local studies, a better characterization of exposure would be an asset to improve our capacity to investigate the impacts of industrial pollution . Finally, intervention studies documenting the possible improvements of the health status of the population after the closure of a plant, or a change in the industrial processes, would be highly informative to improve the knowledge and to help for management (a change in the industrial processes that have been shown to have positive effect in the environment and the health status could be reproduced elsewhere).
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Gastrointestinal parasitism has been identified as a complex disease entity constituting a major impediment to efficient and profitable livestock production particularly in sheep and goats all over the world (1, 2). Parasites cause direct losses due to death and indirectly due to reduced productivity through reduced feed intake, decreased live weight gain and skin wool or mohair quality (3, 4). These include use of anthelmintics, management of grazing land, proper stocking rate and appropriate rotational strategies (5). However, conventional and repeated use of anthelmintics as a means of worm control is now strongly questioned because of increasing development of resistance of parasites to the major anthelmintic groups (6, 7). Conversely, grazing management and biological control are non - chemical methods of parasite control that have proved to be effective (6). Among the alternative methods of control currently available, the breeding of resistant animals in order to improve the host resistance and/or resistance to parasite infestation or infection seems to represent one of the most promising options (811). Therefore, sheep that are well nourished will grow and reproduce faster and are better able to withstand the effects of worm infestation than those on low plane of nutrition (11). Research has shown that increased dietary intake of metabolisable protein and energy with high quality pasture can directly promote host resistance and resilience to worm infection (12). Protein energy deficiencies is a important cause of defective t cell function (13, 14) and t cells have been shown to play vital role in mediating acquired resistance to haemonchosis in sheep (13). Bowie (15) had shown the beneficial effect of treatment with cayenne pepper, garlic powder, and diatomaceous earth on the packed cell volume and the faecal egg count of sheep infected with haemonchus contortus while cassava foliage adequately reduced the adverse effects of gastrointestinal nematode infections in goats, in particular when offered as silage (16). There have been previous attempts aimed at determining the effect of protein of various origins on gastrointestinal parasitism (1719). The relative importance of energy and the dominant effect of protein supply have been partly researched on in the development of resistance to infection (20). This has generated an argument on whether the control of helminths in livestock is more sensitive to protein than to energy . Therefore, the aim of this study was to investigate the effect of varied protein - energy combinations on clinical presentation, performance, haematology and adult worm load of gastro intestinally parasitized, west africa dwarf lambs that have been managed semi - intensively . The study site is located in the rain forest vegetation zone of southwestern nigeria at latitude 7 13, 49 n; longitude 3 26, 11.98 e and altitude of 76 m above sea level . The climate is humid with a mean annual rain fall of 1037 mm and annual mean temperature and humidity of 43.7 c and 82 respectively (meteorology department, ogun osun river basin authority, abeokuta, ogun state, nigeria). Twenty - four newly weaned in 2014 and apparently healthy wad lambs aged 56 months and weighing between 5 and 7 kgs were obtained from the department of animal breeding and genetics, federal university of agriculture, abeokuta . The experimental animals were allowed thirty days of acclimatization prior to commencement of experiment . During this period infected animals were treated with levamisole at 7.5 mg / kg (channelle pharmaceuticals ltd, spain) and diminazine aceturate at 3.5 mg / kg (vetindia pharmaceuticals ltd, india). Experimental animals according to group were tagged and housed in wooden pens with a slatted floor which ensured that animals did not had access to their faeces . All pens were netted with fly - proof nets and fly - proof aluminium netting placed underneath the slatted floor for total recovery of feces from each pen . On day 1 of the experiment, the animals were grazed on nematode infected paddock of stylosanthus hamatus and pennisetum pedicellatum as identified by pasteur and rangeland management department of the federal university of agriculture, abeokuta . This feeding pattern was supplemented by concentrate feed made from wheat offals, cassava and palm kernel cake in varying concentrations, thus grouping the animals into four groups . The crude protein and metabolisable energy contents of the feed supplement was determined as described by aoac (21). Permission for this study was obtained from university experimental animal ethics committee through the college of veterinary medicine representative . The experiment was carried out according to international guiding principles for biochemical research involving animals (22). The lambs were divided by complete randomized design (crd) into four groups (g1, g2, g3 and g4) of six animals each . G1 received supplementary diet low in energy and protein, g2 low energy and high protein, g3 high - energy low protein and group g4, high - energy high protein . Experimental animals were initially allowed access to nematode infected paddocks for a period of 5 hours daily and thereafter fed with concentrate as described above (2.2.2). All groups were observed for clinical signs of gastrointestinal parasitism while faeces and blood were collected on days 0, 30, 60 and 90 of experiment . The body weight was measured using a hanging scale (votilia company, italy). 50 grams of faeces was obtained directly from the rectum after wearing a hand glove . This was used for determination of egg per gram of faeces by the aid of mc master counting chamber (pyser sgi ltd, whitlock australia co. australia). Similarly, 5 ml of blood was collected from the jugular vein of individual animals into edta bottles . This was used for determination of hematological parameters (pcv, hb and rbc) as described by schalm et al ., the animals were humanely euthanized using pentobarbitone sodium (euthatal) to determine the total number and type of worms harboured by individual animal . Significant differences among groups and across days were tested using two - way analysis of variance (anova). The study site is located in the rain forest vegetation zone of southwestern nigeria at latitude 7 13, 49 n; longitude 3 26, 11.98 e and altitude of 76 m above sea level . The climate is humid with a mean annual rain fall of 1037 mm and annual mean temperature and humidity of 43.7 c and 82 respectively (meteorology department, ogun osun river basin authority, abeokuta, ogun state, nigeria). Twenty - four newly weaned in 2014 and apparently healthy wad lambs aged 56 months and weighing between 5 and 7 kgs were obtained from the department of animal breeding and genetics, federal university of agriculture, abeokuta . The experimental animals were allowed thirty days of acclimatization prior to commencement of experiment . During this period, infected animals were treated with levamisole at 7.5 mg / kg (channelle pharmaceuticals ltd, spain) and diminazine aceturate at 3.5 mg / kg (vetindia pharmaceuticals ltd, india). Experimental animals according to group were tagged and housed in wooden pens with a slatted floor which ensured that animals did not had access to their faeces . All pens were netted with fly - proof nets and fly - proof aluminium netting placed underneath the slatted floor for total recovery of feces from each pen . On day 1 of the experiment, the animals were grazed on nematode infected paddock of stylosanthus hamatus and pennisetum pedicellatum as identified by pasteur and rangeland management department of the federal university of agriculture, abeokuta . This feeding pattern was supplemented by concentrate feed made from wheat offals, cassava and palm kernel cake in varying concentrations, thus grouping the animals into four groups . The crude protein and metabolisable energy contents of the feed supplement permission for this study was obtained from university experimental animal ethics committee through the college of veterinary medicine representative . The experiment was carried out according to international guiding principles for biochemical research involving animals (22). The lambs were divided by complete randomized design (crd) into four groups (g1, g2, g3 and g4) of six animals each . G1 received supplementary diet low in energy and protein, g2 low energy and high protein, g3 high - energy low protein and group g4, high - energy high protein . Experimental animals were initially allowed access to nematode infected paddocks for a period of 5 hours daily and thereafter fed with concentrate as described above (2.2.2). All groups were observed for clinical signs of gastrointestinal parasitism while faeces and blood were collected on days 0, 30, 60 and 90 of experiment . The body weight was measured using a hanging scale (votilia company, italy). 50 grams of faeces was obtained directly from the rectum after wearing a hand glove . This was used for determination of egg per gram of faeces by the aid of mc master counting chamber (pyser sgi ltd, whitlock australia co. australia). Similarly, 5 ml of blood was collected from the jugular vein of individual animals into edta bottles . This was used for determination of hematological parameters (pcv, hb and rbc) as described by schalm et al . At the end of the 90-day experimental period, the animals were humanely euthanized using pentobarbitone sodium (euthatal) to determine the total number and type of worms harboured by individual animal . Significant differences among groups and across days were tested using two - way analysis of variance (anova). Lambs in g1 and 3 had anorexia by day 20 post infection and intermittent diarrhea by day 30 in five of the six lambs in either of the groups . However, only intermittent diarrhea was observed from day 60 in 5 out of six lambs in g2 and 4 . The ages and weights of the experimental animals did not differ significantly (p <0.05) among the groups at the onset of experiment (table 1). The% compositions of the concentrate diet fed the experimental animals are detailed in table 2 . Mean (sd) pre - infection ages and body weights of wad lambs fed varied energy- protein densities same superscripts in rows did not differ significantly (p>0.05) among the groups . Ingredient compositions (%) and result of proximate analysis of concentrate diet keys: g1= low energy, low protein, g2= low energy, high protein, g3= high energy, low protein, g4= high energy, high protein total and mean worm counts did not differ significantly (p = 0.309) among the groups (table 3). Similarly, the species of gastrointestinal helminths recovered are presented in table 4 . T. columbriformis was the most prevalent constituting the following; 44% in g1, 44% in g2, 57% in g3 and 43% in g4 (table 4). Total adult worm and mean (sd) adult worm and fecal egg counts by day 90 post - infection of lambs fed varied energy protein densities no (%) distribution of parasites recovered at necropsy by day 42 post infection table 5 contains the results of the mean body weight, egg per gram of faeces, rbc, pcv and hb concentration of experimental animals during the experimental period . Mean weight of all groups increased significantly (p <0.05) across days 30, 60 and 90 post infection . Among the groups, animals in g4 appeared to be heavier than g3 and 2 by day 90 post infection . Mean (sd) weight, fec, pcv, rbc and hb of wad lambs experimentally exposed to nematode infected paddock and fed with supplementary containing varied energy - protein concentrate diet different superscripts in columns and across rows differ significantly (p <0.05). (39) / alphabetic marks represented significant variations in the parameters across the days and groups . Among all groups, the fec did not differ significantly (p <0.05) on day 0, while significant (p <0.01) but irregular pattern of change was observed on days 30, 60 and 90 within the groups . However, g1 was significantly (p <0.05) higher on days 30 and 60 compared to the other groups . Pcv of g4 significantly increased (p<0.05) within the groups and across the days of experiment . The rbc of experimental animals varied significantly (p <0.05) on day 0 among the groups . Hb concentration did not vary among the groups on day 0, but showed significant increase (p<0.05) within the groups for the entire experimental period . Lambs in g1 and 3 had anorexia by day 20 post infection and intermittent diarrhea by day 30 in five of the six lambs in either of the groups . However, only intermittent diarrhea was observed from day 60 in 5 out of six lambs in g2 and 4 . The ages and weights of the experimental animals did not differ significantly (p <0.05) among the groups at the onset of experiment (table 1). The% compositions of the concentrate diet fed the experimental animals are detailed in table 2 . Mean (sd) pre - infection ages and body weights of wad lambs fed varied energy- protein densities same superscripts in rows did not differ significantly (p>0.05) among the groups . Ingredient compositions (%) and result of proximate analysis of concentrate diet keys: g1= low energy, low protein, g2= low energy, high protein, g3= high energy, low protein, g4= high energy, high protein total and mean worm counts did not differ significantly (p = 0.309) among the groups (table 3). T. columbriformis was the most prevalent constituting the following; 44% in g1, 44% in g2, 57% in g3 and 43% in g4 (table 4). Total adult worm and mean (sd) adult worm and fecal egg counts by day 90 post - infection of lambs fed varied energy protein densities no (%) distribution of parasites recovered at necropsy by day 42 post infection table 5 contains the results of the mean body weight, egg per gram of faeces, rbc, pcv and hb concentration of experimental animals during the experimental period . Mean weight of all groups increased significantly (p <0.05) across days 30, 60 and 90 post infection . Among the groups, animals in g4 appeared to be heavier than g3 and 2 by day 90 post infection . Mean (sd) weight, fec, pcv, rbc and hb of wad lambs experimentally exposed to nematode infected paddock and fed with supplementary containing varied energy - protein concentrate diet different superscripts in columns and across rows differ significantly (p <0.05). (39) / alphabetic marks represented significant variations in the parameters across the days and groups . Among all groups, the fec did not differ significantly (p <0.05) on day 0, while significant (p <0.01) but irregular pattern of change was observed on days 30, 60 and 90 within the groups . However, g1 was significantly (p <0.05) higher on days 30 and 60 compared to the other groups . Pcv of g4 significantly increased (p<0.05) within the groups and across the days of experiment . The rbc of experimental animals varied significantly (p <0.05) on day 0 among the groups . Hb concentration did not vary among the groups on day 0, but showed significant increase (p<0.05) within the groups for the entire experimental period . The need to minimize production losses in animal husbandry practices is of paramount importance especially among young animals where ability to thrive and function optimally is threatened by infections like gastrointestinal helminths . The diarrhea observed in all the groups in this study is an evidence of parasitic gastroenteritis (25). T. columbriformis, an important contributor to parasitic gastroenteritis complex accounted for between 43 and 57% of the total worms recovered at necropsy in all the groups and might have been an important contributor to the diarrhea observed . This finding agrees with previous report by chiejina (26) who associated diarrhea in sheep with the non - blood sucking helminths such as t. columbriformis . Such helminths cause proliferative gastrophy, catarrhal inflammation and epithelial ulcerations, thereby resulting in morphologic and functional damage of the gastro - intestinal tract . This subsequently leads to reduced absorption of water and electrolyte and finally diarrhea as a consequence (26, 27). Similarly, the anorexia observed in this study was obvious by day 20 post infection in g1 and g3 lambs fed on low protein diets . In ruminants, cobalt deficiency and a heavy infestation with trichostrongylids this is further corroborated by the previous finding of steel (28) who observed reduced feed in - take when fec was above 3000 epg in sheep infected with trichostrongylids . Animals in g1 and 3 had fec of (3333.33210.82) and 1066.6742.16 at day 60 post infection respectively . The absence of anorexia in groups (g2 and 4), that received high plane of protein is an indication of the ability of high protein diet to mitigate the negative impact of infection . Increased availability of protein has been reported to reduce the degree of anorexia in sheep with t. colubriformis infection (20). (29) attributed reduction in appetite in helminth - infected lambs to be immunostimulatory in origin, which allow for selective feeding particularly for high protein diets . This is necessary because of priority sequestration of amino acid by gastro intestinal tract for repairs, development and expression of local cell mediated responses . This might explain why lambs in g1 and g3 with low protein diets showed obvious anorexia, with high fec at the peak of infection . In all groups, weight changes showed significant (p <0.05) increase despite significant increase in fec especially from days 30 and 60 . The improved weight of animals in g4, followed by g2 compared to other experimental animals attest to the importance of protein and energy diets in improving productivity despite the effect of gastrointestinal parasitism . This finding agrees with the earlier report (19), in wad goats experimentally infected with h. contortus and t. columbriformis and supplemented with dietary protein . Balanced energy - protein supplementation might have afforded opportunity for rumen to function optimally; thus enhancing microbial flora synthesis leading to increase in weight . A 60% increase in protein content of diet was associated with 50% increase in growth rate while 20% increase in energy content resulted in doubling of growth rate over ten weeks period in weaned merino sheep infected with black scour worms (30). The same phenomenon might have been responsible for the significant weight gain in all groups especially those on high plane of protein and energy . (31) observed a strong negative correlation between an initial weight and t. columbriformis worm count after challenge in 8-month - old merino sheep . The significant decrease in fec observed at the latter part of this experiment in all groups indicate that the effect of energy protein supplementation in enhancing immune response appears to be most effective at the latter stages of parasitic acquisition by the host . This observation corroborates an earlier report (20), where effect of protein supplementation became manifest 15 weeks post infection . However, the significant decrease in fec on day 90 post experiment in g1 and g2 compared to g3 and g4 could show the detrimental effects of low energy content on fecundity or oviposition of higher worm load . 18) on xhosa lop - eared goats fed supplementary acacia karroo leaves following experimental haemonchosis . This decrease in fec as seen g1 and g2 with low energy diets in this study was found to be incompatible with research into roles of energy in immune response to gastrointestinal helminth parasitism (3234). The lower fec reported in g1 and g2 with higher worm load and conversely higher fec reported in g3 and g4 with lower worm burden demonstrate positive effects of protein supplementation on worm load . The effects of low energy may have affected negatively on the fecundity of worm in the infected animals . Contrary to research stating that reduced fec in lambs provide a selection criterion for worm resistance, which has been found to be heritable characteristic (3537), the present study thus showed that, the use of reduced fec in lamb for worm resistance might not be true in animal fed on low energy diet . The pcv of all groups did not vary significantly (p <0.05) as the experiment progressed except in g4 . The provision of additional protein to infected animals are able to maintain normal rate of growth suggesting that, if protein supply is high enough, then, it may possible to keep animal performance in the face of larval challenge (38). The recovery of blood sucking helminths such as h. contortus in low number compared to non - blood sucking helminths is a possible explanation for the ability of the animal to maintain normal pcv throughout the experimental period . Equally, the reasons adduced for the improved pcv may hold true for rbc and hb concentration . Furthermore, the presence of parasites such as m. benedini and p. cervi in low number, compared to others might have been responsible for the parameters observed . This is because such parasites do not cause much damage, unless present in large number, which was not the case in this study . Diet low in energy and high in protein as in g2 supplemented lambs appear to be the most preferred diet combination in mitigating the deleterious effects of gastrointestinal helminth infection.
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Primary open - angle glaucoma is described distinctly as a multifactorial optic neuropathy that is chronic and progressive with a characteristic acquired loss of optic nerve fibers . Such loss develops in the presence of open anterior chamber angles, characteristic visual field abnormalities, and intraocular tension that is too high for the continued health of the eye . It manifests by cupping and atrophy of the optic disc, in the absence of other known causes of glaucomatous disease . Thus the clinical diagnosis of poag is commonly based on increase in intraocular pressure, characteristic optic nerve head cupping, and typical visual field defects which are assessed by standard static threshold perimetry, using an automated system such as humphrey field analyzer (hfa). Hfa however does not selectively reveal which structures contribute to the impairment of the visual system observed in glaucoma . It has been suggested that damage to the ganglion cells and/or their axons produce glaucomatous visual field defects . In this context, electrophysiological tests like visual evoked potentials can contribute to detection of glaucomatous optic neuropathy since they are compatible with the functions of retinal ganglion cells, and they make it possible to study different aspects of visual functions . The visual evoked potential is the objective measurement of visual function monitored at the level of the occipital cortex with scalp electrodes . It is recorded with a uniform stimulus check size and a slow reversal rate throughout the field . This paper summarizes many of the studies pertaining to the significance of visual evoked potentials in the assessment of visual field defects in primary open - angle glaucoma . Included are the previous works related to the clinical utilization of veps for the objective assessment of typical visual field defects of poag . The assessment of visual field defects with visual evoked potential has been a hard task . Ever since visually evoked cortical potentials were first used as a diagnostic aid the important question has been whether they could detect visual field defects . In earlier investigations, light - flash stimulators illuminating the entire retina were used and the bioelectrical responses from both hemispheres were compared . Because asymmetries between the hemispheres were also found in normal people only differences of 50 per cent or more between the responses of the right and left hemisphere were considered significant . Later, methods of stimulating the temporal and nasal parts of the retina separately with flash and checkerboard stimulation were introduced . Finally, a sophisticated method of separating the signals from retinal areas stimulated simultaneously was devised . However, there are few reports of the clinical application of these techniques . Since it is believed that elevation of intraocular tension causes pressure on the retinal nerve fibers bundles as they course into the optic nerve and results in the loss of visual function which is known to produce an alteration of the vep waveforms; many earlier studies were interested in correlating vep findings with perimetric defects . However, because the vep is dominated by the central macular responses and reflects mainly macular function, it was altered only when the central visual field was disturbed . Field defects with vep have also been demonstrated using localized retinal stimulation [35]. The latter of the authors have described a technique for producing steady state visual evoked response (ver) to pattern reversal stimulation of retinal areas corresponding to discrete field quadrants . They arbitrarily classified their 21 patients with glaucomatous field defects into three categories according to the size of field defect namely early defects (occupying less than third of quadrant examined)vers from affected quadrant showed a large phase lag compared to normal homonymous quadrant . Moderate defects (occupying third to three quarters of affected quadrant)either no ver was obtained on stimulation of retina corresponding to defective field quadrant or a phase delay was observed . Severe defects (three quarters to the whole quadrant tested)in most cases, no ver was obtained from quadrants tested but in three cases small responses showing a phase shift were observed . They also conducted ver recordings with patient fixating centre of the 22 screen (full - field), the central 8 only and also the centre of the screen when the central 8 was occluded (peripheral). These tests did not show significant phase changes except in four cases with large field defects . The amplitude of the response from the eyes with moderate and severe defects was markedly reduced by comparison with that of the normal eye . Using this technique, an objective assessment of localized visual field defects visually evoked cortical potentials were studied in six patients with a homonymous and six patients with a bitemporal hemianopia by presenting a pattern reversal stimulus separately to a temporal or nasal retinal area and by recording the responses from leads over the hemispheres . Homonymous visual field defects were characterized by a reduction of vecps from the affected hemisphere . Both the topographical position and the dimension and degree of the diminished sensitivity of the visual field are important for changes in the evoked potentials, the nearer to the centre the visual field defect is localized, the larger thus a small relative scotoma located near the centre may affect significant changes on the vep while a large absolute scotoma in the periphery may cause only minor changes in vep . The larger latency increments have been reported when measured in eyes with large field defects but there was no direct relation between field size and latency . The visual field may be nearly intact with a definite increase of latency in the affected eye . Increased pattern vep latency was significantly correlated with both the severity and location of visual field defects and the degree of cupping and pallor of the optic disc in another study . However, some of the earlier works have demonstrated a poor sensitivity of the vep to detection of csg in patients with superior visual field defects due to the dominance of the inferior hemifield signal to the full - field vep response . Mean defect (md) indicative of diffuse nonspecific nerve fibre loss correlated significantly (p <0.05) in eyes with oag in a previous study where flash evoked responses and the visual field indices (vfi) of the octopus g1 program were recorded from 42 eyes of 21 patients, out of which 29 eyes had open - angle glaucoma (oag) and 10 had ocular hypertension (oh). The vep changes observed by some authors in the form of prolonged p100 latency were consistent with the central visual field defects qualitatively and quantitatively . Therefore, it was concluded that the latency of p100 can be a useful quantitative index in the evaluation of glaucomatous visual function damage . The difference in diagnostic sensitivity to glaucoma between vep and visual field changes were studied and the authors have suggested that combination of the two may be a more useful index . The pattern vep was compared to the octopus 2000r automated perimeter in the assessment of central visual function in chronic simple glaucoma (csg) in 90 patients (52 males and 38 females) in two age bands 4060 years and 6180 years vep demonstrated a high detection rate (86.7%) with a relatively low false positive rate of 7.7% (p <0.01). When the two tests were compared, absolute latency and field loss were poorly correlated but interocular differences showed much stronger correlation with the sum of field losses determined with static perimetry . Thus once interindividual variability was eliminated; severity of field loss was mirrored by prolongation of vep latency . In recent years, multichannel pattern visual evoked potentials have begun to be compared to humphrey perimetry in the assessment of central visual function in primary open - angle glaucoma . The multi - channel checkerboard reversal pveps waves to full - field and half - field stimulus of 25 normal persons and 74 patients with primary open - angle glaucoma were recorded and analyzed in a study . The area of visual field corresponding to the area of retina stimulated during multi - channel pveps testing were analysed, straight - line correlation and regression analyses of the various multi - channel pveps parameters and the total db losses were performed . The multi - channel pveps demonstrated that absolute latency and field loss were correlated in the late stage of glaucoma, and absolute amplitude and field loss were not correlated . The authors therefore inferred that in late loss of primary open - angle glaucoma, multi - channel pveps can provide a valuable, objective complement to humphrey perimetry . Vep measurements with presumable stimulation of single neuronal pathways can detect glaucomatous optic nerve damage in a considerable fraction of patients with visual field loss as glaucoma is associated with blue color vision disturbances . Their study included 59 patients (96 eyes) with glaucomatous changes of the optic disc and visual field defects and 58 control eyes of 29 healthy subjects . Four types of pattern vep stimulation (0.9 cycle / degree) were performed in all patients: achromatic, alternating sine - wave stripe pattern (activation of predominantly the magnocellular pathway), isoluminant, red - green stripe pattern (activation of predominantly the parvocellular pathway), and blue grating with yellow background adaptation (activation of the blue - sensitive pathway). In a paired correlation analysis with visual field defects, significant (p <0.05) results were obtained with the perimetric md value for all stimulations and with the neuroretinal rim area of the optic disc which again supports the validity of the vep technique in glaucoma . Correlation coefficients were highest (r = 0.79, p <0.01) for the peak time of the blue - yellow vep . In spite of these results and the fact that there were no other confirmative reports about the usefulness of by - vep, there remains still uncertainty whether the blue yellow - vep becomes pathologic before visual field or optic disc damages appear and whether it is able to predict these defects . To evaluate whether glaucomatous visual field defects could be related to an impaired retinal function, to a delayed neural conduction in postretinal visual pathways, or both; visual field by humphrey perimeter (central 24 - 2 threshold test) and simultaneous recordings of visual evoked potential (vep) and pattern electroretinogram (perg) were assessed in 21 subjects with open - angle glaucoma (poag) and in 15 age - matched controls . Vep in poag eyes showed significantly (p <0.01) delayed p100 latency when compared with controls and correlated with mean deviation (index of global visual field damage, md) (p <0.001) and the p100 amplitudes were also significantly (p <0.01) lower in poag eyes than in control eyes and correlated with md (p <0.001). No significant correlations (p> 0.05) were found between electrophysiological parameters and corrected pattern standard deviation (cpsd), index of localized visual field damage, of 24 - 2 humphrey perimetry . Retinocortical time (rct: difference between vep p100 and perg p50 latencies) and latency window (lw: difference between vep n75 and perg p50 latencies) were significantly (p <0.01) longer in poag eyes than in control eyes and correlated with md (rct: p <0.001; lw: p <0.001). He concluded that in patients with open - angle glaucoma the reduction of the index of global visual field damage (md) could be ascribed to two sources of functional impairment: one retinal (impaired perg) and one postretinal (delayed rct and lw). In the postretinal impairment, a postsynaptic degeneration at the level of the lateral geniculate nucleus could be suggested . To assess the presence of normal or abnormal pattern visual evoked potential (vep) responses in patients with ocular hypertension and open - angle glaucoma (oag), a study was performed on 80 normal control subjects, 68 ocular hypertension patients with intraocular pressure (iop) <18 mmhg under pharmacological treatment and 84 oag patients with intraocular pressure (iop) <18 mmhg under pharmacological treatment veps using high - contrast (80%) 15 checkerboard stimuli with 2 reversals per second were recorded . They showed highly significant positive correlation (p <0.001) between p100 amplitude and hfa24/2 md and md values in their poag patients were negatively correlated with the latency time of p100 . With the multifocal technique, visual evoked potentials (veps) can be recorded simultaneously from many regions of the visual field in a matter of minutes . Recently, the multifocal visual evoked potential technique (mfvep) has generated considerable interest, especially among those seeking objective measures of glaucomatous damage . If both eyes of an individual are normal, then mfveps recorded for monocular stimulation of each eye are essentially identical . However, the amplitude and waveform of the mfvep responses vary across individuals, as well as across the visual field within an individual . These variations are related to cortical anatomy and to the cortical sources contributing to the mfvep . Although there are undoubtedly extrastriate contributions, these contributions are probably smaller for the mfvep than for the conventional vep . The mfvep is not a small version of the conventional vep . To determine the relationship between spatially localized multifocal visual evoked potentials (mfveps) and humphrey visual fields (hvfs) in patients with unilateral field defects, humphrey visual fields and mfveps were obtained from 20 patients with unilateral field losses due to either ischemic optic neuropathy or glaucoma . The amplitude of the mfvep responses was calculated and estimates of the hvf loss in the same regions of the field used for the mfvep were obtained by interpolating the 24 - 2 hvf data . Their results showed that monocular mfvep amplitude decreased with hvf loss, although small mfvep signals were not uniquely associated with poor fields . On average, the monocular mfvep was indistinguishable from noise for field losses between 5 and 10 db, and the interocular ratio of the mfvep amplitudes correlated well with the difference between the hvf values of the 2 eyes . The monocular and interocular results were consistent with a linear relationship between the amplitude of the signal portion of the mfvep response and linear hvf loss . One way to produce this relationship would be if both the signal in the mfvep and linear hvf loss were linearly related to the percentage of local ganglion cells lost . To detect ganglion cell damage with the mfvep requires methods for analyzing the responses and for displaying the results . This method compared the monocular responses from the two eyes of an individual and produced a map of the defects . This map is in the form of a probability plot similar to the one used to display visual field defects measured with automated perimetry . Procedures were described for directly comparing these mfvep probability plots to the probability plots for humphrey visual fields (hvfs). Using the techniques described therein, the relationship between the amplitude of the mfvep and the sensitivity loss of the hvf was discussed . The evidence supports a simple model in which the amplitude of the signal portion, but not the noise portion, of the mfvep response is proportional to hvf loss where hvf loss is expressed in linear, not db, units . It was hypothesized that both the signal in the mfvep, and the sensitivity of the hvf, are linearly related to ganglion cell loss . A theoretical approach was developed which allowed a direct comparison of the efficacy of the mfvep and hvf in detecting glaucomatous damage . In short, when the mfvep has a large snr it will often be superior to the hvf in detecting damage . On the other hand, when the mfvep has a small snr, the hvf will probably be superior . In summary, the authors concluded that the mfvep has a place in the clinical management of glaucoma, although it is not likely to replace static automated achromatic perimetry in the near future . However, this is an evolving technology and the future will undoubtedly see major improvements in the mfvep technique . The multifocal vep (mfvep) technique is still in infancy and there are as yet no studies to determine its reliability compared with other methods of investigation . Another study was conducted to compare latencies of conventional visual evoked potentials (cveps) and multifocal veps (mfveps) in the same patients 75 eyes (47 patients), 75 eyes with suspected glaucoma (46 patients), and 41 control eyes (22 subjects) underwent achromatic automated perimetry and mfvep and cvep testing . The mfvep stimulus was a scaled dart board with 60 sectors; each sector was a pattern - reversing checkerboard . The cvep stimulus was a reversing checkerboard with checks of either 15 minutes or 60 minutes in width . They have shown that the latency of both the mfvep and cvep (conventional vep) bore no obvious relationship to the mean deviation of the visual field in their study . To investigate the changes of color pattern reversal visual evoked potential (cpr - vep) of primary glaucoma using different temporal frequencies cpr - vep was recorded using vision monitor visual electrophysiograph at different temporal frequencies (1, 2, 4, 8, 16, and 32 hz) and different color stimulations (black / white, red / green, blue / yellow) in 41 cases (70 eyes) with primary glaucoma (glaucoma group) and 13 normal subjects (26 eyes) (normal control group) p100 wave amplitudes were compared . In the normal control group, p100 amplitudes declined while the temporal frequency of black / white stimulation was increasing, but they had peaks at 2 hz and 8 hz red / green stimulation and blue / yellow stimulation . In the glaucoma group, cpr - vep p100 declined while temporal frequency was increasing fewer than 3 color stimulations, but had a peak at 8 hz . At 2 hz16 hz, p100 amplitudes were related with the mean defect of humphrey visual field, especially with all 3 color stimulations at 8 hz and with blue / yellow stimulation at 2 hz and 16 hz . P100 amplitude was most different under the 3 color stimulations between the 2 groups at 8 hz . The authors concluded that the changes of cpr - vep p100 amplitude can objectively reflect the glaucoma visual function damage . Cpr - vep p100 amplitude has certain value in studying glaucoma under different color stimulations (black / white, red / green, blue / yellow) at 8 hz, and blue / yellow stimulation at 2 hz and 16 hz . To investigate the difference in color pattern reversal visual evoked potential (cpr vep) between primary open - angle glaucoma (poag) and primary angle closure glaucoma (pacg) patients cpr - vep were obtained in 17 eyes of 12 poag patients, 56 eyes of 41 pacg patients, and 26 eyes of 13 age - equivalent normal persons at an ascending series of temporal frequency (1, 2, 4, 8, 16, and 32 hz), and color stimulation (black / white, red / green, and blue / yellow) p100 wave amplitudes and latencies of these patients were compared, respectively, with those of the normal group . With black / white stimulation, the p100 wave amplitudes were reduced with the increase of temporal frequency in the 3 groups . The p100 wave latencies were extended with the increase of temporal frequency with different color stimulations . The p100 amplitudes were pacg group> normal group> poag group and black / white> blue / yellow> red / green . The p100 wave latencies in the poag group and the pacg group were extended compared with the nc group, but there was no significant difference between pacg group and poag group . Thus they concluded that p100 amplitude of pacg is higher, and poag is lower than normal . The p100 wave latencies of pacg and poag are extended . A very recent study was undertaken to evaluate the color doppler imaging (cdi) and pattern visual evoked potential (p - vep) examinations in primary open - angle glaucoma (poag) patients and investigate the relation between flow velocities measured by cdi and p - vep examination in poag patients, 65 poag patients, and 45 control subjects were investigated for cdi evaluation of the ophthalmic artery (oa), short posterior ciliary artery (spca) and central retinal arteries (cra), and the latency and amplitude of p100 in p - vep were recorded . The differences of cdi and p - vep parameters among poag and control groups were compared . The latency of p100 in vep delayed and the amplitude of p100 decreased in the poag patients comparing with that of the control group . They have found the md values in the poag patients were negatively correlated with the latency time of p100, which was agreed with the previous studies . With an attempt to assess the correlation of visual field indices with vep parameters in primary open - angle glaucoma, we conducted a study on a larger cohort of 100 poag patients and 200 control subjects of central indian population; we observed that our poag patients showed different degrees of visual field impairment detected by a reduction in mean defect (md) and by an increase in pattern standard deviation (psd). The reduced md observed in our poag patients was significantly correlated with the abnormal cortical electrophysiological responses . There was a good significant negative correlation of p100 latency and md and a significant relationship of n155 latency and p100 duration . This finding of significant correlation between the values of md and those of vep parameters is consistent with the results reported in other studies in which abnormal vep responses were related to visual field defects assessed by goldmann perimetry [2, 11, 13, 25, 26], or by static perimetry [11, 13]. Our results are also in close agreement with a recent study which has documented that the md values in poag patients were negatively correlated with the latency time of p100, which also corroborates the findings of previous workers . The mean vep p100 amplitude of poag patients in our study was highly significantly (p <0.001) correlated with value of mean defect (md) in db . Our results correspond with recent study which reported that the md values in their poag patients were positively correlated with the amplitude of p100 . Our results also concur with the findings of previous workers who have put forth similar conclusion . From this paper, it can be concluded that vep is an important visual electrophysiological tool which has been used for the evaluation of visual field defects in primary open - angle glaucoma . Vep is a more objective measure of optic nerve function because it is not influenced by cognitive factors or the motor skills of the subject as compared with the psychophysical tests . Further significant correlations between the magnitude of the vep latencies and the size of visual field defect and optic disc cupping or pallor over the years confirm the validity of vep method in primary open - angle glaucoma . Further, the correlation obtained by us between all the electrophysiological vep parameters and md of humphrey static perimetry suggests that the impaired visual cortical responses observed in glaucoma patients can be revealed by both electrophysiological and psychophysical methods . In addition, the severity of global glaucomatous damage evidenced by reduction in md could depend on the delay in neural conduction from retina to the visual cortex as revealed by the significant correlation between vep latencies and md.
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Patients hospitalized in the cardiovascular surgery department of carlos haya hospital (mlaga, spain) were recruited between february 2007 and june 2008 (n = 15). All patients had an advanced atherosclerotic process and type 2 diabetes for a mean sd of 12 7 years . From patients with clinical stage iv peripheral arterial occlusive disease (paod) and amputation of inferior limbs, two types of vascular biopsy specimens were collected: occlusive popliteal artery (opa) with atherosclerotic plaque and femoral vein (fv). As much of the opa as the fv was obtained from the vascular bundle of each patient . The inclusion criteria were age 1880 years (all patients were> 60 and written informed consent). The presence of atherosclerotic risk factors was evaluated using the european society of cardiology and hypertension definition for hypertension (systolic blood pressure 140 mmhg and/or diastolic blood pressure 90 mmhg), the american diabetes association 2010 definition for type 2 diabetes (repeated fasting glucose levels 126 mg / dl if being treated with oral antidiabetic agents or insulin at the time of the study or if hba1c> 6.5%), the national cholesterol education program - adult treatment panel iii criteria for triglyceride (150 mg / dl) and hdl cholesterol (men <40 mg / dl, women <50 mg / dl) levels, a bmi> 30 kg / m for obesity, and a smoking habit up to 6 months before the hospital admission . Anthropometric and biochemical parameters were sex, age, waist circumference, systolic and diastolic blood pressure, glucose, hba1c, total cholesterol, hdl cholesterol, ldl cholesterol, and triglycerides . Additionally, we measured the homeostasis model assessment index, which is used to quantify insulin resistance and -cell function . The approximating equation for insulin resistance used a fasting blood sample and was derived by (glucose insulin)/405, where glucose is given in milligrams per deciliter and insulin is given in microunits per milliliter . The patients were admitted to the hospital 72 h before surgery, and their treatment often was modified to prepare them for surgery . For all patients, there was a drug washout period of 12 h before blood collection . The therapeutic characteristics of the patients were oral antidiabetic drugs (20.0%), including metformin, gliclazide, or repaglinide (6.7% each); insulin (86.7%); antihypertensive drugs (80.0%), including ace inhibitors (26.7%), angiotensin receptor blockers (20%), calcium channel blockers (20%), -blockers (20.0%), and diuretics (46.7%); hypolipemiant drugs, including statins (26.7%); antiaggregant drugs (80.0%); and anticoagulant drugs (53.3%). Each patient had a stable treatment regimen in the 6 months before the study, which also was an inclusion criterion for this study . The study was approved by the hospital ethics committee, and all patients gave written informed consent . The study protocol complied with the principles of the helsinki declaration . The vascular biopsy specimens, removed immediately after surgery, were immersed in 2-methylbutane and then in liquid nitrogen . Histological studies were performed on 4-mm - thick sections of the vessels, and atheromatous plaque cut in a cryostat at 20c was thaw mounted onto poly - l - lysine treated slides . Tissues were then stained with masson trichrome and photographed under routine light microscopy (leica microsystems ltd . ). Igg and igm antioxldl abs were measured in duplicate as previously described (9,10). In brief, the ldl was isolated from fasting plasma from human blood donors by density gradient ultracentrifugation . Microtiter plates for determination of igg and igm anti - mda - ldl antibodies were coated with either native ldl or mda - ldl and the serum of each patient . The absorbance was read, and the binding of antibodies to mda - ldl (antioxldl abs) was calculated by subtracting the binding of native ldl from the binding of mda - ldl . Pieces of opa and fv vessels were homogenized using the tripure isolation reagent (roche molecular biochemicals, barcelona, spain) according to the manufacturer s instructions in ice and a laboratory batch mixer (t25-ultra3-turrax basic; ika equipment laboratory). Cdna was obtained from 1 g rna using the high capacity cdna reverse transcription kit (formerly the high capacity cdna archive kit; applied biosystems, foster city, ca). Rnas with ratios between 1.7 and 2 were considered adequate for quantification of mrna expression . Recombinant rnasin ribonuclease inhibitor (applied biosystems) was added to prevent rnase - mediated degradation . Gene expression analyses were performed at the mrna level by taqman low density array (tlda). Predesigned taqman probe and primer sets for target genes were chosen from an online catalog (applied biosystems). These were factory - loaded into the 384 wells of the tlda card, which was configured into 8 identical 24-gene sets in duplicate . Twenty - two genes were chosen based on literature reviews of key molecules in inflammation and immunology . Studied genes classified by metabolic pathway involved five microliters of single - stranded cdna (equivalent to 100 ng total rna) were mixed with 45 l nuclease - free water and 50 l taqman universal pcr master mix . After gentle mixing and centrifugation, the 100-l mixture was transferred into a loading port on a tlda card . The card was centrifuged twice for 1 min at 1,100 rpm to distribute the samples from the loading port into each well . It was then sealed and placed in the micro fluidic card sample block of an applied biosystems 7900ht pcr system . The thermal cycling condition was 2 min at 50c and 10 min at 95c followed by 40 cycles of 30 s at 97c and 1 min at 59.7c . Expression levels were measured in duplicate . Only the genes with reproducible amplification curves of both duplicates were analyzed and presented . Tlda cards were analyzed with relative quantitation (rq) documents and the rq manager software for automated data analysis . Gene expression values (rq) were calculated based on the cycle threshold method . Cycle threshold values, defined as the point at which the fluorescence rises above the background fluorescence, were calculated with sds 2.3 software (applied biosystems). A mixture of dna from opa was used as a calibrator, and 18s rrna was the reference for normalization . Results are expressed as mean sd . Relationships between cellular biomarkers and antioxldl ab (plasma igg / igm antioxldl ab levels) were examined by means of the pearson correlation test . Statistical analyses were performed with spss for windows version 17.0 (ibm corporation inc ., somers, ny). The aim of this study was to assess the correlation between plasma igg / igm antioxldl ab levels with specific inflammatory, apoptotic, and lipid metabolism biomarkers in both opa with atheromatous plaque and fv without plaque . The sample size calculation was realized, considering that a correlation coefficient <0.65 could lead to a type 1 error or a false - positive correlation . Thus, based on this aim, the sample size was calculated to have> 70% power (= 0.05) and an expected correlation coefficient of 0.65 . Following this statistical approach, the vascular biopsy specimens, removed immediately after surgery, were immersed in 2-methylbutane and then in liquid nitrogen . Histological studies were performed on 4-mm - thick sections of the vessels, and atheromatous plaque cut in a cryostat at 20c was thaw mounted onto poly - l - lysine treated slides . Tissues were then stained with masson trichrome and photographed under routine light microscopy (leica microsystems ltd . ). Igg and igm antioxldl abs were measured in duplicate as previously described (9,10). In brief, the ldl was isolated from fasting plasma from human blood donors by density gradient ultracentrifugation . Microtiter plates for determination of igg and igm anti - mda - ldl antibodies were coated with either native ldl or mda - ldl and the serum of each patient . The absorbance was read, and the binding of antibodies to mda - ldl (antioxldl abs) was calculated by subtracting the binding of native ldl from the binding of mda - ldl . Pieces of opa and fv vessels were homogenized using the tripure isolation reagent (roche molecular biochemicals, barcelona, spain) according to the manufacturer s instructions in ice and a laboratory batch mixer (t25-ultra3-turrax basic; ika equipment laboratory). Cdna was obtained from 1 g rna using the high capacity cdna reverse transcription kit (formerly the high capacity cdna archive kit; applied biosystems, foster city, ca). Rnas with ratios between 1.7 and 2 were considered adequate for quantification of mrna expression . Recombinant rnasin ribonuclease inhibitor (applied biosystems) was added to prevent rnase - mediated degradation . Gene expression analyses were performed at the mrna level by taqman low density array (tlda). Predesigned taqman probe and primer sets for target genes were chosen from an online catalog (applied biosystems). These were factory - loaded into the 384 wells of the tlda card, which was configured into 8 identical 24-gene sets in duplicate . Twenty - two genes were chosen based on literature reviews of key molecules in inflammation and immunology . Studied genes classified by metabolic pathway involved five microliters of single - stranded cdna (equivalent to 100 ng total rna) were mixed with 45 l nuclease - free water and 50 l taqman universal pcr master mix . After gentle mixing and centrifugation, the 100-l mixture was transferred into a loading port on a tlda card . The card was centrifuged twice for 1 min at 1,100 rpm to distribute the samples from the loading port into each well . It was then sealed and placed in the micro fluidic card sample block of an applied biosystems 7900ht pcr system . The thermal cycling condition was 2 min at 50c and 10 min at 95c followed by 40 cycles of 30 s at 97c and 1 min at 59.7c . Tlda cards were analyzed with relative quantitation (rq) documents and the rq manager software for automated data analysis . Gene expression values (rq) were calculated based on the cycle threshold method . Cycle threshold values, defined as the point at which the fluorescence rises above the background fluorescence, were calculated with sds 2.3 software (applied biosystems). A mixture of dna from opa was used as a calibrator, and 18s rrna was the reference for normalization . Results are expressed as mean sd . Relationships between cellular biomarkers and antioxldl ab (plasma igg / igm antioxldl ab levels) were examined by means of the pearson correlation test . Statistical analyses were performed with spss for windows version 17.0 (ibm corporation inc ., somers, ny). The aim of this study was to assess the correlation between plasma igg / igm antioxldl ab levels with specific inflammatory, apoptotic, and lipid metabolism biomarkers in both opa with atheromatous plaque and fv without plaque . The sample size calculation was realized, considering that a correlation coefficient <0.65 could lead to a type 1 error or a false - positive correlation . Thus, based on this aim, the sample size was calculated to have> 70% power (= 0.05) and an expected correlation coefficient of 0.65 . Following this statistical approach, to characterize histologically the vascular biopsy specimens from patients with atherosclerosis, sections from the vessels were stained with masson trichrome and photographed under light microscopy (fig . The sections corresponding to the opa had soft plaques with a lipid nucleus and calcium deposits . The capsule had few collagen fibers and vascular smooth muscle cells, although with an excess of macrophages and lymphocytes (fig . 1a). In the fv biopsy specimens, we found a normal histology, with the adventitial layer comprising connective tissue as well as collagen and elastic fibers, the media layer comprising smooth muscle and elastic fibers, and the inner layer comprising an elastic membrane lining and smooth endothelium where endothelial cells were located (fig . (a high - quality digital representation of this figure is available in the online issue .) On the other hand, in the patients with paod, different anthropometric parameters (sex, age, bmi, and blood pressure) were not correlated with igg and igm antioxldl abs (table 2). Biochemical parameters showed a negative correlation between igm antioxldl ab and c - ldl plasma levels (r = 0.562, p = 0.05) in opa, but this significance disappeared in fv . Clinical and biochemical variables from patients with paod in addition, we investigated the possible correlation of plasma igg and igm antioxldl ab levels with gene expression of different biomarkers (mrna levels) in patients with paod . The expression was obtained from 46 genes that have an important relevance in all the studied metabolic pathways, and their expression in vessels with a highly atherogenic environment (i.e., opa, fv) were analyzed . Only 11 genes were significantly correlated with antioxldl abs in the biopsy specimens (table 3). The genes altered were those involved in inflammation, apoptosis, cell cycle, cell turnover, transcription factor, and lipid metabolism compared with the housekeeping genes 18s rrna and gapdh . In fv, igg antioxldl ab showed a negative correlation with mmp10 and a positive correlation with mmp2 . On the other hand, there was a positive correlation between igm antioxldl ab and akt1, casp3, pparg, tfpi, nfkb, and vegfa (table 4). In opa, igm antioxldl ab showed a positive correlation with mmp10, akt1, bax, cdkn1a, scarb1, and vegfa, and igg antioxldl ab had no correlation with the studied genes (table 5). Genes with significant correlation with antioxldl abs in the opa and fv biopsy specimens simple linear correlations between different biomarkers and antioxldl abs from fv biopsy specimens simple linear correlations between different biomarkers and antioxldl abs from opa biopsy specimens to assess the association between igg and igm antioxldl abs and drugs, multiple regression models were used to correct for confounding factors . The association analysis did not fall within any of the proposed models (data not shown). The most relevant finding in this study is that igm antioxldl ab showed a significant correlation with different signaling pathways (inflammation, apoptosis, and lipid metabolism) in a highly atherosclerotic environment . To study signaling pathways, there are some important points to consider . The atherosclerotic process is the lipid accumulation in blood vessels, which triggers different responses leading to the plaque development . It has been demonstrated that the atherosclerotic process is accentuated in large arteries where hemodynamic forces are exerted (20). We demonstrate that in both a vein and an artery, plasma levels of igm antioxldl ab correlate with these signaling pathways, indicating that this antibody was expressed in a determined environment (atherosclerotic medium) independently of the analyzed vessel . This event does not occur with igg antioxldl ab . Of all biochemical and anthropometric characteristics of these patients with diabetes, in accord with other studies (9,11), only plasma c - ldl levels showed a negative correlation with igm antioxldl ab . Numerous studies have linked both igg and igm antioxldl abs with atherosclerosis, although it seems that there are more consistent data about the negative association of igm levels with the atherosclerotic process (21). In occluded arteries, only igm levels are positively correlated with the signaling pathways, as argued by many authors (22). In the present study, we analyzed the biomarkers of the different metabolic pathways that have significant correlation with antioxldl ab . With respect to inflammatory biomarkers, it is known that during plaque formation, muscle cells migrate from the media to the intimal layer, where they proliferate and generate growth factors, such as vegfa . In the past decade, vegfa was believed to be a proinflammatory biomarker in endothelial cells involved in the atherosclerotic process (23). In the present study, expression of vegfa is associated with igm antioxldl abs in both fv and opa, demonstrating this relationship . Another inflammatory biomarker, tissue factor pathway inhibitor (tfpi), is a potent regulator of the tissue factor pathway that together with ldl modulates tissue factor function (24). Other proinflammatory biomarkers are the metalloproteinases, which are a family of zinc - dependent endopeptidases, and they are a major contributor to plaque rupture (25). Mmp10 is a key link between inflammation and thrombosis, particularly in situations of increased thrombotic risk (26). In the present study all these findings suggest that igm antioxldl ab is related to the inflammatory process inside an atherosclerotic environment . In relation to lipid metabolism pathway, two biomarkers correlated with antioxldl abs . On the one hand, as such, it depends on the amount of cholesterol in lipid - laden macrophages (27) inside atheroma plaque . In peripheral cells, the lipid uptake by scavenger receptor initiates chronic proinflammatory cascades linked to atherosclerosis . In this sense, the present study demonstrates that scarb1 is positively correlated with igm antioxldl ab levels in opa . In addition, another lipid biomarker is a nuclear hormone receptor, pparg, which also is positively correlated with igm antioxldl ab levels in fv . Pparg agonist - mediated inhibition of the renin - angiotensin - aldosterone system and the thromboxane a2 system as well as endothelial protection may possibly be involved in the inhibitory effects on blood pressure and atherosclerosis (28) in this vessel; thus, the atherosclerotic process is not developed in this environment . Activation of pparg by its ligands could modulate gene transcription, such as nfkb, thereby leading to multiple antiatherogenic and fibrinolytic effects (29). In fact, nfkb also shows a positive correlation with igm antioxldl ab levels in fv . When igm antioxldl ab levels were analyzed in relation with some biomarkers of the apoptosis pathway, we found that akt1, casp3, and bax in fv and only akt1 and bax in opa showed a positive correlation with igm antioxldl ab . Recently, it was demonstrated that in human umbilical vein endothelial cells, oxldl - induced apoptosis was associated with upregulation of proapoptotic bax and casp3 and inhibition of antiapoptotic akt1 accompanied by reciprocal changes in the methylation of promoter regions of these genes (30). In this sense, it could be hypothesized that because of this correlation between these biomarkers and igm antioxldl ab, this antibody has a protective function in atheroma plaque . On the other hand, it was demonstrated that casp3 cleaves specifically with cdkn1a and may trigger the apoptosis pathway (31). The expression of this gene is modulated by the tumor suppressor protein p53 (33). In this study, a positive correlation between cdkn1a and igm antioxldl ab was found in opa . In conclusion, the results of this study show that the gene expression of the apoptosis, lipid metabolism, and inflammation metabolic pathways in the fv and opa vessels is directly related to the levels of igm antioxldl ab . Because this is a cross - sectional study, we could not determine whether igm antioxldl ab has a proinstability effect in plaques, whether it exerts a protective compensatory response, or whether it is merely a correlative marker for the presence of inflammatory cells in patients with diabetes.
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For every living creature, elderliness is the final and inevitable phase of life.1 elderliness is an unstoppable phenomenon that has biological, chronological, and social aspects . The process is not the same for all; it exhibits differences based on the individual involved.2 the elderly population is on the rise worldwide both in terms of number and position in society.3 although the extent of the increase varies with region and age group, the members of today s elderly population mostly reside in developed countries . However, a more rapid increase in the elderly populations of developing countries is expected in the future.4 it is foreseen that in many countries that are categorized as developing, the population of people ages 60 years or above will reach 1.2 billion in 2025 and two billion in the year 2050.5 while there is no particular range that demarcates the advent of elderliness, the most frequently used age limit is 65 years.2 in terms demography and law, the concept of elderliness is used to denote those ages 65 years and older.3 today, japan is the nation with the oldest population . Life expectancy at birth in japan is calculated to be 80 years . Life expectancy at birth is 79 years in countries such as canada, sweden, and switzerland, whereas it is 78 years in countries such as england, france, the netherlands, and italy . Life expectancy at birth is 75 years or older in 37 world nations.6 parallel to these developments around the world, there has been an increase in the elderly population of turkey as well . In the last second half of the 20th century (19502000) in turkey, the percentage of the population that is 60 years of age or older has increased from 3.5% to 5.5% of the total population . This rate is increasing incrementally every day.6 currently, there are approximately four million elderly people in turkey . It is estimated that this number will double in the next 20 years, rising to seven to eight million, and then reach 12 million by the year 2050.6 since ancient times, mankind has sought and continues to seek answers to questions such as these: who is elderly? ; is there a beginning of elderliness? ; when does someone feel old? ; and do the symptoms of elderliness present in the same way for every individual? The famous historian homer emphasizes the experience and wisdom of the elderly when he claims that the youth are ready to harness the ability and experience of the elderly.1 for plato (427347 bc), who draws attention to the individualistic aspect of the period of old age, the lifestyle during adolescence and adulthood is the determining factor in the activity and situation of an elderly individual.1 in different societies, various beliefs concerning the social meaning of aging and attitudes are held, and various customs toward the elderly are observed . In traditional societies, thus, they are perceived as individuals who bear significant social roles in the growth of the second generation and the preservation of culture . In modern societies, they are considered to be individuals whose productivity has come to an end, who are burdens for their families, and who are expected to die as soon as possible.7 people have also different perspectives about aging . There are significant variations in perspectives among different age groups and among different sociocultural and economic structures . The phenomenon of elderliness, which has been present since the beginning of humanity, has been increasing in importance in recent years.8 in societies in which industry is dominant, people are moving toward nuclear families, and the average lifespan is increasing . Consequently, the number of elderly people whose needs for accommodation and social support are met in care and nursing homes is increasing.6 in european countries such as belgium, denmark, france, and england, single individuals who are 65 years or older make up 11% of households . This rate is significantly lower in developing countries, where elderly people live with their children or their grandchildren.6 in eastern societies (thailand, the philippines, singapore, and taiwan) most elderly people live with their children.9 despite everything, japan sustains a strong tradition of respect for social life and for parents as well as its tradition of integrating the elderly into social life.10 in iran, also an asian country, the elderly have started to live alone, particularly in urban areas, because of geographical and social mobility, an increase in the number of nuclear families, a decrease in the number of children, and women s integration into the workforce . However, only a quarter - century earlier, families, particularly those of the lower, lower - middle, rural, and even the upper - middle classes, used to live with their one or two sons . Elevations in social, cultural, religious, and economic situations; an increase in the standards of education and living; and modernization have resulted in radical changes in the family structure . This situation has left the elderly alone.11 this process began in the 1950s in turkey . Society is aging at a steadily increasing rate despite a high birthrate (population aged 65 + years: 7.3%).12 throughout history, respect has been a milestone of our culture . Since time immemorial, ancestors have been respected and the elderly protected . Nevertheless, perspectives on old age vary, and elderliness is a complicated phenomenon . While some perceive elderliness as a problem, others adopt positive attitudes toward it.8 in our country, as in other countries undergoing the aging process, there exists a necessity to reconsider the phenomenon and policies surrounding elderliness . The conception of aging involves the discussion of subjects such as social integration of the elderly beyond mere daily care, the acquisition of lost statuses and roles, increasing functionality, and the efficient use of spare time.9 elderliness elevates the status of an individual in almost every society . In fact, this respect does not stem from age alone; wisdom, experience, and knowledge transmitted from ancestors, all of which are believed to come with age, increase the status of the elderly.13 the youth must get to know and understand adults and the elderly as much as adults need to know and understand the youth . In many conventional understandings, parents perceive caring for children as a social responsibility, and children believe that they must take care of their old parents . This attitude can also be seen in our traditions.13 the basic connection that the elderly have is to their families and particularly to their children . Therefore, it seems that the most significant source of social support is the nuclear family.14 our society needs the elderly population as a balancing element and a source of experience.15 there are differences in perspective among various age groups and among different sociocultural and economic structures . These differences reveal the relationships between the young generation and older generations (the pediatrics geriatrics relationship), which may cause problems such as generational conflicts . In such situations, both groups may despise each other . The youth must get to know and understand adults and the elderly as much as adults need to know and understand the youth . The aim of this study is to evaluate the perspectives on elderliness of students attending different types of high schools in central elazig . Students attending five different types of high schools (private high schools, state high schools, science high schools, anatolian high schools, and vocational schools of health) located in central elazig during the 20092010 school year constitute the population of this study . One class was randomly selected from the first, second, third, and fourth grades of each participating high school . Out of 650 students, 640 (98.4%) participated in the study . Before conducting this study, written research permission was obtained from the elazig provincial directorate of national education, and participants provided their oral consent . A questionnaire in line with the relevant literature was developed by researchers and was administered to students under direct observation in order to collect data about their demographic features and opinions about elderliness . Regarding the questions on elderliness, the question of what the youth know about the elderly was taken into consideration . Open - ended questions were used as well as multiple choice questions so that those completing the surveys could best express their thoughts . The questions were mostly focused on the idea of the elderly and the imminent thoughts of students toward the elderly rather than direct examples of experiences with the elderly . Data gathered from this study were evaluated by using the spss statistics program, and a chi - square test was used to detect significant frequency distributions, medians, standard deviations, and differences between groups . The study faced some limitations because conducting the study with all of the high school students in the city would be difficult in terms of time and cost . Because of this, only five different types of high schools in the city center (excluding districts and villages) were included in the study . The age distribution of participating students was 16.51 1.11 years (minimum 14 years, maximum 20 years). The students sociodemographic characteristics are seen in table 1 . Of all of the students, the mean of the age at which a person was considered elderly by the students was 53.909.392 years . For the question, where should the elderly generally stay?, the answer distribution according to high school type is provided in table 3 . The percentage of those students who would want their parents to live with them was found to be 84.7% in private schools, 90.8% in vocational schools of health, and 93.8% in other types of high schools (state high schools, science high schools, anatolian high schools). In the study, 41.7% of the participating students defined elderliness as being age 60 years or older . Students were asked at which age they thought elderliness began; the mean response was 53.909.392 years . This is the average for the responses, for which the range was 3085 years . The categorization of elderliness varies with the culture of the country and with life expectancy at birth . In a study conducted in manisa, a definition of elderliness similar to ours was found.6 since the group participating in the study comprised adolescents, their definitions of elderliness may be extended to younger age groups . Peace / resting, but 23.6% of students expressed that elderliness means sickness . Young people s association of elderliness with illness may be related to their observations of the elderly around them . The proportion of those who claimed that elderliness means wisdom/ experience was 16.6%, whereas in the study conducted in manisa, 35% of young people stated that elderliness means the end of everything . The proportion who defined elderliness as wisdom was 12.5%.6 in almost every society the senior citizen is an individual who is respected and has some special privileges.11 nurses working in a nursing home in izmir considered elderliness to be a difficult period during which constant care is needed for health and social problems.16 the reason for obtaining different results in our study may stem from interregional and sociocultural differences . Students maintained that the most important difficulty with living with the elderly is intergenerational conflict (30.3%), while an almost equal portion of the students (30.6%) expressed that there are no difficulties at all from living with the elderly . For a questionnaire in which respondents were asked, to what extent do elderly people have certain characteristics?, 34% of those ages 2534 years old stated that the elderly talk nonsense; 44% of respondents considered them to be lazy; and 61% of respondents maintained that elderly people are stubborn.17 the majority of students (83.4%) stated that the elderly should stay with their families . Compared to those attending of other types of schools, more students in vocational schools of health held that the elderly should be in nursing homes (p<0.05). This response may be due to their applied classes, in which they observe sicknesses of the elderly . One out of every ten elderly persons lives alone.18 similarly, in research conducted by bekarolu in trabzon, 66.6% of the elderly living in homes stated that they lived with their children or with their children and spouse, and 21.6% said that they lived with their spouses.19 also, 8% of respondents stated that they lived alone, and 4.6% stated that they lived with other relatives.19 the percentage of those who want their parents to live with them when their parents grow old was 84.7% in private schools, 90.8% in vocational schools of health, and 93.8% in other types of high schools . Subasi and oztek conducted a study of 1,055 households in ankara s cankaya district to investigate the opinions of people over 18 about elderly care.20 in the study, 75.0% of participants stated that children should take care of their parents who are old and cannot look after themselves . They maintained that elderly individuals should be cared for in their own homes.20 in a study conducted by baran et al in 2005 to analyze the relationship between elderly people and their families, 69.5% of individuals who were providing care for the elderly (son, daughter - in - law, daughter, son - in - law, or grandchildren) think that the elderly should be looked after by the son and daughter - in - law or by the daughter and son - in - law.21 on the other hand, the percentage of those who think that the elderly should be taken care of privately for a certain amount of money (3.5%) and the percentage of those who think that public institutions should provide care for the elderly (5.5%) were quite low . Also, 93.8% of the elderly maintained that they were happy to be living with their children.21 our study found that high school students generally perceive elderliness in a traditional manner, and they think that perspectives on elderliness may change through education . Necessary precautions to ease life and care for the elderly staying at home should be taken.
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Despite great advancements in genomics research, dna microarrays are still of limited applicability as diagnostic tests, in part because proteins, not genes, are the ultimate effectors of a biological process . Proteins are often expressed at concentrations and varied structural forms having modifications that cannot be predicted from mrna analysis . There is a great deal of interest in proteomics - based approaches that enable profiling the abundance of proteins, or investigation of protein - protein and protein - drug interactions . The concept of a protein microarray or biochip is attractive for rapid, high - throughput proteomics studies that utilize available sample volumes [1, 2, 3, 4]. Proteins that are present in abnormal concentrations (increased or decreased) in a disease state could be identified and validated as disease markers . Proteomics pattern analysis based on a multiplexed list of biomarkers may well make possible the diagnosis of certain diseases that do not have either effective screening options or biomarkers already detected by conventional immunoassays . Identification of such disease markers will provide valuable information for detection, classification, and prognosis of diseases, as well as targets for treatment outcomes . High - throughput protein chips require arraying a wide range of probes that specifically recognize a single protein in complex mixtures such as serum, plasma, and other biological specimens . These probes are most frequently antibodies or antibody mimics [3, 4, 6, 7]. Given the immense promise of proteomics technologies, there are still limitations that need to be overcome, such as lack of sensitivity and enhanced information about the target . Because there is no pcr equivalent available to amplify proteins, the identification of low - abundance proteins may often be difficult or impossible, and important biomarker information could thus be lost . Rolling - circle amplification (rca) is a unique signal amplification technology that permits detection of multiple proteins with a broad dynamic range on protein microarrays [4, 9]. In this paper, we describe the development of rca - based highly multiplexed and sensitive protein microarray immunoassays for detecting 150 proteins in an elisa format . Application to human serum and plasma samples has established that the rolling - circle amplified protein microarray is a powerful tool for the profiling of expressed proteins in biological specimens . Antibodies (r&d systems, minneapolis, minn; pharmingen, san diego, calif; biosource international, camarillo, calif) were diluted to 0.5 mg / ml in pbs containing 0.05 mg / ml bsa, and 375 pl of each was spotted in quadruplicate onto the slides using a biochip arrayer (packard biosciences, downers grove, ill). Each spot has a diameter of approximately 180 m with a center - to - center spacing of 270 m . Each slide was divided by a teflon mask into 16 subarrays, each with a diameter of 0.65 cm . Within each subarray, 256 spots were printed at known locations, containing 40 different features (antibodies for specific antigens) in quadruplicate (160 spots). The remaining features comprised of controls for antibody immobilization, cy5-labeled mouse igg and internal calibrators (biotin mouse igg). Slides were blocked by adding 30 l blocking buffer (0.5% nonfat dry milk, 0.2% bsa, and 0.05% tween-20 in pbs) to each subarray and incubating for 1 hour at 37c in a humidified chamber, and then washed by immersion in pbs containing 0.5% brij 35 for 2 minutes . A 15 l aliquot of sample (single analyte or mixture of multiple analytes; serum, plasma, or culture supernatant) was immediately added to each subarray, incubated for 30 minutes at 37c, and then washed as described above . After analytes were captured, a 20 l aliquot of the appropriate mixture of biotinylated detection antibodies at 0.1 g / ml each was applied to individual arrays and incubated at 37c for 30 minutes in a humidified chamber . Slides were washed twice for two minutes in pbs containing 0.5% brij 35 . A mouse monoclonal antibiotin antibody - primer conjugate was prepared by derivatization of a mouse monoclonal antibiotin igg (jackson immunoresearch laboratories, inc, west grove, pa) with a 5'-terminal amine - modified oligonucleotide primer, 5'-thiol - aaa aaa aaa aaa aaa cac agc tga gga tag gac at-3', as previously described . The conjugate was annealed with 75 nm circle 1 (cyclic 5'-ctc agc tgt gta aca aca tga aga ttg tag gtc aga act cac ctg tta gaa act gtg aag atc gct tat tat gtc cta tc-3') in pbs containing 0.05% tween-20 and 1 mm edta at 37c for 30 minutes . Rca reaction mixture (20 l) containing native t7 dna polymerase (20 u / ml), 1 mm dntps, ssdna - binding protein 22 g / ml, 1x sequenase buffer, 8% dmso, and 0.05 mm cy5 decorator (5'-cy5-tgt cct atc ctc agc tgg - cy5) was added to each subarray . Slides were incubated at 37c for 45 minutes, then washed twice in pbs containing 0.5% brij 35 and once in pbs, and spin - dried . Arrays were analyzed on an axon genepix 4000b scanner (axon instruments inc, foster city, calif), and fluorescence quantitated by using genepix pro 3.0 quantitation software . It consists of (a) analyte capture by antibody immobilized on microarray, (b) detection by biotinylated secondary antibody, (c) binding of antibiotin antibody - oligonucleotide conjugate pre - annealed with circle 1, and (d) rolling - circle replication to generate long single - strand dna which is hybridized with oligonucleotide decorator . This surface was chosen based on experiments indicating that cyanosilane - activated slides have the advantages of (1) simpler preparation, (2) less - expensive reagents, and (3) more uniformity of bound antibody spot morphology over thiolsilane - coated and cross - linker (such as gmbs) activated glass slides (data not shown). The concentration of capture antibody for printing was optimized by comparing specific signal intensities from the same capture antibody printed on the chip at different concentrations chosen for microarray printing . Specific signals at 0.5 mg / ml reached the point of saturation (data not shown), indicating that antibodies saturated the area available for immobilization in the spot . The uniformity of antibody immobilization on the chip was assessed by measuring the coefficient of variation (cv) from the quadruplicate spots using cy5-labeled anti - isotype antibodies (against the species used in the capture antibodies), previously reported as approximately 10% for each feature on the chip . Rolling circle amplification enables amplification of signals tethered to proteins and nucleic acids [4, 9, 11]. Immunoassays incorporating rca have shown a large increase in sensitivity when compared with the direct assay . The technique consists of protein capture by antibody immobilized on microarrays, detection by biotin - labeled second antibody specific for the captured protein, binding of a universal antibiotin antibody oligonucleotide conjugate pre - annealed with circle, and rca signal amplification where a long single - strand dna is generated by rolling - circle replication in the presence of dna polymerase and nucleotides, with hybridization of the rca product to fluorescent - labeled complementary oligonucleotide probes occurring simultaneously (figure 1). Besides the signal amplification by rca, the sensitivity of the antibody array was further improved by two approaches: (1) choosing optimal capture and detection antibody pairs for the microarray immunoassay and (2) optimizing assay procedures . Optimization of antibodies from different sources (a and b) for choosing optimal pair for immunoassay on protein microarray with examples of (a) il-13 and (b) mcp-2 . For individual features, if the matched antibody pair developed for conventional elisa was available, it was preferentially selected for the chip development . However, in terms of sensitivity and specificity, antibody pairs do not always function equivalently on plastic elisa plates and the glass - based microarrays . Antibody pairs from different manufacturers for the same antigen with similar sensitivity as elisa assays perform quite differently in microarrays . Thus it has become necessary to select optimal pairs for antibody microarrays by comparing all available antibodies and pairs . As an example, figure 2a shows the difference between two matched pairs of antibodies (for elisa) from different sources with specificity for detecting the cytokine il-13 using the antibody microarray . The pair from source a showed strong signal intensity with a sensitivity of less than 100 pg / ml . However, the pair from source b did not show signal even at concentrations as high as 5 ng / ml il-13 . Selecting the antibody pair from source a provided at least 50-fold increase of sensitivity when compared with the pair from source b. alternatively, the elisa antibody pair from the same set is not always the optimal for a microarray immunoassay . Neither of the elisa - matched pairs showed sensitivity better than 100 pg / ml on the microarray slides . However, using a capture antibody from a and a detection antibody from b, the sensitivity for mcp-2 was improved to 10 pg / ml or better, a 10-fold increase in sensitivity relative to a single manufacturer's reagents . For features without matched pairs, monoclonal antibodies with different binding epitopes or polyclonal antibodies were extensively evaluated for sensitivity ranges permitting detection of biologically relevant changes in protein expression . Cross - reactivity of capture antibodies and analytes ((a) and (c)), capture antibodies and detection antibodies ((b) and (d)) on the subarrays 3 (a) and 4 (b). Each subarray was printed with about 35 different capture antibodies (listed on the chart). Cross - reactivity was determined by using different matched secondary antibody and 50 ng / ml or 0 ng / ml of its cognate analyte, which has been described in the text . A shaded square off the diagonal indicates the presence of nonspecific signals (intensity 300). None of these nonspecific signals exceed the intensity of 600 fluorescence intensity units (images are scanned at pmt of 600 and power of 100% on axon scanner). (a) images of detecting 70 proteins in antibody microarray obtained with axon microarray scanner . (b) fluorescence intensities were derived from microarray images with averaging four spots of the same feature . This involved optimizing detection antibody concentrations, conjugate and circle concentrations, incubation temperatures and time, and rca reaction solution composition . Analogous to sandwich elisa assays, the conditions giving the best signal to noise ratio were found to be 0.1 g / ml of detector with assay incubation time of 30 minutes at 37c . Titration of conjugate and circle pre - annealed to conjugate revealed the optimal concentration to be 2 g / ml conjugate with 75 nm circle . Other components, such as the concentration of single - strand dna binding protein (ssb), were also optimized for maximum sensitivity in rca reaction mixtures (figure 3). The analytical sensitivity of the microarray immunoassay was determined by using serial dilutions of each single purified antigen . The sensitivity of detection, defined as the lowest concentration that delivered clear specific signal intensity above detection threshold (mean intensity of the negative control plus 2 sd), was calculated . Sixty - five (43%) of the 150 cytokine features had a sensitivity of 10 pg / ml, 52 (35%) had a sensitivity of 100 pg / ml, 27 (18%) had a sensitivity of 1000 pg / ml, and 6 (4%) had a sensitivity of 1,000 pg / ml (table 1). Sixty - two of the cytokines represented on the chip are unique, with no corresponding commercially available elisa kits . The sensitivity goal for these arrays was adequacy to detect biologically relevant (ie, 2-fold) increases in cytokine level above normal values in clinical fluids including serum and plasma . Although antibodies are highly specific for their respective cognate proteins, it is possible that structurally related proteins might have similar epitopes . Cross - reactivity or nonspecific signals between immobilized capture antibodies and antigens or detection antibodies for 150 features were determined in three different ways . First, the cross - reactivity of individual antigens or detection antibodies was determined by analyzing a single antigen at 0 and 50 ng / ml with its corresponding paired detection antibody . Any signals on the 0 ng / ml array were categorized as cross - reactivity between the detection and capture antibodies . With 50 ng / ml analyte, signals should have only been observed at the specific locations . Other signals after background subtraction (the 0 ng / ml array) were assumed to be cross - reactivity from the analyte . Nonspecific signals were eliminated by segregating all features into multiple subarrays, thus the capture antibodies producing nonspecific signals were physically separated from those particular detection antibodies or antigens . This also allowed simultaneous detection of both components of a biological signaling system, such as a growth factor and its receptor . Residual cross - reactivity and nonspecific signals were first minimized by optimization of washing and blocking conditions . Next, a mixture of several analytes at concentrations of 10 ng / ml each was applied to the chip, followed by detection with the complete detection antibody cocktail . Signals were only observed where both the analyte and its specific detection antibody were present . Then a mixture of all analytes in the subarray at 10 ng / ml each was added to the microarray, but detection was with an incomplete cocktail of antibodies . Signals were only detected on those features corresponding to the specific detection antibodies added . As shown in figure 4, both cross - reactivity between capture antibodies and analytes and cross - reactivity between capture antibodies and detection antibodies in each subarray were minimized . Approximately 99% of nonspecific binding pairs had fluorescence intensity units of less than 300, used as the background signal minimum during the data quantitation . Interferences from biological specimen were screened using normal human serum and single detectors (biotinylated detection antibody) corresponding to each feature on the chip . Only specific signals of the proteins present in the specimen were detected by their associated detector antibody . The antibody microarray was validated by running assays with normal human serum (sigma, st . Signals corresponding to specific proteins were identified (figure 5), which are consistent with their presence with significant level in the normal human serum (table 1). Proteins with low fluorescence signal intensities agree with their very low level in serum (if present), indicating that our antibody microarray provides a specific, robust, and high - throughput approach for global analysis of protein expression . This surface was chosen based on experiments indicating that cyanosilane - activated slides have the advantages of (1) simpler preparation, (2) less - expensive reagents, and (3) more uniformity of bound antibody spot morphology over thiolsilane - coated and cross - linker (such as gmbs) activated glass slides (data not shown). The concentration of capture antibody for printing was optimized by comparing specific signal intensities from the same capture antibody printed on the chip at different concentrations chosen for microarray printing . Specific signals at 0.5 mg / ml reached the point of saturation (data not shown), indicating that antibodies saturated the area available for immobilization in the spot . The uniformity of antibody immobilization on the chip was assessed by measuring the coefficient of variation (cv) from the quadruplicate spots using cy5-labeled anti - isotype antibodies (against the species used in the capture antibodies), previously reported as approximately 10% for each feature on the chip . Rolling circle amplification enables amplification of signals tethered to proteins and nucleic acids [4, 9, 11]. Immunoassays incorporating rca have shown a large increase in sensitivity when compared with the direct assay . The technique consists of protein capture by antibody immobilized on microarrays, detection by biotin - labeled second antibody specific for the captured protein, binding of a universal antibiotin antibody oligonucleotide conjugate pre - annealed with circle, and rca signal amplification where a long single - strand dna is generated by rolling - circle replication in the presence of dna polymerase and nucleotides, with hybridization of the rca product to fluorescent - labeled complementary oligonucleotide probes occurring simultaneously (figure 1). Besides the signal amplification by rca, the sensitivity of the antibody array was further improved by two approaches: (1) choosing optimal capture and detection antibody pairs for the microarray immunoassay and (2) optimizing assay procedures . Optimization of antibodies from different sources (a and b) for choosing optimal pair for immunoassay on protein microarray with examples of (a) il-13 and (b) mcp-2 . For individual features, if the matched antibody pair developed for conventional elisa was available, it was preferentially selected for the chip development . However, in terms of sensitivity and specificity, antibody pairs do not always function equivalently on plastic elisa plates and the glass - based microarrays . Antibody pairs from different manufacturers for the same antigen with similar sensitivity as elisa assays perform quite differently in microarrays . Thus it has become necessary to select optimal pairs for antibody microarrays by comparing all available antibodies and pairs . As an example, figure 2a shows the difference between two matched pairs of antibodies (for elisa) from different sources with specificity for detecting the cytokine il-13 using the antibody microarray . The pair from source a showed strong signal intensity with a sensitivity of less than 100 pg / ml . However, the pair from source b did not show signal even at concentrations as high as 5 ng / ml il-13 . Selecting the antibody pair from source a provided at least 50-fold increase of sensitivity when compared with the pair from source b. alternatively, the elisa antibody pair from the same set is not always the optimal for a microarray immunoassay . Neither of the elisa - matched pairs showed sensitivity better than 100 pg / ml on the microarray slides . However, using a capture antibody from a and a detection antibody from b, the sensitivity for mcp-2 was improved to 10 pg / ml or better, a 10-fold increase in sensitivity relative to a single manufacturer's reagents . For features without matched pairs, monoclonal antibodies with different binding epitopes or polyclonal antibodies were extensively evaluated for sensitivity ranges permitting detection of biologically relevant changes in protein expression . Cross - reactivity of capture antibodies and analytes ((a) and (c)), capture antibodies and detection antibodies ((b) and (d)) on the subarrays 3 (a) and 4 (b). Each subarray was printed with about 35 different capture antibodies (listed on the chart). Cross - reactivity was determined by using different matched secondary antibody and 50 ng / ml or 0 ng / ml of its cognate analyte, which has been described in the text . A shaded square off the diagonal indicates the presence of nonspecific signals (intensity 300). None of these nonspecific signals exceed the intensity of 600 fluorescence intensity units (images are scanned at pmt of 600 and power of 100% on axon scanner). (a) images of detecting 70 proteins in antibody microarray obtained with axon microarray scanner . (b) fluorescence intensities were derived from microarray images with averaging four spots of the same feature . The second approach of improving sensitivity was to optimize the assay procedures . This involved optimizing detection antibody concentrations, conjugate and circle concentrations, incubation temperatures and time, and rca reaction solution composition . Analogous to sandwich elisa assays, the conditions giving the best signal to noise ratio were found to be 0.1 g / ml of detector with assay incubation time of 30 minutes at 37c . Titration of conjugate and circle pre - annealed to conjugate revealed the optimal concentration to be 2 g / ml conjugate with 75 nm circle . Other components, such as the concentration of single - strand dna binding protein (ssb), were also optimized for maximum sensitivity in rca reaction mixtures (figure 3). The analytical sensitivity of the microarray immunoassay was determined by using serial dilutions of each single purified antigen . The sensitivity of detection, defined as the lowest concentration that delivered clear specific signal intensity above detection threshold (mean intensity of the negative control plus 2 sd), was calculated . Sixty - five (43%) of the 150 cytokine features had a sensitivity of 10 pg / ml, 52 (35%) had a sensitivity of 100 pg / ml, 27 (18%) had a sensitivity of 1000 pg / ml, and 6 (4%) had a sensitivity of 1,000 pg / ml (table 1). Sixty - two of the cytokines represented on the chip are unique, with no corresponding commercially available elisa kits . The sensitivity goal for these arrays was adequacy to detect biologically relevant (ie, 2-fold) increases in cytokine level above normal values in clinical fluids including serum and plasma . Although antibodies are highly specific for their respective cognate proteins, it is possible that structurally related proteins might have similar epitopes . Cross - reactivity or nonspecific signals between immobilized capture antibodies and antigens or detection antibodies for 150 features were determined in three different ways . First, the cross - reactivity of individual antigens or detection antibodies was determined by analyzing a single antigen at 0 and 50 ng / ml with its corresponding paired detection antibody . Any signals on the 0 ng / ml array were categorized as cross - reactivity between the detection and capture antibodies . With 50 ng / ml analyte, signals should have only been observed at the specific locations . Other signals after background subtraction (the 0 ng / ml array) were assumed to be cross - reactivity from the analyte . Nonspecific signals were eliminated by segregating all features into multiple subarrays, thus the capture antibodies producing nonspecific signals were physically separated from those particular detection antibodies or antigens . This also allowed simultaneous detection of both components of a biological signaling system, such as a growth factor and its receptor . Residual cross - reactivity and nonspecific signals were first minimized by optimization of washing and blocking conditions . Next, a mixture of several analytes at concentrations of 10 ng / ml each was applied to the chip, followed by detection with the complete detection antibody cocktail . . Then a mixture of all analytes in the subarray at 10 ng / ml each was added to the microarray, but detection was with an incomplete cocktail of antibodies . As shown in figure 4, both cross - reactivity between capture antibodies and analytes and cross - reactivity between capture antibodies and detection antibodies in each subarray were minimized . Approximately 99% of nonspecific binding pairs had fluorescence intensity units of less than 300, used as the background signal minimum during the data quantitation . Interferences from biological specimen were screened using normal human serum and single detectors (biotinylated detection antibody) corresponding to each feature on the chip . Only specific signals of the proteins present in the specimen were detected by their associated detector antibody . The antibody microarray was validated by running assays with normal human serum (sigma, st . Signals corresponding to specific proteins were identified (figure 5), which are consistent with their presence with significant level in the normal human serum (table 1). Proteins with low fluorescence signal intensities agree with their very low level in serum (if present), indicating that our antibody microarray provides a specific, robust, and high - throughput approach for global analysis of protein expression . Immunoassays on microarrays hold appeal for studies requiring the ability to quantify many selected proteins simultaneously . Considerable progress has been made in this area recently in terms of increased assay sensitivity and complexity (ie, degree of multiplexing). Rolling - circle amplification facilitates the use of such arrays with its powerful degree of signal enhancement and simultaneous detection of a large number of defined analytes . Such robust systems will enable routine use of antibody arrays in both research and diagnostic modalities.
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Many debilitating and often fatal disorders, including alzheimer s and parkinson s diseases, the transmissible spongiform encephalopathies, type ii diabetes, and a range of systemic amyloidoses, are associated with the deposition of normally soluble proteins as insoluble aggregates in various types of tissue. (1) although the sequences and native structures of the proteins involved in the different conditions vary greatly, the aggregates found in patients exhibit very similar biophysical properties . These include their fibrillar nature and cross- structure, where -strands are oriented perpendicularly to the axis of the fibril. (1) in many cases the formation of amyloid fibrils requires the monomeric precursor protein to undergo a series of substantial conformational rearrangements . A detailed characterization of the different species involved in these conformational transitions is crucial for developing an understanding of the mechanism behind misfolding diseases . Such species are also key therapeutic targets for developing drugs to treat or prevent amyloid - related diseases . Partially folded states, denatured states, disordered protein ensembles, and molten globules are possible precursors to amyloid fibrils or other aggregates and have therefore been the object of extensive research . Human lysozyme is a small, well - characterized globular protein with a native structure that can be divided into two domains: the domain (residues 1 to 38, and 86 to 130) and the domain (residues 39 to 85) that primarily contain -helical and -sheet secondary structure, respectively(18) (see figure 1a, b). In 1993, pepys and co - workers reported that two variants of human lysozyme are responsible for a hereditary non - neuropathic systemic amyloidosis. (20) given the significant body of data on the structure, dynamics, and folding of both hen and human lysozymes, the link to human disease renders this protein an ideal system to probe the relationships between protein folding and aggregation . The first two single - point mutations found to be linked to lysozyme amyloidosis were i56 t and d67h, and other amyloidogenic and non - amyloidogenic mutations have also been identified more recently (for a review, see dumoulin et al.(21)). The observation that only certain mutational variants form fibrils in vivo raises the question of which factors regulate the amyloidogenicity of this protein . A comparative thermal denaturation study has shown that the two amyloidogenic variants i56 t and d67h are less stable than the wild - type protein (wt)11abbreviations: wt, wild - type; nmr, nuclear magnetic resonance; hsqc, heteronuclear single quantum coherence; uv, ultraviolet; cd, circular dichroism; dsc, differential scanning calorimetry ., suggesting that a destabilization of the native state is, at least in part, responsible for the process of fibril formation . Indeed at ph 5.0 the midpoints of thermal unfolding of the i56 t and d67h variants were reported to be 12 2 c lower than that of the wt protein . However, the naturally occurring and non - amyloidogenic t70n variant also shows a decrease in the unfolding temperature in vitro of approximately 4 c relative to the wt but is not observed to form detectable quantities of fibrils or to give rise to disease in individuals carrying this mutation . In addition, under in vitro conditions, t70n does not form fibrils at rates comparable to the amyloidogenic variants . It therefore appears that the disease - associated mutational variants need to be destabilized enough to allow detectable amyloid fibril formation to take place under physiological conditions but not so destabilized as to trigger clearance by the cell . Abbreviations: wt, wild - type; nmr, nuclear magnetic resonance; hsqc, heteronuclear single quantum coherence; uv, ultraviolet; cd, circular dichroism; dsc, differential scanning calorimetry . (a) representation of the secondary structure of the native state of wt human lysozyme, indicating glycine residues (using dssp(17) on pdb entry 1lz1(18)). Strands are represented by arrows, -helices (a to d) by large boxes, and 310 helices (h1 to h3) by small boxes . The disulfide bridges c6c128, c30c116, c65c81, and c77c95 are shown in purple . (b) tertiary structure of wt human lysozyme with the same color coding as in panel a. all structures are drawn using vmd(19) with the secondary structure defined by dssp. (17) vmd only represents strands comprising three residues and more; therefore, strand 3 (residues 5960) is not shown . The colored spheres represent residues 56 (blue) and 59 (red), sites of the mutations i56 t and i59 (c) normalized signal of the near - uv circular dichroism thermal unfolding traces obtained for the wt (black), i59 t (red) and i56 t (blue) variants, at ph 1.2 . Hydrogen / deuterium (h / d) exchange experiments on the amyloidogenic variants i56 t and d67h have revealed the presence of a partially folded intermediate state that is transiently populated under native conditions and characterized by the unfolding of the -domain and one of the helices (helix c of the -domain). In addition, thermal unfolding experiments carried out in vitro under equilibrium conditions in the presence of a fluorescent dye sensitive to hydrophobic patches (1-anilinonaphthalene-8-sulfonic acid or ans) indicate the presence of partially unfolded species near to the midpoint of thermal denaturation . On the basis of these results, a reduction in the global cooperativity of the variants of lysozyme found in disease has emerged as a key determinant for their amyloidogenicity: the non - cooperative unfolding behavior leads to the formation of partially folded species that are able to associate to form highly organized fibrils, the deposition of which is characteristic of amyloid diseases . Despite the importance of partially folded states in this mechanism, their transient nature and low populations at equilibrium under solution conditions commonly used in in vitro studies (ph> 5) have precluded a direct characterization of these species at high resolution . In this paper, we use low ph conditions to provide such high - resolution structural evidence on the nature of the equilibrium thermal unfolding behavior of human lysozyme . Using nmr spectroscopy in combination with a wide range of biophysical techniques, we studied three variants of human lysozyme that have variable stabilities and propensities to aggregate in vitro, namely the wt protein and the i56 t and i59 t mutants (see figure 1). (29) as stated above, the i56 t variant is linked to familial lysozyme systemic amyloidosis, and the i59 t mutant has been designed to have properties that are intermediate between those of i56 t and the wt lysozymes . Our results reveal that under the acidic destabilizing conditions commonly used to form amyloid fibrils in vitro, human lysozyme is in a dynamic equilibrium between its native conformation and a broad ensemble of non - native conformations: the members of this non - native denatured ensemble contain both molten globular and unfolded regions . Molten globular regions, in which secondary structure elements are present but tertiary interactions are disorganized to varying degrees, are predominant at lower temperatures and unfold gradually, and with local cooperativity, as they become increasingly disordered at higher temperatures . The strategy used in this study exploits low ph conditions to destabilize the native state and to observe partially unfolded states of lysozyme under equilibrium conditions. (30) lowering the ph from 5.0 to 1.2 results in a substantial decrease in the stability of the native state of all three variants, thereby enabling a comparison of the progressive denaturation of the wt protein and the i59 t and i56 t variants at temperatures below 50 c (see figure 1c). Hn backbone hsqc spectra recorded at 20 c are typical of a folded protein, confirming that the native structure is preserved at the lower temperatures in all three variants at ph 1.2 (see figure 2). The temperature at which the tertiary structure is lost differs significantly between the three variants, showing that the i56 t and i59 t single - point mutations have a large and differential effect on the stability of the native state: 48.5 0.3 c for wt, 35.4 0.5 c for i59 t, and 22.6 0.3 c for i56 t lysozyme (see figure 1c, and table s1 in the supporting information). The proteins are stable for days at this ph at low temperature (<35 c), and for several hours at the higher temperatures, enabling detailed nmr analysis to be carried out on the chemically intact protein, and allowing the equilibrium unfolding of all three variants to be compared in detail without the addition of denaturant . Hsqc spectra of i59 t at ph 1.2 recorded at different temperatures . At 20 c, the protein is fully folded (a), whereas at 50 c, the protein is fully unfolded (c). Near the midpoint of the unfolding transition, at 35 c, the protein shows resonances typical of both folded and unfolded states (b). The region boxed in panel (c) indicates the peaks corresponding to glycine residues in the unfolded state . Incubation of 1 mm solutions of wt lysozyme at temperatures near the midpoint of unfolding (47.5 c) and with stirring leads to the essentially complete conversion of the soluble forms of the protein into amyloid fibrils within 45 days . In order to accelerate the aggregation process and avoid prolonged incubation times at elevated temperatures and low ph, which can cause hydrolysis of the protein, aliquots (2% v / v) of preformed fibril seeds were added to the solutions, resulting in more than 95% of the monomeric protein becoming incorporated in fibrils within one day of incubation . The fibrils obtained contain non - hydrolyzed, full length monomeric protein as confirmed by sds - page electrophoresis and have all the morphologic characteristics of amyloid structures: transmission electron microscopy images show that they are long and unbranched, they bind thioflavin - t, an amyloid - specific dye, and they present a high degree of -sheet structure, as indicated by cd (see figure s1 in the supporting information). The observation that amyloidogenic precursors are populated near the temperatures where tertiary structure is lost prompted us to study the nature and relative abundance of such species . Monitoring the unfolding transition of all three lysozyme variants by means of circular dichroism spectroscopy (cd) at different wavelengths reveals that the tertiary structure (near - uv region, 270 nm) is disrupted more readily, i.e. At lower temperatures, than the secondary structure (far - uv region, 222 nm) (see figure 3a and table s1, figure s3, and figure s4a in the supporting information). The difference in melting temperatures detected by near- and far - uv cd increases as the stability of the native state decreases; hence, the temperature difference between the two midpoints is largest for i56 t and smallest for the wt protein . These data show that a simple two - state model fails to describe the complexity of the unfolding process . In addition to the native and unfolded states, at least one more state exists with a secondary structure content similar to that of the native state but with a very significant loss of tertiary structure . Interestingly, the thermal unfolding of the secondary structure is essentially identical for the i56 t and i59 t variants, whereas the tertiary structure is more strongly destabilized for the i56 t than for the i59 t variant (see figure s3 in the supporting information). Thermal unfolding of human lysozyme monitored using cd spectroscopy and dsc: a pseudo - two - state model . (a) equilibrium thermal unfolding of wt, i59 t, and i56 t lysozyme, followed by near - uv (270 nm, red) and far - uv (222 nm, black) cd at ph 1.2 . Thermal melts were fitted to a two - state model (fits in solid lines). For more details, see experimental section and the supporting information . (b) evolution of excess heat capacity with temperature shown for wt (black), i59 t (red), and i56 t (blue) at ph 1.2 . Solid lines represent the experimental data, while dashed lines show curves simulated using the thermodynamic parameters obtained from the fitting the cd data to a three - state model (see the supporting information). The reversibility of the thermal denaturation monitored by dsc is shown in figure s2 of the supporting information . (c) proposed model: scheme of the native state n and the denatured ensemble (dashed green lines) are derived by fitting the near - uv cd first - order transition to a pseudo - two - state model . A three - state model defining native, intermediate, and unfolded states accounts essentially perfectly for the far- and near - uv cd data,(30) and allows the apparent population of each state to be determined for all three variants (see figure s4a, b in the supporting information). In addition, knowledge of the populations of the three states enabled us to deconvolute the far- and near - uv cd data to extract separate spectra of the native, intermediate, and unfolded states (see figure s4c in the supporting information). As expected, the intermediate lacks a well - defined tertiary structure, as indicated by the loss of the native near - uv cd signal, yet preserves an essentially native - like secondary structure signature in the far - uv cd region, indicative of molten globular character (see figure s4c in the supporting information). The population of the intermediate state and the temperature window where it is significantly populated both increase as the stability of the native state decreases . In order to investigate further the nature of the unfolding transition dsc allows the identification of merged, multiple transitions that may not be well - resolved by spectroscopic techniques, provided that these transitions induce a detectable change in both heat capacity and enthalpy. (33) in the present case, the unique heat absorption peak observed by dsc is characteristic of a two - state transition, where the temperature midpoint of the unfolding transition is identical within error to that seen by near - uv cd (see figure 3a, b, experimental section, and supporting information table s1); that is, the observed transition corresponds to the loss of tertiary structure . Furthermore, simulated dsc thermograms, based on the thermodynamic parameters obtained from the three - state fit to the far- and near - uv cd data, show profiles that clearly differ from the experimental ones (compare plain and dashed lines in figure 3b). We therefore conclude that only one conformational transition is detected by dsc that, according to the three - state model, corresponds to the cooperative conversion of the native state into the intermediate state . By contrast, the unfolding of the intermediate does not follow a cooperative first - order transition, but is instead best described as a continuous (second - order) therefore, the dsc data question the validity of a three - state model invoking two first - order transitions . The three - state model defined by the cd data is still useful in an operational sense but needs to be improved with the intermediate and unfolded states replaced by an ensemble of denatured states separated by marginal energy barriers: in this improved model the denatured ensemble consists of a collection of non - native states possessing different degrees of unfolding . The composition of this ensemble changes with temperature, being on average more molten globule - like at low temperatures, and more unfolded as the temperature is increased (see figure 3c). To obtain populations for the native state and the denatured ensemble, we have used the near - uv cd unfolding curve only and fitted it to this pseudo - two - state model (dashed lines in figures 3 and 4). The model that we propose here is, therefore, a significant improvement on the three - state model used until now for human lysozyme . This situation is reminiscent of the thermal unfolding of the molten globule of the structurally homologous protein -lactalbumin whose unfolding does not give rise to any noticeable heat absorption peak and for which a similar denatured ensemble has been proposed . Black dots connected by lines represent the temperature dependence of individual hn hsqc cross - peak volumes of the native state (left), normalized relative to the volume of each peak at 5 c, and the denatured ensemble (right), normalized relative to the volume of each peak at 55 c (see comment for wt in the experimental section). All non - overlapping nmr cross - peaks are shown for the decay of the native state (left), whereas the increases in cross - peak volumes of glycine residues only are shown for the denatured ensemble (right). Glycine residues have been selected because they have all been assigned in the denatured state of all three variants (see figure 5b). Increases in cross - peak volumes for 46 assigned residues of the denatured ensemble of i56 t are shown in figure s5 in the supporting information and show similar results, therefore justifying the fact that only glycines are considered here . The decay of the integrated signal of selected native side chain methyl groups is shown in light blue circles in the left column (see raw data in figure s6 in the supporting information). Full lines represent populations derived by simultaneously fitting far- and near - uv cd data to the three - state model: the populations of the native, intermediate, and unfolded states are indicated by the blue, red, and green lines, respectively (the population of the native state of the i56 t variant is normalized to its value at 5 c). Dashed lines show the results obtained by fitting the near - uv cd data to the pseudo - two - state model: the populations of the native state and the denatured ensemble are shown in blue and green, respectively . To characterize the unfolding process at the level of individual residues, we recorded hn backbone hsqc spectra of all three variants over a wide range of temperatures, acquiring spectra at every 2.5 c between 5 and 55 c . At low temperature, the hn hsqc spectrum of each variant is characteristic of a folded protein with a large number of well resolved and dispersed cross - peaks (see figure 2a). By studying the decrease in volume of non - overlapping backbone cross - peaks as a function of temperature, we observe that the resonances of residues distributed throughout the protein disappear in a concerted fashion, confirming that the unfolding of the native state is highly and globally cooperative (see figure 4, left column). Nmr resonances shifted upfield of 0.5 ppm in 1d nmr spectra are characteristic of buried methyl groups in folded proteins; we observe that these resonances decrease in intensity with increasing temperature similarly to those of backbone amides, showing at the level of individual residues that the secondary and tertiary structures are lost simultaneously in the unfolding of the native state, in agreement with our previous work (see light blue circles on the left column of figure 4, experimental section, and raw data in figure s6 in the supporting information). (30) the relative decreases in the volume of the nmr signals corresponding to the native state agree very well with the population of this state derived from near - uv cd, showing that both techniques probe the disappearance of the highly cooperative native state (see figure 4, left column). At higher temperatures, hn hsqc cross - peaks collapse into the central region of the h spectrum with little chemical shift dispersion as expected for a denatured protein (see figure 2c). Two sets of nmr signals, corresponding to folded and denatured species, are observed at temperatures near the denaturation midpoints identified by cd (see figure 2b). However, only a subset of the resonances from the denatured state is present in the nmr spectra recorded under these conditions, but additional resonances appear gradually as the temperature is increased further (see figures 4, right column, and figure 5). This behavior is exemplified by i56 t (see figure 5a): at 30 c we observe only 26 backbone amide cross - peaks out of 130 residues . As discussed further below, this temperature dependence is similar to that observed for the low ph molten globular state of -lactalbumin, where cross - peaks appear as the temperature is increased. (34) unfolding of the denatured ensemble of human lysozyme monitored by nmr spectroscopy . (a) hn hsqc spectra of the i56 t mutant recorded at different temperatures show the gradual appearance of the peaks from different residues . The lowest contour level was adjusted so as not to display the weak signals of the native state at 30 c, which has a population of ca . . The lowest contour level, the number of contours as well as the increments between them are constant across all temperatures . Changing the lowest contour level displayed does not affect the results qualitatively and only slightly affects the temperature at which a peak first appears; moreover, it affects all residues in a similar way, enabling robust comparisons to be made . (b) region of the hn hsqc spectra (boxed in panel a) containing all resonances from glycine residues in the denatured ensemble . Resonances from the locally folded and unfolded residues are shown in black and red, respectively . The temperatures of disappearance of resonances from the native state, as well as appearance of resonances from the denatured ensemble, are summarized for all three variants in figure s7 in the supporting information . The build - up of cross - peak intensity from the denatured state is markedly non - concerted, especially in the case of the i56 t and i59 t variants (see figure 4, right column). Moreover, the range of temperatures over which nmr cross - peaks from the denatured state appear is closely similar to the range defined by the population profiles of denatured conformations without native tertiary interactions (dashed line) and without secondary structure (full line) determined by cd spectroscopy . This result indicates that nmr is able to distinguish between classes of residues with different unfolding behaviors, ranging from those that are fully unfolded at low temperatures where native tertiary structure is initially lost, to those that become fully unfolded only upon complete denaturation of the secondary structure elements . Residue specific assignments of the denatured state provide structural insights into the locally cooperative unfolding process (see experimental section). To make direct comparisons between the unfolding behavior of the different variants, we focus on a well - resolved region of the spectrum that has been completely assigned in both the folded and denatured states for all three variants . Human lysozyme has eleven glycine residues and, as they are distributed throughout the native fold, these residues act as very valuable probes of the structural properties of different regions of sequence (see figure 1). Glycine resonances cluster in an isolated and well - dispersed region of the hn hsqc spectrum of non - native lysozyme (see squared region in figure 5a) thus enabling us to determine accurately the relative increases in cross - peak volumes of the denatured ensemble as the temperature is increased . As shown in figure 4, the relative volumes of the glycine cross - peaks of non - native lysozyme vary significantly over a wide range of temperatures, showing that the unfolding of non - native states follows a non - cooperative process . Calculation of the expected water exchange rates of amide protons of the unfolded form of human lysozyme (http://hx2.med.upenn.edu/) reveals that the exchange process is very slow and that the magnitudes of the rates do not correlate with the temperature of the appearance of the corresponding peaks in the nmr spectrum, therefore ruling out the possibility that line broadening is due to exchange with water (see figure s8 in the supporting information). (35) expanded plots of the glycine region of the hsqc spectra show the gradual appearance of resonances from the non - native states of the protein as the temperature is increased (see figure 5b). Importantly, the temperature range over which glycine cross - peaks of non - native states appear is observed to differ between wt, i56 t, and i59 t; the range increases as the stability of the native state decreases . As can be seen from figure 5b, this range is around 5 c for wt, 10 c for i59 t, and 27.5 c for i56 t, directly reflecting the increasing degree of global non - cooperativity in the phase transition that accompanies the thermal unfolding of the three variants of lysozyme (see figure 4). The partial assignment obtained for i56 t enables us to identify which parts of lysozyme unfold first . Figure 6 maps the differential and gradual appearance of resonances of locally unfolded states onto the native protein structure . The results clearly show that the -hairpin is the first structural element to unfold, followed by the rest of the -domain, helix c, both 310 helices 1 and 2, and some residues at the chain termini . The rest of the protein, which comprises helices a, b, d and 310 helix 3, unfolds at higher temperatures, although residues located in loops between secondary structure elements tend to unfold at lower temperatures than the elements themselves . For all three variants, the glycine residues that unfold at the lowest temperatures are g48, g55, g72, g68, and g105, all of which are located in the -domain or in 310 helix 2 (see figure 5b) the results suggest that the unfolding process of the denatured ensemble is locally cooperative and structurally similar for all three variants, with the -domain, helix c, both 310 helices 1 and 2, and some residues of the n- and c - termini unfolding first . However, the temperature window over which unfolding takes place, which indicates the degree of global non - cooperativity of the unfolding of the denatured ensemble, is modulated by the single - point mutations of the variants . (a) structural location of individual residues assigned in the hn hsqc spectrum of denatured lysozyme . The color code indicates the temperature of the first appearance of the corresponding cross - peaks in the hn hsqc spectra of the denatured state of i56 t, according to figure 5 . The temperature evolution of chemical shifts, as provided by the temperature series of hsqcs, provides additional information on the denatured ensemble (see figure 7a). The temperature coefficients of amide protons, defined as the slope of a linear fit of h amide chemical shift versus temperature, vary across glycine residues but do not correlate with the temperature at which they appear nor with the protein sequence (see figure 7b). Values more positive than 4.5 ppb / k have been attributed to the existence of intramolecular hydrogen bonds . This is the case for three glycines (g16, g37, and g105), located in the -domain of the protein, whereas the remaining glycines, located in both the - and -domains, have values that are more negative than 4.5 ppb / k . These results suggest that part of the -domain could possess some residual structure at high temperature . However, the interpretation of amide temperature coefficients is not straightforward, especially in the case of chemically exchanging systems like lysozyme at low ph. (39) temperature evolution of h and n chemical shifts of the denatured ensemble . (a) the glycine - rich region of the hsqc spectrum of the denatured ensembles of human lysozyme variants . In each case the top spectrum, in red, corresponds to a temperature of 50 c; spectra were recorded every 2.5 c and are all shown with the same contour levels as in figure 5 . (b) temperature coefficients of h amide chemical shifts of glycine residues of the denatured ensemble . The temperature coefficients are determined as the slope of a linear fit of chemical shifts versus temperature . Results are shown for the wt (black), the i59 t (red), and the i56 t (green) variants . (c) plot of residuals of h and n chemical shifts, after subtraction of the best first - order fit . The p - value shown in the inset of each graph relates to the comparison of fits to first- and second - order polynomial functions . Only residues for which one chemical shift (h or n) has p <0.05 are shown . The temperature evolution of glycine h and n chemical shifts were also analyzed in terms of their deviation from linearity, which reports on possible conformational heterogeneity . For all three variants, we used a p - value analysis to compare the fits to first- and second - order polynomial functions: we observe that nearly all glycine residues show a linear evolution of their h and n chemical shifts with temperature, i.e., do not undergo any important structural changes in the denatured ensemble after becoming visible in the hsqc spectrum (see residuals in figure 7c). The exceptions are g48 in the i56 t and i59 t variants, and g72 in the i56 t variant, where both residues are located in the -domain of the protein: the p - values for g72 in i56 t, and g48 in i59 t, are significantly larger than those for g48 in i56 t, showing that the extent of conformational exchange is most significant for g48 in i56 t . Moreover, deviation from linearity for g48 is larger in i56 t than in i59 t, and non - existent in the wt protein, suggesting that the extent of conformational heterogeneity experienced by g48 within the denatured state correlates with the decrease in native state stability (a similar conclusion holds for g72 for which deviation from linearity is only observed for i56 t). We should note here that the deviation from linearity is minimal and only becomes observable when performing a statistical test: therefore, although the conformational heterogeneity probed by this analysis is statistically significant, it is of rather small magnitude . We attribute this conformational heterogeneity to slight differences in chemical shift, and therefore structure, of g48 and g72 between the denatured states at lower and higher temperature and is most pronounced for g48 in the i56 t variant, as this residue unfolds at lower temperatures . Overall, this analysis shows that, once resonances corresponding to the denatured ensemble appear in the nmr spectrum, their chemical shift temperature evolution is not indicative of major conformational rearrangements . In this report, we have provided a detailed characterization of the energy landscape of monomeric human lysozyme at low ph with the aim of providing key structural insights into the nature of the amyloidogenic species populated under equilibrium conditions . We present here a comparative study of the complete thermal unfolding transition of the three variants i56 t, i59 t, and wt under relatively modest temperatures and in the absence of denaturants . Using low ph conditions has allowed us to manipulate the folding energy landscape by greatly destabilizing the native state and, for the first time for human lysozyme, to observe directly non - native states that are populated in amyloidogenic conditions . The present investigation is therefore distinct from previous studies based on h / d exchange techniques that indirectly detect partially folded species that are transiently populated as a result of fluctuations from the native state . Using a combination of dsc, cd, and nmr spectroscopy, we show that human lysozyme unfolds in a relatively complex process that involves a cooperative transition from the native structure into a denatured ensemble that unfolds progressively with increasing temperature; the cooperative and first - order transition detected by near - uv cd and dsc corresponds essentially exclusively to the loss of tertiary structure . In the denatured ensemble, tertiary contacts are disrupted and secondary structure elements unfold independently, but in a locally cooperative manner . At lower temperatures, the properties of the denatured ensemble are characteristic of a molten globule: it does not give rise to detectable nmr resonances for most of its residues, lacks tertiary contacts as shown by near - uv cd, and retains most of the secondary structure of the native state as seen by far - uv cd . In addition, we observe that the nmr resonances characteristic of fully unfolded conformations do not appear simultaneously at a given temperature but rather become evident gradually (see figures 4 and 5). These observations are consistent with a model in which unfolding proceeds through a continuum of states that are relatively close in energy and separated by marginal barriers, such that they interconvert relatively rapidly (see figure 3c). In agreement with this model, the increase in cross - peak volumes of denatured resonances takes place at higher temperatures than the near - uv cd transition (green dashed lines in figure 4). This behavior is consistent with the observation that no heat absorption peak is detected by dsc; hence, unfolding of the molten globule is not associated with any our observations are in agreement with previous reports on the thermal unfolding of hen egg white lysozyme(43) as well as with numerous previous calorimetric and nmr studies of -lactalbumins, proteins that are homologous to lysozymes; however, whereas -lactalbumin populates essentially 100% of a molten - globular species at 20 c and low ph, the energy landscape of human lysozyme at low ph is significantly more complex, as its native state is more stable and is therefore highly populated at low temperatures . Therefore, -lactalbumins seem to behave at low ph as extremely destabilized mutants of human lysozyme, a phenomenon that can be attributed to the loss of a stabilizing ca ion in the former group of proteins at low ph. (44) a major advance in our study has been to record nmr spectra describing the whole of the unfolding transition, yielding, in particular, a structural description of the states populated in the course of the locally cooperative unfolding of the denatured ensemble . Residue - specific information from nmr spectroscopy has enabled us to map the unfolding process of the denatured ensemble onto the native structure, thereby revealing which parts of human lysozyme unfold first and are therefore of lower conformational stability . Within the denatured state, each secondary structure element unfolds individually in a continuous transition, resulting in the formation of a wide ensemble of partially folded conformations . This unfolding process is structurally similar for all three variants studied here, with the chain termini, the -domain, helix c, and 310 helix 2 from the -domain unfolding at temperatures lower than the rest of the protein (comprising helices a, b, and d and 310 helix 3). Within the -domain, the i56 t variant shows that the -hairpin is the first structural element to unfold . The regions of the protein that unfold first in the denatured ensemble are the ones that are transiently unfolded under native conditions at higher ph (ph 5.0, 37 c), as detected indirectly by h / d exchange techniques: both observations are therefore in agreement and suggest that these regions are of intrinsically lower stability than the remainder of the protein structure . The temperature evolution of amide chemical shifts of the denatured ensemble suggests the presence of some intramolecular hydrogen bonding, and therefore a modest degree of residual structure in the -domain . Once structural elements of the denatured ensemble unfold, however, there is little conformational rearrangement within this locally unfolded conformation, as indicated by the fact that the chemical shift temperature evolution of most glycine residues is essentially linear . Interestingly, both the -domain and helix c have been found to form the core of human lysozyme fibrils prepared at ph 2.0. (31) as we demonstrate here that the -domain and helix c have marginal individual stability, being the first regions of the protein to unfold once the tertiary structure is disrupted, this unfolding process is likely to be at the origin of the formation of amyloid fibrils at low ph . The single - point mutations i59 t and i56 t not only affect the stability of the native state of the protein, as assessed by near - uv cd, therefore influencing the temperature at which the denatured ensemble starts to unfold, but also determine the extent of non - cooperativity in the unfolding of the denatured ensemble, as measured by the temperature window over which structural elements of the denatured ensemble progressively unfold . The more destabilized the native state, the lower the temperature at which the denatured ensemble starts to be populated, and the more pronounced its non - cooperative unfolding . Interestingly, both the native state destabilization and the extent to which cooperativity is lost within the denatured ensemble correlate with the amyloidogenicity of the variant in other solution conditions (ph 5.0), as well as with the expression levels of lysozyme in pichia pastoris. (29) although the results of this study do not directly report on the structural properties of the amyloidogenic state of lysozyme under physiological conditions, they strongly indicate that the propensity of human lysozyme to aggregate is closely related to the shape of the energy landscape of the monomeric protein,(25) that has been probed in the present study . The i56 t and i59 t variants and wt human lysozyme were expressed in aspergillus niger (i56 t) or pichia pastoris (i59 t, wt) and purified as described previously . All experiments were carried out with the protein samples in 50 mm phosphate buffer adjusted to ph 1.2 with hcl . Circular dichroism experiments were carried out using a jasco j-810 spectropolarimeter equipped with a peltier holder . Spectra were recorded using cells of 0.1 and 1 cm path length for the far- and near - uv, respectively, with a protein concentration of ca . Heat - induced unfolding transitions were monitored from 5 to 90 c at a rate of 0.5 c / min or 1 c / min (the data obtained were independent of the heating rate). Data points were acquired every 0.5 c with a response time of 2 s and a bandwidth of 1 nm . Fitting to a two - state model was carried out after normalization of the unfolding transition; the three mutants were fitted simultaneously, with common baselines for the native and the denatured states (see equations in the supporting information). Fitting to a three - state model was carried out for each mutant separately, after normalization of both near- and far - uv transitions (see equations in the supporting information). (33) the cp of the first transition was taken as 1.6 kcal / molk for i59 t and wt, and 1.3 kcal / mol.k for i56 t, according to previous studies. (46) because the theoretical cp calculated for the unfolding of the native state to the completely unfolded state is 1.6 kcal / molk, and therefore very close to the measured cp of the first transition, we assume that the cp for the second transition is much smaller than this value and arbitrarily set it at 0.2 kcal / molk for all three variants. (47) distributing the cp equally over the two transitions (for example with cp = 0.8 kcal / mol.k for both transitions) had very little effect on the fitting, validating our approach to the data analysis . Heat capacities were measured as a function of temperature using a vp - dsc microcalorimeter (microcal llc, northampton, ma), with protein concentrations of ca . 40 mol / l and a scanning rate of 1 c / min, from 10 to 70 c . The baseline obtained by heating a buffer solution was subtracted from the protein unfolding thermogram, and the excess heat capacity (taking the native state as reference) was obtained by subtracting a progressive baseline between the native and unfolded states . The reversibility of the transition was assessed by overlaying the first and second thermal unfolding curves, the latter being obtained by reheating the sample after cooling inside the calorimetric cell . The reversibility was greater than 90% for all variants (see figure s1 in the supporting information). Vant hoff to calorimetric enthalpy ratios were close to unity for the i59 t and wt variants (it was not possible to estimate it for i56 t because of the lack of native state baseline at low temperatures), and data could only be fitted to a two - state model (see equations in the supporting information). N ammonium sulfate and c methanol were used to label the protein produced in pichia pastoris with n and c isotopes, respectively . Fast hsqc schemes for hn 2d spectra were used for monitoring the unfolding process, and spectra were acquired from 5 to 55 c, every 2.5 c.(48) the backbone resonances of the folded state of the i59 t variant were assigned at 25 c using hnca, hncacb, and cbca(co)nh experiments. (49) conventional nmr characterization of the non - native ensemble of lysozyme, that requires several days of measurements at high temperature and low ph (ph 1.2), has previously been limited by the degradation of the sample . Using a procedure based on a 3d version of the n zz - exchange experiments, however, enabled data collection to be achieved in 12 days: the denatured state of the i59 t variant was partially assigned (ca . 82%), by observation of the transfer of magnetization at 35 c between cross - peaks arising from native and non - native states . Some resonances of the i56 t variant could be correlated with those of the i59 t variant by overlaying the spectra of the denatured states, resulting in the assignment of ca . Peak volumes of hsqc cross - peaks were determined assuming gaussian lineshapes, and overlapping peaks were discarded . In figure 4, the traces of the increase in peak volumes for the wt are slightly to the left of the green dashed line, because normalization was carried out relative to a temperature of 55 c, where the far - uv for wt has achieved only 70% of its transition.
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Hepatitis c virus (hcv) is a global healthcare problem, with a prevalence of around 3% worldwide (1); it has six major genotypes (types 1 6). Most infected patients will establish chronicity with long - term complications, including liver fibrosis, cirrhosis, and hepatocellular carcinoma (hcc) (2). The success rates of current treatments with interferon (ifn)-based therapy are highly variable and depend on both host and viral factors (3). Compared to other developing countries, egypt has a high prevalence of hcv, where 20% of egyptian blood donors are seropositive for hcv antibodies (1). Although hcv-4 causes about 20% of chronic hepatitis c in the world, it has not been subjected to enough research, most probably due to its restricted localization to the middle east and africa, where the management strategies for infected patients are not as well developed as for genotype-1, -2, and -3 (4, 5). Micrornas are endogenous small non - coding rnas (22 nucleotides) involved in the regulation of many cellular processes (6). Several host mirnas have been suggested to be involved in hcv entry, the establishment of viral infection, and the multi - step process of chronic hcv infection . The ability of the virus to take over certain cellular mirnas and its persistence are not fully understood (7). The liver - specific mirna, mir-122, positively modulates hcv infection through direct interaction with the 5 utr of the hcv genome and stimulation of hcv translation (8). Remarkably, reagents that can downregulate mir-122 have entered clinical development for hcv treatment (10). Other mirnas have also been reported to physically interact with the hcv genome and attenuate viral replication, namely let-7b, mir-196, mir-199, and mir-448 (11). In hcv - infected patients, lower expression levels of mir-29 have been observed in liver, while its overexpression inhibits viral rna replication in hcv - infected hepatocytes (12). Mir-130a expression has been up - regulated in liver biopsies from hcv - infected patients, as well as in hcv - infected hepatocytes (13). On hcv infection, the virus can modulate the function of certain cellular mirnas involved in ifn production and the innate antiviral immune response, thereby exacerbating the infection (14). In addition, a number of reports have highlighted the importance of host cellular mirnas in modulating responsiveness to exogenous ifn treatment (15). Associated inflammation, fibrosis, and cirrhosis, as well as the initiation and progression of liver cancer (16, 17). In this study, we attempted to identify special characteristics of the egyptian hcv patients of predominantly genotype 4 by performing a selected 94 mirna profile in 50 chronically hcv - infected patients . The correlation between individual mirna expression profiles and clinicopathological variables was assessed for the sake of identifying potential biomarkers related to hcv-4 infection . Fifty patients diagnosed with chronic hcv infection between 2011 and 2012 were enrolled in this study . . The study was reviewed and approved by the cairo university hospital research ethics committee (rec), school of medicine, cairo university . Tissues were divided into two parts; one was used for histopathological examination, and the other was stored in rnalater (qiagen, hilden, germany) at -80c until use . Liver biopsies obtained from normal liver transplantation donors were used as controls . For each biopsy sample, liver fibrosis and activity were scored according to the metavir classification system (18). Patients were considered hcv positive if their serum tested positive in an enzyme - linked immunosorbent assay (elisa; detect - hcv 3, adaltis, milano, italy) for hcv antibodies . All enrolled patients were hcv - rna positive and the viral load was determined using quantitative real - time reverse transcription polymerase chain reaction (qrt - pcr) assay (artus hcv rg rt - pcr, qiagen). Patients who were co - infected with hbv or human immunodeficiency virus (hiv) and/or who had liver cirrhosis were excluded from this study . Liver function tests were conducted to determine patients levels of aspartate aminotransferase (ast), alanine aminotransferase (alt), total bilirubin, albumin, alkaline phosphatase (randox, randox laboratories ltd ., bilharzial antibody titer was determined to assess the bilharzial status of the patients (novatec immundiagnostica gmbh, germany). Hcv genotyping was assessed using the gen - c reverse hybridization strip assay kit (nlm diagnostic, italy). Samples were first homogenized in qiazol lysis reagent, then rna was purified using mirneasy mini kit (qiagen) following the manufacturer s instructions . One microgram of rna was used to prepare cdna using miscript reverse transcription kit (qiagen) according to the supplied protocol . Rna concentrations were quantified using a nanodrop spectrophotometer (nanodrop technologies, wilmington, de, usa). The selected mirnas for this study were previously shown to be associated with liver health and chronic liver diseases . Pcr reaction containing 1x sybr green master mix (qiagen), 200 nm of mirna specific forward primer, and 200 nm of universal primers were performed in 96-well plates using 3 ng in 2.5 l cdna / well . The total reaction volume was 10 l / well with initial denaturation at 95c followed by 40 cycles at 94c for 15 seconds, 55c for 30 seconds, and 70c for 30 seconds . The qrt - pcr experiments were performed on an abi 7500 (applied biosystems, foster city, ca, usa). The applied biosystems 7500 software was used to export the ct values of different mirnas to the microsoft excel program . The relative expression of mirnas was normalized using the mean expression value of all examined mirnas in each sample as a normalization factor, following mestdagh et al . All ct values above or equal to 35 were removed before calculating the mean of the remaining ct values . The mean ct value was calculated for each sample according to the equation 1: the ct and fold changes were calculated according to the equation 2 (fold change = 2): heat - map and two - way clustering analysis were performed with a log2 fold of change using gene - e software (broad institute, inc . ). The chi - square test was used to test the observed distribution of mirna as up- or down - regulated to an expected distribution . The non - parametric mann whitney and kruskal wallis tests were used to analyze differences in mirna values according to categorical clinicopathological features . Fifty patients diagnosed with chronic hcv infection between 2011 and 2012 were enrolled in this study . . The study was reviewed and approved by the cairo university hospital research ethics committee (rec), school of medicine, cairo university . Tissues were divided into two parts; one was used for histopathological examination, and the other was stored in rnalater (qiagen, hilden, germany) at -80c until use . Liver biopsies obtained from normal liver transplantation donors were used as controls . For each biopsy sample, liver fibrosis and activity were scored according to the metavir classification system (18). Patients were considered hcv positive if their serum tested positive in an enzyme - linked immunosorbent assay (elisa; detect - hcv 3, adaltis, milano, italy) for hcv antibodies . All enrolled patients were hcv - rna positive and the viral load was determined using quantitative real - time reverse transcription polymerase chain reaction (qrt - pcr) assay (artus hcv rg rt - pcr, qiagen). Patients who were co - infected with hbv or human immunodeficiency virus (hiv) and/or who had liver cirrhosis were excluded from this study . Liver function tests were conducted to determine patients levels of aspartate aminotransferase (ast), alanine aminotransferase (alt), total bilirubin, albumin, alkaline phosphatase (randox, randox laboratories ltd ., bilharzial antibody titer was determined to assess the bilharzial status of the patients (novatec immundiagnostica gmbh, germany). Hcv genotyping was assessed using the gen - c reverse hybridization strip assay kit (nlm diagnostic, italy). Samples were first homogenized in qiazol lysis reagent, then rna was purified using mirneasy mini kit (qiagen) following the manufacturer s instructions . One microgram of rna was used to prepare cdna using miscript reverse transcription kit (qiagen) according to the supplied protocol . Rna concentrations were quantified using a nanodrop spectrophotometer (nanodrop technologies, wilmington, de, usa). The selected mirnas for this study were previously shown to be associated with liver health and chronic liver diseases . Pcr reaction containing 1x sybr green master mix (qiagen), 200 nm of mirna specific forward primer, and 200 nm of universal primers were performed in 96-well plates using 3 ng in 2.5 l cdna / well . The total reaction volume was 10 l / well with initial denaturation at 95c followed by 40 cycles at 94c for 15 seconds, 55c for 30 seconds, and 70c for 30 seconds . The qrt - pcr experiments were performed on an abi 7500 (applied biosystems, foster city, ca, usa). The applied biosystems 7500 software was used to export the ct values of different mirnas to the microsoft excel program . The relative expression of mirnas was normalized using the mean expression value of all examined mirnas in each sample as a normalization factor, following mestdagh et al . All ct values above or equal to 35 were removed before calculating the mean of the remaining ct values . The mean ct value was calculated for each sample according to the equation 1: the ct and fold changes were calculated according to the equation 2 (fold change = 2): heat - map and two - way clustering analysis were performed with a log2 fold of change using gene - e software (broad institute, inc . ). The chi - square test was used to test the observed distribution of mirna as up- or down - regulated to an expected distribution . The non - parametric mann whitney and kruskal wallis tests were used to analyze differences in mirna values according to categorical clinicopathological features . Liver biopsy and blood samples were collected from 50 chronically infected hcv patients . As expected, 94% of the enrolled subjects in this study were genotype 4, and only 6% were of genotype 1 . Liver biopsy samples revealed that most patients (90%) had mild to moderate fibrosis . Steatosis was mild in 78% and moderate in 12% of the samples (table 1). The log2 fold of change was used to generate expression profile heatmap using gene - e software . Two - way clustering analysis showed an association between the enrolled patients and the expression levels of the studied micrornas (figure 1). Forty - four out of the 94 analyzed mirnas were significantly deregulated; 27 mirnas exhibited at least a 4.0-fold increase, and 17 mirnas exhibited at least a 4.0-fold decrease . To understand the role of the deregulated mirnas in association with hcv infection, their published functions and proposed targets were summarized, as shown in tables 2 and 3 . A number of these mirnas have been shown to be associated with hcv replication, pathogenesis, and associated liver complications . Each column represents a sample and each row shows the fold change of each mirna in comparison to the average of normal samples . The differential regulation is indicated by red for up - regulated genes and blue for the down - regulated ones . Remarkably, our data showed that the liver - specific mirna-122 was strongly down - regulated in the majority of our patients, which contradicts several other previously published papers showing an increase in this mirna correlated with its involvement in hcv infection and replication (20, 21). In contrast, our results were in agreement with spaniel et al . Who found that levels of mir-122 were reduced in japanese patients with chronic hepatitis c (22). Expression levels of seven mirnas (mir-29c, mir-30c, mir-126, mir-145, mir-199a, mir-199a-3p, and mir-222) tended to increase specifically in patients with an increasing grade of inflammatory activity (figure 2). Expression of three other mirnas (mir-95, mir-130a, and mir-142 - 5p) expression was inversely correlated with the serum albumin levels of the enrolled patients . No significant correlations were shown between the expression levels of analyzed mirnas and basic clinical and biochemical characteristics of the studied patients, including age, viral load, serum liver enzymes, afp level, presence of bilharziasis, or steatosis (data not shown). A, mir-23c, p = 0.003; b, mir-30c, p = 0.009; c, mir-145c, p = 0.005; d, mir-199a, p = 0.005 . Five mirnas, namely mir-21 (p = 0.001), mir-23a (p = 0.046), mir-126 (p = 0.045), mir-194 (p = 0.04), and mir-199a-3p (p = 0.02), showed down - regulation in male patients, while only one mirna (mir-638) was significantly down - regulated in female patients (p = 0.009; figure 3a and b). It illustrates the relation between a, the level of expression of mir-199a-3p; and b, mir-638 with gender in the liver tissue of chronic hcv patients . The log2 fold of change was used to generate expression profile heatmap using gene - e software . Two - way clustering analysis showed an association between the enrolled patients and the expression levels of the studied micrornas (figure 1). Forty - four out of the 94 analyzed mirnas were significantly deregulated; 27 mirnas exhibited at least a 4.0-fold increase, and 17 mirnas exhibited at least a 4.0-fold decrease . To understand the role of the deregulated mirnas in association with hcv infection, their published functions and proposed targets were summarized, as shown in tables 2 and 3 . A number of these mirnas have been shown to be associated with hcv replication, pathogenesis, and associated liver complications . Each column represents a sample and each row shows the fold change of each mirna in comparison to the average of normal samples . The differential regulation is indicated by red for up - regulated genes and blue for the down - regulated ones . Remarkably, our data showed that the liver - specific mirna-122 was strongly down - regulated in the majority of our patients, which contradicts several other previously published papers showing an increase in this mirna correlated with its involvement in hcv infection and replication (20, 21). In contrast, our results were in agreement with spaniel et al . Who found that levels of mir-122 were reduced in japanese patients with chronic hepatitis c (22). Expression levels of seven mirnas (mir-29c, mir-30c, mir-126, mir-145, mir-199a, mir-199a-3p, and mir-222) tended to increase specifically in patients with an increasing grade of inflammatory activity (figure 2). Expression of three other mirnas (mir-95, mir-130a, and mir-142 - 5p) expression was inversely correlated with the serum albumin levels of the enrolled patients . No significant correlations were shown between the expression levels of analyzed mirnas and basic clinical and biochemical characteristics of the studied patients, including age, viral load, serum liver enzymes, afp level, presence of bilharziasis, or steatosis (data not shown). A, mir-23c, p = 0.003; b, mir-30c, p = 0.009; c, mir-145c, p = 0.005; d, mir-199a, p = 0.005 . Five mirnas, namely mir-21 (p = 0.001), mir-23a (p = 0.046), mir-126 (p = 0.045), mir-194 (p = 0.04), and mir-199a-3p (p = 0.02), showed down - regulation in male patients, while only one mirna (mir-638) was significantly down - regulated in female patients (p = 0.009; figure 3a and b). It illustrates the relation between a, the level of expression of mir-199a-3p; and b, mir-638 with gender in the liver tissue of chronic hcv patients . Over the last few years, several studies have examined the expression levels of certain mirnas in association with hepatitis c viral infection . Some of the obtained data were provided in the context of hcc (23, 24). In this study, we aimed to identify changes in mirna characteristics of nave egyptian patients with chronic hcv infection . We succeeded in identifying 44 mirnas with at least 4.0-fold change compared to the normal controls . The identified host - altered mirnas may contribute to the chronicity of hcv and mediated pathogenesis . The combined samples clustering with the expression pattern of the studied 94 mirnas could help to cluster the enrolled patients according to the progression of liver chronicity; further investigations are necessary . To develop a better understanding of the role of the deregulated mirnas in association of chronic hcv infection, their suggested functions were manually curated from relevant published literatures (tables 2 and 3). The three liver - specific mirnas analyzed in this study (mir-122, mir-148a, and mir-194) were significantly down - regulated in the majority of the patients . Our results are in agreement with a recent report regarding the reduced level of mir-122 in the tissues of chronic hepatitis c infected patients and chimpanzees . The decrease of mir-122 in our study may have occurred after an initial rise at the beginning of the hcv infection, which was then followed by a decline, since all of our subjects were in the chronic phase of infection . This initial rise of hepatic mir-122 levels followed by a decline was shown to occur in chimpanzees (25). In recent studies, the hepatic mir-122 expression level has been shown to be reduced significantly with the severity of liver fibrosis in patients with chronic hcv infection (26, 27); this might fit with the clinical data of the enrolled patients in this study to some extent . The reduced level of mir-122 was found to be associated with a poor response to inf - based treatment (28). Thus, the selection of patients with chronic hcv infection for effective inf - based therapy may be achieved based on the hepatic expression level mir-122; further investigations on this topic are necessary . Other differentially expressed mirnas in this study have been shown to be associated with liver inflammation to fibrosis and hcc (let-7, mir-21, mir-223 (down - regulated), mir-105, mir147, mir-155, mir-187 (up - regulated) (29). Notably, the down - regulated mir-223 can act as a negative regulator of inflammation during viral infection . Mir-130a, a potential drug target in hcv treatment, was down - regulated in the examined hepatic samples (albeit not below the threshold level we set for this study). It has been reported that mir-130a overexpression can inhibit hcv replication by restoring host innate immune responses and/or down - regulating pro - hcv mir-122 (31). Restoring the expression level of mir-130a in infected patients may be a useful strategy to combat hcv infection . We identified seven mirnas (mir-29c, mir-30c, mir-126, mir-145, mir-199a, mir-199a-3p, and mir-222) that were significantly increased with the inflammation grade of the liver (figure 2). Consistent with our data, it has been reported that members of mir-29 and mir-199 families correlate with the stage of liver fibrosis in hcv patients (32, 33). Identification of possible markers for the inflammation stage can be helpful in monitoring the disease progression and in avoiding the invasive biopsies used in these assessments . Liver fibrosis is the most common final path of chronic liver diseases; this is influenced by the nuclear factor - kappa b (nf-b) signaling pathway (34). Mir-155, which was up - regulated in the present study, has been shown to have a negative effect on regulation of the nf-b pathway through targeting different proteins, including myeloid differentiation primary response gene 88 (myd88). Up - regulation of mir-155 was also reported in hcv - infected cells, leading to repression of the nf-b signaling pathway (35). Meanwhile, mir-16, mir-199a, and mir-223, which were down - regulated in the present study, can regulate the nf-b pathway by targeting key signaling protein genes (36, 37). It was shown previously that c - rel (a member of the nf-b family) was able to negatively regulate mir-1228 by binding directly to its promoter site (38). In the present study, extremely high expression of mir-1228 - 5p it would be interesting to study the effect of increased mir-1228 on the nf-b pathway in infected cells . Our results showed that a distinctive subset of mirnas up - regulated simultaneously in some patients (mir-31, mir-105b, mir-147, mir-149 - 3p, mir-198, mir-302b-5p, and mir-1228 - 5p), in which they exhibited a greater than 200-fold change compared to normal subjects . Further studies are needed to reveal the significance of their expression level and the cellular machinery(ies) governing the mirna - targeted relationship in chronically infected hcv patients . It is interesting to note that a number of the deregulated mirnas investigated in this study and their target genes, which are associated with advanced fibrosis or cirrhosis, play a critical role in the initiation and progression of hcc (tables 2 and 3). In addition, three mirnas, namely mirna-18a, mir-18b, and mir-21, can promote cell proliferation and invasion and contribute to evasion of the host immune system . The role of several up - regulated mirnas in association with chronic hcv infection is not yet fully understood (table 3). In conclusion, many of the dysregulated mirnas in this study are associated with chronic liver disease and subsequent complications . This may reflect the potential mechanism(s) of persistent hcv infection to drive normal hepatocytes to malignancy via changing expression of several mirnas, as a layer of gene expression control, in the host cell . Moreover, the identified expression profiles of some examined mirnas might offer important points to consider for the treatment of nave patients and management of chronically infected hcv patients in egypt and around the world.
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Over the last three decades, candida species has emerged as an important cause of health care associated and opportunistic infections . The increased use of intravenous catheters, total parenteral nutrition, broad spectrum antibiotics, and cytotoxic chemotherapy and an increase in the population of immunocompromised patients have contributed to the increase of these infections . Expression of virulence factors like germ tube formation, adhesins, phenotypic switching, thigmotropism, and biofilm formation and the production of hydrolytic enzymes contribute to the pathogenesis of candidiasis . Although most infections are attributed to c. albicans, the shift towards treatment resistant non - albicans candida (nac) species is evident in recent years [4, 5]. The problem of emergence of nac spp . Becomes more acute because different species of nac exhibit varying degrees of resistance, either intrinsic or acquired or both, to commonly used antifungal drugs . Isolated from various clinical types of candidiasis . In india, c. tropicalis is the most common cause of health care associated candidemia . The increased isolation of c. tropicalis from various clinical types of candidiasis is of concern because of its ability to develop rapid resistance to fluconazole . Among candida spp ., expression of virulence factors may vary depending on the infecting species, geographical origin, type of infection, the site and stage of infection, and host reaction . Knowledge of these virulence factors will be an important tool to understand pathogenesis of candidiasis and in addition will help explore new antifungal drug targets for improved therapeutic regimens . A review of the available literature has revealed a dearth of information regarding the epidemiology, pathogenesis, virulence factors, and antifungal susceptibility patterns of c. tropicalis . Therefore the present study was taken up with an aim to study the virulence factors and antifungal susceptibility profile of c. tropicalis isolated from various clinical specimens . The present study was conducted in the department of microbiology, rural medical college and hospital of pravara institute of medical sciences, loni, maharashtra, and is part of a phd thesis . The protocol of the study was approved by the institutional ethics committee (registration no . Pims / phd / rc/2013/24). A total of 125 c. tropicalis isolated from various clinical samples were included in the study . C. tropicalis was identified by hicandida identification kit and colony color on hichrome candida agar (himedia laboratories pvt . Ltd ., mumbai, india). The virulence factors studied were exoenzymatic activity (coagulase, phospholipase, and proteinase), biofilm formation, and haemolysin production . Coagulase production by c. tropicalis was detected by the method of yigit et al . . Approximately 0.1 ml of an overnight culture of c. tropicalis was aseptically inoculated into a tube containing 500 l of rabbit plasma . The tubes were incubated at 35c and observed for clot formation after 2, 4, 6, and 24 h. the presence of a clot that could not be resuspended by gentle shaking indicated positive coagulase test . Staphylococcus aureus atcc 25923 and s. epidermidis atcc 14990 were used as positive and negative controls, respectively . The phospholipase activity of c. tropicalis was detected by the method of samaranayake et al . . Approximately 5 l of standard inoculum of test strain containing 10 candida cells / ml was aseptically inoculated onto egg yolk agar . The plates were dried at room temperature and then incubated at 37c for 48 h. the plates were examined for the presence of precipitation zone around the colony . The presence of precipitation zone indicated expression of phospholipase enzyme . C. albicans atcc 10231 was used as positive control . The phospholipase index (pz) was defined as the ratio of the diameter of the colony to the total diameter of the colony plus the precipitation zone . A pz value of 1 denoted no phospholipase activity; pz <1 indicated phospholipase production by the isolate . To minimize experimental error, the assay was conducted in duplicate on three separate occasions for each isolate . Approximately 10 l of standard inoculum (10 candida cells / ml) was aseptically inoculated on to 1% bsa plate . The plate was incubated for 5 days at 37c . C. albicans atcc 10231 was used as positive control . After incubation, further proteinase activity was inhibited by adding 20% trichloroacetic acid and the plate was stained with 1.25% amidoblack . A zone of proteolysis surrounding the colony that could not be stained with amidoblack indicated proteinase activity . The proteinase index (prz) was measured in terms of the ratio of the colony to the diameter of unstained zone . A prz value of 1 indicated no proteinase activity; prz <1 denoted proteinase expression by the isolate . The lower the prz value, the higher the proteinase activity . To minimize experimental error, haemolytic activity of c. tropicalis was screened on sheep blood sabouraud dextrose agar plate by the method described by manns et al . . Approximately 10 l of standard inoculum (10 candida cells / ml) was aseptically inoculated onto the medium . The culture plates were incubated at 37c for 48 h. c. albicans atcc 90028 was used as the control strain . Streptococcus pyogenes (lancefield group a) and streptococcus sanguis were used as positive controls for beta and alpha haemolysis, respectively . Haemolytic activity (hz) was calculated in terms of the ratio of diameter of the colony to that of the translucent zone of haemolysis (in mm). The ability of c. tropicalis isolates to form biofilms was assessed by the tube method described by yigit et al . . Colonies of c. tropicalis from sabouraud dextrose agar were inoculated in saline and incubated overnight at 37c . 0.5 ml of this saline suspension was added into screw capped conical polystyrene tubes containing 5 ml of sabouraud dextrose broth supplemented with glucose (final concentration of 8%). Presence of visible adherent film on the wall and at the bottom of the tube indicated biofilm formation . Ring formation at the liquid interface was not considered as an indication of biofilm production . Staphylococcus epidermidis atcc 35984 and c. albicans atcc 10231 were used as positive and negative controls, respectively . The antifungal susceptibility testing of c. tropicalis isolates was performed using hicomb minimum inhibitory concentration (mic) test (himedia laboratories pvt . The antifungal agents tested were amphotericin b (range 0.00232 g), fluconazole (range 0.016256 g), itraconazole (range 0.00232 g), and ketoconazole (range 0.00232 g). The inoculum was prepared by inoculating 3 - 4 colonies of the c. tropicalis isolate to be tested in saline . The suspension was inoculated on the agar plate containing rpmi 1640 supplemented with 2% glucose by lawn culture method using tipped cotton swab . The antifungal strips were aseptically placed on the media with the help of forceps and the plates were incubated at 35c for 2448 h. c. albicans atcc 90028 and c. parapsilosis atcc 22019 were used for quality control . The results of antifungal susceptibility test were interpreted as sensitive (s), dose - dependent susceptible (dds), and resistant (r). Interpretative criteria for azoles were those recommended by the clinical laboratory standard institute (clsi) [14, 15]. Due to the lack of defined breakpoints for amphotericin b arbitrary values based on the studies of other researchers majority of the isolates were obtained from urine samples (38.4%) followed by vaginal swabs (28.8%). Indwelling urinary catheters, use of antibiotics, geriatric patients, and diabetes mellitus were risk factors found associated with c. tropicalis uti . Pregnancy, uncontrolled diabetes, and use of low dosage azole maintenance regimen were predisposing factors for c. tropicalis vulvovaginitis . Icu stay, total parenteral nutrition (tpn), prior exposure to fluconazole, and diabetes mellitus were the major risk factors for candidemia . Coagulase production was noted in 18 (14.4%) isolates . Of these, only 2 isolates showed coagulase expression within 4 h of incubation . Maximum isolates demonstrated resistance to fluconazole (71.2%) followed by ketoconazole (68%). Fluconazole resistance was more common in isolates obtained from cases of candidemia, opc, and vulvovaginal candidiasis . Miscellaneous samples included foley's catheter tips, ear swab, endotracheal tube, and pleural fluid . The increased frequency of candida infections to a certain extent coincides with advances in the field of medicine . Once dismissed or ignored as nonpathogenic, commensal, or contaminant have emerged as potential pathogens . Among nac spp . C. tropicalis alone, or in association with other species, is implicated more frequently in human infections . In this study, c. tropicalis was most commonly isolated from urine samples . Paul et al . Reported c. tropicalis as the most prevalent nac spp . Causing candiduria . In the study by jain et al ., c. tropicalis was the predominant cause of candiduria in catheterized icu patients . The major risk factors for candiduria included indwelling catheters, recent use of antibiotics, advanced age, and diabetes mellitus . Use of broad spectrum antibiotics helps in colonization by candida by suppressing normal bacterial flora of gut and lower genital tract . Diabetes not only impairs host immunity but also increases candida colonization by promoting stasis of urine in neurogenic bladder . In recent years, many studies have shown an increased prevalence of vvc due to nac spp . In our study, pregnancy, uncontrolled diabetes, and use of low dose azole maintenance regimen were major predisposing factors for c. tropicalis vulvovaginitis . Widespread and inappropriate use of antifungal therapy in the form of self - medication, long term maintenance dosage, and use of a single dose oral and topical azole results in eradication of c. albicans and selection of nac spp . That are resistant to commonly used antifungal drugs . Opc occurs in up to 90% of hiv infected individuals during the course of infection . In recent years like c. tropicalis, c. glabrata, and c. krusei have been increasingly recovered from hiv patients with opc . Predominance of c. tropicalis among nac spp . As a causative agent of opc in hiv infected individuals was also noted in studies by other researchers [16, 25]. The increased isolation rates of nac spp . From blood stream infections along with a gradual shift in the antifungal susceptibility profile are documented in many recent studies . In our study icu stay, tpn, prior exposure to fluconazole, and diabetes mellitus were risk factors identified to be associated with c. tropicalis candidemia . The increased use of fluconazole is considered a major cause for increase of c. tropicalis candidemia . Studies of various researchers from different parts of india have reported c. tropicalis to be the most prevalent nac spp . . Extensive research on these virulence factors is focused on c. albicans, which is considered the most pathogenic member of the genus . However, quite a few research articles refer to virulence factor production in nac spp . In the present study, biofilm formation is of utmost importance as these organisms not only colonize medical devices, but also lead to resistant health care associated infections . These enzymes facilitate adaptation to distinct types of infection and enhance survival of the pathogen . Most of the studies on exoenzymes are focused on phospholipases and secreted aspartyl proteinases (sap). Coagulase binds plasma fibrinogen and activates a cascade of reactions that induce clotting of plasma . In our study coagulase production rodrigues et al . Reported high coagulase activity in c. tropicalis (82.6%). Haemolysin secretion followed by iron acquisition facilitates deeper tissue invasion by candida . In the present study, 30.4% of c. tropicalis showed haemolytic activity . Reported high haemolytic activity in c. tropicalis isolated from hiv infected individuals . Among extracellular hydrolases, proteinases and phospholipases play major a role in host tissue invasion, colonization, and progression of infection . Phospholipases facilitate the invasion of the host mucosal epithelia by hydrolyzing one or more ester linkages in glycerophospholipids . In our study investigators like thangam et al . Reported high phospholipase activity in c. tropicalis isolates among nac spp . Screening of phospholipase production in biofilm forming isolates can be used as an important parameter to differentiate invasive strains from noninvasive colonizers . Proteinases are capable of degrading host epithelial and mucosal barrier proteins such as collagen, keratin, and mucin . They also aid candida to resist cellular and humoral immunity by degrading antibodies, complement, and cytokines . This observation was in agreement with other researchers like deorukhkar and saini, mane et al ., and dost et al . The clsi standardized broth microdilution method is complex and labor intensive to use as a routine method . Alternative methods like disc diffusion and etest have been adapted for sensitivity testing of candida spp . By resource resistance rates for the azole group of antifungal drugs were more as compared to amphotericin b. azole resistance in c. tropicalis is insufficiently investigated . In the present study, c. tropicalis isolates were found to be more resistant to fluconazole . Resistance to fluconazole sanglard and odds described overexpression of cterg11 gene associated missense mutation to be responsible for the acquired azole resistance in c. tropicalis . The increase in the rate of fluconazole resistance in c. tropicalis is of concern because this species is one of the most commonly isolated nac spp . And fluconazole is the most common antifungal agent used for the treatment of various types of candidiasis . C. tropicalis capable of exhibiting certain virulence factors like biofilm formation and phospholipase production had higher rates of resistance to fluconazole . As compared to bacterial biofilms, candida biofilms are resistant to many antimicrobial agents; the removal and replacement of infected medical device are required for effective treatment . Increased incidence of systemic candidiasis along with antifungal resistance has become an important healthcare issue worldwide . C. tropicalis exhibit a great degree of variation not only in their pathogenicity but also in their antifungal susceptibility profile . The identification of virulence attributes specific for each species and their correlation with each other will aid in the understanding of the pathogenesis of infection . The importance of early and accurate identification of infecting candida species along with susceptibility testing for timely institution of appropriate therapy cannot be overstated.
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Enlargement of upper - body adipose tissue depots, either visceral or truncal subcutaneous depots, in obese individuals is a risk factor for metabolic dysfunction in diabetes, obesity and atherosclerosis whereas lower - body adiposity appears protective . Although men tend to store lipid more in the visceral depot and women in the lower - body, both men and women demonstrate marked variability regarding regional fat distribution . Epidemiological studies indicate that a suboptimal intrauterine environment including poor maternal nutrition, predisposes to diabetes, visceral obesity and metabolic syndrome suggesting that adiposity levels and fat distribution phenotype could be programmed prenatally . Current research efforts are focused on elucidating the alterations in adipose tissue development, hormone levels, and epigenome (reviewed in) that may lead to adult obesity . However, there are limited data on the influence of poor maternal nutrition on the variation in maturation of fetal adipose tissue from different depots that may contribute to the development of specific distinct body fat distribution phenotypes later in life . Adipose tissue mass is a function of adipocyte size (hypertrophy) and number (hyperplasia). Fat cell number depends on the abundance of adipocyte precursor cells and ability of adipocyte progenitor cells (preadipocytes) to form new fat cells through proliferation and adipocyte differentiation (adipogenesis). Studies in mice and pigs show that the formation of the pool of adipocyte precursors through commitment of mesenchymal stem cells to adipocyte lineage and the preadipocyte proliferation starts prenatally within the mural compartment of the adipose tissue vasculature (a progenitor niche), which further governs adipocyte differentiation . Adipogenesis in perirenal adipose tissue during fetal development and early postnatal life in sheep undergoes profound modifications . Adipogenesis starts with a phase of intense proliferative activity of primordial adipose tissue followed by adipocyte differentiation characterized by dominant features of brown adipose tissue (pronounced expression of the specific marker ucp1) in late gestation to meet the increased need for heat production associated with the changes in the temperature from ~40c in the womb to the lower temperature of the extrauterine environment . After birth, there is a gradual transformation of brown to white phenotype of adipogenesis characterized by a complete loss of ucp1 expression to adapt to the new diet containing higher amounts of lipids . Thus, the fetal and early postnatal periods appear to be a critical time for the enrichment of the adipocyte precursor pool and to accomplish functional adjustments . To improve understanding of effects of maternal suboptimal nutrition on adipose tissue function, we examined the impact of moderate maternal nutrient reduction (mnr) on in vitro adipocyte differentiation in adipose - derived stromal - vascular cells (ascs) from omental, subcutaneous abdominal and femoral adipose tissue depots in control normally grown baboon fetuses (ctr) of well - nourished mothers and fetuses of mothers fed 70% global diet of ctr from 30 d pregnancy to term (mnr), a nutrient challenge that leads to intrauterine growth restriction (iugr) and a pre - diabetic phenotype by puberty . We hypothesized that decreased fetal nutrient availability would accelerate the brown - to - white alteration in differentiation of adipose tissue in a fetal sex dependent manner . Baboon (papio species) singleton pregnancies were studied at the southwest national primate research center at the texas biomedical research institute (tbri). Healthy female baboons of similar body weights (1015 kg) were randomly assigned to outdoor group cages and maintained in social groups of 1016 with a vasectomized male . At the end of the acclimation period (30 days) pregnancy was dated initially by timing of ovulation and changes in sex skin color and confirmed at 30 days of gestation (0.16 g; term, ~184 days) by ultrasonography . The model of 30% global maternal nutrient reduction from 30 days of pregnancy to term has been described in detail . The pregnant baboons were fed purina monkey diet 5038 containing protein 15.7%, fat 6% by acid hydrolysis, and glucose 0.29% (the full composition of monkey diet 5038 can be found at http://labdiet.com/pdf/5037-5038.pdf). Mothers were allowed to recover from surgery and returned to their group housing . Paired fetal adipose tissue samples from omental (om), subcutaneous abdominal (sca), and subcutaneous femoral (scf) regions were collected from twenty near - term (165 days gestation (dg)) baboon fetuses . We studied ctr fetuses of ad libitum fed mothers (5 female and 5 males) and fetuses of mothers fed the 70% global diet eaten by ctr (mnr, 5 female and 5 males). All procedures were approved by the tbri and university of texas health science center, san antonio institutional animal care and use committees and studies were conducted in aaalac accredited facilities . Tissues were removed from the fetus under aseptic conditions, placed in hank's buffered salt solution and shipped at room temperature to the pennington biomedical research center . Within ~24 h from collection, adipose tissue was digested enzymatically and adipose derived stromal - vascular cells (ascs) isolated as previously described . Cultures were expanded in 10% fetal bovine serum (fbs) and third passages were frozen until samples from all fetuses were available for batch processing in a single assay . Frozen adipose tissue culture samples were thawed and further expanded in culture plates coated with a soluble extract of engelbreth - holm - swarm tumors (e - c - l; millipore, cat no . Cells were switched to a differentiation cocktail comprised of dmem - f12 (1:1) medium supplemented with 3% fbs, 10 mg / ml transferin, 33 m biotin, 17 m calcium pantothenate, 0.5 m insulin, 0.1 m dexamethasone, 0.2 nm tri - iodo - thyronine, 60 m indomethacin, 1 m roziglitazone, and 0.5 m ibmx (the last three components for the first 3 days only) for 9 days . Rna was isolated and cleaned using rneasy kit (qiagen, valencia, ca). Qrt - pcr was used to determine the relative gene expression levels of fatty acid binding protein 4 (fabp4, hs01086177_m1), adiponectin (hs00605917_m1), peroxisome proliferator - activated receptor gamma (ppar, hs01115513_m1), uncoupling protein 1 (ucp1, hs00222453_m1), and t - box 15 (tbx15, hs00537087_m1) after asc differentiation . Tbp (hs99999910_m1) was used as an internal control . Of note, all the primers were from applied biosystems (life technologies, us). Fifty ng of cdna template per sample was amplified on the abi prism 7900ht by qpcr . The gene expression was determined in relation to the mean value for the subcutaneous abdominal acs using the ct method . At the end of the differentiation protocol, asc cells were harvested in a non - denaturing buffer containing 150 mm nacl, 10 mm tris ph 7.4, 1 mm egta, 1 mm edta, 1% triton - x 100, 0.5% igepal ca-630, 1 m phenylmethylsulfonyl fluoride (pmsf), 1 m pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 m leupeptin, and 2 mm sodium vanadate and frozen . Next, the samples were thawed, needled, and centrifuged at 14,000 g at 4c for 10 minutes . Three males and three females from each ctr and mnr, which has the largest, smallest and the median values of the respective mrna levels were selected for the western blotting . Samples from the three depots of the individual fetuses were pooled and 50 g of the samples were loaded on to the 10% polyacrylamide gel . Protein from brown adipose tissue of mice (30 g) was also loaded as a positive control for ucp1 . Proteins were then transferred to a pvdf membrane and were probed with antibodies that recognize ucp1 [kindly donated by dr . Gettys, pennington biomedical research center, baton rouge, la; see more details in, ppar (santa cruz #sc-22022, dilution 1:200; run on a separate blot), fabp4 (r&d - af804, 1:2000), peroxisome proliferator - activated receptor gamma, co - activator 1 alpha (pgc1; abcam - ab77210, 1:1000), cytochrome c oxidase subunit 4 (coxiv; cell signaling-4844s, 1:1000), and tubulin (loading control; cell signaling-2148s), followed by secondary antibody conjugated with horseradish peroxidase . Signals were detected by enhanced chemiluminescence, and quantitated using alphaeasefc analyzer software and normalized to tubulin in the corresponding blot . At necropsy white adipose tissues from om, sca and scf depots of male and female baboon fetuses were fixed in 4% paraformaldehyde overnight and paraffin embedded . Images from 5 fetuses from each sex in both treatment groups were collected using a hamamatsu nanozoomer digital slide scanning system (hamamatsu city, japan). Adipocyte lobules were manually outlined and adipocytes within these clusters counted using 20x magnification and image j (nih) software by an investigator blinded to the tissue source . Expression of adipogenesis - related genes was analyzed by two - way anova, in which we used 1) maternal diet, depot and maternal diet depot interaction and 2) maternal diet, sex and maternal diet sex interaction as fixed effects and the baboon fetus i d as a random effect, followed by tukey adjustment for the pair - wise comparisons . The effect of maternal diet on protein expression was analyzed for each sex using student s t - test . The effect of maternal diet and adipose tissue depot and their interaction on adipocyte number per unit area (an indirect inverse measure of adipocyte size) were analyzed for each sex and then combined by two - way anova . Sas (version 9.1; sas institute, cary, nc) was used for analysis . Baboon (papio species) singleton pregnancies were studied at the southwest national primate research center at the texas biomedical research institute (tbri). Healthy female baboons of similar body weights (1015 kg) were randomly assigned to outdoor group cages and maintained in social groups of 1016 with a vasectomized male . At the end of the acclimation period (30 days) pregnancy was dated initially by timing of ovulation and changes in sex skin color and confirmed at 30 days of gestation (0.16 g; term, ~184 days) by ultrasonography . The model of 30% global maternal nutrient reduction from 30 days of pregnancy to term has been described in detail . The pregnant baboons were fed purina monkey diet 5038 containing protein 15.7%, fat 6% by acid hydrolysis, and glucose 0.29% (the full composition of monkey diet 5038 can be found at http://labdiet.com/pdf/5037-5038.pdf). Mothers were allowed to recover from surgery and returned to their group housing . Paired fetal adipose tissue samples from omental (om), subcutaneous abdominal (sca), and subcutaneous femoral (scf) regions were collected from twenty near - term (165 days gestation (dg)) baboon fetuses . We studied ctr fetuses of ad libitum fed mothers (5 female and 5 males) and fetuses of mothers fed the 70% global diet eaten by ctr (mnr, 5 female and 5 males). All procedures were approved by the tbri and university of texas health science center, san antonio institutional animal care and use committees and studies were conducted in aaalac accredited facilities . Tissues were removed from the fetus under aseptic conditions, placed in hank's buffered salt solution and shipped at room temperature to the pennington biomedical research center . Within ~24 h from collection, adipose tissue was digested enzymatically and adipose derived stromal - vascular cells (ascs) isolated as previously described . Cultures were expanded in 10% fetal bovine serum (fbs) and third passages were frozen until samples from all fetuses were available for batch processing in a single assay . Frozen adipose tissue culture samples were thawed and further expanded in culture plates coated with a soluble extract of engelbreth - holm - swarm tumors (e - c - l; millipore, cat no . Cells were switched to a differentiation cocktail comprised of dmem - f12 (1:1) medium supplemented with 3% fbs, 10 mg / ml transferin, 33 m biotin, 17 m calcium pantothenate, 0.5 m insulin, 0.1 m dexamethasone, 0.2 nm tri - iodo - thyronine, 60 m indomethacin, 1 m roziglitazone, and 0.5 m ibmx (the last three components for the first 3 days only) for 9 days . Rna was isolated and cleaned using rneasy kit (qiagen, valencia, ca). Qrt - pcr was used to determine the relative gene expression levels of fatty acid binding protein 4 (fabp4, hs01086177_m1), adiponectin (hs00605917_m1), peroxisome proliferator - activated receptor gamma (ppar, hs01115513_m1), uncoupling protein 1 (ucp1, hs00222453_m1), and t - box 15 (tbx15, hs00537087_m1) after asc differentiation . Tbp (hs99999910_m1) was used as an internal control . Of note, all the primers were from applied biosystems (life technologies, us). Fifty ng of cdna template per sample was amplified on the abi prism 7900ht by qpcr . The gene expression was determined in relation to the mean value for the subcutaneous abdominal acs using the ct method . At the end of the differentiation protocol, asc cells were harvested in a non - denaturing buffer containing 150 mm nacl, 10 mm tris ph 7.4, 1 mm egta, 1 mm edta, 1% triton - x 100, 0.5% igepal ca-630, 1 m phenylmethylsulfonyl fluoride (pmsf), 1 m pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 m leupeptin, and 2 mm sodium vanadate and frozen . Next, the samples were thawed, needled, and centrifuged at 14,000 g at 4c for 10 minutes . Three males and three females from each ctr and mnr, which has the largest, smallest and the median values of the respective mrna levels were selected for the western blotting . Samples from the three depots of the individual fetuses were pooled and 50 g of the samples were loaded on to the 10% polyacrylamide gel . Protein from brown adipose tissue of mice (30 g) was also loaded as a positive control for ucp1 . Proteins were then transferred to a pvdf membrane and were probed with antibodies that recognize ucp1 [kindly donated by dr . Gettys, pennington biomedical research center, baton rouge, la; see more details in, ppar (santa cruz #sc-22022, dilution 1:200; run on a separate blot), fabp4 (r&d - af804, 1:2000), peroxisome proliferator - activated receptor gamma, co - activator 1 alpha (pgc1; abcam - ab77210, 1:1000), cytochrome c oxidase subunit 4 (coxiv; cell signaling-4844s, 1:1000), and tubulin (loading control; cell signaling-2148s), followed by secondary antibody conjugated with horseradish peroxidase . Signals were detected by enhanced chemiluminescence, and quantitated using alphaeasefc analyzer software and normalized to tubulin in the corresponding blot . At necropsy white adipose tissues from om, sca and scf depots of male and female baboon fetuses were fixed in 4% paraformaldehyde overnight and paraffin embedded . Images from 5 fetuses from each sex in both treatment groups were collected using a hamamatsu nanozoomer digital slide scanning system (hamamatsu city, japan). Adipocyte lobules were manually outlined and adipocytes within these clusters counted using 20x magnification and image j (nih) software by an investigator blinded to the tissue source . Expression of adipogenesis - related genes was analyzed by two - way anova, in which we used 1) maternal diet, depot and maternal diet depot interaction and 2) maternal diet, sex and maternal diet sex interaction as fixed effects and the baboon fetus i d as a random effect, followed by tukey adjustment for the pair - wise comparisons . The effect of maternal diet on protein expression was analyzed for each sex using student s t - test . The effect of maternal diet and adipose tissue depot and their interaction on adipocyte number per unit area (an indirect inverse measure of adipocyte size) were analyzed for each sex and then combined by two - way anova . Sas (version 9.1; sas institute, cary, nc) was used for analysis . Male mnr fetuses had lower weight and abdominal circumference than ctr males (762 24 g vs. 884 49 g, p = 0.03 and 13.5 0.3 cm vs. 15.5 0.6 cm, p = 0.01, respectively). Body weight and abdominal circumference were similar between mnr and ctr female fetuses (752 43 g vs. 794 40 g, p = 0.2 and 13.9 0.6 cm, vs. 14.7 0.5 cm, p = 0.2, respectively). The crown - rump length was similar in ctr and mnr fetuses of both sexes (males: ctr, 27.4 0.9 cm vs. mnr, 26.4 0.8 cm, p = 0.5 and females: ctr, 25.3 1.9 cm vs. mnr, 26.4 1.6 cm, p = 0.7). Although the maternal diet and depot each had no effect on the gene expression of terminal adipogenic genes, we found a significant interaction between diet and the fetal sex (figure 1); i.e. Maternal suboptimal nutrition increased the expression of adipogenesis - related genes in male but not female fetuses . Specifically, while expression of adipogenic genes in ctr female and male fetuses was similar, expression of the classical adipogenic transcription factor ppar and its targets fabp4, adiponectin, and to a remarkable degree ucp1 was higher in male mnr compared to female fetuses (figure 1). Likewise, we found a significant interaction between diet and the fetal sex regarding tbx15 gene expression (figure 1). Specifically, the expression of tbx15 was higher in ctr female vs. male fetuses (p = 0.03). Mnr females showed lower tbx15 expression than female ctr (p = 0.03) as opposed to males, in whom mnr was associated with a trend to increased expression (p = 0.08). Thus, the expression of tbx15 tended (p = 0.09) to be lower in female vs. male fetuses in the mnr group . In males (figure 2a), all proteins related to white (ppar and fabp4) and brown (ucp1 and pgc-1) adipogenesis as well as mitochondrogenesis (coxiv) in in - vitro differentiated ascs from pooled om, sca and scf adipose tissue samples were higher in mnr than ctr fetuses . In contrast, some of these proteins (ppar, ucp1 and coxiv) were unchanged and others (fabp4 and pgc-1) were lower in mnr than ctr female fetuses (figure 2b). The adipocytes per unit area are less abundant in mnr in both sexes (p = 0.02) combined indicating that mnr promotes adipocyte hypertrophy (table 1). This effect of the maternal diet was primarily evident in males in whom the difference in adipocyte number per field reached borderline significance (p = 0.08) but not in females . However, the female fetuses tended to have a higher number of adipocytes in the omental compared to both subcutaneous depots (p = 0.09) indicating that subcutaneous adipocyte size tend to be larger than the size of omental adipocytes . We found no interaction between maternal diet and depot effects on the adipocyte number per field . Male mnr fetuses had lower weight and abdominal circumference than ctr males (762 24 g vs. 884 49 g, p = 0.03 and 13.5 0.3 cm vs. 15.5 0.6 cm, p = 0.01, respectively). Body weight and abdominal circumference were similar between mnr and ctr female fetuses (752 43 g vs. 794 40 g, p = 0.2 and 13.9 0.6 cm, vs. 14.7 0.5 cm, p = 0.2, respectively). The crown - rump length was similar in ctr and mnr fetuses of both sexes (males: ctr, 27.4 0.9 cm vs. mnr, 26.4 0.8 cm, p = 0.5 and females: ctr, 25.3 1.9 cm vs. mnr, 26.4 1.6 cm, p = 0.7). Although the maternal diet and depot each had no effect on the gene expression of terminal adipogenic genes, we found a significant interaction between diet and the fetal sex (figure 1); i.e. Maternal suboptimal nutrition increased the expression of adipogenesis - related genes in male but not female fetuses . Specifically, while expression of adipogenic genes in ctr female and male fetuses was similar, expression of the classical adipogenic transcription factor ppar and its targets fabp4, adiponectin, and to a remarkable degree ucp1 was higher in male mnr compared to female fetuses (figure 1). Likewise, we found a significant interaction between diet and the fetal sex regarding tbx15 gene expression (figure 1). Specifically, the expression of tbx15 was higher in ctr female vs. male fetuses (p = 0.03). Mnr females showed lower tbx15 expression than female ctr (p = 0.03) as opposed to males, in whom mnr was associated with a trend to increased expression (p = 0.08). Thus, the expression of tbx15 tended (p = 0.09) to be lower in female vs. male fetuses in the mnr group . In males (figure 2a), all proteins related to white (ppar and fabp4) and brown (ucp1 and pgc-1) adipogenesis as well as mitochondrogenesis (coxiv) in in - vitro differentiated ascs from pooled om, sca and scf adipose tissue samples were higher in mnr than ctr fetuses . In contrast, some of these proteins (ppar, ucp1 and coxiv) were unchanged and others (fabp4 and pgc-1) were lower in mnr than ctr female fetuses (figure 2b). The adipocytes per unit area are less abundant in mnr in both sexes (p = 0.02) combined indicating that mnr promotes adipocyte hypertrophy (table 1). This effect of the maternal diet was primarily evident in males in whom the difference in adipocyte number per field reached borderline significance (p = 0.08) but not in females . However, the female fetuses tended to have a higher number of adipocytes in the omental compared to both subcutaneous depots (p = 0.09) indicating that subcutaneous adipocyte size tend to be larger than the size of omental adipocytes . We found no interaction between maternal diet and depot effects on the adipocyte number per field . This study investigated the impact of decreased fetal nutrient availability on differentiation and functional properties of adipose tissue depots in male and female baboon fetuses . Our nonhuman primate model of development in a precocial species mnr male fetuses weighed significantly less (approximately 14%) than ctr indicating development of iugr, while the decrease in fetal weight in females was only 5% in absolute terms and was not statistically significant . The greater slowing of growth in male than female fetuses has been extensively reported and is usually attributed to the faster growth rate in normal male vs. female fetuses . Thus, male baboon fetuses may have experienced a greater relative degree of nutritional deprivation . The lower abdominal circumference in mnr vs. ctr male fetuses but comparable crown - rump lengths in both groups suggest that the mnr male fetuses may have preferential reduction in total adipose tissue rather than lean mass similar to findings from previously published furthermore, rodent studies on effects of poor maternal nutrition show that body weight changes in the offspring are largely accounted for by variation in body fat . In addition, nutrient deprivation exerted differential effects on adipocyte differentiation between male and female fetuses . Specifically it stimulated mostly brown adipogenesis in male fetuses as judged by the 26-fold increase in ucp1 gene expression and the modest 1.5-fold increase in the expression of its transcriptional regulator and a marker of white adipogenesis ppar, and its target genes fabp4 and adiponectin . These results are similar to the findings of substantial upregulation of ucp-1 and a concomitant modest increase in several transcriptional regulators [pgc-1, ppar, and type 2 deiodinase (dio2)] in response to adrenergic stimulation of adipose tissue in male mice . Although ppar binding sites are present in both the ucp-1 and fabp4 promoters, the ppar co - activator pgc-1 activates only ucp-1 . Additional findings of higher protein expression of ucp-1 together with that of pgc-1 and of the marker of mitochondrial content coxiv (brown adipocytes have a high mitochondrial content) in differentiated ascs of male mnr vs. ctr fetuses provides further support of the overall predominant increase in brown over white adipogenesis . The expression of ucp1 and pgc-1 is in part dependent on adrenergic stimulation by norepinephrine released by sympathetic efferents in white adipose tissue through the stimulatory 1-, 2-, and 3-adrenergic receptors and the inhibitory 2-adrenoreceptors (reviewed in). We have previously shown in this mnr model that there is a decrease in 1- and no change in 2-receptor levels in the fetal liver at term but changes in adipose tissue in this model have not been analyzed . However, in vitro studies show that sex hormones differentially affect adrenergic receptor expression in 3t3-l1 preadipocytes and adipocytes and maternal under - nutrition during early phases of gestation in male sheep decreases plasma testosterone levels . These data suggest a potential role of sex hormones in regulating brown adipogenesis through modulation of the adrenergic systems . The reduced adipose tissue mass in the face of comparable adipocyte size in iugr male fetuses compared to females lends support to the idea of smaller pool of adipocytes and their precursor cells . Given that development of adequate pool of brown adipocytes is a major requirement to provide sufficient thermogenesis for neonatal survival, the overall stimulation of adipogenesis and establishment of more brown than white adipocyte phenotype appear to be adaptive mechanisms to attain the critical number of brown adipocytes . In contrast to findings in males, mnr did not affect fetal growth and did not stimulate adipogenesis in female fetuses . Indeed there was a decrease in protein expression of fabp4 and pgc-1. Glucocorticoids are potential mediators that could explain this phenomenon since they are a major factor regulating terminal differentiation in a wide range of fetal tissues . We have shown increased activity of the fetal hypothalamo - pituitary adrenal axis and elevated fetal circulating cortisol in this mnr model . Although there are no fetal sex differences in circulating cortisol, we have demonstrated increased local production of cortisol at term that is fetal sex specific, being increased in female adipose tissue but not male and in male liver but not female liver . Glucocorticoids enhance recruitment of stem cells towards the adipocyte lineage and cause adipocyte hypertrophy in primary in vitro differentiated porcine adipocytes . A recent study reports that increased expression and activity of 11 beta - hydroxysteroid dehydrogenase type 1 (11-hsd1), an enzyme that converts inactive cortisone to the active glucocorticoid cortisol, leads to suppression of genes characteristic of brown adipose tissue . We recently demonstrated increased activity of 11-hsd1 in female but not male adipose tissue in this mnr model at term . We hypothesize that increased local production of cortisol stimulates prenatal differentiation by switching adipocyte differentiation from the fetal brown fat - like to white fat type . If this occurs too late in the mnr males, or too early in the mnr females it may be maladaptive, predisposing to obesity and insulin resistance . However, further studies are required to identify the molecular mechanisms underlying these interesting sex differences . An alternative mechanism may involve genes encoding transcription factors regulating embryonic and fetal development and pattern specification based on data from transcriptional profiling studies in rodents and humans showing depot- and sex - dependent differences in their expression . The expression of several developmental genes, tbx15, glyp4, and hoxa5, correlates with levels of obesity (body mass index) and fat distribution (waist - to - hip ratio). We focused on tbx15 as it is expressed predominantly in brown adipose tissue and in those white adipose depots that are capable of giving rise to brown - in - white adipocytes . Also, sirna - mediated silencing of tbx15 expression in primary preadipocyte cultures from epididymal white and interscapular brown adipose tissue from 129/sv mouse pups down - regulates the adipogenic genes (ppar and fabp4) and the brown phenotypic marker genes (prdm16, pgc-1, coxiv, ucp1) in brown adipocytes . Our findings of decreased expression of tbx15 gene in females and a trend for increased expression in male fetuses from mnr mothers could explain the trend for decreased adipogenesis in females and, in part, the enhanced white and particularly brown adipogenesis in male fetuses . It is noteworthy that the ctr females show increased tbx15 expression compared to ctr males . This corresponds to the higher expression of ucp1 in adult women compared to men suggesting a possible contribution of tbx15 to development of brown phenotype of white adipose tissue in adulthood . Recent evidence shows a relationship of ucp1 mrna abundance with a member of the homeobox group of developmental genes, hoxa1 in the perinatal period . Furthermore, hoxa2 gene [located adjacent to hoxa1 gene on chromosome 7 and thus theoretically its expression will overlap with that of hoxa1 both spatially (same adipose tissue sites) and temporally (same fetal period)] is expressed more in men compared to women . Together, these data suggest that both hoxa1 and hoxa2 may be additional candidates for the sex - dependent regulation of fetal brown adipogenesis . Future studies of sex - differences in ontogeny of expression of an extended panel of embryonic patterning and developmental genes are warranted to gain a better understanding about their role as mediators of sex - hormone related intrinsic identity of preadipocytes and subsequent sex - specific adipose tissue programming events . It remains to be shown how these responses of adipocyte differentiation affect adipose tissue function and metabolic health in adulthood . Studies investigating the dynamics of adipocyte cellularity with weight gain, using a cross - sectional design or obtained longitudinally by serial biopsies of inguinal adipose tissue depots suggest an oscillatory pattern of adipose tissue remodeling, involving simultaneous and repetitive cycles of hyperplasia, hypertrophy, and hypoplasia (decreased adipocyte number), presumably reflecting proliferation and differentiation of adipocyte precursor cells, development of mature adipocytes, and apoptosis, respectively . Interestingly, the rate of enlargement of adipocytes (hypertrophy) is proportional to the difference between the lipid load and the storage capacity of adipocytes, which likely depends on both adipocyte number and metabolic properties . The high rate of adipocyte hypertrophy and low contribution of hyperplasia in white adipose tissue predispose some individuals to increased susceptibility to apoptosis, increased initiation of a local inflammatory response (infiltration of adipose tissue with immune cells and increased secretion of pro - inflammatory molecules by immune cells and adipocyte precursor cells). Given that maternal under nutrition appears to reduce the preadipocyte pool in males but to maintain or increase the abundance of preadipocyte in females suggest that males may be preconditioned to develop adipocyte hypertrophy, and hence local inflammation, more readily than females . This predisposition may be further enhanced by a potential catch up postnatal growth observed in iugr . In support, a study of developmental ontology in ovine fetuses and early newborns shows that the intrauterine nutritional environment elicits a lower inflammatory response prenatally with higher local inflammation in adipose tissue in offspring suggesting a possible role of inflammation in mediating, the long - term unfavorable metabolic consequences of poor maternal nutrition . An important question that arises is whether the increased expression of ucp1 and/or increase in brown versus white adipose tissue could potentially lead to increased energy expenditure in these mnr male fetuses and how to reconcile this possibility with the increased risk of obesity documented in small for gestational age newborn babies . Brown adipocytes are only present in large numbers during the perinatal and early postnatal periods . It is not known whether these brown adipocytes transdifferentiate into white adipocytes or are lost due to increased cell death . Also, it is not known whether the brown - to - white transdifferentiated adipocytes during early postnatal growth retain a higher expression of 3-adrenoreceptors which appears to be critical for the appearance of brown adipocytes in response to cold stimulation or emerge de novo as a new population . Lastly, there is no consensus yet whether the expression of ucp1 or the increased brown versus white adipose tissue is fundamental to body weight regulation . Further longitudinal studies of the dynamic changes in brown features of postnatal adipocytes are needed to fill in these gaps of knowledge . In conclusion, the parallel evaluation of white and brown adipogenesis in fetal adipose tissue development suggest that the control of adipogenesis and the establishment of brown / white adipocyte phenotype may be an important target for nutritional reprogramming of adipogenesis and thermogenesis in response to suboptimal nutrition . Our data support the emerging view that challenges in pregnancy can have differential effects in the presence of a male or female fetus as shown here in the different response of the female mnr fetus which adapts while the male fetus continues on the fetal brown adipose tissue track in a relatively non - adaptive fashion . Our findings further reinforce the need to observe and compare responses according to fetal sex when assessing developmental programming of adiposity in response to sub - optimal maternal nutrition.
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Inflammation is a physiological reactionwhich involves cellular and biochemical responses, which is not only symptom for common diseases but also known to be an early phase for some serious diseases such as alzheimer's disease, cancer, heart vascular diseases etc . Nonsteroidal anti - inflammatory drugs (nsaids) like ketoprofen, ibuprofen, aceclofenac, and so forth under current clinical usage for the treatment of inflammation, algesia and pyresis are associated with major drawbacks of gastrointestinal disorders like dyspepsia, gastric ulcers, and so forth, due to the direct contact of free carboxylic group with the gastric mucosa [3, 4] and due to decrease in production of prostaglandins in tissue . In order to overcome these drawbacks, there is an urgent need for design and synthesis of new chemical entities with excellent anti - inflammatory response and minimum side effects . Hydrazones constitute a versatile compound of organic class having the basic structure r1r2c = nnr3r4 [7, 8]. Two nitrogen atoms of hydrazone are nucleophilic but the amino type nitrogen is more reactive, whereas the carbon atom possesses both characters, that is, nucleophilic and electrophilic . The active centers of hydrazine, that is, carbon and nitrogen, are mainly responsible for the physical and chemical properties of the hydrazones and, due to the reactivity toward electrophiles and nucleophiles, hydrazones are used for the synthesis of organic compound such as heterocyclic compounds [9, 10]. The general method for the synthesis of the hydrazones is the reaction of hydrazine with carbonyl compounds such as aldehydes or ketones in solvents like ethanol, methanol, butanol [1113], and so forth . Hydrazones and their derivatives are known to exhibit interesting diverse biological activities like antioxidant, anti - inflammatory [15, 16], anticonvulsant [17, 18], analgesic [19, 20], antimicrobial [2123], anticancer [24, 25], antiprotozoal, antiparasitic, cardioprotective, antidepressant, antitubercular [30, 31], anti - hiv, and trypanocidaletc . Hydrazones are also used to couple with certain drugs and the bonds based on hydrazones are stable at the neutral ph . The hydrazone schiff bases of aroyl, acyl, and heteroaroyl compounds are known to have an additional donor site, that is, c = o, which make them more versatile and flexible . This versatility has leaded the hydrazones to emerge as good chelating agents that can form a variety of complexes with different transition metals . Some hydrazones have been introduced by the researchers as potent drugs, such as nifuroxazide, an intestinal antiseptic [34, 35], dihydralazine as hypertensive, and gyromitrin, a toxin . A series of benzothiazine n - acylhydrazones 1(a h) was designed by structural modification of piroxicam and synthesized and evaluated for the anti - inflammatory and antinociceptive activities (figure 1). The pharmacological screening revealed that compounds 1(a h) have exhibited better activity than standard drug piroxicam . Compounds 1f and 1 g were identified as new anti - inflammatory and antinociceptive agents which were capable of inhibiting cell recruitment by 70% and 80%, respectively, at dose of 100 mol / kg, p.o in zymosan- and carrageenan - induced peritonitis . A novel series of 6-substituted-3(2h)-pyridazinone-2-acetyl-2(p - substituted / nonsubstituted benzal)hydrazine 2(a i) was synthesized and evaluated for their analgesic and anti - inflammatory activity (figure 2). The activity was evaluated by using carrageenan - induced paw oedema assay and indomethacin was employed as standard for comparison of results . The results revealed that 6-substituted-3(2h)-pyridazinone-2-acetyl-2(nonsubstitutedbenzal)hydrazones, that is, 2a, 2b, and 2c, were found to be most potent anti - inflammatory agents . Compound 2a, 6-[4-(3-chlorophenyl)piperazine]-3(2h)-pyridazinone-2-acetyl-2(p - substituted / nonsubstituted benzal)hydrazine, was found to be slightly better than standard drug indomethacin . A novel series of n-(substituted benzylidene)-2-(n-(4h-1,2,4-triazole-4-yl) benzamido)acetohydrazide derivatives 3(a j) was synthesized and evaluated for the anti - inflammatory and antimicrobial activity (figure 3). Indomethacin was used as standard drug and carrageenan - induced paw oedema method was employed for the anti - inflammatory screening of the test compounds . It was found that compounds 3f, 3 g, and 3j substituted with 4-chloro, 4-dimethylamino, and 4-nitro exhibited 66.7, 61.7, and 63.2% inhibition, respectively, at 50 mg / kg, oral dose, whereas standard drug indomethacin had shown 64.2% inhibition at 10 mg / kg, oral dose . Synthesis of a novel series of some amidine and hydrazone derivatives 4 was reported (figure 4). The synthesized compounds were further subjected to evaluation of anti - inflammatory and analgesic activities . Anti - inflammatory activity evaluation was carried out using carrageenan - induced rat paw oedema assay and compound 4a was found to have good anti - inflammatory activity . A series of nineteen pyrazine n - acylhydrazone (nah) derivatives 5(a s) was (figure 5) designed by molecular simplification of the prototype (lassbio-1018), a nonselective cyclooxygenase inhibitor . Synthesis of the designed compounds was carried out and they were evaluated for their anti - inflammatory and analgesic activities in several animal models of pain and inflammation like writhing test, formalin test, hot plate test, zymosan - induced peritonitis, capsaicin - induced ear edema, and freund's adjuvant - induced arthritis model . Thalidomide (tnf- inhibitor), celecoxib (cox-2) inhibitor, and indomethacin (selective cox-1 inhibitor) were employed as standard drugs . Results enlightened that compound 5o (2-n-[(e)-(3,4,5-trimethoxyphenyl)methylidene]-2-pyrazinecarbohydrazide, lassbio-1181, had better pharmacological activities and can be used as a lead compound for development of new analgesic and anti - inflammatory agents . A novel series of isatin derivatives, that is, isatin-3-[n2-(2-benzalaminothiazol-4-yl)]hydrazones 6(a j), was synthesized and evaluated for anti - inflammatory, analgesic, and antipyretic activities (figure 6). Carrageenan - induced rat paw oedema model was used for evaluation of anti - inflammatory activity and indomethacin was employed as standard drug . It was found that the compounds 6f, 6h, and 6j substituted at fifith position with methyl, chloro, and nitro groups, respectively, exhibited significant anti - inflammatory activity . Synthesis of a novel series of orally active n - phenylpyrazolyl - n - glycinyl - hydrazone derivatives, 7a g (figure 7) and 8 (figure 8), was carried out . Synthesized compounds were evaluated for their in vivo analgesic and anti - inflammatory activities and in vitro inhibition of tnf- (tumor necrosis factor). Compounds 7a, (e)-2-(3-tert - butyl-1-phenyl-1h - pyrazol-5-ylamino)-n-((4-(2-morpholinoethoxy)naphthalen-1-yl) methylene)acetohydrazide, and 7f, (e)-2-(3-tert - butyl-1-phenyl-1h - pyrazol-5-ylamino)-n-(4-chlorobenzylidene)acetohydrazide, were known to possess anti - inflammatory activities when compared to the standard drug used, that is, sb-203580 . N) (figure 9) was reported and evaluated for their in vitro anti - inflammatory, antimicrobial and antioxidant activities . Albumin denaturation studies were carried out in order to evaluate the anti - inflammatory activity by employing diclofenac sodium as standard . Compounds 9c, 9d, and 9e were reported to have good anti - inflammatory activity due to presence of 4-nitro, 4-methyl, and 2-hydroxy groups, respectively, whereas 9e was found to be the most active anti - inflammatory agent . Synthesis and biological evaluation of some new benz[b]thiophene derivatives like thiadiazole, pyrazoline, oxadiazole and diaryl pyrazoles was carried out . N-(3- chlorobenzo[b]thiophene-2-carbonyl)-3-methyl-4 - 9 substituted phenylhydrazono pyrazolin-5-one 10(a - e) were subjected to anti - inflammatory and antimicrobial evaluation (figure 10). Compound 10b substituted with 2-nitro-4-methyl was found to have 50.25% inhibition which is near about the standard drug diclofenac sodium i.e. 51.88% . Synthesis of zinc complexes 11, [zn(lassbio-466)h2o]2 of salicylaldehyde 2-chlorobenzoyl hydrazone (h2lassbio-466), and 12, [zn(hlassbio-1064)cl]2 salicylaldehyde 4-chlorobenzoyl hydrazone (h2lassbio-1064), was carried out . The complexes were further subjected to evaluation for peripheral and central nociception and acute inflammation in animal models . Both of the complexes exhibited anti - inflammatory activity . Complex 11 has shown activity in both phases of formalin test like indomethacin and indicated its ability to inhibit nociception associated with the inflammatory response, whereas h2lassbio-466 was active only in first phase of formalin test . Synthesis of some new hydrazide derivatives of 2-napthoxy acetic acid, nicotinic acid [47, 48] and napthlene-1-acetic acid has been reported . Out of these hydrazide derivatives most active antimicrobial compounds carrageenan induced rat paw oedema model was employed for the evaluation of theses active compounds using diclofenac sodium as standard drug . Results revealed that naphthalen-1-yloxy)-acetic acid [1-(2-bromo-4-cyano - phenyl)-ethylidene]-hydrazide 13 (figure 11) has shown percent inhibition of 20.90% at a dose of 50 mg / kg . The nicotinic acid hydrazide derivatives, 14a and 14b, substituted with nitro group at meta and ortho positions, respectively, were found to be the most active anti - inflammatory agents (figure 12). The percent inhibition of compounds 14a and 14b was found to be 37.29% and 35.73%, respectively, at the dose of 20 mg / kg and 34.17% and 25.12%, respectively, at the dose of 50 mg / kg, whereas percent inhibition of diclofenac sodium was found to be 38.85% . The conclusion drawn from the results was that the substitution of nitro group and halogens contributed to anti - inflammatory activity . Synthesis of benzophenone semicarbazone (bsc) 15a and acetophenone semicarbazone (asc) 15b was carried out (figure 13). The anti - inflammatory activity was determined on swiss albino mice by using carrageenan - induced mice paw oedema model . Both of the compounds were screened at two different doses, that is, 25 mg / kg and 50 mg / kg (p.o . ). Compound 15a showed 36.6% and 46.6% of inhibition at 25 mg / kg and 50 mg / kg, respectively, whereas compound 15b showed 34.6% and 41.5% inhibition . Diclofenac sodium was used as standard drug which showed 70.29% inhibition at 10 mg / kg (p.o . ). From the above observations, it was concluded that both of the test compounds possessed anti - inflammatory activity . A novel series of 2-[4-(substituted benzylideneamino)-5-(substituted phenoxymethyl)-4h-1,2,4-triazol-3-yl thio]acetic acid, 16(a all the newly synthesized compounds were evaluated for in vivo anti - inflammatory and analgesic activities . Among the series 16d, 16e, 16j, and 16k showed significant activity with 63.4%, 62.0%, 64.1%, and 62.5% edema inhibition, respectively, as compared to standard drug diclofenac 67.0% after the third hour . Compounds 16 g, 16h, 16i, and 16l showed good anti - inflammatory activity, whereas compounds 16a c and 16f were found to be the least active . The results enlightened the effect of electron withdrawing moiety, that is, chloro group on the anti - inflammatory activity . A series of bis - hydrazone derivatives, that is, (z)-n,n-(1-(4-substituted phenyl)ethene-1,2-diyl)bis(4-substituted benzhydrazide), 17(a d), was synthesized by reaction of 2-chloro-1-(4-chloro phenyl)ethanone or 2-bromo-1-(4-bromophenyl)ethanone with acid hydrazides (figure 15). All the synthesized compounds were evaluated for the anti - inflammatory, analgesic, and ulcerogenic activities . Formalin - induced rat paw oedema model was selected and ketoprofen was employed as standard drug . All compounds were found to exhibit good anti - inflammatory activity with percent inhibition of 68.4% and 61.4% of compounds synthesis of a series of hydrazone derivatives, that is, n-(substituted benzylidene)-3-cyclohexylpropionic acid hydrazide, 18(a inducible nitric oxide synthase (inos) and nf-b were selected for determination of anti - inflammatory activity . Compounds 18c, 18e, and 18i were more active than the other synthesized compounds which proved that there is a positive correlation between the inhibitions of inos activity and functional groups, that is, methyl, fluoro, and isopropyl, on phenyl ring . Inhibition of nf- b mediated transcription was seen in human chondrosarcoma (sw1353) cells and compounds 18a, 18c, and 18h have shown inhibition with ic50 values of 6.9, 7.7, and 6.4 g / ml, respectively (figure 17). This observation correlated that methyl and chloro groups have a considerable influence on the inhibition of nf-b mediated transcription . The other compounds 18d, 18e, 18 g, and 18i inhibited the nf-b activity to a lesser extent with ic50 values in the range of 1014 g / ml . Compounds 18b, 18d, and 18j were found to be comparatively less active than other compounds . From the results, it was concluded that substitutions at para position of phenyl ring had significant influence on anti - inflammatory activity . By application of molecular hybridization approach design, and synthesis of thirty two furoxanyl n - acylhydrazones(furoxanyl - nah) synthesized compounds were evaluated for their in vitro as well as in - vivo analgesic and anti - inflammatory activities . The in vitro anti - inflammatory activity was evaluated by decrease in nf-b activation and interleukin-8 inhibition by using a human pathway - specific reporter cell system (ht-29-nf-b - hrgfp) whereas carrageenan induced paw oedema was used for in vivo evaluation of anti - inflammatory activity . Furoxanyl - nah 19 (figure 17)and benzofuroxanyl - derivative 20 (figure 18) were reported to have orally anti - inflammatory and analgesic activities without interleukin-8 inhibition . Furoxanyl - nah derivative 21a (figure 19)was emerged as a structural lead to develop new lipoxygenase (lox) inhibitors . The active derivatives 19, 21a and 21b were found to be less mutagenic and were proposed as candidates for the further clinical studies . Synthesis of a series of novel acyl - hydrazones bearing 2-aryl - thiazole moiety was carried out . The synthesized compounds were screened for invivo anti - inflammatory activity by evaluating three parameters, that is, nitric oxide synthesis, phagocytes activity, and acute phase bone marrow response, in acute experimental inflammation . Compounds 22c, 22e, 22f, 24b, and 26b were found to have a good inhibitory effect on the acute phase marrow response by reducing the absolute leukocytes count due to the lower neutrophils percentage . Compound 22c (figure 20) with 2-phenyl - thiazole and [2-(4-methylphenyl)-4-methylen]-thiazole hydrazine moieties was proved to be a more potent inhibitor of the acute phase bone marrow response than meloxicam, the anti - inflammatory standard drug . Phagocytic activity was assessed by calculating phagocytic index (pi) and the phagocytic activity (pa). All the newly synthesized compounds reduced pi significantly and compounds 22a (figure 20), 22b (figure 20), 22d (figure 20), 23b (figure 21), 24b (figure 22), 25 (figure 23) and 26b (figure 24)were observed as more potent inhibitors than meloxicam . Pa was significantly reduced by the compounds 22a, 22d, 23b, 24b, 23c, 25 and 26b from which 22a, 22d, 20 and 26b were found to be more potent inhibitors than meloxicam . The no synthesis was significantly reduced by 22a, 22b, 23c, 26a, and 26b and they all, except for 26a, displayed a stronger inhibitory activity than meloxicam . Synthesis of a novel series of phthalic anhydride based substituted benzylidene - hydrazide derivatives, 27a all the synthesized derivatives were screened for in vivo anti - inflammatory and analgesic activities by carrageenan - induced rat paw oedema and tail immersion methods, respectively, using diclofenac sodium as standard drug . The results revealed that derivatives 27d, 27e, and 27h (figure 25) have shown potent anti - inflammatory activity with percentage inhibition of 58.6%, 61.4%, and 64.0%, respectively, which is comparable with standard drug diclofenac sodium, that is, 68.0% . The reaction time of derivatives 27d and 27h that was found to be 8.91 0.21 and 9.09 0.03, respectively, after 90 minutes which is comparable with reaction time of diclofenac sodium (10.93 0.01) after 90 minutes has shown analgesic potency of these derivatives . Hydrazone derivatives are well known to have various important pharmacological activities and are used for synthesis of a wide variety of medicinally active compounds . This review paper summarizes the anti - inflammatory potential of hydrazone derivatives and the effect of substitutions of different groups on the anti - inflammatory activity . This summarized study is an attempt to bring about the anti - inflammatory activity for awaking the safe use of this important chemical moiety with minimal or no ulcerogenic effects in future.
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Prior to the nation s independence, the health care system in ghana under colonial rule was organized primarily for the benefit of the elite few . The development of public health services in ghana (formerly the gold coast) dates back to the 1880s, when the gold coast medical department was established to provide rudimentary services to european colonial government officials . In the 20 century, a colonial health policy of prevention for the indigenous population was recognized through the establishment of the sanitary branch in 1909, and the medical research institute in 1917, which trained paramedical personnel . The colonial government provided free health services to civil servants, but those not in civil service and their families were left to secure their own health care at their own expense . When ghana attained independence under kwame nkrumah in 1957, a national health service (nhs) similar to that of britain s was established to extend free care to the entire population at government health facilities . The nhs system remained in place until shortly after 1966, when the nkrumah government was overthrown in a military coup . A system for health service fees was introduced through the hospital fees decree in 1969, later amended by the hospital fees act in 1971, but the fees were never fully implemented . Fees were eventually imposed under the hospital fees regulation of 1985, when economic growth slowed and rising inflation crippled ghana s economy and forced the country to turn to the international monetary fund and the world bank for financial assistance . As ghana s economy worsened, government expenditures on health care, which averaged 2.3 percent of the gross domestic product in 1998 - 2002, declined to around 1.9 percent in 2003 - 2004 . Declining government expenditures on health care shifted the burden of health costs onto the ghanaian population through health user fees (a system also known as cash and carry) that required patients to pay money upfront to health facilities before services were provided . The cash and carry system created financial barriers to health services for the majority of the nation s poor who could not afford to pay for even basic health care, including reproductive health services . While the cash and carry system was in effect, disparities in health care access and health status between ghana s rural and urban populations became more pronounced . Even though some services, such as antenatal and postnatal care and immunizations, were to be exempted from health user fee payments, in practice they often were not, and the out - of - pocket costs had a negative impact on people s overall health, especially on birth outcomes among the most vulnerable . Many studies found that in ghana and elsewhere, the number of patients treated at government health facilities dropped immediately following the introduction of health user fees . The majority of poor people in northern ghana faced the biggest barrier to health care due to high out - of - pocket costs . Eliminating the financial barriers to health care for the majority poor in ghana, especially those in northern ghana, was the government s primary public health goal when it introduced the national health insurance scheme (nhis) after passage of the national health insurance act (nhia) by ghana s parliament in 2003 . Access to health services through programs such as the national health insurance scheme in ghana allows pregnant women the opportunity to visit health facilities for antenatal and delivery services that they could not otherwise afford . The nhis, which started in 2003 on a limited basis in four of ghana s ten regions, was fully implemented nationwide by 2005 . The program requires all adults in ghana aged18 and older to enroll in the nhis and pay premiums that range from the equivalent of $2.50 up to $50.00 annually, depending on each enrollee s financial and employment status . The scheme provides coverage for children 17 years and younger without premium payments, provided the child s parents are fully registered in the program . Pregnant women with low incomes are also provided free coverage under the nhis to enable them to access antenatal services and skilled care at delivery . Although membership in the nhis had declined in some of ghana s districts, there was a 118 percent increase in membership among the indigent population in northern ghana between 2007 and 2008 . Overall, nhis participants in northern ghana increased from 143,460 to 863,099 users between 2005 and 2008, a 502 percent increase in just three years . The increase in enrollment among the poor in northern ghana ensured that a majority of pregnant women would be able to access skilled care at the hospital rather than deliver at home . The nhis has been operational in ghana for a decade now, supporting increased access to skilled care at health facilities for the majority of indigent pregnant women in northern ghana . However, research has not adequately tracked the program s utilization, particularly service utilization and birth outcomes among the poor in northern ghana . Most studies of the nhis have focused on enrollment and access; very few address birth outcomes . Particularly absent are studies that compare birth outcomes under the cash and carry system with those under national health insurance in sub - saharan africa, especially in ghana . This study used sampled birth records abstracted from birth registry folders at the tamale teaching hospital (tth), the primary referral hospital for the entire northern sector of ghana . Tamale, the regional capital of the northern region, is the fourth largest city in ghana . It has a population of a little less than 380,000, of which 51 percent are female and 49 percent male . Birth outcome data at tth are recorded in the labor and maternity ward s delivery folders at the time of birth . The validity of these records is ensured through cross - checking by a head nurse and the supervising obstetrician . For this research, the delivery folders for the years 2000 - 2003 (when cash and cary was in effect) and 2008 - 2011 (after full implementation of nhis) were arranged chronologically, and selected in a systematic sampling method that examined and abstracted birth records of each day s deliveries . Days with fewer or no birth records were accommodated by over - sampling records from the day before or after . The sampled birth records were analyzed using stata, version 11.2 (stata corp, college station, tx). The primary aim of this study was to examine trends in lbw among infants delivered under the cash and carry system, compared to the nhis . Chi - squared tests were used to determine changes in prevalence (and significance) of lbw . Analyses were performed for each of the selected variables in both periods 2000 - 2003 and 2008 - 2011 . The dependent variable birth weight (in grams) of live births which is a continuous variable, was coded as a dichotomous variable so that lbw (<2,500 g) and normal birth weights (2,500 g) yielded the desired outcomes in repeated measurements . The independent variables maternal age, parity (number of times a woman has given birth), maternal hemorrhage (blood loss), miscarriage (including induced abortion), type of birth (vaginal or caesarian section), fetal heart rate, and gender were selected because they have been documented in previous research to be associated with birth weight . Deliveries during the cash and carry period were used as a proxy for lack of access to insurance and professional antenatal care prior to childbirth, while deliveries under nhis represented access to health insurance and at least four antenatal care visits prior to childbirth at tth . It had already been established that in ghana more than 98 percent of pregnant women receive antennal services under the nhis; the majority of women (85% or more) receive at least the four antenatal visits recommended by the world health organization prior to delivery . The administration at tamale teaching hospital in ghana granted permission for the abstraction of the birth records from the hospital s labor and maternity ward . The mean maternal age was 27 (sd=6), with a range of 14 to 50 years . Table 1 presents a descriptive outcome of total delivery records of live births analyzed in this study . Characteristics of all live births under cash and carry and nhis systems at tamale teaching hospital in northern ghanacash and carrynhis note . Nhis = national health insurance scheme; lbw = low birth weight; ml = milliliters; bpm = beats per minute . Trends in the prevalence of lbw among all infants born in 2000 - 2003 and 2008 - 2011 are presented in table 2 . A higher prevalence of lbw was observed among young mothers (aged 18 - 24) compared to mothers aged 25 and older . The prevalence of lbw among younger mothers ranged from 17.5 percent to 36.8 percent [p<0.001] under cash and carry, compared to 16 percent and 22 percent [p>0.05] under nhis . Prevalence of lbw in live birth infants during the cash and carry and nhis systems of care, and by selected maternal and other variables in northern ghana first - time mothers (parity = none) under cash and carry were also significantly more likely to deliver low birth weight infants, with prevalence ranging from 17% to 35.8% [p<0.001], compared to the lbw prevalence among first - time mothers under nhis of 17% to 21.9%, [p>0.05]. There were no significant changes observed in trends for lbw among mothers with prior childbirth experience (parity = one or more) under either cash and carry or nhis . In 2000 - 2003 (cash and carry), caesarean deliveries comprised 11to 17 percent of total deliveries, compared to 2008 - 2011 (nhis) when they accounted for 18 to 22 percent . The prevalence of lbw among caesarean deliveries under cash and carry went from 14 percent in 2000 to 19 percent in 2003, compared to 15 percent in 2008 and 27 percent in 2011 under nhis . However, vaginal deliveries showed mixed results for the prevalence of lbw in both periods . In 2000, the lbw prevalence for vaginal births was 26 percent; it decreased significantly to 15 percent [p<0.001] by 2003 . In 2008, the lbw rate was 14 percent; this increased to 19 percent by 2011, but the change was not statistically significant . Trends in lbw among infants with normal fetal heart rates of 130 - 140 beats per minute prior to birth significantly decreased from 25 percent [p<0.001] in 2000 to 15.6 percent [p<0.01] in 2003; lbw in this category increased from 15 percent [p>0.05] in 2008 to 20.8 percent [p<0.05] in 2011, which was not statistically significant . This study examined trends in lbw among infants born during the cash and carry period compared to infants born under the nhis . Associations between lbw and factors such as maternal age, parity, caesarean delivery, and infant s gender have all been well documented in other research . The majority of infants in this study approximately 85 percent were delivered by mothers 18 to 34 years old . Mothers aged 18 to 24 in both the cash and carry and nhis systems were more prone to deliver lbw infants compared to older mothers . This suggests that regardless of the mother s insurance status at delivery, her age was a factor in her infant s birth weight . The current study showed that more than one - third of infants were born to first - time mothers, who experienced significantly higher prevalence of lbw in both the cash and carry and nhis periods . However, there were no substantial differences in the prevalence of lbw among infants born to mothers with prior birth experience (parity = one or more) in 2001 - 2003 (cash & carry) compared to infants born in 2009 - 2011 (nhis) by mothers with similar parity . This suggests that delivery under nhis, which guaranteed access to antenatal care, did not translate into a reduction in lbw births among multiparous mothers . This is also confirmed by research on multiparous women and lbw conducted in other african countries and the us . More than 90 percent of all infants from the cash and carry and the nhis periods were born to mothers with no history of miscarriages or prior experience of induced abortion . Year - to - year trends showed that during the cash and carry period, mothers with no history of miscarriage gave birth to infants with a significantly higher prevalence of lbw in 2000, which decreased by as much as 38 percent by 2003 . Under the nhis, there was a lower prevalence of lbw in 2008, which increased by about 50 percent by 2011 . This indicates that access to health services under nhis had little impact on infants birth weight for mothers with no history of miscarriage . The improvement in birth weights among mothers with no history of fetal loss during cash and carry is consistent with similar findings observed among african - born black women in the us . Caesarean deliveries in the former period generally constituted less than 15 percent of total births (except 17% in 2003), which the who recommends should be the upper limit for caesarean deliveries compared to all births . Under nhis, more deliveries occurred by caesarean, which increased from 18 percent in 2008 to 22 percent by 2011 . The increased use of caesarean sections may have resulted from several factors including the availability of insurance coverage, which provided an incentive for compromised pregnancies to be surgically delivered . The observed increased use of caesarean deliveries under the ghanaian insurance program was similar to that seen in a large east african hospital . These results highlight the possibility that when patients pay for care out - of - pocket, fewer opt for the more expensive caesarean procedure . The significant increase in the prevalence of lbw among caesarean - sectioned infants observed under nhis could be explained by the availability of nhis making it possible for more mothers with compromised pregnancies (and therefore prone to having lbw infants) taking advantage of caesarean delivery to prevent adverse pregnancy outcomes . Infants with normal fetal heart rates of 130 - 140 bpm were generally born at higher birth weights than infants who had abnormal fetal heart rates . The observed association between normal pre - delivery fetal heart rates and higher birth weights in this study is consistent with the results of other research . More than half of all infants born under cash and carry and nhis were male . Even though this study revealed mixed trends on birth weights related to selected variables under the cash and carry and nhis periods, the large sample size and the comparability of the birth records increased the robustness of the study . However, there are some significant limitations in this research, including the fact that hospital - based data in sub - saharan africa generally exclude those women who choose to deliver at home . At - home delivery is still a common practice in northern ghana and elsewhere in africa despite the availability of insurance and skilled care . Data on mothers who opted for the services of traditional birth attendants (tba) even after accessing antenatal care services at the hospital were not included in this research . Other important variables that have been shown to affect birth weight, such as gestational age and mother s weight gain during pregnancy, were not available and therefore not included in this analysis . Though the delivery folders generally capture a range of birth outcome data at the time of delivery, information such as mother s income, educational level, employment status, marital status, and religion are not recorded in the delivery folders . These factors might have provided important additional information about trends in birth weights during the cash and carry and nhis periods . Despite its limitations, the hospital - based delivery data set showed that overall trends in birth weight outcomes were not significantly impacted by the introduction of nhis compared to cash and carry . However, younger and first - time mothers delivered more lbw babies under cash and carry compared to nhis, and more lbw babies were delivered by caesarian under nhis . Although northern ghana is an economically deprived part of the country, it is also possible that women with higher socioeconomic status self - selected to deliver at the hospital during cash and carry . This research reveals important information about the birth weights of infants born at northern ghana s major hospital . However, the limitations discussed earlier make a strong argument for further research on birth outcomes, especially birth weights . Future research should incorporate the key factors that limited this study, including: mothers socioeconomic status; gestational age; maternal weight gain during pregnancy; and the number of antenatal care visits for each mother prior to delivery . If available, data from at - home births should also be included to help clarify causation and increase the generalizability of the research . There is a consensus in public health research that insurance coverage, which reduces the financial barriers to health care services, improves general health outcomes . Understanding the differences in the prevalence of low birth weight between the cash and carry and nhis systems in northern ghana is important for public health policy makers | there, especially as the country hopes to meet the united nations millennium development goals (mdg) by the end of 2015 . Since birth weight is an important predictor of infants surviving their first year of life, the variables revealed by this research to be related to lbw under nhis should help guide maternal and child health policies, particularly as they relate to health facilities, tbas, antenatal services, and nutritional guidelines to improve birth outcomes . The mortality rate for children under five years remains high in ghana, especially in the northern region . By understanding the factors that affect lbw, the country can focus its resources and efforts to ensure that infants are born at normal birth weights, which is a well documented indicator for their survival.
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Although various prostheses have been used for anterior chest wall reconstruction, selection of the procedure depends on the surgeons experience . The case of a patient who underwent reconstruction of the anterior chest wall using a titanium plate sandwiched between two polypropylene mesh sheets is reported . A 79-year - old woman was referred to our department with a diagnosis of recurrent chondrosarcoma . The first operation for sternal chondrosarcoma included sternal resection and reconstruction with polypropylene mesh and a musculocutaneous flap . However, 18 months after the first operation, computed tomography revealed five tumors located on the anterior chest wall and another tumor located in the subcutaneous tissue on the right chest wall . The tumors were considered metastatic lesions, with no evidence of enlarged mediastinal lymph nodes or distant metastases on radiographic examination . Thus, it was judged that complete resection was possible, and the patient underwent subtotal sternectomy, total resection of the body and partial resection of the manubrium sternii, together with partial resection of the 1st5th ribs and costal arch, with a surgical margin of more than 1.0 cm for each tumor . This resection left a defect measuring 17 14 cm on the anterior chest wall . Reconstruction of the defect was undertaken with a titanium plate (titanium mini mesh sheet, 01 - 13155, 132 82 mm; thickness 0.5 mm, stryker leibinger & co., germany) sandwiched between two polypropylene mesh sheets . The lowermost and the uppermost layer consisted of a polypropylene mesh, and the sheet was fixed to the manubrium and each rib with absorbent suture . The middle layer was a titanium plate, which was fixed to the manubrium and costal arch directly by absorbable #2 polyfilament braided suture and pulled toward each rib stump (fig . 1). . No paradoxical movement of the rib cage was noted during respiration in the postoperative period . Twelve months after operation, the patient had maintained excellent range of motion without instability or lordosis (fig . B chest wall defect after subtotal sternectomy and resection of the 1st5th ribs and costal arch . C the middle layer consists of a titanium plate fixed to the manubrium and costal arch, pulled to each rib stump . The lowermost layer is a polypropylene mesh sheet . D the uppermost layer consists of a polypropylene mesh sheet fixed to the manubrium and each ribfig . 2postoperative chest x - ray and computed tomography scans showing the titanium plates secured to the manubrium and ribs surgical images . A local recurrent tumors on the chest wall . B chest wall defect after subtotal sternectomy and resection of the 1st5th ribs and costal arch . C the middle layer consists of a titanium plate fixed to the manubrium and costal arch, pulled to each rib stump . The lowermost layer is a polypropylene mesh sheet . D the uppermost layer consists of a polypropylene mesh sheet fixed to the manubrium and each rib postoperative chest x - ray and computed tomography scans showing the titanium plates secured to the manubrium and ribs sternal tumors are uncommon; however, they are of different pathological types, such as sarcoma and metastatic tumors of the breast, thyroid, kidney, and colon . King et al . Recommended a 4-cm free margin for highly aggressive primary tumors and 2-cm margins for metastatic, benign, or low - grade malignancies to avoid local recurrences . In any case, complete resection of the sternal tumor results in a wide defect on the anterior chest wall . The ideal prosthetic material should be easily available, durable, easily usable, adaptable, rigid, resistant to infection, translucent to radiographs, and of low cost . Generally, polypropylene mesh sheets or polytetrafluoroethylene patches (e - ptfe) covered with a musculocutaneous flap are used . However, their rigidity is insufficient to protect intrathoracic organs . Various prostheses have been used, with sufficient rigidity, such as sandwiched polypropylene mesh and stainless steel mesh, methyl methacrylate sandwiched between polypropylene mesh, titanium plate - supported methyl methacrylate sandwich, titanium plate with gore - tex dual mesh, and composix mesh . However, methyl methacrylate is not easy to handle and is difficult to adapt to the shape of the patient s chest . Titanium mini mesh sheet has strong rigidity, no plasticity, translucency to radiography, magnetic resonance imaging (mri) compatibility, and biocompatibility . We think that the combination of a metal material and a mesh is an appropriate prosthesis, because of its durability, ease of use, adaptability, rigidity, and translucency to radiography . The advantages of the present procedure are based on the easy use of the titanium plate, irrespective of the shape of the defect and the physiological nature of the material . The titanium plate is used to provide protection for intrathoracic organs, while the polypropylene mesh is flexible in both vertical directions and thus allows movement of the chest wall during breathing . In conclusion, the procedure with the titanium plate sandwiched between two polypropylene meshes achieved good fixation and flexibility . In patients who require extensive anterior chest wall and sternal resection,
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Many patients experience immense pain prior to their death . However, the intravenous or oral dosage of opioids for pain control results in unacceptable sedation . Intrathecal injection of opioids has already been successfully utilized as a front - line treatment for cancer pain refractory to traditional treatment . Intrathecal morphine has been found to be nonsedative or minimally sedative in multiple studies without the negative effects of parenteral or oral opioids . Cancer pain refers to symptoms resulting from inflammation, compression, and neurological and ischemic damage at various sites . According to the clinical symptoms reported, many patients who have somatic pain also have neuropathic pain . Although opioids appear to be effective in overall pain control, neuropathic pain resulting from major dysfunction of the somatosensory system may be less likely to respond to opioid therapy . In these conditions, however, increased opioid doses are associated with unsatisfactory negative effects . Therefore, the combination of opioids with other drugs, such as local anesthetics, must be considered . Morphine exerts effects against pain by binding to the,, and opiate receptors, which stimulates potassium influx, giving rise to postsynaptic neuron membrane hyperpolarization in the dorsal horn of the spinal cord . Voltage - sensitive calcium influx is decreased, thereby decreasing the neurotransmitter release from presynaptic terminals . Ropivacaine, an amino - amide local anesthetic, blocks the generation and conduction of nerve impulses through blockade of sodium influx . Studies have indicated that ropivacaine promotes the effects of intrathecal opioids . Despite existing documentation and studies for the usage of intrathecal ropivacaine mixed with morphine, few studies have utilized an intrathecal continuous injection of ropivacaine with morphine to treat cancer pain . The objective of this study was to determine the efficacy and safety of continuous ropivacaine and morphine injection using intrathecal access ports in patients suffering from cancer pain refractory to traditional treatment modalities . The institutional ethics committee was used to assign patients and provide advice regarding ethical issues . The study was approved by the institutional review board, which also confirmed informed consent by the patients . This study included thirty - six terminal cancer patients between november 2010 and september 2013 (table 1). Randomization numbers were generated using an automated and validated system to assign treatment arms, and the assignments were concealed from patients and investigators . Constant morphine injection was given to group m patients through intrathecal access for pain administration, whereas constant morphine and ropivacaine injection was given to group r patients . All 36 patients of the study had received treatment for pain that had proven refractory to traditional therapies such as transcutaneous electrical nerve stimulation, physiotherapy, pharmacotherapy (anti - inflammatory drugs, nonsteroidal drugs, tricyclic antidepressants, oral / transdermal opioids, anticonvulsants, and antispasmodics), and psychotherapy . Patients were chosen prior to catheter placement according to the following inclusion criteria: intractable cancer pain unmanaged by high dosage of oral or parenteral analgesics, intolerability to surgery for pain, or general failure to relieve pain; emotional stability of the patient and family members; and presence of an accountable and competent care provider . The patients were psychiatrically evaluated before the initial port implant and no progressive psychiatric disease was found . They were told about the availability of an intrathecal catheter for pain management (with dosages administered by a responsible and capable caregiver) days ahead of the catheter implantation . In addition to verbal instruction, they were presented with the intrathecal catheter and port system and informed of the implantation treatment in written form, particularly with regard to the advantages and potential side effects of intrathecal therapy . They were also advised in writing of complications and related blood tests, particularly coagulation screening, c - reactive protein and white cell count . Clindamycin (600 mg) was administered intravenously 2 h before catheter insertion for antibiotic prophylaxis . Intrathecal catheterization combined with implantation of a subcutaneous infusion port (celsite, b. braun, france) was conducted in the sterile condition in an operating room . The patient was put in the lateral decubitus position and then draped in a sterile fashion . Lumbar puncture was performed at the interspace between l2 and s1 using an 18-gauge tuohy needle . Approximately 1530 cm of the catheter was introduced intrathecally according to the location of the pain under fluoroscopic guidance . The catheter was moved through the subcutaneous tissue between the lumbar incisions and port pocket and then attached to the port . The port pocket was established through a bone structure (the base of the ribs was usually chosen). The port was placed into a subcutaneous pocket organized in the chosen area and attached to the fascia . Access to the port was via percutaneous injection using a special noncoring needle that was connected to a patient - controlled analgesia (pca) device with a total volume of 100 ml and a rate of 0.5 ml / h . The original intrathecal morphine dosage was obtained from the preoperative morphine injection using an oral - intrathecal ratio of 300: 1 . The starting dose of ropivacaine was 0.375 mg / ml, and the total dose per day was 4.5 mg . The dose was titrated every 24 h until pain was reduced or therapy - limiting side effects were identified . One day before leaving hospital, the constant intrathecal dosage was selected to be the optimal dosage for placing intrathecal port . We analyzed the demographic statistics (e.g., sex and age) of the patients, cancer categories, location of pain, morphine dosage history, use of oral medications, and pain intensity before implantation of the port as reported using the numerical rating scale (nrs) in which 0 means no pain and 10 means the greatest pain . The nrs score and intrathecal morphine and ropivacaine consumption on postsurgical days 1, 3, 7, and 15 were evaluated . The barthel index, a disability scale with scores from 0 (completely dependent) to 100 (completely independent), was used to evaluate the functional status of the patients . Data regarding technical aspects of the procedure (such as catheter tip location, injection interspace, and device - related complications) and nonpharmacological methods used to reduce cancer pain were obtained . The day before hospital discharge, the use of extrasystemic opioids and adjuvants was also recorded . The paired - sample t - test and two - way anova were used for data comparisons . Thirty - six patients completed the trial: 17 in group r and 19 in group m. in group m, 11 male and 8 female patients were studied, with an average age of 64.0 years (4085 years). In group r, 14 male and 3 female patients were studied, with an average age of 62.6 years (4985 years). In group m, nociceptive pain was present in 11 patients, whereas a mixture of neuropathic - nociceptive pain was present in 8 patients . In group r, nociceptive pain was present in 7 patients, whereas mixed neuropathic - nociceptive pain was present in 10 patients . Physicians were asked to rate the pain characteristics based upon complaints from patients, with nociceptive pain defined as aching, squeezing, or pressure - like sensations; neuropathic pain was characterized as tingling, burning, or electrical sensations . The mean duration of hospital care was 12 days for patients in group m and 15 days for those in group r. the doses of preoperative systemic opioids (intravenous and oral oxycodone, intravenous morphine, and transdermal fentanyl) were summarized as the oral morphine equivalent dose . Additional usage of opioid treatment was also documented to summarize gross opioid usage on a daily basis and presented as the variation in preoperative opioid dosage . The preoperative oral morphine consumption was between 45 and 600 mg / day (mean: 200.8 mg / day) in group m. the preoperative oral analgesic consumption was between 6 and 750 mg / day (mean: 223.7 mg / day) in group r. the mean preoperative doses of oral morphine in groups m and r were similar (p = 0.688). The daily dosage of intrathecal morphine and ropivacaine was adjusted at a certain time based on the pain degree and the bolus dosage required in the last 24 h. a significant increase was observed in intrathecal morphine administration on postoperative days 3, 7, and 15 in comparison with day 1 . In group m, the mean doses of intrathecal morphine on postoperative days 1, 3, 7, and 15 were 0.67 0.48 mg, 1.02 0.64 mg, 1.44 0.86 mg, and 2.36 1.56 mg per day, respectively . In group r, the mean doses on postoperative days 1, 3, 7, and 15 were 0.75 0.66 mg, 0.93 0.80 mg, 1.09 0.99 mg, and 1.23 1.10 mg per day, respectively . The mean doses of intrathecal morphine on postoperative days 1, 3, and 7 in groups m and r were similar (p = 0.688, p = 0.697, and p = 0.207, resp . ). However, the mean dose of intrathecal morphine on postoperative day 15 in group r was significantly lower than that in group m (p = 0.005). In group r, the mean doses of intrathecal ropivacaine on postoperative days 1, 3, 7, and 15 were 4.5 mg, 6.10 2.10 mg, 6.96 2.48 mg, and 8.71 6.54 mg per day, respectively . The average nrs scores for the 19 patients in group m on the preoperative day and postoperative days 1, 3, 7, and 15 were 8.17 0.51, 4.78 1.0, 3.78 0.80, 3.06 0.94, and 2.50 1.04, respectively . The average nrs scores for the 17 patients in group r on the preoperative day and postoperative days 1, 3, 7, and 15 were 7.78 0.73, 3.67 1.37, 2.56 1.34, 2.06 1.16, and 1.33 0.77, respectively . The average nrs scores on the preoperative days were similar in groups m and r (p = 0.069). The average nrs scores on the postoperative days 1, 3, 7, and 15 decreased gradually in both groups m and r (f = 40.26, p <0.001 and f = 30.62, p <0.001). However, the average nrs scores on postoperative days 1, 3, 7, and 15 were statistically lower in group r than in group m (f = 37.38, p <0.001) (figure 1). The barthel index scores on the preoperative day and the 15th postoperative day were 53.61 6.82 and 63.06 7.70, respectively, in group m. the barthel index scores on the preoperative day and the 15th postoperative day were 55.0 6.86 and 68.33 6.64, respectively, in group r. the barthel index score on the preoperative day was similar in groups m and r (p = 0.472). However, the barthel index score on the 15th postoperative day was higher in group r than in group m (p = 0.017) (figure 2). Most intrathecal catheters were implanted at the l2 - 3 interspace in both group m (72.2%) and group r (61.1%), and most of the catheters reached t10 in both group m (55.6%) and group r (44.4%). No patient experienced respiratory depression . In group m, one patient experienced transient urinary retention and three experienced nausea and vomiting . In group r, one patient experienced transient urinary retention, one constipation, and one nausea and vomiting . Ropivacaine was added to the opioid injection without any significant dermal numbness or decreased sensation . There were no complications or adverse effects serious enough to require intrathecal port removal or treatment changes . The first study of intrathecal morphine in a cancer patient was performed by tung et al . In 1980 . The present findings are in agreement with existing studies demonstrating a decrease in pain upon intrathecal drug delivery . Our findings illustrate that administration of ropivacaine and intrathecal morphine through an indwell injection port is both efficacious and safe for severe cancer pain . Simultaneously, this study demonstrated a larger decrease in nrs rates using intrathecal morphine and ropivacaine compared to intrathecal morphine alone . Our findings demonstrate that intrathecal ropivacaine combined with morphine is safe; adverse effects were mild and rare . The mechanism of action of intrathecal opiates is under debate . As an opiate receptor agonist, morphine blocks the activity of some cells in the substantia gelatinosa of the medullary dorsal horn . As a result, intrathecal narcotic management decreases cutaneous pain transmission at the dorsal horn and transmission of nociceptive impulses through ascending pathways . Several studies have found that intrathecal morphine management is an efficient approach for analgesia in humans and animals . However, studies have large increases in the required dosage of intrathecal morphine with increasing cancer pain on postoperative days . Among patients with insufficient pain management on intrathecal morphine, our study showed that the mean dose of intrathecal morphine on postoperative day 15 in patients receiving both morphine and ropivacaine was significantly lower than that in patients treated with morphine alone . The combined intrathecal treatment is useful when insufficient pain reduction is realized with intrathecal monotherapy, especially in neuropathic pain patients . Different pain statuses have various potential mechanisms; as a result, a medication with a particular mechanism tends to only be efficient for one or two specific pain conditions . Thus, combining agents tends to be useful to treat multiple aspects of the pain, generating synergistic efficacy for pain management . Intrathecal morphine targets medullary opioid receptors, whereas intrathecal ropivacaine functions at the dorsal root and the nerve roots to promote pain management . Ropivacaine reversibly blocks sodium influx, thereby hindering pain signal transmission through a and c fibers . Ropivacaine may increase opioid efficacy synergetically: (1) decreasing voltage - sensitive calcium influx, thus promoting the opioid - mediated presynaptic inhibition of neurotransmitter release from terminals of a and c fibers [2, 14] by decreasing the conformational transformation of medullary opioid receptors (,, and receptors). Our findings show higher benefit for pain management and quality of life for inpatients with cancer pain when using the combined treatment . Based on our analysis, the recommended daily dosage of intrathecal ropivacaine to realize pain control was 4.515.3 mg . Because we could reduce pain with a low dose of ropivacaine, the observed pain control may reflect synergy between ropivacaine and opioids . To our knowledge, this is the only study with a standard, quantitative assessment of changes in the nrs score and quality of life when the combination of intrathecal morphine and ropivacaine is used to reduce cancer pain . In our study, the average nrs scores on postoperative days 1, 3, 7, and 15 were significantly lower in patients treated with ropivacaine and morphine than in those treated with morphine alone . The barthel index ratings indicate that the quality of life on the 15th postoperative day was significantly higher in patients treated with morphine and ropivacaine when compared with those injected with morphine alone . Nausea and vomiting tended to be the most common and were observed in 15.7% (3 of 19) of morphine - only patients and 5.8% (1 of 17) of patients treated with ropivacaine and morphine; these symptoms are frequently reversible with ondansetron . Urinary retention is also relatively common with intrathecal opioids . In our patients, urinary retention often resolved in a few days or weeks, and constipation was not observed, as most patients treated with systemic opioids in this research were treated with a bowel stimulant, a stool softener, or laxatives prior to intrathecal management . Constipation occurred in 1 patient in group r. somnolence and sedation are rare adverse effects for medullary opioids . Infection, respiratory distress, motor dysfunction, seizures, weight increase, reduced sexual impulses, and port malfunction are also possible risks but were not observed in our series . Because of the above risks and system implantation costs, proper selection of patients prior to subcutaneous intrathecal port transplantation is critical . The following selection criteria are recommended: (1) great pain despite oral narcotic management or unsatisfactory narcotic side effects at the dosage required to manage pain; (2) medication for the underlying disease, for example, radiation tumor treatment or surgical tumor treatment, that has not caused the pain; and (3) neuroablative processes for pain reduction that were refused by patients or deemed as unsatisfactory by physicians . In addition, the outcome with the intrathecal port is greatly determined by the attitude of the patient and family; a peaceful and friendly environment is important . The implantation of intrathecal catheters connected to subcutaneous injection ports was appropriately indicated for patients in this study on the grounds of life expectancy, expense, and drug / dosage requests . First, the effective sample size was small, as the study included only 36 patients . Second, long - term complications, such as formation of granulomas and intrathecal infection, were not assessed because of the short follow - up period . Third, mild negative effects, such as sedation or pruritus, may have been underestimated, as the patients showed pharmacological adverse effects in a passive manner in the present study . Thus, it is not clear whether opioid - mediated side effects were decreased by the intrathecal treatment . Intrathecal morphine and ropivacaine management in connection with a transplantable subcutaneous port is a safe and efficient approach to provide treatment of intractable cancer pain . The extra intrathecal ropivacaine enhances pain management and increases the quality of life . The usage of intrathecal ropivacaine is therefore deemed to be safe and acceptable . Large - scale prospective random trials are necessary to assess the advantages and safety of intrathecal ropivacaine.
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In iran, as in many countries in the world, bursate nematodes belonging to the trichostrongyloidea superfamily are of major veterinary importance in small ruminant production systems (1, 2). Ostertagia species and marshallagia marshalli are the most prevalent nematodes infecting sheep and goats (3). Iran is a semi dry country but climatologically can be divided into 4 different zones . Zone 4 (central and salt deserts) is neither suitable for animal husbandry nor fit for human habitations . The other three zones include: caspian zone (zone i), mountain plateau zone (zone ii) and the persian gulf lowland (zone iii). The combined sheep and goat populations in these 3 zones are 52 million and 26 million, respectively (2, 4). Recent reports indicate that anthelminthic resistance also occurs in some sheep nematodes, especially in teladorsagia circumcincta, in iran (5, 6). Accurate identification of sheep nematodes is a critical point in epidemiological studies and monitoring of drug resistance in flocks (7 - 9). However, due to a close morphological similarity between the eggs and larval stages of many of these nematodes, such identification is not a trivial task (7, 10). There are a number of studies showing that ribosomal dna (rdna), especially the internal transcribed spacer (its) 1 and 2 regions, are suitable regions for phylogenetic investigations and valuable targets to design probes or define markers for the identification of bursate nematodes (8, 10 - 13). Some studies have also successfully used the intergenic spacer (igs) fragment for species - specific identification of strongyle nematodes (14, 15). Due to genetic diversity and also intraspecific variations among nematodes from different geographical populations (9, 16), it seems advantageous to have complete sequences of the genetic targets to be used in species identification from all target species from a specific geographical region . In this study, five common bursate nematodes (haemonchus contortus, trichostrogylus colubriformis, teladorsagia circumcincta, marshallagia marshalli, and nematodirus oiratianus) were collected from sheep of zones ii and iii of iran . The objective was to compare the its1, its2 and igs regions of these nematodes in order to choose best target for specific identification methods . H. contortus, t. colubriformis, t. circumcincta, m. marshalli, n. oiratianus adult male nematodes were collected from the gastrointestinal tract of sheep slaughtered from khuzestan, chaharmahal and bakhtiari provinces in south west iran . Nematodes were washed in phosphate buffered saline (pbs) and adult male nematodes were identified morphologically according to the keys of soulsby (17) and then stored in 70% ethanol until being used . Before dna extraction, the nematodes were removed from ethanol, dried and washed in distilled water and stored for 12 days in 1.5 ml tube at 20 c . High pure pcr template preparation kit (roche diagnostics) was used to extract dna from a single male nematode of each of the species following the manufacturer s instructions . Available genbank 28s and 18s sequences of h. contortus (am039742), t. colubriformis (aj920350 and am039743.1), nematodirus battus (aj920360 and am039752) and t. circumcincta (af044934.1) were used to design primers . To amplify the its1, 5.8s and its2 regions in one reaction, the primers were based on conserved sequences at the 3end of 18s rdna and 5end of 28s rdna (table 1). The igs primers were designed from conserved sequences at the 3end of the 28s rdna and 5end of 18s rdna (table 1). The pcr was performed on 50 l total volume and included 1x pcr buffer (promega), 1u taq polymerase (promega), 30 pmol/50 l of each primer (sigma), 200 m of each dntp(promega), 3.5 mm mgcl2 and approximately 2 ng per 4 l of genomic dna in an automated thermocycler (thermohybaid, msc), (18) under the following conditions: 5 min incubation at 95 c to denature double - strand dna, 35 cycles of 45 s at 60 c (annealing step), 45 s at 72 c (extension step), and 45 s at 94 c (denaturing step). Finally, pcr was completed with an additional post - amplification extension step for 10 min at 72 c . The pcr products were analyzed on a 1% agarose gel in 1 tae buffer and visualized using sybr - green dye and uvp image analyzer (biospectrumac imaging system). To purify the pcr products, 40 l of each pcr reaction mix was loaded on a 1% low melting point agarose gel . The specific amplified fragments bands (800 - 1000bp in its and 600 - 1500 bp in 28s, igs and ets) were cut out and purified with promega dna purification system kit . Pgem - t easy vector cloning kit (promega) was then used following the manufacturer s instructions to clone the purified pcr products . The vectors are prepared by cutting the pgem-t easy vectors, with ecori and adding a 3 terminal thymidine to both ends . Insertional inactivation of the alpha - peptide allows recombinant clones to be directly identified by blue / white screening on indicator plates . Pureyield plasmid miniprep system (promega) was used for plasmid purification before being sent for sequencing (gatc - biotech, germany). Multiple alignments of the its1, its2 and igs sequences for each species were then used to compare and calculate similarity scores between species . In this step rdna sequence of some non - iranian nematode isolates were also included (table 2). Clustalw2 sequence alignment tool (embl - ebi - http://www.eb-i.ac.uk/tools/msa/clustalw2/) was used for all alignments and calculation of similarity score . H. contortus, t. colubriformis, t. circumcincta, m. marshalli, n. oiratianus adult male nematodes were collected from the gastrointestinal tract of sheep slaughtered from khuzestan, chaharmahal and bakhtiari provinces in south west iran . Nematodes were washed in phosphate buffered saline (pbs) and adult male nematodes were identified morphologically according to the keys of soulsby (17) and then stored in 70% ethanol until being used . Before dna extraction, the nematodes were removed from ethanol, dried and washed in distilled water and stored for 12 days in 1.5 ml tube at 20 c . High pure pcr template preparation kit (roche diagnostics) was used to extract dna from a single male nematode of each of the species following the manufacturer s instructions . Available genbank 28s and 18s sequences of h. contortus (am039742), t. colubriformis (aj920350 and am039743.1), nematodirus battus (aj920360 and am039752) and t. circumcincta (af044934.1) were used to design primers . To amplify the its1, 5.8s and its2 regions in one reaction, the primers were based on conserved sequences at the 3end of 18s rdna and 5end of 28s rdna (table 1). The igs primers were designed from conserved sequences at the 3end of the 28s rdna and 5end of 18s rdna (table 1). The pcr was performed on 50 l total volume and included 1x pcr buffer (promega), 1u taq polymerase (promega), 30 pmol/50 l of each primer (sigma), 200 m of each dntp(promega), 3.5 mm mgcl2 and approximately 2 ng per 4 l of genomic dna in an automated thermocycler (thermohybaid, msc), (18) under the following conditions: 5 min incubation at 95 c to denature double - strand dna, 35 cycles of 45 s at 60 c (annealing step), 45 s at 72 c (extension step), and 45 s at 94 c (denaturing step). Finally, pcr was completed with an additional post - amplification extension step for 10 min at 72 c . The pcr products were analyzed on a 1% agarose gel in 1 tae buffer and visualized using sybr - green dye and uvp image analyzer (biospectrumac imaging system). To purify the pcr products, 40 l of each pcr reaction mix was loaded on a 1% low melting point agarose gel . The specific amplified fragments bands (800 - 1000bp in its and 600 - 1500 bp in 28s, igs and ets) were cut out and purified with promega dna purification system kit . Pgem - t easy vector cloning kit (promega) was then used following the manufacturer s instructions to clone the purified pcr products . The vectors are prepared by cutting the pgem-t easy vectors, with ecori and adding a 3 terminal thymidine to both ends . Insertional inactivation of the alpha - peptide allows recombinant clones to be directly identified by blue / white screening on indicator plates . Pureyield plasmid miniprep system (promega) was used for plasmid purification before being sent for sequencing (gatc - biotech, germany). Multiple alignments of the its1, its2 and igs sequences for each species were then used to compare and calculate similarity scores between species . In this step rdna sequence of some non - iranian nematode clustalw2 sequence alignment tool (embl - ebi - http://www.eb-i.ac.uk/tools/msa/clustalw2/) was used for all alignments and calculation of similarity score . Its1 and its2 were identified from the whole its1 - 5.8s - its2 sequenced fragment for each species . The length of its1 and its2 fragments ranged between 382 - 400 and 231 - 248 bp, respectively (fig . 1; table 3). The igs was identified from the whole 28s - igs - ets-18s sequenced fragment for each species and ranged between 179 (partial sequence) 457 (complete sequence) bp (fig . 2; table 3). Sequence data of the its1, its2 and igs of h. contortus (africa), t. circumcincta (uk) and its1 of t. colubriformis (uk), also sequenced as part of another study in university college dublin, was also included in the final comparison (table 2, 4 & 5). Similarity score percentage in the its1, its2 and igs regions are summarized in table 4 & 5 . Because of incomplete sequences in the its2 and igs of t. colubriformis (uk) it was not included in the analysis . Overall, the highest similarity in the its1 sequences was detected among t. circumcincta and m. marshalli (94%) and the lowest between n. oiratianus and h. contortus (africa) (67%).the iranian and uk t. circumcincta and t. colubriformis its1 sequences were 100% identical . The similarity score percentage between the iranian nematodes and equivalent species obtained from genbank ranged between 96% - 99% (table 4). In the case of the its2 sequences the similarity score percentage among the iranian nematodes and the equivalent species obtained from genbank ranged between 96% - 100% (table 4). The highest similarity among the genera overall was detected between t. circumcincta and m. marshalli (88%) and the lowest similarity was between n. oiratianus and h. contortus (africa) (64%). A high similarity was found between t. colubriformis and m. marshalli (99%) and low similarity between t. circumcincta (uk) and all other species . The highest similarity was between t. circumcincta and m. marshalli (78%) and lowest similarity was between n. oiratianus and t. circumcincta (47%) (table 5). Previous studies have indicated that sequences of the its and igs regions of rdna are useful targets to find genetic markers (7 - 9, 15) for the differentiation between nematode species . In the present study we have shown that there are differences in the its regions between the five species that could be utilized for specific identification (table 4). On the other hand the igs region may be a less suitable molecular target for some taxa, compared with the its regions, as there can be considerable length variation in the igs sequence within individual organisms (9). The extent of sequence similarities of the igs region among the iranian species in this study ranged from 47 to 99% . A very high sequence homology (99%) was observed between t. colubriformis and m. marshalli, whereas the similarity between t. circumcincta (uk) and all other species was low (15 - 28%). Low levels of similarity between related species and extensive sequence homology between species of different genera was also observed in horse cyathostomins (19). Most studies have found that the sequence variation in both the its1 and its2 sequences within species is quite small (9, 12). However, there are limited data available on the genetic variation among different nematode population from different geographical regions . In the case of h. contortus, 15 nucleotide variations were found in the its1 region between isolates from iran and africa . The its1 of iranian t. circumcincta and t. colubriformis were completely identical to the respective uk isolates and is in general agreement with results from hoste et al . Similarly, the its2 sequences of the two h. contortus isolates showed 8 nucleotide variations, including 6 nucleotide substitutions and 2 nucleotide insertions / deletions . In the case of t. circumcincta both the its1 and its2 sequences are convenient targets for the species - specific identification of iranian bursate nematodes . Due to some sequence variations that may occur between geographically different isolates (of the same species), it is advisable to obtain sequences from local isolates before design of specific identification methods.
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Statins or 3-hydroxy - methylglutaryl coenzyme - a (hmg - coa) reductase inhibitors are a potent class of cholesterol synthesis inhibitors and an established therapy for primary and secondary prevention of coronary artery diseases . Several large studies confirmed reduction in cardiac mortality and morbidity through the treatment with statins . These clinical benefits were reported to be majorly due to lowering of low - density lipoprotein cholesterol (ldl - c). A linear relationship between ldl - c and cardiovascular (cv) event rates the study showed landmark reduction in the rate of major coronary event by over 50% after use of statins . This reduction was associated with 2-mmol (78 mg / dl) reduction in ldl - c levels . Later several other studies such as the cholesterol treatment trialists meta - analyses, the treating to new target, heart progression study (hps), cholesterol and recurrent events (care) trial and long - term intervention with pravastatin in ischaemic disease (lipid) studies also confirmed the findings of 4s . Accumulating evidence have suggested that statins, in addition to ldl - c lowering, exhibited properties of plaque stabilization and endothelial homeostasis; anti - inflammatory, antioxidant, anti - proliferative and immunomodulatory effects; normalization of sympathetic outflow; and prevention of platelet aggregation . Pleion, which means more, and tropy meaning response or stimuli . Though pleiotropic effects are defined as a single gene affecting multiple systems or determining more than one phenotype, lately statin pleiotropy is referred as statins exerting multiple pharmacological activities . The pleiotropic effect is the class effect of statins and is exhibited by all statins acting on non - hepatic tissues, including hydrophilic pravastatin, which penetrates poorly through the cell membranes of non - hepatic cells . The rapidity with which statins produce beneficial effects has suggested involvement of mechanisms other than its lipid - lowering activities . Over the last more than 10 years, several clinical trials indicated that the benefits of statins extend beyond their effects on cholesterol, supporting the theory of statin pleiotropy [table 1]. Clinical studies elucidating statin pleiotropy the program on the surgical control of the hyperlipidemias (posch) study reported the benefits of cholesterol lowering after partial ileal bypass surgery after almost 10 years, whereas most statin trials showed benefits at much earlier time points (e.g., within 5 years). Comparing benefits after 5 years for all lipid - lowering trials, non - statin trials no longer were aligned to the same cholesterol: mortality risk reduction slope, whereas all statin trials aligned to this slope . In the posch study, that the 34% reduction in ldl - c within the first 3 months post ileal surgery was although earlier unnoticed could be explained by additional non - lipid effects of statins . In the west of scotland coronary prevention study study, cox regression analyses of data highlighted that pravastatin treatment decreased ldl - c down to 24% and further reducing ldl - c levels did not show additive any benefit, and such phenomenal reduction in coronary heart disease risk was surprising in the pravastatin - treated group as such benefits could not be expected with decrease in ldl - c within this range . This strongly supported non - lipid - lowering theory of statins . In another statin trial with 3000 subjects with unstable angina or non - q - wave acute myocardial infarction, aggressive treatment with statins effectively reduced recurrent ischemic events within 16 weeks after acute coronary events, reduced serum ldl- by 40% and relative risk by 16% . According to the investigators, this period was too short to expect changes in lesion size and plaque stability of such magnitude . Therefore, it was assumed that these early changes were due to the direct cellular effects on the vascular wall and improvement of endothelial function with statins . Other studies that suggested the involvement of a non - lipid - lowering pleiotropic mechanism of statins were large prospective trials, such as the hps and anglo - scandinavian cardiac outcomes (ascot) trials . These studies demonstrated that relative risk reduction with statin treatment was independent of baseline lipid values . The hps trial reported that absolute benefits were related directly to the individual vascular risk of the subjects . Patients with low hdl - c and high vascular risk, such as subjects with diabetes, significantly benefited from statin therapy possibly through lipid - independent mechanisms . In 2003,, reported a randomised trial with hypertensive subjects (n = 10,305) with> 3 cv risk factors . The subjects received either atorvastatin 10 mg / day or placebo and their total cholesterol level was measured to be <250 mg / dl . The study was halted after a median follow - up of 3.3 years as there was a 36% reduction in the risk of myocardial infarction and fatal coronary heart disease . These very early benefits with large reduction in coronary heart disease events were incredible and highlighted the involvement of mechanisms other than the lipid - lowering effects of stains . This was supported by earlier studies such as the myocardial ischaemia reduction with aggressive cholesterol lowering study (miracl) and the pravastatin or atorvastatin evaluation and infection therapy trial (prove - it) where early clinical benefits were seen in subjects with coronary heart disease . This rapid time course of event reduction in high - risk subjects with recurrent coronary ischemia suggested non - lipid - lowering effects . In 2006, vyas et al ., reported over 35% reduced risk of sudden cardiac death or ventricular arrhythmia development due to the plaque - stabilizing effect of statins, increased coronary blood flow and microvascular myocardial perfusion . In 2004, cannon et al ., reported the prove - it trial, comparing the effect of intensive therapy with atrovastatin (80 mg) daily versus moderate therapy with pravastatin (40 mg / day) in 4162 subjects with acute coronary syndrome . The authors reported that subjects were benefitted from intensive therapy than standard therapy, leading to separation of event curves within 3 weeks of treatment . Also, a decrease in hazard ratio was apparent as early as 6 months from treatment . In a sub - study of the prove - it trial, gibson et al ., in the year 2009 reported the benefit of intensive statin therapy in clinical outcomes in subjects undergoing percutaneous coronary intervention for acute coronary syndrome . In 2007, patti et al ., reported a randomized trial where 12-h pretreatment with 40 mg / day atorvastatin prior to coronary angioplasty showed improvement in clinical outcomes in subjects with acute coronary syndrome no reduction ldl - c in levels are commonly observed, suggested that the anti - inflammatory and pleiotropic properties of statins may be of clinical importance . In 2009, an observational study showed that statin users hospitalized for acute coronary syndrome were not only at a lower risk of developing in - hospital atrial fibrillation but also had a significantly lower risk of developing ventricular arrhythmias, cardiac arrest and/or death . In these subjects, intensive statin therapy had reduced the risk of major adverse cv events compared with moderate - dose statin therapy . The reduction in the incidence of target vessel revascularization was independent of ldl - c and c - reactive protein (crp) lowering, suggesting the statin pleiotropic effect of high - dose therapy . Scientists have been intrigued with the magnitude- and timing - reduced cv events in some clinical trials and asserted that these rapid effects mediated by statins could not be explained solely on the basis of the lipid - lowering mechanism of statins . High - sensitivity c - reactive protein (hscrp) is a non - specific marker of inflammatory diseases, and has been recognized as an independent risk predictor of cardiovascular diseases . Several studies have demonstrated lowering of hscrp upon treatment with statins and this effect was independent of lowering of ldl - c . In patients with high crp values, higher coronary deaths occurred in spite of low ldl - c levels . The air force / texas coronary atherosclerosis prevention study reported lowering of crp values through treatment with lovastatin, and improvement in cardiac outcomes was achieved with lowering of crp levels independent of ldc - c levels . In 2005, nissen et al ., reported the existence of an independent correlation between low levels of crp and atheroma progression rate from a post - hoc analysis of data of a clinical study . According to the authors, regression of atheroma size was more rapid and significant with greater reduction in crp levels . However, less regression in atheroma size was observed despite greater reduction in ldl - c levels . In the primary prevention also treatment with statin benefitted patients with high hscrp levels and low ldl - c levels and other cv risks . Subjects in the early or mild stages of heart failure benefitted from statins and the benefits were reported to be due to anti - inflammatory effects and improvement in endothelial function with statin treatment . The presence of inflammatory components in mediating cv diseases were supported by a retrospective study, the corona (controlled rosuvastatin multinational trial in heart failure) trial, and patients benefitting from lowering of hscrp levels suggested a non - lipid effect of rosuvastatin . The finding was further confirmed by the multicenter cosmos (coronary atherosclerosis study measuring effects of rosuvastatin using intravascular ultrasound in japanese subjects) trial . These trials with rosuvastatin showed significant reduction in plaque volume after treatment with rosuvastatin independent of ldl - c reduction, suggesting non - lipid - lowering effects . In a double - blind study with 58 subjects with coronary artery disease, comparable reduction in ldl - c occurred through treatment with high- (80 mg / day) than low - dose atorvastatin (10 mg / day) plus ezetimibe (10 mg / day). Statins are known to lower cholesterol by reversibly inhibiting hmg - coa reductase, the well - known and widely established mechanism of action of statins . Inhibition of cholesterol synthesis occurs as a result of prevention of mevalonate from producing hmg - coa, since mevalonate is not an immediate precursor of cholesterol synthesis and also acts as precursor for several other key molecules needed for normal functioning of cellular processes . In addition to cholesterol synthesis, mevalonate is required for production of non - steroidal isoprenoid intermediates such as the farnesyl pyrophosphate, geranylgeranyl pyrophosphate, isopentanyl adenosine, dolichols and polyisoprenoid side chains of ubiquinone and heme - a . These isoprenoid intermediates are integral to the post - translation modification and activation of several intracellular / signaling proteins such as the -subunit of heterotrimeric g - proteins; heme - a; nuclear lamins; and small gtp - bound protein ras and ras - like proteins such as rho, rab, rac, ral or rap . Post - translational isoprenylation, that is, attachment of isoprenoid chains helps these proteins in their covalent attachment, sub - cellular localization and intracellular trafficking . Both ras and rho proteins switch from their gdp - bound inactive state to the gtp - bound active state [figure 1]. These signaling proteins play an indispensable role in multiple cellular processes cell signaling, cell differentiation and proliferation, myelination, cytoskeleton dynamics and endocytotic / exocytotic transport [figure 2]. Rho protein cycles between a cytosolic, inactive gdp - bound and an active, membrane, gtp - bound state . Inhibition of mevalonate synthesis by statins prevents membrane targeting of rho and its subsequent activation of rock . This cycle is controlled by several cofactors, including guanine nucleotide exchange factors, gtpase - activating proteins, and guanine nucleotide dissociation inhibitors . An important step in the activation of rho gtpases is posttranslational isoprenylation, which allows translocation of rho to the cell membrane and subsequent activation mevalonate pathway for cholesterol biosynthesis showing the effects of inhibition of hmg - coa reductase by statins . Statins decrease the isoprenylation of signaling molecules, which leads to modulation ([increase]/[decrease]) of various signaling pathways . Enos: endothelial nitric oxide synthetase; et-1: endothelin-1; etc: electron transport chain; nadph: nicotinamide adenine dinucleotide phosphate (reduced); pai-1: plasminogen activator inhibitor-1; t - pa: tissue - type plasminogen activator statins have been reported to prevent isoprenylation of rho- and rho - associated kinases (rock) at doses that were used to lower ldl - c levels . Rho / rock are involved in controlling important functions such as regulation of contraction, migration and adhesion of vascular smooth muscle cells . Statins by inhibiting rho / rock activation increase the bioavailability of nitric oxide (no). The rac protein has been involved in cardiac hypertrophy, actin cytoskeleton remodeling and generation of reactive oxygen species, and statins benefit by inhibiting rac1 . Inactivation of rho by statins was reported to upregulate peroxisome proliferator - activated receptor (ppar) expression and induce ppar- transcription activity in macrophages, and inhibit lipopolysaccharide . Matrix metalloproteinases (mmps) are zinc - dependent endopeptidases that promote angiogenesis, intimal proliferation, vessel wall remodeling and extracellular matrix degradation through enhanced migration of vascular smooth muscle cells . The role of rho / rock pathway in the secretion of mmp-2 through stimulation of endothelin-1 and angiotensin - ii has been confirmed with use of small interfering rna and the rock pathway inhibitor, y-27632, which inhibited mmp-2 release . Statins decrease inflammatory cells in atherosclerotic plaques and increase plaque stability through combined reduction of lipids, macrophages and mmps . Other cellular molecules inhibited by statins include nuclear factor-b (nf-b), monocyte chemoattractant protein-1 and hscrp . Under normal physiological conditions, nf - kb resides in the cytoplasm bound with inhibitor i - kb (ib). In response to inflammatory stimuli, the nf - kb then translocates into the nucleus and induces the expression of inflammatory cytokines, monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 . Statins decrease the levels of reactive oxygen species and prevent dislocation of ikb from nf - kb, resulting in inactivation of nf - kb . Statins have been reported to reduce the expression of monocyte chemoattractant protein-1, which lowers the interaction between monocytes and the vascular wall, monocyte chemotaxis, and growth and proliferation of macrophages . Antigen - presenting cells express major histocompatibility complex - ii constitutively, and its expression in human endothelial cells and monocytes increases in the presence of interferon-. Various statins reduce the expression of major histocompatibility complex - ii on antigen - presenting cells, leading to decreased major histocompatibility complex - ii - mediated activation of t - cells . In addition, statins reduce the production of various inflammatory markers such as tumor necrosis factor-, interleukin-1 and chemotactic cytokines . Statins were reported to disrupt the oxidative stress / inflammatory cycles by decreasing lipid peroxidation and release of inflammatory mediators . Statins improve endothelial function by inhibiting the isoprenylation of rac and rho, and activation of the rock pathway . Inactivation of the rock pathway stabilizes the mrna of endothelium - derived nitric oxide synthase (enos) and activates the akt / pi3k cascade, leading to high enos activity, increased no production and bioavailability . Protein kinase akt has been associated with improvement of myocardial aerobic metabolism and increase in the levels of angiopoetine (protein growth factor), which accelerate the process of angiogenesis . Inactivation of the ras signaling pathway was associated with prevention or reversal of cardiac remodeling in humans . In a preclinical study, atorvastatin reduced the synthesis of collagen, -1-procollagen mrna expression and expression of profibrotic peptide connective tissue growth factor in cell cultures of rat and human cardiac fibroblasts . Rosuvastatin administration was reported to decrease the levels of tnf- and mmp-2 and increased the circulating levels of bone marrow - derived stems cells . Statins also have inhibitory effects on release of mmp-1 and mmp-3 from macrophages and endothelial cells, and modulate remodeling processes . In endothelial cells, statins activate protein kinase akt leading to stimulation of enos activity, increased no synthesis and neoangiogenesis . In the central nervous system, statins regulate sympathetic and vagal outflow by increasing endothelial no synthesis, and reduce the expression of angiotensin - ii and endothelial receptors . Statins by normalizing sympathetic outflow showed beneficial effects in hypertensive patients with a history of mi and cerebral ischemia . Statins attenuate the cytokine - mediated proliferation of vascular smooth muscle cells in coronary artery smooth muscle cells, arrest the cell cycle between g1s phase transitions and inhibit smooth muscle cell proliferation through modulation of ras and rho activity . Atorvastatin inhibited serotonin - induced mitogenesis and migration by inhibiting gtp - rhoa formation in pulmonary artery vascular smooth muscle cells, which was reversed by geranylgeranyl pyrophosphate but not farnesyl pyrophosphate . The anti - platelet and antithrombotic activities of statins have been attributed to reduction of thromboxane a2, tissue factor expression and ppars . In heart failure statins activate phosphoinositide-3 kinase and akt, and increase the circulating levels of bone marrow - derived endothelial progenitor cells, which may contribute to improved cardiac regeneration and/or repair . In 2012, bouitbir et al ., reported statins as a new activating factor of cardiac mitochondrial biogenesis and antioxidant capacities . Mitohormesis is defined as a pre - conditioning or adaptive response of mitochondria to low levels of oxidative stress induced by physical exercise, reduced caloric uptake and glucose restriction . This pleiotropic effect of statins was attributed to increased resistance / stress defense against higher levels of oxidative stress and damage to mitochondria and cardiac tissues . Use of antioxidants prevented statin - induced mitohormesis since antioxidant vitamins had decreased the clinical benefits of the simvastatin niacin combination in subjects with coronary artery diseases and low levels of hdl - c in the study by brown and co - workers . Therefore, some of the cardioprotective mechanisms of statins could be attributed to statin - induced mitohormesis . Treatment with simvastatin has been reported to reverse the pulmonary vascular effects of cigarette smoke, including pulmonary hypertension, smoke - induced emphysema, smoke - induced small arterial remodeling and emphysema in guinea pigs exposed to cigarette smoke for 6 months . In cancer, preclinical studies have shown anti - proliferative, pro - apoptotic, anti - invasive and radiosensitizing properties of statins . Various lipophilic statins such as lovastatin, simvastatin and fluvastatin exerted direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes, indicating potential use of statins in breast cancer . In 2008, bifulco et al ., suggested that statins may have potential to treat various other neurological disorders, including alzheimer's disease, parkinson's disease, multiple sclerosis and primary brain tumors as antioxidant, anti - inflammatory, anti - platelet activities and effects on the nitric oxide synthase system have been confirmed previously . In vitro studies, including a few animal studies, have suggested that statins may increase bone mass by enhancing bone morphogenetic protein-2-mediated osteoblast differentiation and activity . In addition, statins also exerted beneficial effects in rheumatoid arthritis by inhibition and downregulation of these cytokines and chemokines . In 2009, glynn et al ., reported an analysis of the jupiter, which showed that rosuvastatin nearly halved the risk for symptomatic venous thromboembolism vte in apparently healthy patients and the effects were ascribed to the pleiotropic effects of statins . A recent meta - analyses including one randomized and nine observational trials was conducted on nearly 1 million people who were on statin therapy . The results showed a 32% decrease in the risk of vte, 41% decrease in deep vein thrombosis and 30% decrease in pulmonary embolism . In women with polycystic ovary syndrome, statins have been reported to directly or indirectly block oxidative stress - mediated increase in thecal interstitial cell proliferation, steroidogenesis and insulin resistance . In seasonal and avian h5n1 influenza, administration of statins showed modulatory activities on various cytokines and increased the levels of other potential immunomodulatory molecules . The beneficial effect of statins in influenza was partly ascribed to their cardioprotective, anti - inflammatory and immunomodulatory effects . A global consensus on the concept of statin pleiotropy could not be reached as yet and remains a hot topic for debate . The pathophysiological implications of blockade of mevalonate production in the pathway leading to synthesis of cholesterol, that is, the mevalonate pathway suggested multiple mechanisms of statins . Over the years, analyses of several clinical studies, including the landmark hps and ascot - lla trial, reported findings with statins that were inexplicable with the lipid - lowering mechanism alone . The most probable mechanism of statin pleiotropy is prevention of isoprenoids synthesis as decrease in levels of circulating isoprenoids have been reported with treatment with statins.
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This is due to climatic factors, poor environmental sanitation and cultural habits in this region, which provide conducive atmosphere that allows transmission of the parasite throughout the year.1 it has been estimated that about 90% of the annual 500 million cases of malaria occur in the sub - saharan africa with 80% of the 1.5 - 3.0 million annual deaths due to malaria.1 most studies from sub - saharan africa showed that about 25 million pregnant women are at risk of malaria infection every year2 while it is estimated that 40% of the world's pregnant women are exposed to malaria infection during pregnancy.3 in nigeria, at least 50% of the population has malaria infection annually with under - five children and pregnant women at greater risk of the debilitating effects of the infection.3 malaria accounts for 30% of childhood mortality and 11% of maternal deaths in nigeria.1 in many african countries, malaria is holo - endemic and non - pregnant female adults have a significant level of immunity against malaria . However, during pregnancy, these women experience a considerable decline in their levels of immunity to malaria4 and several studies have reported that 1 and 2 pregnancies are associated with a higher prevalence of malaria in the first half of pregnancy in women living in endemic malarious areas.567 malaria is an important cause of maternal anaemia, intrauterine growth retardation, intrauterine death, still birth, premature delivery, low birth weight (lbw), perinatal and neonatal morbidity and mortality and post - partum morbidity.489 in sub - saharan africa, poor nutrition, micronutrient imbalances (especially vitamin a, zinc, iron and folate), human immunodeficiency virus (hiv) co - infection, poverty and limited access to effective primary healthcare and emergency obstetric services exacerbate the impact of malaria in pregnancy.8 women are particular at risk of cerebral malaria, hypoglycaemia, pulmonary oedema and severe haemolytic anaemia . Foetal and perinatal loss has been documented to be as high as 60 - 70% in non - immune women with malaria.3 these complications are commoner in primigravidae than multigravidae.34 the renewed interest in protecting and promoting both maternal and child health has led to the three pronged approach of tackling malaria in pregnancy, namely: intermittent preventive treatment of malaria using an effective antimalarial drug to address the heavy burden of asymptomatic infections among pregnant women living in areas of moderate to high transmission of plasmodium falciparum; the use of insecticide treated nets by all pregnant women and effective case management of malaria illness and anaemia.10 intermittent preventive treatment of pregnancy (iptp) involves the administration of therapeutic doses of an antimalarial drug to a population at risk whether or not they are known to be infected, at specific point intervals usually with the aim of reducing morbidity and mortality.11 in 2002, world health organization (who) developed a strategic framework for the control of malaria during pregnancy in africa . The document recommends that pregnant women receive at least two doses of sulphadoxine - pyrimethamine (sp) as intermittent preventive treatment during the second and third trimesters during the routine antenatal visit while chemoprophylaxis is no longer recommended for a no of reasons including the difficulty in the delivery of this strategy, poor adherence with weekly drug dosing and rising rate of resistance to most of the chemoprophylaxis regimens including chloroquine.12 recently, ipt has been shown to be better than malarial chemoprophylaxis and has replaced it.3411 the objective of this study was to determine the influence of the use of intermittent preventive treatment of malaria with sp during pregnancy on the prevalence of malaria in pregnancy and the outcome of pregnancy . The outcome of this would help to strengthen the use of intermittent preventive treatment of malaria by the pregnant women during routine antenatal care in this centre being a new and emerging teaching hospital . One of the guidelines in the routine management of pregnant women in our centre is to send blood sample of every pregnant woman with clinical diagnosis of malaria to the laboratory for examination for malaria parasite . The national iptp coverage is about 16.6% in the urban areas and 8.5% in the rural areas while the coverage is 14% for the south - west region.13 ipt of malaria with sp (500 mg sulphadoxine and 25 mg pyrimethamine) was given in the second and third trimester by direct observed therapy (dot) under the supervision of nurses at the antenatal clinic and usually documented in the case record along with the number of doses the pregnant women had taken . Pregnant women on ipt of malaria with sp are usually instructed to return to the clinic in case of any febrile illness . This was a descriptive cross - sectional study carried out at the maternity department of the ekiti state university teaching hospital, ado - ekiti, one of the referral centres in south western nigeria . Forty consecutive pregnant women at term, who gave an informed consent form or thumb - printing, were recruited into the study on each clinic day during the period of study . The social class of each woman was determined by adding the scores from the husband's occupation and woman's level of education as described by olusanya et al.14 the data were analysed using statistical package for social sciences (spss) software, version 15 . About 4,200 women recruited for the study participated by filling the questionnaires giving a response rate of 100% . Table 1 showed that 3,136 (74.7%) pregnant women received iptp while 1064 (25.3%) did not receive this . Out of the 3,136 women that received the iptp drug, 700 (22.3%) of them received one dose of iptp while 2436 (77.7%) received two doses of the iptp drug . Majority (76.9%) of the women that received iptp drug were multiparous while 23.1% of them were nulliparous . The age range of the respondents was between 19 - 41 years with a mean age of 31.19 4.88 years, the parity of the women was para 0 - 4 with a mean parity of 1.61 1.09, their gestational age at booking ranged from 9 - 34 weeks with a mean gestational age of 21.13 4.56 weeks and the gestational at presentation in labour was between 32 - 43 weeks with the age at 38.83 1.54 . The duration of labour was between 5 - 12 hours with a mean duration of 8.63 1.53 and the birth weight of their babies ranged from 2.2 - 4.4 with a mean birth weight of 3.27 0.40 . The socio - demographic characteristics of the pregnant women involved in the study table 2 showed the comparison of the characteristics between the women who received iptp and the women who did not receive the treatment . The comparison of demographic characteristics and obstetric outcome between pregnant women who used iptp and those who did not use iptp there was no significant difference in the mean age, mean gestational age at booking and delivery between the women that used iptp drug and those who didn't use but there was significant difference in the mean parity, birth weight and duration of labour of the women . More women who are multiparous, yoruba and with higher educational status and social class were associated with increased use of iptp during pregnancy in this study with p value of 0.001, 0.023 and 0.001, respectively . Table 3 showed the prevalence of malaria in pregnancy among the women who had iptp and those who did not have iptp . The prevalence of malaria in pregnancy among women who had iptp and those who did not have iptp there was a higher prevalence of malaria in pregnancy among women who did not receive intermittent preventive treatment of malaria compared to women who used it and p = 0.0001 . Table 4 showed the prevalence of malaria in pregnancy among the women who had a single dose of iptp and those women who had double dose of iptp . The prevalence of malaria in pregnancy among women who had single dose of iptp and those who had double dose of iptp among the women who used iptp and had malaria in pregnancy, the prevalence of malaria was higher in those who had a single dose of the drug compared to those who had two doses with p = 0.0001 . Table 5 showed the outcome of pregnancy among the women who had iptp and those who did not have during pregnancy . The outcome of pregnancy among women who had iptp and those who did not have iptp the mean gestational age at presentation in labour were comparable between the women who received iptp and those women who did not receive even though the women who received iptp presented at a higher gestational age in labour, however this was not significant, p value = 0.122 the duration of labour was higher in women who did not use iptp and the birth weight of babies was also lower in them, p values are 0.011 and 0.0001 . The present study revealed that more than two - thirds of the pregnant women studied had access to iptp of malaria and took the recommended two doses of sp . The high coverage recorded in this study is similar to that reported in previous studies done among pregnant women in beua and ibadan by takem et al.,12 and falade et al.,15 respectively, but higher than that in previous hospital based studies among pregnant women in tanzania and kenya, respectively, by nganda et al.,16 and van ejik et al.,17 this higher coverage was due to the fact that the study was done in the antenatal clinic where the drug was being administered by direct observed therapy . There was a low prevalence of clinical malaria among women who used intermittent preventive treatment compared to those who did not use it and this was statistically significant . Even among the women that took the drug, those that took two doses of sp experienced a lower prevalence of clinical malaria compared to those who took one dose of the drug . This is comparable to reports by takem et al.,12 and mbonye et al.,18 who reported a reduction in the prevalence of peripheral parasitaemia and parasite density among women of all parities . The increased use of intermittent preventive treatment with increasing level of education found in this study is not surprising since those with higher educational status are more health conscious and can easily apply health education programs to their daily living . This finding is consistent with report in previous studies by takem et al.,12 and marchant et al.,19 women in the higher social class and parity were also associated with greater tendency to use the intermittent preventive treatment drug . This is because women in that class tend to know and appreciate the benefits of the preventive measures against malaria in pregnancy like the use of insecticide treated nets and intermittent preventive treatment amongst others . Women who are multiparous comply more with antenatal instructions including the use of prescribed drugs compared to women who are primigravidae women since they would have discovered the benefits associated with these drugs in their previous pregnancies . This is similar to finding earlier reported by marchant et al.,19 but not consistent with that of takem et al.,12 who reported that the effect of socio - economic status on the use of iptp was close to null in their studies . Women who received intermittent preventive treatment during pregnancy had better outcome of their pregnancy . Low birth weight and prematurity are the greatest risk factors for neonatal mortality and a major contribution to infant mortality.15 in this study, babies born to mothers who received intermittent preventive treatment of sp on the average weighed more than babies born to women who did not received the drug . They also had shorter duration of labour which reduced the length of time the babies had to be exposed to the stress of labour . The birth weight of babies in this study may not only be attributed to the low prevalence of malaria in pregnancy in them since other factors such as socio - economic level may also play a role but this was statistically significant . This is similar to reports from falade et al.,15 mbonye et al.,18 and shulman et al.20 high educational status and gravidity are associated with the use of iptp among pregnant women attending antenatal clinic in this study and that use of sp as intermittent preventive treatment of malaria during pregnancy has been accepted by women as a malaria control strategy in pregnancy . However, much work still need to be done to improve the uptake of the drug especially among the pregnant women who are of low educational status and low parity, who incidentally had a higher prevalence of clinical malaria in this study and to ensure that all pregnant women received the recommended two doses of the intermittent preventive treatment of sp during pregnancy . In conclusion, the results from the study also showed that iptp of malaria during pregnancy with sp is beneficial in improving pregnancy outcome and is widely used by the pregnant women attending antenatal clinic though not optimal . Therefore, health education programs for pregnant women in this area should be intensified in women of low educational status and low parity to improve the uptake among them . Direct observation therapy system (dots) can also be employed in which the pregnant women would be asked to take the drug under the observation of the nurses in the antenatal clinic . This would help in improving the compliance rate since some women may get the drug and not use it . Government should be encouraged to make the drug available in the various antenatal clinics to be distributed to pregnant women free or at subsidized rate as part of efforts at improving maternal and child health to achieve the millennium development goals (mdgs) four and five.
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The main - stay therapy for this condition is chronic oral anticoagulation, however, in patients with a high bleeding risk profile or prior life - threatening bleed, conventional therapy may not be ideal . New advanced technologies such as transcatheter closure of the left atrial (la) appendage (laa) provides clinicians an alternative therapy to reduce the stroke risk in patients with contraindications to chronic oral anticoagulation . In this case, we present a 66-year - old male with chronic af who underwent laa closure and was found to have a large thrombus attached to the device a year later . This case is valuable for dealing with thrombus formation even after one year device closure . Several studies of the percutaneous transcatheter delivery of dedicated laa occlusion devices have shown promising results that offer an alternative to warfarin therapy for selected patients . Despite the encouraging results of several studies about the procedure, additional studies are needed to verify the safety and effectiveness of the devices . As in the present case, thrombosis might be a possible late complication and information to guide imaging monitoring and treatment is lacking . A 66-year - old male with an extensive past medical history including hypertension, hyperlipidemia, diabetes, metastatic prostate cancer treated with prostatectomy along with radiation and chemotherapy, previous ischemic stroke, known severe 3-vessel coronary artery disease with coronary bypass grafting surgery after myocardial infarction, preserved ejection fraction of 50%, and chronic af on anti - coagulation with warfarin . The patient suffered a fall resulting in left cerebellar hemorrhage, left temporal lobe subarachnoid hemorrhage as well as encephalomalacia of posterior right frontal lobe due to intraparenchymal hemorrhage . Due to his inability to tolerate long - term anticoagulation and high chads2 score of 5, he underwent percutaneous laa closure using a 16 mm amplatzer muscular ventricular septal defect closure device (aga medical corp ., plymouth, mn, usa). He was not a candidate for lariat device (sentreheart, redwood city, ca, usa) due to prior cardiac surgery . The device was deployed with no complications and no residual flow . The patient was placed on life - long daily aspirin 81 mg and a three - month course of clopidogrel 75 mg . Transthoracic 2d - echocardiogram performed post - procedure, 45 days, and at 6 month follow - up was unremarkable for device complications . However, the 1 year follow - up transthoracic 2d - echocardiogram revealed a spherical echo dense structure measuring 2.38 1.42 cm suggestive of clot in the left atrium (fig . 1, 2, and 3, supplementary movie 1 and 2). Transesophageal echocardiogram demonstrated a bilobed echo dense mass in the la, the smaller being 1.53 1.87 cm and the larger mass being 2.45 2.33 cm (fig . 4 and 5, supplementary movie 3). Repeat transthoracic echocardiogram six months later did not reveal any evidence of residual thrombus on contrast imaging . His warfarin was subsequently discontinued and he has been maintained on daily 81 mg aspirin . He has remained asymptomatic from a cardiac and neurological standpoint and is regularly followed by cardiology . Af is the most common sustained cardiac arrhythmia and increases the risk for stroke.1) laa is the site of around 7590% of thrombus formation in non - valvular af, and thus obliteration or exclusion of this structure, at least in theory, should lead to stroke risk reduction.2) three devices have been specifically designed for laa occlusion: the percutaneous left atrial appendage transcatheter occlusion (plaato), the watchman (atritech, boston scientific, natick, ma, usa) laa system, and the amplatzer cardiac plug (acp) (aga, st . Jude medical, minneapolis, mn, usa).3) the plaato device has been discontinued for commercial reasons . The embolic protection in patients with atrial fibrillation trial compared closure of the laa with the watchman device with long - term warfarin therapy and found it was non - inferior to standard anticoagulant therapy for stroke prophylaxis in high - risk af patients.4) recently, the asa plavix feasibility study with watchman left atrial appendage closure technology study found that laa closure with the watchman device can be safely performed without a warfarin transition, and is a reasonable alternative to consider for patients at high risk for stroke but with contraindications to systemic oral anticoagulation.5) a variety of amplatzer septal occluder devices (aga, st . Paul, mn, usa) have been available for many years and used extensively for closure of atrial and ventricular septal defects and other cardiac shunts . Eventually, a novel device, the acp, was developed for the purpose of closing the laa . Similar to prior trans - catheter laa closure devices, the acp system is delivered into the left atrium via a trans - septal approach . The relatively short length of the acp device allows for implantation into shallow laa variants, which gives it an advantage over the plaato and watchman devices that require a deeper laa anatomy because of their longer profiles.3) the incidence of thrombus formation on devices is relatively uncommon with prior studies showing an incidence for the watchman device thrombus formation between 24%.5)6)7) in a study by urena et al.,8) none of the 52 patients developed a device thrombus on the amplatzer device; however, there have been two case reports from europe.9)10) prior studies demonstrate high stroke risk score, high pre - procedural platelet count, and low left ventricular ejection fraction as independent risk factors for the formation of thrombus on the acp device.7) transthoracic echocardiogram has a lower sensitivity to identify la thrombus . Use of contrast - enhanced echocardiography significantly improves the sensitivity for identifying intra - cardiac masses when standard imaging does not reveal diagnostic information.11) individuals who will benefit from laa closure are probably those who are at the highest risk for both stroke and hemorrhagic complications; therefore, patient selection is of paramount importance in order to realize the optimal device and antithrombotic strategy . As in the present case, thrombosis might be a rare, but possible complication of device - based antithrombotic therapy, and additional studies are needed to know if the current practice of treating patients only with double - antiplatelet therapy before endothelization of the device is sufficient, or if one should use oral anticoagulation in the first 3 months as is advised in biological prostheses . In addition, periodic surveillance by echocardiography with contrast should be recommended to monitor the formation of thrombus in all types of devices . The frequency, duration, and type of echocardiogram (transthoracic or transesophageal) for thrombus formation monitoring still needs further research . Three chamber view movie clip showing a stalk with pedunculated thrombus contrast - enhanced echocardiogram movie clip showing enhanced view of thrombus transesophageal echocardiogram movie clip confirms a large bi - lobed thrombus
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Asphyxiation by an inhaled foreign body is a leading cause of accidental death among children younger than 4 years . In a recent series of 103 children with foreign body aspiration (fba), 64% of the patients were boys and the majority (73%) was younger than 3 years of age . The most common symptoms were sudden choking crisis (74%) and paroxysms of cough (73%). The most sensitive and specific clinical features were choking (86%) and witnessed aspiration episode (89%), respectively . Available chest radiographs revealed radio - opaque objects in 27% of patients . In another series, fba was suspected by the parents in 59% of patients while witnessing of choking episode was the most important historical event to pinpoint an early diagnosis of fba in children . Most aspirated foreign bodies are organic materials (81%), nuts, peanuts (59%), seeds, and fruits being the most common . Although jellies may also be aspirated in the lungs of small children consuming sweets, there are no reports describing characteristics of gummy or jelly sweets fba in the literature . Today, haribo0 is the biggest manufacturer of gummy and jelly sweets in the world, with its products mainly consisting of gummi bears, other jelly sweets and liquorice . We present a case of chewing gummi bear particles aspiration in a 5-year - old child . This is the first time that an extended haribo lung is described after a secret a previously healthy 5-year - old girl presented with a 24-hour history of sore throat, chest pain, and shortness of breath at the pediatric intensive care unit, university hospital, heraklion, greece . On physical examination a posteroanterior chest radiograph revealed right lung collapse and emphysema of the left lung, with tracheal deviation and mediastinal shift [figure 1]. Thoracic computed tomography scanning showed extensive multiple obstructions of the distal airways of the right lung which were initially suggestive of disseminated fba [figure 2]. Emergency bronchoscopy was performed and multiple small chewing gummi bear (haribo) particles impacted in the orifices of the right main bronchus and right lobar and segmentalinic bronchi were successfully removed and aspirated . Next day chest radiograph result was normal and the patient was discharged uneventfully without any complication . Chest radiograph showing tracheal deviation (black arrows), mediastinal shift (white arrow), left - sided hyperinflation, and low lung volumes and diffuse haziness on the right consistent with atelectasis ct scan displays multiple obstructed lobar and segmentalinic bronchi (black arrows) in the atelectatic right lung . Sudden onset of cough (72%), dyspnea (64%), and wheeze (60%) are the predominant symptoms and signs . The majority of foreign bodies (88%) lodge in the bronchial tree (right - sided 52%), with the remainder catching in the larynx or trachea . Only 11% of the foreign bodies are radio - opaque on radiograph, with chest radiographs being normal in 17% of children . Obstructive emphysema (53%) and normal chest radiograph (34%) are the most frequent radiological findings . Clinical and radiological findings of pneumonia and atelectasis are significantly more common in the groups with negative bronchoscopy or with delayed diagnosis . However, in toddlers with unexplained acute respiratory distress with refractory parenchymal infiltrates, unrecognized fba should be considered . Although rigid bronchoscopy is the traditional diagnostic gold standard, the use of computerized tomography, virtual bronchoscopy, and flexible bronchoscopy is increasing . Aspiration of gummi bears may cause a silent choking episode leading to life threatening severe respiratory complains, even in children older than 4 years . In a recent healthy lifestyle in europe by nutrition in adolescence (helena) cross - sectional survey girls selected more fruit juice, water, herbal infusions, and sweets . Gummy and jelly sweets have become a clear favorite, attracting a loyal fanbase which is constantly growing throughout the world . The chewing gummi bear (haribo, bonn, germany), a dancing bear molded from fruit gum [figure 3], has inspired a million different variant innovations in size, animal, shape, color, and flavor . This is the first time that a lung filled with gummi bears is described after fba in a child (medline search). The gummi / gummy bear is a dancing bear molded from fruit gum foreign body asphyxiation and ingestion needs a focus on education of parents and child caregivers regarding age, appropriate food, risk of play with small items, but also of older children consuming gummy or jelly sweets . Legislation for gummy sweets could be extended to children up to the age of 6 years, and to similar products marketed for children . In conclusion, we have reported our experience with a first case of a haribo lodged in the right main bronchus and right lobar and segmentalinic bronchi . Aspiration of gummy or jelly sweets may cause a silent choking episode leading to life threatening severe respiratory complains . Clinicians should keep a high index of suspicion for silent asphyxia episodes in children consuming gummy or jelly sweets, even in those older than 4 years old . Labels on these products should now include warnings for the danger of suffocation in all age's children.
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Bats were sampled in january and may 2007 at 6 sites in ghana: the center of accra (urban habitat); the wooded outskirts of accra (savannah habitat); and forested habitats at pra, kibi, adoagyiri, and oyibi (a plantation with woodland / forest border). Bats were captured by using 618-m mist nets; roosting eidolon helvum were captured by using nets on poles . A sample size of 59 would provide 95% confidence of finding at least 1 lbv - seropositive bat in a large population (> 5,000), given a seroprevalence of 5% and assuming random sampling (3). Captured bats were manually restrained and anesthetized by intravenous injection; 0.21.0 ml of blood was collected from the propatagial vein before the bat was released . Blood was centrifuged in the field at ambient temperature at 3,000 rpm for 15 min . Two species, epomophorus gambianus and e. helvum, were caught in sufficient numbers (117 and 66, respectively) for reasonable inferences to be made about lbv seroprevalence rates (table). A standard approach was used to calculate 95% confidence intervals (cis) for seroprevalence (3). Because of the relatively short distances between study sites and the likelihood of bats mixing between these sites, bats of each species were considered to belong to single populations . All but 3 e. helvum were derived from a colony in accra, whereas e. gambianus were derived from all habitat types . * phylogroup 1 contains challenge virus standard (cvs), genotype 1; phylogroup 2 contains lagos bat virus (lbv), genotype 2; ci, confidence interval; nt, not tested . Gambianus was caught in all habitats, including at the city colony and in plantations . Bat serum samples were tested for virus neutralizing antibody against classical rabies virus (challenge virus standard) by using a standard fluorescent antibody virus neutralization (favn) test (5). Antibodies to lbv were measured by using a modified favn test (6). Because positive bat antiserum from naturally infected bats was not available, for positive controls we used human rabies immunoglobulin, lbv - positive rabbit serum, and serum from mice vaccinated with human diploid cell vaccine . All samples were analyzed in duplicate and serially diluted by using a 3-fold series (representing reciprocal titers of 9, 27, 81, and 24319,683) (6). The modified favn test requires a cut - off threshold, which in prior bat lyssavirus studies has been a titer of 27, to avoid false - positive results (6,7). The first 121 samples collected were tested against the challenge virus standard; no tested bat was seropositive at 1:3 dilutions . A mean antibody titers to lagos bat virus (lbv) in 6 species of fruit bat in ghana . An lbv - specific modified fluorescent antibody neutralization test was used to determine the level of antibody in bats; it used two 3-fold serial dilutions and derived a mean dilution at which the bats neutralized lbv . 1, epomops franqueti; 2, epomophorus gambianus; 3, epomops buettikoferi; 4, eidolon helvum; 5, hypsignathus monstrosus; 6, nanonycteris veldkampii . Levels of specific virus neutralizing antibodies against lbv were higher in e. helvum (seroprevalence 37%, 95% ci 24%49%) than in e. gambianus (3%, 95% ci 0%7%). Of 6 epomops buettikoferi, no sex differences in e. helvum seroprevalence were evident (1.0, p>0.9). Because of the high level of seropositivity in e. helvum, we attempted to determine a possible case reproduction rate (r0) for lbv infection in this species by using the equation r0 = 1/x *, where x * = proportion of susceptible hosts in a population (9). We assumed that infection with each virus within the bat populations is endemic, stable, and randomly dispersed; that all seropositive animals have lifelong immunity that is detectable serologically; and that seropositivity is to 1 virus . On the basis of these assumptions, r0 = 1.6 (95% ci 1.32.0). Previous studies have suggested that healthy bats develop antibodies to other lyssavirus infections (7,10,11), which may reflect past exposure, rather than survival from clinical disease . Lbv likely co - evolved with its natural megachiropteran host until a genetic stasis had been reached in which the virus this situation would result in high seroprevalence rates after a wave of virus circulation in a colony . Nine seropositive bats (8 e. helvum, 1 e. buettikoferi) were apparently healthy pregnant females . These results support theories that lyssaviruses are endemic within specific bat populations, that they may not cause high mortality rates, that exposure rates of lbv between megachiroptera in old world african bats are high, and that bats may breed successfully after lbv exposure (7,8). The number of high reciprocal titers against lbv (figure 1) and the history of lbv isolation in e. helvum suggest that lbv circulates in megachiroptera in ghana . However, further work is needed to determine the specific phylogroup 2 virus and its prevalence within specific bat populations . No previous estimate of r0 for genotype 2 lyssavirus has been calculated, and although anamnesis may lead to no detectable antibodies in bats with immunity and a consequent underestimate of r0, this value indicates the potential r0 and is comparable to values previously estimated for lyssavirus infections in bats (7,11). More detailed analysis relating to age - specific infection and survival rates or mode of transmission was precluded by the difficulty in determining the age of adult bats, the lack of juveniles in the sample, and the cross - sectional sample used . The underlying cause of the difference in seroprevalence between e. gambianus and e. helvum with respect to lbv infection is unclear . Possible explanations include differential susceptibilities to infection; virus host adaptation; different contact with the virus, including a recent epidemic in the e. helvum colony; or different population ecology . E. helvum resides in high - density populations (hundreds of thousands) (figure 2, panel a) and migrates annually, compared with e. gambianus, which resides in less dense colonies of tens or hundreds (4). E. helvum commonly forms large colonies in african cities in close proximity to humans and domestic animals and is a food source in west africa (figure 2, panel b). No investigations into infections of humans were made during these investigations, but lyssavirus infections in humans in africa are underdiagnosed (12). Despite reduced pathogenicity of lbv in the laboratory, it has been isolated from dogs, cats, and a mongoose (2). Conversely, mokv has caused a fatal case of encephalitis in a human (1). Lbv and mokv each have a substitution in the r333 glycoprotein residue (1). Although it is not the only protein to determine the pathogenicity of lbv, the r333 substitution still remains an important marker of rabies pathogenicity . In conclusion, the high seroprevalence to lbv in this population may pose a substantial public health risk because e. helvum is widely distributed in africa and a food source in west africa.
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The calcium - sensing receptor (car), a g protein - coupled receptor that signals through gi, gq and sometimes g12/13 pathways, is best known and understood for its role in regulating the secretion and synthesis of parathyroid hormone in response to extracellular ca in the parathyroid glands . Like many other g protein - coupled receptors, the car signals through a defined set g proteins, but also interacts with other proteins that probably give it its unique signalling personality. This receptor was first cloned as an extracellular ca sensor using a xeno - pus oocyte expression system . Since that time, it has been detected in most epithelial and mesenchymal cell types including renal and gastrointestinal epithe - lium, endothelial cells, keratinocytes, breast tissue, osteoblasts, cardiac myocytes and cells of the central and peripheral nervous systems where it can be expressed in non - polarized cells and polarized cells on either the apical or basolateral membrane . The car appears to have distinct functions in these different cell types, although the functions are not precisely defined . For example, the car can either stimulate cell division (rat-1 cells) or inhibit division and promote differentiation (colonocytes or parathyroid cells) [36]. Additionally, its biologic effects in tissues such as the kidney or parathyroid glands cannot be explained completely on the basis of known second messenger signalling effects [1, 7]. The original model of signalling by g protein - dependent receptors was relatively simple . The receptors and g proteins through which the intracellular second messenger systems are activated are attached to the plasma membrane of the cell . These proteins could be relatively free in the membrane or loosely associated with each other . Upon activation of a receptor, the protein interactions change, g proteins are activated, which in turn activate effector molecules such as enzymes or ion channels, to generate intracellular signals (fig . Protein interactions have been developed and as more precise analysis of cell signalling has become possible, the inadequacy of the original model has become clear through demonstration that g protein - coupled receptors interact with many intracellular proteins in addition to g proteins . These interacting proteins include rgs proteins (regulator of g protein signalling), scaffolding and structural proteins, ion channels, additional signalling proteins, chaperone and trafficking proteins and others that may not have defined functions yet [811]. Receptors that act through similar sets of g proteins may have different signalling or biologic effects in different regions of a cell and in different cells . This finding suggests that they may have common activities based on the g proteins with which they interact, but that they may also have distinct functions based on the unique sets of other proteins with which they interact as well as their unique locations within a cell . Although no one receptor in a native tissue has been characterized completely, enough work has been done with different receptors including the 2-, -adrenergic and metabotropic glutamate receptors in various experimental systems to indicate that such a scenario is not only plausible, but likely (fig . 1) [911]. Four scenarios in which all g protein - coupled receptors interact with g subunits that regulate standard second messenger generation and other common proteins (grey triangle, e.g. Arrestin). They also interact with additional different proteins that give them unique signalling personalities . In a the receptor shown in b interacts with an accessory protein (e.g. A ramp) as well as another unique protein . The receptor shown in c has a long third intracellular loop that interacts with a unique protein (octagon), and a long c - terminus with a pdz domain that binds a pdz protein that itself brings additional proteins into the complex . In the scenario shown in d, the c - terminus of another membrane protein (e.g. A channel) interacts with the c - terminus of the receptor, and the receptor binds an additional protein . Most work on the signalling pathways controlled by the car has focused on traditional g protein - coupled pathways, gi, gq, and in some cases g12 or g13[1, 1216] (fig ., the car inhibits adenylyl cyclase and activates extracellular signal - regulated kinase (erk), through gq it activates phos - pholipase c, increases cai and dag (diacyl glycerol) levels, and activates phospholipase a2, and through g12/13, it activates rho and phospholipase d . Although the full physiologic significance is not understood, activation of the car initiates ca oscillations via a gq - dependent mechanism that when prolonged or forming a plateau, inhibit adenylyl cyclase activity co - ordinately with gi activation . This system serves as an active turn - off system for camp signals and depends on the forms of adenylyl cyclase expressed in different tissues . In the intestine, the car inhibits cholera toxin and e. coli heat stable enterotox - in - stimulated fluid secretion via activation of cyclic nucleotide phosphodiesterases and inhibition of nkcc1 (sodium, potassium, 2cl transporter-1) activity . In keeping with studies of other g protein - coupled receptors, the car transactivates, the epidermal growth factor receptor presumably via a matrix metal - loproteinase [19, the car is expressed on apical or basolateral membranes of epithelial cells where it is likely to come into contact with distinct sets of proteins that should give the car different signalling characteristics and biologic effects . Similarly, the car is expressed in many different cell types with different functions, so its signalling and biologic functions could vary from cell type to cell type . A schematic diagram of the principal second messenger signalling pathways that have been described for the car . Most of these studies pathways were identified in heterologous expression systems, and may not all exist in all cells where the car is expressed at all times . A good example of the unique signalling and physiologic activities of the car is found in the distal nephron of the kidney where activation of the car results in significant na, k, ca, mg, cl and h2o loss . Although the car acts via gi, gq and g12/13, its biologic effects in the distal nephron cannot be explained solely on this basis . A number of g protein - coupled receptors that also act via gi, gq and g12/13 are also expressed in the distal nephron, including those for angiotensin ii (at1), bradykinin (b2) and endothelin (etb). All of these receptors inhibit camp production, stimulate phospholipase c (with increases in cai and protein kinase c activity, and decreases in pip2 levels), and stimulate phop - spholipase a2 (with increases in 20-hete and other arachidonic acid metabolites). In all studies and experimental systems, activation of the car results in physiologically significant inhibition of na transport in the distal nephron, while activation of the at1 receptor (presumably at1) can stimulate or inhibit transport depending on the concentration, experimental system and study and the effects are minor [2131, 31]. These differences between the responses of the distal nephron to ca and aii indicate that the two receptors have distinct effects on cells despite the fact that they stimulate production of the same second messengers . The unique signalling characteristics and biologic effects of the car may be explained by its interactions with proteins in addition to g protein subunits (table 1). G protein - coupled receptors have four domains that are exposed to the intracellular space and that are available to interact with other proteins, three intracellular loops that connect transmembrane (tm) domains 1 and 2 (intracellular loop 1 or ic1), tm domains 3 and 4 (ic2), tm domains 5 and 6 (ic3) and the c - terminus . The intracellular loops, particularly ic2 and 3 interact with g proteins as well as other proteins including proteins involved in signalling, such as the arrestins or spinophilin . In g protein - coupled receptors, the size of the loops and c - termini are highly variable . In the car, ic1 and 3 are 14 aa, ic2 is 24 aa, all relatively short and the c - terminus is 220 aa, relatively long . Because of its size, the c - terminus is easier to use for techniques to identify interacting protein such as yeast two hybrid cloning . For the car, as well as the better - characterized metabotropic glutamate receptors (both have a similar structures), interacting proteins have generally been identified using yeast two hybrid assays with tissue - specific cdna libraries and the c - terminus as bait . The interactions are verified with co - immunopre - cipitation and the functional interactions are characterized in relatively generic expression systems, such as hek-293 cells . Although these car - interacting proteins have not been characterized completely in native tissue, enough data exist to believe that the interactions are real, and that the biologic functions they contribute are also likely to be real . Interactions with other proteins have been identified presumptively based on the fact that they are receptor interacting proteins and affect the behaviour of the car . A number of these interacting proteins such as -arrestin or receptor kinases are common to many other g protein - coupled receptors, or are involved in trafficking and degradation, and so are unlikely to be responsible for distinct signalling features of the car . Consequently, we will focus on three sets of proteins, receptor activity modifying proteins (ramps), filamin, a potential scaffolding protein, and two inwardly rectifying potassium channels, kir4.1 and kir4.2 that through their interactions with the car, could begin to explain some of its unique signalling characteristics . Proteins with which the car interacts . Table 1 lists the car interacting proteins that have been described so far alphabetically in the first column . The third column lists the experimental basis for the interaction of a protein with the car, y2h for yeast two hybrid, co - ip for co - immunoprecipitation, gst for gst pull - down assays and functional if expression or mutagenesis studies indicate that the interaction is functionally significant . The fourth column (car domain) the fifth column states whether co - localization studies of the car were performed with the interacting protein . Native means that studies were performed in native tissue, and het means that studies were performed in heterologous expression systems . The affinity of the car for its agonists or calcimimet - ics (agents that sensitize the car to activation by agonists), appears to depend on the cell type where it is expressed . In the parathyroid gland and parathy - roid cells, the ec50 for ca is approximately 1.0 mm, while for the car expressed in cultured cells, the ec50 for ca is on the order of 3.5 mm [32, 33]. In ker - atinocytes, the ec50 for induction of a differentiation marker, involucrin (presumably a car - mediated event), by extracellular ca is approximately 0.1 mm . Parathyroid cells are forty times more sensitive to cinacalcet for inhibition of parathyroid hormone secretion than are thyroid c cells to stimulation of calcitonin release . This variability in car responsiveness could be explained by its interaction with different sets of intracellular proteins or accessory proteins such as ramps, a family of proteins that affect trafficking, glycosylation, ligand specificity and second messenger production by the receptors with which they interact . Ramp1 was discovered using xenopus oocyte expression cloning to identify an accessory protein for the calcitonin - like receptor (clr) that could explain differences in ligand binding and sig - nalling observed in vivo and in different expression systems . Ramp1 is a 148 aa protein with a long extracellular domain, a single tm domain and a short cytoplasmic tail that interacts with both the calcitonin receptor (ctr) and clr . In these settings, ramp1 acts as a chaperone permitting cell surface expression and alters the affinity as well as the selectivity of these receptors for agonists . Ramp2 and ramp3 were subsequently identified using database searches and have similar structures and effects on the ctr and clr . Ramp3 has a c - terminal pdz domain, so it may have different functions from ramp2 and 3 . The ramps interact with their target receptors via their extracellular n - termini and tm domains . This family of proteins is relatively new and has not been studied extensively, so their tissue distributions, subcellular localizations and catalogue of receptors with which they interact are not fully defined, although ramps are expressed in all tissues studied to date [35, 36]. The level of ramp expression changes in physiologic states and disease models, so receptor activity could be modulated by this mechanism [36, 37]. Most work has focused on the ctr, the clr and their ligands calci - tonin, adrenomedullin, calcitonin gene - related pep - tides (cgrp) 1 and 2 and amylin . Adrenomedullin and the cgrp affect vascular tone and blood pressure [38, 39]. Ramps interact with other class 2 gpcrs, the vasopressin / pituitary adenylate cyclase - activating peptide (vpac)-1, parathyroid hormone1, parathyroid hormone 2 and glucagons receptors, but the functional and physiologic consequences of these interactions are not known . The car, a class 3 g protein - coupled receptor, interacts with ramp1 and ramp3 . The initial observation was that the car could not reach the cell surface in cos7 cells . Subsequently, the investigators found that cos7 cells do not express any of the known ramps . Interaction of the car with one of these ramps is required for cell surface expression . In the absence of ramp1 or 2, the car is trapped in the endoplasmic reticulum in an immature core gly - coslyated form . The ramps facilitate its exit from the endoplasmic reticulum, and its transit to the golgi where it is glycosylated and then moves to the cell surface . The functional consequences of interaction with ramp1 or ramp3 were not tested, so it is possible that interaction with the two different ramps leads to different car activation kinetics . The fact that ramp3 has a c - terminal pdz domain means that it could also contribute to localization of the car in the cell and with other signalling proteins . For example, ramp-3 interacts with n - ethylmaleimide - sensitive factor (nsf) via its pdz domain . Interaction with nsf is required for recycling of the agonist - occupied clr and 2-adrenergic receptor [9, 35]. At least two groups identified filamin as a car interacting protein that could serve as a scaffold for other signalling proteins [40, 41]. Filamin is a homodimer made up of 280 kd proteins that contain n - terminal actin - binding domains, 24 96 aa igg - like repeats, a c - terminal dimerization domain (repeat 24) and two hinge regions [42, 43]. Filamin was originally described as an actin cross - linking protein that provides mechanical strength to the actin cytoskeleton . Subsequently, it was found to interact with a number of tm proteins (many of them signalling proteins) and anchor them and the plasma membrane to the cytoskeleton . A partial list of these proteins includes integrins, ca and k channels, a subset of g protein - coupled receptors (the d2 dopamine receptor, the ctr, some metabotropic glutamate receptors, and the car), and the insulin receptor [11, 42]. Filamin also interacts with intracellular signalling proteins including map kinases, rho gtpases, rho guanine nucleotide exchange factors (gefs), rho kinase, smads and phosphatases [42, 43]. Filamin appears to be involved in organization of these proteins in the cell and in their trafficking in that under the correct experimental conditions, loss of filamin or interference with its interaction with the protein of interest results in altered membrane expression and lost or reduced function [44, 45]. A problem in understanding the function of filamin is that its distribution in various differentiated cells has not been fully determined . Despite the fact that filamin is generally considered a cytoskeletal protein, most of it is found in the soluble fraction of hepatocytes, endothelial cells and presumably other cell types [4648]. This distribution suggests that filamin may have a role in processes, such as protein trafficking in addition to anchoring proteins to the cytoskeleton and plasma membrane . One recent study indicates that in polarized epithelial cells, fil - amin may be preferentially expressed in the apical membrane, suggesting that in polarized cells, fil - amin's scaffolding function may be more important at the apical surface . The mid - portion of the car c - terminus interacts with the region of filamin that contains repeats 1517 and hinge 1 based in studies utilizing yeast two hybrid, co - immunoprecipitation and gst - fusion protein - binding assays . In the absence of filamin or when the interaction of filamin and the car is blocked, the car does not activate erk or jun n - terminal kinases (jnk) appropriately [40, 41, 50]. This interaction is also important for car - mediated rho activation because expression of peptides that interfere with the car - filamin interaction block car - mediated rho activation . The simplest explanation for these observations is that filamin contributes to cell surface expression of the car . This is certainly the case, but even in the absence of filamin some car reaches the cell surface . Evidence that fil - amin also provides an important scaffolding function comes from studies of ca or phe - induced car - mediated intracellular ca oscillations . The oscillations stimulated by phe require g12/13, rho, trpc1, an intact cytoskeleton and filamin, while the ca - stimulated oscillations persist with disruption of the interactions of filamin and the car [12, 52]. Although the precise mechanism by which filamin is involved in car - mediated inhibition of parathyroid hormone secretion on the apical surface of parathy - roid cells is not defined, an intact cytoskeleton is required . If filamin is primarily found on the apical surface of epithelial cells, its interaction with the car may be important for those cell types (e.g. Renal proximal tubule, intestinal and parathyroid cells) where the car is expressed on the apical surface . The c - terminus of the car interacts with and inactivates two inwardly rectifying k channels, kir4.1 and kir4.2 in the kidney that are expressed in the distal nephron (thick ascending limb of henle and distal convoluted tubule) as well as other tissues . Four kir subfamilies, kir2.x, kir4.x, kir5.x and kir7.x, are expressed on the basolateral membrane of the distal nephron where the car is also expressed [5558]. A number of channels undoubtedly contribute to the distal nephron basloateral k conductance, but studies indicate that the biophysical properties of a component of it are compatible with homomeric kir4.1, kir4.2, or heteromeric kir4-kir5.1 channels [55, 59, 60]. These channels are probably involved in recycling k for na, k - atpases (and possibly h, k - atpases) and regulating membrane potential . G protein - dependent signalling systems regulate kir channels by a number of mechanisms that involve protein protein interactions and second messengers including release of g subunits that interact directly with kir3 channels (girk) to activate them, and inhibition of kir3 channels by rgs (regulator of g protein signalling) proteins [6169]. The 2 adren - ergic and dopamine d2 and d4 receptors interact directly with heteromeric kir3 channels (kir3.1/3.4 and kir3.1/3.2), but the physiologic consequences of these interactions are not defined . Control of inositol lipid metabolism by plc and pi kinases regulate kir channel activity by determining the level of pip2, a lipid that is required for channel activity . The car interacts directly and selectively with kir4.1 and kir4.2 as demonstrated with yeast two hybrid assays and co - immunoprecipitation . The channels can be co - immunoprecipitated from kidney cortex, and kir4.2 and the car can be co - immuno - precipitated from heart and liver . The reason for this association may be for regulation of channel activity by direct interaction with the receptor, to localize pip2 metabolism near the channel, (the car interacts with and regulates pi4-kinase and regulates plc) or for trafficking to affect the stochiometry of the receptor channel complex [54, 71]. Kir4.1 and kir4.2 have c - terminal pdz domains, and may be organized in multi - protein complexes on that basis . Although frequently overlooked in the category of interacting proteins, g protein - coupled receptors interact with each other to form homodimers and with other receptors to form heterodimers . The car forms homodimers in the er via interactions of cystienes in the extracellular domain and this dimerization is important for cell surface expression [7274]. Interaction of the three intracellular loops of the car with other proteins has not been specifically demonstrated, but can be inferred from functional studies and knowledge of the behaviour of the potential interacting proteins . The car must make contact with the g protein subunits through which it acts, gi, gq and g12/13 . Mutagenesis of the second and third intracellular loops and naturally occurring mutants demonstrate that these loops contribute to activation of, and presumably interact directly with gq and gi because mutants are defective in plc activation, a gq - dependent event [75, 76]. These loops are usually sites of phosphorylation by g protein receptor kinases (grks) in other receptors, and sites for interaction with proteins like arrestin and spinophilin . Arrestins then bind to the phosphorylated receptor, block interaction with g proteins and initiate termination of the signal as well as internalization of the receptor [9, 10]. Arrestins are also capable of activating the erks via src kinase in a g protein - independent manner . Based on the fact that over - expression of grk 2 or 3 and -arrestins 1 or 2 reduces car signalling, these proteins probably interact with the car, but the sites of phosphorylation and of interaction were not defined, and interaction was not specifically demonstrated directly . Precisely how interactions with grks and arrestins affect car signalling and trafficking have not been studied . In addition to filamin and the k channels, kir4.1 and kir4.2, a number of other proteins interact with the car c - terminus . The c - terminus is phosphory - lated by, and so must interact with protein kinase c. this phosphorylation at thr 888 results in reduced intracellular ca store release . Pkc - mediated car phosphorylation may also be important for mediating the effects of -arrestins because signalling by a mutant car that lacks pkc phospho - rylation sites is not affected by expression of -arrestins . The degradation of the car is mediated by the ubiquitin system through interactions with the e3 ubiquitin ligase, dorfin and amsh (associated molecule with the sh3 domain of stam), a deubiqui - tinating enzyme [79, 80]. Both interactions were initially identified using yeast two hybrid cloning and co - immunoprecipitation or gst pull - down assays . As expected and consistent with effects of ubiquitination of other g protein - coupled receptors, over - expression of dorfin resulted in reduced levels of car protein while a dominant negative dorfin construct increased car protein . Dorfin can mediate degradation of immature car protein from the er . Although expression of amsh would be expected to increase car protein expression, it had the opposite effect . The car interacts with several other proteins, but the region of the car responsible for the interaction is not defined . Caveolin, a protein that also interacts with filamin, co - immunoprecipitates with, and co - localizes with the car in parathyroid cells [81, 82]. Caveolae are membrane microdomains enriched in cholesterol and glycosphingolipids that in contrast to lipid rafts also contain caveolin . These structures concentrate the components of g protein signalling systems, and caveolin may be involved in stable membrane expression of these proteins as well as endocytosis and sorting . Precisely how its presence in caveolae or its interactions with caveolin affect car function are not defined, but the correlation of reduced caveolin and car expression in parathyroid adenomas suggests that the interaction may be important functionally . This arrangement of proteins could result in a signalling module that regulates the metabolism of inositol lipids in a coordinated manner and in a limited region of the cell to precisely control processes such as channel activity, or it could contribute to protein trafficking that can also depend on inositol lipid metabolism [9, 54, 69]. Regulators of g protein signalling (rgs) proteins have been implicated in car signalling through expression studies, but interactions of the car or a car - based signalling complex with specific rgs proteins have not been demonstrated [16, 71]. G protein - coupled receptors act not only through het - erotrimeric g proteins, but though additional proteins that may interact directly with the receptor or be brought into proximity by scaffolding or structural proteins . A long - standing question has been why so many receptors exist to signal through the same g proteins . Part of the answer is that signalling by receptors through subsets of g proteins represents a common characteristic of many receptors, but that the unique signalling and biologic characteristics of receptors are determined by the additional restricted, and possibly unique proteins with which an individual receptor interacts . Studies of proteins that interact with the car are less advanced than studies of 2-, -adrenergic or metabotropic glutamate receptors, but some common themes are becoming clear . G protein - coupled receptors interact with each other in the er, an interaction that is important for trafficking and cell - surface expression . These receptors also interact with a restricted set of g proteins, although the location for the initial interaction is not established . Signalling by this class of receptors involves rgs proteins that participate in signal termination, can initiate signals (e.g. Rho gef), and that can contribute to the specificity of g protein - receptor interactions . G protein - coupled receptors interact with grks and second messenger - dependent kinases (e.g. Protein kinase c and protein kinase a), and arrestins as a part of the signal termination process and for receptor traffick - ing . The placement of these receptors in cells is specific and is determined by interactions with other proteins that may have structural, scaffolding, signalling, or mixed functions, and that may contain motifs such as pdz domains . Many interacting proteins such as arrestins have multiple functions that include sig - nalling and trafficking, so these aspects of receptor biology may not be separable . Based on current information, several interactions may distinguish the car from other gi, gq and g12/13-coupled receptors . Filamin appears to serve as a scaffolding protein that also affects car traffick - ing . The scaffolding function is probably important in all cells, but particularly so on the apical surface of polarized cells were filamin is enriched . Although the significance of the interaction with ramps1 and 3 is not fully established, these proteins have the potential to affect localization and signalling by the car . The ability of the car to interact directly with ion channels (kir4.1 and kir4.2) in tissues could permit tight control over channel activity through direct protein protein contact or with minimal dispersion of second messengers in the cell . Undoubtedly, additional interacting proteins will be discovered that will provide a more complete understanding ofthe function of the car and other similar receptors.
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Both fields have long recognized that the cutaneous surface is inhabited by myriad bacteria, fungi, and viruses and that these microbial communities are intricately linked to human health and disease (chiller et al ., 2001; fredricks, 2001; grice and segre, 2011; kong, 2011; leyden et al ., while many oft - studied organisms are pathogens, commensal microbes also play a significant, if perhaps often unappreciated, role in human health . Still open to question is the extent to which commensals provide direct benefit or simply prevent fellow harmful microbes from establishing residence . Three major questions that have been addressed with different methods over the decades are: (1) what microbes are present on the skin surface? (2) how does microbial diversity contribute to health and disease states? And (3) how do dermatologic practices alter microbial diversity? . Extensive reviews on the current state of skin microbiome research have been recently published (grice and segre, 2011; kong, 2011). This review represents a non - comprehensive historical overview of the> 5 decade long exploration into the relationships between resident and transient as well as commensal and pathogenic skin microbes . Finally, we discuss anticipated progress in the future and potential impact of external factors including antimicrobials on the healthy skin microbiome . Cool, acidic, desiccated healthy skin is a rather inhospitable environment for microbial growth . Epidermis represents a formidable physical barrier, resisting penetration by microorganisms while retaining moisture and nutrients inside the body (madison, 2003). Moreover, skin is a continuously self - renewing organ, with squames (and adherent bacteria) constantly shed from the skin surface as a result of terminal differentiation (elias, 2005). Although these obstacles would seemingly resist establishment of microbial inhabitants of the skin and skin appendages, we estimate that ~1 billion bacteria inhabit a typical square centimeter of skin covering the surface and extending down into the appendages and glands (grice et al ., 2008). The microbiota is generally conceived of as two groups: (1) resident microbes belong to a relatively fixed group of microorganisms that is routinely found in the skin, and that reestablishes itself after perturbation . Resident microbes are often considered to be commensal, meaning that the microbes are not harmful and may provide benefit to the host . (2) transient microbes do not establish themselves permanently on the surface, but rather arise from the environment and persist for hours to days . Resident and transient microbes are not pathogenic under normal conditions if proper hygiene is maintained and if the normal resident flora, immune responses, and skin barrier function are intact . However, after perturbation, resident and/or the transient bacterial populations can colonize, proliferate and produce disease . For example, staphylococcus epidermidis is a skin commensal but can be an opportunistic pathogen in immunocompromised hosts (otto, 2009). In addition, staphylococcus aureus can be a resident microbe in asymptomatic carriers, yet is also an important pathogen . Microbial diversity varies across the niches comprising the average adult human s 1.8 m of skin . For example, hairy, moist underarms lie a short distance from smooth dry forearms, but these two niches are ecologically distinct as are their resident microbial communities (grice et al ., 2009). Intrinsic factors including age, genetic makeup, and immune reactivity also influence the composition of skin microorganism communities . Environmental factors such as climate and extrinsic factors like hygiene may also have profound effects on microbial communities . The topography of human skin varies at a microscopic as well as a macroscopic level . Distinct habitats are characterized by differences in skin thickness and folds and densities of hair follicles and glands . Cutaneous invaginations and appendages, including sweat glands (eccrine and apocrine), sebaceous glands, and hair follicles, are likely to be associated with their own unique microbiota (figure 1). For example, sebaceous glands secrete lipid rich sebum, a hydrophobic coating that protects and lubricates hair and skin . While sebum generally serves as an antibacterial coating, propionibacterium acnes hydrolyses triglycerides present in sebum, releases free fatty acids that promote bacterial adherence, and then colonizes sebaceous units (marples et al ., 1971). This example shows how humans and their commensal microbial communities have evolved together to provide mutual benefit . Historically, culture - based approaches have been the standard for characterizing microbial diversity . However, some bacteria (including treponema pallidum, for example) require fastidious growth conditions and are notoriously difficult to isolate . Other bacterial species, e.g. Staphylococcus aureus, grow readily under standard culture conditions and subsequently overcrowd more fastidious bacteria . Comprehensive skin microbial surveys performed decades ago were limited by the ability to provide the appropriate growth conditions required to culture and isolate fastidious microbes . Despite these limitations, pioneering studies used cultivation techniques to identify members of skin microbial communities, including s. epidermidis and other coagulase - negative staphylococci . Other microorganisms that are generally regarded as skin colonizers include those of the phylum actinobacteria (the genera corynebacterium, propionibacterium, and brevibacterium) and the genus micrococcus . Demodex mites are microscopic arthropods, which feed on sebum and colonize sebaceous areas of the face (figure 1). The role of commensal viruses has not been studied, and investigations are currently limited by the available molecular and microbiological means to identify and characterize viruses . Research characterizing skin microflora was international, including early luminaries such as mary j. marples of new zealand, albert kligman and james j. leyden at the university of pennsylvania, david taplin at the university of miami, and william c. noble of the university of london . Kligman published one of the earliest comprehensive reviews on the subject in 1954, launching the field with the idea that a great deal more remains to be learned about the forces which control the bacterial ecology of the surface of the skin (pillsbury and kligman, 1954). We had the opportunity to interview noble and leyden about their perspectives on the history of this field . Mary marples brought ecological theory to the study of skin microflora making the analogy: in the soil the densest populations of microorganisms are in the rhizosphere, the region that surrounds plant roots . The comparable region in the skin is the hair follicle(marples, 1969). In marples one thousand - page the ecology of the human skin, numerous subjects affecting skin bacterial load and diversity are discussed and depicted with homunculi, scaled models of the human body illustrating the total volume of sweat produced, density and types of hair, regional distribution of eccrine sweat glands . Figure 2 is an adaptation of one of these homunculi, highlighting the body regions are that considered to be the main reservoirs (termed headquarters) and the distribution (termed range) of propionibacterium acnes (figure 2). The research of william c. noble, editor of the skin microflora and microbial skin disease, contributed to our understanding of how skin and nasal microbes play important roles in the epidemiology of bacterial and fungal infections, particularly in the hospital setting . When noble completed his phd in 1964, the field of microbiology in dermatologic conditions was largely confined to atopic dermatitis associated with staphylococcus aureus and acne with propionibacterium acnes . It soon became clear from our own studies and that of others that skin was a microbial habitat in its own right (noble, 1993). In describing his research interests, he stated, it should be evident that the research which i and my students followed was not always in a straight line . If it was plotted out it would resemble a plant, a stem rooted in dermatology but with many side shoots. Noble s work moved from microbiology to dermatology to public health . Noble described early baseline studies, obtaining samples from representative individuals in dutch villages to calculate s. aureus nasal carriage . When transporting the thousand bacterial cultures back to england, noble simply explained the public health relevance to the customs officer, who replied very good, sir. Many things were easier at that time local nurses went into schools to look for fungus in children s hair and no one questioned the nurses authority with their white coats and a swab in their hand . As an example of how the understanding of skin microbiology impacted public health needs, noble described frequent outbreaks of erythrasma in a mental health hospital where patients in this overrun facility only had water to bathe once a week build a new water tower was the simple scientific solution (somerville et al ., 1970). When a senior microbiologist learned that noble would move to an institute of dermatology he remarked s so much to do. And in fact, one of noble s salient comments during our interview was on the extent to which he enjoyed tremendous intellectual freedom . Not only were these young men exposed to completely novel microbial organisms, they were also exposed to harsh battle conditions . Taplin described foot lesions associated with pseudomonas cepacia in the toewebs of troops training in swamp water (taplin et al ., 1971). Taplin expanded these findings to characterize and distinguish tropical - immersionfoot versus warm - water - immersion - foot syndrome . Both of these immersion disorders caused a large number of foot casualties among combat forces in vietnam, but had different clinical presentations, histopathology, and preventative measures (allen and taplin, 1973). Taplin recapitulated warm - water - immersion - foot disorder with five volunteers who stood in florida swamp water for 36 to 72 hours . While recovery generally required 4 to 5 days in hospital, taplin and colleagues were instrumental in introducing topical applications of silicone grease as an effective prophylaxis against warm - water - immersion - foot . Leyden had a prolific career working closely with kligman for many years where we were simultaneously doing a hundred things . Underlying their productive research was intellectual freedom, financial support, and, perhaps as importantly, pure enjoyment . They described ecologic shifts in the cutaneous microflora related to long - term antibiotic usage as well as age - related changes . They analyzed a full range of skin changes associated with human life: skin microflora in diaper dermatitis, atopic dermatitis, acne vulgaris, dandruff . Their skin microbiology research was wide - ranging from gram - negative folliculitis as a complication of antibiotic therapy in acne to axillary odor . Leyden and kligman were prodigious writers, as prominently reviewed in a jid article in 1976 (kligman et al ., 1976). During the interview, leyden paid homage to other influential investigators in the field of skin microbiology including peter williamson, richard marples (son of mary marples), gerd plewig, bill noble, sydney selwyn, sam shuster, keith holland, howard maibach, and raza aly . Leyden recounted to us that at one point an 88-year - old kligman turned to him and said, we had a lot of fun, did nt we? Leyden responded, we had more fun than any two people would ever dare to dream . As these luminaries recognized, microbes flourish in the context of a large community and only a minority of bacteria thrive in isolation . Weeds species that flourish under the typical nutritional and physiological conditions used by diagnostic microbiology laboratories . For example, hair follicles and sebaceous glands represent anoxic environments, harboring anaerobic microorganisms that are often slow growing and that require special conditions for growth and during sample transport and processing . The development of molecular techniques to identify and quantify microbial organisms has revolutionized our view of the microbial world and ushered in another gold rush in studying skin microbes . Genomic characterization of bacterial diversity relies on sequence analysis of the 16s ribosomal rna gene, which is present in all bacteria and archaea but not in eukaryotes . The 16s rrna gene contains variable regions, enabling taxonomic classification, and conserved regions, serving as binding sites for pcr primers . Importantly, an organism does not need to be cultured to determine its type by 16s rrna sequencing (dethlefsen et al . Genomic approaches to characterize skin bacteria have revealed a much greater diversity of organisms than that revealed by culture - based methods, predominantly from four different phyla: actinobacteria, firmicutes, bacteroidetes and proteobacteria . Our group and others have demonstrated that the proportion of these bacterial phyla is dependent on the physiology of the skin site, with specific bacteria being associated with moist, dry and sebaceous microenvironments (figure 3) (costello et al ., 2009; the most diverse skin sites are the dry areas, with mixed representation from all four phyla . A surprising feature of the microbiota of dry sites, as captured by molecular analysis, is the abundance of gram - negative organisms, which were previously thought to colonize the skin only rarely, as contaminants from the gastrointestinal tract . As shown in figure 4, the microbiome of the antecubital fossa, back, nare, and plantar heel are more similar to the same site on another individual than to a different site on the same individual . In this sense, the ecological niche, or skin site, is a greater determinant of the microbiota composition than the individual genetic variation among healthy volunteers . Some sites on an individual are similar, such as different sebaceous regions that share common ecological features . Molecular analysis of skin microbiota has also revealed that the temporal variability of the skin microbiome is dependent on the site sampled . In healthy adults, skin niches including the nares, glabella, and external auditory canal demonstrate relative stability as compared to dry regions such as the inner forearm and the heel . In general, contralateral sites on the same individual are more similar to each other than to a corresponding site on another individual . Several studies have assessed skin microbial communities with molecular typing of 16s rrna genes with interesting results . An early genomics - based skin microbial study demonstrated that the microbial community composition of the palmar surface of the hand was significantly affected by handedness, time since last hand washing, and an individual s sex (fierer et al ., 2008). The knight group later examined whether the residual skin bacteria left on objects could be matched to the individual who touched the object (fierer et al ., 2010). The authors showed that skin - associated bacteria identified on inanimate objects could be linked to specific individuals . Investigators have compared the skin microbiome in infants delivered vaginally and by c - section just after birth . While vaginally delivered infants acquired bacterial communities resembling their own mother s vaginal microbiota, c - section infants harbored bacterial communities more similar to those found on the skin surface (dominguez - bello et al ., 2010). A recent study of the evolution of the infant skin microbiome demonstrated that cutaneous microbial communities become more diverse with increasing age (capone et al ., 2011). In 2008, the nih common fund launched an integrated human microbiome project to characterize the microbial diversity of 5 body sites (skin, nares, oral cavity, gut and vagina from women) of 250 healthy volunteers, the largest multi - body site study of the human microbiome to date . This project aims to take advantage of new, high - throughput dna sequencing technologies to establish a baseline to empower future clinical studies examining associations between changes in the microbiome and health / disease (peterson et al ., 2009). The project has already begun to serve as a tremendous resource for human microbiome investigators with regards to sequence data, technology and analytical tool development, and discussions on the social, legal, and ethical implications of this type of research . Dna sequencing studies enable researchers to concurrently search for a microbial contribution toa clinical disorder in the important context of the microbial community . Atopic dermatitis (ad), a chronic relapsing disorder that affects ~15% of us children, is a classic example of a disorder commonly associated with s. aureus microbial colonization and infection . The prevalence of ad has doubled or tripled in industrialized countries over the past three decades with no clear cause . This observation raises the intriguing possibility that environmental differences in industrialized countries, e.g. Reduction in parasitic infections, reduction in common childhood diseases, increase in hygiene practices, increase in antibiotic use, etc ., modulate the gene classic ad manifests at stereotypical sites, including the antecubital fossa and the popliteal fossa, sites that harbor similar organisms when compared to other body sites . Common effective treatments for ad include topical or systemic antibiotics, and steroids as well as dilute bleach baths to lower the bacterial load . Our group and others are currently investigating the interaction among different species of staphylococcus spp ., the microbial communities en toto, and disease course (hanifin, 2009). Future studies may provide insights into the potential role of s. aureus in the onset of ad and the therapeutic potential of selectively modifying the skin microbiome . Genomic approaches to studying skin microbes have also been utilized in acne vulgaris and plaque psoriasis . 16s - based sequencing has been used to study acne vulgaris, indicating that healthy follicles harbor p. acnes exclusively and acne lesions harbor a combination of s. epidermidis, corynebacterium spp ., and predominant p. acnes . Future skin microbiome studies of acne vulgaris may expand our understanding of how p. acnes behaves within communities of skin bacteria . In light of the common use of topical and systemic antibiotics in the treatment of acne, investigations in the acne skin microbiome may provide a deeper understanding of the mechanisms of antibiotic - resistance in cutaneous microorganisms . Lesional skin from plaque psoriasis patients exhibited increased bacterial diversity, including increased streptococcus and less p. acnes, than skin from healthy controls and nonlesional skin in psoriasis patients (gao et al ., 2008). Chronic wounds, affecting diabetic, elderly, and immobile individuals, are an example where commensal skin organisms invade and become pathogenic after breaching the skin barrier . Although bacteria do not cause the initial wounding event, they are thought to contribute to the lack of healing and persistent inflammation that is associated with chronic wounds . However, molecular studies have not yet identified a unique organism that colonizes wounds of the same etiology (for example, in diabetic foot ulcers or venous leg ulcers). This is in contrast to burn wounds, in which a particular microbial species, such as streptococcus pyogenes, enterococcus spp . Or pseudomonas aeruginosa is usually readily identifiable (grice et al ., 2010; martin et al ., 2010). Much remains to be learned about the possible role of microbes in skin diseases and how microbial communities interact with underlying genetic and environmental variations to potentially lead to human diseases . Many common skin disorders are associated with certain age groups, a specific body distribution, and/or particular microorganisms . Whether this specificity is driven by the endogenous microbial community structures dna sequencing studies enable researchers to concurrently search for a microbial contribution toa clinical disorder in the important context of the microbial community . Atopic dermatitis (ad), a chronic relapsing disorder that affects ~15% of us children, is a classic example of a disorder commonly associated with s. aureus microbial colonization and infection . The prevalence of ad has doubled or tripled in industrialized countries over the past three decades with no clear cause . This observation raises the intriguing possibility that environmental differences in industrialized countries, e.g. Reduction in parasitic infections, reduction in common childhood diseases, increase in hygiene practices, increase in antibiotic use, etc . Classic ad manifests at stereotypical sites, including the antecubital fossa and the popliteal fossa, sites that harbor similar organisms when compared to other body sites . Common effective treatments for ad include topical or systemic antibiotics, and steroids as well as dilute bleach baths to lower the bacterial load . Our group and others are currently investigating the interaction among different species of staphylococcus spp ., the microbial communities en toto, and disease course (hanifin, 2009). Future studies may provide insights into the potential role of s. aureus in the onset of ad and the therapeutic potential of selectively modifying the skin microbiome . Genomic approaches to studying skin microbes have also been utilized in acne vulgaris and plaque psoriasis . 16s - based sequencing has been used to study acne vulgaris, indicating that healthy follicles harbor p. acnes exclusively and acne lesions harbor a combination of s. epidermidis, corynebacterium spp ., and predominant p. acnes . Future skin microbiome studies of acne vulgaris may expand our understanding of how p. acnes behaves within communities of skin bacteria . In light of the common use of topical and systemic antibiotics in the treatment of acne, investigations in the acne skin microbiome may provide a deeper understanding of the mechanisms of antibiotic - resistance in cutaneous microorganisms . Lesional skin from plaque psoriasis patients exhibited increased bacterial diversity, including increased streptococcus and less p. acnes, than skin from healthy controls and nonlesional skin in psoriasis patients (gao et al ., 2008). Chronic wounds, affecting diabetic, elderly, and immobile individuals, are an example where commensal skin organisms invade and become pathogenic after breaching the skin barrier . Although bacteria do not cause the initial wounding event, they are thought to contribute to the lack of healing and persistent inflammation that is associated with chronic wounds . However, molecular studies have not yet identified a unique organism that colonizes wounds of the same etiology (for example, in diabetic foot ulcers or venous leg ulcers). This is in contrast to burn wounds, in which a particular microbial species, such as streptococcus pyogenes, enterococcus spp . Or pseudomonas aeruginosa is usually readily identifiable (grice et al ., 2010; martin et al ., 2010). There is a general agreement that microorganisms likely contribute to various skin disorders . Much remains to be learned about the possible role of microbes in skin diseases and how microbial communities interact with underlying genetic and environmental variations to potentially lead to human diseases . Many common skin disorders are associated with certain age groups, a specific body distribution, and/or particular microorganisms . Whether this specificity is driven by the endogenous microbial community structures another question is whether indigenous skin microorganisms provide some benefit to the host, and whether these microbes are truly symbiotic, or commensal . Skin microbes contribute to the skin innate defense by producing antimicrobial peptides . In a recent example of host and microorganism cooperating to combat invasion by pathogens, the commensal skin bacteria s. epidermidis was shown to inhibit nare colonization and biofilm formation by s. aureus . In combination, a subset of s. epidermidis expressing an endopeptidase and an antimicrobial peptide synergistically interfered with s. aureus colonization (iwase et al ., 2010). This example raises several important points for consideration, including complex microbial interactions and potential co - evolution of the host and resident microorganisms . In this 75 anniversary series, richard gallo reviews the relationship between the commensal microbes, potential pathogens, and the host response including immune cells and expression of innate defense proteins [ref needed]. His laboratory has elegantly demonstrated that commensal s. epidermidis affects inflammation and enhances keratinocytes expression of antimicrobial peptides to augment skin defense against infection (lai et al ., 2010; lai et al ., 2009). As our understanding of the roles of skin microbes expands, we may identify functions of skin microorganisms beyond contributing as protectors against pathogens . Skin microbes may educate our skin immune system analogous to that observed with effects of gut microbes on the development of the gut - associated lymphoid tissue in germ - free mice . Although molecular approaches to surveying bacterial diversity provide a less biased representation of skin microbiota than culture - based assays, they are not without their limitations . First and foremost, dna sequencing does not distinguish living from dead organisms . Since skin is an organ that is exposed to the environment, it is difficult to determine which of the identified species are transient and which species are members of the resident community . Moreover, 16s rrna gene sequencing cannot distinguish between methicillin - sensitive and methicillin - resistant staphylococcus spp . Future genomic studies will move from 16s rrna surveys of skin bacteria to 18s rrna surveys of fungi and then on to full metagenomic sequencing, simultaneously assessing the full genetic material of the microbial community . The current limitations of metagenomics are both the limited dna starting material and the development of computational methods to analyze the complex results . Antimicrobial therapies are a mainstay of dermatologic practice, but they have associated risks that are not fully characterized . Recent studies of the gut microbial community have demonstrated that treatment with ciprofloxacin resulted in profound and rapid loss of microbial diversity . Some but not all members of the microbial community began to recover within a week after completion of treatment . However, even after 10 months, the gut never returned completely to its baseline composition, suggesting that antibiotic perturbation may shift the community to an alternative state (dethlefsen and relman, 2011). Expanding on this idea, vancomycin - resistant enterococci (vre) were able to displace the normal recovering commensal organisms if mice were pre - treated with antibiotics . Similarly in human guts, colonization by vre preceded bloodstream infection in patients undergoing transplantation (ubeda et al ., 2010). These findings raise additional questions for skin biology, including exploring the impact of topical, repeated, or continuous use of antibiotics on microbial community stability and on beneficial microbes . As the current arsenal of effective antimicrobial weapons against potential pathogens is gradually diminished by antibiotic resistance, it is vital to explore not only antimicrobial chemicals derived from microorganisms, but also the possibility that prebiotics and probiotic microbial organisms themselves may offer promise as viable alternatives . Our current society paradoxically strives to colonize our guts with probiotic yogurts, but sterilize our skin with hand sanitizers . Rather than direct skin application of live bacterial cultures, we may need to identify emollients or other bacterial products to promote the growth of commensals . With continued expansion of our knowledge of the skin microbiota, these questions will guide future research efforts directed towards understanding the complex interactions governing the host microorganism relationship . Although molecular approaches to surveying bacterial diversity provide a less biased representation of skin microbiota than culture - based assays, they are not without their limitations . Since skin is an organ that is exposed to the environment, it is difficult to determine which of the identified species are transient and which species are members of the resident community . Moreover, 16s rrna gene sequencing cannot distinguish between methicillin - sensitive and methicillin - resistant staphylococcus spp . Future genomic studies will move from 16s rrna surveys of skin bacteria to 18s rrna surveys of fungi and then on to full metagenomic sequencing, simultaneously assessing the full genetic material of the microbial community . The current limitations of metagenomics are both the limited dna starting material and the development of computational methods to analyze the complex results . Antimicrobial therapies are a mainstay of dermatologic practice, but they have associated risks that are not fully characterized . Recent studies of the gut microbial community have demonstrated that treatment with ciprofloxacin resulted in profound and rapid loss of microbial diversity . Some but not all members of the microbial community began to recover within a week after completion of treatment . However, even after 10 months, the gut never returned completely to its baseline composition, suggesting that antibiotic perturbation may shift the community to an alternative state (dethlefsen and relman, 2011). Expanding on this idea, vancomycin - resistant enterococci (vre) were able to displace the normal recovering commensal organisms if mice were pre - treated with antibiotics . Similarly in human guts, colonization by vre preceded bloodstream infection in patients undergoing transplantation (ubeda et al ., 2010). These findings raise additional questions for skin biology, including exploring the impact of topical, repeated, or continuous use of antibiotics on microbial community stability and on beneficial microbes . As the current arsenal of effective antimicrobial weapons against potential pathogens is gradually diminished by antibiotic resistance, it is vital to explore not only antimicrobial chemicals derived from microorganisms, but also the possibility that prebiotics and probiotic microbial organisms themselves may offer promise as viable alternatives . Our current society paradoxically strives to colonize our guts with probiotic yogurts, but sterilize our skin with hand sanitizers . Rather than direct skin application of live bacterial cultures, we may need to identify emollients or other bacterial products to promote the growth of commensals . With continued expansion of our knowledge of the skin microbiota, these questions will guide future research efforts directed towards understanding the complex interactions governing the host microorganism relationship.
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Helicobacter pylori infection is the main known cause of gastritis, gastroduodenal ulcer and gastric cancer . After 30 years of experience in h. pylori treatment pylori antibodies are detected in blood few weeks after infection (active infection), in case of past exposure or after antimicrobial eradication . Moreover, individuals vary considerably in their antibody responses to h. pylori antigens . Therefore, the urea breath test (ubt) is considered to be a gold standard when using 37 kbq (1 ci) of c - urea for the c - ubt, the patient is not exposed to more radiation than is acquired from the natural environment in 1 day as almost all the ingested radioactivity is excreted from the body (urine and breath) within 72 - 120 h. thus, c - ubt use in children is justified without any fear of radiation phobia . The eradication rate of h. pylori by the standard 10-day triple therapy (a proton pump inhibitor [ppi], clarithromycin and amoxicillin; each given orally twice daily) is decreasing due to antibiotic resistance, mainly to clarithromycin . In contrast, the sequential 10-day therapy (a ppi and amoxicillin; each given orally twice daily for 5 days; followed by a ppi, clarithromycin and tinidazole; each given orally twice daily for another 5 days) is promising and achieves higher eradication rates . H. pylori infection has been found to be associated with low iron stores and iron deficiency anemia . In iron deficiency anemic h. pylori - infected patients, eradication of the infection could be effective in improving anemia and iron status . The difference from baseline to endpoint of hemoglobin, serum iron and serum ferritin was significantly different between anti h. pylori treatment plus oral iron and oral iron alone . Serum ferritin level is positively correlated with the total iron stores in the absence of inflammation where low serum ferritin reflects depleted stores . This study was therefore designed to investigate, which h. pylori eradication regimen; sequential or standard; more effectively improves the associated iron status and iron deficiency in children . The study protocol was approved by the research ethics committee and was performed in accordance with the international guidelines of medical research . The study was conducted on recently - diagnosed h. pylori positive; otherwise apparently healthy; intermediate school male children (12- 15 years). They were tested for h. pylori immunoglobulin g (igg) antibody and those positives were subjected to ubt to detect active infection . Children who consumed acid inhibitors, bismuth compounds or antibiotics during the previous 4 weeks or those with known allergy to antibiotics were excluded . Written informed consent was obtained from fathers of all subjects . Serum ferritin was measured for 18 boys, then they were randomized 1:1 into two groups (n = 9). One group received the standard eradication therapy consisting of rabeprazole 20 mg, clarithromycin 250 mg and amoxicillin 500 mg each administered orally twice daily for 10 days . The second group received the sequential eradication therapy consisting of rabeprazole 20 mg and amoxicillin 500 mg for 5 days, followed by rabeprazole 20 mg clarithromycin 250 mg and tinidazole 500 mg for another 5 days, each administered orally twice daily . Boxes containing a number of these tablets sufficient for the treatment duration were provided to each child . Six weeks after completion of eradication therapy, h. pylori status was assessed using ubt and serum ferritin was measured to estimate effects of eradication therapy on it . The preparations used were: rabeprazole (pariet, 20 - 40 mg, janssen - cilag, eisai co., ltd ., tokyo, japan), clarithromycin (claridar, 15 - 30 mg / kg) and amoxicillin (amoxydar forte 20 - 40 mg / kg), dar al dawa, naur, jordan and tinidazole (fasign, 50 - 75 mg / kg, pfizer ltd ., serum h. pylori igg level was measured by the enzyme linked fluorescent assay (elfa) test with biomrieux hpy - vidas system (marcy letoile, france). Three milliliter of blood was obtained from each subject and the sera were stored at -80c until analysis . The assay principle combines a two - step enzyme immunoassay sandwich method with a final fluorescent detection (elfa). It has a sensitivity of 98.10%, specificity of 90.82% and was interpreted as follows: <0.75 is negative, 0.75 to <1.00 is equivocal and 1.00 is positive . After overnight fast, boys swallowed 37 kbq (1 ci) of encapsulated c urea / citric acid solution (helicap, kibion ab, uppsala, sweden) with 25 ml water . Subjects exhaled into the breath card until its color indicator changed from orange to yellow . The breath samples were measured using the heliprobe analyzer and the activity was measured for 240 s. values <25 counts per minute (cpm) were defined as heliprobe 0 (not infected), 25 - 50 cpm as heliprobe 1 (equivocal) and> 50 cpm as heliprobe 2 (infected). Serum ferritin was quantitatively measured using the architect ferritin reagent kit (abbott laboratories, abbott park, il, usa), which combines a two - step chemiluminescent microparticle immunoassay technology with flexible assay protocols, referred to as chemiflex . In the first step, after washing, anti - ferritin acridinium labeled conjugate is added in the second step . Pre - trigger and trigger solutions are then added to the reaction mixture; the resulting chemiluminescent reaction is measured as relative light units (rlus). A direct relationship exists between the amount of ferritin in the sample and the rlus detected by the architect optical system . Normal range of serum ferritin in children aged 12 - 15 years old is 20 - 200 ng / ml . The statistical package for the social sciences (spss 18, ibm corp ., new york, usa) program was used . The chi - square () test was used for comparison of nominal data . Numeric values were given as mean values standard error of mean analysis was performed for paired data using two - tailed student's t - test . A value of p <0.05 was considered to be statistically significant . The study protocol was approved by the research ethics committee and was performed in accordance with the international guidelines of medical research . The study was conducted on recently - diagnosed h. pylori positive; otherwise apparently healthy; intermediate school male children (12- 15 years). They were tested for h. pylori immunoglobulin g (igg) antibody and those positives were subjected to ubt to detect active infection . Children who consumed acid inhibitors, bismuth compounds or antibiotics during the previous 4 weeks or those with known allergy to antibiotics were excluded . Written informed consent was obtained from fathers of all subjects . Serum ferritin was measured for 18 boys, then they were randomized 1:1 into two groups (n = 9). One group received the standard eradication therapy consisting of rabeprazole 20 mg, clarithromycin 250 mg and amoxicillin 500 mg each administered orally twice daily for 10 days . The second group received the sequential eradication therapy consisting of rabeprazole 20 mg and amoxicillin 500 mg for 5 days, followed by rabeprazole 20 mg clarithromycin 250 mg and tinidazole 500 mg for another 5 days, each administered orally twice daily . Boxes containing a number of these tablets sufficient for the treatment duration were provided to each child . Six weeks after completion of eradication therapy, h. pylori status was assessed using ubt and serum ferritin was measured to estimate effects of eradication therapy on it . The preparations used were: rabeprazole (pariet, 20 - 40 mg, janssen - cilag, eisai co., ltd ., tokyo, japan), clarithromycin (claridar, 15 - 30 mg / kg) and amoxicillin (amoxydar forte 20 - 40 mg / kg), dar al dawa, naur, jordan and tinidazole (fasign, 50 - 75 mg / kg, pfizer ltd ., co., tadworth, surrey, uk). Serum h. pylori igg level was measured by the enzyme linked fluorescent assay (elfa) test with biomrieux hpy - vidas system (marcy letoile, france). Three milliliter of blood was obtained from each subject and the sera were stored at -80c until analysis . The assay principle combines a two - step enzyme immunoassay sandwich method with a final fluorescent detection (elfa). It has a sensitivity of 98.10%, specificity of 90.82% and was interpreted as follows: <0.75 is negative, 0.75 to <1.00 is equivocal and 1.00 is positive . After overnight fast, boys swallowed 37 kbq (1 ci) of encapsulated c urea / citric acid solution (helicap, kibion ab, uppsala, sweden) with 25 ml water . Subjects exhaled into the breath card until its color indicator changed from orange to yellow . The breath samples were measured using the heliprobe analyzer and the activity was measured for 240 s. values <25 counts per minute (cpm) were defined as heliprobe 0 (not infected), 25 - 50 cpm as heliprobe 1 (equivocal) and> 50 cpm as heliprobe 2 (infected). Serum ferritin was quantitatively measured using the architect ferritin reagent kit (abbott laboratories, abbott park, il, usa), which combines a two - step chemiluminescent microparticle immunoassay technology with flexible assay protocols, referred to as chemiflex . In the first step, after washing, anti - ferritin acridinium labeled conjugate is added in the second step . Pre - trigger and trigger solutions are then added to the reaction mixture; the resulting chemiluminescent reaction is measured as relative light units (rlus). A direct relationship exists between the amount of ferritin in the sample and the rlus detected by the architect optical system . Normal range of serum ferritin in children aged 12 - 15 years old is 20 - 200 ng / ml . The chi - square () test was used for comparison of nominal data . Numeric values were given as mean values standard error of mean analysis was performed for paired data using two - tailed student's t - test . A value of p <0.05 was considered to be statistically significant . Two boys from the sequential therapy group were poorly compliant, considered drop - outs and excluded . 16 were included in the on - treatment analysis, with nine and seven in the standard and sequential groups, respectively . Six weeks after completion of therapy, h. pylori test on c - ubt was negative in 5 (55.6%) of nine patients in the standard and in 4 (57.1%) of 7 patients in the sequential groups . This eradication rate although slightly higher with sequential therapy, it is non - significant (p = 0.949) [table 1]. To exclude any direct effects of drugs (i.e., not through h. pylori eradication) on serum ferritin, it was measured post - treatment for all children (i.e., whether ubt was negative or positive) and non - significant differences were found [figure 1]. As shown in table 2, serum ferritin in the same group (standard or sequential), non - significantly differed before and after therapy . Between the two groups, serum ferritin after therapy, the difference between both groups was also non - significant [figure 2]. Effect of h. pylori eradication therapy, standard and sequential (n=9 and 7 respectively), on urea breath test (expressed as percentages of the total number of cases) effect of helicobacter pylori eradication therapy, standard and sequential (n=9 and 7 respectively), on mean serum ferritin after treatment (mean sf after tx) in case of success and failure of h. pylori eradication (negative and positive urea breath test respectively) effect of h. pylori eradication therapy, standard and sequential (n=9 and 7 respectively), on serum ferritin (ng / ml) effect of helicobacter pylori eradication therapy, standard and sequential (n=9 and 7 respectively), on mean serum ferritin after treatment (mean sf after tx) irrespective of h. pylori eradication it is used to diagnose active h. pylori infection in igg - positive subjects and to detect successful eradication after therapy . The current study showed that h. pylori eradication rate although slightly higher with sequential therapy, it was non - significant compared with that of standard therapy . Serum ferritin non - significantly differed between the two therapy groups and in the same therapy group before and after treatment . In a systematic review, it was mentioned that that while the majority of the randomized clinical trials (rcts) have shown superior eradication rates with sequential therapy, the largest rct from latin america did not find a significant difference between the standard and sequential regimens . It was concluded that sequential therapy has good efficacy; however, further trials other than those from asia and italy are required to assess its superiority over existing regimens before recommending it as the first line of treatment for h. pylori infection . In children with h. pylori infection, sequential therapy resulted in a higher eradication rate than standard therapy although the difference was of borderline statistical significance . In an italian study, eradication rate was found to be significantly greater with sequential than standard treatment; however, this may be due to the additional antibiotic (tinidazole) in sequential therapy . Incomplete follow - up in this study makes results non - generalizable to other countries . H. pylori infection was not significantly related to low serum ferritin or iron deficiency anemia . Moreover, h. pylori infection had a non - significant negative effect on serum ferritin in children and no significant difference in prevalence of iron deficiency was found between h. pylori - positive and negative groups . However, in another study, serum ferritin was significantly lower and iron deficiency was significantly higher in h. pylori - seropositive than in seronegative children . The benefit of h. pylori eradiation for iron deficiency anemia has been extensively studied, but data are still equivocal . In a double - blind randomized trial among non - iron - deficient asymptomatic h. pylori - infected children living in the contiguous united states, no effect of h. pylori eradication regarding changes in iron stores was found . However, those who had their infection eradicated by the quadruple sequential eradication at follow - up had a significantly larger increase in serum ferritin from baseline . In contrast, h. pylori infection was found to be correlated with lower serum ferritin and iron deficiency . After eradication, serum ferritin increased and approximately half of persons resolved their iron deficiency . This controversy may be explained by a seroepidemiologic study in school children in pondicherry in india that mentioned that response to anti - h . Pylori therapy in children with iron deficiency could establish the causal relationship between iron deficiency anemia and h. pylori infection only after other causes of iron deficiency such as hookworm infestation and poor iron intake have been identified and treated . A limitation of the present study is that it was conducted on a relatively small number of male children . H. pylori eradication rates in children were non - significantly different in sequential versus standard therapy . Moreover, serum ferritin was non - significantly different between both therapies and in the same therapy group before and after treatment . Further studies enrolling more markers of iron deficiency are required to precisely assess the relationship between h. pylori eradication therapy and iron status and to determine which regimen, sequential or standard, is considered superior in this respect.
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If there is any litmus test for measuring the familiarity with japanese popular culture, seirogan might be a good one . Looking and tasting like charcoal, as a renowned japanologist recalled, this soft, pellet - shaped, anti - diarrhoeal herbal medicine is a symbol of katei jbiyaku (), a generic over - the - counter pill that any japanese household stocks and uses to treat minor digestive disturbances in everyday life . Intimately and unmistakably linked to the idea of travel, seirogan is currently manufactured by more than thirty different pharmaceutical companies in japan . Alone sold roughly 41 million bottles of seirogan (or 2.8 billion yen in sales revenue) from 1995 to 2004, and the pill is now exported to canada and the united states (us) as well as korea, taiwan, and china . Given the embeddedness of seirogan in the everyday lives of the japanese, which can be roughly approximated to the ubiquity of aspirin in western countries, it is hardly surprising that nationwide consternation erupted between 1998 and 2000 in the wake of the allegation that wood creosotes the pill s main ingredient increase the likelihood of liver cancer . A dependable travel companion for japanese people, from a middle school student participating in a shgaku - ryk (school trip) to a professional flight attendant travelling globally, seirogan tells us much more than a simple meteoric success story of a well - advertised, over - the - counter drug . What is especially intriguing to historians is the less than subtle way that seirogan conceals its past, which is as obtrusive as its characteristic odour, in its very name . In kanji, one of the japanese writing scripts, seirogan () currently represents efficacious - dew - drops. Before 1949, however, seirogan () had a totally different meaning, and the secret lies in the nature of kanji and its use of chinese characters . Dew, but it also refers to russia as a country . Combined with the chinese character, sei (), meaning conquer, seirogan before 1949 meant conquer - russia - pill, representing strong hostility against russia and subsequently against the soviet union . Only after 1949, with the guidance of the ministry of health and welfare (kosei - sho,), who feared the potential political fallout of this rather unpleasant designation of an immensely popular medicine, pharmaceutical manufacturers began to use the homonym, sei (), thus keeping the same pronunciation, but changing the word s meaning radically from a hostile and imperialistic one (conquer russia) to an innocuous even aesthetic one (efficacious dew). Despite its significance as the symbol of the medicalisation and militarisation of japanese society in the twentieth century, historians have paid little, if any, attention to seirogan . A freelance journalist, machida shinobu, is probably the most prolific and authoritative writer on the study of seirogan; his pioneering collection of materials on seirogan is exemplary, but his analysis of it solely in the context of japanese traditional medicine does not reflect the larger social and cultural changes of modern japan . Tanaka satoshi superbly analyses the labels and iconography of various japanese drugs, but his treatment of seirogan is rudimentary . When addressing this topic, historians of pharmacy have invariably paid disproportionate attention to the technical aspects of wood creosote . What professional historians have left as a lacuna has been filled by various urban legends . Originating during the time of the russo - japanese war of 1904 and 1905, seirogan is said to be a pharmaceutical product commissioned by the meiji emperor . In preparation for the war, according to one of these legends that made its way even into a newspaper in korea, the meiji emperor allegedly issued an imperial ordinance searching for the ultimate cure for dysentery, which accounted for numerous japanese deaths during the sino - japanese war ten years before . The drug that proved particularly effective against diarrhoea, so we are told, was named seirogan, the conquer - russia - pill, and has remained a companion of the japanese people . Of course this narrative is not entirely true, but it seems convincing and persuasive among the lay public primarily for two reasons . First, it fits nicely into the still prevalent, conventional narrative of the russo - japanese war (19045); in particular, it jives with the image of the jingoistic rioters who stormed hibiya park on 5 september 1905 upon hearing news of the humiliating treaty of portsmouth at the close of the russo - japanese war, where japan would receive no outright gains of land, let alone a war indemnity from russia . Second, it aligns with the interpretation of japanese imperialism emphasising formal or informal governmental intervention and manipulation . In other words, as a medical product entrenched in the ideological milieu of wartime japan, seirogan seems to illustrate how a medical product was appropriated as an imperial ideological tool in rallying the nationalist spirit of the japanese people through its medicinal and symbolic efficacy and how the government played a critical role in the process . But the story is much more complicated than has been suggested or believed, not to mention the lack of documentation substantiating the nationwide competition or an imperial ordinance from the meiji emperor . In this article i aim to untangle the process through which seirogan became deeply entrenched in the everyday life of the japanese people in the years from 1905 to 1945 . Recognising that pharmaceutical medicine is a medium through which one can achieve a deeper understanding of people s everyday lives in relation to state and society, i maintain that seirogan reveals unique relationships between the state, emerging commercial manufacturers, and the voracious and patriotic japanese people . In particular, i argue that it was not the japanese government that realigned the relationship of the people to the state by enlisting pharmaceutical manufacturers, and thus instilling a sense of militarism in the minds of the ordinary japanese; rather, it was pharmaceutical manufacturers who took advantage of existing patriotic fervour for their commercial interests and, in the process, reinforced the ideology of militarism on a quotidian level . When the japanese swallowed the pill, they were not just digesting a highly effective pharmaceutical product, they were consuming a cure for empire . This pecuniary inculcation and appropriation of patriotic feelings and pride by pharmaceutical companies is evident in the language of contemporary advertisements for seirogan . Carefully worded and impressively drawn for maximum commercial impact, visual images with normative overtones, a contributor to the society s shared daydreams, as we are told by roland marchand with regard to the american case . And increasingly in the last decade, historians of medicine have emphasised the crucial roles played by pharmaceutical companies, not merely as a thermometer that measured and reflected social and cultural realities, but as a thermostat that actively transformed social imagery and created favourable market conditions . Nancy tomes, for instance, emphasised in her fielding h. garrison lecture of 2004 the predominant effectiveness of pharmaceutical advertising and marketing activities in the united states for the past century that simply overpowered the movements of consumer activists and physician reformers in defining medical culture and policies . Geographically closer to japan, sherman cochran similarly highlighted the foundational role of chinese medicine men both chinese entrepreneurs selling western - style drugs and those selling traditional chinese medicines in creating a burgeoning consumer society in china during the first half of the twentieth century through their conscious adoption of various marketing schemes and media, including of course carefully concocted advertisements . Japan as a maturing nation - state and fledgling empire was no exception to the global ascendance of commercial advertisement as a new source of common vocabulary and to the diligent appropriation of new technologies by pharmaceutical companies . As susan burns superbly illustrates in her groundbreaking article, patent medicine advertisements in meiji and taish japan signified visual icons that did more than merely sell the products. In trying to carve out their own market in a hostile commercial environment, pharmaceutical manufacturers such as tsushima juntend, morishita hiroshi yakub, and yamazaki teikokud often amplified and propagated the governmental vision of health, nation, and subjecthood in their advertisements . Yet, as burns shows us, manufacturers were not merely ventriloquising the official ideology: their messages asserted that bodily health can be attained through the market, circumventing the regulatory power of medicine professionals and institutions and the state that sustained them. This competing yet complementary relationship between the state and pharmaceutical manufacturers during the meiji (18681912), taish (191226), and early shwa (192689) eras is replicated in the advertisements for seirogan . But before we turn to the colourful and evocative world of advertisement for seirogan in the 1930s, where the distinctive strategies of nakajima pharmaceutical in osaka transformed the drug into a symbol of empire and patriotism, we first need to tackle the vexing enigma of seirogan: who were the inventors of the drug? When was seirogan first manufactured in japan? This is a rather simple question, but one that has been contested for a long time due to the pill s popularity and obvious commercial potential . Aside from the urban legend recounted in the introduction, there is a claim from the leading manufacturer of seirogan, the aforementioned taik pharmaceutical from osaka . According to the official company history, seirogan was originally introduced in japan in 1902 when it was manufactured and sold by nakajima saichi under the name of chy - seirogan (: loyal - and - brave - conquer - russia - pill). The company website lists the sales licence issued by the osaka district office, but does not provide any evidence proving that the sales licence was indeed for seirogan and that they were the manufacturer who supplied the government with the pills, making the overall claim vulnerable to contestation . Totsuka michitomo (), an army physician and instructor at the army medical college, was experimenting with select cultures of typhoid fever and e. coli when he accidentally discovered that creosote was effective in reducing diarrhoea . Totsuka brought this to the attention of one of his students, shiraiwa rokur (), who had been studying bacteriology, and together they created the pill for the use of japanese soldiers . Machida mentions in his book that he has documentation from the national diet library, but he does not specify which documents he consulted . Given the lack of a smoking gun document that can substantiate the real inventor of seirogan or kureostogan (creosote pill), as it was called within the military, kasai (or kawanishi) kenji (, 18681927) emerges as the most likely candidate . According to mizokami kuniyoshi, the editor of the diary of army physician mizokami sadao (), a participant in the russo - japanese war, kasai was the creator of the pill . A graduate of the medical faculty of tokyo imperial university and a veteran army physician of the sino - japanese war, kasai helped totsuka to create a workable pellet called kureostogan . Although mizokami does not refer back to any primary source, the circumstantial evidence supporting kasai s candidacy is strong . In 1906 kasai was ordered to study internal disease in germany, and at the completion of his two - year research sojourn in munich, he submitted six theses for a doctoral degree of medicine to kyoto imperial university . Of notable interest are two of his theses written in german on the efficacy of creosote as a medicinal ingredient: kureosto no ch ni oyobosu say ni tsuite [on the use of creosote on intestines]; kureosto no chiryteki y ni tsuite [on the therapeutic application of creosote]. It would not be too far - fetched to postulate that kasai made his name known during the russo - japanese war for his work related to the creosote pill and that, for this notable achievement, he was given the honour of studying in germany with the support of the ministry of war . Kasai, an expert on parasitic diseases, especially schistosomiasis, assumed the position of chief sanitation officer of the south manchurian railway company (mantetsu), the notorious quasi - governmental spearhead organisation of the japanese empire in manchuria, where he stayed until he eventually rose to become the first president of the medical college of south manchuria . If the creosote pill was the invention of this army physician, when was seirogan first introduced to the soldiers in the battlefields in manchuria? The detailed day - to - day diary of army staff physician mizokami sadao evinces that, on 27 april 1904, kureostogan was distributed for the first time, two and a half months after the onset of the war . Later, in the entry of 28 may 1905, mizokami includes a report of dysentery pill consumption in the month of may . Among the 170 soldiers in the third company, 145 consumed entire doses while twenty of them took two - thirds and five of them took only one - third of the allocated doses . What is interesting in this entry is that the name of the pill has been changed from kureostogan to seirogan in the meantime . Machida claims that the pill s official designation remained kureostogan through the end of the war, but the diary of mizokami claims otherwise, which is confirmed by the imperial army s own supplementary expense report demonstrating that, as early as june 1904, the pill was called seirogan . Also significant to our discussion of seirogan along with the issue of its inventor and adopted name is that its extensive use from 1904 was noticed even by outside observers from europe . Quoting the magazine chemist and druggist as its source, the times of london reported on 19 november 1904 one of the heaviest consumptions of drugs and medicines in the war in the article titled japanese war drugs: at least 50,000 cases of medicines and medical requisites have been shipped from japan to the port of dalny, whence they are distributed among the troops in manchuria beechwood creosote is one of the most interesting articles in use; it is supposed to prevent dysentery, which is very prevalent in manchuria, and the medical department decided to give each soldier a pill containing 0.25 creosote at a meal, or three pills a day . Each soldier s requirements are nicely packed in small tins, each containing 90 pills, or a month s supply; they are labelled russian expedition pills. At the busiest time 2,000,000 pills a day were made by the army s tablet and pill works in tokio . During february the government bought all the available stocks of creosote, and consequently the price has advanced from 1.50 yen per pound (the normal market price) to 4 yen! At least 50,000 cases of medicines and medical requisites have been shipped from japan to the port of dalny, whence they are distributed among the troops in manchuria beechwood creosote is one of the most interesting articles in use; it is supposed to prevent dysentery, which is very prevalent in manchuria, and the medical department decided to give each soldier a pill containing 0.25 creosote at a meal, or three pills a day . Each soldier s requirements are nicely packed in small tins, each containing 90 pills, or a month s supply; they are labelled russian expedition pills. At the busiest time 2,000,000 pills a day were made by the army s tablet and pill works in tokio . During february the government bought all the available stocks of creosote, and consequently the price has advanced from 1.50 yen per pound (the normal market price) to 4 yen! The production of seirogan in the army s own tablet and pill factory discredits the claim by taik pharmaceutical that they (or their predecessor, nakajima pharmaceutical) were the manufacturers of the army supplies . 2,000,000 pills a day, which was equal to a daily supply of three pills to over 700,000 soldiers and the nearly tripled price of creosote in the global market in other words, how serious was the damage from dysentery and related diseases for the japanese army? The japanese army had struggled with diseases such as dysentery, typhoid fever, and beriberi (kakke in japanese) ever since meiji japan introduced universal conscription in 1872 and started building up its military capacity . During the sino - japanese war of 18945, only 1,594 died in active combat on the front lines; the remaining 11,894 soldiers, or eighty - eight per cent, died from sickness . 949 out of 1,306, or seventy - two per cent, died from diseases, but the fatality rate was still alarming to the japanese army leaders who were to shoulder the task of expansion into manchuria and eventually into the chinese mainland . Thankfully, the return of a cohort of military - sponsored physicians from germany in the late 1880s and early 1890s provided the japanese government with sorely needed experts . With mori rintar (better known by his pen name, mori gai, and his eventual position as army surgeon - general) as the most renowned example of this cohort, these military physicians, who had been exposed to cutting - edge hygiene and bacteriology in berlin and munich, were determined to fight the disease enemy from multiple angles . On one hand, the military adopted various preventive measures, such as the modernisation of the rationing system, the improvement of the hygienic situation in the barracks, and the provision of a clean potable water system . On the other hand, military medical personnel strove to provide curative methods to treat diseases, and seirogan was developed in this context . While the vanity of germany - educated bacteriologists sometimes had a disastrous impact on the lives of the japanese soldiers for instance, ogata masanori and mori rintar firmly believed that beriberi was caused by germs, disputing takaki kanehiro s idea that the cause of beriberi would be found in a diet based on white rice the combination of preventive and curative measures dramatically reduced fatality from diseases . Of the 84,435 army soldiers who died in the russo - japanese war, only 23,093 or twenty - seven per cent died from sickness . In other words, the fatality rate from diseases dropped from eighty - eight per cent to twenty - seven per cent in the short span of ten years . This notable achievement was striking enough for louis livingstone seaman, an american surgeon - major, to dedicate his 1906 book, the real triumph of japan: the conquest of the silent foe, to the medical and sanitary officers of the japanese army . As a foreign military attach to the second imperial army of japan on the mongolian frontier during the russo - japanese war, seaman witnessed how cutting - edge hygienic medical facilities and techniques such as a state - of - the - art water - testing outfit, microscopes, and a portable x - ray apparatus were working seamlessly in the japanese army . As a result of this innovation, seaman observes, malaria is malaria and typhoid is typhoid in the japanese army, and not fever, caused by inappropriate and irritating rations, because every case there is differentiated under the microscope and otherwise . Diseases are not guessed at. Just as a foreign observer was inspired by the medical prowess of japanese army physicians and their medicinal products, at the close of the war returning japanese soldiers brought home stories of their stunning victories over the russians along with stories of seirogan . Among them, the most famous though unsubstantiated was the story of peasants in manchuria who were willing to give up a chicken for a single pellet of seirogan . Nishimura, the author of the guni no mitaru nichiro sens of 1934, introduces yet another story in which a local resident in the hida () region voluntarily gave his seirogan that he himself had saved for many years after his service in manchuria to a dying visitor to his village . Imbued with a mythic aura, seirogan, the cure for empire, finally came home . It has often been believed that the victory of japan over russia the first defeat of a european power by non - europeans intensified a sense of belonging among the japanese people while elevating the status of the military in the society and minds of the japanese . This narrative of enthusiastic popular nationalism instilled and fuelled by the war, however, has recently been challenged by a new interpretation that emphasises the plurality and diversity of japanese society before, during, and after the war . Most notably, naoko shimazu, with her careful analysis of private materials such as diaries of japanese conscripts and her emphasis on local dimensions of wartime mobilisation and commemoration, argues that the wartime japanese society of 19045 was anything but a unified society of fervent patriots under the direct guidance of a strong central government; instead, it was a war - weary and ambivalent populace . Of course, shimazu does not necessarily believe that all of the japanese were despondent . If anything, some segments of society were vociferously patriotic, such as the pro - war factions, the commercial sector (including the media) which saw war as a lucrative bedfellow, as well as those (mostly in the middle classes) in society who could afford to partake in the patriotic bubble of the wartime economy . Japanese pharmaceutical manufacturers were one of such commercial opportunists who lost no time in capitalising on the patriotic fervour . An advertisement for seiro (), a product of pharmaceutical manufacturer hirose - shten () from 1906, is a clear example of this trend (figure 1). Some segments of society were vociferously patriotic, such as the pro - war factions, the commercial sector (including the media) which saw war as a lucrative bedfellow, as well as those (mostly in the middle classes) in society who could afford to partake in the patriotic bubble of the wartime economy . A proud army officer is holding the kyokujitsu - ki, the flag of the japanese imperial army and navy, and the glorious laurel of seiro . To his right, one finds a quick description of the origin of this drug with important keywords highlighted in block letters: at the time of the russo - japanese war, the japanese government bestowed largess (400,000 yen) upon the army surgeon commander and the army surgeon - general; with that money, the army created a group of thirty - six pharmacologists who invented the drug . The advertisement does not stop at touting the army s achievement and the company s alleged connection to the military . If you do not know this pill, warns the caption, you are not a japanese citizen. The final finesse is a classic lie of a merchant: this drug is not for commercial profit; it is to celebrate and remember the glorious victory. Who would dare to refuse such a noble product? Purchase of this drug was after all tantamount to the confirmation of one s sense of belonging and loyalty to the state . Similar to seiro in its use of a uniformed man, ganso seirogan (original conquer - russia - pill) from nippon iyakuhin seiz, played the card of authenticity this is original when the company introduced its product in 1909 . To ward off rivals and to give the impression of genuineness, matsumoto jun (, 18321907), the first army surgeon - general of meiji japan (figure 2). On one hand, as the first army surgeon - general and as part of the very first generation of japanese physicians who had been educated with western medical science in nagasaki, matsumoto bespoke the genesis of modern medical science and its products in japan . Simultaneously, dressed in military uniform and endowed with a long beard, his physical appearance corporealised the authenticity of ganso seirogan . The fact that matsumoto was in office from 1873 to 1879 and therefore had nothing to do with the development of seirogan was simply ignored . The relatively simple process of concocting this anti - diarrhoeal pill enticed even a hardcore revolutionary activist into the market of seirogan . Kimoto bonjin (, 18881947), a famous anarchist and one of the founding members of the anti - establishment society suiheisha (organisation of levellers), began manufacturing and selling seirogan in his house in 1920 . Educated at osaka dental school (osaka shika igaku senmon gakk, now osaka dental university), kimoto must have possessed a workable level of pharmacological knowledge and put this into use to raise money for the cause of egalitarianism . His venture was commercially viable until his production and sale of the drug was put under strict governmental control in 1941, apparently due to his political orientation . However, kimoto s pharmaceutical business put bread on the table for his family for over twenty years . The irony of an anarchist making a living by selling a conquer - russia - pill might give the impression that there was a surge of interest in the drug during or immediately after the russo - japanese war . In contrast to what some historians have long argued, however, no major newspapers and magazines, such as tokyo puck, which was saturated with patent medicine advertisements, ever advertised seirogan in the 1910s . Given this strange absence of seirogan in the major venues of commercial advertisements, one might be tempted to claim that the extreme popularity and name recognition of seirogan made it unnecessary to advertise it broadly during much of the 1910s and the first half of the 1920s . But the reality was that the name seirogan was not ingrained in the world of pharmaceutical manufacturers, not to mention the minds of the japanese people who had already lost interest in remembering the russo - japanese war in the era of taish democracy (191226). For instance, nihon meiyaku ichiran, a registry of the most famous drugs in japan published in 1934, lists over 480 pharmaceutical products, but does not include seirogan in its extensive list . In this respect, the rise of seirogan to the level of a nationally renowned medical product was the work of nakajima saichi drugstore which, in recognising the changing geopolitical situation of the 1930s, mobilised the power of the advertisement and cleverly appropriated the potential of the military image for the benefit of its own commercial success . Little is known about nakajima drugstore or its owner nakajima saichi, except that he was based in osaka, the traditional epicentre of pharmaceutical production and distribution since the tokugawa era (16001868). But there should be no contention that nakajima drugstore initiated the advertising campaign of seirogan and in the process indelibly imprinted the brand and image of seirogan in the minds of the japanese people . In fact, until 1923, when the first national advertisement of the nakajima drugstore s chy - seirogan (loyal - and - brave - conquer - russia - pill) was featured in yomiuri shinbun, a japanese newspaper, there was no significant advertisement campaigning of seirogan (figure 3). Neither did the advertisement immediately develop into a full - scale bombardment of newspapers and magazines with images of seirogan . Between 1923 and 1925, nakajima drugstore briefly tried advertising with the tokyo - based yomiuri shinbun, but it was merely experimental; there was only one advertisement in 1923 (24 december), four in 1924, and six in 1925 . Moreover, instead of helping nakajima drugstore to gain recognition among japanese consumers, this early phase of advertising that patently failed to reflect the pulse of the society almost cost nakajima drugstore its entire business of manufacturing and selling seirogan . On 19 october 1925 the embassy of the soviet union in tokyo sent a strongly worded request to the ministry of foreign affairs of japan, asking them to pay attention to the advertisement published in yomiuri shinbun and to take measures to stop such a campaign . Similar. The letter argues in a very diplomatic way that the publication of such claim and the very fact that such a name is given to a merchandise of any kind is contrary to the condition of the normal relations established between the two countries. Faced with this acerbic reaction from the soviet union, the ministry of the foreign affairs of japan in turn mobilised administrative power to block such advertisements . Not only did the ministry alert the bureau of patents so that, on 6 january 1926 the registration of the trademark of nakajima s chy - seirogan was cancelled on the grounds that it was against international customs and public order, it also made sure that the decision was confirmed in a court ruling on 28 june . In a response sent to the soviet embassy on 15 november 1926, almost a year after the initial request from the soviet side, therefore, the ministry could proudly claim that it had solved the matter permanently and even included as evidence a copy of the advertisement of another former seirogan manufacturer (published in osaka asahi shinbun on 19 october 1926) who cleverly registered the trademark of shinrogan (befriending - russia - pill). The russians might have been satisfied with this solution, but it was not the end of chy - seirogan, let alone the term seirogan . Unlike what the ministry of foreign affairs insinuated to the soviet embassy, the court ruling of 28 june did not prohibit the manufacturing and selling of the drug itself; rather, what it specified was the cancellation of the trademark only, based on the fact that seirogan was a commonly accepted name, thus ironically making the name seirogan available to anyone . Nakajima drugstore did not, however, resume its marketing campaign immediately after the unintended acquiescence of the government . On the one hand but, more significantly, they must have realised (rather belatedly) that with the overall social sentiment of the taish period, with its emblematic dedication to democracy and internationalism, it was not favourable for them to market a drug that was based solely upon the mythic image of japanese soldiers during the war that had ended twenty years earlier . The voluntary (or maybe involuntary) marketing hiatus did not last long, though . In september 1931 the infamous manchurian incident, which would eventually usher in the so - called fifteen - year war (from 1931 to the surrender of japan on 15 august 1945) in asia, broke out and a discussion about it subsequently flooded the japanese media . In a new geopolitical situation where the notion of war became relevant and imminent to japan as a whole, the state found the victory of the russo - japanese war a reusable historical asset that could rekindle popular enthusiasm for military conquest in manchuria . Enabling the state to achieve this task was what historian shimazu calls the commemoration industry, a certain commercial sector that acutely recognised and was determined to capitalise on the wind of social and market change blowing through japan and asia through pageantry, exhibitions, and films . The principal vehicle for popularizing the memory of the russo - japanese war in the 1930s. At this new historical juncture where the memory of the russo - japanese war was being resurrected from the dustbin of history, nakajima drugstore lost no time in taking advantage of the changing geopolitical and domestic situation and immediately resumed its advertising campaign of seirogan in osaka asahi shinbun, the leading newspaper of the kansai region, in september 1931 . The pace of the still - sporadic marketing efforts of the early 1930s accelerated to full speed in 1937 with the outbreak of the second sino - japanese war in china . In 1938 there were thirty - eight listings with osaka asahi shinbun, evenly distributed throughout the year, and nakajima drugstore kept a similar frequency until 1941, when it added tokyo asahi shinbun as well and ran thirty advertisements with it in 1941 alone . Admittedly, the absolute number of the advertisements from 1937 to 1941 may seem to indicate that the inundation of images of seirogan in japanese newspapers is an exaggeration . Even at the height of the advertisement spree but the effectiveness of the nakajima advertisements resided not in their frequency but, rather, in three distinctive and innovative features: the appropriation of the authority of the military, the extensive use of explicit illustrations, and the re - interpretation of overarching wartime ideologies . These aspects made the drug resonate uniquely with the japanese public who were now more receptive to and familiar with militaristic imagery than ever before . In its advertisements, first and foremost, nakajima drugstore broke from the convention of using authoritative figures in the sales of patent medicine . Just as they do today, medical doctors frequently appeared in drug advertisements to affirm the medical efficacy of pharmaceutical products, and modern japan was not immune to this tried - and - proven tactic . Berutsu - gan, a patent medicine of suzuki nippon - seiyakusha of tokyo, is one such example that relied almost exclusively on the fame of doctors . In the advertisement that appeared on 8 april 1938 in osaka asahi shinbun, suzuki nippon - seiyakusha used erwin baelz, arguably the most well - known german doctor in japan who taught at tokyo imperial university over a quarter century (18761902) and had been a court physician to the japanese imperial household, to make this cure - all drug appear authentic (figure 4). Of course, nippon - seiyakusha did not reveal in the advertisement that baelz had passed away in 1913 and could not possibly have given permission to the company to use his transliterated name in japanese (berutsu) as the brand of a miracle drug that allegedly cured such diverse diseases and symptoms as syphilis, intoxication, constipation, and general fatigue ., nakajima drugstore elevated this tradition of using external authority to a whole new level by appropriating the ultimate authority available in japan the imperial army and navy, and by extension, the emperor of japan himself . Rikukaik goy yaku (a drug adopted and consumed by the imperial army, navy, and air force). In the main body of the advertisement, one could almost always find blatantly self - congratulatory text giving the aura of historical authenticity . Not only has this drug served our imperial army for over thirty years since the russo - japanese war, it has been adopted and endorsed in abundant quantities by many quarters since the recent manchurian incident and the second sino - japanese war . All those who fight on the home front should take this opportunity to keep this drug on hand and recognise its medical efficacy . Not only has this drug served our imperial army for over thirty years since the russo - japanese war, it has been adopted and endorsed in abundant quantities by many quarters since the recent manchurian incident and the second sino - japanese war . All those who fight on the home front should take this opportunity to keep this drug on hand and recognise its medical efficacy . This strategy of inventing tradition as a way to concoct an alleged connection with the military was visually enhanced by the extensive use of explicit images of soldiers on duty . From a young recruit beaming with a bright smile with seirogan in his right hand to a soldier out on patrol on a snowy night, soldiers of the japanese imperial army, faithful to their duties throughout the four seasons, were prominently featured in the advertisements of nakajima drugstore . A navy sailor in his emblematic white uniform and a fighter jet flying over the japanese archipelago more importantly, nakajima drugstore actively rehashed, reformulated, and reintroduced contemporary wartime ideology, using readily available and widely understood symbols and icons . In the process, of course, nakajima drugstore did not forget to cleverly ensconce their commercial interests in the languages of anti - communism, condescension towards china, and the new order system . In short, drawn from a reservoir of familiar images and symbols of anti - russianism, anti - communism was the most easily discernible message conveyed in the advertisements of seirogan . As michiko ikuta noted in her article, as early as the late tokugawa era (16001868), perceptions of russia in japan were anything but positive; whereas the dutch and other europeans were perceived in light of their religious and intellectual challenges, russians, due to their geographical proximity to japan, were predominantly portrayed as menacing intruders . This negative image of russia was further amplified and reinforced during the course of the russo - japanese war . In the process of creating and propagating a favourable image of japan both within and without the japanese archipelago, the japanese authorities, often with the explicit support of american and british press, successfully demonised their enemy . The subsequent sovietisation of russia further denigrated the image of russia among the japanese . And the nakajima drugstore did not hesitate to co - opt the widely recognised image of the soviet demon . As is seen in the advertisement posted in osaka asahi shinbun on 20 july 1939, communism personified by the soviet union was a red demon (aka - ma in japanese) that japan was heavenly mandated to destroy (figure 5). Rhyming with the call for action against communism was the exhortation to destroy the disease demon (by - ma) by using a drug, seirogan . The comparison cannot be misunderstood just as imperial japan was destined to destroy the red demon, seirogan was invented to kill the disease demon. Figure 5:osaka asahi shinbun, july 20, 1939 . Along with fervent anti - communism, a strong condescension toward china was a recurring theme of advertisements for seirogan that any japanese could easily decipher the meaning of . A tall japanese soldier was typically juxtaposed with a chinese child, usually a girl, holding the child by the hand, carrying him or her on his back, or patting him or her on the head . Most prevalent, however, was the image of a japanese soldier offering seirogan to a needy and eager chinese child . The advertisement of 17 february 1937, with the title praying for the health of strong imperial soldiers is one such example (figure 6). Here, a japanese soldier with a smile is handing out pellets of seirogan to a chinese child, whose head and right hand only appear in the bottom half of the advertisement . The child is speaking to the japanese soldier in broken japanese: japan, soldier, strong . Japan as an imperial power needs to help china, an immature society, and the easiest thing to do to respond to this call of duty is to purchase seirogan, an undeniable symbol of japanese vitality . The most outlandish attempt to appropriate the wartime ideology was nakajima drugstore s invention of the slogan, new order of health (kenk no shintaisei) in parallel to the government s new order system (shintaisei). As is well documented by historians, the new order movement from 1938 was designed to reintegrate the japanese people into the well - oiled machine of wartime imperialism . Using the concept of a new order of health, the nakajima drugstore encouraged the japanese to consider not only their attitudes in relation to their own bodies, but also toward the government and its soldiers, a refashioning that nakajima drugstore was ready to commercially exploit . The advertisement of 2 november 1940 in this respect is exceptional both in its form and content (figure 7). Moving away from the usual reliance on explicit images of masculine soldiers, this advertising piece titled new order of my life (boku no seikatsu shintaisei) takes the form of an eight - panel comic illustrating an exemplary everyday life under the new order that every japanese person should strive to follow . The comic starts with the daily practice of paying respect at the shinto shrine. Then, the male protagonist substitute food, and he does not forget to remind his colleague that today is a non - smoking day. Thinking of the war front, he pushes himself to work harder . It seems like i forgot to do something when he hears, please take a look at this, daddy, a timely reminder from his son who is holding a letter saying thank you, soldiers. Yes, i ve been meaning to send a comfort kit (imonbukuro) to the front. The advertisement ends with a scene of mom, dad, and son all smiling while putting seirogan into a comfort kit . As with other advertisements from nakajima drugstore, the message does not require even a moment of deliberation not only does seirogan signify the culmination and completion of the daily activities under the rubric of the new order movement, it also functions as a medium that links the home front to the battlefield, thus providing a space for japanese consumers to contribute to and associate themselves with the war efforts . Figure 7:osaka asahi shinbun, 2 november 1940 . Historian marchand argues, advertisements were secular sermons, exhortations to seek fulfilment through the consumption of materials goods. And as secular sermons preached during wartime japan, the advertisements of nakajima drugstore s seirogan extolled the consumption of this anti - diarrhoeal pill as the faithful fulfilment of the patriotic duty of imperial subjects . How successful was the advertising campaign? Did japanese consumers actively respond to the patriotic yet commercial exhortations? Unfortunately, there are no official sales figures available from the 1930s and the first half of the 1940s, but we can ascertain the enduring success of the drug from circumstantial evidence . First of all, in 1949, as previously mentioned in the introduction, the japanese government, in consideration of its fragile relations with the soviet union, ordered manufacturers of seirogan to change the kanji character that corresponds to sei from righteousness. With no request from the soviet union, as was the case in 1925, this decision by the licensing department of the ministry of health and welfare was most likely a proactive effort to demilitarise the popular over - the - counter drug that had gained a wider consumer base through the wartime period . On a related note, the administrative guidance that required all manufacturers to change the kanji character only, not the actual name of seirogan itself, should be interpreted as reflecting the embeddedness of seirogan in the minds of japanese consumers . Furthermore, the vehement opposition from small - scale manufacturers of seirogan against the attempt of taik pharmaceutical to register the name of seirogan with a new kanji character as a trademark evinces the commercial success or at least the commercial viability of seirogan in the transwar years . As previously explained, taik pharmaceutical is the successor of the nakajima drugstore . Shibata otojir, who founded shibata pharmacy in 1940, acquired the sales rights for chy seirogan from nakajima drugstore in april 1946 and subsequently changed the name of his company to taik pharmaceutical in november of the same year . In 1949, likely following the administrative guidance of the ministry of health and welfare, taik pharmaceutical changed the name of its flagship drug chy seirogan to nakajima seirogan (with a new kanji), and then, in november 1954, registered seirogan as a trademark . In response to this effort to monopolise the over - the - counter anti - diarrhoeal product, a group of small pharma - manufacturers formed the national coalition for the free use of seirogan (seirogan jiy shiy kantetsu zenkoku dmeikai) and strongly argued that seirogan should not be monopolised by a single company because it was originally invented by the imperial army and the name had become a common noun rather than a brand . The fierce legal battle that engulfed the japanese pharmaceutical world in the 1950s and 1960s came to an end only in march 1974 when the japanese supreme court declared that seirogan is a common noun and thus cannot be used as a trademark . The postwar contention over the use of the name seirogan unmistakably indicates that this stomach pill had rooted itself deeply into the minds of the japanese people in the 1930s and 1940s . And this imprint was mostly the result of the advertising campaign of nakajima drugstore, which took advantage of the changing geopolitical situation in east asia and the rise of militarism in japan from 1931 . By riding upon the image of the imperial army, using explicit graphics, and cleverly appropriating the official ideology of the wartime period in its advertisements, nakajima drugstore accentuated meanings bestowed on seirogan and created a tangible medical product that ordinary japanese consumers could depend upon and associate themselves with . An icon of the health, vitality, and military prowess of japan, seirogan ultimately prescribed what it took to be a truly patriotic japanese person soon after general douglas macarthur was relieved of his duty as supreme commander of the allied powers in japan in april 1951, howard m. handleman (191394), a well - respected veteran war correspondent and the far east director of the international news service, sent japanese publisher dai nippon ybenkai kdansha a ten - page article reflecting on the legacy of macarthur in postwar japan . In this article, which was subsequently translated and published in the july issue of japanese magazine kingu as general macarthur and seirogan, handleman invites readers to the day when macarthur first landed in japan, a country still bristling with more than two million men under arms and still resentful for the blood it had lost to the conqueror whom macarthur symbolized. Almost everyone believed in august 1945 that it would be difficult, if not impossible, to occupy and democratise japan . But it looks that way only to those who did nt know the story of the dysentery pills, retorts handleman . Macarthur learned a lesson from those dysentery pills near the yalu that he never forgot, and the lesson he learned made him completely confident in 1945 that he could move into japan without any trouble. What was the story and what was the lesson? By now, the story should be a familiar one . During the russo - japanese war, japanese soldiers were dying en masse from dysentery, but they were using every opportunity to avoid taking the foul - smelling dysentery pills they crunched the pills into the ground with their boots, they dropped them in their pockets, they threw them into bushes. Given the seriousness of the situation, the highest field command held a staff conference to find a way to force soldiers to take the pills, and the solution was to issue the order in the name of the emperor . Macarthur, who was in manchuria as an aide - de - camp to his father, lieutenant general arthur macarthur, jr ., did not anticipate that a mere wrapping of the order in the words of the emperor would persuade japanese soldiers . The order was issued; the soldiers took their pills; the army revived to whip the russians; macarthur was impressed. According to handleman, it was a momentous day for macarthur and eventually for japan as well macarthur was impressed so much that more than 40 years later, when his armies had defeated another japanese army, he felt absolutely certain that the order of another emperor to accept the conquerors without resistance would be obeyed to the letter. And we all know what it meant to the postwar us occupation policy in japan; the emperor was exonerated of war responsibility, and the position of japan was transformed from a quasi - totalitarian enemy state into an ally in the pacific . Did seirogan spare the life of the shwa emperor and change the course of history? Maybe in a convoluted way . Intriguing in its own right, the issue of seirogan in its relation to macarthur, let alone its impact on macarthur s vision of postwar japan, was not the main focus of this article . Rather, the unexpected story is introduced here as a snippet that shows in what ways this seemingly insignificant dysentery pill has been intimately connected to the lives of the japanese people in the twentieth century . As i argued in this article, seirogan exemplifies a culmination of the convergence of a governmental initiative to enhance military capabilities, the commercial ingenuity of pharmaceutical manufacturers to profit from an expanding imperial culture, and a consumer response to patriotic exhortations . But the matrix that seirogan allows us to glimpse is not a typical top down governmental method of mobilising private sectors to manipulate public opinion for the cause of external imperialist expansion and domestic stability . In the eyes of the japanese government, at least throughout the 1920s, jingoistic pharmaceutical manufacturers were more of a nuisance that could easily disrupt international relations than an effective tool of rallying ordinary japanese people under the banner of the empire . Even with the government s indifference, if not hostility, pharmaceutical companies quickly recognised and exploited the pecuniary opportunities that seirogan and its symbolism provided to them once the tide turned favourable from 1931 . In turn, japanese consumers reacted to these commercial sermons carefully anchored in patriotic and militaristic discourses and images by opening their wallets . In other words, seirogan reveals an inverted power relation among the state, commercial sectors, and imperial citizens in the wartime japanese empire, where pharmaceutical companies were at the driving seat . As noted, the striking capability of pharmaceutical manufacturers to adapt to changing operational environments did not stop with the demise of the japanese empire . The postwar metamorphosis of seirogan from a symbol of empire into a contextless icon of health and travel in japan further evinces the continued resilience and creativity of drug makers . This clever marketing tactic, which effectively bleached the stain of militarism off the label of seirogan, also explains the seemingly ironic popularity of seirogan as the symbol of the quality of japanese - made medicines among korean and chinese consumers who are often extremely bothered by japan s historical amnesia . On a general level, seirogan thus shows the possibility that an entirely new perspective can be cast onto a familiar question using a commodity as a mediating material . As andrew gordon proved in his groundbreaking monograph that rewrote a history of modern japan through a sewing machine, a commodity, especially a mass - produced consumer product, is a powerful medium that highlights an intricate web of connections between society and culture, between production and consumption, and between local and global . In this respect, a medicinal product is even more illuminating, for it also touches upon the dimensions of body, disease, and unavoidable control, and its efficacy does not discriminate between the bodies of the colonisers and the colonised.
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The ultimate goal of periodontal therapy is to arrest the progress of periodontal disease and regenerate the lost periodontal support . Hyaluronic acid (ha) is a high molecular weight, nonsulphated glycosaminoglycan component which is produced during various phases of cell's life cycle . It contributes in tissue hydrodynamics, cell migration, and proliferation, and improves healing properties of the tissue . Its physiological, structural, and biochemical properties prove that it provides elasticity and stability to tissues and is critically beneficial in tissue regeneration . Pharmacologically, the content of ha in the tissues is often broken down by the blood stream or by lymphatic drainage and its turnover rate is found to be 2030% . After being broken down, ha was discovered in 1934 from cow's eye by karl meyer and john plamer and was first used in 1942 by endre balazs as a substitute for egg white in bakery products . The chemical structure of ha shows alternate units of n - acetyl glucosamine and d - glucorounic acid, both of the components linked through glycosidic bonds . Ha acts as an antioxidant by scavenging reactive oxygen species, which helps in the regulation of immune response implying its anti - inflammatory properties . It has also been reported that ha shows osteoinductive properties, which is useful for treatment of periodontal disease . Other beneficial effects have also been seen for the treatment of recurrent apthous ulcer, for treating gingival lesions, and promote healing in extraction socket . Ha has also reported as a diagnostic biomarker of inflammation in gingival crevicular fluid and repair of tissues . Recent investigations have indicated that ha induces mineralization of dental pulp cells through cd44 cell surface glycoprotein and is considered to be a principle ligand for receptor cd44 . Hyaluronic acid was found to have extensive actions in various periodontal therapies such as topically applied in subgingival regions reduces microbial activity, bone regeneration in deep periodontal bony defects, guided bone regeneration, nonsurgical treatment of peri - implantitis pockets, peri - implant maintenance of immediately placed implants, and gingival augmentation in mucogingival surgery . Ha may act as a scaffold for other molecules such as bone morphogenic protien-2 and platelet derived growth factor - bb, used in guided bone regeneration techniques and tissue engineering research . Ha when applied to patients with chronic periodontitis showed reduction in bleeding on probing (bop), probing pocket depth (ppd), and clinical attachment level, and hence, can be used as an adjunct to scaling and root planning . Ha gel, injections, or oral (by mouth) should not be used in patients with allergies . A 24-year - old female reported to the department of periodontics with her esthetic concern and complaint regarding the loss of interdental papilla, i.e. Black triangle, in the anterior maxillary region [figure 1]. Patient was concerned for esthetics; class ii type of interproximal tissue loss without any bone loss and no extra oral deformities were detected . A written informed consent was taken from the patient before the procedure, and ethical clearance certificate was obtained from the institution . Preoperative: black triangle between maxillary right central incisor and lateral incisor nordland and tarnow proposed a classification using three reference points, i.e., contact point, facial and apical extent of cementoenamel junction (cej), and interproximal extent of cej (icej), and was classified into the following four classes: normal: interdental papilla occupies embrasure space to the apical part of the interdental contact point . Class i: tip of interdental papilla occupies space between the interdental contact point and the most coronal part of cej . Class ii: tip of interdental papilla lies at / or the apical to the icej but coronal to the apical most part of the cej on facial aspect . Class iii: tip of interdental papilla lies at level with or apical to the facial cej . The loss of interdental papillae can be due to many reasons such as gingivitis, oral hygiene procedures with trauma, tooth shape with abnormal anatomy, or improper contours of the restoration . Inclusion criterion for the study were: age range: 2075 yearsmaxillary anterior teethplaque index less than 20%teeth should not have caries and no fixed prosthesis or orthodontic appliancenonsmoker individualsno systemic disease that may affect the periodontal statusavoid consumption of drugs causing gingival overgrowth . Age range: 2075 years maxillary anterior teeth plaque index less than 20% teeth should not have caries and no fixed prosthesis or orthodontic appliance nonsmoker individuals no systemic disease that may affect the periodontal status avoid consumption of drugs causing gingival overgrowth . Patient was completely informed about the study . After introducing a local anesthetic agent, less than 0.2 ml of the ha gel was injected at the sites 23 mm apical to the coronal tip of the papilla [figure 2]. The patient was instructed not to brush on the day of treatment but maintain oral hygiene, and after 24 hours, using a soft bristle toothbrush at the anterior teeth to start their routine oral hygiene measures and avoid using dental floss at the sites of treatment . The patient was on follow - up for 3 weeks and was recalled for a total of 4 times . After first follow - up, 3 weeks later, the treatment patient did not show any improvement, therefore; another shot of 0.2% ha injection was given . Measurements of the black triangle were done using photography . Injecting 2% hyaluronic acid into interdental papilla after 3 months, photographs were taken and comparison was done using these images . This technique resulted in significant gain in papillary volume and esthetic improvements were notable [figure 3]. Because using 0.2% ha for the reconstruction of lost interproximal tissue gave significant changes, it can be used as a noninvasive method, which will reduce the use of surgical procedures for regenerating soft tissues in upcoming years . This study can be limited by the amount of the interproximal tissue loss, depending upon the size of the black triangle, which might require surgical intervention to gain complete coverage . The main advantage of this study is that it is nontoxic to the patient and there is reduced discomfort after the procedure as compared to surgical procedure done . Furthermore, this study can be elaborated by more number of patients depending upon the size and type of the black triangle . Becker et al . Concluded that ha gel is a synthetic material and can be used with no drug interference and is a safe material, which significantly decreases the interdental black triangle in the esthetic zone . Vedamurthy reported ha to be a dermal filler and applied it for soft tissue augmentation, observing significant improvements . Monheit et al . Discussed the inherent properties of ha that make them ideal for cosmetic surgeries . Studied gingival augmentation with an autologous cell ha and reported significant results with the complete coverage . Found that antioxidant capacity of ha is inversely proportional to the severity of inflammation and can be used as a biomarker in periodontitis . It is acceptable that injecting ha to periodontal wound sites had shown significant effects in periodontal tissue regeneration . Engstrm et al . Reported bone regenerative effects of ha in nonsurgical and surgical groups and showed no statistical difference when evaluated on radiographs in the nonsurgical group; however, there was remarkable decrease in the height of alveolar bone after oral prophylaxis in both the nonsurgical and surgical group . Ha when involved with soft and hard tissues showed negligible effect on the immune system of the patient . Stated enhanced accelerating capacity of new bone formation in the infrabone defects when combined with autologous bone graft . This study indicates possible improvements in regenerating lost interdental papilla and removal of black triangle by injecting ha into the lost papilla using a nonsurgical approach . Because of its property of modulating periodontal wounds, this nonsurgical approach limits the use of surgical procedures for regenerating lost papilla, and hence, it is a noninvasive method and also reduces patient discomfort . Therefore, this study demonstrates ha to be a nonsurgical approach for regenerating lost papilla and gave significant and satisfactory results . To overcome the limitations of using of ha for regenerating lost papilla, this study need to be elaborated with more number of patients depending upon the size of the black triangle.
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Cerebral palsy (cp) is the most common cause of neurological disability in children1, affecting approximately 1 in 1,300 live births2 . At an early age, abnormal hand postures such as thumb adduction and/or flexion with limited wrist extension are the primary manifestations of hand involvement . Daily living activities and functional independence are affected by increased tonus of the upper limb, impaired posture and function3, 4 . In children with cp, neurodevelopmental treatment (ndt) is the most widely used treatment approach, which aims to maximize the child s potential to improve motor functions while preventing musculoskeletal complications5, 6 . In the literature, it has been reported that intensive neurodevelopmental treatment is effective for improvement of gross motor function, hand functions, and the quality of upper - extremity movement7, 8 . Virtual rehabilitation is a relatively new method that has been adopted by physical therapists who specialize in cp treatment . In recent years, there has been growing interest in therapeutic use of the nintendo wii and wii fit (wii) as virtual rehabilitation tools . Although limited in number, some studies have suggested that use of these devices, could result in significant therapeutic gains in motor function for children with cerebral palsy9, 10 . The theories of motor control and motor learning emphasize the importance of including motivating, repetitive, purposeful exercises with games and fun to support development in the rehabilitation of children with cp11, 12 . During standard physiotherapy sessions, it can be difficult to motivate a child and encourage participation in activities . Therefore, physical therapists need various motivational techniques and methods to gain the most benefit from exercises applied to improve limb use13 . Virtual reality games can be motivating and entertaining for the paediatric age group, thereby holding the interest of the child during treatment10, 14, whilst also improving upper extremity skills and control4, 10, skills required for daily living activities15, and speed of upper extremity use14 . In the literature, there are studies that have evaluated use of thenintendo wii and similar games consoles for improvement of upper extremity functions in patients with various diagnoses14, 16,17,18, but there are very few that have investigated the effectiveness of use of technological systems to improve upper extremity functions in children with cp . The current study aimed to determine the effects on quality of upper extremity skills, movement speed of the upper extremity, disability, and functional independence as a result of adding virtual reality games to a neurodevelopmental treatment program . A total of 33 children with spastic hemiparesis were evaluated, and 30 were included in this study between june and september 2014 (fig . 1fig . The inclusion criteria were a) aged between 6 and 15 years, b) level 13 of the manual ability classification system, c) level 1or 2 of the gross motor function classification system16, 19, 20, d) ability to grasp and release an object, and e) no history of surgery or botulinum toxin application to the upper extremity in the previous 6 months . Parents or legal guardians read and signed an informed consent form before the study, and the marmara university health sciences institute ethics committee approved the study (02.05.2014 - 13). Flow chart describing the progression of participants through the trial the children were divided into two groups on the basis of arrival at the clinic . We used simple randomization with a random number generator (115, group 1; 1630: group 2). The first 15 children were assigned as group 1, and the program applied was ndt+wii . The second 15 children were assigned as group 2 and received ndt5, 6 only . Treatment in both groups was applied by a physical therapist for 45 minutes per session, twice a week, for 6 weeks . The patients in group 1 played virtual reality games of tennis, baseball, and boxing (each game 5 minutes) for 15 minutes in addition to the standard neurodevelopmental treatment in each treatment session, wich was focus on the hemiplegic hand . Two experienced physical therapists selected the games for moving to the hemiplegic upper extremity . The ndt program for both groups was applied according to each child s individual needs and included tonus regulation, support of sensation, perception and motor development, facilitating normal movement for upper extremity activities, upper extremity functional skills training, and daily living activities such as getting dressed and eating . Before starting the nintendo wii games, the patients were informed about the content of each game and had one practice session, during which the children were supported verbally and physically until they achieved the correct movements . In this study, use of the upper extremity was evaluated using the quality of upper extremity skills test (quest), which has been tested for validity and reliability in children with neuromotor dysfunction . This test indicates four domains: dissociated movement, grasp, weight - bearing, and protective extension21 . Hand function speed was assessed with the jebsen taylor hand function test (jthft), which has been shown to be reliable and normative for children22, 23 . In this study, the subtests to assess hand function included turning over cards 3 5 inches (7.5 cm 12.5 cm) in size, simulated feeding, picking up small common objects, stacking checkers, picking up large objects, and picking up large heavy objects . The time (in seconds) taken to perform seven fine motor hand tasks was measured . Hand disability was assessed using the abilhand - kids test, which has been found to be valid and reliable in children with cp . Turkish translation of the abilhand - kids test was performed by the authors . In this test, 21 mainly bimanual, activities are rated by the children s parents on a 3-point scale (0=impossible, 1=difficult, 2=easy), describing their child s perceived difficulty in performing each activity . This is a useful tool for assessing functional status in children with neurodevelopmental disabilities; it includes 18 items scored on a 7-level ordinal scale . The reliability and stability of the scale has been proven25, 26 . In the current study, all assessments were performed twice: once at the start of the treatment program and again at the end of the 6-week program . In addition, a user satisfaction questionnaire was administered to the patients in group 1 . This questionaire assessed parameters related to virtual reality games including the level of difficulty of the game, motivation, cognitive abilities, and physical effort of the player27 . Baseline characteristics were compared using the independent samples t test and the chi- square test . A comparison of the groups was made using the independent samples t - test for the jebsen taylor hand function test and the mann - whitney u test for the quality of upper extremity skills test, abilhand - kids test, and weefim with an intention - to - treat analysis of the difference between the baseline and final scores with a 95% confidence interval . The paired sample t - test was used to compare jthft outcomes before and after treatment . While the wilcoxon signed - rank test was used to compare other outcomes before and after treatment . The mann - whitney u test was used to compare improvements and differences between the groups . From the 33 patients with cp who were referred, 30 were included in the study for evaluation and were divided into 2 groups (fig . 1). There were no significant differences between the groups with respect to demographics, clinical features, or baseline test scores (tables 1table 1.the demographics and clinical features of the participantsndt+nw (n=15)ndt (n=15)age (years) (mean sd)9.53 3.049.73 2.86*gender (f / m) (n)7/89/6**macs (medianscore- range)2 (range 13)2 (range 13)*effected extremity (right / left)8/710/5**gmfcs9 cases-29 cases-2 * 6 cases-16 cases-1sd: standard deviation, f: female, m: male, ndt+nw: neurodevelopment treatment+nintendo wii, ndt: neurodevelopment treatment, macs: manual ability classification system, gmfcs: gross motor function classification system . * t - test . Sd: standard deviation, f: female, m: male, ndt+nw: neurodevelopment treatment+nintendo wii, ndt: neurodevelopment treatment, macs: manual ability classification system, gmfcs: gross motor function classification system . * * test following the 6-week treatment program, statistically significant improvement was seen in the ndt+wii group in the scores for all domains of quest (p=0.002, p=0.003, p=0.017, 0.005), jthft (p=0.001), abilhand - kids (p=0.001), and weefim self - care (p=0.026) scores . Statistically significant improvement was determined in the dissociated movements domain of the quest (p=0.007), jthft (p=0.001), abilhand - kids (p=0.002), and weefim - self - care (p=0.007) scores in the ndt group (table 2table 2.results of outcomes for two groups before and after the treatmentndt+nw groupbefore mean (sd)after mean (sd)mean change (sd)quest dissociated movements (score)80.1 (7.73)85.6 (8.54)5.5 (4.40)*quest grasps (score)42.2 (18.76)47.1 (16.64)4.9 (3.61)*quest weight bearing (score)69.2 (19.46)72.7 (19.60)3.5 (5.79)*quest protective extension (score)72.9 (14.78)77 (12.66)4.0 (4.56)*jthft (s)40.4 (16.44)32.9 (14.88)0.7 (0.99)*abilhand - kids (logits)0.56 (2.34)1.33 (2.04)0.73 (1.16)*weefim (score)46 (8.23)46.7 (7.51)7.5 (4.82)*ndt groupquest dissociated movements (score)81.4 (10.70)86.4 (8.78)5.0 (4.94)*quest grasps (score)53 (16.45)55.7 (15.30)2.7 (4.62)quest weight bearing (score)75.4 (17.07)77.3 (15.43)1.8 (7.15)quest protective extension (score)71 (23.52)74 (23.36)2.9 (6.84)jthft (s)31.5 (9.57)29.9 (8.83)0.4 (0.27)*abilhand - kids (logits)1.07 (1.90)1.51 (1.70)0.60 (0.63)*weefim (score)48.3 (7.27)48.9 (7.14)1.5 (1.93)*ndt+nw: neurodevelopment treatment+nintendo wii, ndt: neurodevelopment treatment, quest: quality of upper extremity skills test, jthft: jebsen taylor hand function test . * significantly different from pre - treatment p<0.05). Ndt+nw: neurodevelopment treatment+nintendo wii, ndt: neurodevelopment treatment, quest: quality of upper extremity skills test, jthft: jebsen taylor hand function test . * significantly different from pre - treatment p<0.05 the within - group analysis showed that the jthft score of the ndt+wii group was superior to that of the group receiving neurodevelopmental treatment only (p=0.000). There were no significant differences between groups with respect to the other test scores . The results of the user satisfaction questionnaire completed by the ndt+wii group are shown in table 3table 3.user satisfaction questionnary: answers in frequencies (n=15)12345wii: presentationi could see all things in the game very well---78i found the moving things / objects in the game very interesting-1 - 95the things i saw in the game were very attractive---213the game reacted good on my movements-2256i could hear all sounds very well-1 - 410the sounds i heard out of the game were very attractive-1 - 410i could not hear where all sounds out of the game did come from6333-wii: level of difficultythe game was too hard35 - 7-i still must learn a lot, before i can play this game-7152i had the feeling i could win---69the game was too easy-825-player: motivationi would find it nice if i could play the game together with more children at the same time----15i liked it that the game was getting more difficult-2265the game was so attractive that i lost all count of time---411i would not like to play this game more often1012 - 2wii training is less fun than regular physiotherapy----15player: cognitive capabilitythis game was easy to understand-1 - 68this game was easy to play11 - 85it was very logical playing the game by moving my arm / hand1 - 275my experience with playing the game looks like real life playing13 - 56player: physical efforti found it hard to play the game by moving my hands39 - 3-i became more tired from this game than from the regular physiotherapy26 - 52by playing the game, i learned new movements-1 - 311i think i could learn new movements by playing the game more often-1 - 3111-completely disagree, 2-slightly disagree, 3-neutral, 4-slightly agree, 5-completely agree . Five children found boxing and playing tennis and baseball too easy, and seven children found the games easy . In this study, the effects of ndt and ndt+nw on upper extremity functions, speed, manual ability, and level of independence in activities of daily living were comparatively investigated in the children with hemiparetic cp . In the study, improvements were achieved in functional ability, speed, manual ability, and the dissociated movements subgroup of the quest results in the group in which ndt (45 min) was applied for 6 weeks . On the other hand, improvements were observed in the results of all hand functions, speed and manual ability, and all subgroup scores of the quest scale in the group in which ndt+nw (30 min+15 min) was applied . The satisfaction questionnaire applied to the ndt+nw group showed that the children were extremely satisfied with the practice . Higher success rates were achieved in terms of speed in the group in which nintendo wii was added to ndt when compared with the ndt only group . Ndt is a global therapeutic approach that addresses all requirements of a child and aims to increase the child s functionality and participation in daily life . Although this approach is not a therapeutic modality only for hand functions, it does include some activities in the session that address daily use of the hand5, 7, 8 . In the literature, there are few studies that contain sufficient evidence and show the healing effect of the ndt approach on upper extremity functions of children28 . However, the absence of a comparable no treatment control group is a limitation decreasing the power of our results . Data from the literature suggest that the use virtual reality positively affects participation of children in treatment and provides motivation and adaptation27, 29 . In previous studies focusing on the upper extremity in cp, the study periods were 430, 625, 31, 32 and 8 weeks12, 33 . Therefore, as in the literature, we preferred a study period of 6 weeks to compare nintendo wii and ndt practices . On the other hand, time spent on virtual reality generally varied between 20 minutes and 3, 5 hours, 35 days per week in the studies conducted using virtual reality13, 30, 33,34,35 . As it has also been suggested in the literature, we thought that the nintendo wii, as a game - based approach, might be a useful part of rehabilitation for children with cp . Therefore, in our study, we evaluated the contribution of the nintendo wii to the upper extremity when used for a period of time period (15 min) during a routine ndt treatment session14 . Applied 15 minutes of nintendo wii game play in addition to 30 minutes of conventional neurologic treatment twice a week in children with cerebral palsy and obtained significant differences in eye - hand coordination and visual motor speed in comparison of a virtual reality training group with a conventional neurological physical therapy group36 . The children in our study also regarded the virtual reality games as a reward given at the end of ndt practices, but not as a treatment, which may be accepted as a condition that may increase motivation and cooperation . It was also clinically observed that the subjects in our study were more motivated to participate in the activities during the ndt session . This was also observed in the satisfaction questionnaires completed by the ndt+nw group at the end of the study . Since computer games have recently become an important from of entertainment, which may be addictive, in the lives of children lives, this interest and positive motivation in the children were expected . In addition, the effort of the children to move quickly during nintendo wii practice to score in the game and play better versus the computer might be the reason for the higher scores in the ndt+nw group when compared with the ndt only group . It might be accepted as a limitation that we obtained no records of the scores obtained by the children in the games at the beginning and end of the study . Comparison of the scores between the beginning and end of treatment might objectively show the development of the child s ability to play the game . On the other hand, a powerful aspect of our study is that all cases had homogeneously hemiplegic type cerebral palsy . The quest has been used to measure the results in studies on virtual reality35, 37 . In our study, the posttreatment quest scores of all participants in the ndt+nw group were better compared with those of the ndt group (p<0.05). We think that achieving a statistically significant increase in ndt+nw group with regard to weight bearing and protective extension scores at the end of 6 weeks might be related to the effort of the children to move their arms to better grasp and use the nintendo wii game console . During the practice, it was also clinically observed that some children were unable to firmly grasp nintendo wii game console at the beginning of treatment, but were better able to grasp and control the wii nunchuk at the end of 6-week treatment period . The posttreatment increase in quest grasps scores especially supports this clinical observation . In our study, jthft scores improved in both groups when compared with those for the to pretreatment period . Although there was no statistically significant difference between the two groups in terms of jthft results, it was observed that the ndt+nw group completed the test 6 seconds earlier, on average, at the end of the 6-week treatment period . This outcome indicates that the use nintendo wii games was more effective than ndt in increasing the speed of movement . In general, an important question about virtual reality practices is about whether achieved gains contribute to real life activities . In our study, among the cases tested by a method that includes measurements obtained using functional real life activities, such as the jthft, the better results in the nintendo wii group represent evidence showing that gained obtained by virtual environment therapies might be transmitted to real life . There are also some other studies presented in the literature demonstrating that similar successful results have been achieved regarding the transmission of through virtual reality therapy skills gained here to real life15, 33 . In our study, independence of the children was evaluated with the weefim, which revealed similar developments in both groups in terms of eating, care, bathing, and the upper and lower trunk dressing fields . There is one study in the literature that included a case with hemiplegic cp and showed results similar to our findings in terms of improvement in daily self - care activities15 . Furthermore, a posttreatment improvement was observed in both groups in terms of the scores of the abilhand - kids which was previously used in studies in which virtual reality was applied to measure specific daily life skills of the hand31, 35, while no significant difference was present between the groups . We could measure these functional gains in our study only at the questionnaire level . We suggest that magnetic resonance imaging of this kind of gain might increase the objectivity and power of further studies . The part of the user satisfaction questionnaire concerning the difficulty of the nintendo wii games, all children in the ndt+nw group in our study stated that they could win the games in spite of their physical limitations . This situation may indicate that the games strongly motivated the children who tried the virtual reality games, in spite of their physical limitations . Some children stated that the games were more tiring than the other methods applied in ndt . Although they were very tired, all children stated that playing with the nintendo wii was much more fun when compared to conventional therapy . In addition, all cases specified that the games were attractive and that they lost track of time during therapy . We interpreted this situation as indicating that supporting traditional therapies, which become boring and monotone for children during long rehabilitation periods in children with cp, with virtual reality games might increase the motivation of children in therapy . Virtual reality games, which have become a part of daily life, make children perform desired activities voluntarily and in a more motivated way . These results suggest that the children were very pleased with the virtual reality games added to therapy . The game console used in this study relies mostly on use a controller, it was observed that the children with cp sometimes lost control of the controller due to excitement - related increased spasticity and that the device failed to sense their movements . We think that further studies are required that, include a higher number of participants and use more current systems with sensors sensitive to not only movements of a controller but also all body movements . In conclusion, application of nintendo wii virtual reality games combined with ndt had benefical effects on upper extremity functions, upper extremity speed, and independence level in daily life activities in the children with hemiplegic cp . The increase in speed was more prominent in the ndt+nw group than in the ndt group . Since the nintendo wii increases motivation and provides on opportunity for enjoyable therapy, we think that it increases adaptation of children to rehabilitation programs and that it may be added to conventional treatment programs . Use of virtual reality as a treatment modality for improving functionality in children is considered a rich study field in which further results may be obtained from randomized studies with larger sample sizes and control groups.
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The proteolytic process of myofibrilar proteins plays a major role during post mortem meat tenderisation . The first of the systems initiates the breakup and destabilisation of the myofibrilar structure, while the second one affects partial protein degradation leading to their further proteolysis and development of tender meat . It is believed that the calpain proteolytic system plays the main role in the post mortem proteolysis and the process of meat tenderisation and a special role in this regard is assigned to -calpain [1, 2]. The post mortem protein proteolytic process in bovine meat is also significantly influenced by calpastatin (calpain inhibitor) which is less abundant in pork (more tender) than in beef (less tender) [3, 4]. There are many elements influencing the activity of meat proteolytic enzymes, nevertheless, special attention is paid to genetic factors [57]. Page et al . Described two mutations in the -calpain gene (capn1) which are associated with bovine meat quality . Transversion in exon 9 (c3709 g) and transition in exon 14 (a4558 g) were significantly correlated with bovine meat tenderness in piemontese angus as well as jersey limousine breeds measured 48 h post mortem . Polymorphism in intron 17 of this gene was significantly correlated with meat tenderness measured on days: 7, 14 and 21 post mortem in brahman breed . Experiments conducted by juszczuk - kubiak revealed a correlation of the rflp / foki polymorphism of the capn1 gene (intron 14 and exon 6rflp - hpych4iv / agei) with bovine meat quality, including its tenderness (exon 6rflp - hpych4iv / agei) and capn2s (3utr rflp / mboii) gene, i.e. A regulatory subunit of m - calpain with the above - mentioned traits (meat and fat content in valuable cuts, tenderness, colour, taste, consistence, ph). Recently, polymorphism of a new snp in the 30utr region of the bovine -calpain small subunit (capns1) gene (exon 11rflp / mboii) has been discovered . Therefore, it was considered appropriate to investigate its impact on the course of proteolysis and the process of bovine meat tenderisation . Analyses were performed on the thoracic and lumbar (m. longissimus thoracis and lumborum) longest muscles collected from 76 bulls of four cattle breeds (holstein - friesian, polish red, hereford and limousine) slaughtered at the age of 612 months . Capn1s gene polymorphism identification of bovine muscles was performed with the assistance of the pcr - rflp technique employing mboii endonuclease . Primers with capn1s - f 5-cctcactgtctgtcccttcc-3 and capn1s - r 5-acacaaatgttgggcttgg-3 sequences amplifying 332 bp were used in the experiment . The identification of the -calpain gene was performed in the 3utr axon 11 region . Samples for analyses were collected 45 min post mortem and proportions of myosin heavy chain (mhc) isoforms in fractions of rinsed myofibrils were determined employing methodology of mozdziak et al . . Changes in protein proportions of muscle tissue were evaluated 45 min post mortem and later: after 48, 72 and 240 h of cold storage using one - way electrophoresis (sds - page) in 15% polyacrylamide gel with addition of 8 m urea in which the acrylamide to bis - acrylamide ratio was 199:1 . From among muscle tissue proteins, particular attention was paid to the protein with the molecular weight of approximately 3700, 2400, 200, 103 and 37 kda . Meat tenderness measurements were conducted using an instron type 1140 apparatus with a warner - bratzler attachment in accordance with methodology given by grze et al . . Prior to value measurements of the shear force, samples were cut into slices 2530 mm thick, vacuum - packed and heated in a water bath at the temperature of 8081c to reach inside temperature of 72c and kept at the above temperature for the period of 90 min . Following their thermal treatment, samples were cooled for about half hour to room temperature and cuboids were excised measuring 10 10 40 mm which were subsequently cut perpendicularly to muscle fibres . Additionally, sensory tenderness assessment according to linear scale [15, 16] was carried out where the score of 10 points corresponded to very tender and 1 point very tough meat . Tenderness analyses, both instrumental and sensory, were conducted on days: 2, 4 and 10 of the meat cold storage ageing . Statistical calculations were based on the analysis of variance, whereas the significance of differences was calculated using tukey test at the significance level of = 0.05 using for this purpose statistica v. 8.0 software . Depending on genotypes, the determined proportions of native titin (t1 of 3700 kda m.w .) In the muscle tissue of the experimental cattle ranged from 2.53 to 3.21% (table 1) and were relatively low in comparison with data reported in other experiments [17, 18] where these proportions fluctuated from 7 to 10% . However, the above values referred to proteins of rinsed myofibrils . Since our experiments were carried out on meat proteins without their prior fractionation, the obtained values could be lower . At the same time, however, it is worth emphasising the fact that a relatively large share (from 3 to 5%) occurred in the 2400 kda m.w . This could indicate that the process of titin degradation in the meat of the examined bulls was relatively well advanced and its total proportion determined on the basis of the entire band comprising titin t1 and t2 ranged from 8 to 9%.table 1share of selected proteins in bovine lt&l muscle depending on genotype of -calpain (capn1s) and storage time in chilled room (%) genotypes mboiinmolecular weight of proteins (kda) and storage time of meat (45 min, 48, 96 and 240 h)37002400200103approx . 3745 min48 h96 h240 h45 min48 h96 h240 h45 min48 h96 h240 h45 min48 h96 h240 h45 min48 h96 h240 htotal563.13.33.13.03.43.94.14.514.015.515.516.13.123.133.143.057.05.95.33.7ct (1)253.23.73.52.53.23.63.94.413.715.915.916.03.193.093.243.157.35.85.34.1cc (2)263.23.12.83.03.74.04.24.614.215.215.216.13.143.183.163.066.76.05.23.4tt (3)82.52.63.34.23.54.75.04.614.115.015.316.22.843.082.792.747.35.95.33.6n number of animals in the groupmean values marked with different small letters differ in columns (p 0.05)mean values marked with different capital letters differ in rows (p 0.05) share of selected proteins in bovine lt&l muscle depending on genotype of -calpain (capn1s) and storage time in chilled room (%) n number of animals in the group mean values marked with different small letters differ in columns (p 0.05) mean values marked with different capital letters differ in rows (p 0.05) investigations conducted by huff - lonergan et al . Showed that the majority of native titin (t1) was degraded during the period of 2 weeks . Fritz and greaser maintained that titin was still present even after 16 days of cold storage . In our studies, the recorded changes in t1 and t2 proportions during 10-day meat cold storage were small, although the proportion of t2 kept increasing gradually confirming observations made by ho et al . . The above researchers claim that this band could be noticed on meat protein separations even after 28 days of ageing both in meat after electrical stimulation and in meat which was not subjected to such treatment . In the discussed trial, the highest protein share of 2400 kda m.w . Was observed in homozygotes but differences between homozygotes and heterozygote amounted only to 0.220.23% and were not statistically significant (= 0.05) (table 1). The recorded small changes in the proportions of the two above - mentioned titin bands could have been associated with the sex of animals . It is evident from investigations conducted by huff - lonergan et al . That the degradation process of this protein is slower in the meat of bulls which were analysed in this experiment in comparison with the meat of steers . The same researchers also suggest that the degradation process in meat of young bulls is similar to that observed in the raw material obtained from cows . The protein proportion of 200 kda m.w . Corresponding to myosin heavy chains (mhc) and titin degradation products [22, 23] increased during the cold storage period from the initial 14% to the final level of over 16% (table 1). Statistically significant differences were determined for the heterozygote and homozygote cc . In the first case, they concerned differences between the first date of assessment (45 min) and the remaining dates, whereas in the second only between the extreme terms of examination (table 1). As evident from experiments conducted by sawdy et al . And morzel et al ., myosin heavy chains undergo post mortem degradation the effect of which can also be the process of meat tenderisation . Band at simultaneous possibility of mhc degradation makes it possible to assume that this increase could have been associated with the advancing titin degradation since products of its degradation are also observed in the band with the same molecular weight [22, 26]. The proportion of protein of 103 kda m.w.which corresponds to -actinin, the main protein of z line during 10-day cold storage practically remained unchanged and differences observed between the initial and final values did not exceed 0.07%, irrespective of genotype (table 1) indicating that this protein did not undergo degradation during the cold storage of the examined muscle tissue and this is in keeping with the findings of other researchers [17, 22]. The above researchers reported lack of degradation and noticeable changes in the -actinin band during the first 1418 days of ageing storage at the temperature of 4c . A similar phenomenon was also observed in case of application of electrical stimulation of bovine carcasses . The results of experiment conducted by purintrapiban et al . And goll et al . Reveal that -actinin is not degraded by calpains, although following interactions with other enzymes the process of its degradation is possible . In the course of meat cold storage, changes were recorded in protein proportions of 37 kda m.w . Which, with respect to molecular weight, corresponds to troponin t [2931]. This protein, from the point of view of molecular weight, may correspond to the glyceraldehyde 3-phosphate dehydrogenase beginning with the 45th minute until the 240th hour post mortem, the share of this protein decreased from 7.03 to 3.70% (table 1). The process of degradation of this protein was similar for the heterozygote and homozygotes, although its course was most dynamic (decline in proportions of about 50%) for the tt homozygote and the slowest for the heterozygote . Among the analysed genotypes of the capn1s gene, statistically significant differences were observed in proportions of this protein between the analysed dates of ageing . In the case of the tt homozygote, these differences concerned extreme values, whereas for the heterozygote and cc homozygote, differences were recorded between three dates of cold storage . Protein following 240 h storage was observed for cc homozygote and differences between its proportions for this genotype and the heterozygote were statistically significant (table 1). This appears to indicate similar relationships regarding changes of this protein to those observed in the case of gapdh in pork meat [24, 32]. The above researchers maintain that its degradation may explain changes in the overall acceptability including meat taste, smell and tenderness during cold storage at 4c . The above remark does not rule out the participation in this process of t troponin whose degradation is associated with increased meat tenderness [2931]. When analysing the course of transformations taking place in animal muscle tissues after slaughter, which are associated with proteolysis and exert influence on its tenderness, attention is frequently focused on the character of muscle fibre metabolism [18, 33, 34] emphasising that the course of proteolysis is usually faster in muscles with the majority of type mhc 2a fibres in comparison with the red fibres (mhc 1). That is why in the course of these experiments, relationships between the changes the proteolytic process and the type of muscle fibres were analysed determining their proportions with the assistance of sds - page electrophoresis . Appear to indicate that degradation changes of muscle tissues take place faster in light fibres in comparison with the red ones, although results published by renand et al . Seem to contradict these findings . One of the factors that can disturb these relationships may be the rate of the glycolytic process . Rapid rate of glycolysis, which is supported by the occurrence of type 2a and 2b mhcs, leads to the development of meat defects . Glycolytic processes are slightly faster in them than in the type 1 red fibres rarely found in bovine muscles . In the discussed experiment, the highest (38.08%) proportion of the type 2a mhcs was determined for the heterozygote of the capn1s gene and differences between values determined for the heterozygote and homozygotes were statistically significant (table 2) ranging from 7.01 to 3.88% . Statistically significant differences also occurred for proportions of the type 1 mhcs between heterozygote for which their lowest level was determined (30.19%) and homozygotes (cc40.20% and tt39.01%, table 2). The observed similar course of muscle protein degradation changes recorded for the compared genotypes accompanied by significant differences in mhc polymorphism may indicate that muscle fibre metabolism was a decisive factor in the meat tenderisation process.table 2share of mhc isoforms in lt&l muscle depending on -calpain genotype (capn1s)genotypes mboiinmhc 2amhc 1total7634.2036.21ct (1)2938.0830.19cc (2)3631.0740.20tt (3)1134.2039.01see table 1 share of mhc isoforms in lt&l muscle depending on -calpain genotype (capn1s) meat tenderness during ageing was evaluated at three terms (48, 96 and 240 h) by measuring the value of the shear force (n / cm) and sensorially employing a linear 10-point scale . Together with the progressing process of proteolysis, starting with the 48th hour post mortem, a gradual drop in the shear force value was observed . After 48 h of ageing, the highest tenderness was determined for the cc homozygote, while already from the 4th day, the lowest shear force values were determined for heterozygote . On the last (10th) day of investigations, statistically significant differences were observed between the tenderness determined for the cc homozygote and heterozygote . Meat from the latter genotype was characterised by the highest tenderness (table 3). When investigating the relationship of the calpain l and ll gene polymorphism with meat tenderness, costello et al . Also determined its highest value for heterozygote . After 14 days of ageing, it amounted to 39.05 n / cm for the calpain l gene (exon 9, genotype ga) and to 43.80 n / cm for the gene of calpain ii (regulatory unit, genotype ab).table 3values of shear force (n / cm) and results of sensory evaluation of lt&l muscle tenderness (scores) depending on -calpain genotype (capn1s)genotypes mboiinshear force value of meat (n / cm)nsensory evaluation of meat (scores)48 h96 h240 h48 h96 h240 htotal69148.6133.9108.2714.24.95.7ct (1)27146.0132.099.1274.24.76.1cc (2)31153.7135.5117.0334.14.85.5tt (3)11140.8134.2105.4114.75.65.6see table 1 values of shear force (n / cm) and results of sensory evaluation of lt&l muscle tenderness (scores) depending on -calpain genotype (capn1s) sensory assessment of meat tenderness reflected the results of the shear force value measurements . The highest scores corresponding to tender meat were given to the meat that was ageing for 10 days, though they were not the highest in absolute terms (maximum score was slightly above 6 points) (table 3). These results appear to indicate that the meat of 6 to 12-month old cattle requires the same length of the ageing period to reach the desired tenderness as that of older cattle and this is in agreement with observations made by dransfielda et al . And koczaka et al . On the 10th day of ageing, the highest scores were awarded to heterozygote (ct) but differences between it and the remaining genotypes of the capn1s gene were not statistically significant (table 3). When analysing shear force values and scores allotted to meat samples by sensory evaluation, statistically significant differences were observed between them taking into consideration cold storage time . These differences were observed when analysing all samples, irrespective of their genotype in relation to the capn1s gene and when its impact was taken into consideration . However, in the first case, statistically significant differences were observed between each of the analysed dates . In the case of genotypes, they usually referred to differences between the 2nd and 10th day of meat cold storage . Statistically significant impact was observed of the capn1s gene on meat tenderness of young slaughter cattle . Meat tenderness improvement was associated with a significant decrease in the proportions of about 37 kda m.w . The recorded drop in proportions of the first group of proteins could have been associated with troponin t and gapdh degradation, whereas the increase in the second band with titin degradation and appearance of products of its decomposition . Heterozygote of the capn1s gene was characterised by the highest share of the type 2a mhc isoform and the lowest of the type 1 . A similar course of degradation changes of muscle proteins for the compared genotypes at simultaneous significant differences in mhc polymorphism may indicate that this polymorphism associated with the impact of the capn1s gene was a decisive factor which determined meat tenderness.
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The polymerization of a light - cured material will result in shrinkage of the restoration . These are believed to cause microleakage, postoperative sensitivity, recurrent caries and eventual loss of the restorations . After exposure of the restoration to oral fluids, some relief from the curing shrinkage may arise from water uptake . The water that diffuses into the material causes a gradual expansion, up to a certain equilibrium value which will contribute to relaxation of shear stresses . In contrast to the rather rapid polymerization contraction and stress development, the hygroscopic relief will proceed slowly and might even take days . Hygroscopic expansion may compensate for the curing shrinkage thereby improving the marginal quality of the restoration and closing of the gap [57]. The rate and magnitude of hygroscopic expansion of a resin material depends on several variables such as the nature of the resin, the type of filler, filler loading, filler matrix adhesion and the volumetric ratio between the filler and matrix [810]. Results from a 7-day - study showed that the hygroscopic expansion of composites reached equilibrium after approximately four to six days depending on the materials investigated . Whereas, in another study, an increased water absorption was observed during the first month for all composite resins with a further small increase up to six months . The linear hygroscopic expansion of conventional and resin - modified gic liners was measured for up to one week . It was observed that the dimensions of the conventional gics did not show any significant change after 30 minutes of immersion, while the resin - modified cements exhibited changes up to 24 hours and remained constant for the next week . The resin modified gic liners showed a significantly higher expansion than the conventional cements, a finding which was confirmed by other studies . It was also reported that at six months, the mean change in linear expansion was 0.16%, 0.66% and 0.32% for the microfilled composite, polyacid - modified composite resin and dual curing composite, respectively; concluding that the hygroscopic expansion of the polyacid - modified composite resin material was significantly greater than that of the composite resin tested . Investigations of linear hygroscopic expansion of the resin - modified gics are limited, especially regarding their long - term expansion . The aim of this study was to investigate the long - term linear hygroscopic expansion of resin - modified gics in comparison with those of a conventional gic, a polyacid - modified composite resin and two composite resins . The magnitude of the hygroscopic expansion of specimens which were made using bulk insertion techniques was also investigated . The materials used in this study were fuji cap ii, fuji ii lc, photac - fil aplicap, vitremer, dyract, tetric and z 100 (table 1). The moulds (64 mm) used for specimen preparation were of the split - body type, constructed of stainless steel . Before use, a dry ptfe (poly tetra fluoro - ethylene) separating film the mould was placed on a clean glass plate that had been coated with a ptfe film . The mould was overfilled with the restorative material and another glass plate was placed over it with firm pressure . Each layer of the material was light cured separately using the visilux 2 (3 m, usa) curing unit for the recommended time . Fuji cap ii was inserted into the mould in one bulk and the specimens were left for 10 minutes at 37c and 100% humidity . Five specimens were made for each material . To assess the effect of a bulk insertion technique on the expansion of the light - cured materials, five more specimens were made for each of these materials . To prepare these specimens, the material was applied in bulk until the mould was overfilled and light - cured from both sides simultaneously using two light - curing units visilux ii and caulk max; l.d . Any flash of excess material was removed so that the surfaces of the specimen were flush with the surfaces of the mould and were perpendicular to the long axis of the specimen . Lapping was carried out in as short a time as possible without using water to eliminate the effect of hydration . After lapping, the specimen was removed and stored in a separate plastic bottle containing distilled water at 37c for up to one year . Prior to storage, the length of each specimen was measured three times using a digital micrometer (mitutoyo, mitutoyo corporation, japan) and the mean of the three readings was used for calculation of the percentage change in the length of the materials . The specimens were removed from the water after 24 hours, one week, one month, three months, six months, nine months and one year following preparation and dried using tissue paper . The length of the specimens was again measured as mentioned above and the linear expansion of the materials was presented as the percentage of change of the specimens in relation to the baseline measurement . The mean hygroscopic expansion and standard deviation (sd) were calculated for each material at various time intervals . Paired sample t - test with bonferroni correction was employed to assess the difference between the means of the two groups . Photac - fil aplicap showed the highest expansion values followed by fuji ii lc and vitremer; whereas, fuji cap ii exhibited the least expansion . Bulk inserted specimens showed higher expansion than layered specimens (p<0.001) except for z100 . This could be explained by the fact that z100 was the only material that exhibited similar expansion for both layered and bulk inserted specimens (0.325 vs 0.330). Paired sample t - test showed that the time to reach a constant level differs for each material . For instance, fuji cap ii, fuji ii lc and tetric achieved the constant length at three months, while this time was six months for photac - fil aplicap and z100 and nine months for vitremer and dyract . The interaction between time, material and method of preparation as well as the interaction between time and method of preparation was not significant (p=0.330 and p=0.151, respectively). However, the interaction between time and material was significant (p<0.001), which could be elucidated by the difference in the hygroscopic expansion slope of materials (fig 1). Dimensional changes of restorative materials caused by hygroscopic expansion may be determined by a variety of equipment and test methods such as hydrostatic or archimede s principle; model cavities cut in brass, in which the hygroscopic expansion of the material was expressed by the displacement force generated due to water sorption; the ability of materials in reducing the marginal gap; measuring the relaxation of setting shrinkage shear stress; measuring the length of specimens by means of an electric or a computer - controlled laser micrometer or a microscope . In the present study, a micrometer with an accuracy of one micron was used to measure the change in the length of cylindrical specimens (64 mm). The procedure proved to be uncomplicated and the equipment was inexpensive . In addition, the advantages of using specimens with the above dimensions were that they simulated relatively large dental restorations . In this study, unlike the conventional gic, the resin - modified cements showed significant expansion . As mentioned earlier fuji cap ii, fuji ii lc and tetric reached a constant length after about three months while more time was necessary for photac - fil aplicap and z100 (six months). The finding that a relatively long time was required for the composite materials to reach a constant length is in agreement with that of momoi and mccabe, who observed an increase in the displacement force due to water uptake during the six - month test period . They suggested that such an increase indicated that there were regions within the specimens that were not fully saturated . In another study, however, after seven days of storage in water at 37c, all composite materials showed significant hygroscopic expansion that did not significantly increase further until the 30-day storage . It was observed that the dimensions of conventional gics did not show any significant change after 30 minutes of immersion in water, while the resin modified cements exhibited changes up to 24 hours and remained constant for the next week . The composite resin used demonstrated a significantly continuous increase during the period of the study (one week). These results indicate that composite resins require a longer time than the resin - modified gics to reach a constant length . The higher linear expansion for the resin - modified gics observed in the present study is in agreement with the result of recent investigations . Irie et al reported that the resin - modified gic liners showed significantly higher linear expansion than the conventional cements, the magnitude of which was in the range of 2.4% 6.3% . In kimishima et al s, vitremer showed slightly less expansion (4.98%) than fuji ii lc (5.44%), which is in line with the results of the present study . This high linear expansion of resin - modified gics could be attributed to the presence of hydrophilic resin hema in resin - modified cements . The higher linear expansion associated with dyract, in comparison with that of composite resins, could also be explained by the fact that this material also contained some hydrophilic monomers . It was reported that at six months, dyract showed a linear expansion (0.66%) greater than that of either a light - cured composite (0.16%) or a dual - cured composite resin (0.32%). In addition, it was shown that fuji ii lc produced the greatest (38 mpa) and most rapid rise in lateral stress brought about by hygroscopic expansion . Z100 and tetric produced a linear rise up to six and four mpa, respectively . These findings are congruent with the results of the present study where dyract showed a hygroscopic expansion intermediate to that of the resin - modified gics and composite resins . The results currently recorded for fuji cap ii must be viewed with caution . In the present study, the specimens made from fuji cap ii were maintained at 37c and 100% relative humidity for 10 minutes . Therefore, the first measurement was performed approximately 20 minutes after mixing (taking into account the time required for lapping). Since conventional gics undergo setting shrinkage immediately after setting, this delay in measurement might have had an effect on the results . For all other materials which were all of the light - curing type, the measurements were carried out within 5 minutes after curing . Except for z100, the difference between the linear hygroscopic expansion of the layered and bulk inserted specimens was generally significant, with the bulk inserted specimens showing less expansion . This finding is in agreement with that of bowen et al, who reported that when a composite resin was placed in bulk it had more shrinkage, less hygroscopic expansion and some degree of residual shrinkage . The higher shrinkage of a material placed in bulk; therefore, might account for the lower hygroscopic expansion observed in this study . However, it is known that restorations that are inserted in bulk show wider marginal gaps and more microleakage . As discussed earlier, the setting shrinkage of restorative materials results in marginal gap formation, microleakage and probably recurrent caries . Since the expansion occurs sometime after the shrinkage has taken place, the expansion will not lead to the re - establishment of any broken adhesive bonds and perfect closure . During its earliest stages, expansion may simply cause a closing up of the contraction gaps caused by setting shrinkage . Continued expansion, however, may cause development of an outward pressure against the cavity walls . Estimation of the magnitude of this pressure may enable its clinical significance to be inferred . Feilzer et al suggested that in the clinical situation, a slight positive stress is preferable to a tensile stress as it may improve the marginal integrity of a restoration . They found that when the experiments ended after 15 hours, a further build - up of compressive stress was observed with the resin - modified gics . Momoi and mccabe reported that the hygroscopic expansion of composite resins during a 6-month study resulted in pressure values of 3.3 to 14.5 mpa dependent on the material . They suggested that positive pressure of a similar magnitude pushing against the cavity walls may be capable of putting the supporting tooth tissues under considerable stress . Using the results of this study and the modulus of material elasticity provided by another study, the magnitude of the pressure generated from the expansion of the materials tested may be calculated from the following equation: stress = straine, where e is the modulus of elasticity of a material and strain is the expansion (%) of the material at a given time . Such calculations gave pressure values of 124.55 mpa for photac - fil aplicap, 111.33 mpa for vitremer, 103.41 mpa for fuji ii lc, 44.66 mpa for dyract, 34.95 mpa for z100, 11.04 mpa for tetric and 0.52 mpa for fuji cap ii; each calculation was made using the values obtained at the time when the material reached its constant length . These values are much higher than those reported by momoi and mccabe, watts et al these studies employed a model cavity design, in which the materials were inserted in a brass mould and the pressure required to displace the specimens was calculated . The presence of such close contact between the brass and test material might have led to a lesser water absorption, hence less hygroscopic expansion than that recorded in the present study . The resin - modified gics exhibited the highest linear hygroscopic expansion among the materials tested . Photac - fil aplicap showed the highest hygroscopic expansion between the resin - modified cements . The time to reach a constant level at one year, however, its linear expansion was significantly higher than the composite resins.
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Dermatology and cardiology at a first glance seem to be very distinct medical fields, but the relationship between them is shown by the adverse effects of amiodarone, the key antiarrhythmic drug . Amiodarone has been used by physicians for over 50 years to treat supraventricular and ventricular cardiac arrhythmias, and it often provides the last treatment option due to its high efficacy . However, prolonged amiodarone therapy unfortunately carries an extended risk of adverse effects, mainly involving the thyroid gland, liver, lungs, eyes, and skin . The incidence in the population of patients with prolonged use of this drug reaches nearly 75% according to various sources . The main skin changes induced by amiodarone are phototoxic and photoallergic reactions, as well as hyperpigmentation . In most cases although the dermatological complications usually do not influence the outcome of the therapy and rarely cause the discontinuation of treatment, they have great impact on patient quality of life . Phototoxic reactions are the most common dermatological adverse effect of amiodarone therapy, affecting 2575% of patients on long - term treatment . Photoallergy is considerably less likely to occur, but the risk also increases with prolongation of the therapy . Amiodarone and its active metabolite, desethylamiodarone, cause decrease of med (minimal erythema dose) in the range of ultraviolet radiation type a (uv - a 320400 nm), which can reach up to 50% of the correct values . In individual cases there may also be a reduction of med for the ultraviolet type b radiation (uv - b 290320 nm). The mechanism of the reaction seems to be connected with the creation of active metabolites due to radiation, such as oxygen free radicals, which in turn leads to destruction of dna particles, cell membranes, and oxygenation of lipids . Ferguson et al . Demonstrated phototoxic effects of amiodarone and desethylamiodarone by carrying out in vitro tests with the use of photohemolysis, dna synthesis inhibition assay in pha - stimulated lymphocytes, and the killing of macrophages obtained from mouse peritoneal fluid . Skin changes usually occur after at least 4 months of therapy and with the minimal cumulative dose, which is 40 g . They have the typical erythematous or eczematous appearance with accompanying pruritus in the parts exposed to sunlight, usually on the hands, face, and neck . The eruptions are less prominent on the chin, lower lip, and behind the ears . The symptoms begin several minutes after exposure to the sunlight, continue up to 24 hours and usually subside by around 48 hours, but in some cases they persist up to 72 hours . Phototoxic and photoallergic reactions might even occur a few months after the withdrawal of amiodarone due to its long elimination time, which on average takes 3540 days, and in obese patients can take 100 days for complete excretion . In the literature there are also descriptions of prolonged photosensitivity cases (up to 15 years following the discontinuation of therapy). The extent of the reactions depends mostly on the individual sensitivity of the skin to sunlight, but it is always proportional to the exposure time . The basis of therapy is withdrawal of the medication and restoration of skin lipid layers . It also includes the use of medium strength topical glucocorticosteroid, oral non - steroidal anti - inflammatory drugs, and intensive sunscreen protection . Prevention of acute reactions caused by sunlight in patients treated with amiodarone should include avoidance of frequent sun exposure on the skin and the proper application of external sun protection products with high protection factors (spf 50 or 50 +), reapplied not less frequently than approximately every 2 hours . Sunscreen should provide the full spectrum of protection, including a uv - a radiation . Particularly effective are creams containing zinc oxide or titanium dioxide . Glass commonly used in windows is permeable to uv - a radiation and does not provide protection to people in buildings and cars . In contrast to the phototoxic reactions, which do not depend on the skin type, hyperpigmentation usually occurs in patients with skin type i phototype . In these patients, long - term therapy with amiodarone leads to characteristic blue - grey discolorations, located mostly on the face, ears, and palms of the hands (figure 1). Histopathological examination of the skin shows yellow - brown deposits of lipofuscin aggregates within macrophages, mast cells, endothelial cells, smooth muscle cells, keratinocytes, and fibroblasts . Some authors report that the hyperpigmentation is likely due to the amiodarone deposits instead of lipofuscin . Amiodarone binds to phospholipids and creates insoluble compounds that do not follow the regular degradation pathways, which leads to their accumulation in cellular lysosomes . The discovery of increased amounts of iodine or drug metabolites in granules proves that amiodarone or desethylamiodarone are present in the affected skin . In addition to the deposits of lipofuscin, histopathological examination revealed the presence of inflammatory cells around the microvessels . Hyperpigmentation usually occurs 20 months into amiodarone treatment regime and sometimes after longer periods of time . The doses of the drug used are higher in patients with hyperpigmentation (400800 mg per day) compared to patients with only phototoxic reactions . The changes have blue - grey discoloration, but in 10% of patients it takes on yellow - brown pigmentation . Full remission is commonly achieved after a few months to several years, which is the consequence of slow elimination of amiodarone and its metabolites from tissues . Unfortunately, even after drug withdrawal for a few years, some changes may persist . Prophylaxis includes avoidance of direct excessive sunlight exposure to the skin and use of sunscreen products with high protection factors (spf 50 or higher) against uv - a and uv - b radiation . Switching from amiodarone to another antiarrhythmic drug could also be very beneficial . There have been individual reports on the efficacy of vitamin b6 at 300 mg per day . In contrast, there are reports of spectacular efficacy of cosmetic laser application, which could possibly be very beneficial in patients with too many cardiovascular complications to allow amiodarone withdrawal [1517]. There are accounts of very successful therapy with uv - b rays, leading to higher tolerance to sunlight and prolongation of possible exposure times without adverse effects . Amiodarone is considered to be a cause of pseudoporphyria, which is a disease with clinical picture similar to this of porphyria cutanea tarda, but without any identified biochemical changes in the metabolism of porphyrins . Pseudoporphyria is usually manifested by tense bullae, erosions, scars, postinflammatory hyperpigmentation, and increased skin fragility, especially in the sun - exposed regions . A very rare adverse reaction to amiodarone treatment is linear iga bullous dermatosis, and it is thought that amiodarone can exacerbate the course of this disease . Characteristic changes include the presence of well - differentiated blisters located either on healthy skin or on erythematous and edematous areas . Location of the changes does not correspond to the sun - exposed parts of the skin . The amiodarone molecule contains a large amount of inorganic iodine, which may exacerbate certain skin diseases, such as dermatitis herpetiformis and psoriasis . Moreover, there have been reports of post - amiodarone pseudo - purulent changes, such as acne or blister - purulent lesions characteristic of iodism . Less common dermatological complications of amiodarone include hives, pruritus, erythema nodosum, purpura, and the most severe variant of erythema multiforme toxic epidermal necrolysis . The acute complication of treatment with intravenous amiodarone administration can take on the form of necrosis of the skin and soft tissue due to venous extravasation . The risk is related to low ph (3.54.5) of the solution and the content of solvents, such as benzyl alcohol and polysorbates . A few cases of basal cell carcinoma associated with chronic use of this drug have been reported . Most of the changes were observed on the site of a preexisting acute phototoxic reaction . Complications of chronic amiodarone therapy compel the prescribing physician to inform the patient about all possible adverse reactions, methods of avoiding them, and the need for specialist advice if symptoms occur . Any doctor approached by a patient with skin lesions secondary to amiodarone must be aware of the risks of stopping treatment, because it can exacerbate the severity of arrhythmias and even lead to sudden cardiac death . Cooperation between cardiologists and dermatologists will undoubtedly facilitate risk stratification and ensure that optimal decisions will be made to provide maximum safety of the patient, while at the same time reducing the adverse effects.
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A 53-year - old man presented with transient acute onset of left - sided numbness and speech disturbance one week prior to admission . Transient ischemic attack or acute cerebral infarction were possible diagnoses, so mri and mr angiography were performed, showing an occlusion of his right mca at the proximal m1 segment without any acute ischemic lesion or infracted area on mri, including diffusion - weighted images . The next day, a conventional angiogram was taken and revealed a tapered occlusion at proximal m1 segment of right mca, collateral pathways through perforating arteries, rich pial collateralization from distal anterior cerebral artery, and a shift of the watershed zone (fig . These findings were compatible with the long - standing stage of the occlusion of mca rather than the acute occlusion . He also has an incidental saccular aneurysm at left posterior communicating artery (p - com) origin . The brain single photon emission computed tomography (spect) showed mildly decreased perfusion in right middle cerebral artery territory and the vascular reserve was also decreased mildly after acetazolamide injection (fig . An echocardiogram and myocardial spect excluded cardiac embolus as the etiology of the occlusion . Although the patient had no past medical history, untreated hypertension and diabetic mellitus were found during evaluation . In this setting of presumably chronic occlusion the patient was initially treated with antiplatelet and circulating drug and carefully observed for two weeks . After two months, the left p - com aneurysm was managed by the microsurgical aneurysmal neck clipping and there were no developing or new neurologic symptoms after surgery . Immediate postsurgical computed tomography angiogram (cta) showed persistent right mca occlusion and complete aneurysm clipping (fig . The brain cta of 21 months after neck clipping developed recanalization of the previously occluded right mca . A subsequent conventional angiogram confirmed nearly complete patency of the right mca with focal mild stenosis, normal blood flow through mca, and the normalization of the shift of watershed zone (fig . Our patient experienced late spontaneous recanalization and restoration of blood flow by an unknown mechanism within 21 months, without any neurologic deficit . Spontaneous recanalization of the occluded mca has been a common finding in acute ischemic stroke . Several studies demonstrated that most recanalization may occur in the acute phase of stroke, within approximately 48 hours after onset [2, 5]. A recent meta - analysis of thrombolytic therapy attempted to quantify spontaneous recanalization in ischemic stroke . Spontaneous recanalization occurred in 21.4% of patients within 24 hours and in 52.7% of patients by a week . Although the analysis of these data had limitations, including the variety of times examined, but the suggestion is that the natural history of cerebral embolus is dissolution and spontaneous recanalization over a period of time . Like these studies, spontaneous recanalization of mca, in which occlusion is apparent in the acute phase have occasionally occurred in the subacute phase of stroke . But late recanalization in the chronic phase has not been reported to the best of our knowledge . Unlike recanalization of occluded artery in acute stroke, the spontaneous recanalization of a long - standing occlusion of extracranial artery has been only anecdotally reported [7 - 9]. The mechanism by which recanalization of chronically occluded carotid arteries occurs is still little known . In 1999, suggested the possibility of occlusions resulting from ulcerated plaque thrombosis presenting long - term recanalization by thrombolysis . Colon et al ., in 1999, published a series of four cases of spontaneous recanalization of the internal carotid artery, which through imaging examinations and intraoperative finding, proved to be a hypertrophy of vasa vasorum, causing reperfusion of the distal to the occlusion . Another possible mechanism is that, in the case of myointimal hyperplasia or atherosclerotic disease, the neovascularization is induced, which in the long term can allow perfusion distal to vessel occlusion . Persistence of some embryonic vessels can also account for the complete non - occlusion of the whole internal carotid artery segment, allowing action of varied mechanisms for vessel recanalization . Nowadays some groups have been following patients with carotid occlusions, with the aim of better evaluating the natural history of such lesions . Verlato et al ., in 2000, published a cohort study including 41 patients with carotid occlusion . The mean follow up periods were 44.5 months . In one case with asymptomatic carotid occlusion of 41 patients, spontaneous recanalization was identified at three years after the diagnosis, remaining without symptoms for the whole period . The mechanism of late recanalization of chronic occlusions of mca could be one as remarked above or not . Unlike the extracranial carotid artery, the intradural cerebral artery has a little chance to the hypertrophy of vasa vasorum or the revascularization within a plaque due to the nature of intracranial artery, including the lack of vasa vasorum and the smaller caliber . In our case, we cautiously speculate that the recanalization process could be that the repeated embolic formation and accumulation were progressed to complete obstruction for some period of time in focal thrombotic area . After antiplatelet therapy, the thrombolysis occurred gradually instead of repeated embolic accumulation . Finally, spontaneous recanalization occurred at this late stage . The angiographic changes in our patient illustrate that the chronic mca occlusion were spontaneously recanalized by an unknown mechanism within 21 months, with a good clinical outcome . The incidence, mechanism, and ideal management of this extraordinary finding remain unclear . Through this rare case, further investigation and studies into the underlying mechanism of spontaneous recanalization should be performed.
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Food allergy policies in public places and stories in the popular press are evidence of the rising awareness of food allergies as an important public health risk . In its most severe form, ige - mediated food allergies can lead to anaphylaxis, which is potentially fatal . For example, self - reported prevalence in the us is 9.1% (8% for children) and 5.3% for respondents with a physician diagnosis [2, 3]. In canada, the canadian estimates, however, were based on a nonrepresentative sample that underrepresented vulnerable populations (i.e., low income families, immigrants, and aboriginal peoples) [5, 6]; a second national survey undertaken to address this limitation indicated that the prevalence of self - reported food allergy for immigrants who have lived in canada for less than 10 years (herein referred to as new canadians) was substantially lower than their canadian born counterparts: 3.2% . These researchers also investigated perceived prevalence of food allergy in the same populations and found that perceived prevalence was observed to surpass systematic estimates by up to 30% . The gap between perceived and actual risk of food allergy suggests that the public's perception of true risk of allergy is, in fact, inflated . Moreover, new canadians were found more likely to rate the risk of food allergy as high compared to nonimmigrants or immigrants who have lived in canada for over 10 years, despite the substantial differences in measured prevalence . In light of the above, this paper used a qualitative research design to unpack the perceptions and experiences of new canadians directly affected by food allergy by addressing the following objectives: (1) to understand how new canadians perceive food allergies and their associated risks and (2) to investigate how new canadians manage and cope with food allergies . Waterloo region, ontario, canada, was chosen as the research site as it is one of the fastest - growing areas in the province with the 9th highest proportion of visible minorities of all census metropolitan areas across canada . Qualitative methods have been used with several populations at risk of anaphylaxis: children [1113], adolescents [14, 15], their parents [13, 14, 16], allergists, and the general public [18, 19]. First, key informants (an allergist, public health nutritionist, and public health planner) recruited through telephone and e - mail were interviewed in person (averaging 30 minutes) as representatives of key constituencies able to provide an information - rich connection to the research topic, as well as access to the wider food allergies community . Service providers and health workers have been shown to be rich and largely untapped sources of information with important insights to issues of health and migration . Each interview covered topics specific to the key informant's observations and knowledge of food allergies from their professional work experience . Second, in - depth interviews with affected individuals were conducted with new canadians who either had a physician - diagnosed food allergy of their own or were a parent of a food allergic child . The focus was on individuals of east and southeast asian descent as 50% of the immigrant participants in the canadian national prevalence survey were born in an asian country [4, 5]. East and southeast asians are also two of the most common visible minorities in waterloo region . Participants (n = 18) were recruited using kijiji (an online network for posting local classified advertisements), the interviewed key informants, and contacts made with local cultural centres . 2012 to jan . 2013) until saturation was reached (i.e., until no new themes emerged from the interviews). Participants were also asked to complete a short demographic survey to gather additional information on their food allergy and length of time in canada . 78% of the sample was female and 72% of all participants were under 30 years old (table 1). Just over half the participants have lived in canada for less than 10 years (n = 10). The most common self - reported allergen was shellfish (n = 12) and the majority of participants were over 5 years old when their allergy was diagnosed (n = 15). Each interview was conducted by the primary author and audio - recorded for transcription and subsequent thematic analysis using qsr international's nvivo 9 . A theme code set was created both deductively (based on the research objectives and interview guides) and inductively (themes emerging from the interview transcripts). Coding was assessed for inter- and intra - rater reliability, achieving 81% agreement with a second coder at the graduate level . Key themes that were identified were those that appeared with the greatest frequency as discrete units of text in the interview transcripts . Three key themes emerged from the key informant interviews, the first being appropriate and informed diagnosis . New canadians are concerned with receiving confirmation of their allergy and getting reassurance that their problem is not life - threatening . While the diagnosis process is straightforward, it can be difficult to educate new canadians about how to avoid a reaction as well as the importance of using epinephrine in the form of an epi - pen: understanding what the issue is, is tough enough for patients that have grown up in canada . To really appreciate and understand what a food allergy is, what it involves, and the potential risks, i think for immigrants it is even tougher because in most countries where they have immigrated from, allergy didn't exist . Or if it did exist, it was a really, really small issue . (key informant 1) understanding what the issue is, is tough enough for patients that have grown up in canada . To really appreciate and understand what a food allergy is, what it involves, and the potential risks, i think for immigrants it is even tougher because in most countries where they have immigrated from, allergy didn't exist . Or if it did exist, it was a really, really small issue . (key informant 1) the second theme that emerged is the challenge of shaping a safe school environment for food allergic children . Placing bans on foods in schools can be very restrictive, making it difficult for people to find food alternatives, especially if cost is a concern . A restrictive diet can also make it more difficult to ensure that children are meeting nutritional needs: it becomes very restrictive for the students who aren't allergic . So it is really concerning when foods are starting to be banned from the classroom, because soon, if there are more and more allergic students, many foods will be banned and it's very hard for people to find those foods and for there to be foods that are within the right price range, especially for people with lower income . So this is a major issue, and we really strive towards educating that it should be an allergy safe environment as opposed to an allergy free environment, because it really, in our opinion, is almost impossible to do that . (key informant 2) it becomes very restrictive for the students who aren't allergic . So it is really concerning when foods are starting to be banned from the classroom, because soon, if there are more and more allergic students, many foods will be banned and it's very hard for people to find those foods and for there to be foods that are within the right price range, especially for people with lower income . So this is a major issue, and we really strive towards educating that it should be an allergy safe environment as opposed to an allergy free environment, because it really, in our opinion, is almost impossible to do that . (key informant 2) furthermore, studies have shown that food allergic children have been known to experience stigma and/or social exclusion, particularly in the school setting . How allergic children are being perceived by their peers, especially if an incident gets reported in the media, was raised by key informants: i was really disturbed by the fact that you know [a story about an allergic child] was in the paper, and it was naming the school and the age of the child and the parent, so people know who this child is i don't think it is fair to anyone involved, but it is definitely not fair to the child and we don't want to be causing that, so i just want to make it as normal as possible for them . (key informant 2) i was really disturbed by the fact that you know [a story about an allergic child] was in the paper, and it was naming the school and the age of the child and the parent, so people know who this child is i don't think it is fair to anyone involved, but it is definitely not fair to the child and we don't want to be causing that, so i just want to make it as normal as possible for them . (key informant 2) four key themes emerged from interviews with affected individuals: perceived prevalence, perceived etiology, management and coping, and quality of life . Initially, however, participants were asked to describe the emotions and feelings they experienced when they had their first allergic reaction . Many were surprised or even shocked to learn of their allergy (n = 12):yeah, i was actually quite surprised and so were my parents because we had been around people with allergies before, like my friends and stuff, but none of them had such a severe reaction and especially because it was so unexpected at such a young age . (participant 15: female raised in singapore and japan, <5 years in canada, allergic to peanut and shellfish)participants also felt disappointment (n = 7) and even sadness (n = 1) when they were diagnosed because they either found it difficult to give up a kind of food they liked or were upset that previous allergic reactions could have been prevented: i would just feel sad, like i just think that it is a kind of sickness . I just feel it is a misfortune right, why do we have a son that has food allergy? (participant 4: female from china, <10 years in canada, son is allergic to peanuts and beans) i was actually quite surprised and so were my parents because we had been around people with allergies before, like my friends and stuff, but none of them had such a severe reaction and especially because it was so unexpected at such a young age . (participant 15: female raised in singapore and japan, <5 years in canada, allergic to peanut and shellfish) i would just feel sad, like i just think that it is a kind of sickness . I just feel it is a misfortune right, why do we have a son that has food allergy? (participant 4: female from china, <10 years in canada, son is allergic to peanuts and beans) one of the predominant perceptions regarding food allergies is that prevalence is lower in asia than it is in north america . Current evidence supports this perception, although it is speculated that rates of prevalence in more modernized regions of asia, such as hong kong, are comparable to rates in north america [24, 25]. This perception was also held by participants, as 83% of them thought that food allergies were less common in their birth place: well i realized especially like in grade four and grade five, all my friends could not eat peanuts, so we couldn't even bring peanuts to school, so that is when i realized, oh food allergies are pretty common here in canada . Whereas i never really thought about it back in the philippines . (participant 7: male from philippines, <20 years in canada, allergic to shrimp and shellfish)it is a lot rarer in china . Over here there is a lot of emphasis [on it], like one of the first things you learn in school is don't bring any peanuts . (participant 12: female from china, <20 years in canada, allergic to shrimp and prawns) well i realized especially like in grade four and grade five, all my friends could not eat peanuts, so we couldn't even bring peanuts to school, so that is when i realized, oh food allergies are pretty common here in canada . Whereas i never really thought about it back in the philippines . (participant 7: male from philippines, <20 years in canada, allergic to shrimp and shellfish) it is a lot rarer in china . Over here there is a lot of emphasis [on it], like one of the first things you learn in school is don't bring any peanuts . (participant 12: female from china, <20 years in canada, allergic to shrimp and prawns) 78% of participants could not speculate on the cause of their allergy . However, when asked to speculate on the etiology of food allergy more generally, they cited examples related to diet, genetics, and the hygiene hypothesis . For example: it might be because chinese people eat everything, so from their ancestry they kind of don't have those allergies, because they start to eat anything like from years ago, but here people only eat certain kinds of food . They don't eat animals, like dogs and stuff, and so it is really hard to say . (participant 14: female from china, <5 years in canada, allergic to peanuts, beans, fish, and shrimp) it might be because chinese people eat everything, so from their ancestry they kind of don't have those allergies, because they start to eat anything like from years ago, but here people only eat certain kinds of food . They don't eat animals, like dogs and stuff, and so it is really hard to say . (participant 14: female from china, <5 years in canada, allergic to peanuts, beans, fish, and shrimp) participants also found it harder to manage their allergy in their birth place (table 2). Participants attributed this difference to lower levels of awareness and, subsequently, less respect and understanding of their needs for accommodation . Food allergy impacts the quality of life of not only the allergic individual, but also their family . 39% of respondents reported that the impact of the food allergy on their quality of life was negligible (table 3); that is, they adapted quickly to their food allergy and never felt any serious disappointment from being allergic . Impact on their quality of life as it took some time to accept the allergy and learn how to manage everyday life . Three participants reported being significantly impacted by the allergy; two had multiple allergies and one had tried various remedies, both chinese and western medicines, to alleviate symptoms . A third who reported significant quality of life impacts had been teased by family and friends: my parents started helping me to treat my allergy back when i was fifteen or sixteen . (participant 1: female from hong kong, <10 years in canada, allergic to lychee and lobster) my mom, she is not allergic to anything, so she is always like why are you allergic? There is a phrase that she says [often]. She always says my sister will eat anything she cooks, but i am always the weird kid that [she] always has to be careful with, [because there are] so many limitations to the food that i eat . (participant 3: male raised in hong kong, <20 years in canada, allergic to shellfish and peanut)the presence of a strong support system can make a positive difference in coping . 72% of participants said their family and friends are supportive because they understand the severity of their allergy and are willing to make accommodations (table 4). However, 39% of participants reported that their family was initially skeptical of their food allergy: my parents started helping me to treat my allergy back when i was fifteen or sixteen . It is like a drink that is similar to ginseng . It is supposed to make your metabolism better . (participant 1: female from hong kong, <10 years in canada, allergic to lychee and lobster) my mom, she is not allergic to anything, so she is always like why are you allergic? There is a phrase that she says [often]. She always says my sister will eat anything she cooks, but i am always the weird kid that [she] always has to be careful with, [because there are] so many limitations to the food that i eat . (participant 3: male raised in hong kong, <20 years in canada, allergic to shellfish and peanut) a lot of people are skeptical . My relatives [will say something] like are you sure that you are allergic to it? Maybe you should try without knowing it and see if it is a psychological thing . (participant 3: male raised in hong kong, <20 years in canada, allergic to shellfish and peanut)participants had to prove their allergy to their relatives by showing their physical reaction when they consumed an allergen . My relatives [will say something] like are you sure that you are allergic to it? Maybe you should try without knowing it and see if it is a psychological thing . (participant 3: male raised in hong kong, <20 years in canada, allergic to shellfish and peanut) misinformed perceptions about oral immunotherapy were also raised, as participants had experienced situations in which they were encouraged by family members to eat an allergen . The thinking was that regular exposure to the allergen would eventually cure them of their condition:[my dad] is like just keep eating, because whenever there were shrimps or like at home, my dad would always kind of force me to eat it, and he would be just like . You can like overcome it if you just slowly like try to build immunity against it but i kind of didn't want to do it, just because i was afraid of getting a reaction . (participant 7: male from philippines, <20 years in canada, allergic to shrimp and shellfish) [my dad] is like just keep eating, because whenever there were shrimps or like at home, my dad would always kind of force me to eat it, and he would be just like oh it is all in your head . You can like overcome it if you just slowly like try to build immunity against it but i kind of didn't want to do it, just because i was afraid of getting a reaction . (participant 7: male from philippines, <20 years in canada, allergic to shrimp and shellfish) interestingly, some participants had no prior knowledge of what a food allergy was, even if they had one, until they came to canada: r (r refers to the question asked by the researcher): when you were growing up were you familiar with what food allergies are? P (p refers to the participant's response): no, i actually wasn't . It was only when i came to canada that i had heard that people have allergies to peanuts and stuff like that . I didn't know how severe it can get, that people can actually get it, and so forth . (participant 11: male from thailand, <20 years in canada, allergic to fish and shellfish)further, even if they were familiar, there was no mechanism in their native language to describe it . For example, 28% of participants could say food allergy in chinese (mandarin and cantonese), but there was no equivalent chinese term for anaphylaxis . The same observation was made by participants who spoke malay, thai, and japanese . It was speculated by some participants that familiarity with food allergies in asia may increase over time, but at this point it has not been recognized as a medical condition, at least not in the general population . R (r refers to the question asked by the researcher): when you were growing up were you familiar with what food allergies are? P (p refers to the participant's response): no, i actually wasn't . It was only when i came to canada that i had heard that people have allergies to peanuts and stuff like that . I didn't know how severe it can get, that people can actually get it, and so forth . (participant 11: male from thailand, <20 years in canada, allergic to fish and shellfish) a key aspect of managing and coping with food allergies is information . Approximately 56% of participants said that their physician was their go - to source for information . Three of the four mothers with an allergic child identified their allergist as being extremely helpful . Community and public health services were mentioned less frequently (14% of total mentions). Personal internet research was the preferred method for learning more about food allergies with 67% of participants having conducted internet searches at least once: thank god we have internet . I can't remember the website i went to, but i do a lot of research . (participant 4: female from china, <10 years in canada, son is allergic to peanuts and beans) thank god we have internet . I can't remember the website i went to, but i do a lot of research . (participant 4: female from china, <10 years in canada, son is allergic to peanuts and beans) shellfish was the most common self - reported allergen amongst participants (table 1)., the risk of peanut and tree nut allergy is much higher in individuals from western countries [5, 2628]. While the influence of birth place in development of shellfish allergy is still unknown, it is likely that multiple environmental factors are contributing to this phenomenon, including gender, living environment (urban versus rural), and age of intake . Researchers are only beginning to find potential links between age of fish intake and allergic disease that may also be the case of shellfish allergy [28, 29]. Interestingly, twelve participants were 10 years or older when diagnosed (table 1). This is in sharp contrast to the high rates of allergic children in north america who are diagnosed at a much younger age (between 2 and 4 years). While reasons for this are unclear, contributing factors may include a lower level of awareness in new canadians and limited availability of epinephrine autoinjectors in asia [30, 31]. In countries such as the philippines, limited accessibility to health care in parts of asia may partially explain why several participants never opted to have their food allergy confirmed by a doctor, a challenge to determining prevalence based on self - report . These findings support the hypothesis that new canadians may have a lower level of awareness of food allergies and, hence, their inflated perception of risk . The difference in their level of awareness was evident in discussions that touched on their first allergic experience, the etiology of food allergies, and the lack of policies in their birth place to protect them in public places . Furthermore, similar findings have been found in the reverse situation; parents of expatriate children with a food allergy in singapore reported that differences in food preparation and food labeling in asia caused the greatest rise in stress levels after moving to singapore . Most schools in asia do not take precautionary measures to protect food allergic children and legislature on food labeling is rare . By understanding factors that influence risk perception such as familiarity and personal ability to affect risk, policy makers can anticipate how the public will respond . Furthermore, health professionals should be aware that there is a wide variation in the quality of information on food allergy distributed to patients when diagnosed overseas, if they receive any information at all . Hence, health professionals and leaders in food allergy advocacy should be encouraged to tailor comprehensive, online resources that are accessible to new canadians . In terms of management and coping, participants relied on food labels and prior experience with allergens to stay safe . This is concerning as oral immunotherapy is not yet widely accepted as a treatment [34, 35]. Moreover, this practice put participants in an uncomfortable situation, which could have possibly led to accidental exposure and/or issues of management anxiety . Social isolation / exclusion also emerged as participants who had lived with their food allergy in asia expressed frustration as they fended for themselves, especially if they were the only one in their social circle with a food allergy . Qualitative research studies are by definition constrained by sample size but allow for a deeper exploration of lived experience . The reliance on self - reported food allergy in this study is typical of research in this field but does present a potential bias on the part of respondents . Further, not all countries in east and southeast asia were represented in the sample, so transferability of research findings to other regions may not be possible . Given the level of diversity within asia itself, we recognize that the treatment of our participants as a homogenous group is a limitation of this study . These findings indicate that food allergy among new canadians is an important (and potentially growing) public health issue deserving of attention, given the reported impacts on social isolation / exclusion and quality of life, as well as the dearth of resources available . This exploratory research raises several issues deserving of more research: health literacy levels in immigrant populations, the wider variety of allergens that immigrants may be reacting to, and further research to understand the impacts of food allergies in other ethnocultural groups in canada as immigrants represent a large and growing proportion of the canadian population . Subsequently, further research will be needed to inform and facilitate change at the policy level in order to protect allergic individuals in canada and abroad.
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Austocystin d was tested for cytotoxicity after 72 h treatment in a panel of cell lines . As indicated in table 1, austocystin d displayed potent cytotoxicity, with gi50 values below 10 nm for three cell lines, and marked selectivity, with greater than 10 000-fold selectivity between the most sensitive cell line (mcf7) and the least sensitive cell line (mes - sa). The selectivity of austocystin d was not easily explained by tissue origin, as demonstrated by a comparison of mcf7 and mcf10a breast cell lines, where a selectivity of greater than 1000-fold was observed for mcf7 cells (figure 2a). Furthermore, the selectivity and activity was unlike that of chemotherapeutic agents such as doxorubicin and etoposide, both 50-fold more toxic to mcf10a cells, or the structurally related aflatoxin b1, which did not display selectivity or potency, with gi50 values in each cell line above 5 m (figure 2). The differential selectivity to that of doxorubicin and etoposide indicates that austocystin d may have the potential to overcome resistance to these agents, and the differential potency to aflatoxin b1 indicates that these structurally related natural products (figure 1) may not share a related mechanism of action or cellular activity . Austocystin d potency and selective cytotoxicity is distinct from doxorubicin, etoposide, and aflatoxin b1 . Dose - dependent growth inhibition of tissue - matched transformed, mcf7, and nontransformed, mcf10a, cells after 72 h treatment with (a) austocystin d, (b) doxorubicin, (c) etoposide, and (d) aflatoxin b1 . Vertical bars represent sem, representative experiments shown . Since austocystin d was previously shown to display selective cytotoxicity to cells overexpressing the abc transporter mdr1, we investigated the possibility that austocystin d interacts directly with this transporter . Mdr1 drug efflux activity is accompanied by atp hydrolysis and therefore can be monitored in vitro by measuring the production of inorganic phosphate from membranes containing mdr1. (6) similar to known mdr1 substrates, verapamil and vinblastine,(1) austocystin d stimulated atpase activity in this assay, suggesting that it is also likely to be an mdr1 substrate (figure 3a). Furthermore, cyclosporin a, a known inhibitor of the substrate - stimulated mdr1 atpase activity,(7) fully inhibited austocystin d - stimulated atpase activity . (a) effect of austocystin d (ausd) and mdr1 substrates verapamil (ver) and vinblastine (vin) on the atp hydrolysis by membranes containing mdr1 in the presence (dashed lines) or absence (solid lines) of 1 m mdr1 inhibitor cyclosporin a (csa). Austocystin d, verapamil, and vinblastine each activated mdr1 atp hydrolysis that was inhibitable by cyclosporin a. (b) effect of 10 m austocystin d, 40 m verapamil, and 10 m cyclosporin a on the retention of calcein in hct-15 cells . Austocystin d behaved similarly to verapamil and cyclosporin a to inhibit calcein - am efflux and thereby increase intracellular calcein fluorescence relative to vehicle (dmso)-treated cells . (c, d) effect of austocystin d and verapamil on (c) calcein - am efflux and (d) growth in hct-15 cells . Vertical bars represent sem, representative experiment shown . To determine whether austocystin d also modulated drug efflux activity in cells, we measured the abundance of the fluorescent dye calcein in live cells. (8) in this assay, the nonfluorescent calcein acetoxymethyl ester (calcein - am) is added to cells and is hydrolyzed to calcein by cellular esterases . Calcein - am is an mdr1 and mrp1 family substrate and thus is actively effluxed from cells expressing those transport proteins, resulting in low levels of retained fluorescence. (9) mdr1 and mrp1 family substrates or inhibitors prevent calcein - am efflux, leading to high levels of retained fluorescence . Similar to mdr1 substrate verapamil and inhibitor cyclosporin a, austocystin d inhibited efflux of calcein - am, indicating that it can also modulate drug efflux activity in cells (figure 3b). Although austocystin d modulated abc transporter activity in vitro and in cells, this activity does not account for the cytotoxicity of austocystin d. verapamil and austocystin d both act as mdr1 substrates with similar cellular activity, yet verapamil is more than 1000-fold less cytotoxic than austocystin d (figure 3c, d). Furthermore, we evaluated a panel of cell lines for abc transporter activity in relation to austocystin d sensitivity . We measured calcein - am efflux as an indication of the presence of functional mdr1 or mrp1 family members and hoechst 33342 efflux as an indication of the presence of functional bcrp family members . As indicated in table 2, sw620 cells lacked activity for known abc transporters yet were sensitive to austocystin d. taken together, the lack of correlation between verapamil and austocystin d cytotoxicity and abc transporter activity provides strong evidence that the cytotoxicity of austocystin d is not due to its interaction with abc transporters . <50% of mcf7 cells displayed bcrp activity . Because of its structural similarity to aflatoxin b1, a cyp - activated mutagen,(11) we investigated whether the selective cytotoxic activity of austocystin d was due to cyp - activated dna damage . Austocystin d and aflatoxin b1 share a vinyl ether moiety, critical in aflatoxin b1 for its biological activity (figure 1). This moiety in aflatoxin b1 undergoes epoxidation catalyzed by cyp enzymes, and the resulting epoxide forms dna adducts. (11) to detect cellular dna damage, we established an in - cell western assay measuring the phosphorylation levels of histone h2ax . Histone h2ax is phosphorylated by the kinase atm at serine 139 in response to dna double strand breaks . This signal occurs in response to dna - damaging agents such as bleomycin and camptothecin and is a sensitive readout of dna damage response pathways. (14) in this assay, we observed that known dna damage agents camptothecin, doxorubicin, and bleomycin induced dose - dependent phosphorylation of histone h2ax after 4 h of drug treatment (figure 4a, figure s1a, b). We subsequently determined that austocystin d induced the phosphorylation of histone h2ax in cell lines for which it was cytotoxic (figure 4a, b). Austocystin d caused a cell - line selective induction of the dna damage response that matched its cell - line selective cytotoxicity . (a) in - cell western showing the effect of austocystin d, doxorubicin, bleomycin, and dmso (vehicle) on levels of phosphorylated histone h2ax (ph2ax) after 4 h treatment of hct-15 cells . (b) in - cell western data showing the effect of austocystin d on ph2ax in sw620, hct-15, and hela cells following 1 h of treatment . Vertical bars represent sem . To investigate the likelihood that austocystin d required cyp activation to induce the dna damage response and be cytotoxic, we treated cells with both ketoconazole, a cell - permeable inhibitor of cyp 3a4 activity,(5) and austocystin d. ketoconazole fully prevented austocystin d cytotoxicity and induction of the dna damage response, demonstrating that cyp activity is required for austocystin d s antiproliferative activity (figure 5a, b). This effect was specific to austocystin d and did not result from general inhibition of the dna damage response (figure 5c). Inhibition of cyp or hydrogenation of austocystin d abrogates austocystin d dna damage and cytotoxicity . (a) inhibitory effect of the cyp inhibitor ketoconazole on hct-15 cell growth inhibition of 0.5 m (light blue), 1 m (blue), and 2 m (dark blue) austocystin d. (b) inhibitory effect of 10 m ketoconazole on austocystin d - induced phosphorylation of histone h2ax in hct-15 cells following 1 h ketoconazole pretreatment and 4 h austocystin d treatment . (c) specific inhibition of 10 m ketoconazole (ket) on 10 m austocystin d - induced phosphorylation of histone h2ax in sw620 cells in comparison to 20 m camptothecin . (d, e) comparison of austocystin d and dihydro - austocystin d for (d) growth inhibition after 72 h treatment of mcf7 cells and (e) induction of phosphorylation of histone h2ax after 4 h in mcf7 cells . The epoxide forms a direct covalent interaction with guanine nucleotides, causing dna damage. (11) we sought to probe the involvement of an austocystin d epoxide intermediate by hydrogenation of the vinyl ether, since the aflatoxin b1 epoxide intermediate is known to be short - lived under physiological conditions,(15) and we therefore expected the austocystin d epoxide to also be short - lived . We reduced the vinyl ether of austocystin d, yielding dihydro - austocystin d(16) (3, figure 1) and observed that dihydro - austocystin d displayed only very weak cytotoxicity and induction of the dna damage response, further supporting the importance of metabolic activation for the mechanism of action of austocystin d (figure 5d, e). To support the cellular data indicating that austocystin d induced dna damage following cyp activation, we applied an in vitro reconstituted dna damage assay . In this assay, supercoiled plasmid dna is incubated with compound in the presence or absence of human liver microsomes, a rich source of cyp enzymes, and an nadph regenerating system to generate insitu the nadph that is required for the cyp activity . The resulting nicked dna can be detected by agarose gel electrophoresis . In this assay, austocystin d caused direct dna damage at 100 nm, consistent with the compound s gi50 in cells (figure 6 and table 1). This in vitro dna damage activity required cyp activity, as increased nicked dna was observed only in the presence of both liver microsomes and the nadph regenerating system required for activity of cyp enzymes in vitro . (a) dna agarose gel showing the effect of 1 m austocystin d on converting supercoiled plasmid dna to nicked dna after treatment with different additives . (b) dna agarose gel showing the potency of austocystin d - induced dna nicking . Austocystin d was originally discovered more than 30 years ago in an effort to identify and characterize secondary metabolites in maize meal cultures of aspergillus ustus (bainier) thom . And church. (16) more recently, it was reported to act as a selectively cytotoxic agent to certain cancer cell lines with an apparent selectivity toward cells expressing mdr1. (3) we sought to elucidate the mechanism of action of austocystin d to uncover a potentially useful approach to selectively target chemoresistant cancer cells that act by expression of abc transporters . Our approach involved the investigation of two possible leads: that abc transporter activity might be required for austocystin d antiproliferative activity and that austocystin d might act by a mechanism similar to aflatoxin b1, a well - characterized structurally related compound that is activated by cyp enzymes to induce dna damage . We began by confirming the cell - line selectivity of austocystin d and found it to be remarkably potent and selective, displaying over 1000-fold selectivity between sensitive and resistant cell lines . In comparison to the chemotherapeutic agents doxorubicin and etoposide, austocystin d displayed a reverse sensitivity pattern between the breast cancer cell line mcf7 and the proliferative nontransformed breast cell line mcf10a with the advantage of being more selective for the cancer cell line . Austocystin d was also only weakly active against the normal human umbilical vein endothelial cell line (huvec), suggesting the possibility of selectively targeting cancer cells, particularly in the setting of resistance to standard chemotherapy . Further characterization of a subset of cell lines revealed that abc transporter activity did not correlate with austocystin d antiproliferative activity . For example, sw620 cells were highly sensitive to austocystin d with a gi50 of 27 nm but did not display abc transporter activity . To investigate the possibility that austocystin d induced dna damage in cells, we measured the induction of phosphorylation of histone h2ax, which occurs in response to dna damage . Austocystin d induced histone h2ax phosphorylation, and this activity fully correlated with its cellular cytotoxicity . The histone h2ax phosphorylation and antiproliferative activity could be fully inhibited by treatment with ketoconazole, a cyp inhibitor with highest potency toward cyp 3a4, or reduction of austocystin d to dihydro - austocystin d, a form that was not expected to undergo cyp - catalyzed epoxidation like aflatoxin b1. (11) therefore, a mechanism emerged in which austocystin d is activated by cyp enzymes to induce dna damage leading to cell death . Further support for this mechanism of action came from in vitro experiments in which we directly observed dna damage in the presence of liver microsomes expected to contain cyp enzymes . While austocystin d and aflatoxin b1 share a similar mode of action in vitro and in the ames test,(4) namely, cyp - activated dna damage, the compounds differ dramatically in both potency and cell - line selectivity . In the comparison between mcf7 and mcf10a, aflatoxin b1 displayed little selectivity and poor potency, while austocystin d was highly selective and potent, being 1000-fold more active against mcf7 cells . Although both compounds contain a vinyl ether moiety, the remainder of the structures may encode cyp enzyme specificity or sensitivity, or differences in dna recognition . Alternatively, the reactivity of the vinyl ether or epoxide may vary between the compounds, leading to differences in rate of epoxide formation or covalent interaction with cellular dna . One possibility for the difference in activity between austocystin d and aflatoxin b1 is the absence of a tertiary alcohol in aflatoxin b1 adjacent to the site of epoxidation . This hydroxyl is present in aflatoxin m1, a metabolite of aflatoxin b1. (19) neal et al . Directly compared the cytotoxicity of aflatoxins b1 and m1 and reported differences; the human b lymphoblastoid cell line mcl-5 that stably expresses cyps 1a2, 2a6, 3a4, and 2e1 and microsomal epoxide hydrolase was sensitive only to aflatoxin b1, while the untransfected line, chol, was sensitive only to aflatoxin m1. (20) a comparison of the biological activity of austocystin d to aflatoxin m1 will help reveal the importance of the tertiary alcohol to differences between austocystin d and aflatoxins . In addition, an investigation into cyp selectivity and hepatoxicity will help reveal distinctions between austocystin d and aflatoxins and the potential clinical utility of austocystin d. despite its initial discovery as an mdr+ cell selective agent, we have determined here that the unusual selectivity of austocystin d is mediated by cyp activation, rather than by a direct interaction with abc transporters . This unanticipated finding is likely explained by coordinate expression of cyps and abc transporters by the pregnane xenobiotic receptor (pxr) or the constitutive androstane receptor (car) nuclear hormone receptors . These receptors induce the expression of a coordinated cellular detoxification program in response to xenobiotic small molecules, and each is known to upregulate both abc transporters and cyp enzymes . Thus, our findings support a model whereby transcriptional upregulation of abc transporters by pxr / car can lead to a multiple drug resistance phenotype and that this transcriptional program also induces tumoral expression of cyp enzymes . This model is also supported by the observations that pxr agonists lead to multiple drug resistance and that chemotherapeutics prone to multiple drug resistance induce pxr - mediated transcription . These findings suggest a context - dependent cytotoxicity strategy for the design of new therapeutics for multiple drug resistant cancers, where the co - regulation of mdr and cyp enzymes creates a vulnerability to cyp - activated prodrugs that are directly activated in the tumor tissue . Previous cyp - activated anticancer agents, such as cyclophosphamide, have relied on metabolic activation of the drug in the liver . This is typically associated with systemic toxicity as the activated drug enters circulation. (25) in the present work, we show that austocystin d has significant cancer cell autonomous activity, suggesting that the molecule or derivatives could be suitably activated directly within tumors . Due to the coordinate expression of cyps and abc transporters such as mdr1, the natural product austocystin d or compounds with a similar mechanism of action have the potential to target multiple drug resistant tumors, and thus may address a significant clinical need . Austocystin d was isolated as previously described,(27) and dihydro - austocystin d was synthesized as described in the supporting information and has previously been synthesized. (16) all other compounds were obtained from commercial suppliers . Huvec cells were obtained from lonza, and other cell lines were obtained from atcc . Cells were plated in 96-well plates at densities ranging from 2500 to 10 000 cells / well 24 h prior to the addition of compounds . Compound stocks in 100% dmso were 3-fold serially diluted in 100% dmso in 96-well u - bottom plates to generate 1000 dose curves . These were subsequently diluted 1:100 in rpmi-1640 medium and then diluted 1:10 into the wells plated with cells for a final dmso concentration of 0.1% . The cell - titer blue viability assay (promega) gi50 values for compounds were determined relative to dmso control samples measured at 0 h (0% growth) and 72 h (100% growth). Drug - stimulated mdr1 atpase activity was determined by measuring inorganic phosphate release. (6) human mdr1 and control membranes (bd biosciences) were prepared in assay buffer (50 mm tris - mes ph 6.8, 1 mm egta, 1 mm dithiothreitol, 25 mm kcl, 2.5 mm sodium azide) with and without cyclosporin a (50 m final concentration). Compounds were added to membranes and incubated for 10 min at 37 c, and the atpase reaction was initiated by the addition of atp . The final concentrations of membrane, atp, and dmso in the reactions were 200 g / ml, 4 mm, and 1%, respectively . The reactions were stopped by the addition of sds solution containing antifoam a (final concentration of 3%) after 0 and 60 min incubation periods . Two equal volumes of 35 mm ammonium molybdate in 15 mm zinc acetate:/10% ascorbic acid (1:4) were subsequently added followed by a 20 min incubation at 37 c and measurement of absorbance at 800 nm . The released inorganic phosphate was determined by comparison to a phosphate standard curve at 800 nm . The mdr1-specific atpase activity was calculated by comparing atpase activity from mdr1 membranes to that of control membranes . Mdr1/mrp cellular activity was determined by measuring calcein - am efflux. (28) cells were plated in triplicate wells at a density of 5 10 cells per six - well 2 days prior to the assay . For the assay, cells were treated with compounds for 60 min at 37 c followed by the addition of calcein - am (0.25 m). After 30 min at 37 c, the cells were washed twice with pbs, trypsinized, suspended in cold pbs, and analyzed with a facs calibur equipped with a 488 nm argon laser (bectondickinson). In figure 3c, the mean fluorescence of each histogram was calculated for austocystin d and verapamil at the indicated doses in duplicate, and the average of two samples was plotted with graphpad prism5, as above . Dmso was set to 100% abc transporter activity, and 100 m verapamil was set as 0% abc transporter activity for normalization . Bcrp cellular activity was determined by measuring hoechst 33342 efflux. (10) cells were trypsinized from semiconfluent 150 cm plates, resuspended at 10 cells / ml in media (rpmi-1640, 10% fbs), and incubated with 5 g / ml hoechst 33342 at 37 c . After 45 min, cells were washed once with cold hanks balanced saline solution supplemented with 1 mm hepes and 2% fcs (hbss+) and resuspended in media (rpmi-1640, 10% fbs) with 10 m fumitremorgin c at 37 c . After 20 min, cells were washed with cold hbss+, resuspended in cold hbss+ containing 2 g / ml propidium iodide (pi) to exclude nonviable cells, and analyzed with a facs lsrii (bd biosciences) equipped with 360 nm uv and 488 nm argon lasers . Cells were plated in 96-well black - walled plates at 10 000 cells / well 24 h prior to the addition of compounds . Compound stocks in 100% dmso were 3-fold serially diluted in 100% dmso in 96-well u - bottom plates to generate 1000 dose curves . These were subsequently diluted 1:100 in rpmi-1640 medium and then diluted 1:10 into the wells plated with cells for a final dmso concentration of 0.1% . After 4 h at 37 c, cells were fixed in 4% paraformaldehyde for 20 min, washed briefly in 0.1% tween-20, and permeabilized in 0.1% triton - x 100 in pbs . After 20 min, cells were incubated in blocking buffer (licor biosciences, odyssey blocking buffer) for 1 h and incubated overnight with antiphospho - h2ax antibody (cell signaling technology, phospho - h2a.x ser139) at 1:400 in blocking buffer . The cells were subsequently washed and incubated with goat - anti - rabbit ir800cw (licor biosciences) at 1:800 in blocking buffer and draq5 (biostatus limited) at 1:10 000 . Signals were detected with a licor odyssey infrared scanner (licor biosciences) at 700 and 800 nm . Each well was normalized within the well by taking the ratio of 800 nm/700 nm . Ratios of duplicate or triplicate wells were averaged and normalized to the in - plate controls 0.1% dmso (0% induction) and 10 m etoposide (100% induction)., cells were pretreated with 10 m ketoconazole for 1 h prior to the 4 h treatment with austocystin d or camptothecin . Supercoiled plenti6 plasmid dna (120 g / ml) was incubated at 37 c with compound (100 nm, 500 nm, or 1 m; 1% dmso final concentration) in the absence or presence of human liver microsomes (0.25 mg / ml; cellzdirect) and an nadph regenerating system (1.5 mm nadp, 12.5 mm glucose-6-phosphate, 0.25 u / ml glucose-6-phosphate dehydrogenase, 100 mm phosphate ph 7.5, 5 mm mgcl2). After 4 h, the reaction was stopped by addition of an equal volume of phenol / chloroform (1:1) solution . After vigorous vortexing, the emulsion was separated by centrifugation, and the dna was recovered from the aqueous phase by using a gel extraction kit (qiagen). The purified dna was then subjected to electrophoresis in tae buffer using a 0.8% agarose gel, and the bands were photographed using biorad gel doc.
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Burkholderia is a ubiquitous organism that occupies many diverse ecosystems such as soil, water, and even the human respiratory tract . The burkholderia cepacia complex (bcc) consists of nine closely related burkholderia species that have been widely studied and used for various purposes including biological control of plant pathogens, bioremediation of organic compounds, and plant growth promotion . There are several pathogenic strains of burkholderia that cause diseases in plants and animals . Among those that are pathogenic, burkholderia pseudomallei is the causative agent for melioidosis, and b. mallei is responsible for glanders disease and was used as a biological weapon in world war i; both remain a potential threat today . For this study we used the opportunistic pathogen burkholderia vietnamiensis, which causes diseases in immunocompromised individuals and patients suffering from cystic fibrosis, as the model organism . B. vietnamiensis has minimal growth requirements and easily adapts to a range of nutritional conditions enabling it to survive in unfavorable conditions . The most common factors that have been studied are the quorum - sensing molecules which help cell - to - cell communication and adherence [5, 6]. According to previous studies many proteins associated with pathogenicity of burkholderia species are expressed in very low amounts within the bacterial cell . In previous studies designed to identify these virulence factors, the proteins were overexpressed in e. coli and amplified before their analysis [79]. In this study mass spectrometry was used to identify these low abundance virulence factors without prior amplification . Recently, riedel et al . This involved analysis of the digested peptides using a maldi tof - tof mass spectrometer . In our study, both gel - based lc - ms / ms and gel - free multidimensional protein identification technology (mudpit) analyses were used to identify proteins from b. vietnamiensis . By combining both methods, the main objectives of the present experiments are to achieve high coverage of the opportunistic pathogen burkholderia vietnamiensis g4 proteome expressed in a minimal medium and to identify the expressed proteins, including low abundance virulence factors, using mass spectrometry . Minimal media and late log phase collection were chosen to favor enhanced production of virulence factors and to provide a the proteome of the organism was divided into extracellular, cell surface, cell wall, and intracellular protein fractions . Gel - based and gel - free proteomics which have been used to identify proteins from many sources including pathogenic bacteria, cancer cells, and different tissue types were used . For the gel - based approach, a 1d gel separation and lc - ms / ms analysis was applied . By initially fractionating the sample, cutting each fraction's whole gel lane into 16 pieces, and digesting slices individually, we generated more protein identifications than spot selection on a 2d gel as indicated in the maldi - tof ms approach . Clearly, those authors could have run maldi on every spot, resulting in additional ids . This method involved two types of hplc separation, namely, strong cation exchange (scx) followed by reverse phase separation . B. vietnamiensis g4 strain (100% homologous to ay268153 gene bank dna sequence accession number) was initially obtained from the maier lab culture collection . One liter of mineral salt medium (msm - a minimal medium) containing 2% glucose as the sole carbon and energy source was used to grow the bacterium at room temperature according to methods described in the literature . The bacterial cells were harvested in the late exponential phase when the optical density of the culture reached about 1.6 at = 600 nm . Total cellular protein from the bacteria was determined by the lowry assay using the calibration curve plotted with standard bovine serum albumin (bsa - sigma) [13, 14]. Extracellular proteins were separated from the bacterial culture using previously described methods [10, 15] with slight modifications . Briefly, the bacterial culture was centrifuged using a beckman j2 - 21 centrifuge at 5000 rpm for 20 min . The cell pellet was used for the fractionation of intracellular, cell wall, and cell surface proteins . One hundred ml of the bacterial supernatant (extracellular fraction) was mixed with ice cold 10% (w / v) trichloroacetic acid (tca - sigma) and kept overnight at 4c . Precipitated proteins were washed with 95% ethanol, air dried and divided into two portions . One portion was dissolved in protein sample buffer (buffer s-250 mm tris [ph = 6.8], 30% glycerol, 8.0% sodium dodecyl sulfate (sds), 1% -mercaptoethanol and a trace amount of bromophenol blue as the coloring agent) to run protein gel electrophoresis . The second portion was dissolved in urea buffer (8 m urea in 50 mm ammonium bicarbonate) for mudpit analysis . The cell pellet obtained after the removal of the supernatant was washed twice with 0.9% nacl and centrifuged again for 20 min at a speed of 5000 rpm . The pellet was then washed with tris / hcl (ph = 7.5) and centrifuged again . One gram of the cell pellet was suspended in 10 ml of tris / hcl buffer and sonicated using a branson sonifier 450 probe sonicator for 20 min . To analyze the cell wall proteins, the protein pellet was dissolved in protein sample buffer (buffer s) for sds - page separation and urea buffer for mudpit analysis . The supernatant, after removing the cell wall pellet, was used to analyze intracellular proteins . The proteins present in the supernatant were precipitated using a 3-fold volume of ice - cold acetone and kept overnight at 20c . The precipitated proteins were separated by centrifugation at 5000 rpm for 20 min . The precipitate was washed, air dried, and dissolved in protein buffers for sds - page and mudpit analysis . Five grams of the bacterial cell pellet was suspended in 100 ml of 0.2 m glycine / hcl with stirring at 20c for 1 h. in the acidic medium, all the soluble proteins present in the surface of the cell will move into the solution . The cellular debris was removed by centrifugation with the soluble proteins remaining in the supernatant . The supernatant was extracted and neutralized with 0.1 n naoh and proteins were precipitated with ice - cold acetone (three volumes) and kept at 20c overnight . The protein pellet was used to analyze cell surface proteins using gel - based lc - ms / ms and mudpit mass spectrometric techniques . For sds - page and in - gel digestion, 50 mg of protein fractions were dissolved in the protein sample buffer (buffer s) and boiled in a water bath for 5 min . Samples were spun down and loaded into the 50 l well in a biorad (12% tris - hcl) precast ready gel (biorad, hercules, calif, usa). Protein gels were run by using 1x running buffer (buffer r-259 mm tris base, 2 m glycine, 1% (w / v) sds diluted with distilled water) at a constant voltage of 85 v for 20 min followed by 150 v for 40 min (until the dye runs off the gel). A single lane of stained gel was cut into 16 pieces of approximately the same size and transferred into 16 wells of a 96-well plate to perform in - gel tryptic digestion . Gel bands were destained and tryptically digested using a perkinelmer multipprobe ii plus ex robotic liquid handling system according to methods indicated in the literature . After extraction, the peptides were transferred into 500 l eppendorf tubes and dried down using a savant speed vac concentrator . Each concentrated peptide solution (10 l) containing ~50 g of protein was loaded into individual hplc autosampler vials and analyzed by lc - ms / ms . Proteins were reconstituted in 100 l of 8 m urea in 100 mm nh4hco3 . Reduction of the proteins was carried out using 2 l of 100 mm dithitheritol (dtt) with incubation for 15 min at room temperature . Reduced proteins were alkylated by adding 3 l of 100 mm iodoacetamide and incubated for 15 min in the dark at room temperature . Enzymatic digestion was carried out using 12 l of 0.3 g/l endoproteinase lys c (princeton separation, freehold, nj, usa) and incubated for 2 - 3 h at 37c . The solution was diluted with 650 l of 100 mm nh4hco3, 50 l acn and 9 l of 100 mm cacl2 . Twelve microliters of 0.1 g/l trypsin (sigma, saint louis, mo, usa) was then added to the diluted sample mixture, mixed thoroughly and incubated overnight at 37c . Digested peptides were cleaned and desalted using spec pt c18 solid phase cartridge (ansys diagnostics lake forest, calif, usa). The resulting peptides were dried under vacuum and redissolved in 0.5% formic acid before the mudpit analysis . Silica capillary columns (100 m i.d .) Were packed with methanol / zorbax c18 stationary phase slurry, using a bomb loader, up to about 7 cm . The packed column was connected with the commercial instrument according to the method established in the lab [18, 19]. The hplc system was connected to the linear ion trap ltq mass spectrometer (thermo fisher, san jose, calif, usa) to carry out peptide analysis . The digested peptides from the gel bands were eluted in a buffer gradient consisting of buffer a (h2o with 0.1% formic acid) and buffer b (acn with 0.1% formic acid) at a flow rate 400 l / min into the ltq . The peptides were eluted with a linear gradient from 0 to 50% buffer b over 30 min intervals after the initial 10 min washing step with buffer a. the concentration of buffer b was increased from 50 to 98% over 10 min followed by a 5 min wash with 5% b. spectra were scanned over a mass / charge range of 4001500 mass units . Xcalibur data - dependent software was set to fragment the seven most intense peaks as described previously . For mudpit analysis first, a silica column (100 m i.d .) Was packed with 5 m poly - hydroxyethyl a strong cation exchange resin (polylc; thenest group, southborough, mass, usa) to 6 cm in length . A t - junction was used to apply voltage through a gold electrode . For the mudpit analysis, 13 different solvent / salt steps were used . A 10 l (~50 m) sample the sample was loaded into the scx column using an isocratic gradient of 95% buffer a and 5% buffer b for 70 min . To elute the nonbonded peptides a solvent gradient similar to that of reverse phase separation was applied over a period of 125 min . A 4 min pulse of 10% buffer c (250 mm ammonium acetate, 5% acn, 0.1% formic acid) was applied to the lc system followed by a gradient of 550% solution b over 65 min, 5098% b over 5 min and 5 min wash with 98% b. the percentage of buffer c was increased in 10% increments up to 100% so as to form a step elution gradient . The last step of the elution was 50% of buffer d (1.5 m ammonium acetate, 5% acn, 0.1% formic acid) for 4 min followed by reverse phase gradient with extended washing time at 98% buffer b to elute strongly bound peptides . Data from all 13 steps were combined including the loading and initial washing step to get comprehensive coverage of all the peptides eluted from the column . The sequest database search algorithm (thermo fisher, san jose, calif, usa; version 27) was used to interpret the ms / ms spectra obtained from the lc - ms / ms and mudpit runs . The sequest database search algorithm correlates experimental data with theoretically generated spectra from a known protein sequence in a database [21, 22]. All spectra were matched to a protein database created using all web - available sequenced burkholderia protein databases and common contaminants such as trypsin and human keratin peptides . The burkholderia database consists of b. vietnamiensis, b. cenocepacia, b. thailandensis, b. cepacia, b. multivorans, b. pseudomallei, b. mallei, and b. xenovorans . Protein sequences were obtained from ncbi (http://www.ncbi.nlm.nih.gov/), sanger institute (http://www.sanger.ac.uk/) and expasy (http://www.expasy.org/). In the sequest database search, the following criteria were used: the minimum x - correlation factor (xcorr) was set to 1.8, 2.5, and 3.5 for singly, doubly, and triply charged peptides, respectively . The delta correlation score (dcn) was set to 0.08 which indicates a significant difference between the best - matched peptide reported and the next best - matched peptide . Carbamidomethylation (57) of cysteine was used as a static modification and oxidation of methionine (16) was used as a dynamic modifications for the sequest search [23, 24]. We solely report the proteins with two or more peptide matches obtained from the sequest database search algorithm . To confirm protein identification all data were also analyzed with a second algorithm using the web available x - tandem (http://www.thegpm.org/) software [26, 27]. To run x - tandem, first all the data containing.dta files were combined using a lab developed perl script sub_append.pl and append.pl . For the x - tandem analysis, parent ion mass tolerance of the peptide was set at 1.5 and fragment ion mass tolerance was set to 0.5 . The same static and dynamic modifications were selected for the x - tandem analysis as for the sequest analysis ., portland, ore, usa) was used to compare and validate sequest and x - tandem data . Proteins with two or more peptide identifications based on the combination of results from both database search algorithms were taken as true identifications . For the scaffold analysis, the following probability values were selected: 95% for peptide identification calculated using peptideprophet software and 99% for protein identification with the additional requirement that protein should contain at least 2 peptides per protein . Proteins identified from the reverse database were eliminated from the total number of proteins identified in both gel - based and gel - free methods, and the false discovery rate (fdr) was ~1% of the total protein detected using the abovementioned methods . At least two biological replicates and three technical replicates were analyzed from each protein fraction . All proteins identified using these criteria are listed in the supplemental material (see supplementary material available at doi:10.1155/2011/701928). Proteins from b. vietnamiensis grown in mineral salt medium, with 2% glucose as the carbon source, were fractionated into 4 different fractions, extracellular; intracellular; cell surface, and cell wall proteins . By combining both gel - based and gel - free proteomic analysis data, e. coli was added into the protein database as a distractor to further validate the protein identifications, and only 7 proteins were identified as e. coli proteins, increasing the confidence of protein identification . The much smaller number of hits from the distractors supports the identity of the proteins for the targeted organism . Phosphorylation was used as a possible posttranslational modification in the database search algorithms . For most of the data sets, this is likely due to the fact that that sample preparation did not include the enrichment of phosphorylated peptides and no ms experiments were performed to identify phosphorylation . Figure 1 summarizes the total number of proteins identified using both gel - based and gel - free proteomic analysis . The functionality of the proteins identified from each cellular fraction will be analyzed in the following sections according to the overlap indicated in the venn diagram . The functionality of these common proteins was determined by using the dbget search function at the kyoto university bioinformatics center's genome.jp website (http://www.genome.jp/dbget/). Figure 2 shows the identified protein categories of the proteins common to all four fractions . Most of them are housekeeping proteins such as ribosomal proteins, metabolic enzymes, and chaperones . Several hypothetical and uncharacterized proteins were also found in all four cellular fractions indicating the incompleteness of the gene annotation of b. vietnamiensis and the bcc in general . It is known that bacterial virulence factor production is controlled by quorum sensing which occurs when the cells reach a high density at a late exponential phase . Extracting proteins at this stage helps us to identify more proteins associated with pathogenicity as well as proteins associated with metabolism which is clearly indicated in the data presented in the supplemental material . Also in the early stationary phase, bacteria sense nutrient limitation and release stress related proteins . Three stress - related proteins termed universal stress - related proteins (e.g, ospa domain protein) were found common to all four cellular fractions of this bacterium . The exact functions of these proteins are not known but related studies with e. coli showed that they may assist cell division under nutrient - limiting conditions . One important protein found to be common to all four fractions is the protein ecotin . Ecotin binds the active site of a protease to inhibit its activity and helps bacteria survive phagocytosis within a host cell . The ecotin protein has been isolated only in burkholderia species pathogenic to mammalian cells such as b. pseudomallei and b. vietnamiensis, but not the plant pathogen b. fungoforum [34, 35]. There are four possible combinations in which the same identified proteins will be common to only three cellular fractions; the four possible combinations of three fractions are intracellular / extracellular / cell wall, intracellular / extracellular / surface, intracellular / surface / cell wall, and extracellular / surface / cell wall fractions . Figure 1 shows that 26 proteins were found to be common to the fractions intra / extra / cell wall, 17 proteins were found to be common to the intra / surface / cell wall fraction, 22 were found in intra / extra / surface fractions and most of these common proteins were associated with the transport system of the cell . These transport systems link the outside environment with the cell interior by spanning the cell wall . When the protein fractionation is carried out, these transport proteins can be distributed in all the fractions . There can be fraction carry over when the separation is carried out to give common proteins in the fractions . Among the proteins found to be common in these fractions is the lema (lesion manifestation a) family of proteins which is involved in the two component secretory system of the bacteria . This is a membrane protein which has a signal sequence, usually 2030 amino acids long and interacts with a signal recognition particle . Most bacteria have specific sequences for these signaling molecules, and in this proteomic study we found the lema family protein in cell wall / extra / surface fractions . The sequence of the protein was blast searched through the nonredundant protein database using the ncbi website and it was found that the protein sequence is specific to burkholderia species . The peptide containing the first 17 amino acid residues at the n - terminus is specific to b. vietnamiensis g4 strain . Figure 3 shows the spectrum of the doubly charged peptide dlqaqlegtenr which belongs to the lema protein . A series of y ions and corresponding b ions can be observed in the spectrum . The strong h2o loss peak is consistent with the presence of a hydroxyl side chain (threonine) in the peptide . In addition to this peptide, five more unique peptides of lema were identified in the extracellular fraction of b. vietnamiensis g4, giving 35% sequence coverage of the protein . The n terminus of this lema is predicated to orient outside the bacterium and finding this in all extracellular / surface / cell wall fraction supports this prediction . These lema proteins associate with gaca (global activator a) to form the two - component sensory system that regulates the production of n - acyl homoserine lactones in bacteria . The n - acyl homoserine lactones (ahls) are associated with bacterial cell - to - cell recognition generally known as quorum sensing . Several studies have shown that ahl - dependent quorum - sensing supports interspecies communication among the bacteria . This has been observed in cystic fibrosis patients where both burkholderia and pseudomonas can colonize in the lungs of the patients and the production of extracellular proteases and siderophores from burkholderia is stimulated by the presence of pseudomonas in the biofilm [40, 41]. The structure of these ahl differ in the length of their n - linked side chain, the nature of the substitution at the 3-carbon position and the presence or absence of one or more unsaturated bonds within the side chain . All species that belong to the bcc can produce c6-ahl and c8-ahl molecules and in addition b. vietnamiensis can produce c10-ahls . Direct involvement of ahl molecules in the pathogenicity of the bacterium is not known; however, it has been reported that the presence of these signaling molecule is important to obtain full virulence of the bacteria . The presence of the proteins that regulate the production of these quorum - sensing molecules in the cell surface, cell wall, and extracellular protein fractions suggest that bacteria are attempting to regulate these signaling molecules to control the production of some of the extracellular proteases . Most proteins found only in intracellular and extracellular, and intracellular and cell surface fractions belong to the transport proteins such as dna - binding proteins and membrane - associated proteins like efflux pumps . These proteins can span the membrane and during the protein extraction parts of the protein can distribute in the two cellular fractions . Forty - one proteins were found to be common in both cell wall and intracellular fractions . When the cellular fractionation was carried out, there can be many intracellular proteins associated with the cell debris, and those will appear in both intracellular and cell wall fractions . Most of the proteins found to be common to intracellular and cell wall fractions are related to metabolism and few are found to associate with the membrane . B. vietnamiensis g4 is well known for its ability to metabolize toxic organic contaminants such as aromatic hydrocarbons . Carboxymethylenebutenolidase, an enzyme responsible for biodegradation of fluorobenzoate and benzoate, was found to be common in both intracellular and cell wall protein fractions, with three unique peptide identifications that cover 28% of the protein sequence . This finding is consistent with the ability of b. vietnamiensis to degrade toxic molecules such as aromatic hydrocarbons . The ability of a motile bacterium to swim toward or away from specific environmental stimuli, such as nutrients, oxygen, or light provides cells with a survival advantage, especially under nutrient - limiting conditions . This behavior is called chemotaxis and is mediated by changing the direction of the bacteria by briefly reversing the direction of rotation of the flagellar motors . Chemotaxis sensory transducers are membrane proteins produced by bacteria as a sensory molecule in order to capture certain molecules . Methyl - accepting chemotaxis sensory transducer protein was found in both the cell wall and extracellular fractions . These sensory molecules are important for bacterial survival and play a role in the pathogenicity of most bacteria, especially those that are susceptible to antibiotics . Several stress - related proteins were also found in these cellular fractions indicating the nutrient limitation of the growth medium . Many hypothetical proteins and proteins with unknown functionality were also found in all cellular fractions . From the proteomic analysis of b. vietnamiensis using both gel - based and gel - free methods, many proteins were identified as specific to only one cellular fraction . Many anabolic and catabolic enzymes were present in all four cellular fractions indicating the increased cellular metabolism at the late exponential phase . Proteins related to genetic information processing such as nucleotide transcription and translation were also found in high abundance . This illustrates the rapid cell multiplication with this culture medium and is represented also in the presence of several proteins involved in cell division in these cellular fractions . Membrane or membrane - associated proteins are important in the pathogenicity and for the survival of the bacterium in the environment . Many membrane - associated proteins were identified that are specific to one of the four cellular fractions . There were ion transport / binding proteins such as abc transporters, membrane bound porins, lipoproteins, and peptidoglycan - associated proteins, sensory proteins, and some electron transport proteins . As mentioned earlier, b. vietnamiensis is known to degrade various organic contaminants [43, 45]. Bisphenol a is an emerging environmental contaminant, especially as a water pollutant known as an endocrine disruption chemical [46, 47]. In this proteomic analysis we found arylesterase which has the functionality to degrade bisphenol a. arylesterase was identified with 5 unique peptides which cover 28% of the protein sequence . Figure 5 shows the fragmentation pattern of the peptide idysvanasvsgdttsggr from the protein arylesterase . This is a doubly protonated peptide with a 2.78 xcorr score and e value of 4.27 . A predominant y ion series can be found in the spectrum . The protein sequence was blast searched to check for homology, and it was found that the sequence is species specific . The closest protein homologue to the b. vietnamiensis g4 arylesterase was found to be lysophospholipase a from b. cenocepacia, which has 88% sequence similarity with this protein sequence . This arylesterase can be used to distinguish b. vietnamiensis g4 from other burkholderia species in a mixed culture system . The presence of these proteins in a cell culture illustrates the ability of b. vietnamiensis to degrade environmental pollutants . Different types of stress - related proteins were found specific to all four fractions indicating the nutrient limited status of the culture medium . The cell wall fraction contained three stress - related proteins including water stress and hypersensitive response protein . These proteins are present in most plant pathogenic bacteria and are produced when the plant defense mechanism acts against the invading bacteria . Another important class of proteins found specific to the extracellular fraction is the signaling molecules . Some are signal peptidases and some are antigens that induce the immune response in the host cells . All these categories of proteins illustrate that important cellular mechanisms of b. vietnamiensis are active and detectable by using mass spectrometry - based proteomic studies . Most of previous proteomics studies of virulence factors were carried out by expressing proteins of interest in e. coli and analyzing them individually . In the present study we show that mass spectrometry - based proteomic studies can be used to directly detect at least some of these low abundance virulence factors present in the pathogenic bacteria species [4852]. Many of these identified virulence factors were found in cell surface, cell wall, or extracellular fractions of bacteria . Some of the virulence factors identified by this analysis include outer membrane lipoprotein, flagellin which is responsible for motility, and outer membrane porins that are responsible for many antibiotic resistance properties of bacteria . Among the other virulence factors identified, phasin helps granule formation under nutrient - limiting conditions, superoxide dismutase, and catalase are reactive against reactive oxygen species, and adhesin helps to attach to the host [4, 54]. Several secretion proteins were identified including hlyd which is responsible for secretion and transporting hemolysin, which is embedded in the gram - negative inner cell membrane . In the present study we show that mass spectrometry - based proteomic studies can be used to detect these low abundance virulence factors present in the pathogenic bacteria species [4852]. Proteins were extracted from four different cellular fractions: extracellular, intracellular, cell wall, and cell surface proteins . Different precipitation methods were used to separate the proteins into these cellular fractions . As compared to the whole proteome analysis, fractionating the proteome into different segments allows in depth analyses and if interested, one fraction at a time can be analyzed to study a specific protein . A bottom - up proteomics approach was used to identify the proteins found in the four cellular fractions . Both gel - based lc - ms / ms and gel - free mudpit (lc / lc - ms / ms) methods were used to analyze proteins from b. vietnamiensis . A total of 803 proteins were identified using the gel - based method, and a total of 775 proteins were identified using the gel - free mudpit method . By combining both gel - based and gel - free proteomic analysis, more than 1200 unique proteins were identified from b. vietnamiensis g4 . Previous reports on b. cenocepacia, which is a close relative of b. vietnamiensis g4, identified 390 proteins by 2d gel separated protein analysis using maldi - tof / tof . This indicates that the combination of both gel - based and gel - free methods provides a more comprehensive coverage of the proteome compared to either individual method . The existence of this type of knowledge on the proteins expressed at a specific stage of the bacterial life cycle will help future research work based on this burkholderia species . Many proteins identified were related to specific functionality of b. vietnamiensis, for example, biodegrading proteins . Also, many known virulence factors were identified from this analysis, which indicates the applicability of mass spectrometry - based proteomic studies to identify possible pathogenic strains.
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Ovarian cancer is the eighth most frequent malignant neoplasia and the fifth most common cause of death from malignant tumor growth in women in the us . The frequency of ovarian cancer increases in each decade of life, with the highest rate of diagnosis occurring in women that are 75 years of age . Early diagnosis of ovarian tumors is difficult since tumors with diameters less than 5 cm cannot be recognized by bimanual pelvic examination [3, 4]. However, pelvic examination, ultrasonography, detection of tumor markers (such as ca 125, ca 15.3, ca 72.4, and ca 19.9), as well as color doppler imaging have been shown to be useful in the diagnosis of ovarian cancer, despite the limitations of these methods in differentiating between benign and malignant tumors . Nitric oxide (no) is a biological messenger synthesized from l - arginine by nitric oxide synthase (nos). The endothelial and neuronal isoforms of nos (enos and nnos, resp .) Are constitutively expressed in many cell types, however, inducible nos (inos) is only induced in leukocytes, endothelial cells, and other specific cell types after stimulation by bacterial endotoxins or cytokines, resulting in higher concentrations of no . Production of no is a mechanism by which activated endothelium can lyse tumor cells, however, it can also regulate tumor growth and metastasis depending on its concentration [8, 9]. Data from previous studies also suggest that no is both cytotoxic and cytostatic against microorganisms and malignant cells [10, 11] with synthesis of no by malignant cells causing no - mediated apoptosis . As a free radical, no can react to produce peroxynitrites which can directly and indirectly cause dna damage . If produced for a long period of time, excess no production can lead to mutations and ultimately to cancer [13, 14]. In addition to increasing the metastatic potential of tumor cells via mutations in the dna, no production by neoplastic cells promotes angiogenesis, an essential process for the growth and maintenance of tumors [15, 16]. Understanding the role of inos in ovarian cancer would provide valuable insight into the development of additional therapeutic options . The aim of the present study was to identify differences between inos expression and the local production of no in patients with varying stages and grades of ovarian cystic tumors . Therefore, levels of intracystic no metabolites and expression of inos were analyzed in tumor sections from patients with nonneoplastic, benign, or malignant ovarian tumors . This study enrolled 63 randomly selected women who received pelvic mass outpatient services from the discipline of gynecology and obstetrics of the federal university of tringulo mineiro (uftm), uberaba, brazil . These patients underwent surgery for an adnexal mass between february 1996 and february 2007, and informed consent was obtained from patients to allow their tissue to be used for examination and related experiments . Candidates for exploratory surgery were characterized by one or more of the following criteria: cysts with 1 thick septum (> 3 mm) or 2 thin septa, a cyst diameter 7 cm, persistence or increase in the cyst or ovarian volume over a minimum of two follow - up periods, the presence of vegetation or calcification, a solid or predominantly solid tumor, ascites, elevated serum levels of tumor markers, or a resistance index 0.4 as detected by color doppler imaging [3, 5]. Inclusion criterion was the anatomicopathological finding of an ovarian tumor (primary neoplastic or nonneoplastic tumor). Exclusion criteria were adnexal torsion, cyst rupture, metastasis of another primary tumor, or previous chemotherapy . The anatomicopathological evaluation and staging of all cases were performed according to guidelines published by the world health organization (who) and the international federation of gynecology and obstetrics (figo) [17, 18]. Patients were divided into 3 groups according to the classification of tumor type: nonneoplastic (n = 15), benign (n = 28), or malignant (n = 20) (which included cystadenomas of borderline malignancy). Characterization of patient groups is presented in table 1 . Cystic fluid samples were aseptically collected by punction immediately following resection of tissue . The collected fluids were immediately stored on ice until centrifugation (180 g, 15 minutes) was performed . Cell supernatants were transferred to fresh tubes, and the cell pellets were resuspended in phosphate - buffered saline (pbs). The levels of no metabolites (nitrite plus nitrate) in cystic samples were determined by enzymatically reducing the nitrate present with nitrate reductase as previously described . Briefly, 50 l of nondiluted sample was incubated with an equal volume of reductase buffer (0.1 m potassium phosphate (ph 7.5), 1 mm nadph, 10 mm fad, 4u of nitrate reductase / ml) for 20 hours at 37c . A standard nitrate curve was obtained by incubating sodium nitrate (10 to 200 m) with reductase buffer . Briefly, the samples were incubated with an equal volume of freshly prepared griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride in 5% phosphoric acid). Absorbance at 550 nm was determined using a multi - well plate reader (multiskan mcc/340mkii, flow laboratories) and the results were reported as micromoles of no3 + no2 . Specimens obtained from surgical resection were fixed in 10% formalin before being processed in paraffin . Sections stained with hematoxylin - eosin were reviewed by a pathologist and a representative section for each case was selected for immunohistochemical analysis . Selected sections were deparaffinized, rehydrated, and heated in a microwave oven in 0.01 m citrate buffer (ph 6.0) for 30 minutes . Endogenous peroxidase activity was blocked by 3% hydrogen peroxide for 10 minutes, followed by a wash with pbs . The sections were incubated overnight at 4c with an anti - inos rabbit polyclonal antibody (santa cruz biotechnology, 1:200). The conjugate used was the avidin - biotin peroxidase detection solution (dako cytomation lsab and system - hrp). The signal was visualized using diaminobenzidine (dako cytomation liquid dab and substrate chromogen system, dako). Slides were counterstained with harris's haematoxylin, dehydrated, cleared, and mounted . A skin sample with chronic granulomatous inflammation known to be positive for inos was used as a positive control . Two independent observers evaluated the sections and the intensity of staining was evaluated subjectively using the following designations: 0 (no signal), 1 (weak), 2 (medium), 3 (strong). When scores of multiple tissue stainings were combined, scores that were 1 were labeled weak intensity, and scores 2 were labeled strong intensity . For immunohistochemical staining, the concordance between staining intensity scores was calculated according to the following classifications: kappa <0.4: slight concordance; kappa 0.4 and <0.8: moderate concordance; kappa 0.8 and <1: strong concordance; kappa = 1: perfect concordance . The fisher's exact test was used to compare inos immunohistochemistry results and to assess the relationship between inos expression and no levels with the stage and grade of the ovarian cancer samples analyzed . Data for nitrate levels were expressed as the mean + / standard deviation (sd) and values were compared by anova followed by the tukey test for individual comparisons . The correlation between intracystic nitrate levels and inos immunolabeling was tested using spearman's rank correlation coefficient . Sixty - three randomly selected women receiving pelvic mass outpatient services from the discipline of gynecology and obstetrics of the federal university of tringulo mineiro (uftm) were enrolled in this study . For those diagnosed with nonneoplastic tumors (n = 15), 10 (66.7%) had serous ovarian cysts and 5 (33.3%) had functional cysts (corpus luteum, follicular, and theca lutein cysts). The 28 patients diagnosed with benign neoplasias included 11 (39.3%) with serous cystadenoma, 6 (21.4%) with mucinous cystadenoma, 6 (21.4%) with mature teratoma, 3 (10.7%) with cystadenofibroma, as well as 1 (3.6%) with serous- and 1 (3.6%) with mucinous - cystadenoma associated with brenner's tumor . For the 20 patients diagnosed with malignant neoplasias, the cases included 7 (35%) serous adenocarcinoma, 3 (15%) granulosa cell tumor, 3 (15%) mucinous cystadenoma of borderline malignancy, 2 (10%) mucinous cystadenocarcinoma, 1 (5%) endometrioid adenocarcinoma, 1 (5%) granulosa cell tumor associated with brenner's tumor, 1 (5%) anaplasic adenocarcinoma, 1 (5%) immature teratoma with epidermoid carcinoma, and 1 (5%) serous cystadenoma of borderline malignancy . In the ovarian cancer patient group, the number of each tumor stage resected included 5 (25%) of i - a, 1 (5%) of i - b, 2 (10%) of i - c, 1 (5%) of iii - a, 10 (50%) of iii - c, and 1 (5%) of iv . In the iii - c and iv stages, five complete surgeries, four satisfactory citorreductions and only 2 unsatisfactory citorreductions were performed . Patients receiving adjuvant first - line chemotherapy received either a combination of cisplatin, epirubicin, and cyclophosphamide for epithelial tumors, or cisplatin, etoposide, and bleomycin for granulosa cell tumors of stages i - b . Of the 12 carcinomas, 5 (41.7%) were well differentiated, 4 (33.3%) were moderately differentiated, and 3 (25%) were poorly differentiated . Five patients were diagnosed with malignant ovarian tumors had died before their follow - up . There were sufficient samples to perform inos immunohistochemistry on 15 nonneoplastic tumors, 21 benign tumors, and 18 malignant tumors (figures 1 and 2). The results are summarized in table 2 . Samples with strong inos staining (scored 2) were more frequently found in ovarian cancer samples than in nonneoplastic (p = .0014) or benign neoplasia samples (p = .0003). Samples of malignant ovarian cancer tissue were further classified into well - differentiated (n = 11) or moderately / poorly differentiated (n = 7) tumors . For the well - differentiated tumors, 8 (72.7%) presented strong staining of inos while the other 3 presented weak or medium staining of inos . For the moderately / poorly differentiated tumor samples, all showed weak / medium intensity of inos . Overall, well - differentiated tumors presented a higher frequency of strong inos expression compared to moderately / poorly differentiated carcinomas (p = .004; fisher's exact test). No statistically significant correlation was found between the intensity of inos staining and tumor stage . Of the 5 (27.8%) patients diagnosed with malignant ovarian tumors that had died by the time of follow - up, only one of the samples previously collected from those five individuals showed strong expression for inos . Cystic fluid samples were collected at the time of surgical resection and were subsequently tested for no metabolite levels . Cystic fluids from 1 nonneoplastic tumor, 1 benign neoplasia, and 2 malignant ovarian tumors were not tested due to the viscous consistency of those fluids . The mean levels of no metabolites detected in the malignant tumor samples (75.7 m, n = 18) were significantly higher (p = .045) than the no metabolite levels for benign ovarian tumors (38.5 m, n = 27). However, statistically significant differences were not detected between no levels of malignant neoplasia samples versus nonneoplastic samples (40.9 m, n = 14) (figure 3). To examine whether intracystic no metabolite levels could be predictive for tumor stage, patient samples were divided into two groups: those with no metabolite levels <80 m and those with no metabolite levels> 80 m . The value of 80 m was derived from the median value of no detected in malignant tumor samples (81.6 m). No stage ii samples had detectable levels of no metabolites . For stage i samples, 6 (85.7%) had no metabolite levels> 80 m, and only 1 (14.3%) had no metabolite levels <80 m . In contrast, 3 of stage iii / iv tumor samples (27.3%) had no metabolite levels> 80 m and 8 (72.7%) had no metabolite levels <80 m . These results indicate that intracystic no metabolite levels> 80 m were significantly more frequent in stage i samples than in stage iii / iv samples (p = .0498; fisher exact test). However, there was no significant correlation between no metabolite levels and the grade of tumor differentiation . Correlations between no production and inos expression in the collected tumor tissues are summarized in table 3 . Differences in inos expression between nonneoplastic, benign and malignant ovarian neoplasias suggest a role for no in ovarian carcinogenesis . Our experiments revealed greater expression of inos in malignant ovarian neoplasias than in benign or nonneoplastic tumors . There were two cases of ovarian cancer that did not show inos immunoreactivity, while approximately one - third of benign and nonneoplastic tumor samples were positive for inos expression . Our results are consistent with other studies that have shown that a majority of ovarian malignant neoplasias present nos activity, while inos is detected at lower levels in patients without cancer . Although we hypothesize that inos may be a marker to detect malignant disease, it is not considered by others to be a marker exclusive to malignant disease . Expression of nos in malignant tissue derived from gynecological, breast, central nervous system, gastric, and colorectal tumors has been reported, suggesting its role in cancer progression [2529]. A positive correlation between inos expression and an increased density of tumor microvessels in human colorectal cancer was shown by cianchi et al ., and inos expression has been associated with increased vascularization and tumor invasion in endometrial malignant neoplasia . Correspondingly, patients with lung cancer, prostate cancer, or cervical cancer treated with no inhibitors showed antivascular activity . These data support the hypothesis that the inhibition of inos may provide a new therapeutic option for the treatment of ovarian cancer . Recently, it was demonstrated that inos expression in serous and low - grade carcinomas was significantly higher than in nonserous and high - grade carcinomas . Advanced stage tumors expressed low levels of inos and were associated with a shorter mean survival time although this was not determined to be a statistically significant correlation . In our study, patients with malignant tumors had significantly higher levels of intracystic no and inos expression in well - differentiated carcinomas compared to moderately / poorly differentiated carcinomas . In addition, positive inos expression in ovarian carcinoma had been identified as a positive disease - related survival indicator, and was found to be consistent with the high levels of inos activity and no production of nonmetastatic cells versus metastatic cells . In contrast to our findings, a separate study found that patients with advanced ovarian serous tumors express inos and cox-2 and experience a shorter disease - free interval and survival rate . In addition, patients negative for inos expression presented a complete clinical response to a first - line treatment of chemotherapy . A separate study found a positive correlation of no synthesis with tumor progression in a breast cancer model . In a study by taveres - murta et al ., increased levels of no metabolites in the tumor microenvironment were found in patients with ovarian cancer, but not in patients with benign neoplasia . Similarly, supernatants of cell cultures obtained from well - differentiated, malignant ovarian tumors were found to contain higher levels of no metabolites compared to cell cultures from patients with poorly differentiated tumors . However, to our knowledge, this is the first study to evaluate both intracystic no production and inos expression from the same tumor tissue . Our results suggest that intracystic no is not produced by tumor cells since no significant correlation was found between the levels of no metabolites detected and the intensity of inos expression detected by immunohistochemical staining of the tumor tissue . Instead, an analysis of no production from effusions (ascitic, cystic, or pleural fluids) of ovarian malignant tumors showed a significant correlation between the percentage of macrophages present and detectable levels of no metabolites, suggesting that macrophages play a significant role in the production of no in the tumor microenvironment . An association between increased intracystic leukocyte infiltrates and no production has previously been associated with ovarian cancer . No produced by ovarian carcinoma cell lines has also been shown to correlate with the extent of tumor cell apoptosis observed . These results, in combination with our data, suggest that the level of inos expressed by tumor cells and infiltrating leukocytes accounts for the intracystic no production detected although it cannot be ruled out that the inos associated with ovarian tumors could also be expressed by immune cells [36, 37]. Studies using activated macrophages treated with nos - specific inhibitors have shown an inhibition of no production and induced cytotoxicity, suggesting that activated macrophages may mediate no - dependent cytotoxicity . High concentrations of no can mediate cytotoxic activity against tumor cells, while low concentrations have been associated with angiogenesis . Correspondingly, high concentrations of no have not been found to be maintained in many malignant neoplasias . These opposing actions of no have been attributed to factors such as differences in the isoform of nos expressed, the level of nos expression, and the type of cell involved in either in vitro or in vivo systems . In ovarian cancer cell lines, high levels of no donors or strong expression of the inos suppressed survivin levels (a human gene that is part of the inhibitor of apoptosis family), and have been shown to induce apoptosis . It is hypothesized that no signaling could contribute to therapy resistance in epithelial ovarian cancer by modulating survivin expression since low levels of no are associated with resistance to carboplatin- and paclitaxel - induced apoptosis . The ability to modulate nos gene expression may represent an opportunity to control the growth and metastasis of tumors in vivo . In this study, increased expression of inos and increased production of no metabolites were detected in the tumor microenvironment of ovarian cancer samples compared to benign and nonneoplastic ovarian tissue samples . For ovarian cancer samples, inos expression correlated with the extent of tumor differentiation and intracystic no metabolite levels correlated with the tumor stage . We hypothesize that the absence of a correlation between no production and inos expression indicates that different cell types are involved in inos expression, and that controlling no production and inducing nos may represent valuable strategies in the prevention of benign tissue transitioning into well - differentiated malignant ovarian tumors.
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Epidermoid and dermoid cysts are benign lesions developing from abnormal epithelial components of ectodermal tissue formed during the fetal period, or implanted epithelium arising after trauma or surgery . These lesions, which can be seen anywhere in the body, occur in the head and neck area in approximately 7% of cases . Those in the oral cavity are mostly in the floor of the mouth (in the sublingual, submental or submandibular areas) and in various other localizations including the labial, lingual or buccal mucosa . Their incidence in the oral cavity makes up for 1.6% of the total occurrences and they constitute less than 0.01% of all the cystic lesions of the oral cavity . These cysts are termed epidermoid if they are enclosed in epithelium only, if they comprise skin appendages and teratoid if they include other tissues like muscle, cartilage or bone . Surgical excision is sufficient for cure . Even though reports of epidermoid cysts in the head and neck, especially those in the floor of the mouth, soft palate, lips, or the lingual and buccal epithelium can be found in the literature, but there is no report of an intratonsillar epidermoid cyst . We intend to report the case of a patient who underwent tonsillectomy for diagnostic purposes because of an epidermoid cyst arising from the tonsil and confirmed by histology . A 42-year - old female came to our clinic for sore throat and difficulty in swallowing . Ent examination showed marked hypertrophy of the right tonsil in comparison with the contralateral one, and a smooth - surfaced mass near the upper pole of the tonsil [figure 1]. A magnetic resonance imaging (mri) examination was performed, which evidenced a protrusion of the clearly hypertrophic tonsil into the nasopharynx [figure 2]. A right tonsillectomy was performed for diagnostic purposes after having obtained the patient's informed consent . It was interesting to note that these cavities contained keratin in a lamellar arrangement and their epithelium was of a squamous character [figure 3]. The patient was discharged on the first post - operative day in excellent condition; her follow - up, reaching 10 months, was entirely uneventful . View of the asymmetrical hypertrophy of the right tonsil with a mass arising in its upper pole view of the hypertrophic right palatine tonsil in t2-weighted mri cystic cavity within the tonsillar tissue lined with keratinized epithelium . Dermoid cysts have been classified by meyer in 1955 as true dermoid cysts, epidermoid cysts and teratoid cysts . A true dermoid cyst is lined with keratinized epithelium and contains skin appendages that could be described as hair follicles or sebaceous glands . An epidermoid cyst, on the other hand, is lined with simple squamous epithelium and its wall does not contain fibrous structures or skin appendages . A teratoid cyst may contain tissues ranging from simple squamous epithelium to ciliated cylindrical respiratory epithelium; its contents may be of ectodermal, mesodermal or endodermal in origin . A theory widely accepted today on the etiology of these lesions is their development from the epithelial remnants remaining isolated during the closure of the first and second branchial arches in the midline . Another theory is the development of cysts from abnormal inclusion of cells during surgery or trauma . Even though the fact that our patient was aged 42 years would seem to favor the latter mechanism, she presented no history of surgery or trauma . A study on 1495 cases by new and erich shows that their location is most frequently anal (44.5%) and ovarian (42.1%). Cases of cysts in the head and neck area are only 7% of the total body . In a study of 103 patients with diagnosis of epidermoid and dermoid cyst of the head and neck, 46.6% of these were orbital, 23.3% buccal and submental, 12.3% nasal, 10.7% cervical and 2.9% labial . Various publications also report epidermoid cysts of the oral cavity in the soft palate, the uvula and the sublingual area . The male / female ratio of the patients with a diagnosis of epidermoid cyst is 3/13 and the age range of the large majority is 10 - 35 years . Especially the latter fact leads to the thought that cyst formation could be stimulated by hormonal influence during puberty . The fact that our patient was a 42-year - old female would also support such an idea . The appearance of epidermoid cysts on mri is variable according to their fluid contents and protein density . Often, though, they exhibit a low signal intensity with t1a sequences and a high signal intensity with t2a sequences . The mri performed in our patient showed a marked hypertrophy of the right tonsil, which was protruding into the nasopharynx . The fact that the appearance of the cyst in the mri examination was not cystic could be due to the high protein density of its contents . . It should be excised without opening because its contents could have an irritating effect on the surrounding fibrovascular tissue . A malignant evolution has only been seen in the teratoid type and was reported to have an incidence of 0.5% . A tonsillectomy was performed in our patient; the cyst was excised within its capsule and the follow - up during 10 months was entirely uneventful . While the expansion of lymphoid follicles within the tonsil is the most frequent cause of their hypertrophy, asymmetric hypertrophy must lead to the suspicion of diseases like tonsillar tumor, atypical infection, granulomatous disease or tumors of the parapharyngeal area . In a series of 49 patients with asymmetrical tonsils with normal neck examination and normal overlying mucosa, only two (4.8%) a diagnostic tonsillectomy is indicated as asymmetrical tonsillar hypertrophy carries a potential risk of neoplastic transformation . Our patient also had an asymmetrical appearance of the tonsils . Also, the mass covered by normal mucosa and arising from the upper pole was suspicious for the presence of tumor and the patient was subjected to diagnostic tonsillectomy . In conclusion, epidermoid cysts, a rare occurrence in the head and neck area, can also be found inside the palatine tonsils and cause asymmetrical hypertrophy.
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Chemotherapy - induced hair loss is a feared side effect of cancer treatment (katsimbri et al . Scalp cooling during administration of chemotherapy prevents hair loss (e.g., ridderheim et al . Cooling can be achieved by means of a cap that is pre - cooled in a freezer or that exchanges coolant with a reservoir . The hair preservative effect of scalp cooling is attributed both to reduced blood flow by vasoconstriction and to reduced reaction rates in the body at the level of the hair follicle . When the scalp is cooled, vasoconstriction is induced, and through this, blood flow to the hair follicle is decreased . This reduced blood flow results in a decrease of the total amount of cytotoxic drug available for uptake in the hair follicle . (2007), and it was found that cooling the scalp to 20 c reduces local blood flow down to 20% of normal . A further decrease in temperature did not result in a further decrease in local blood flow . Based on this, it might be expected that there is a limit in temperature below which the effectiveness of scalp cooling will not increase anymore . However, the hair preservative effect of scalp cooling is also attributed to reduced cell metabolism . Because of reduced temperatures during scalp cooling, cellular drug uptake and damage are assumed to be lower, and with this, hair follicles are thought to be less susceptible to cytotoxic drugs . (2003) showed that in vitro doxorubicin uptake in kidney cells at 37 c was 4.5 times higher than drug uptake at 4 c. in addition, decorti also showed that doxorubicin uptake is dependent on extracellular concentration . Unfortunately, no studies have been performed to quantify these processes in the human hair follicle . For a better understanding of the functioning of scalp cooling, it is important to quantify the contribution of reduced drug uptake and drug damage, if any, on the hair preservative effect of scalp cooling . To this end, we investigated experimentally the relationship between doxorubicin exposure and cell damage at different temperatures using normal human epidermal keratinocytes . Chemotherapy disrupts the rapidly dividing keratinocytes (the cells that actually produce the hair shaft) in the hair follicle (cotsarelis and millar 2001). Therefore, the use of keratinocytes as an in vitro model is indicative for chemotherapy - induced damage to the hair follicle . Moreover, human keratinocytes are commercially available and they are easy to cultivate, which means that with this model, a wide range of boundary conditions can quickly be investigated . The goal of the experiments was to assess the effects of temperature and chemotherapy on keratinocyte survival . Pooled neonatal normal human epidermal keratinocytes (nhek) were obtained from cambrex bio science verviers, belgium (catalog number cc-2507). These cells have a doubling time of approximately 24 h. cells were cultured in t-75 flasks at a seeding density of 3,500 cells cm under an atmosphere of 95% air and 5% co2 at 37 c using 15 ml of keratinocyte general medium (kgm2; cambrex bio science verviers, catalog number cc-3107). Medium was refreshed the day after plating, and afterwards every other day until cells reached 7080% confluence . Then, cells were harvested using 6 ml of trypsin solution and subsequently cryopreserved in liquid nitrogen at a density of 1.2 10 cells per milliliter using 80% kgm2, 10% fetal bovine serum, and 10% dimethyl sulfoxide (dmso). For the experiments on the effect of temperature and chemotherapy on keratinocyte survival, third passage cells were plated in 24-well plates at a cell density of 6,000 cells per square centimeter . Cells were incubated at 37 c for a period of 24 h to allow the cells to recover from handling . Concentrations of doxorubicin in kgm2 (0.01, 0.04, 0.1, 0.5, 1.0, 3.0, or 10.0 g ml) were prepared . Plates were incubated for 4 h at either a low temperature (tl = 10 c), a medium temperature (tm = 22 c), or a high temperature (th = 37 c). For each combination of temperature and doxorubicin concentration, a sample size of eight cells receiving medium without any doxorubicin were used as a control group for each specific temperature . At the end of the exposure time, doxorubicin was removed, and cells were washed with 500 l phosphate - buffered saline (pbs). Fresh medium (250 l per well) was added to each well, and plates were then incubated at 37 c for a post - exposure time of 72 h, after which, a viability assay was performed as described below . The damage to the nhek cells exposed to different temperatures and doxorubicin concentrations was determined by a colorimetric mtt (tetrazolium) viability assay . The assay is based on the observation that viable cells have the ability to metabolize a water - soluble tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (mtt) into a product termed purple formazan (mosmann 1983; edmondson et al ., 1988; sgouras and duncan, 1990). The purple formazan can be solubilized, and the optical density of the solute can be determined using a spectrophotometric technique . The resulting absorbance is directly proportional to the number of cells, and this linearity extends over a wide range of cell numbers (mosmann 1983). A standard mtt solution was prepared by dissolving 5 mg ml of mtt (mtt formazan, sigma aldrich, zwijndrecht, the netherlands) in pbs . The standard mtt solution was added to complete medium (kgm2) in a ratio of 1 to 10 to obtain a final solution of 10% mtt solution and 90% kgm2 . A volume of 220 l of this solution was added to all test wells, and the plates were left to incubate at 37 c and 5% co2 for a period of 45 min . After this period, the mtt / medium solution was removed, and 200 l of a solution of 90% dmso and 10% triton x was added to extract the purple formazan salt from the cells . Plates were sealed and kept in dark for 30 min, after which, 100 l of each well was transferred to a new well of a 96-well plate . The optical density (od) was measured using an automated spectrophotometric plate reader (synergy ht, biotek instruments inc ., winooski, vt, usa) set to 570 nm and using a reference wavelength of 650 nm . Blanks (100 l of dmso / triton x solution only) were used as an extra control . For each well, the corrected optical density od(570650 nm) was provided by the plate reader . Mean optical density and standard error of each temperature and concentration group [odt, c se(odt, c)] and their respective control group [odt,0 se(odt,0)] were calculated . Based on these values, the viability (s) for each temperature and concentration group can be calculated as: 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s_{{\text{t, c}}} = \frac{{{\text{od}}_{{\text{t, c}}}}} {{{\text{od}}_{{\text{t, c}}}}} $$\end{document} an analysis of variance (anova) was performed using statistical software (spss version 15.0, spss inc ., the anova test is used to investigate whether significant differences in mean values exist between two or more groups . In this study, we want to investigate what the effect of temperature on the one hand, and the effect of doxorubicin concentration on the other hand, is on cell survival . Using a general linear model, the viability is statistically modeled as: 2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$s_{t, c} = \mu + \alpha t + \beta c + \gamma (t \cdot c) + \varepsilon $$\end{document} this means that we investigated whether temperature, concentration, and an interaction term are significantly relevant for describing our data . Where appropriate, a post hoc analysis was used using tamhane s t2 criterium (based on unequal variances) to test for differences between various doxorubicin concentrations and various exposure temperatures . A significance level of p microscopic photographs of cells at selected concentrations are shown in fig . 1 . Here, we can see that the control groups of all temperatures show similar cell counts . With increasing concentration g ml (c4), a clear difference is visible between the high temperature group and both the medium and low temperature group . It can be seen that there is a difference in cell morphology between 0.04 g ml (c2) and 0.5 g ml (c3). Cells in the latter group, and in groups with higher doxorubicin concentrations, show enlarged and flattened cell shape and increased granularity . These are characteristics of senescence or aging, which is known to be caused by doxorubicin (roninson 2003). This figure shows the cell viability as a function of doxorubicin concentration for different doxorubicin exposure temperatures . In this figure, we can see that the viability of keratinocytes as a function of doxorubicin concentration shows a decreasing s - curve with increasing concentrations . Viability levels are slightly above 1 for low concentrations (0.01 g ml), and with increasing concentrations, viability gradually drops towards zero for high concentrations (10 g ml). For different temperature groups, cell viability at a specific concentration is always lowest for the high temperature group . In the mid - section of the concentration range (i.e., 0.11 g ml), the differences between the high temperature group (th) and the two lower temperature groups (tl and tm) are more pronounced . Cell viability for these lower temperature groups decreases more slowly than for the high temperature group, until at a concentration of 1 g ml, a rapid drop in cell viability is visible . The difference between the individual temperature groups at low and high concentrations is therefore small . C1 = 0.01 g ml, c2 = 0.04 g ml, c3 = 0.5 g ml, c4 = 3.0 g ml, c5 = 10 g ml.figure 2.cell viability as a function of doxorubicin concentration at different temperatures . Delta: tl (10 c), filled square: tm (22 c), and open circle: th (37 c). C1 = 0.01 g ml, c2 = 0.04 g ml, c3 = 0.5 g ml, c4 = 3.0 g ml, c5 = 10 g ml . Delta: tl (10 c), filled square: tm (22 c), and open circle: th (37 c). Data points show the mean and standard error of eight replicates . The results of a significance test are shown in table 1 . From an anova analysis, we found that the effect of doxorubicin concentration on nhek viability was highly significant [f(6,161) = 165.213, p <0.001], as was the effect of temperature on nhek viability [f(6,161) = 37.054, p <0.001]. The effect size of doxorubicin (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta _ {\text{p}}^2 $$\end{document}; partial eta squared) was higher than the effect size of temperature (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta _ {\text{p}}^2 $$\end{document} = 0.88 compared to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta _ {\text{p}}^2 $$\end{document} = 0.36). For all temperature groups, increased doxorubicin concentration has a significant decreasing effect on cell viability . This effect was highly significant for the high temperature group [f(6,49) = 119.400, p <0.001], the intermediate temperature group [f(6,49) = 33.166, p <0.001], and the lowest temperature group [f(6,49) = 68.225, p <0.001] based on these results, a post hoc analysis was performed for each temperature group to check where a significant transition in cell viability as function of doxorubicin concentration occurs . This transition point may be viewed as the inflection point of the s - curve . For the low and medium temperature group the anova analysis also revealed a significant interaction between doxorubicin and temperature on cell viability [f(6,161) = 2.754, p = 0.002], although the effect is smaller than that of temperature and doxorubicin (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\eta _ {\text{p}}^2 $$\end{document} = 0.199). This significant interaction term shows that the effect of temperature on cell viability is not the same at the levels of different doxorubicin concentrations . For low and high doxorubicin concentrations, there is hardly any influence of a lower temperature on cell survival . In the mid - section of the graph it can be seen that significant differences exist between tl and th for concentrations of 0.1 g ml and up, except for a concentration of 1 g ml . At this concentration, the significance level is not high enough, but still, a trend is visible (p <0.1). Between tm and th, significant differences exist for concentrations of 0.5 g ml and up, except for a concentration of 1 g ml where no significant difference is visible . The only significant difference between tl and tm exists at a concentration of 10 g ml, although a trend is visible at a concentration of 0.1 g ml . Table 1.results of an anova analysis for determining the significance between different temperature groups at a specific doxorubicin concentrationdoxorubicin (g ml)f valuep valuetl vs thtm vs thtl vs tm0.01f(2,21) = 0.3150.733 0.04f(2,21) = 1.9580.166 0.1f(2,21) = 15.187<0.001p <0.01 p <0.1 * 0.5f(2,21) = 31.605<0.001p <0.001p <0.001 1f(2,21) = 4.380<0.05p <0.1 * 3f(2,21) = 12.908<0.001p <0.01p <0.01 10f(2,21) = 22.587<0.001p <0.001p <0.05p <0.05p values are shown with their respective significance level . Trends (p <0.1) are marked with an asterisk . Results of an anova analysis for determining the significance between different temperature groups at a specific doxorubicin concentration p values are shown with their respective significance level . We examined the role reduced metabolism has on the hair preservative effect of scalp cooling . For this, a controlled in vitro experiment was conducted to assess the damage exerted by doxorubicin on human epidermal keratinocytes for a range of chemotherapy concentrations and exposure temperatures . The effects of temperature and doxorubicin on cell damage were determined from cell viability measurements (mtt assay). We found that increased doxorubicin concentrations have a significant decreasing effect on keratinocyte cell viability . Viability was close to 1 for low concentrations and close to 0 for high concentrations . In between we also found that reduced temperature has a significant increasing effect on keratinocyte cell viability . The two cooled temperature groups (10 c and 22 c) show a higher cell viability for each doxorubicin concentration than the 37 c group . At the mid - range of doxorubicin concentrations (0.041 g ml), the effect of reduced temperature is more pronounced than in the extreme values of doxorubicin concentration . This observation is confirmed by the fact that an interaction term of doxorubicin concentration and exposure temperature was significant . The increased effect of reduced temperature was significant for doxorubicin concentrations of 0.1 g ml and higher . We found no significant differences between the low (10 c) and medium (22 c) group . The results of the experiment are in line with the hypothesis of scalp cooling . In this hypothesis, the reduced temperature caused by scalp cooling will result in lower perfusion and a lower cellular metabolism . With this, the supply, uptake and damage of a chemotherapeutic agent will be diminished . For low doxorubicin concentrations (0.01 and 0.04 g ml), the mtt assay for each temperature group showed a higher viability than the respective control group . Because of the fact that each specific temperature group has its own control group, it is unlikely that this effect is caused by a measurement error . This means that apparently, cells are metabolically more active when exposed to a small amount of doxorubicin . It is possible that cells in the control group are close to reaching confluence at the time that viability is determined . As cells reach confluence, therefore, the rate at which cells divide will become lower, and hence, their metabolic activity will decrease . Another possibility is that a small amount of doxorubicin will stimulate cells to repair damage exerted to the cells . Hence, it may actually result in larger cell growth compared to a control group receiving no doxorubicin . At this point, however, we have no indication for which explanation may be true . Another remarkable result is that there is no significant difference between the medium and low temperature group . Thus, it seems that there is a limit in effect of reduced temperature . In a pilot study to determine optimal doxorubicin exposure temperature, we found that an exposure temperature of 26 c also showed increased cell viability (data not shown), and this increase did not differ from other temperature groups . During scalp cooling, a skin temperature of approximately 20 c based on the results of our experiment, one might expect that the limit in effect of reduced temperature during scalp cooling may be reached for a temperature as high as 26 c. this means that during scalp cooling, the effect of reduced metabolism is already at its maximum . However, care has to be taken when the results of an in vitro experiment are generalized . Therefore, further studies on the effect of reduced temperature on cell damage are needed to exactly define this limit . Our current study confines the boundary conditions in doxorubicin concentration and exposure temperature that can be used in these further studies . Recently, it was shown that there is a limit in perfusion during scalp cooling (janssen et al . The findings of the current study indicate that there is no difference in cell survival between 22 c and 10 c. based on these findings, we can conclude that there is an optimal temperature in scalp cooling.
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Knee osteoarthritis (oa) is one of the most common musculoskeletal disorders worldwide, and a leading cause of knee pain and disability in the elderly . The number of patients (aged 40 years) in japan with knee oa diagnosed from radiological findings is estimated to be approximately 25.3 million, and approximately 8 million patients are estimated to exhibit oa symptoms but do not have a definitive diagnosis1 . Therefore, there is an urgent need to reduce the progression and incidence of knee oa in japan . The medial tibiofemoral compartment of the knee is the part most commonly affected by oa2 . The peak force is higher in the medial compartment of the knee than in the lateral compartment during walking and other weight - bearing activities3, 4 . Excessive knee load is a significant risk factor for an increased risk of structural progression4, 5 . A high knee adduction moment (kam) reflects increased compressive forces acting on the medial aspect of the knee6 and is widely considered a surrogate measure of medial knee compression7 . Lateral wedge insoles (lwis) are one of the treatment options frequently recommended in clinical guidelines8 for the management of medial knee oa . Lwis have been shown to reduce kam7 and pain9, and to prevent or delay the progression of medial knee oa10, 11 . On the other hand, medial wedge insoles (mwis) tend to increase kam7, 12 . The efficacy of lwis may be limited because they fail to reduce kam or increase kam in certain persons13, 14 . However, no study has evaluated the effects of lwis and mwis, or has identified nonresponders to those types of therapy . The possible explanations for nonresponders are variations in the foot alignments and gait patterns . It is recommended that the assessment of patients with knee oa should include foot evaluations because these patients have been shown to exhibit a more pronated foot type compared with controls15 . In addition, previous studies have reported that patients with knee oa demonstrate changes in gait patterns, such as increased toeing out16, 17, reduction in gait speed18, and leaning of the trunk in the direction of the stance leg19, to reduce kam during gait . Therefore, to more accurately investigate the effects of a wedged - insole intervention, unilateral weight bearing was analyzed as a movement task that assumed the mid - stance during gait according to a previous study20 . Thus, the primary purpose of this study was to evaluate the immediate effects of lwis and mwis under unilateral weight - bearing conditions in healthy young adults . The secondary purpose was to examine whether variations in foot alignment influence the changes in kam . It was hypothesized that kam in subjects who have normal foot alignment would significantly decrease under the lwi condition than under the barefoot (bf) and mwi conditions . The subjects were 30 healthy young adults without disease and/or present illnesses that would affect unilateral standing (mean age, 21.2 1.9 years; mean height, 1.62 0.08 m; mean weight, 55.6 9.5 kg; and mean body mass index [bmi], 21.1 2.2 kg / m). Before the experiments, the purpose of this study was explained to the participants and informed consent was obtained from them . This study was approved by the ethics committee of human research, hiroshima international university (no . 13 - 4). Before motion analysis, foot alignment measurements were performed for all subjects by using the foot posture index (fpi). The fpi is a six - item foot posture assessment performed with the subject standing and relaxed in the bipedal position21 . The six items of fpi include talar head palpation, curves above and below the lateral malleoli, calcaneal angle, talonavicular bulge, medial longitudinal arch, and forefoot to rearfoot alignment . Each item was scored on a five - point scale between 2 and + 2, which provided a total sum of all items between 12 (highly supinated) and + 12 (highly pronated). All subjects were assessed by using fpi as follows: normal foot (total score between 0 and + 5), pronated foot (total score between + 6 and + 12), and supinated foot (total score between 12 and 1). A three - dimensional motion analysis system that included eight infrared cameras (vicon mx; vicon motion systems, oxford, uk) and two force plates (amti, watertown, ma, usa) were used to record the kinematics and kinetic data at the sample frequency of 100 hz . Kinematic and kinetic data were low - pass filtered with a fourth - order butterworth filter with cutoff frequency at 6 and 15 hz, respectively . A total of 40 reflective markers were placed at the following landmarks on both sides of the subjects: the acromion process, olecranon, radial styloid process, anterior superior iliac spine, posterior superior iliac spine, superior aspect of the greater trochanter, medial and lateral femoral condyles, midpoint between the greater trochanter and the lateral femoral condyles, medial and lateral malleoli, midpoint between the lateral knee joint line and the lateral malleolus, posterior distal aspect of the calcaneus, posterior proximal aspect of the calcaneus, lateral calcaneus, sustentaculum tali, and head of the first and fifth metatarsals . These anatomical markers were used to construct anatomical coordinate systems for the trunk, pelvis, thigh, shank, and foot segments . In addition, a marker was attached to the right scapula to distinguish the right and left sides . This study involved a within - subject design where every subject was tested under three conditions . All subjects were instructed to stand on their left leg under three different conditions . The lwis had a base height equal to that of the fifth metatarsal and were medially inclined, and the outside was 7 mm thicker than the inside . The mwis had a base size equal to that of the fifth metatarsal and were medially inclined, and the inside was 7 mm thicker than the outside . The insoles were made of high - intensity silicon rubber (nakamura brace, ohda, japan), with a hardness of durometer type a (shore a) 40 . Following a previous study22, the subjects were asked to stand on one leg with a toe - out angle of 0 and to flex the contralateral hip to 30 while maintaining the contralateral hip at neutral abduction / adduction and rotation, with their hands on their abdomen . Before collecting experimental data, the subjects practiced standing on one leg under each condition several times, to become familiar with the task . They were then instructed to stand on one leg while looking straight ahead for 10 s. the three conditions were applied in a random order, and measurements were repeated three times for each condition . Data analyses were done by using the bodybuilder software (vicon motion systems, oxford, uk). The coordinates of each joint center were calculated according to methods described in previous studies23, 24 . An eight - link segmental model was developed to calculate the hip, knee, and ankle kinematic and kinetic data by using inverse dynamics according to the techniques described by vaughan et al25 . Anthropometric parameters for mass, center of mass, and moment of inertia for each segment were obtained from the report by okada et al26 . A knee joint moment was calculated by using the tibial coordinate system with the origin in the knee joint center . In this study, kam was defined as the external knee adduction moment after normalization to the subject s body mass (nm / kg). Furthermore, a knee - ground reaction force lever arm (kla) was calculated, defined as the perpendicular distance between the ground reaction force and the knee joint center in a laboratory frontal plane, based on the report by hinman et al27 . In this study, changes in kam and kla between the lwi and mwi conditions were independently examined . The change as the difference was calculated between each subject s kam and kla when using lwis or mwis, and their kam and kla in the bf condition . The percentage change was calculated as follows:(kam or kla under the lwi or mwi condition / kam or kla under the bf condition) 100 . The above equation expresses the change in kam or kla as a percentage of the value under the bf condition . Although data during a 10-s duration was recorded for each condition, the time between 5 and 6 s was used for the analysis to eliminate the initial unstable phase associated with the transition from the initial stance to standing on one leg22 . The mean value during this 1-s duration was calculated for all variables, and the mean value of three trials was determined for each subject . All statistical analyses were performed with ezr on r commander v1.10 (easy r, saitama, japan). The normality of data distribution was assessed by using the shapiro - wilk test . To compare the three conditions, repeated - measures one - way analysis of variance with a post hoc tukey s test was used to evaluate the differences that were normally distributed, or the kruskal - wallis test with a post hoc steel - dwass test for those that were not . Similarly, to compare the changes in kam and kla between the lwi and mwi conditions, the paired t - test or wilcoxon signed - rank test was used to evaluate the differences . Pearson s correlation or spearman s rank correlation was also used to examine the associations between the changes in kam and kla . Concerning the reliability of the fpi, the results showed an intraclass correlation (icc1,1) of 0.87 . Therefore all subjects (n = 30) were classified into three subgroups according to the left foot alignment results of the fpi . The characteristics of the three groups are summarized in table 1table 1.the characteristics of the three groups based on fpinormal group (n = 18)pronated group (n = 6)supinated group (n = 6)age (years)21.1 1.321.3 1.021.3 1.2height (m)1.61 0.081.63 0.071.61 0.06body weight (kg)54.8 9.156.0 9.657.7 11.9bmi (kg / m)20.9 1.821.0 2.822.0 3.1values are expressed as mean sd . The subjects age, height, body weight, and bmi were similar between the subgroups . Values are expressed as mean sd table 2table 2.kam and kla for three conditions (n = 30)bflwimwikam (nm / kg)0.43 0.080.42 0.080.45 0.08kla (mm)127.6 12.6126.7 12.7130.7 12.7bf: bare foot, lwi: lateral wedge insole, mwi: medial wedge insole, kam: knee adduction moment, kla: knee - ground reaction force lever arm . Values are expressed as mean sd shows the value and standard deviation of kam and kla under the bf, lwi, and mwi conditions . On the other hand, concerning the changes in kam and kla as a percentage of the value under the bf condition, both of these variables significantly decreased under the lwi condition compared with the mwi condition (table 3table 3.the change in kam and kla under the lwi and mwi conditions (n = 30)lwimwikam (%) 99.6 (96.4102.7)104.6 (101.7107.0) * kla (%) 99.9 (97.1102.4)102.2 (101.0104.6) * values are expressed as median (iqr). * bf: bare foot, lwi: lateral wedge insole, mwi: medial wedge insole, kam: knee adduction moment, kla: knee - ground reaction force lever arm . Values are expressed as mean sd values are expressed as median (iqr). * p <0.05 under the lwi condition, the change in kam positively significantly correlated with the change in kla (r = 0.356, p <0.05). However, under the mwi condition, there was no significant association between the change in kam and kla (r = 0.307, p = 0.11). The changes in kam and kla under the lwi and mwi conditions between the subgroups are presented in table 4table 4.the change in kam and kla under the lwi and mwi conditions between the subgroups lwimwikam (%) normal (n = 18)98.9 4.4104.2 5.7 * pronated (n = 6)94.4 8.8108.6 6.4 * supinated (n = 6)101.8 2.6102.7 4.1kla (%) normal (n = 18)99.2 3.0101.3 3.7 * pronated (n = 6)99.7 5.4104.8 3.8supinated (n = 6)100.9 (98.5103.2)103.6 (102.4105.5)values are expressed as mean sd or median (iqr) * p <0.05 . In the normal and pronated groups, the change in kam significantly decreased under the lwi condition compared with the mwi condition (p <0.05). However, it was not significantly different between conditions within the supinated group . In the normal group, the change in kla also significantly decreased under the lwi condition compared with the mwi condition (p <0.05). In contrast, it was not different between conditions within the pronated and supinated groups . Values are expressed as mean sd or median (iqr) * p <0.05 in this study, we evaluated the immediate effects of lwis and mwis under unilateral weight bearing conditions in healthy young adults . The findings from our study revealed that both the kam and kla were not significantly different between the three conditions . However, the change in kam and kla significantly decreased in the lwi condition compared with the mwi condition . These variables ware normalized to the value in the bw condition, which suggests that the lwi may have effects of decreasing the kam and kla, whereas the mwi may have effects of increasing those variables . Additionally, in the lwi condition, the change in kam was positively correlated with the change in kla . Therefore, this study has demonstrated that the primary mechanism of the biomechanical effects of lateral wedges is related to a reduced kla . Our results showed that kam and kla were not significantly different between the three conditions . These results did not agree with those of previous kinematic studies that demonstrated that lwis significantly reduced kam in patients with knee oa28, 29 . The difference in findings can be assumed to be due to a difference in the measurement tasks . Most of the previous studies analyzed kam during gait, particularly a value of peak during the stance phase . On the other hand, takacs and hunt20 reported that kam increased significantly with a contralateral pelvic drop during unilateral weight bearing . To eliminate the influence of gait speed and patterns that affect kam in the present study, we also selected unilateral weight bearing for the measurement tasks . Therefore, the lack of a difference between the three conditions may have been caused by variations in individual motor control . We therefore conclude that the common lwi and mwi conditions could not affect kam and kla during unilateral weight bearing when compared with the bf condition . Under the lwi condition, a previous study reported that lwis of approximately 5 reduced the peak kam and knee adduction angular impulse27 . Furthermore, a stepwise regression analysis demonstrated that the reduction in the peak kam may have been the result of a decreasing kla27 . Similar to their findings, our results demonstrated that the change in kam mildly correlated with the change in kla under the lwi condition . In brief, the reduction in kla with lwis may be the central mechanism explaining the load - reducing effect, which also led to the reduction in kam with lwis . However, there was no association between the change in kam and that in kla under the mwi condition . A previous study demonstrated that there were no significant changes in either the first or second peak kam during the gait stance phase with the addition of medial arch supports in patients with knee oa30 . Moreover, the researchers observed considerable individual variations in response to the arch support across participants . On the other hand, in the present study, we observed that the changes in both kam and kla significantly increased under the mwi condition compared with the lwi condition . These results suggest that although mwis may cause an increase in kam and kla, the increase in kam does not necessarily correlate directly with the increase in kla . As a result of the fpi classification, the changes in kam and kla significantly decreased under the lwi condition than under the mwi condition in the normal group . In contrast, the change in kam significantly decreased under the lwi condition than under the mwi condition in the pronated group; however, the change in kla was not significantly different under the two conditions . These results suggest that persons with a pronated or flat foot receive a beneficial effect with a reduction in kam; however, this could not be closely correlated to the reduction in kla . A previous study reported that the reduction in the peak kam correlated with a reduction in peak hip adduction during the stance phase27 . Another previous study revealed that healthy subjects could decrease the first peak of kam by 65% compared with a normal gait by walking while leaning their trunks toward the stance limb31 . Therefore, the present study suggests that the mechanism of reducing kam can be potentially explained by not only the decrease in kla but also another kinematic or kinetic change such as a response of the hip, pelvis, or upper trunk . Neither the change in kam nor that in kla was significantly different between the lwi and mwi conditions in the supinated group, indicating that lwis or mwis could not change these values in persons with a supinated or hollow foot . Furthermore, this suggests that such persons do not experience positive effects on kam, which is considered a surrogate measure of medial knee compression . Therefore, the present results provide important insights that could help clinicians decide whether lwis will be efficacious for patients with medial knee oa by using a simple clinical examination . First, because our study did not clarify the influence on gait, the findings need to be interpreted with caution . Second, as our study involved healthy participants, we can only draw conclusions about the effects of lwis without the limitations of diseases . The purpose of the present study was to evaluate the inherent effect of lwis and mwis on kam without possible confounding parameters such as the severity of knee oa and gait speed . Third, our decision to categorize participants by using the fpi resulted in a decrease in the sample size per group . The findings from our study suggest that not only changes in kla but also those of another factor may have an influence on the change in kam in some persons with a particularly pronated or flat foot . Therefore, in addition to the assessment of the foot alignment by using the fpi, future research should be performed with a larger number of subjects, and should aim to examine additional kinematic and kinetic factors that indicate decreased kam . Furthermore, longitudinal studies are needed to examine both the immediate and long - term effects of lwis for knee oa patients . Despite these limitations, our findings suggest that the assessment of foot alignment would assist in the identification of persons who may respond positively to lwi treatments.
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A significant number of these admissions are attributable to a small number of complex patients with other comorbidities who do not engage well with mainstream services . Assertive outreach teams have been used in the field of psychiatry to engage patients who are poorly compliant . This study examines whether an alcohol assertive outreach team (aaot) can engage with this group and reduce hospital admissions . The aaot is a multidisciplinary team with medical, psychiatric, substance misuse, psychology, nursing and social work specialists . The team worked with patients with the highest number of alcohol - related admissions and case managed in a community setting for 6 months . The admission and emergency department attendances of the cohort were compared for the 3-month period before and after the intervention . Christo inventory for substance misuse services (ciss) scores were determined pre and post the intervention period . The total number of admissions in 3 months fell from 151 prior to the intervention period to 50 following the intervention . Emergency department attendances also fell from 360 in 3 months to 146 following the intervention period . An aaot model appears to reduce hospital admissions and emergency department attendances in a complex group of patients that display high alcohol - related admissions . In the uk nhs, a continued rise in alcohol - related admissions is clearly seen . The current costs for alcohol - related admissions in england are estimated at 1.2 billion.1 within this general rise, there is a cohort of patients who frequently attend the emergency department and have high levels of repeated hospital admissions . This group have complex physical health, psychiatric and social needs, but the most common factor is high levels of alcohol use.2 within this group, there are a high proportion of dual diagnosis patients: those with both mental health and alcohol problems . Unfortunately, they do not interact well with mainstream services and higher death rates are seen.3 previous studies have looked at strategies to impact patients who are frequent attenders at emergency departments . Patient care plans have been used to reduce emergency department attendances with mixed results.46 these plans involve a specific management plan that is implemented when the patient attends the emergency department . Case management strategies have also been used.7 8 this involves an ongoing clinical interaction which extends beyond the hospital into the community . Case management involves multidisciplinary teams usually comprising psychiatric, social work, substance misuse, nursing and medical specialists . In mental health services, these are multidisciplinary teams which work with people with complex health needs, drug, alcohol, mental health and housing issues . They have been shown to be effective in patients with high levels of inpatient bed use.9 these teams are commonly targeted at people who engage poorly with services . Assertive outreach teams are typically defined by workers with a low caseload, usually 10 patients per worker, assertive engagement mechanisms and a high proportion of contact within the community.10 recent work has demonstrated the use of assertive treatment methods to improve engagement in alcohol services and to improve rates of patients entering recovery.11 in view of the high levels of alcohol - related admissions seen in the north west of england, the north west chief executives challenge, led by david dalton and other experts, developed strategies to improve the quality of alcohol care . The two initiatives modelled were the development of an alcohol assertive outreach team (aaot) and a 7-day alcohol specialist nurse service.12 an aaot was developed to work with two separate cohorts of patients . The first group are patients with alcohol - related liver disease who are increasingly using hospital services and exceed the threshold of two admissions wholly attributable to alcohol . The aim of this study is to establish whether an aaot is an effective model for reducing hospital admissions and emergency department attendances in the top 30 most frequently admitted patients . The study was carried out between january 2011 and july 2012 at the salford royal nhs foundation trust . The study was a retrospective cohort analysis of the effects of assertive outreach methods on the admission rates pre and post the intervention period . The aaot comprised a consultant in emergency medicine, a consultant psychiatrist specialising in addiction disorders, an emergency department nurse, a social worker, a psychologist, an alcohol worker, a support worker and an administrator . In the uk, a national audit system exists to record the number of alcohol - related admissions to each hospital . A database was created to identify the adult patients with the highest number of alcohol - related unscheduled care admissions . Patients were case managed for a period of 6 months; after this time the database was refreshed . A new cohort of patients was then case managed for the subsequent 6 months . To case manage the patients, case profiles were initially formed by information gathered from the patient, the acute trust, mental health trust, community alcohol team, primary care services, council and criminal justice services . The face - to - face interaction as part of the case management was organised in the community away from the hospital . Common strategies were fast access to alcohol detoxification; appropriate referral to outpatient specialities and support in getting there; alcohol and psychological support; facilitating housing solutions; and robust responses to violence and aggression . A part of the case management involved the production of individual care plans that would be implemented when a patient attended the emergency department . The plans were immediately available to the emergency department staff at the point of triage and helped produce a consistent coordinated response whenever patients interacted with the hospital . The primary outcome measure was the number of admissions per month in the 3-month period immediately before and after the 6 months of case management intervention . Emergency department attendances were also recorded during these periods . At the start and end of the 6-month case management period, christo inventory for substance misuse services (ciss) scores were calculated.13 this is a subjective score that reflects a patients social functioning, psychological and general health, compliance, drug and alcohol use and criminality . The score is a 020 score, with lower scores reflecting better outcomes (above 11 reflects high problem severity). The score has been previously validated.13 ethics approval was not required as this study fulfils the criteria for service evaluation . Statistical analysis was performed with non - parametric statistical analysis as the ciss scores, admissions and attendance rates were not normally distributed . Medians are quoted and the wilcoxon rank sum test was used for comparing effects between groups . The team was established and began to case manage patients in april 2011 . Due to delays in fully establishing the team, only 24 patients were case managed in the initial cohort . A further 30 patients were case managed in the 6-month period starting in october 2011 . A significant reduction in hospital admissions was seen in the 3 months following case management as compared with the 3-month period immediately prior to the start of case management . The total number of hospital admissions were 151 prior to case management, reduced to 50 following the case management period (w=2070, p<0.001). A similar significant reduction was also seen in the total number of emergency department attendances in the 3-month periods before and after intervention (360 reduced to 146, w=2215, p<0.001). The white bar represents the 6-month period in which the team actively case managed 54 patients . There was a significant reduction in the ciss scores at the start as compared with the end of the case management period (median 11 reduced to 8, p<0.001). There were no deaths in the study group during the period of case management and up to 3 months after . However, one patient left the local area and contact was not able to be maintained . There was loss of engagement with a second patient who did remain within the local area . This study demonstrated that case management using the alcohol assertive outreach model can reduce emergency department attendances and hospital admissions . This study is unique in that the focus is on patients with high level of alcohol admissions rather than frequent emergency department attendances alone . While these groups share similar complexity, this is a slightly different cohort than the traditional group of emergency department regular attenders . Our pilot work demonstrated a lower level of homelessness in this group in contrast to our most frequent emergency department attenders . Our high admissions group had more stable accommodation and we were able to remain in contact and engage in the outreach aspect of the team's work . However, we had no exclusion criteria and homeless patients were included in the study . A second reason for addressing a high admissions group rather than frequent emergency department attenders was the perceived cost benefit of reducing admissions rather than emergency department attendances alone . Based on national indicators and length of stay costs, an average alcohol - related admission costs a primary care organisation 1824 and an alcohol - related emergency department attendance costs 80.14 in the 3-month period after the end of case management alone, we demonstrated a reduction in admissions from 151 to 50 . Taking into account the reductions in admissions and attendances seen during the first year of the team alone, the cost reductions cover the 300 000 required to establish the service . These initial cost reductions do not take into account any ongoing reduction in admissions and subsequent savings . Previous studies using care plans and case management strategies have shown mixed outcomes in emergency department frequent attenders . Studies from the uk, australia and the usa have all demonstrated reduction in emergency department attendances,2 4 5 8 15 but other teams have reported no benefit6 16 or even an increase in emergency department use.7 there is less evidence on the effects on admission rates . A uk study reported a good impact on admission rates,4 but this was not repeated in other healthcare systems . A us randomised control trial reported no impact on admissions with case management;8 however, increased admissions were seen in an australian trial.7 traditional clinical approaches to medical, alcohol and psychiatric disorders are effective when a single disorder is identified . Unfortunately, when complex comorbidity is present, patients tend to fall between the gaps between services and commonly present and are admitted to inpatient hospital beds . We found that through effective multidisciplinary working, the aaot could more effectively support these complex individuals . The most important factor that increased the effectiveness of the team was the creation of an extensive case profile for each patient from a wide ranging variety of information sources . This permitted the team to plan effective interventions which supported the patient's medical, psychiatric, substance misuse and social difficulties in a coordinated fashion . As with all projects that impact the frequent users of emergency departments, there has to be concern that patients are not simply being diverted to other emergency departments . This is true especially with homeless emergency department regular attenders who commonly move between areas . One undoubtedly had moved out of the area and was likely to have attended other emergency departments . The second patient remained within the local area but the team was not able to engage with him . For the rest of the patients, the ongoing community engagement allowed us to be confident that they remained within the locality of the hospital . Indeed, the reduction in the ciss scores that are demonstrated point to improved functioning and a reduction in problem severity . The postintervention ciss score remains high in comparison with other settings such as abstinence based treatment centres or outpatient alcohol services, but this reflects the severity of the cohort that this group comprises . Assertive outreach is a uk term to refer to the north american assertive community treatment for psychiatric care . It is more successful when hospital use is high.9 assertive approaches to engagement in uk community alcohol work have demonstrated to be beneficial . Passetti et al11 set up a clinic which targeted patients with a history of disengagement . They used an assertive approach and saw more people completing withdrawal treatment and entering an aftercare placement . We needed to modify the original model to adapt to our local application, but maintained the fidelity of the assertive approach.10 this was defined by a small caseload with 10 patients per worker, backed by a multidisciplinary team with psychiatry, nursing and substance misuse workers, and uses assertive engagement mechanisms and focuses on community engagement . We used a preintervention and postintervention comparison whereby all patients were used as their own control . Some of the effects could be attributed to a natural reduction in emergency department attendances and admissions over time . This effect has been previously noted in two us studies,6 8 but it is not a consistent effect with studies demonstrating no reduction16 or even an increase in emergency department attendance and admissions over time.7 a multicentre randomised controlled trial would be needed to adequately power a study to address this issue . We took a pragmatic approach in using a prestudy and poststudy to describe the effects of alcohol assertive outreach work in reducing admissions in this cohort . The main clinical findings of this study describe an aaot approach to the support and management of a cohort of patients who demonstrate high numbers of alcohol - related admissions to a uk inner city hospital . This approach appears to reduce admissions, emergency department attendances and improves social functioning . What is already known on this topica complex group of patients with high levels of repeat alcohol - related admissions are well recognised, but difficult to impact what this study addsan alcohol assertive outreach team can engage with this complex patient group and reduce hospital admissions how might it impact on clinical practice in the foreseeable futurealcohol assertive outreach teams provide a solution for reducing alcohol - related admissions in the highest users of inpatient services a complex group of patients with high levels of repeat alcohol - related admissions are well recognised, but difficult to impact an alcohol assertive outreach team can engage with this complex patient group and reduce hospital admissions alcohol assertive outreach teams provide a solution for reducing alcohol - related admissions in the highest users of inpatient services
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A renal leiomyosarcoma is an uncommon neoplasm of the kidney, accounting for approximately 1% of all malignant renal tumors, and has a poor prognosis [1, 2]. Its symptoms, such as nonspecific abdominal discomfort, dull pain, and oliguria, are common in other conditions; therefore, a renal leiomyosarcoma is difficult to diagnose preoperatively . In this case, the condition had been preoperatively misdiagnosed four years earlier as a renal cell carcinoma before it was diagnosed as a primary renal leiomyosarcoma . Furthermore, it took several months to diagnose a recurrent renal leiomyosarcoma in the same patient because of the ambiguous, nonspecific symptoms . She visited our clinic complaining of diffuse abdominal pain, and colonoscopy showed a submucosal tumor or an extrinsic mass involving the descending colon . There are no reported cases of a renal leiomyosarcoma invading the retroperitoneal colon and mimicking a submucosal lesion . A 57-year - old woman visited our gastroenterology department complaining of diffuse lower abdominal discomfort . The preoperative diagnosis was a renal cell carcinoma, but she was subsequently diagnosed with a primary renal leiomyosarcoma on pathologic examination (fig . Regular follow - ups with a urologist and abdominal computed tomography (ct) showed no recurrence for three years . After another year, she returned to the hospital complaining of diffuse abdominal discomfort . At that time the findings of a general physical examination were unremarkable, except for diffuse abdominal sensitivity . The laboratory test results were as follows: leukocyte count, 6,770/mm; hemoglobin level, 12.0 g / dl; and platelet count, 276,000/mm . The levels of aspartate aminotransferase (12 iu / l), alanine transaminase (10 iu / l), alkaline phosphatase (82 iu / l), and total bilirubin (1.2 mg / dl) were within the reference range . Colonoscopy revealed a hemispheric lesion similar to a submucosal tumor in the descending colon, approximately 30 cm above the anal verge (fig . The larger lesion was located in the peritoneum, adjacent to the anterior portion of the descending colon, and the smaller lesion was located in the retroperitoneum, adjacent to the posterior portion of the descending colon . Pet - ct showed a large omental mass on the left side of the abdomen, with a small solid area of increased glucose metabolism and hypermetabolism (maximum standardized uptake value of 13.7), in the posteromedial portion of the mid - descending colon (fig . 4). A laparoscopic tumorectomy and segmental resection of the descending colon were performed . Examination of the gross specimen revealed a firm white mass approximately 10 9 cm and a 2 2 cm mass invading the descending colon (fig . The number of mitoses was between 10 and 19 in each of 10 high - power fields; hence, the histologic grade was high . The microscopic examination (20 magnification) of a section stained with hematoxylin and eosin revealed a solid tumor mass, well demarcated by a capsule with a peripheral zone . Fascicles of spindle cells with elongated and blunt - ended nuclei were visible at 100 magnification (fig . The patient then received two cycles of mesna, adriamycin, ifosfamide, and dacarbazine (maid) chemotherapy at the oncology department . Leiomyosarcomas are cancers of the smooth muscle cells, which can arise at any location . The uterus, the retroperitoneum, and the intra - abdominal cavity are common primary sites . This tumor is most commonly seen in the fifth and the sixth decades of life, and the male - to - female ratio is 1:2 . High - grade leiomyosarcomas frequently metastasize, and the most commonly affected organ is the lung . Prognosis is poor, and the five - year survival rate is 29 - 36% . In most reported cases, presentations are nonspecific and the results of physical examinations are normal . Radiologic features are nonspecific; therefore, it is difficult to diagnose this cancer preoperatively . Four years earlier, our patient had also been diagnosed preoperatively as having a renal cell carcinoma based on radiologic examination, the physical examination findings having been nonspecific . Leiomyosarcomas are distinguished from leiomyomas by cellular pleomorphism, an increased mitotic rate, and the presence of cellular necrosis . Furthermore, in this case, our pathologist conducted immunohistochemical staining, which is useful in the diagnosis of leiomyosarcoma . Immunohistochemical staining revealed positive findings for vimentin and smooth muscle actin and desmin . If a leiomyosarcoma specimen is found to have a pleomorphic component on immunohistochemical staining, it is difficult to distinguish it from a malignant fibrous histiocytoma . However, such heterologous differentiation was not observed in this case [6, 7]. A good prognosis can be expected only with complete surgical excision (the treatment of choice) [8 - 10]. Irradiation therapy and chemotherapy do not alter the course of the disease and are not successful in metastatic lesions . A tumor size of less than 5 cm, low histological grade, absence of lymph node metastasis, and radical excision of the mass are all associated with an improved prognosis . To have ensured a better prognosis in this particular case, a radical nephrectomy should have been performed as the first operation . Unfortunately, as the patient had been thought to have a renal cell carcinoma in the preoperative state, she instead underwent a simple left nephrectomy . A submucosal tumor is defined as an elevated or polypoid mass covered with normal mucosa . Several colorectal diseases, such as lipoma, leiomyosarcoma, lymphangioma, and carcinoid tumor, can be detected as lesions mimicking submucosal tumors . We also confirmed the lesion in the descending colon as being caused by extrinsic compression by performing a ct scan . As observed in this case, recurrence of a leiomyosarcoma in the retroperitoneum needs to be considered in the differential diagnosis of submucosal tumors in the colon . Thus, we report this unusual, but interesting, case of recurrence of a rare renal leiomyosarcoma.
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Endometrial carcinoma is the most common gynecologic malignancy, with an estimated 40 100 incident cases and 7470 deaths in 2009 . The current standard of care for patients with endometrial cancer consists of surgical staging, including total hysterectomy, bilateral salpingo - oophorectomy, and pelvic and para - aortic lymph node dissection . Depending on the surgical stage and histological grade, the adjuvant treatment may include radiation therapy and/or chemotherapy . In approximately 10% of patients, however, there is a relative lack of consensus regarding the most appropriate management strategy for these patients . Patients with clinically occult stage ii disease usually have undergone simple hysterectomy rather than radical hysterectomy, which is then followed by adjuvant radiation therapy . Patients with clinical stage ii disease may be treated with preoperative (neoadjuvant) radiation therapy followed by simple hysterectomy, or alternatively, primary radical hysterectomy and possible adjuvant tumor - directed radiation therapy . It is especially advocated for patients having co - morbidities that limit their candidacy for radical surgery, including obesity . In all patients with clinical stage ii disease, neoadjuvant radiotherapy can decrease the size and extent of bulky cervical disease, making it easier to obtain clear surgical margins . Further, it avoids the increased morbidity associated with lymphadenectomy followed by radiation therapy, as is seen in cervical cancer . Conversely, advocates of primary treatment by radical hysterectomy cite the ability to debulk large tumor prior to attempting radiation therapy, to clarify the origin of the primary tumor as cervical or endometrial, and to clarify the stage and grade of disease prior to initiating adjuvant therapy . Most of the published literature on preoperative treatment is with low dose rate (ldr) brachytherapy [912]. Due to the relative rarity of this clinical scenario, there is no published data on dose, fractionation or pathological response to high dose rate (hdr) intracavitary brachytherapy in the neoadjuvant setting . The goal of this study was to evaluate pathological response, tolerance, and outcome with this latter approach . Twelve women clinically diagnosed with clinical stage ii endometrial carcinoma from 1999 - 2010 were treated with preoperative radiation therapy . All patients had biopsy - confirmed endometrial cancer and had pretreatment ct scan and/or mri for staging . The dose of external beam (ebrt) was 45 - 50.4 gy in 25 to 28 fractions followed by hdr brachytherapy . The hdr brachytherapy was done with placement of a smit sleeve and tandem and ring applicator . Prior to 2004, three patients received a dose of 4 gy in four fractions, but since that time all patients received doses between 5 - 5.5 gy in three fractions . Initially, for the first six patients, the dose was prescribed to point a with orthogonal film based dosimetry . The last six patients were treated with mri or ct based planning with the clinical target volume being the entire uterus and cervix (fig . 1). Demographics, tumor histology, and pathological response to neoadjuvant hdr brachytherapy prior to simple hysterectomy in twelve patients with clinical stage ii endometrial carcinoma histological and figo grading from endometrial biopsy, not surgical specimen eqd2 equivalent dose in 2-gray increments, pr partial response, cr complete response microscopic residual disease, ned no evidence of disease, awd alive with disease sagittal (left) and coronal (right) view showing clinical target volume (red) covered by prescription isodose line of 100% (red) simple hysterectomy with lymph node sampling was performed four to eight weeks after the last brachytherapy treatment . The total doses from ebrt and brachytherapy were summated and normalized to a biologically equivalent dose of two gy per fraction (eqd2) using the linear quadratic model and 10 gy . All patients were followed every three months for the first two years, and every six months thereafter, with vaginal cytology at each surveillance visit . The median age was 57 (range 41 - 76) with median body mass index (bmi) of 35 (range 29 - 45). All patients completed the planned course of preoperative radiation therapy with a median eqd2 dose of 65.0 gy (range 56.3 - 75.4 gy). All patients had simple hysterectomy with lymph node sampling with uneventful recovery from the operation . Complete pathological response (pcr) was seen in two patients (17%) while five patients (42%) had only microscopic residual disease confined to either the endometrium or cervical glands . Two patients were found to have para - aortic disease and received adjuvant para - aortic radiation . Two patients with high grade pathology (one each with serous and clear cell histology) also received adjuvant chemotherapy after surgery . At a median follow up of 37 months (1 - 91 months), one patient has developed recurrence at the vaginal apex six months after completing initial therapy and is alive with the disease at 91 months, while another developed a lung recurrence at 28 months . One patient has died of comorbidities at 86 months with no evidence of the disease at last follow up . Two - year actuarial disease - free and cause - specific survivals were 88% and 100%, respectively; these were calculated for only those patients with at least two years of follow - up . The goals of this neoadjuvant therapy are to shrink the primary disease, to treat pelvic nodes and parametria, and to make the patient more suitable for simple hysterectomy . There is no published experience with hdr intracavitary brachytherapy as preoperative treatment for endometrial cancer . The american brachytherapy society in its published guidelines for hdr brachytherapy for endometrial cancer have not recommended a dose or fractionation schedule for preoperative hdr brachytherapy for stage ii endometrial cancer, likely due to the lack of published data . Multiple studies have shown equivalency of hdr and ldr brachytherapy for cervical cancer [14, 15]. The advantages of hdr include short treatment time, ability for optimization, and a lack of radiation exposure to healthcare personnel . Since 2004, our institution has followed a fractionation schedule of three fractions of 5 - 5.5 gy to deliver a dose equivalent to 65 - 70 gy . The schedule has been well tolerated, has shown a good response rate, and has not revealed significant morbidity . More recently, we have integrated three - dimensional planning for hdr brachytherapy, which helps to conform better the dose to the tumor while reducing the dose to critical organs (fig . 1). The results of this small series are comparable to published studies using ldr brachytherapy . In a series of 74 patients with stage ii endometrial cancer treated at the university of kentucky (uk) with a combined dose of 65 gy, the complete pathological response rate was seen in 31% and the residual disease confined to endometrium in 29% [10, 11]. In our study, which had a median eqd2 dose of 66.31 gy, pcr was seen in 22%, with superficial residual disease confined to the endometrium in 33% . In the uk series, para - aortic nodal disease was noted at the time of surgery in five patients, compared to two patients in our series . Patients with cervical involvement are at increased risk of both pelvic and para - aortic nodal involvement; since only the pelvic nodes are treated during the whole pelvic radiation therapy given preoperatively, para - aortic nodal sampling at the time of surgery should be considered . The optimal management for clinical stage ii disease that provides a better outcome, in terms of disease control and morbidity, it is difficult to study prospectively because of the comparative rarity of this clinical scenario . In a retrospective analysis of 184 consecutive patients with clinical or pathologic stage ii endometrial cancer treated with definitive intent with either preoperative or postoperative radiation therapy, only the grade and histology were found to be independent predictors of disease free survival . Since a subset of these patients are not ideal candidates for radical surgery because of medical co - morbidities, advanced age, and obesity, the preoperative radiation therapy followed by simple hysterectomy may be a better option . Ours is the first published series using hdr and shows the hdr fractionation schedule, as done in our series, is well tolerated and would be an option for patients treated with neoadjuvant radiation therapy.
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Ernst fuchs was the first in 1906 to report both the clinical and pathologic features of a consistent number of patients with a chronic low grade anterior chamber inflammation, heterochromia, and cataract . Nowadays fhi is one of the most common forms of anterior uveitis, accounting for up to 8% of endogenous uveitis seen in referral center [24]. Usually unilateral, it is characterized by the presence of diffusely distributed small, white, stellate, or rounded keratic precipitates, low grade inflammation in anterior chamber, absence of posterior synechiae, diffuse iris stroma atrophy with or without heterochromia, and variable vitreous inflammation [1, 411]. It is not associated with systemic diseases and it is not or poorly responding to corticosteroid therapy . Fhi affects both sexes equally, and the prognosis is usually good . The onset is between 29 and 44 years of age [4, 7, 911, 1317]. Iris atrophy and heterochromia are its most peculiar findings; although not pathognomonic, they are due to atrophy and depigmentation of all iris layers (anterior border, stroma, and pigmented epithelium). Usually the lighter eye is affected, although bilateral cases have been described as well as inverse heterochromia . The natural history of the disease is characterized by a slow progression over time, without substantial reduction of visual acuity until the development of significant vitreous opacities or cataract . A late or wrong diagnosis may lead to severe ocular complications, mainly related to a useless long - term corticosteroid therapy . The most frequent complications of fhi are cataract (70% of patients) and glaucoma (25%) [6, 7]. Glaucoma is often difficult to manage with medical therapy and may lead to a surgical intervention not always associated with good results . The visual prognosis of patients who undergo cataract surgery with intraocular lens implantation is usually good, with complete visual function recovery [1820]. The purpose of the present study was to evaluate the clinical and epidemiological findings, the prevalence and incidence of complications, and the long - term visual prognosis of italian patients with fhi . A retrospective analysis of the clinical charts of patients referred to the ocular immunovirology service of sapienza university of rome, rome, italy, from january 2003 to december 2012 was performed . Inclusion criteria was a diagnosis of fhi, which has been made upon several of the following clinical features: (i) small to medium - sized keratic precipitates involving the whole endothelial surface; (ii) a chronic inflammation in anterior chamber, usually 2 + according to sun criteria; (iii) diffuse iris stromal atrophy with or without heterochromia; (iv) lack of posterior synechiae unless there was a history of ocular surgery; (v) absence of snowbanks or choroidal / retinal infiltrates despite the presence of vitreous cells . One - hundred and fifty - eight patients, 80 females (50.6%) and 78 males (49.4%), fulfilled the criteria and were included in the study . After recording a detailed ocular and medical history, each patient underwent a full ophthalmological examination including best - corrected visual acuity (bcva), slit - lamp biomicroscopy, bilateral indirect ophthalmoscopy and biomicroscopy of the fundus, and goldmann applanation tonometry . In some unclear cases, in order to exclude other forms of uveitis, we performed additional laboratory examinations, including a complete blood cell count, erythrocyte sedimentation rate, tuberculin skin tests or quantiferon - tb gold, syphilis serology, serum biochemical analysis, chest x - ray, and angiotensin converting enzyme determination . For all the patients, we have recorded the following: age at uveitis diagnosis, age at fhi diagnosis, age at presentation at referral center, unilateral or bilateral involvement, clinical features at presentation, clinical course, forms of treatment, ocular complications at our first examination or occurring during the follow - up (when available), and visual prognosis . Thirty - five patients were observed only once, while 123 subjects (124 eyes) were followed up for more than 3 months (mean follow - up: 30.74 26.9 months; range: 3119 months). Ocular complications and the need for medical and surgical treatment were evaluated on those last patients . A further evaluation to assess the long - term visual prognosis was done on 50 patients who had a follow - up longer than 36 months (mean: 63.47 20.38 months; range: 36119 months). The statistical analysis was performed using the student's t - test and the test . The average age at uveitis diagnosis was 27.19 10.61 years (range 761 years), while that of fhi was 29.22 11.31 years (range: 861 years). The average age at our first examination was 32.69 10.97 years (range: 864 years). The mean interval from uveitis diagnosis to examination in our referral center was 2.12 1.7 years . There was no significant difference between the average age at uveitis onset between males (27.74 10.54 years, range: 761 years) and females (26.66 10.73 years, range: 850 years) (t = 0.64; p = 0.52). In 29 patients (18.35%) fhi was diagnosed in children (before 16 years of age) and in 1 of those before 7 (0.6% of all the patients, 3.4% of the pediatric ones). Fhi was diagnosed after 60 years of age in one patient only, during a routine eye examination . Fifty - five patients (34.8%) had a positive history of rubella, while seventy - three patients (46.2%) had no history of rubella and were not investigated serologically . Thirty patients (18.98%) had no history of rubella and were serologically tested: all (100%) were positive for rubella igg antibody . Blurred vision and floaters were the most frequent presenting symptoms (86 patients, 54.5%, and 64 patients, 40.5%, resp . ). Other symptoms recalled by the patients included hyperemia (25 patients, 15.8% of the cases), photophobia (24 patients, 15.1%), and pain (21 patients, 13.2%). Thirty - six patients were asymptomatic (23%), and the diagnosis was made during a routine eye examination . The clinical findings of patients with fhi at our first observation are reported in table 2 . One hundred and thirty - six eyes (86%) showed a bcva 0.6, 16 (10.1%) between 0.2 and 0.5, and 6 eyes (3.8%) 0.1 . Glaucoma was the leading cause of a severe visual loss in 50% of those cases (3 eyes, 1.88% of the eyes), followed by cataract, severe vitreous opacities, and corneal scar in 1 eye each (0.6%). The most common clinical findings were small to medium - sized keratic precipitates (95.6% of the eyes). According to sun criteria no to moderate inflammation in anterior chamber (cells from 0 to 2 +) was observed in 158 eyes (99.37%), and 1 eye only presented 3 + cells (0.6%). Sixty eyes (37.7%) presented iris nodules: koeppe nodules in 48 cases (30.2%), busacca in 2 (1.2%), and both types in 10 eyes (6.3%). Iris stroma atrophy was found in 138 eyes (86.8%), but only 61 (38.3%) presented heterochromia . In one patient anisocoria was observed in 4 eyes (2.4%) and no patient had posterior synechiae . In 32 eyes (20.12%) we found an increased intraocular pressure (iop): 27 have been already diagnosed and treated, while 5 were unaware of the condition (3.78% of all the patients with a normal iop before our observation). Fourteen eyes (8.8%) had undergone cataract surgery with intraocular lens (iol) implantation elsewhere . Among the remaining 144 patients (145 eyes), 83 eyes presented a posterior subcapsular cataract (57.24%) and 4 eyes (2.75%) a white cataract . Vitreous opacities were found in 145 eyes (91.2%), where in 72 eyes (45.28%) they were 2 + . Optic disc changes were found in 27 eyes (17%): 15 eyes (9.4%) presented a cup / disk ratio> 0.6, 7 (4.4%) a pale optic disc, 2 (1.2%) both of these changes, and 3 (1.9%) an optic disc hyperemia . Serum antitoxoplasma igg antibody was positive in 2 of these patients (25% of those with retinal scars), but none of the patients had a history of symptomatic ocular or systemic toxoplasmosis . Epiretinal membranes were found in 3 eyes (1.9%), retinal tear in 1 eye (0.6%), and a retinal hole in another one (0.6%), respectively . Four patients with fhi (2.5%) presented a systemic disease prior to uveitis onset that might confuse the diagnosis, because of their possible association with uveitis: 2 (1.25%) were affected by psoriasis, 1 (0.6%) by celiac disease, and 1 by acute rheumatic disease . Two other patients (1.25%) developed a systemic disease potentially associated with uveitis during follow - up: one, serologically positive for rubella, developed an optic neuritis in fhi unaffected (and completely normal) eye, 19 years after fhi diagnosis, and was diagnosed as having multiple sclerosis on a nuclear magnetic resonance results and neurologic consultation; another patient developed hodgkin's lymphoma 2 years after fhi onset . Among 119 eyes which were followed up for a mean period of 30.74 26.92 months, 29 (24.36%) which at baseline have had no cataract or an initial subcapsular cataract without significant reduction of visual acuity presented a bcva reduction <5/10 because of cataract onset / progression . Iop increase developed in 15 of 91 eyes (16.48%), with an incidence of 0.06 eye / year . Epiretinal membranes were quite rare (2 eyes; incidence: 0.006 eye / year). Figures 1 and 2 show the kaplan - meier curve for assessing the risk of developing cataract and increased iop . The overall risk for a visual acuity reduction 0.4 was 0.001 eye / year . The only significant risk for a visual acuity reduction 4/10 was an iop increase at presentation (p = 0.02). During our follow - up, 54 eyes (43.5%) had no therapy . Twenty - nine eyes (23.38%) were given a course of systemic corticosteroid therapy: 12 eyes (9.75%) to try to lower vitreous opacities and 17 (13.8%) to prevent and control postoperative (cataract and glaucoma) inflammation . Sixty - five eyes (52.4%) received topical corticosteroid therapy and 40 eyes (32.25%) mydriatics, mostly immediately after a surgical procedure (25 cases of cataract, 11 cases of glaucoma). Forty - seven eyes (37.9%) received iop lowering drops . Comparing the therapeutic regimen given before and during our follow - up, we have observed a significant decrease in the need for medications: topical steroids from 82.2% to 52.4% of the eyes (= 23.76; p <0.001) and systemic steroids from 58.8% to 23.38% (= 30.79; p <0.001). These drugs were discontinued in all the cases during our follow - up (= 6.33; p = 0.012). In twenty - five patients (25 eyes) phacoemulsification and in the bag iol implantation was performed by us (17.4% of the phakic patients at our first examination). Bcva at the end of follow - up was 0.8 in all the cases (mean 0.88 0.04). Capsulotomy was performed in 13 patients (33.33% of all the patients who have undergone cataract surgery): in 7 out of 25 eyes operated by us (28%) and in 6 out of 14 eyes (42.85%) operated elsewhere (= 0.34, p = 0.55). Trabeculectomy was performed in 14 eyes (8.8% of all the patients; 33.3% of those with iop increase), in 11 cases (6.9%) by us . The mean bcva at the end of follow - up of eyes undergoing trabeculectomy was 0.61 0.42, in 3 eyes less than 0.5 and in 2 less than 0.1 . Both of these presented a very low preoperative visual acuity (light perceptions and 0.1, resp . ). In 50 patients (50 eyes), followed up for an average period of 63.47 20.38 months (range: 36119 months), we have observed a bcva at baseline 0.6 in 45 eyes (90%) and 0.1 in 3 eyes (6%). At the end of follow - up one eye (2%) presented a bcva 0.1, because of a corneal scar already present at the first examination, while all the others (98%) had a bcva 0.6 . At the final examination 2 eyes (4%) presented, compared to baseline, a bcva reduction of 0.3 (cataract) and five (10%) presented an increase of bcva of 0.3: in three cases after cataract surgery, in one after trabeculectomy, and in 1 because of vitreous opacities reduction after anti - inflammatory therapy . Fhi is a quite common type of uveitis and its diagnosis relies of clinical findings [412]. This is why diagnosis is often delayed, because general ophthalmologist might miss to analyze properly the specific clinical features such as small or medium - sized keratic precipitates distributed on the whole endothelial surface, iris atrophy or heterochromia, low or moderate chronic inflammation in anterior chamber, absence of synechiae, and variable vitreous involvement . In our series norrsell and sjdell reported a period of 3 years between the onset of the first ocular symptoms and the diagnosis, while fearnley and rosenthal reported a delay of 6.7 years [9, 10]. This finding could be easily explained by the absence of heterochromia, which has been considered in the past the hallmark sign of fhi . It is of note that only 38% of patients had heterochromia at our first observation, performed on average 5 years after symptoms onset . Described heterochromia in 39% of cases while older studies reported higher prevalence (7075%) [4, 911]. The lower prevalence of heterocromia found in tertiary referral center for uveitis might be due to the possibility that only the most difficult - to - diagnose cases are sent for referral, while the true prevalence of heterochromia in general fhi population might be higher . Iris atrophy is definitely more common than heterocromia in italy (87% of the eyes). Although two series from brazil and spain reported a low frequency of iris atrophy (18% and 14.8%) [8, 11], this finding can be found in 48 to 100% of patients from different countries [4, 6, 811, 14, 16]. Therefore iris atrophy should be considered a more appropriate clinical feature to be associated with fhi . The mean age of uveitis diagnosis was found to be 27 years, lower than reported by other authors (29.5 to 44.5 years) [4, 6, 811, 16], with no significant differences between genders, as already described [7, 9, 10]. Our study has confirmed that fhi can also appear before 16 years of age in almost 20% of the patients . Tappeiner reported on fhi onset in childhood in 8% of german patients, 26% of whom were before 7 years of age: this last finding seems to be more rare in italy (0.63% of the patients; 3.4% of the pediatric ones). Several theories have been proposed and, among the possible causes, some viral (rubella, cytomegalovirus) and parasitic (toxoplasma gondii) agents were suggested . Quentin and reiber detected the presence of anti - rubella antibodies, using the goldmann - witmer index, in 100% of fhi patients . Birnbaum et al . Demonstrated a significant decline in the prevalence of fhi after the introduction of a rubella vaccination program . In our series 34.8% of patients had a positive history of rubella infection and an additional 19%, who did not remember any rubella infection, tested positive serologically . Cytomegalovirus dna has been also found by pcr in aqueous humor of patients with a clinical diagnosis of fhi, both in asia and in europe . The association with toxoplasma gondii was also described, but it is controversial [5, 30, 31]. The presence of chorioretinal scars in patients with fhi has been reported for decades, from 0% in a large group of chinese patients to 28% in brazil, a country where toxoplasmosis is endemic . Only 25% of our patients with fhi and chorioretinal scars tested positive serologically for igg antitoxoplasma antibody: it seems therefore reasonable to agree with kreps et al . Who have reported that the findings of the last decade show that rubella virus is the major but likely not sole blurred vision and floaters were the most frequent presenting symptoms (54.5% and 40.5% resp ., in our patients), similar to turkish population, but significantly lower than in sweden (71%) and england (83.9%) [9, 10]. Although the classic description of fhi does not include a ciliary reaction, redness photophobia and pain have been recorded at the uveitis diagnosis in almost 15% of our cases . Twenty - three percent of italian patients with fhi was completely asymptomatic, and the diagnosis was made during a routine eye examination . A bilateral involvement was found in one of our patients (0.6%) only, while this frequency varies in the literature from 0% to 21% . Norrsell and sjdell have described a worse prognosis in bilateral cases, with more frequent cataract extraction and pars plana vitrectomy . Small to medium - sized keratic precipitates are typical of fhi [411] and have been found in 96% of our patients . At their first observation they were either equally diffuse on the whole endothelium (76.3%) or mainly located in the central and inferior part (23.7%) a recent study has shown a close association between endothelial precipitates and cmv - dna in the aqueous humor, suggesting that the endothelial cells may be the target of a viral infection . The presence of iris nodules (37.7% in our patients, mostly koeppe's one: 30.2%), with no posterior synechiae and with a low to moderate inflammation in anterior chamber (cells 2 + in 99.4% of the our cases), is similar to the clinical findings reported in literature and should strongly suggests the diagnosis of fhi . Vitreous inflammation frequency has been reported from 14.8 to 92.6% in different series [4, 6, 811, 14, 16, 34]. It was found by us in 91.2% of patients, 45% of whom showed vitreous opacities> 2 + . Again this frequency can be attributed to each author's affiliation, working with general population or in referral center for uveitis . Nevertheless it is important to stress that vitreous opacities, even 2 +, can be easily found in fhi, especially in long - standing cases, and might be a confounding finding leading to a wrong diagnosis of intermediate or posterior uveitis, especially when the iris atrophy and heterochromia are not clearly detectable . In such cases the presence of a normal foveal reflex and the absence of choroidal, retinal, or vascular lesions, of snowballs in the far periphery, are elements that should lead to diagnose fhi . Cataract becomes mature on average 8 years after first subcapsular opacities have been detected but can also develop very rapidly . In our study, a posterior subcapsular cataract was observed in 57.2% of phakic eyes at the first examination, while the overall prevalence was 63.5%, similar to turkish patients (69%). The highest frequencies of cataract in fhi were described by liesegang (90%) and tabbut et al . Visual prognosis in fhi after cataract surgery is better than that reported in other forms of uveitis [35, 36]. Fourteen patients before our observation and 25 patients during our follow - up (total: 39 patients, 24.7% of all the cases) underwent cataract surgery with iol implantation, confirming previous results with a final visual acuity 0.8 in all cases . The clinical characteristics of fhi such as low anterior chamber inflammation, absence of posterior synechiae and macular edema, and benign long - term prognosis make this uveitis an ideal candidate for iol implantation . In contrast, chronic inflammation in fhi might justify the high prevalence of posterior capsule opacification . In 33.3% of patients who underwent cataract surgery, this percentage was higher than that found in senile cataract (20 to 40%) [37, 38], but it seems possible to lower the opacification rate with a meticulous removal of all the cortex and probably a more aggressive treatment tailored on a precise knowledge of the preoperatively inflammatory status . In fact we have observed a lower prevalence of posterior capsule opacification after cataract extraction and in the bag iol implantation comparing our patients (28%) to those operated elsewhere (42.8%). The prevalence of an increased iop and/or secondary glaucoma in patients with fhi is variable, with literature data ranging between 6.3% and 59% of cases [4, 10]. The onset of iop increase is difficult to determine because it might remain unrecognized for a long period of time . In our patients the prevalence of increased iop was 20.1% and the incidence, during a mean follow - up of 30 months, was 0.06 eye / year . So at the end of our observation, the prevalence of iop alterations was 33.87% . La hey et al . And jones reported in fhi a failure of medical therapy in 37% and in 73% of the cases, respectively [12, 16]. According to fearnley and rosenthal, glaucoma surgical approach in fhi patients is useful in 47% of eyes, while liesegang performed glaucoma filtering surgery in 66% of their patients [9, 14]. In our study, 11 eyes during follow - up and 3 before our first observation underwent trabeculectomy (8.8% of all the patients, 33.3% of the patients with iop elevation) with an incidence of 0.03 eye / year . In these patients the mean postoperative visual acuity was 0.61 0.42, while the only 2 patients who had a final visual acuity equal to or less than 0.1 had a preoperative visual acuity of light perceptions and 0.1 . Our study has demonstrated that the incidence of visual decrease 0.4 is very low in medium term follow - up (0.001 per eye / year). Al - mansour et al . Reported a worsening of visual acuity only in 10% of the eyes compared to baseline, while most of the eyes had a visual acuity unchanged or improved . In our patients the risk of developing a reduction in visual acuity is significantly related to the presence of an increased iop or glaucoma at the first observation (odds ratio 10.11, p = 0.02), similar to al - mansour et al . Longer than 5 years we have observed a final visual acuity> 0.6 in 98% of the patients, with one patient only presenting a poor visual acuity related to a previous corneal scar . Fhi is often misdiagnosed and patients are treated with topical, systemic, and peribulbar steroids . Peribulbar or topical steroid treatment has a minimal effect on this inflammation and it might promote the onset of posterior subcapsular opacity and elevated iop . The referral of patients in tertiary eye care center can significantly lower the amount of corticosteroids therapy (p <0.001 for both topical and systemic route of administration) and of immunosuppressive drugs (p = 0.012). These might potentially reduce the incidence of cataract and glaucoma, which are typical side effects of such a therapy, and, more importantly, of immunosuppressives - related systemic side - effects . In conclusion in italian patients the average age at presentation is lower than in other countries, and 18% of patients are aged 16 or less . Therefore in children with unilateral, chronic anterior uveitis, it is essential to exclude fhi to avoid the incidence of therapy - related side - effects . The clinical findings of fhi do not differ from those reported in other countries, but particular attention should be paid for iris atrophy and vitreous opacities, which resulted in being more frequently encountered than heterochromia . Our study confirmed that fhi has a good long - term visual prognosis, despite the significant incidence and prevalence of cataract and glaucoma; the only significant risk factor for visual loss is iop increase . During cataract management it is important to remove all cortical residuals and follow adequately the patient in the postoperative period to reduce the incidence of posterior capsule opacification . A correct and prompt diagnosis allows to avoid unnecessary and potentially dangerous therapy . A proper uveitis, and its complications
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However, the iron overload in various organs leads to serious problems such as metabolic, endocrine, and growth abnormalities in such patients despite the expanded use of iron - chelator drugs (3 - 5). Patients with thalassemia intermedia (ti) although the rate of iron loading is slower in ti than in tm, patients with ti can eventually develop complications similar to those of the patients with tm, including hepatic, endocrine and cardiac dysfunction (6). Moreover, in patients with transfusion independent thalassemia, serum ferritin was a poor predictor of liver iron concentration; therefore, chelation therapy may be postponed in patients with ti (7). Hepcidin is a small peptid hormone secreted by hepatocytes to regulate plasma iron concentration and distribution in different tissues (8, 9). Chronic excess of hepcidin causes iron restricted anemia (9), while the hepcidin deficiency leads to iron overload with iron deposition in the liver and parenchyma (10). Increased plasma and stored iron stimulate hepcidin production, which in turn blocks dietary iron absorption and consequently iron loading .conversely, hepcidin is suppressed in iron deficiency (11). Hepcidin concentration is significantly higher in chronically transfused patients than in non - transfused ones, presumably due to iron overload (12 - 16). Despite using iron chelatorin patients with thalassemia, iron overload is one of the main mortality factors in such patients and hepcidin targeted therapeutics can help with better management of iron overload in patients with thalassemia (17). The current study aimed to determine the correlation between serum ferritin and hepcidin levels in patients with tm and ti, also compare serum hepcidin and ferritin levels between the two groups of patients . The current cross - sectional study was conducted in hematology research center of shiraz university of medical sciences, shiraz, iran, in 2013 . The study subjects included 90 patients, 50 tm and 40 ti, selected by systematic random sampling from the 500 tm and 280 ti patients registered at the thalassemia clinic of tertiary state hospital affiliated to shiraz university of medical sciences . This is a referral center with 45 beds for patients with thalassemia in southern iran . This study was approved by medical ethics committee of shiraz university of medical sciences (grant number: 3652, approval date: 21.03.2011). The diagnosis of thalassemia was based on complete blood count (cbc), hb electrophoresis and clinical history . In the tm group, blood transfusion had been started before two years old and all of them were on regular blood transfusion every - two to four weeks . In the tm group, inclusion criteria were age above 10 years old, under regular follow up and regular blood transfusion, and iron chelation therapy . In the ti group, inclusion criteria were transfusion independence and taking hydroxyurea (hu) 10 - 15 mg / kg / day for a duration of at least ten years . Patients with the history of any recent infection or surgery, congestive heart and liver failures as well as positive viral hepatitis were excluded from the study . A 2.5 ml sample of venous blood withdrawn from each participant was added to the tubes containing clot activator . Plasma centrifuge was done immediately and 2 ml serum was separated and preserved at -20c, then carried by cold box to the laboratory to check serum hepcidin and ferritin levels . Also, 2 ml sample of venous blood was withdrawn from each patient and added to the tubes of cbc containing ethylenediamine tetraacetic acid edta anti - coagulant . Complete blood count test was performed immediately with peripheral blood smears and then stained with wright stain and the rate of nucleated red blood cells (nrbc) per 100 white blood cells (wbc) was counted . Fasting serum ferritin was checked by enzyme linked fluorescent assay elfa method, using mini vidas (biomerievx s.a, france) machine, and hepcidin was measured by enzyme linked immunosorbent assay elisa method (competitive assay), using elisa reader dynex (usa) machine with drg hepcidin kit (eia 4 705). According to the direction of hepcidin kit manufacturer, the normal range of hepcidin in 5% - 95% of people with the age range of 23 to 67 years is from 13.3 to 54.4 ng / ml . The normal range of ferritin was considered as 20 - 300 g / lin males 10 - 100 g / l in females before menopause, 20 - 200 g / l in females after menopause and 150 - 500 test of normality was done by shapiro - wilk test . Among all the evaluated variables, only age had normal distribution . Descriptive data were summarized as mean and standard deviation, as well as median and interquartile range (iqr), where appropriate . Correlation between serum hepcidin and quantitative variables in each of the two groups of patients with tm and ti was evaluated by spearman correlation test . Quantitative data between the two groups of patients were compared by student t - test and mann - whitney test . Considering the correlation coefficient of 0.68 for urinary hepcidin and serum ferritin levels reported by origa et al . (14) in 44 patients with thalassemia including 22 tm and 22 ti, = 0.05 and = 0.01, the sample size was estimated as 29 patients in each group . For higher accuracy, sample size test of normality was done by shapiro - wilk test . Among all the evaluated variables, only age had normal distribution . Descriptive data were summarized as mean and standard deviation, as well as median and interquartile range (iqr), where appropriate . Correlation between serum hepcidin and quantitative variables in each of the two groups of patients with tm and ti was evaluated by spearman correlation test . Quantitative data between the two groups of patients were compared by student t - test and mann - whitney test . Considering the correlation coefficient of 0.68 for urinary hepcidin and serum ferritin levels reported by origa et al . (14) in 44 patients with thalassemia including 22 tm and 22 ti, = 0.05 and = 0.01, the sample size was estimated as 29 patients in each group . For higher accuracy, the mean age of the patients with tm and ti were 21.4 6.1(from 11 to 33 years) and 23.7 6.1 years (from 8 to 38 years old) respectively, and there was no statistically significant difference between the two groups (p = 0.076). Considering gender distributions, 21 (43.8%) and 22 (55%) of the subjects in the tm and ti groups were female, respectively; there was no statistically significant difference (p = 0.392). The median iqr for serum hepcidin levels were significantly higher in the tm compared to ti group (87.6, 43.9 vs. 51.8, 23.4, p <0.001). In addition, the median iqr for serum ferritin levels were 2208 (3761), and 465 (632) in the tm and ti groups respectively; the difference was statistically significant (p <0.001) (table 1). Table 2 indicates the results of correlation between serum hepcidin levels and the evaluated parameters for the subjects with tm and ti . No statistically significant correlations were found between serum hepcidin with any of the evaluated variables in the two groups . Also there were no statistically significant differences in serum hepcidin levels between males and females in patients with tm and ti (p = 0.107 and p = 0.861 respectively). Considering the cut - off point of 54.4 ng / ml for serum hepcidin levels, 44 (91.7%), and 18 (45%) subjects had hepcidin levels higher than normal range in the tm and ti groups, respectively (p <0.001). Abbreviations: iqr, interquartile range; sd, standard deviation; nrbc, nucleated red blood cells; wbc, white blood cell . Data are presented as mean sd, median (iqr) or male / female . Data are presented for n = 40 . Statistically significant . In thalassemia major, it was pre - transfusion hemoglobin abbreviations: nrbc, nucleated red blood cells; rs, spearman s rho correlation coefficient; ti, thalassemia intermedia; tm, thalassemia major; wbc, white blood cell . In thalassemia major, it was pre - transfusion hemoglobin . Determining hepcidin concentrations in subjects with iron - loading anemias may be useful to identify the patients at higher risk of iron toxicity due to severely decreased hepcidin levels (17). Based on the current study results, 91.7% of the subjects with tm had serum hepcidin levels above normal compared to the patients with ti, 45% . Also, the median of serum hepcidin levels were significantly higher in the tm compared to ti group that was similar to those of the previous studies (14). In iron loading anemias not treated by frequent transfusions, such as beta thalassemia intermedia and congenital dyserythropoietic anemias, urinary and serum hepcidin decrease due to high erythroid signals . Hepcidin deficiency in turn allows excessive iron absorption and development of systemic iron overload (12 - 14, 18). Also, it could be due to more iron overload resulted from regular blood transfusion in the patients with tm compared to ti . As observed in the current study, ferritin levels were significantly higher in the subjects with tm . Hemoglobin levels were significantly lower in the subjects with tm than in ti, which may be due to the time of blood sampling in the subjects with tm, immediately before transfusion . During transfusion intervals, it is known that in patients with ti, there is also increased risk of iron over load due to increased intestinal iron absorption triggered by chronic anemia, ineffective erythropoiesis and decreased serum hepcidin (19, 20). The current study showed higher erythropoietic activity in the subjects with ti than tm, since the median value for nrbc was significantly higher in ti compared to tm group . In ti group, since all subjects were on hu therapy for a long time, erythropoietic activity was expected to be suppressed and consequently hepcidin level was expected to be higher . But it did not occur and it seems that hu at a dose of 10 - 15 mg / kg / day was not enough to suppress bone marrow activity and make erythropoiesis ineffective . However, previous reports documented that hu with the same dose was effective in decrease or cessation of the need for regular blood transfusion, as well as increasing hb levels in the patients with thalassemia (21 - 24). The current study showed no significant correlation between serum hepcidin and ferritin levels as a marker of iron overload in any of the tm or ti groups . This result was in accordance with the previous reports that demonstrated the dominant role of erythropoiesis compared to iron overload in regulation of hepcidin in patients with thalassemia (25 - 27). (15), the current study found no statistically significant correlation between hepcidin and pre - transfusion hemoglobin levels in tm patients . In a longitudinal study on 31 patients with tm . The strong point of the current study was large sample size compared to those of the previous reports; however, there were some limitations in the study, since molecular erythropoietic activity was nit evaluated . In conclusion, based on the current study results, there was insignificant correlation between serum hepcidin and ferritin levels as a marker of iron overload in any of the two groups of patients with tm and ti, which supported the results of previous studies that regulation of hepcidin in patients with thalassemia is more affected by erythropoeitic activity than by iron overload . Also serum hepcidin levels were significantly higher in the tm than in ti group, which could mainly result from higher erythropoeitic activity in ti . In the subjects with ti, it is believed that low dose hu (10 - 15 mg / kg / day) therapy for a long time could increase hb levels and lead to transfusion - independence, but probably not high enough to suppress bone marrow activity and make erythropoiesisin effective . Increased awareness of hepcidin levels in the patients with thalassemia could be helpful to diagnose and manage iron burden in such patients.
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Acuaria hamulosa), is a species of acuariidae family and etiological agent of cheilospirurosis in birds such as chickens, turkeys, pigeons, and guinea fowls [1, 2]. Cheilospirura hamulosa has cylindrical body with two triangular lips and 4 cuticular cordons that extend near posterior extremity . The c. hamulosa is located under gizzard cuticle mainly in koilin or muscular wall of the host [1, 2]. This nematode has indirect life cycle . The grasshoppers (melanoplus, oxyanitidula, and spathosternum parasinifrum), beetles, and weevils are intermediate hosts for c. hamulosa and birds acquire their infections by eating contaminated arthropods containing infective third - stage larvae . Due to its high pathogenicity in poultry, the c. hamulosa has veterinary and public health importance . Cheilospirura hamulosa can cause several complications such as granulomas and nodules that lead to anemia, impotence, and mortality in chickens . Cheilospirura hamulosa may cause zoonosis and has been recovered from a nodule on the conjunctiva of a filipino farmer . The previous studies in rural areas of iran indicated that the infection of this worm is common among free - range chickens, whereas there is no published data on partridges in iran . The main goal of the present study was to survey prevalence of cheilospirura hamulosa infection in partridges (alectoris chukar) using morphological - based methods, in taleqan county of iran . This area is located in alborz province and its height is 1900 m above the sea level . Latitude and longitude of taleqan are 36 degrees 15n and 50 degrees 46e, respectively . Every year from the end of september to the beginning of february a license is issued by the environment protection agency (epa) of iran for hunting partridges . Every hunter has permission to hunt the maximum of three partridges weekly . For this research, the gizzard of each partridge was removed from the alimentary tract and delivered to the helminthology laboratory of shahid beheshti university of medical sciences . The gizzards were examined macroscopically and then dissected in a 0.85% nacl solution (normal saline) to remove cuticle . The worms were rinsed in normal saline and fixed in alcohol - glycerin (70% alcohol, 50 ml; glycerin, 50 ml) solution . Helminthes were counted and identified under light microscopy (zeiss, germany) and stereoscopic microscope (zeiss, germany) and traced by camera lucida (zeiss, germany). The morphological identification of the nematodes to the species level was done according to methods described by skrjabin et al . . Out of a total of 100 partridges examined, the prevalence of c. hamulosa was 30% with a mean intensity of 3.9% and range of infection of 112 . A total of 116 worms were recovered from partridges . Among the recovered helminthes, 60.3% were female and 39.7% male . The specimens of c. hamulosa were found free under the gizzard cuticle, partially or fully burrowed in the walls (figure 1). Microscopic description was based on 10 adult worms, five males and five females (table 1). The average length of adult males was 12.2 0.67 mm and their average width was 0.3 0.06 . The mean of cordons length was 9.2 0.28, ratio between cordons and body length was 1: 1.33 (figure 2, (a1)(a3)), long spicule slender was 1.44 0.08 mm in length, short spicule shaped like a chopping knife was 0.62 0.11 mm long, and ratio between long and short spicules was 1: 2.3 . The ratio between mean of long and short spicules length and body length were 0.11 mm and 0.04 mm, respectively . The mean length of caudal alae was 0.42 0.02 and its width was 0.32 0.02, with tail 0.43 mm long (n = 1). In each worm there were ten pairs of caudal papillae: three couples were observed in precloacal, two pairs in adcloacal, and five couples in postcloacal (figure 2, (b1)(b3)). The mean length and width of females were 17.5 2.14 and 0.39 0.04 mm, respectively . The mean of cordon length was 12.96 0.72, and ratio between cordons and body length was 1: 1.68 . The females were amphidelphic, and their vulva is located slightly posterior to the middle of the body at 2.29 0.92 from the posterior end, circular sphincter 0.064 0.065, tail 0.44 (n = 1) long (figure 3, (a1)-(a2) and (b1)-(b2)). Embryonated eggs were 0.0450.055 (0.051) long and 0.0280.03 (0.029) wide (figure 4). The nematode c. hamulosa reported herein is reported for the first time from partridges in iran . The partridge (alectoris chukar) is the most important bird hunted in iran . In the present study, prevalence of c. hamulosa in the partridge was 30% with a mean intensity of 3.9% and range of infection of 112 . The reported prevalence of this nematode by menezes et al . In brazil was 14.3% in ring - necked pheasants (phasianus colchicus) with a mean intensity and range of infection of 1.5, 1 - 2, respectively . In domestic chickens (gallus g. domesticus), the prevalence, mean intensity, and range of infection were 26.7%, 4 and 112, respectively . A 2-year study in kashmir, india, on the prevalence of the nematode c. hamulosa in indigenous fowl the prevalence of c. hamulosa in guinea fowls (numida meleagris galeata pallas) from ghana and chickens in zimbabwe and cuba was 37.8%, 46.6%, and 84.6%, respectively [1113]. The reported prevalence of c. hamulosa on native fowls from golestan province in north of iran was 4% which is significantly lower than our result, because the study population is completely different from our study . The nematodes of the genus acuaria (cheilospirura) are parasitic among different families of birds and they are located under the koilin layer usually in the cardiac or pyloric regions . Also in our study the specimens of c. hamulosa were found under the gizzard cuticle, partially or fully burrowed in the walls of the organ (figure 1). Most of the reported lengths for male and female c. hamulosa are within the range of 914 and 1525, respectively ([3, 14] and [1, 7, 8]) (table 1). In this study, male worms were smaller than the female worms in average body length, overall cordon, glandular oesophagus, and muscular oesophagus length (table 1). Moreover, in our study, the long spicules were smaller and the length of short spicules was longer compared with previous studies reported by cram [1, 3], yamaguti, and gomes et al . Average length of male worms was higher than those reported by gomes et al . . In our study, the female worms were smaller in maximum body length than those described by cram and yamaguti [3, 7] but still were bigger than those described by gomes et al . . The female of c. hamulosa in our study had smaller muscular and glandular oesophagus than those reported by gomes and yamaguti [7, 8], whereas values obtained for the length of tail and egg size were bigger compared to two studies mentioned above (table 1). We observed 10 pairs of papillae in c. hamulosa as reported by cram and gomes et al . ; however, in the precloacal region, 2 pairs of papillae were unclear (figure 2, (b1)(b3)). Due to the significant pathogenic effects of these nematodes (cheilospirura spp .) In poultry and wild bird population and very limited prevalence data of these helminthes in iran, further study will be needed on different aspects of acuarioidea family including pathogenesis and their prevalence in other avian species . In the present study, we report for the first time the isolation and morphological characterization of cheilospirura hamulosa from partridges in iran . The morphological characters described in this study
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Newborn infants who sustain an acute intrapartum hypoxic ischemic insult of sufficient magnitude to result in long - term neurologic sequelae invariably have recognizable clinical encephalopathy during the first days of life . It is important to identify infants at high risk for hypoxic ischemic encephalopathy in order to provide them proper treatment soon after birth [3, 4]. However, most newborns with perinatal asphyxia have unpredictable course; development of hypoxic ischemic encephalopathy and neurodevelopmental outcome cannot be reliably predicted[1, 2, 4]. Measurements of hydroxybutyrate dehydrogenase, brain - specific creatine kinase, neuron- specific enolase, lactate dehydrogenase, and interleukin-6, in serum or cerebrospinal fluid may have some value as markers of hypoxic severe tissue hypoxia causes the accumulation of intermediary metabolites excreted by the kidneys, notably lactate[7, 8], which can be measured readily by proton nuclear magnetic resonance (1h nmr) spectroscopy[9, 10]. It was previously reported that increases in urinary lactate excretion could be detected by 1h nmr spectroscopy in newborn infants with perinatal complications . In the present study, we measured urinary lactate and creatinine concentrations within the first 6 and 24 hours after birth and determined sensitivity and specificity of the ratio of urinary lactate to creatinine for the early identification of infants in whom hypoxic ischemic encephalopathy is likely to develop . We studied 50 consecutive newborn infants with perinatal asphyxia who were born in our hospitals between october 2006 and june 2007 . Inclusion criteria consisted of gestational age at least 36 weeks and over 2 kg birth weight with perinatal asphyxia and some degree of hypoxic ischemic encephalopathy . Perinatal asphyxia was defined as the presence of at least three of the following conditions: intrapartum distress as indicated by fetal bradycardia with a heart rate of less than 100 beats per minute, late decelerations, or absence of heart - rate variability, thick meconium - stained amniotic fluid, apgar score of 6 or less at five minutes; need for resuscitation for more than one minute with positive - pressure ventilation and oxygen immediately after birth and arterial blood ph value of 7.20 or less or a base deficit of at least 14 mmol per liter within the first hour after birth . Ischemic encephalopathy was classified as mild, moderate, or severe on the basis of the staging system described by santa and sarnat . This system assesses the infant's level of consciousness, muscle tone, cranial nerves, primitive reflexes, spontaneous motor activity, autonomic function and seizures . Ischemic encephalopathy was classified as mild if hyperexcitability or hypotonia persisted without seizures for at least 24 hours after birth; as moderate if the infant was lethargic and had hypotonia, weak primitive reflexes, and seizures; and as severe if the infant had frequent seizures, apnea, flaccid weakness, or coma . The exclusion criteria were maternal drug addiction, congenital infections, or perinatal infections, including chorioamnionitis and more than 42 weeks gestation and development of acute renal failure . The control group consisted of 50 normal, full - term newborns who met the following criteria: no maternal illness, normal results of fetal monitoring, apgar score of at least 8 at one and five minutes, and a normal course during the first week of life . The infants in both groups were examined daily during the first week after birth by a single examiner who did not know the results of the urinary testing . The study was approved by ethics committee of kurdistan university of medical sciences and written informed consent was obtained from the parents of the infants . Urinary 1h nmr spectroscopy: spot urine samples were collected within 6 hours and again 24 hours after birth and were immediately centrifuged . The supernatants were stored at 80c for later assay . The urine samples were prepared for analysis by adding 0.05 ml of deuterium oxide to 0.45 ml of urine contained in a 5-mm nmr tube . The methyl proton signal of creatinine with a chemical shift set at 3.06 ppm was selected as an internal standard, and the resonance was assigned for lactate and other metabolites . The peak heights for lactate (1.34 ppm) and creatinine (3.06 ppm) were determined, and the ratio of lactate to creatinine was calculated . For statistical analysis, birth weight, gestational age, and sex were similar among the normal infants, and the infants with asphyxia that developed hypoxic ischemic encephalopathy (table 1). All infants with asphyxia developed hypoxic ischemic encephalopathy . The disease was judged to be mild in 17, moderate in 21, and severe in 12 patients . Characteristics of 50 normal newborn infants and 50 infants with asphyxia and hypoxic ischemic encephalopathy p values are for comparisons between the infants with hypoxic ischemic encephalopathy and normal infants levels of arterial blood gas were measured within the first hour after birth l / c: lactate/ creatinine ratio ratio of lactate to creatinine in urine: the mean ratio of l / c in urine within six hours after birth was 3.32 in the infants who subsequently developed hypoxic ischemic encephalopathy- a value that was 11 times as high as that in normal infants (0.30.08, p<0.0001) (fig . Urinary l / c ratio of 0.48 or higher had 96.1 percent sensitivity and 100 percent specificity in predicting the development of hypoxic ischemic encephalopathy . Among the infants who developed hypoxic ischemic encephalopathy, there was a significant trend for the ratio to increase with the severity of the hypoxic ischemic encephalopathy in six hours: 2.20.5 in the infants with mild encephalopathy, 4.21.5 in those with moderate encephalopathy, and 3.43.3 in those with severe encephalopathy . L / c ratio within six hours after birth in a normal infant, and an infant with asphyxia and hypoxic ischemic encephalopathy the mean ratio of l / c in urine within 24 hours after birth was 1.50.55 in the infants who subsequently developed hypoxic ischemic encephalopathy - a value that was 5 times as high as the ratio in normal infants (0.30.08, p<0.0001). Urinary l / c ratio of 0.48 or higher had 98 percent sensitivity and 100 percent specificity in predicting the development of hypoxic ischemic encephalopathy . There was a significant trend for the ratio to increase with the severity of the hypoxic ischemic encephalopathy in 24 hours: 1.10.5 in the infants with mild encephalopathy, 1.51.5 in those with moderate encephalopathy, and 23.3 in those with severe encephalopathy . Sensitivity and specificity of the urinary l / c ratio at 6 hours after birth was 100 and 96 subsequently, and at 24 hours after birth sensitivity was 100 and specificity 98 (table 2). Sensitivity and specifity of urinary lactate / creatinine ratio at 6 hr and24 hr after birth ppv: positive predictive value npv: negative predictive value in our study, the newborn infants with asphyxia were examined before hypoxic ischemic encephalopathy developed . We selected only infants with asphyxia and hypoxic ischemic encephalopathy . Within six hours after birth, urinary l / c ratios were much higher in the infants that developed hypoxic ischemic encephalopathy . These results suggest that the urinary l / c ratio within six hours after birth is related to the occurrence and degree of hypoxic ischemic encephalopathy . Chao - chang found the same result; it is to be noted that in our study number of infants with asphyxia and hypoxic ischemic encephalopathy was almost four times more than in their study . This suggests that the biochemical derangement detected in the urine after perinatal asphyxia is more pronounced within a few hours after birth than later . The study of chao - chang showed in infants with asphyxia the urinary l / c ratio within the first six hours after birth was also significantly related to the neurodevelopmental outcome at one year of age . Kant showed that urinary l / c ratios are much higher in babies with thin meconium, and meconium staining was indicator of perinatal asphyxia of newborns . Our study shows that urinary l / c ratio, determined by 1h nmr within six hours after birth in infants with perinatal asphyxia, can be used to identify most of the infants who will develop hypoxic the salient abnormality in our study was a marked increase in the urinary l / c ratio within six hours after birth in newborn infants with asphyxia and hypoxic ischemic encephalopathy . Urinary lactate may result from systemic tissue hypoxia, skeletal - muscle ischemia, or renal injury during asphyxia[2, 8 13]. In our study, conventional indicators (apgar scores, arterial - blood ph, and base deficits) could not be used to predict the development of hypoxic ischemic encephalopathy, although a multivariate model that incorporated a combination of these markers was somewhat predictive in other studies[1, 14 15]. Most studies of perinatal asphyxia have measured biologic markers (brain - specific creatine kinase, hypoxanthine, erythropoietin, and lactate dehydrogenase) in serum or cerebrospinal fluid, but the tests are usually performed several days after birth, when the infants may already have hypoxic these tests may be useful as markers of tissue injury, but they offer little information that can be used to identify newborn infants at high risk for hypoxic ischemic encephalopathy . Ischemic encephalopathy that may cause high urinary lactate excretion in newborns are acquired diseases (eg, necrotizing enterocolitis) and congenital metabolic disorders (eg, pyruvate dehydrogenasedeficiency, glucose-6-phosphatase deficient glycogenosis, pyruvate decarboxylase deficiency, propionyl coenzyme a carboxylase deficiency, and methylmalonic - aciduria)[18, 19]. Ischemic brain injury in newborn infants, most of these conditions are readily distinguishable from asphyxia . In addition, 1h nmr can also be used to detect these metabolic disorders . As a limitation of the study we could not follow patients after discharge from nicu and compare growth and development in the two groups for determination relationship between urinary l / c ratio and their growth and development . We suggest further study to compare relationship between urinary l / c ratio at birth and later growth and development . Our study showed that the urinary lactate / creatinine ratio in newborn infants with asphyxia is useful for predicting the development of hypoxic ischemic encephalopathy . The l / c ratio may therefore be useful in identifying infants most likely to benefit from intervention.
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Diabetic nephropathy (dn) is a common complication of type 2 diabetes mellitus (dm) and is the leading cause of end - stage renal disease (esrd). Microalbuminuria is an early sign of dn, which correlates with and can predict the progression of renal damage and cardiovascular morbidity [15]. The discovery of biomarkers for the earlier stages of dn would enable early intervention to reduce the impact of this chronic vascular complication . Diabetic nephropathy is associated with altered vascular structure, endothelial dysfunction, and disrupted homeostasis and angiogenesis [4, 6, 7]. Abnormal glomerular angiogenesis in patients with dn is associated with glomerular hypertrophy and results in glomerular capillary injury and urinary albumin excretion [8, 9]. Thus, the mechanisms for the development of abnormal angiogenesis in dn involve a complicated interplay between pro- and antiangiogenic factors . Two families of growth factors, angiopoietin / tie-2 and vascular endothelial growth factor (vegf)/vegf receptor (vegfr), are thought to be associated with the development of dn . Vegf increases vascular permeability and is mitogenic for endothelial cells, acting early and at most points of the angiogenic cascade . Within the angiopoietin family, angiopoietin-1 (ang-1) and angiopoietin-2 (ang-2) are the best - studied ligands for tie-2 receptors . Ang-1 signaling via tie-2 is involved in capillary sprouting, endothelial cell survival, and vascular remodeling . Several studies suggest that ang-2 signaling, in combination with vegf, leads to sprouting angiogenesis, while ang-2 signaling in the absence of vegf causes vessels to regress . Hence, selective upregulation of vegf and ang-2 may lead to aberrant proliferation of leaky, friable vessels . Emerging evidence suggests that vegf and angiopoietin are critical in glomerular physiology and in the pathogenesis of glomerular disease in dm [1417]. Previous studies have reported upregulated plasma levels of vegf and ang-2 in human and animal dn [10, 16, 1820]. Notably, circulating ang-2 levels associated with albuminuria have been reported in chronic kidney disease (ckd) and systemic lupus erythematosus (sle). Transgenic mice with inducible overexpression of ang-2 in podocytes have been shown to develop significant increases in albuminuria, which, in turn, correlates with and predicts the progression of renal damage in dm . Increased ang-2 levels in patients with dm, little is known about the urinary levels of these angiogenic factors in different stages of dn, and potential correlations between them and albuminuria have not yet been studied . In the present study, we measured urinary ang-2, ang-1, and vegf levels to elucidate the possible correlation between urinary angiogenic factors and renal damage in patients with various phases of type 2 dm . The retrospective study included 113 insulin - dependent patients with type 2 dm recruited from the department of endocrinology and nephrology at union hospital (wuhan, china) between december 2012 and march 2014 . Type 2 dm was defined according to established who criteria (i.e., fasting blood glucose (fbg) 7.0 mmol / l, postprandial blood glucose 11.1 mmol / l, or symptoms of dm with random blood glucose 11.1 mmol / l). All patients maintained a stable body weight for at least 3 months before beginning the study . Patients with acute vascular events or hospitalization (defined as stroke, myocardial infarction, unstable angina, or coronary or peripheral revascularization within the last 3 months); high - range hypertension (160/100 mmhg); current infection; or evidence of neoplastic, hepatic, or significant renal disease (requiring dialysis) within 3 months prior to enrollment were excluded from the study . Diagnosis of dn was made according to the criteria of kidney disease outcomes quality initiative (kdoqi). Based on the urinary albumin excretion rate (uaer) at baseline, patients were classified as having normoalbuminuria (dn1 group: uaer <20 g / min), microalbuminuria (dn2 group: uaer 20200 g / min), or macroalbuminuria (dn3 group: uaer> 200 g / min). Thirty subjects undergoing routine health checks were recruited into the control group (nc). The study protocol was approved by the medical ethics committee of union hospital, tongji medical college, huazhong university of science and technology, and was conducted according to the principles of the declaration of helsinki . Morning preprandial levels of fasting blood sugar (fbg), serum creatinine, triglyceride (tg), high - density lipoprotein cholesterol (hdl - ch), low - density lipoprotein cholesterol (ldl - ch), and total cholesterol were measured with a full automatic biochemical analyzer (hitachi 7150, tokyo, japan). Glycosylated hemoglobin (hba1c) was measured with a d10 hemoglobin testing system (bio - rad laboratories, hercules, ca, usa), using a cation exchange hplc and an immunoturbidimetric assay method (roche / hitachi 902 cobas system). Twenty - four h urine samples were collected for the determination of ang-1, ang-2, and vegf levels and the estimation of uaer . Urinary protein quantitative measurements (24 h upqm) were obtained for all patients . Renal function was assessed by glomerular filtration rate (gfr) detection with single photon emission computed tomography (spect) (ge millennium mg dualhead, ge healthcare, milwaukee, wi, usa, usa). Levels of angiogenic factors (ang-1, ang-2, and vegf) in serum and urine were measured by enzyme - linked immunosorbant assay (elisa) in frozen serum samples and in urine samples . In brief, monoclonal antibodies specific for ang-1 (raybiotech, norcross, ga, usa), ang-2 (raybiotech), and vegf (neobioscience technology co., shenzhen, china) were precoated onto microplates . Ang-1, ang-2, or vegf present in a sample was bound to the wells by the immobilized antibody . The wells were washed, and biotinylated anti - human antibody specific for ang-1, ang-2, or vegf was added . After the removal of unbound antibodies, the wells were washed, and a 3,3,5,5-tetramethylbenzidine (tmb) substrate solution was added . The tmb solution changed in color from blue to yellow, in proportion to the bound concentration of ang-1, ang-2, or vegf . The absorbance of the solution in each well was measured by a microplate reader (bio - tek elx800; vt, usa) at a wavelength of 450 nm . All samples were examined in duplicate, and mean values were used for statistical analysis . Statistical analysis was performed using a commercially available statistical software package (spss for windows, version 18.0; spss, chicago, il, usa). One - way analysis of variance (anova) with tukey's post hoc test was used to compare groups of normally distributed data . Categorical data were analyzed using the chi - square test, and pearson's or spearman's correlation coefficients were used to test associations between variables . A stepwise multiple regression analysis was performed to identify the predictors of ang-2 (dependent variable). All tests were two tailed, and values of p <0.05 were considered statistically significant . No significant differences were found in age, gender, bmi, diastolic blood pressure (dbp), total cholesterol, ldl - ch, hdl - ch, or tg among the three diabetic groups and the control group . However, systolic blood pressure (sbp), hba1c, and fbs were higher in the diabetic patients than in control subjects . Hba1c appeared to be lower in the dn3 group than in the dn1 and dn2 groups after long - term treatment with hypoglycemic drugs; however, no significant differences in hba1c and fbs were found among the three diabetic groups . Serum creatinine was significantly higher, and gfr was significantly lower, in the dn3 group than in the dn1 and dn2 groups (p <0.001). Both uaer and 24 h upqm increased progressively from the dn1 to the dn3 groups (p <0.001). Serum levels of ang-2 were markedly increased in diabetic patients compared with values in the control group (p <0.001; figure 1(a)). Moreover, serum ang-2 was significantly higher in patients with macroalbuminuria (dn3) than those in the dn1 and dn2 groups (p <0.001; figure 1(a)). Diabetic patients exhibited higher levels of urinary ang-2 than controls (p <0.001; figure 1(b)), and urinary ang-2 increased in a stepwise manner with increasing degrees of albuminuria in the three diabetic groups (p <0.001; figure 1(b)). No significant difference was found in serum ang-1 levels between any of the four groups (data not shown). However, urinary ang-1 levels were significantly higher in the dn1 group than in the control group (p <0.05; figure 1(c)) and lower in the dn3 group than in the control group (p <0.001). In addition, patients in the dn1 and dn2 groups had significantly higher urinary ang-1 levels than those in the dn3 group (p <0.001; figure 1(c)). No significant difference was found in serum vegf among the groups (data not shown); however, subjects with dm exhibited significantly higher urinary vegf levels than the control subjects (p <0.001; figure 1(d)). Moreover, urinary vegf was significantly higher in patients with macroalbuminuria (dn3) than in the dn1 and dn2 groups (p <0.001; figure 1(d)). No difference was observed in urinary vegf in diabetic patients with or without microalbuminuria . Table 2 and figure 2 summarize the results of the analyses undertaken in patients with dn . Serum levels of ang-2 were significantly positively correlated with urinary ang-2 and vegf levels (all p <0.001), as well as with uaer, 24 h upqm (both p <0.001), and serum creatinine (p <0.001). In addition, serum ang-2 was negatively correlated with gfr (p <0.001). Similarly, urinary ang-2 was strongly correlated with urinary vegf, uaer, 24 h upqm, and serum creatinine (all p <0.001). A negative correlation was found between gfr and urinary ang-2 (p <0.001). No significant correlations were found between serum or urinary ang-2 and age, bmi, dbp, sbp, hba1c, fbs, hdl - ch, ldl - ch, total cholesterol, or triglyceride (data not shown). In these subjects, stepwise multiple regression analyses including uaer, gfr, age, bmi, dbp, sbp, fbs, hba1c, and serum ang-2 identified that uaer (p <0.001) and gfr (p = 0.001) are significant predictors of urinary ang-2 . This study is the first to investigate urinary levels of ang-1, ang-2, and vegf in human type 2 dm with varying uaers . (1) urinary ang-2 increased in a stepwise manner in type 2 dm patients with various degrees of kidney damage (normoalbuminuria, microalbuminuria, and macroalbuminuria). This alteration was accompanied by increased urinary vegf, as well as early increased and later decreased urinary ang-1 . (2) urinary ang-2 levels in normoalbuminuria patients increased prior to changes in albumin levels . (3) among the angiogenic growth factors, urinary ang-2 was strongly associated with degree of albuminuria and gfr in type 2 dm patients . Alterations in the vegf and ang-1/ang-2 system have been reported to play an important role in the pathobiology of glomerular disease in dm [2, 1417, 20, 26] and chronic kidney disease (ckd) [5, 2729]. Most of those studies focus on circulating levels of angiogenic growth factors . In this study, we evaluated simultaneously the levels of ang-1, ang-2, and vegf in both serum and urine . We found that urinary ang-2 increased stepwise with albuminuria levels than ang-1 and vegf at a greater rate . This observation is likely due to the tubular pathophysiological changes, which occur before the glomerular stage of disease . This suggests that the serum and urinary ang-2 are related to subclinical tubular impairment and may be an earlier measurable marker of renal involvement before the onset of albuminuria . Ang-2 has been linked to increased microvascular permeability, and podocyte overexpression of ang-2 was shown to produce albuminuria in transgenic mice . Circulating ang-2 levels correlating positively with proteinuria have been reported in human sle and ckd . In our type 2 dm patients, we found that albuminuria is a significant predictor for urinary ang-2 levels after adjustments were made for those possible confounders . The association between urinary ang-2 and albuminuria suggests that upregulation of ang-2 may destabilize glomerular endothelial cells and directly or indirectly affect podocytes, leading to the deterioration of glomerular filtration barrier function [5, 31]. Abundant ang-2 protein was detected by immunohistochemical staining in glomeruli in endothelial cells alongside capillary loops in renal biopsies from patients with dm (data not shown). Taken together, these findings suggest that high glucose - induced glomerular endothelial damage may lead to secretion of ang-2, and elevated expression of ang-2 in the glomerular endothelium may further increase albuminuria through the damaged glomerular filtration barrier . The positive correlation with serum creatinine and the negative correlation with gfr suggest that increased ang-2 may be associated with the development of renal impairment . Blood ang-2 levels rise in line with the decline in renal function in type 2 dm and ckd, and this inverse correlation may predict long - term mortality in patients with ckd [27, 33]. In our study these observations suggest that urinary ang-2 levels increase in parallel to the deterioration of renal function . In our study, elevated urinary ang-2 was correlated with serum ang-2, suggesting that urinary levels of ang-2 may be representative of local production and release of ang-2 into the circulation in patients with dn . Alternatively, as dn progresses, decreased gfr leads to higher serum ang-2, which allows greater penetration of the glomerular barrier and leads to proteinuria . However, the multivariate analysis showed that serum ang-2 is not a predictor for urinary ang-2 . Based on the correlation between serum ang-2 and hba1c, accumulation of advanced glycation end product (age) in endothelial cells subjected to hyperglycemia may upregulate serum levels of ang-2 and vegf [34, 35]. However, no correlation was found between urinary and/or serum ang-2 and hba1c in our study . However, the possibility can be not excluded that increased urinary ang-2 levels are a consequence of mechanisms that are unassociated with the accumulation of ages in the glomerular endothelium . We unexpectedly found that urinary ang-1 was significantly higher in dm patients with normoalbuminuria and lower in those with macroalbuminuria than in control subjects . This observation, also reported by rizkalla, showed upregulation of ang-1 in the early phase of the disease and progressive downregulation of renal ang-1 expression in experimental dm [17, 26]. Ang-1 is produced by glomerular podocytes [36, 37] and plays an important role in maintaining the structure and integrity of the glomerular filtration barrier . Further studies will seek to determine whether this increase is a short - term response of podocytes to hyperglycemia or will reduce the vascular permeability through endothelial cell glycocalyx layer modifications or other mechanisms . The decrease in ang-1 in subjects with macroalbuminuria suggests that ang-1 production is attenuated at the later stage of dn, which may be associated with a decreased number or function of podocytes, or both ., we found no differences in serum ang-1 levels in groups with varying degrees of albuminuria . However, dessapt - baradez reported decreased ang-1 levels in mice with streptozotocin - induced type 1 dm, which was accompanied by marked albuminuria, nephromegaly, hyperfiltration, glomerular ultrastructural alterations, and aberrant angiogenesis . The differential expression pattern of ang-1 in serum may be due to the different subjects and varying stage of dn . Urinary vegf was increased in our study in all diabetic groups even at the normoalbuminuric stage though serum vegf was unaltered . These findings are in agreement with previously published findings [10, 14, 18, 40]. However, some centers have reported an increase in plasma vegf in type 2 dm [19, 41, 42]. These differences likely reflect the different populations being studied, as well as variations in study design . The results of this study show that urinary ang-2 increases in a stepwise manner in type 2 diabetic patients with varying degrees of kidney damage . Ang-2 measurement in urine is a useful, noninvasive tool for the evaluation of renal involvement in the course of dm, especially in normoalbuminuric patients . Further investigations with a larger sample size and a prospective design are required to confirm the potential application of ang-2 as a useful biomarker for the early detection of diabetic nephropathy.
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Tissue - organ bath systems have been widely used in ex vivo studies for cerebrovascular function by measuring the forces in strips or rings of isolated vessel segments9,19,20,23,25), whereas the transcranial window methods have been developed to study the vessel function in vivo in live experimental rodents18), in particular, in leptomeningeal arteries and arterioles (only to a depth of 250 m from the cortical surface)17,24). The ex vivo studies would have several advantages over the in vivo cranial window methods in the following respects: 1) this method allows dose - escalating and/or antagonism studies, 2) biochemical degradation and/or metabolism of applied agents is negligible, 3) the effects of nonvascular cells (i.e., glial and neuronal cells) can be removed, and 4) studies can be performed under physiologic as well as pathophysiological conditions . Larger pial arteries, where blood pressure and blow flow rate are tightly regulated, have been utilized in ex vivo studies largely because of the easiness of their preparation23,25). In addition, dacey and duling5) first demonstrated that small arterioles (i.e., perforating arterioles) served as an ex vivo experimental system though there were more technical difficulty as compared with the large vessel preparation . The " vessel conduit system " utilizing intracerebral perforating arterioles isolated from rat brain has provided as a gold standard ex vivo model for many years4,5,7,9,12,13). Though this method with an organ bath setting is well suited for microvessel experiments, especially in terms of minimal mechanical injury during preparation5,9), utilization of larger vessels (i.e., leptomeningeal arteries form mouse brain) has not been addressed . For example, anatomically, intracerebral large subarachnoid arteries [as middle cerebral artery (mca)] of rodents show a short interbranch distance relative to vessel diameter, which makes it difficult to find a long (0.5 - 1 mm), branch - free segment feasible for cannulation . In this study, we developed the method for isolation of the olfactory artery from the mouse brain and characterization of vascular reactivity . Male c57/bl6 mice (10 to 14 weeks of age) were purchased from the jackson laboratory (bar harber, me, usa) and anesthetized with sodium pentobarbital (30 mg / kg, i.p . ). Briefly, the brain was rapidly removed cautiously without damaging vessels within the circle of willis and basal large arteries . The removed brain was faced to ventral surface up for the olfactory artery (oa)2) dissection in cooled (4) chamber with 3-(n - morpholino) propanesulfonic acid (mops)-buffered saline (in mmol / l: 144 nacl, 3.0 kcl, 2.5 cacl2, 1.4 mgso4, 2.0 pyruvate, 5.0 glucose, 0.02 ethylenediaminetetraacetic acid, 1.21 nah2po4, and 2.0 mops) containing 1% dialyzed bovine serum albumin . Of note is that the oa is also named internal ethmoidal artery (iea) (fig . Both oas were separated from the parenchyma and in turn an unbranched oa vessel segment (approximately 500 m or longer) was prepared for cannulation . The isolated vessel was carefully transferred with a wiretrol ii 5 - 10 l micropipette (drummond scientific co., broomall, pa, usa) from dissection chamber to a temperature controlled cannulation chamber (2.5 ml) which was mounted on a stage of an inverted microscope (zeiss tv 200, thornton, new york, usa). The holding and perfusion pipettes used for cannulation were fabricated by pulling and shaping glass tubes (drummond scientific co.; 2.13 mm o.d ., 1.63 mm i.d ., and 1.19 mm o.d ., 1.02 mm i.d ., respectively; 15-cm length) using a microforge (stoelting co., wood dale, il, usa). The collecting pipettes were also fabricated from the same glass as the holding pipettes (2.13 mm o.d . The final holding pipette diameter was 80 - 90 m and perfusion pipette diameter was 45 - 50 m . The vessel was cannulated at one end with the concentrically mounted perfusion (inner) and holding (outer) pipette system . All experiments were performed at a fixed intravascular pressure (60 mm hg) applied through the perfusion pipette without intraluminal flow (fig ., the bath temperature was raised from room temperature to 37.5. we discarded vessels if there was a pressure drop . We equilibrated the cannulated artery in the organ bath for 45 minutes . Before the experiment, vessel responsiveness was tested by applying 50 mm kcl . If the vessel showed good response, we further investigated vessel responses to other vasoactive agents after washing out kcl thoroughly . If the vessel showed a poor response to 50 mm kcl (less than 15% diameter change), we discarded the vessel . The organ bath was continuously circulated with mops buffer using a peristaltic pump (model 203, scientific industries, bohemia, ny, usa) at a rate of 0.5 ml / min . The cannulation pipettes are mounted in custom made plexiglas holders, which have side ports to allow intraluminal pressure application and measurement as well as suction to aspirate the vessel ., michigan city, in, usa) and displayed on a monitor . For measurement of the vessel diameter, a computerized diameter tracking system (diamtrak software, tim neild, adelaide, australia) was used . We applied all drugs dissolved in mops at 37.5 sequentially from low to high concentrations . After the vessel responsiveness was confirmed with kcl, we added phenylephrine (pe; sigma, st . Louis, mo, usa), followed by prostaglandin pgh2 (u-46619; sigma) or endothelin-1 (et-1; sigma, st . After applying of kcl and pe, we waited until the vessel diameter returned to the initial baseline by washing out the former drug completely at least for 20 minutes . For the dilation activity study, we induced submaximal constriction with pe 510 m. if the vessel showed good response with pe 510 m (more than 8% diameter change), we applied acetylcholine (ach; sigma, st . Louis, mo, usa). If the vessel showed poor response less than 8% diameter change with pe 510 m, we discarded the vessel because it could be difficult to detect vasodilatory effect clearly . Values were expressed as meanstandard error of mean where n indicated the number of vessels studied . One - way repeated - measure analysis of variance (anova) was performed to test for differences between concentration - response of kcl relationships . Agonist comparisons for concentration - response curves were analyzed with a two - way anova with repeated measures using the proc mixed general linear models associated with the statistical analysis software (sas 9.3, sas institute, inc ., the log (ec50) values (concentration producing 50% of the maximal response) were calculated by nonlinear regression analysis of concentration - response curves of each agonist (prism 5.0, graph pad software inc ., san diego, ca, usa). We used the equation to calculate the percent of constriction as [(dinitial - ddrug)/dinitial]100%, where dinitial is the diameter just before applying vasoconstrictor and ddrug is the diameter after vasoconstrictor . Percent dilation was calculated using the equation [(ddrug - dbase)/(dinitial - dbase)]100%, where ddrug is the diameter in drug for vasodilator and dbase is the diameter in drug for submaximal constriction and dinitial is the diameter just before applying vasoconstrictor (pe 510 m). Male c57/bl6 mice (10 to 14 weeks of age) were purchased from the jackson laboratory (bar harber, me, usa) and anesthetized with sodium pentobarbital (30 mg / kg, i.p . ). Briefly, the brain was rapidly removed cautiously without damaging vessels within the circle of willis and basal large arteries . The removed brain was faced to ventral surface up for the olfactory artery (oa)2) dissection in cooled (4) chamber with 3-(n - morpholino) propanesulfonic acid (mops)-buffered saline (in mmol / l: 144 nacl, 3.0 kcl, 2.5 cacl2, 1.4 mgso4, 2.0 pyruvate, 5.0 glucose, 0.02 ethylenediaminetetraacetic acid, 1.21 nah2po4, and 2.0 mops) containing 1% dialyzed bovine serum albumin . Of note is that the oa is also named internal ethmoidal artery (iea) (fig . Both oas were separated from the parenchyma and in turn an unbranched oa vessel segment (approximately 500 m or longer) was prepared for cannulation . The isolated vessel was carefully transferred with a wiretrol ii 5 - 10 l micropipette (drummond scientific co., broomall, pa, usa) from dissection chamber to a temperature controlled cannulation chamber (2.5 ml) which was mounted on a stage of an inverted microscope (zeiss tv 200, thornton, new york, usa). The holding and perfusion pipettes used for cannulation were fabricated by pulling and shaping glass tubes (drummond scientific co.; 2.13 mm o.d ., 1.63 mm i.d ., and 1.19 mm o.d ., 1.02 mm i.d ., respectively; 15-cm length) using a microforge (stoelting co., wood dale, il, usa). The collecting pipettes were also fabricated from the same glass as the holding pipettes (2.13 mm o.d . The final holding pipette diameter was 80 - 90 m and perfusion pipette diameter was 45 - 50 m . The vessel was cannulated at one end with the concentrically mounted perfusion (inner) and holding (outer) pipette system . All experiments were performed at a fixed intravascular pressure (60 mm hg) applied through the perfusion pipette without intraluminal flow (fig ., the bath temperature was raised from room temperature to 37.5. we discarded vessels if there was a pressure drop . We equilibrated the cannulated artery in the organ bath for 45 minutes . Before the experiment, vessel responsiveness was tested by applying 50 mm kcl . If the vessel showed good response, we further investigated vessel responses to other vasoactive agents after washing out kcl thoroughly . If the vessel showed a poor response to 50 mm kcl (less than 15% diameter change), we discarded the vessel . The organ bath was continuously circulated with mops buffer using a peristaltic pump (model 203, scientific industries, bohemia, ny, usa) at a rate of 0.5 ml / min . The cannulation pipettes are mounted in custom made plexiglas holders, which have side ports to allow intraluminal pressure application and measurement as well as suction to aspirate the vessel . The plexiglas holders were mounted on a microscope mounted v - track system9). The artery was observed with the aid of a video system (dagemti inc . For measurement of the vessel diameter, a computerized diameter tracking system (diamtrak software, tim neild, adelaide, australia) was used . The vessel diameter and pressure data were recorded and digitally stored continuously . We applied all drugs dissolved in mops at 37.5 sequentially from low to high concentrations . After the vessel responsiveness was confirmed with kcl, we added phenylephrine (pe; sigma, st . Louis, mo, usa), followed by prostaglandin pgh2 (u-46619; sigma) or endothelin-1 (et-1; sigma, st . After applying of kcl and pe, we waited until the vessel diameter returned to the initial baseline by washing out the former drug completely at least for 20 minutes . For the dilation activity study, we induced submaximal constriction with pe 510 m. if the vessel showed good response with pe 510 m (more than 8% diameter change), we applied acetylcholine (ach; sigma, st . Louis, mo, usa). If the vessel showed poor response less than 8% diameter change with pe 510 m, we discarded the vessel because it could be difficult to detect vasodilatory effect clearly . Values were expressed as meanstandard error of mean where n indicated the number of vessels studied . One - way repeated - measure analysis of variance (anova) was performed to test for differences between concentration - response of kcl relationships . Agonist comparisons for concentration - response curves were analyzed with a two - way anova with repeated measures using the proc mixed general linear models associated with the statistical analysis software (sas 9.3, sas institute, inc ., the log (ec50) values (concentration producing 50% of the maximal response) were calculated by nonlinear regression analysis of concentration - response curves of each agonist (prism 5.0, graph pad software inc ., san diego, ca, usa). We used the equation to calculate the percent of constriction as [(dinitial - ddrug)/dinitial]100%, where dinitial is the diameter just before applying vasoconstrictor and ddrug is the diameter after vasoconstrictor . Percent dilation was calculated using the equation [(ddrug - dbase)/(dinitial - dbase)]100%, where ddrug is the diameter in drug for vasodilator and dbase is the diameter in drug for submaximal constriction and dinitial is the diameter just before applying vasoconstrictor (pe 510 m). The baseline diameter of mouse oas in this study was 1203.5 m (n=28) at 60 mm hg . Application of a depolarizing agent of kcl showed a dose - dependent vasoconstriction (n=12) (fig . We found there were two populations of vessels: 1) the vessel segments (n=15) having constriction by 8% in response to 110 m pe (28.63.8%) (fig . 4), or 2) the vessel segments (n=5) having constriction by <8% in response to 110 m pe (2.60.6%), though all vessel segments revealed substantial vasoconstriction to kcl . U-46619 induced a maximal constriction (49.46.3%, n=9) at a concentration of 110 m (fig . 4). The et-1, known as a potent vasoconstrictor, showed most significant and prolonged vasoconstriction in most of the oas tested with the greatest potency and efficacy among the vasoconstrictors tested (43.74.7% at 110 m, n=8) (fig . Next, we examined the vasodilatory responses of the isolated oas under the condition that the vessels were sub - maximally pre - constricted in the presence of 510 m pe . We found that both ach and bk (110 - 110 m) induced vasodilation in a dose - dependent fashion . The maximal vasodilation was observed when the vessels were treated with 110 m ach (54.76.3%, n=8) or 110 the baseline diameter of mouse oas in this study was 1203.5 m (n=28) at 60 mm hg . Application of a depolarizing agent of kcl showed a dose - dependent vasoconstriction (n=12) (fig . We found there were two populations of vessels: 1) the vessel segments (n=15) having constriction by 8% in response to 110 m pe (28.63.8%) (fig . 4), or 2) the vessel segments (n=5) having constriction by <8% in response to 110 m pe (2.60.6%), though all vessel segments revealed substantial vasoconstriction to kcl . U-46619 induced a maximal constriction (49.46.3%, n=9) at a concentration of 110 m (fig . 4). The et-1, known as a potent vasoconstrictor, showed most significant and prolonged vasoconstriction in most of the oas tested with the greatest potency and efficacy among the vasoconstrictors tested (43.74.7% at 110 m, n=8) (fig . Next, we examined the vasodilatory responses of the isolated oas under the condition that the vessels were sub - maximally pre - constricted in the presence of 510 m pe . We found that both ach and bk (110 - 110 m) induced vasodilation in a dose - dependent fashion . The maximal vasodilation was observed when the vessels were treated with 110 m ach (54.76.3%, n=8) or 110 in the present study, we recorded the vascular reactivity of isolated mouse olfactory artery ex vivo which constricted in response to various vasoconstrictors, including kcl, pe, et-1, and a thromboxane agonist (prostaglandin pgh2; u-46619). Moreover, this isolated vessel demonstrated vasodilation in a dose - dependent manner when treated with vasodilatory agents including acetylcholine and bradykinin . Our findings suggest that the isolated olfactory artery would provide as a useful ex vivo model to study the molecular and cellular mechanisms of vascular function underlying cerebrovascular disorders and the direct effects of such disease - modifying pathways on cerebrovascular function utilizing pharmacological agents and genetically modified mouse models . Leptomeningeal arteries have distinct features morphologically and functionally as compared with perforating arterioles and capillaries in the brain . For example, leptomeningeal arteries (also called subarachnoid and pial arteries) are surrounded by the cerebrospinal fluid and have multiple layers of vascular smooth muscle cells (vsmcs), while smaller branches of cerebral vessels like perforating arterioles is consist of a single layer of vsmcs6,10). An inner elastic lamina is seen in the major leptomeningeal arteries but not in the perforating arterioles10). In mouse model of cerebral aneurysm, we can see the typical anatomic feature of large leptomeningeal artery at oa16). In fact, smaller perforating arterioles are closely communicated with surrounding glial cells and neuronal parenchymal elements, which leads to distinct vascular reactivity to vascular stimuli . In one hand, large leptomeningeal arteries are more sensitive to sympathetic nerve stimuli26), whereas small arterioles are more reactive than that of large arteries to a certain condition such as an elevation of pco2 or hypercapnia11,15). On the other hand, large cerebral arteries are more sensitive to changes in blood pressure while cerebral blood flow in these vessels remains unchanged via an autoregulatory mechanism15). Thus, because of their anatomical and functional differences between large leptomengeal arteries and small perforating arterioles, one cannot extrapolate the findings from the large arteries to those of the small arterioles, or vice versa . Dacey and duling5) initially developed an elegant ex vivo experimental system where perforating arterioles isolated from rat brain was utilized . Since genetically modified mouse models (designed to either deplete or overexpress target proteins) have been more widely used in the many setting of neurovascular disease, ex vivo experiments utilizing such genetically modified mice are desired . However, the ex vivo experimental technique, in particular, using large leptomeningeal arteries of mice has not been fully established largely due to the technical difficulty in isolation of cerebral vessels without physical stress and cannulation of these leptomeningeal arteries . Though one study reported that perforating intracerebral arterioles demonstrates spontaneous tone and responses to vasoactive reagents3), we observed that these perforating arterioles from c57bl6 mouse (the same type of mouse that coyne et al.3) used in the study) often had many small branches of arterioles; therefore, it was difficult to maintain the intraluminal pressure (60 mm hg) consistent throughout the experiment . Similarly, we found a large vessel segment of the proximal middle cerebral artery had the same issue . We, therefore, sought to examine what vessel segment of large leptomeningeal arteries in c57bl6 mouse (the most common source strain of genetically engineered mice) would be more feasible for ex vivo studies without such technical problems . We tried to find out unbranched intracerebral large artery that was easy to handle with proper size during vessel isolation and cannulation . During preliminary anatomical dissection study, oas, which was also named ieas had fewer branches and was suitable size to manipulate2,8,27). Also oa was remarkably constant in its origin, size and course in rat2) and it was similar in mouse, we found the olfactory segments from mouse brain showed slightly smaller diameter than that of the vessel segments from the proximal mca (40 - 90 m)1,22) before pressure loading . After cannulation and incubation with an intraluminal pressure of 60 mm hg, these vessels passively dilated up to 1203.5 m in diameter by 45 min post incubation, which is also smaller to mca diameter of 142.57.9 m14). In contrast, it was reported that isolated perforating arterioles showed spontaneous constriction under the same bathing medium as our experimental condition during this period of incubation after cannulation3,5), indicating functional difference between leptomeningeal and perforating vessels . This notion is further supported by previous studies demonstrating that pial arteries and perforating arterioles responded differently to reperfusion after transient ischemia17). We found the isolated olfactory artery was capable to dilate as well as constrict in response to vasoactive agents (fig ., vascular reactivity of the olfactory artery to these vasoactive agents is to some extent comparable to that of the mca of mouse brain1,21,22). For example, the potent vasoconstrictor et-1 induced vasoconstriction by 43.74.7% at a concentration of 110 m in the olfactory artery (fig . Interestingly, we found though most of the selected olfactory arteries (15 of 20) displayed consistent vasoconstrictor response 110 m of pe, the rest of vessels showed little response to pe . This variable response could be possible that the distribution of -adrenergic receptors varies between the isolated olfactory arteries . The limitations of this study are small number of experimental vessels and we added chemical agents only extraluminally . Moreover, our laboratory processes require learning period to be accustomed due to meticulous manipulation of vessels and handling of fine glass tube . Despite these limitations, our study suggests that isolated mouse oa is a useful ex vivo model of large leptomeningeal artery . In the present study, we were able to isolate the olfactory arteries from mouse brain without causing apparent traumatic damage and measure the vessel reactivity to vasoactive agents, suggesting that mouse olfactory arteries can be used to study the molecular and cellular mechanisms of cerebrovascular function.
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Her medical history included a spine operation due to tuberculous spondylitis of the lumbar spine about 10 years ago . The initial brain computed tomography (ct) scan (brilliance 64-multislice ct scanner, philips medical system, ma) demonstrated a well - defined intra - axial mass in the cerebellum (fig . The mass was mainly located in the right cerebellar hemisphere, displacing the cerebellar vermis to the left side and compressing the right side of the 4th ventricle and resulting in mild obstructive hydrocephalus . The mass was composed of a peripheral isoattenuation area and a central low attenuation area, which was the central necrotic portion in the precontrast ct scan (fig . The isoattenuation area of the mass demonstrated heterogeneous dense enhancement after the injection of contrast materials . Some vascular or linear enhancing portions were observed, but the central low attenuation portion was scarcely enhanced . One day after admission, brain magnetic resonance imaging (mri) (twin speed 1.5 t mr scanner, ge healthcare, wi) was performed . The mass was hypointense on precontrast t1-weighted images (repetition time [tr]/echo time [te], 400/-8 ms) (fig . (tr / te, 3700/-1200 ms), the mass showed an intermediate signal intensity at the periphery, and central hyperintensity (fig . The outer surface margin was comparatively smooth and slightly lobulated, but the inner surface margin was very irregular . After contrast material injection, the periphery showed heterogeneous enhancement as did the brain ct images (fig . Gradient echo images (tr / te, 500/-15 ms) revealed multiple bleeding foci in the periphery as dark signal intensity foci (fig . F - fluorodeoxyglucose positron emission tomography / computed tomography (f - fdg pet / ct) demonstrated hypermetabolism at the tumor periphery . There was a metabolic defect within the central area of the tumor, which was correlated to the central necrosis of the tumor (fig . Macroscopically, it was a relatively well - circumscribed and firm mass with areas of necrosis and hemorrhage . Microscopically, it had a biphasic pattern with areas of gliomatous and sarcomatous differentiation (fig . The gliomatous component consisted of atypical glial cells with pleomorphic nuclei and frequent mitotic figures . Geographic necrosis and endothelial cell proliferation the tumor predominantly consisted of spindle cell proliferation forming sarcomatous areas with intersecting fascicular or storiform patterns similar to fibrosarcoma . Extensive necrosis, frequent mitotic figures (> 80/10 hpfs) and abnormal mitoses were observed in the sarcomatous component (fig . The gliomatous component showed immunoreactivity for glial fibrillary acid protein (gfap), in contrast with lack of immunoreactivity for gfap in the sarcomatous component (fig . Abundant collagen and reticulin fibers were found between individual tumor cells in the sarcomatous component using masson - trichrome and reticulin staining (fig . Almost all previously reported cases of gliosarcoma were located in the supratentorial area, especially in the peripheral region of the temporal lobe (2). Gliosarcoma occurs less commonly in the frontal, parietal or occipital lobes, and in the corpus callosum . (3) showed similar mri findings to supratentorial gliosarcoma such as peripheral location of the cerebellum, broad base on the meninges, and multiple lesions . Other brain and spinal gliosarcoma cases usually had a medical history of radiation therapy for other cerebellar tumors . Quite a number of gliosarcomas have been reported as secondary tumors after radiation therapy for histologically different primary cns tumors (5 - 9). Among several possible mechanisms for gliosarcoma histogenesis, the transformation theory, which states that part of a glial tumor is dedifferentiated to primitive cells and transformed into sarcomatous components, supports the idea that radiation therapy might induce a dedifferentiation of the glial cell to the sarcomatous cells in cases with previous cns tumors (4, 6, 8, 10, 11). Compared to the previous literature, our case had no history of previous radiation therapy for a cns tumor . In addition, its location was in a relatively central portion, without abutting the meninges . Pathologically, gliosarcoma is characterized by a biphasic tissue pattern with alternating areas of glial and mesenchymal differentiation (7). The sarcomatous component shows malignant changes of spindle cells with features of fibrosarcoma or malignant fibrous histiocytoma . Areas of mesenchymal differentiation into cartilage, bone, fat and smooth or skeletal muscle may be associated . In our case, although no specific mesenchymal differentiation was seen, the tumor was predominantly sarcomatous and had a mixture of clearly malignant mesenchymal gfap - negative areas and gfap - positive glial areas . Therefore, we were able to exclude the possibility of glioblastoma with a florid fibroblastic proliferation . Radiologic findings of gliosarcomas in the previous literature are variable and sometimes the tumors are very aggressive . A gliosarcoma may appear as a well - defined hyperdense mass with heterogeneous or ring enhancement due to a fibrous component . In contrast, a glioblastoma usually shows low or intermediate attenuation in a precontrast ct scan . Vasogenic edema often accompanies glioblastoma, but central necrosis is less common due to the predilection of such tumors to develop in a peripheral location . Dural involvement is not uncommon due to its peripheral location (3, 5, 12). On mri, gliosarcoma appears as a heterogeneous mass both in t1- and t2-weighted images, with strong peripheral enhancement and central hemorrhage or necrosis (5). Extracranial metastasis is also common in gliosarcomas, resulting in a worse prognosis (8, 10). In our case, the imaging findings were similar to those of an infratentorial glioblastoma, which usually presents as an aggressive heterogeneous tumor with hemorrhage, necrosis, a cystic portion or calcification, and surrounding peritumoral edema (15). But, it is known as a less aggressive and slower growing tumor than a supratentorial glioblastoma, and sometimes there is a lack of peritumoral edema, as in our case (16, 17). In addition, as the most common incidence of both tumors occurs between the 5th and 7th decades of life, it was very difficult to make the differential diagnosis . Common differential diagnoses for gliosarcoma are glioblastoma, metastasis, brain abscess or other infratentorial intraaxial tumors such as hemangioblastoma or medulloblastoma (5, 12, 15 - 17). In conclusion imaging findings were so similar to those of an infratentorial or supratentorial glioblastoma that it was difficult to distinguish from a glioblastoma . Though a rare entity, gliosarcoma should be considered in the differential diagnosis of infratentorial tumors with radiological features of glioblastoma and metastasis in elderly patients.
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In the last three decades, a large volume of studies have established that oxidized low - density lipoprotein (oxldl) is a useful marker for cardiovascular diseases (cvds) (see [16]). The measurement of oxldl correlates with the presence of cvds and indicates that oxldl is a potential prognostic marker for future health events . Although the pathological aspects of oxldl have been well studied, the formation, distribution, and overall fate of oxldl in vivo remain unclear . The ldl receptor discovered by goldstein and brown has central roles in the systemic and cellular metabolism of cholesterol . Cholesterol accumulates in atherosclerotic lesions even in patients with familial hypercholesterolemia having genetically impaired ldl receptor . This observation raised the question of how lipid - laden foam cells are formed without ldl receptor . They also suggested the importance of recognition of modified ldl by specific receptors . By using acetylated ldl as the model ligand, they demonstrated the presence of new receptors that facilitate foam cell formation, which were called scavenger receptors . Scavenger receptor type a (sr - a), the first scavenger receptor to be identified, is responsible for the recognition and uptake of acetylated ldl and oxldl [9, 10], but sr - a is not the sole receptor for modified ldl . More than 10 scavenger receptors have been identified so far [1113]. In 1984, steinbrecher and his colleagues proposed that oxidative modification promotes foam cell formation from macrophages and that the oxidative modification of ldl can be achieved through the interactions of ldl with endothelial cells in vitro . Oxldl induces a number of proatherosclerotic effects, including endothelial activation and smooth muscle proliferation [1419]. Berliner et al . Reported that the oxidative modification of ldl through coculture with endothelial cells had stimulatory effects on many types of cells and that these effects are due to the oxidized phospholipids generated in oxldl . The accumulation of oxldl in the foam cells in atherosclerotic lesions from human coronary arteries, carotid arteries, and aortas of watanabe heritable hypercholesterolemic (whhl) rabbits has been demonstrated by immunohistochemical analysis using anti - oxldl monoclonal antibodies [2023]. Since the clearance of oxldl from the circulation is so efficient, oxldl was believed initially to be absent in blood . Anti - oxldl monoclonal antibodies were applied to sensitive elisa procedures to measure very small amounts of oxldl in circulating blood [2528]. Several research groups, including ours, have demonstrated that the plasma oxldl level in patients with cvds is significantly higher than the level measured in healthy subjects [21, 2931]. The elisa procedures introduced by these groups were different so that the values of the oxldl measurements cannot be directly compared; however, they all reported an increase in oxldl levels in patients with acute myocardial infarction (ami), cerebral infarction, or those receiving hemodialysis . The details of the elisa procedures used and the clinical observations have been summarized previously . It has been long believed that ldl is modified in vessel wall and subsequently accumulates in the tissue . However, tsimikas et al . Showed that plasma oxldl increases concomitant with the regression of atherosclerotic lesions, suggesting that oxldl can be transferred between vessel wall and the circulation . Recently, the importance of the plasma oxldl level as a plausible predictive marker for secondary prevention has been discussed [3336]. To understand the role of oxldl in atherogenesis, it is important to clarify the behavior of oxldl in vivo during atherogenesis, particularly how oxldl is formed, how it moves around tissues, and how it is metabolized . In this paper, we briefly review recent advances regarding dynamics and kinetics of oxldl in vivo and suggest several possibilities for future research . We and others started to measure human plasma oxldl levels using sandwich elisa procedures [2527]. The combination of two antibodies, one for oxidized phosphatidylcholines (oxpc) and the other for human apolipoprotein b (apob), enables accurate and efficient detection of oxldl; as low as 1 ng / ml of oxldl can be measured . In the last 15 years, many clinical studies were conducted using these techniques . Oxldl levels increase in the plasma of patients with several pathological conditions, including cvds [21, 29, 30, 33], cerebral infarction, and hemodialysis for the treatment of chronic renal failure [25, 28, 38]. This evidence strongly suggests the involvement of in vivo oxldl in atherosclerosis and indicates that oxldl levels may increase in patients with symptoms of atherosclerosis . In addition, recent studies demonstrated that temporal rises and falls in plasma oxldl levels occur under certain conditions . Acute myocardial infarction (ami) is caused by total occlusion of a coronary artery following the rupture of an atherosclerotic plaque and acute thrombosis . Reported that the plasma oxldl level in patients with ami increased by approximately 3.5 fold than in control subjects . However, this increase in the plasma oxldl level appeared to be a temporal change during followup studies on the ami patients . The increased plasma oxldl levels dropped to near the normal range by the time the subjects were discharged from the hospital . The plasma oxldl levels of these patients increased for the first few days following onset of the disease, then returned to normal range within 30 days . Similar temporal changes in plasma oxldl levels have been reported in patients with acute infarction of the coronary arteries and after percutaneous transluminal coronary angioplasty (ptca) treatment [31, 39, 40]. Ami is induced by spontaneous plaque rupture, and the plaque can be further damaged by following ptca treatment with a balloon catheter and stent . Increased plasma oxldl levels after these acute events are partially due to the release of oxldl from the atherosclerotic plaques upon rupture . The time - course behavior of oxldl during the early stages of atherogenesis was investigated . To utilize apolipoprotein e knockout (apoe - ko) mice, the lesion area was less than 1% of the whole aortic surface at 10 weeks, but it gradually increased after 20 weeks, and it grew up to 32% by 40 weeks of age (figure 1). The oxldl level was 0.006 ng / micro g ldl at 6 weeks, but it was elevated to 0.042 ng / micro g ldl at 20 weeks and then decreased by 60% to 0.018 ng / micro g ldl at 28 weeks . This temporal rise in oxldl occurred before the size expansion of atherosclerotic lesions in the aorta . This observation suggests that the oxldl is generated during atherogenesis in apoe - ko mice in vivo . Reduction in the plasma oxldl level coincided with the increase in the development of the lesion and accumulation of oxldl in the atherosclerotic intima . Studied plasma oxldl levels (oxpc / apob ratios) during the regression of atherosclerosis in adult cynomolgus monkeys and new zealand white rabbits using anti - oxpc and anti - apob monoclonal antibodies . Plasma oxldl levels decreased during the progression of lesions under a high - fat diet and increased during the regression of these lesions after the diet was changed to normal . This observation also suggests that oxldl can be transferred between the intimal regions and the circulation . Atherosclerotic lesions can regress under certain conditions, such as a low - fat diet regimen or treatment with a lipid - lowering drug . In human studies, increases in plasma oxldl levels was reported in healthy volunteers fed a low - fat diet . After 37 healthy women took low - fat low - vegetable diet for 5 weeks, oxpc / apob ratio increased by 27%, while total cholesterol did not change . In miracl study, patients with unstable angina pectoris or ami were treated with atorvastatin (80 mg / day) for 16 weeks . The rupture of a plaque could cause the rapid release of oxldl into the circulation, but this may not be the only way to transfer oxldl from the lesions into circulation . According to these recent studies, without tissue damage, oxldl may possibly be equilibrated between the plasma and tissues (figure 2). In addition, trapping circulating oxldl by injecting anti - oxldl antibodies or soluble form of the scavenger receptor lox-1 induced regression of atherosclerotic lesions [45, 46]. Although that lipoprotein metabolism in rodents may have some difference from that of humans, it is nice if the regression therapies of atherosclerosis become available . One of the major issues regarding oxldl is the site of oxldl formation in vivo . The most common way to prepare oxldl is to incubate isolated ldl fractions with micromolar concentrations of copper sulfate for 324 hours . The copper ion induces lipid peroxidation chain reactions, and, subsequently, the chemical modification of the apob protein side chains with reactive lipid peroxidation products, such as 4-hydroxynonenal (4-hne), acrolein, and malondialdehyde (mda). The presence of adducts of these oxidized products on apob protein has also been immunologically confirmed in atherosclerotic lesions [4749]. Although concentrations of metal ions are typically low in the plasma, including copper ion, sufficient amounts of reducing compounds and proteins exist in the plasma that can protect ldl from oxidation . However, whether copper ion - induced ldl oxidation occurs under physiological conditions remains unclear . Cell culture - dependent modification of ldl has been used to prepare minimally modified ldl (mm - ldl), which is one form of oxidatively modified ldl [14, 15]. The chemical modification of mm - ldl is moderate judging by increases in thiobarbituric acid - reactive substances (tbarss) and mobility in agarose gel electrophoresis, so that mm - ldl binds to ldl receptor rather than scavenger receptors . However, mm - ldl showed strong inflammatory effects on the cells in vessel wall tissues [1517, 50]. Mm - ldl contains substantial amounts of oxpc, and oxpc is believed to be partially responsible for these biological effects [5153]. Sandwich elisa system that uses the anti - oxpc monoclonal antibody binds as competently to mm - ldl as to copper - induced oxldl . Circulating oxldl may be qualitatively similar to mm - ldl, since oxldl can escape clearance system by the scavenger receptors and is immunologically positive to measurement (figure 3). Heinecke et al . Reported that chlorotyrosine and nitrotyrosine, which are generated by the myeloperoxidase-(mpo-) dependent modification of proteins, accumulate in atherosclerotic lesions and in circulating ldl [55, 56]. Mpo, an enzyme that is secreted from macrophages and neutrophils, is involved in antibacterial host defense through the generation of reactive oxygen species . Mpo could oxidize ldl particles, and the resulting oxldl is taken up by macrophages to form foam cells . Since mpo is able to modify ldl even in the presence of plasma in vitro, mpo secreted by the macrophages and/or neutrophils of atherosclerotic lesions could induce oxldl formation . According to this hypothesis, when the foam cells were cultured for 7 days, 7-ketocholesterol and lipofuscin - like fluorescent materials were formed in the intracellular vesicles of the foam cells . Since oxidative changes were inhibited by antioxidants and by the lysosomotropic agent chloroquine, they proposed that ldl can be oxidized within the lysosomes of macrophages . We recently found the presence of oxldl in human gingival crevicular fluid (gcf). Periodontitis, a major cause of tooth loss in many countries, is a chronic infectious disease that leads to the destruction of the connective tissue attachments and alveolar bone . Recent studies have suggested that a correlation between atherosclerosis and periodontal disease exists [59, 60]. Gcf is a plasma exudate from the microcirculation of gingival tissue, which is located in the tiny space between the gingiva and tooth, and gcf secretion increases in patients with periodontal disease . Gcf contains several plasma proteins, including cytokines, hormones, and protective proteins to bacterial infection [6264], but the protein profile of gcf is different from that of peripheral plasma . Using paper points, up to 1 l of gcf samples can be easily collected without damaging the tissue, and the lipoprotein and cytokine concentrations can be investigated . Surprisingly, the oxldl level in the gcf samples was 14-fold higher than in the plasma oxldl levels obtained from the same subjects . We demonstrated that oxldl induces il-8 production in ca9 - 22 human gingival epithelial cells in culture, suggesting that oxldl in gcf could function as an inflammatory stimulant . If oxldl in gcf is simply transferred from circulation, the ratio of oxldl to ldl (same as oxldl levels) should be the same as that in plasma . These results suggest that the oxldl in gcf could be formed in periodontal pockets or local gingival tissues rather than simply being transferred from the plasma . It is an interesting view point that oxldl could be generated in extra - arterial tissues . Scavenger receptors are a set of receptors that bind to oxldl but not to native ldl particles . Macrophages and related cells, such as kupffer cells, express some of the scavenger receptors, such as sr - a and cd36, but it is now apparent that other types of cells such as endothelial cells also possess other scavenger receptors [1113]. Currently, scavenger receptors are considered to be a set of pattern recognition receptors that are expressed by the innate immune - defense system against various pathogens [67, 68]. Oxldl and/or the oxidized phospholipid components of oxldl are also recognized by toll - like receptors 2 and 4 [69, 70], indicating an overlap of the modification structures on oxldl and nonself patterns recognized by defense system . Scavenger receptors recognize oxldl particles, because their modified structures are similar to pathogen - related epitopes thereafter, oxldl is removed from the milieu by endocytosis . This process is a part of self - defense responses, however, it may cause accumulation of massive amount of lipids inside macrophages . In vivo evidence of oxldl accumulation in foam cells using an anti - oxpc monoclonal antibody, oxpc accumulation was clearly observed in the cytoplasmic space of foam - cell macrophages in the aortic lesions of whhl rabbits and atherosclerotic lesions in human coronary arteries obtained from an autopsy specimen . Oxpc and apob strictly colocalized in the foamy macrophages of the lipid - rich core of atherosclerotic lesions . Apob is a large protein with a molecular weight of approximately 500 kda . During oxidative modification, aggregation of ldl with conjugated apob however, partially degraded apob fragments were found in atherosclerotic lesions . On examining the ldl fraction extracted from lesions of the human carotid artery by western blotting with anti - apob monoclonal antibody, the size of apob proteins in the ldl extract was 100200 kda (figure 2(b)). Partial degradation of oxldl in macrophages was observed using macrophages in vitro, whereas acetylated ldl is completely degraded in the macrophages [71, 72]. Cross - linking of apob protein formed in oxldl may contribute to this resistance to proteolysis . Oxldl is possibly processed by lysosomal enzymes in foam cells . For future research, investigating whether plasma oxldl is partially degraded in patients with ami during the acute phase would be interesting . Since the temporal rise and fall of plasma oxldl levels after plaque rupture are believed to be caused by release from atherosclerotic lesions, such information would be an important clue for elucidating the origin of oxldl in the circulation . Another possibility that should be considered is whether lp(a) is a circulating oxldl . A prospective clinical study that examined the relationship between oxldl and future cardiovascular events showed a close overlap of plasma oxldl level and lp(a) concentration . The human ldl fractions were further separated into subfractions by density, and oxpc was found to be enriched with the subfractions that contained lp(a). Lp(a) is a unique ldl particle in which apob protein binds to another protein containing repeated kringle domains, apolipoprotein(a), through a disulfide bond . Apolipoprotein(a) has some similarities with plasminogen, but the physiological function of lp(a) is largely unknown . When radiolabeled oxldl is injected into rats via the femoral vain, most of the radioactivity is cleared from circulation within 3 minutes, and more than 70% of radioactivity goes to the liver . Oxldl has been documented to accumulate in atherosclerotic lesions in the aorta and coronary arteries of patients with cvds; however, the possible roles and pathological changes that occur in the liver of patients with cvds have not been studied in depth . The proteolytic capabilities of liver cells may possibly be different from those of the macrophages in the vessel wall . Kupffer cells, the macrophage - like cells of the liver, express scavenger receptors, including sr - a . However, it was surprising that the clearance rate of oxldl from circulation in sr - a knockout mice was similar to that in normal mice . This suggests that there might be alternative receptors or systems for the uptake of oxldl in liver cells . Recently, li et al . Reported that both kupffer cells and sinusoidal endothelial cells in the liver endocytose oxldl and mm - ldl, and this process is mediated by the novel scavenger receptor stabilin-2 . Stabilins, specifically stabilin-1 and stabilin-2, were initially found as receptors for hyarulonan [77, 78]; however, they were identical to feel-1 and feel-2, which are reported to be endothelial scavenger receptors . These two stabilins are expressed in the sinusoidal endothelial cells of the liver but show different ligand specificities . Stabilin-1 binds to the both mildly and highly oxidized forms of ldl, while stablin-2 selectively binds to the highly oxidized ldl . When nonparenchymal cells, which contain both sinusoidal endothelial cells and kupffer cells, were incubated with these two forms of oxldl, the uptake of mildly oxidized form of ldl was observed only in endothelial cells, whereas both cells were capable of taking up highly oxidized ldl . The co - localization of oxldl and stabilins has been demonstrated by immunoelectronmicrography . These observations suggest that endothelial cells might have a significant role in the metabolism of circulating modified ldl . If mm - ldl represents the circulating form of oxldl, endothelial cells may be an important site of accumulation, metabolism, and further modification of mm - ldl (figure 4). Certain scavenger receptors, such as sr - a, cd36, and lox-1, have been extensively studied to determine their relationship to foam cell formation and atherogenesis . However, other receptors for oxldl may possess different tissue distributions and functions . Among scavenger receptors, srec and cl - p1 have been reported to be selectively expressed in endothelial cells [80, 81], but the physiological functions of these special receptors have not been clarified . To elucidate the clearance of oxldl, studies on the contribution of endothelial cells and the specialized receptors are required . Recent studies have suggested the plasma oxldl concentrations may change under prepathological and postpathological conditions . Oxldl may be transferred between tissues and plasma and does not merely accumulate in the lesions but is equilibrated between the tissues and circulation . Oxldl can be formed in various sites in addition to the tissue of the vessel wall . However, many unknowns remain to be elucidated regarding the metabolic fate of oxldl in the liver . A recent study pointed out that stabilins may have an important role in the recognition and clearance of oxldl and mm - ldl from circulation . The receptors working in the liver may be different from those of cells in vessel wall tissues . Further studies are needed to understand the in vivo behavior of oxldl and elucidate the contributions of oxldl and oxidative stress to the mechanism of atherogenesis.
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Cutaneous squamous cell carcinoma (cscc) is the second most common type of non - melanoma skin cancer (nmsc) globally, with a constantly increasing incidence . The known risk factors for cscc include aging, fair complexion, homosexual intercourse, chronic skin ulcers, burn scars, immune suppression, and exposure to ultraviolet light and chemical carcinogens . The vast majority of csccs are curable with simple surgical resection, and prognosis is usually favorable . However, approximately 4% of the patients develop metastatic disease and 1.5% eventually die from the disease . There are few treatments for patients with advanced csccs, primarily due to the lack of knowledge regarding genomic alternations that induce metastatic cscc . Therefore, it is important to identify specific molecules involved in metastatic cscc that can potentially become targets for diagnosis and enable the development of new therapy strategies . Angiogenesis, the formation of new blood vessels from pre - existing vasculature, plays a key role in tumor development, invasion, and metastases formation . Angiogenesis is a complicated process stimulated by a variety of factors, among which vascular endothelial growth factor (vegf) is crucial . Vegf is one of the most potent endothelial cell growth factors; it promotes formation of new blood vessels and stimulates vascular permeability of endothelial cells . Vegf initiates intracellular signal transduction pathways by specifically binding 2 transmembrane vegf receptors on endothelial cells . While initiating intracellular signal transduction pathways, vegf supplies the newly formed blood vessels with nutrients and oxygen . This induces aggressive local growth and metastases formation, resulting in poor prognosis and low survival rates of cancer patients . The vegf gene is located at chromosome 6p21.3 and consists of 8 exons and 7 introns . It is very polymorphic, with at least 30 functional single - nucleotide polymorphisms (snps) in the 5-untranslated region, 3-untranslated region (utr) and promoter . Single - nucleotide polymorphisms (snps) of vegf that can be found in the promoter or other regulatory regions may regulate vegf expression or activity . The involvement of vegf polymorphisms in cancer progression has been demonstrated in several types of tumors [1518]. Vegf polymorphisms have previously been described in solid carcinomas, containing lung cancer, non - hodgkin s lymphoma, cervical cancer, colorectal cancer, esophageal squamous cell carcinoma (escc) [10, 23], and oral squamous cell carcinoma (oscc) [9,2426]. The impact of the vegf snps on clinical outcomes of patients with cancer has been previously described . However, the impact of vegf polymorphism during follow - up has been much less studied . The -1154460c> t snp (rs833061) and -1154g> a snp (rs1570360) are located at the promoter region and may regulate promoter activity . However, the effect of vegf gene -460c> t snp (rs833061) and -1154g> a snp (rs1570360) on cscc risk and prognosis remains unclear . In the current study, the -460c> t snp (rs833061) and -1154g> a snp (rs1570360) within the vegf gene were detected in cscc patients; a regular follow - up was conducted for all the patients . This study aimed to further clarify the potential association of these 2 snps with the cscc risk and to evaluate the prognostic impact of vegf - related snps on cscc patients . They were treated in the department of surgery at shandong provincial hospital affiliated to shandong university between 2010 and 2014; the group included 55 men and 45 women . The ages of patients ranged from 34 to 76 years old, with the average age of 56.0410.41 years old . The control group contained 124 healthy subjects who came from our hospital s physical checkup center during the same period of time . Written permission was obtained from all the participants and the study was approved by the research ethics committee of shandong provincial hospital affiliated to shandong university . Peripheral venous blood samples (5 ml) were acquired from all the participants and stored at 20c until the extraction of genomic dna . Genomic dna was isolated with the qiaamp dna mini kit (qiagen, hamburg germany) according to the manufacturer s protocol . The primers of -1154460c> t were 5-cgagagtgaggacgtgtgtg-3(forward) and 5-attggaatcctggagtgacc-3(reverse); the primers for -11541154g> a were 5-tcctgctccctcctcgccaatg-3(forward) and 5-ggcggggacaggcgagcatc-3(reverse), purchased from sangon biotech (shanghai, china). The pcr reaction was performed in a 20-l reaction mixture containing 40160 ng dna template, 0.25 units taq dna polymerase (sangon biotech), 250 m each deoxyribonucleotide triphosphate (dntp) (sangon biotech), 0.5 m forward primer, 0.5 m reverse primer, and 1pcr buffer with 1.2 mm mgcl2 . The pcr reaction was carried out in a thermocycler (mj research, usa) and reaction conditions consisted of 94 c pre - denaturizing for 5 min, 94c denaturizing for 45 s, 61c annealing for 45 s, 72c extension for 45 s, and 72c continuous extension for 7 min after 35 cycles . The amplified products were digested with bshl236i and mnli restriction endonuclease (takara, japan) incubated at 37c for 16 h. the pcr and digestion products were analyzed with 3% agarose gels . Follow - up files were created for all the cscc patients upon admission, including complete pathological, clinical, and follow - up data . A regular follow - up of 2~5 years for 100 patients was performed by hospital visit, treatment, and telephone interview . Follow - up start time was defined as the discharge time of patients after system treatment and the follow - up deadline was january 2015 . Follow - up time was counted monthly until death or the end of the follow - up time . Hardy - weinberg equilibrium (hwe) was calculated with hardy - weinberg equilibrium theory (p = allele frequency, q=1p, p+q=1). The different distributions of allele frequencies and genotypes in the case and control group were analyzed using the chi - square test . Logistic regression analysis was used to calculate the odds ratios (ors) and 95% confidence intervals (cis). Kaplan - meier method was used to plot survival curves and the statistical differences were analyzed with the log - rank test . They were treated in the department of surgery at shandong provincial hospital affiliated to shandong university between 2010 and 2014; the group included 55 men and 45 women . The ages of patients ranged from 34 to 76 years old, with the average age of 56.0410.41 years old . The control group contained 124 healthy subjects who came from our hospital s physical checkup center during the same period of time . Written permission was obtained from all the participants and the study was approved by the research ethics committee of shandong provincial hospital affiliated to shandong university . Peripheral venous blood samples (5 ml) were acquired from all the participants and stored at 20c until the extraction of genomic dna . Genomic dna was isolated with the qiaamp dna mini kit (qiagen, hamburg germany) according to the manufacturer s protocol . Genotyping of the vegf gene -460c> t and -1154g> a polymorphisms were detected using pcr the primers of -1154460c> t were 5-cgagagtgaggacgtgtgtg-3(forward) and 5-attggaatcctggagtgacc-3(reverse); the primers for -11541154g> a were 5-tcctgctccctcctcgccaatg-3(forward) and 5-ggcggggacaggcgagcatc-3(reverse), purchased from sangon biotech (shanghai, china). The pcr reaction was performed in a 20-l reaction mixture containing 40160 ng dna template, 0.25 units taq dna polymerase (sangon biotech), 250 m each deoxyribonucleotide triphosphate (dntp) (sangon biotech), 0.5 m forward primer, 0.5 m reverse primer, and 1pcr buffer with 1.2 mm mgcl2 . The pcr reaction was carried out in a thermocycler (mj research, usa) and reaction conditions consisted of 94 c pre - denaturizing for 5 min, 94c denaturizing for 45 s, 61c annealing for 45 s, 72c extension for 45 s, and 72c continuous extension for 7 min after 35 cycles . The amplified products were digested with bshl236i and mnli restriction endonuclease (takara, japan) incubated at 37c for 16 h. the pcr and digestion products were analyzed with 3% agarose gels . Follow - up files were created for all the cscc patients upon admission, including complete pathological, clinical, and follow - up data . A regular follow - up of 2~5 years for 100 patients was performed by hospital visit, treatment, and telephone interview . Follow - up start time was defined as the discharge time of patients after system treatment and the follow - up deadline was january 2015 . Follow - up time was counted monthly until death or the end of the follow - up time . Hardy - weinberg equilibrium (hwe) was calculated with hardy - weinberg equilibrium theory (p = allele frequency, q=1p, p+q=1). The different distributions of allele frequencies and genotypes in the case and control group were analyzed using the chi - square test . Logistic regression analysis was used to calculate the odds ratios (ors) and 95% confidence intervals (cis). Kaplan - meier method was used to plot survival curves and the statistical differences were analyzed with the log - rank test . There were 55 men and 45 women in the patient group; 46 patients were under 60 years old and 54 patients were 60 years old or older (average age: 56.0410.41 years old; range: 3476 years old). There were 40 poorly - differentiated cases (the percentage of differentiated cells <50%), 34 moderately - differentiated cases (the differentiated cells between 5075%), and 26 well - differentiated cases (the differentiated cells> 75%), according to broders classification . Thirty - five cases with lymph node involvement and 65 cases without lymph node involvement were identified . The control group contained 124 healthy subjects attending the hospital physical checkup center during the same period . There were 66 males and 58 females in the control group; 59 subjects were under 60 years old and 65 patients were 60 years old or older (average age: 53.7911.89 years; range: 3078 years). Age and sex in the two groups showed no significant differences (p>0.05) (table 1). Genotypes of the vegf-460c> t and -1154g> a were successfully detected in all the dna samples using the pcr - rflp method . The homozygous t / t genotype remained uncut at 175bp; the homozygous c / c genotype was completely digested into 155 bp and 20 bp fragments; 175 bp, 155 bp, and 20 bp fragments corresponded to the heterozygous c> t genotype (figure 1). The 206-bp pcr product of vegf-1154g> a was digested with mnli restriction endonuclease and the g allele the homozygous a / a genotype was digested into 184bp and 22bp fragments; the homozygous g / g genotype was completely digested into 150 bp, 34 bp and 22 bp fragments; all 4 fragments (184 bp, 150 bp, 34bp, and 22 bp) corresponded to the heterozygous g> a genotype (figure 2). The distribution of the vegf-460c> t snp genotype in patients is presented in table 2 . The genotype frequencies of cc, ct, and tt in the well - differentiated group was 46.15%, 38.46%, and 15.38%, respectively; in the moderately - differentiated group, the genotype frequencies of cc and ct were 44.11% and 55.89%, respectively; the genotype frequencies of cc and ct were 95.00% and 5.00%, respectively, in the poorly - differentiated group . There were hardly any carriers of homozygote tt in either moderately- or poorly - differentiated grades . Differences between the 3 groups were significant (p<0.001). The genotype distribution of vegf-1154 g> a was also associated with histological differentiation of cscc (p<0.001). The genotype frequencies of gg in the well - differentiated group, the moderately - differentiated group, and the poorly - differentiated group were 96.15%, 47.06%, and 25.00%, respectively; the genotype frequencies of ga in the well - differentiated group, the moderately - differentiated group, and the poorly - differentiated group were 4.00%, 47.06%, and 60.00%, respectively . The genotype distribution of vegf-460c> t and vegf-1154 g> a showed no significant correlation with age, sex, or lymph node metastasis (all p>0.05) (table 2). The vegf-460c> t and -1154g> a genotype and allele distribution of the cscc patients and of the control group are presented in table 3 . The genotype distribution of the vegf-460c> t and -1154g> a polymorphism did not deviate from hardy - weinberg equilibrium (p>0.05). The ct and cc genotype frequencies of vegf-460c> t in the control group were 50.81% and 41.13%, which were significantly different from those in the patient group (31.00% and 65.00%, respectively) (ct vs. cc, or=0.39, 95%ci: 0.220.68, p=0.001). Compared to the cc genotype, the tt+ct genotype significantly decreased the risk of cscc (or=0.36, 95% ci: 0.210.63, p<0.001). The c allele frequencies in the control group and patient group were 66.53% and 80.50%, respectively; while the t allele frequencies in the control group and patient group were 33.47% and 19.50%, respectively; the differences in the 2 groups were statistically significant (t vs. c, or=0.48, 95%ci: 0.310.75, p=001). Comparing the genotype distributions of the vegf gene -1154 g> a polymorphism in cscc patients to that in the controls revealed no significant differences (p>0.05) and the differences in allele frequencies were also not statistically significant . The follow - up deadline was january 2015; the longest follow - up time was 60 months and the shortest follow - up time was 24 months . Eleven patients were lost during the follow - up period; the follow - up rate was 89% . For vegf-460c> t snp, the mean survival time of cscc patients with cc, ct, and tt genotype were 28.92, 53.54, and 58.75 months, respectively . The cc genotype was an adverse factor that negatively affected the survival time of cscc patients when compared with heterozygote ct and homozygote tt (p<0.001, figure 3). For vegf-1154 g> a snp, the mean survival time of cscc patients with gg, ga, and aa genotypes were 41.19, 36.41, and 23.88 months, respectively; compared to the gg and ga genotypes, the aa genotype had a significant correlation with decreased overall survival time (p=0.009, figure 4). There were 55 men and 45 women in the patient group; 46 patients were under 60 years old and 54 patients were 60 years old or older (average age: 56.0410.41 years old; range: 3476 years old). There were 40 poorly - differentiated cases (the percentage of differentiated cells <50%), 34 moderately - differentiated cases (the differentiated cells between 5075%), and 26 well - differentiated cases (the differentiated cells> 75%), according to broders classification . Thirty - five cases with lymph node involvement and 65 cases without lymph node involvement were identified . The control group contained 124 healthy subjects attending the hospital physical checkup center during the same period . There were 66 males and 58 females in the control group; 59 subjects were under 60 years old and 65 patients were 60 years old or older (average age: 53.7911.89 years; range: 3078 years). Age and sex in the two groups showed no significant differences (p>0.05) (table 1). Genotypes of the vegf-460c> t and -1154g> a were successfully detected in all the dna samples using the pcr - rflp method . The homozygous t / t genotype remained uncut at 175bp; the homozygous c / c genotype was completely digested into 155 bp and 20 bp fragments; 175 bp, 155 bp, and 20 bp fragments corresponded to the heterozygous c> t genotype (figure 1). The 206-bp pcr product of vegf-1154g> a was digested with mnli restriction endonuclease and the g allele was replaced by the a allele with a loss of digestion site . The homozygous a / a genotype was digested into 184bp and 22bp fragments; the homozygous g / g genotype was completely digested into 150 bp, 34 bp and 22 bp fragments; all 4 fragments (184 bp, 150 bp, 34bp, and 22 bp) corresponded to the heterozygous g> a genotype (figure 2). The distribution of the vegf-460c> t snp genotype in patients is presented in table 2 . The genotype frequencies of cc, ct, and tt in the well - differentiated group was 46.15%, 38.46%, and 15.38%, respectively; in the moderately - differentiated group, the genotype frequencies of cc and ct were 44.11% and 55.89%, respectively; the genotype frequencies of cc and ct were 95.00% and 5.00%, respectively, in the poorly - differentiated group . There were hardly any carriers of homozygote tt in either moderately- or poorly - differentiated grades . Differences between the 3 groups were significant (p<0.001). The genotype distribution of vegf-1154 g> a was also associated with histological differentiation of cscc (p<0.001). The genotype frequencies of gg in the well - differentiated group, the moderately - differentiated group, and the poorly - differentiated group were 96.15%, 47.06%, and 25.00%, respectively; the genotype frequencies of ga in the well - differentiated group, the moderately - differentiated group, and the poorly - differentiated group were 4.00%, 47.06%, and 60.00%, respectively . The genotype distribution of vegf-460c> t and vegf-1154 g> a showed no significant correlation with age, sex, or lymph node metastasis (all p>0.05) (table 2). The vegf-460c> t and -1154g> a genotype and allele distribution of the cscc patients and of the control group are presented in table 3 . The genotype distribution of the vegf-460c> t and -1154g> a polymorphism did not deviate from hardy - weinberg equilibrium (p>0.05). The ct and cc genotype frequencies of vegf-460c> t in the control group were 50.81% and 41.13%, which were significantly different from those in the patient group (31.00% and 65.00%, respectively) (ct vs. cc, or=0.39, 95%ci: 0.220.68, p=0.001). Compared to the cc genotype, the tt+ct genotype significantly decreased the risk of cscc (or=0.36, 95% ci: 0.210.63, p<0.001). The c allele frequencies in the control group and patient group were 66.53% and 80.50%, respectively; while the t allele frequencies in the control group and patient group were 33.47% and 19.50%, respectively; the differences in the 2 groups were statistically significant (t vs. c, or=0.48, 95%ci: 0.310.75, p=001). Comparing the genotype distributions of the vegf gene -1154 g> a polymorphism in cscc patients to that in the controls revealed no significant differences (p>0.05) and the differences in allele frequencies were also not statistically significant . The follow - up deadline was january 2015; the longest follow - up time was 60 months and the shortest follow - up time was 24 months . Eleven patients were lost during the follow - up period; the follow - up rate was 89% . For vegf-460c> t snp, the mean survival time of cscc patients with cc, ct, and tt genotype were 28.92, 53.54, and 58.75 months, respectively . The cc genotype was an adverse factor that negatively affected the survival time of cscc patients when compared with heterozygote ct and homozygote tt (p<0.001, figure 3). For vegf-1154 g> a snp, the mean survival time of cscc patients with gg, ga, and aa genotypes were 41.19, 36.41, and 23.88 months, respectively; compared to the gg and ga genotypes, the aa genotype had a significant correlation with decreased overall survival time (p=0.009, figure 4). Angiogenesis is an important process for tumor development, invasion, and metastasis; it is stimulated by a variety of factors . Recently, several vegf snps have been identified and their important effects have attracted considerable attention . Correlation between vegf snps and susceptibility or prognosis in various forms of cancers has been reported in many studies . In the present study, the -460 c> t and -1154 g> a polymorphisms of vegf in 100 cscc patients and 124 healthy subjects were observed . In order to analyze the relation of vegf gene polymorphisms with cscc risk, the patients were stratified by age, sex, histopathological grade, and lymph node involvement . The results demonstrate that the genotype distribution of vegf-460 c> t shows no correlation with age, sex, or lymph node metastasis (all p>0.05), but there was association with histological differentiation of cscc (p<0.001). A significant difference was found between the genotype distribution and allele frequency of the vegf gene -460 c> t polymorphism of the control group and that of the patients with cscc . Furthermore, a higher percentage of the t allele and the tt homozygote were distributed in cscc patients compared to the controls . Therefore, we can reason that there was a much lower cscc risk for individuals with the ct heterozygote genotype in their vegf-460 c> t region, indicating that the vegf-460 c> t polymorphism could act as a candidate genetic biomarker in cscc patients . The association of the vegf-460 c> t polymorphism with clinical outcomes in cscc patients has not been evaluated . However, there were several studies concerning vegf-460 c> t polymorphism in other cancers . Indicated that the t allele and tt genotype of vegf-460 c> t are significantly associated with oscc . Borase et al . Demonstrated that the t allele and tt genotype of vegf-460 c> t can greatly increase the risk for oscc patients . In the analysis of a correlation between clinical characteristics and the vegf gene -1154 g> a polymorphism, the results demonstrate that the genotype distribution of vegf-1154 g> a shows no relation with age, sex, or lymph node metastasis (all p>0.05), but there was an association with histological differentiation of cscc (p<0.001). The decreased frequency of the vegf-1154 a allele (aa and ga genotypes) in cscc patients did not differ significantly from that of the control . In a previous study, kmmerer et al . Demonstrated that the genotype distribution of vegf-1154 g> a in oscc patients and controls had no significant difference . Unal et al . Examined 89 healthy subjects and 57 patients with laryngeal squamous cell carcinoma; they found that the -1154 gg genotype of vegf appears to be a higher risk for laryngeal scc patients . Prognostic factors play an essential role when come to dividing patients into different risk groups in order to predict outcomes and provide adequate therapy strategies . In the current study, the relationship between vegf gene -460 c> t polymorphism, -1154 g> a polymorphism, and the survival of patients with cscc was examined for the first time . The results demonstrate that the tt genotype of -460 c> t is a protective factor in comparison to genotypes of ct and cc, positively affecting the survival time of cscc patients (p<0.001). The aa genotype of -1154 g> a has a significant correlation with the decreased overall survival time in cscc patients when compared with homozygote gg and heterozygote ga (p=0.009). Observed that the vegf-460 c> t snp had no significant impact on survival in advanced osccs, while the -1154 aa genotype was associated with significantly worse survival in the univariate analysis . In contrast, supic et al . Found that the -1154 gg genotype may be a prognostic marker associated with significantly worse survival in advanced - stage oscc patients with a similar sample size . Other studies revealed the -1154 gg genotype to be an adverse survival factor in colorectal cancer, but it was associated with a higher overall survival of the 1154 a allele in advanced breast cancer . To sum up, results of clinical studies are inconsistent, possibly due to differences in ethnic groups studied, tumor locations, and sample sizes . In this study, only 2 snps in the promoter were analyzed, which might be a limiting factor . The results of 1 specific snp may be influenced by the other snps in the angiogenesis pathway . In addition, the sample sizes of the present study are somewhat small, even though similar studies were previously performed . Hence, more studies, including larger sample sizes and more snps, distributed not only in the promoter but also in the 5 and 3 un - translated region, should be conducted . Our data suggest that the vegf gene -460 c> t polymorphism is related to the risk of cscc, and both vegf gene -460 c> t polymorphism and -1154 g> a polymorphism are correlated with the prognosis of cscc patients . Therefore, the vegf gene -460 c> t polymorphism and -1154 g> a polymorphism may serve as a potential genetic marker for the risk and prognosis of cscc.
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Masturbation is considered a normal sexual behavior and occurs in 9094% of males and 50 - 60% of females at some point during their lives . However, masturbatory behaviors in children and infants area less commonly addressed issue in the literature and most findings are form case reports or case series . Childhood masturbation (cm) or gratification disorder, first described in 1909 by still, is characterized by the stimulation of the genitalia and has been reported to begin at 2 months of age with a peak of occurrence at 4 years and adolescence . The diagnosis of this condition is difficult since masturbatory activity in this age group usually does not involve genital manipulation . The most commonly reported manifestations of cm include dystonic posture, grunting, rocking, facial flushing and sweating, friction of thighs, and pressure on the perineum . Although there are no definite criteria for the diagnosis of cm, studies have reported several common features in these children such as no alteration in consciousness, cessation with distraction, normal examination, normal electrocardiography (eeg), and normal neuroimaging . The concept of cm is deemed a normal behavior by many authors, although the boundary between normal and abnormal conditions as well as the role of environmental and individual factors is unclear . Genital irritation due to any causes as well as sexual abuse is well - determined associations . The onset of cm is reported to be associated with a genitourinary tract infection or a stressful life event, and sleep difficulties have been more common among these children . Cm is commonly misdiagnosed as epilepsy, movement disorders such as dystonia or dyskinesia, abdominal pain, or colic . Children may be referred due to suspected seizures or movements by other physicians or description of strange episodes or attacks by parents . Reports show that many of these children have undergone extensive investigations such as magnetic resonance imaging, electroencephalography (eeg), intravenous pyelography, small - bowel biopsy, and gastrointestinal barium swallow and some cases have been treated with antiepileptic agents before the establishment of a diagnosis . Several studies have suggested home video taping as a helpful tool in making the diagnosis . We have followed up children with masturbatory behaviors and collected data for the past 3 years . This study is the first and only study in iran to investigate the largest series of cm cases . We aimed to probe into the association between the presence of mucus in urine and cm in otherwise healthy children and evaluate its value as a diagnostic test . This cross - sectional study recruited all outpatient infants and children suspected of seizure or epilepsy referred to the pediatric neurology clinic of imam khomeini hospital, a major referral university hospital in the iranian capital, tehran, between june 2008 and september 2011 . The diagnosis of gratification disorder was established through direct observation during hospitalization or home video tapes . Of 623 children referred to the clinic within the study period, 359 were found to have cm and were considered group a. the rest of the children (n=264) were diagnosed with various other disorders and were assigned to group b. informed written consent was obtained from the parents, and the study protocol was approved by the ethics committee of tehran university of medical sciences . The diagnosis of masturbation was made if the child had several episodes of at least one of the following characteristic masturbatory activities, provided the cessation of the episode with distraction and no change in consciousness: 1) color change as flushing and sweating, 2) extremity posturing such as arm twisting or extension, clenched fists, hip flexion or extension and/or hip adduction, and pressing the suprapubic region against something or applying manual pressure, 3) head and neck posturing such as staring, neck twisting, and open mouth, and 4) vocalization as quiet grunting and/or heavy breathing and tachypnea . Masturbatory behaviors in the children were observed, and the presence of each of these 4categories of activities was recorded . The rest of the collected data included demographic characteristics, a detailed history, complete clinical and neurodevelopmental examinations, laboratory findings (particularly urine analysis), and eeg . Patients with developmental delay, any tanner stages of puberty, neurodevelopmental disorder, urinary tract infection (positive urine culture), or kidney stone were excluded from the study . A clean - catch urine sample was collected from each child for analysis and culture after the diagnosis of masturbation was confirmed . Statistical analysis was performed using statistical package for the social sciences (spss), version 16 . Fisher s exact test was used to compare the study parameters between the 2 groups, and the pearson s correlation test (cohen s kappa [] values) was utilized to determine the association between mucus in urine and the clinical findings of masturbation (the score of the masturbatory activity). Data on 623 children below 12 years of age and at a mean age of 40 months (359 in group a and 264 in group b) were analyzed . Characteristics of the two groups there were no significant differences between the 2 groups regarding these characteristics . Mucus in urine (microscopic finding) was positive in 357 (99.44%) of the 359 children in group a, while only 22 (8.3%) of the 264 children in group b were mucus positive in urine analysis . A significant correlation was found between the presence of mucus in urine and the 4 main categories of masturbatory behaviors (p<0.001). The mean score of masturbatory activities was 1.240.50 in the infants (24 months old) and 1.400.63 in the children (> 2 years old). Eeg was found to be abnormal (pike slow, 2 - 3 htz and multi - spike pattern = abnormal eeg) in 201 of the 359 children (55.98%) in group a and in 131 of the 264 children (49.62%) in group b. no significant correlation was seen between abnormal eeg and masturbatory behaviors (p=0.32). Masturbation was mostly performed in prone and lateral decubitus positions in both of the children and infants . The most common category of masturbatory activity was self - manipulation in the children and the seizure - like pattern in the infants . Age was not associated with the masturbation score or the existence of any of the 4 main categories of masturbatory activities (p=0.15 in the infants <24 months old and p=0.93 in the children> 24 months old). In addition, no relation was seen between sex and the masturbation score (p=0.88 in the male infants <24 months old and p=0.47 and in the male children> 24 manths old). Cm is commonly recognized as a variant of normal behavior and can occur at any age . However, its diagnosis is difficult due to a variety of manifestations which can be misdiagnosed as several other medical disorders as well as the absence of typical genital manipulation in this age group . The present study analyzed one of the largest series of these children and is the only report from iran . To the best of our knowledge, this is the first study to assess the diagnostic value of a laboratory test in this condition . Three groups of children with masturbatory activity have been referred to us during the past3years in the pediatric neurology clinic of imam khomeini hospital: children with only masturbatory behaviors, children with suspected seizures or movements with normal eeg, and finally children with epileptic movements and abnormal eeg . The mean age in the 359 children with masturbation in our study was 40 months . Most of the subjects were older than 2 years, with a peak at 3.5 years . In addition, the score of masturbatory activity was higher in the children older than 2 years . It seems that children aged between 3 and 4 years begin to explore their body and learn that the stimulation of the genitalia provides a pleasurable sensation, hence the peak of this diagnosis at this age group . Although definite conclusions on sex differences in cm are difficult to make, in general it is believed that females masturbate less often than do males . However, in several previous studies, masturbation was more frequently reported in girls than in boys . In our study, the female - to - male ratio was 7:1 . It is not possible to conclude a higher prevalence of cm in girls only due to clinical / epidemiological findings of small series, particularly since several factors such as social and cultural beliefs and anatomical differences may play a role in these reported sex differences . It may be assumed that masturbatory behaviors in boys create less worry in parents to seek medical help due to the cultural and social backgrounds; this creates the referral bias, which can be more dominant in small study populations . Epilepsy and cm may coexist, which will render the diagnosis even harder . In our study, 201 of the 359 children were proved to have simultaneous epilepsy by eeg . Nonetheless, the rest of the case group commonly had been previously misdiagnosed as epilepsy and movement disorders, with many of them being already on antiepileptic medications . Consistently, other studies have reported frequent cases of cm misdiagnosed as epilepsy and some other medical disorders . Misdiagnosis is mostly due to the nonspecificity of the presentations as there may be merely repeated adduction of thighs or episodes of staring, shaking, or eidetic imagery or unvocalized speech with imagery individuals . Infants may seem unhappy during the episode and their jerky spasms may cause confusion with epileptic infantile spasm . Clinicians should include cm in the differential diagnosis of children referred for an evaluation of suspected seizure, epilepsy, or movement disorders or any strange behavior described by parents . Normal eeg during the fits, lack of response to antiepileptic agents, reviewing videotape recordings, and obtaining detailed history may shed light on the diagnosis of seizure - like episodes as masturbatory behaviors . This may prevent the unnecessary extensive investigations to which many of these children have been subjected in the past before the establishment of the diagnosis of cm . Once the diagnosis is made, management will be as simple as offering reassurance to the parents and providing them with education about the condition and behavior - modification methods . Apart from analyzing the epidemiological and clinical characteristics of the children with cm, we also tested a hypothesis in our study based on the author s years of clinical experience about this concept . We examined the association between the presence of mucus in otherwise normal urine analysis and cm to see whether it can be used as a complementary diagnostic test in this condition . Mucus in urine may be positive in other clinical conditions such as urinary tract infection and stones, which were ruled out in our study . Secreted from the vesical glands of the genitourinary tract, mucus in not generally seen in the urine samples of children under the age of 12 . According to our findings, the frequency of positive mucus in urine was significantly high among the children and infants with cm when compared with the other group . Moreover, it was significantly correlated with the presence of masturbatory behaviors in the 4 studied categories . Considering the direct observation of the mentioned masturbatory activities as the main method for the definite diagnosis of cm it is deserving of note that all the children with cm were set up for follow - up visits; however, the noncompliance of the parents precluded a sufficient data collection for analysis and report . Nevertheless as a whole, it was observed that parental education and instructions such as changing diapers more frequently, using cotton underwear, and distracting the child and engaging him / her in other activities during the episodes led to a reduced occurrence of the condition in most of the followed cases and the urine mucus test was found to become negative in all the cases in whom the masturbatory behaviors were stopped completely due to the parents report . Our findings suggest that the presence of mucus in urine can be used as an alternative laboratory test in cm in children below 12 years old and even in infants (24 months old).
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Autografted adipose tissue is potentially an excellent soft tissue space - filler because it is autologous, abundant, and biocompatible . Adipose tissue autografts have been used for facial plastic surgery, treatment of burn patients, breast implants and to improve phonatory closure of vocal folds [15]. However, the survival of adipose grafts can be quite variable and their volume is often decreased significantly after several months . An important determinant of long - term adipose autograft survival is thought to be short - term survival during the postimplantation period . Before a new blood supply to an implanted tissue is established, diffusion limits the delivery of oxygen and glucose and the removal of metabolites including co2 and lactate, particularly to the more central parts of a graft . These factors will impact the survival of key cell types present in the implant, including adipocytes, preadipocytes, and stem cells . The poor survival of adipose autografts reported in many studies may therefore relate to effects of limited mass transport (which by definition includes diffusion, convection, and other means of translocating molecules) during the postimplant period [68]. Ultrasound (us) technology has been used in biotechnology for improving of cell viability via its ability to increase mass transport [911]. Us enhances molecular motion via thermal and nonthermal effects (cavitation, microstreaming), and has been tested previously as a means for increasing blood flow and improving tissue healing and regeneration [12, 13]. Some previous applications of low - intensity ultrasound (lius) have been in the context of cartilage and bone regeneration or tissue engineering, where it was shown that lius stimulated increased cellular activity [14, 15]. In vivo, lius has been shown to improve re - ossification of slow - healing adult calvarial bone . Lius seems to have several mechanisms by which it increases mass transport, including cavitation (increases cell membrane permeability), acoustic microstreaming (enhances convection), and thermal warming . There is also some evidence that lius can have effects on gene expression and cell differentiation that might relate to direct effects on cellular mechanotransduction [1820]. In c2c12 cells, lius stimulation enhanced differentiation of c2c12 into osteoblast and chondroblast lineages via erk1/2 signaling . Lius therefore seems to be a promising modality to explore for its potential to improve adipose tissue graft viability by promoting cell survival . If it can increase the viability of adipose autograft tissue, it could potentially be applied in a noninvasive manner to autograft sites . For initial studies, we characterized the effect of lius on dye diffusion and temperature in tissue culture plates and then on proliferation, metabolic activities, and gene expression of cell and tissue models grown in these same plates . For cells, we used c2c12 muscle cells in 2d culture because this is a well - known cell type that responses to lius stimulation inducing differentiation of the cells [19, 21]. For tissue, we used lipoaspirated human abdominal adipose, which was cleaned and rendered into organoids . These, together with evidence for decreased expression of a marker for cell injury (tumor necrosis factor - alpha, tnf-) after lius, suggest that lius may have a role in promoting adipose autograft survival . (tucson, az). Multiwell tissue culture plates (12-well) were from becton dickinson (franklin lakes, nj). Two configurations were designed to visualize the effects of lius stimulation on diffusion (figures 1(a) and 1(h)). Two microliters of trypan blue dye were loaded in the center of wells containing 3 ml of dmem media (figures 1(b), 1(e), and 1(i)). For controls, the wells were left for 3 or 10 minutes without treatment (figures 1(c) and 1(d)). For stimulated groups, an ultrasound probe was placed 2 mm above the bottom of the indicated well (figures 1(a) and 1(h)), and 22 khz lius at a power level of 30 mw / cm (level 3 on unit) was applied for 3 (figures 1(f) and 1(j) or 10 minutes (figure 1(g)). Diffusion patterns were documented photographically . Acoustic pressure was measured using a miniature high sensitivity [211 db re 1 v/pa] hydrophone (type 8103, brel & kjaer sound and vibration measurement, denmark) calibrated over the frequency range from 0.1 hz to 180 khz . The high - impedance hydrophone signal was routed through a signal - conditioning charge preamplifier (model 2525, brel & kjaer, denmark) using 30 db of gain . Voltage at the preamplifier output was sampled with a pc - based oscilloscope (picoscope 4224, pico technology, uk). Acoustic measurements were carried out using the same setup as described for the dye visualization experiments . Wells of the plate were filled with 3 ml of deionized water . The emitting us transducer was mounted vertically facing the center of well 1 (figure 2(a)), and the hydrophone was immersed centrally in wells 2, 3, and 4 . To allow for background correction, the signal acquisition for each well was carried out twice: with and without ultrasonic excitation . Pressure (p) was calculated from acquired voltage readings (e) based on the manufacturer - provided charge sensitivity of the hydrophone (s = 92 10 pc / pa) and the output voltage sensitivity of the preamplifier (vu = 30 mv / pc) as c2c12 cells were seeded at 50,000 cells per well and incubated for 24 hours to permit cell attachment to the plate . On day 2, lius at 30 mw / cm was applied to a central well, as shown in figure 1(a), for 3 minutes or 10 minutes per day for 6 days . This regimen was chosen based on pilot experiments showing that the effects of lius on cell permeability are quite persistent, with changes lasting as long as 6 days . The stimulations were conducted at room temperature in a sterile hood with the culture plate firmly taped to the bench surface and then the cultures were returned to a standard 37c co2 incubator . All procedures involving human tissues were followed by the mgh guideline for human subject handling . Human abdominoplasty specimens (n = 2) were obtained from 2030 year - old female donors by excision and were then further processed by lipo - aspiration and washed with saline to collect smaller sized pieces of (~25 mm) adipose tissue, which we term organoids . The whole process took 4 - 5 hours and the samples were kept at 4c during such time . The adipose organoids were then placed in 12-well tissue culture plates (500 mg of tissue with 3 ml of culture media). Indirect lius was applied by placing the probe in culture medium in a middle well (figure 1(h), marked as us), while the tissue samples were placed in the outer wells (figure 1(h)). For 6 days, lius stimulation was applied for 3 minutes with a power level setting of 30 mw / cm . In the control plates, cell growth was measured by counting cells using a hemocytometer (vwr, bridgeport, nj). Two independent experiments (n = 2) with each in quadruplicate were performed (n = 4) (total n = 8). Glucose consumption and lactate production were measured on days 2, 4, and 6 by analyzing the culture media with stat critical care xpress analyzer (nova biomedical, waltham, ma). This assay measures the remaining glucose and production of lactate in the culture medium . Total dna content was measured using the picogreen assay (molecular probes, eugene, or). The adipose tissue was suspended in an extraction buffer (1 n nh4oh, 0.2% triton x-100) and treated with a bead beater (biospec products, bartlesville, ok). Next, the samples were centrifuged at 10,000 g for 10 minutes to remove debris, and the extract was used for the picogreen assay following the manufacturer's instruction . Cell viability was measured by reduction of alamarblue, a colorimetric indicator of reduction / oxidation . The cultured adipose organoids (500 mg) were incubated in 2 ml culture media and 10% (v / v) of alamarblue solution was added . The rate of reduction / oxidation of alamarblue was measured at 0, 2, 4, 6, 12, and 24 hours . At each time point, the absorbance of the culture medium was measured at 570 and 600 nm . The metabolic rate was calculated by an equation provided by the manufacturer using the measured absorbance . Total rna from the adipose specimens was isolated using the rneasy mini kit (qiagen, ca). Briefly, 1 g of total rna was incubated with oligo - dt and reverse transcription was performed . After the reverse transcription, 200 ng of the cdna were used for real - time pcr using taqman gene expression assay systems (applied biosystems, foster city, ca). Taqman universal pcr mix, tnf- and gapdh primers (assays - on - demand products) and cdna were mixed, and the pcr reaction was performed at 50c for 2 minutes, 95c for 10 minutes, and 40 cycles of 95c for 15 seconds and 60c for 1 minute . Gapdh was used as an endogenous control and the average cycle threshold (ct) of 3 replicates was used for the calculation . The fold difference of target genes was determined relative to the gapdh gene (relative expression = 2[(ctsample ctgapdh)]). Adipose organoids were fixed in 10% formalin for 24 hours, dehydrated with a graded series of ethanol, embedded in paraffin, and then sectioned at 5 m thickness . The c2c12 cells were fixed in 10% formalin for 2 hours and permeabilized with 0.1% triton - x100 for 30 minutes to increase antibody binding . The cells were incubated with 10% horse serum for 40 minutes at room temperature and incubated for 1 hour at 37c with antitropomyosin (sigma, st . Louis, mo) at a concentration of 1: 100 diluted in pbs containing 0.5% tween 20 and 1.5% horse serum . Fluorescence - conjugated secondary antibodies (fluorescence conjugated - anti - mouse igg, vector laboratories, ca, 1: 200 dilution) were used . A fluorescence microscope (nikon eclipse e600, japan) statistical analysis was performed using single factor anova with scheffe post - hoc test (p <.05). Lius enhanced the movement of dye (compared to controls) in wells adjacent to the lius probe for the two plate configurations shown in figures 1(a) and 1(h). In both instances, the dye in the wells adjacent to the lius moved away from the lius probe . After 3 minutes the entire aliquot of dye appeared to have translocated (figures 1(f) and 1(j)), while after 10 minutes the dye was more dispersed throughout the well, with the most concentrated dye in a radial orientation relative to the lius well (figure 1(g)). In the configuration where some wells were not adjacent to the lius probe (figure 1(h), wells 3, 4, 5 & 6), there was less effect of the lius on movement and diffusion of the dye . A hydrophone was used to measure the acoustic pressure generated by the ultrasound probe and examples of the recorded acoustic waveforms are shown in figure 2 . The well containing the lius probe (well 1) had an acoustic pressure of 1500 pa . The wells 2, 3, and 4 showed significant attenuation of the acoustic pressure relative to the well containing the probe . Pressure in those wells was correlated with their distance from the probe (well 2: 341 pa, well 3: 228 pa, and well 4: 150 pa). C2c12 cells grown with lius stimulation showed significantly increased proliferation (figure 3(a)) in a dose - dependent manner . Following 6 days of daily stimulation, the 10 minutes group showed a twofold increase in total cell number to control (p <.05). Cells with lius stimulation also showed higher lactate production at days 4 and 6 compared to controls (figure 3(b)). C2c12 cells stimulated with lius showed positive staining for tropomyosin (a muscle protein) but controls were negative for the stain (figure 4). Although the stimulated group had higher expression of tropomyosin than the nonstimulated groups, there was no significant difference in expression of tropomyosin in the 3 or 10 minutes stimulated samples . Following 6 days of daily lius stimulation for 3 minutes, total dna assays of the adipose organoids showed significantly higher growth in the wells 3 and 4 compared to the control (wells 3 and 4, p <.05). Wells 3 and 4 had mean dna levels that were double of that of the controls . Glucose levels in the culture media of wells 1 and 2 were reduced following lius, indicating enhanced glucose uptake and thus increased metabolic activity compared to the control (figure 5(b), p <.05). Cell viability tested by alamarblue reduction showed similar viability in all samples (figure 5(c)). First, in the nonstimulated group and in samples from wells 5 and 6, there appeared to be damaged adipocyte profiles and smaller diameter adipocytes (figures 6(e)6(g)). There was also an apparent increase in the fraction of stromal cells relative to the number of mature adipocytes (also seen for well 3, figure 6(c) and normal tissue figure 6(h)). The shape of the mature adipocytes appeared to be best preserved for organoids from wells 1, 2, and 4 . Expression of the tnf- gene in the lius stimulated group was lower than control for wells 1 and 2 (figure 7, p <.05). Dye spread data indicated that lius enhances mass transport of dye molecules (in this case trypan blue, which has a molecular weight of 961 da). After 10 minutes of lius, increased transport of the dye was observed, producing a remarkable radial dispersion (figure 1(g)) in wells adjacent to the lius probe . It is likely that the radial pattern is the result of reflection of the lius energy from the walls of the plastic tissue culture plate . These effects are probably highly dependent on the geometry of the setup and are not expected to appear in the ultimate clinical use of lius; however, they do serve as a reminder that the local levels of the ultrasound are influenced by local variations in tissue acoustic impedances . It also indicates that the lius intensity experienced by samples in the test wells was not uniform, though it does appear to have been affected as a function of proximity to the probe . Acoustic pressure measurement using hydrophone clearly demonstrated that wells adjacent to the probe received more lius stimulation than the wells located far away from the probe (figure 2). This data suggests that depending on location of the probe one can expect variable effects of lius, which eventually may be possible to manipulate cellular responses . In conventional 2d cell culture systems, culture medium is typically refreshed every 2 - 3 days; therefore, it is believed that nutrient and gas supply are not limiting factors for cell growth . It is not clear whether the increased metabolic activity, proliferation, and increased tropomyosin expression of lius - stimulated c2c12 cells can be explained by increased mass transport, since this is not thought to be a limiting factor for 2d cell culture . Another possibility is that mechanotransduction signaling in response to lius - induced shear stress could have stimulated changes in the c2c12 cells, similar to the mechanism proposed in lius experiments with chondrocytes . For example, zhou et al . Demonstrated that mechanotransduction of acoustic pulsed energy of ultrasound by skin fibroblasts is mediated by integrin receptors and the erk 1/2 and rhoa signaling pathways . In the context of using lius to enhance autograft survival, the possibility that the lius can directly activate signaling pathways in implanted cells needs to be taken into account . Tropomyosin expression was higher in the lius - stimulated groups than the controls, suggesting that lius stimulates differentiation of c2c12 cells under these conditions . Both the 3- and the 10-minute stimulated groups showed similar expression of tropomyosin, perhaps because longer exposure may predominantly stimulate cell proliferation rather than cell differentiation . The enhanced proliferation and uptake of glucose by the adipose organoids, as well as some indications of intact tissue morphology and reduced tnf- expression, the size of the organoids ranged from 25 mm in diameter and thus the interior of cells is beyond the distance of 0.2 mm that is deemed sufficient for diffusion of molecules in a tissue culture situation [25, 26]. In this study, we evaluated a single dose of lius on the organoids: 3 minutes of stimulation, applied once daily at an intensity of 30 mw / cm for 6 days following previous studies (data not shown). Obviously there are many parameters that can be adjusted, and further study is needed to optimize intensity, frequency, and duty cycle of the lius depending on cell and tissue types . The in vitro model presented here may be useful for evaluating some of these parameters prior to in vivo studies . Tnf- was used as an indicator of tissue damage, since release of this cytokine is typically associated with tissue injury . But tnf- also modulates other cellular function such as recruiting mesenchymal stem cells in myocardial infarction and induction of apoptosis in chondrocytes . The lius - related reduction in tnf- secretion seems to indicate that the lius had a beneficial effect on organoid survival . Young and dyson reported that us - treated tissue contains less macrophages (meaning less tnf-) than control . However, it has also been reported that lius stimulation of bone can increase tnf- levels participating in fracture healing . This discrepancy may be due to different stimulation settings (indirect versus direct stimulation), different stimulation intensity (22 khz, 1 mhz, 3 mhz), or different cell and tissue types (adipose tissue versus bone). Lius has been used to enhance bone healing, repair of damaged muscle, and differentiation of mesenchymal stem cells into osteoblasts or chondrocytes [19, 32, 33]. It will be important to test for in vivo effects of lius on damaged soft tissue to assess whether our in vitro results can be confirmed in vivo . Nonthermal effects such as microbubbles and microstreaming generated by lius can enhance ionic transport, permeability of the cell membrane and cellular activity [16, 34]. In our study, upon lius stimulation, c2c12 muscle cells clearly showed higher cell proliferation and differentiation than the nonstimulated groups . In addition, we obtained preliminary evidence that lius can influence the viability of adipose organoids in an in vitro organ culture model . Our data suggests that indirect lius stimulation may enhance c2c12 cell proliferation, metabolic activity, and differentiation of cells and tissues . It may be possible to implement lius stimulation to increase survival of implanted soft tissue.
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Hydroxyl radical attack on dna most frequently leads to base damages that result in generating a range of derivatives [13]. Aerobic organisms, within the course of evolution, developed a range of adaptive mechanisms inducing synthesis of anti - oxidative enzymes and/or enzymes repairing oxidative damages of dna [57]. Oxidative damage to nucleic acid has been associated with a number of pathologies including cancer and neurodegenerative and cardiovascular diseases [810]. To date, more than 20 different types of oxidative modifications of bases have been identified . Of all the modified bases, the processes which repair 8-oxogua are perhaps best understood, and may be regarded as a template for the processes which repair other lesions . To combat the deleterious biological effect of the presence of 8-oxogua, cells have developed specific mechanisms to remove this lesion from cellular dna . In mammalian cells, the first level of this protection is human mut t homologue (hmth1) which hydrolyses 8-oxodgtp (a potential substrate for dna polymerase), thereby eliminating it from the nucleotide pool . The second level of defence is specific glycosylases that initiate base excision repair (ber). Finally, human mut y homologue (hmyh) removes adenine that is mis - paired with 8-oxogua . Most recently, we proposed that nucleotide excision repair (ner), which involves the removal of a lesion - containing oligonucleotide, may compliment the go system, based upon evidence that oxidative dna damage may be repaired by this route . However, there is very little evidence that 8-oxodg is a direct product of dna repair itself (ie, released as the deoxynucleoside, rather than the base, from dna). It is generally accepted that products of cellular repair of oxidatively damaged dna, such as modified bases and nucleosides: 8-oxo-7,8-dihydroguanine (8-oxogua) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodg) are excreted in urine . An assay was performed to analyze levels of 8-oxogua and 8-xoxdg in urine samples with regard to diet . In case of the examined group, a conclusion was drawn that diet does not determine excretion of these biomolecules in urine . In another study, different amounts (up to 25 mg) of 15n labeled oxidatively modified dna were absorbed orally by volunteers . Throughout 2 weeks, blood and urine samples were collected . No 15n 8-oxogua or 8-oxodg were detected either in urine and or in dna of monoclonal cells in venous blood obtained from the same subjects taking part in the study, demonstrating that diet has no influence on the level of these damages . In both known works by faure et al . And erhola et al ., no rise of levels in 8-oxodg excretion in urine was observed despite unequivocal evidence for mass reduction of treated tumors . In certain reports, a clear rise of 8-oxodg in urine excretion was observed after radio - chemotherapy or radiotherapy itself [2123]. However, measuring excretion of repair product in urine exclusively may be misleading since it provides no information on the oxidative state of the organism (damage rate vs. repair rate) in cellular dna and reports only the mean value of damage repair in the past . The aim of the work was to investigate whether the levels of markers of oxidative damages of dna: excreted in urine 8-oxo-7,8-dyhydro-2-deoxyguanosine (8-oxodg) and 8-oxo-7,8-dyhydroguanine (8-oxogua) as well as the level of 8-oxodg in dna of venous blood leukocytes differ in a population of healthy subjects when compared with cancer patients . Hydroxyl radical attack on dna most frequently leads to base damages that result in generating a range of derivatives [13]. Aerobic organisms, within the course of evolution, developed a range of adaptive mechanisms inducing synthesis of anti - oxidative enzymes and/or enzymes repairing oxidative damages of dna [57]. Oxidative damage to nucleic acid has been associated with a number of pathologies including cancer and neurodegenerative and cardiovascular diseases [810]. To date, more than 20 different types of oxidative modifications of bases have been identified . Of all the modified bases, the processes which repair 8-oxogua are perhaps best understood, and may be regarded as a template for the processes which repair other lesions . To combat the deleterious biological effect of the presence of 8-oxogua, cells have developed specific mechanisms to remove this lesion from cellular dna . In mammalian cells, the first level of this protection is human mut t homologue (hmth1) which hydrolyses 8-oxodgtp (a potential substrate for dna polymerase), thereby eliminating it from the nucleotide pool . The second level of defence is specific glycosylases that initiate base excision repair (ber). Finally, human mut y homologue (hmyh) removes adenine that is mis - paired with 8-oxogua . Most recently, we proposed that nucleotide excision repair (ner), which involves the removal of a lesion - containing oligonucleotide, may compliment the go system, based upon evidence that oxidative dna damage may be repaired by this route . However, there is very little evidence that 8-oxodg is a direct product of dna repair itself (ie, released as the deoxynucleoside, rather than the base, from dna). It is generally accepted that products of cellular repair of oxidatively damaged dna, such as modified bases and nucleosides: 8-oxo-7,8-dihydroguanine (8-oxogua) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodg) are excreted in urine . An assay was performed to analyze levels of 8-oxogua and 8-xoxdg in urine samples with regard to diet . In case of the examined group, a conclusion was drawn that diet does not determine excretion of these biomolecules in urine . In another study, different amounts (up to 25 mg) of 15n labeled oxidatively modified dna were absorbed orally by volunteers . Throughout 2 weeks, blood and urine samples were collected . No 15n 8-oxogua or 8-oxodg were detected either in urine and or in dna of monoclonal cells in venous blood obtained from the same subjects taking part in the study, demonstrating that diet has no influence on the level of these damages ., no rise of levels in 8-oxodg excretion in urine was observed despite unequivocal evidence for mass reduction of treated tumors . In certain reports, a clear rise of 8-oxodg in urine excretion was observed after radio - chemotherapy or radiotherapy itself [2123]. However, measuring excretion of repair product in urine exclusively may be misleading since it provides no information on the oxidative state of the organism (damage rate vs. repair rate) in cellular dna and reports only the mean value of damage repair in the past . The aim of the work was to investigate whether the levels of markers of oxidative damages of dna: excreted in urine 8-oxo-7,8-dyhydro-2-deoxyguanosine (8-oxodg) and 8-oxo-7,8-dyhydroguanine (8-oxogua) as well as the level of 8-oxodg in dna of venous blood leukocytes differ in a population of healthy subjects when compared with cancer patients . Analysis of daily excretion of 8-oxogua and 8-oxodg with urine was done in a study group consisting of 222 patients with malignant cancer (iii and iv degree of clinical stage). Leukocytes from peripheral blood samples for analysis of 8-oxodg level were obtained from 179 patients from among the study group . Control peripheral blood samples for analysis of 8-oxodg in leukocytes were obtained from 134 healthy volunteers . From this group, 85 urine samples were taken for the measurement of daily excretion of 8-oxogua and 8-oxodg in urine . The patients had various malignant tumors:, head and neck cancer (n=45), breast cancer (n=32), colon cancer (n=25), lung cancer (n=37), uterine cancer (n=15), ovarian cancer (n=39), testicular cancer (n=7), prostate cancer (n=11), gastrointestinal cancer (n=11). The patients were asked to abstain from vitamin supplementation for at least 1 month before the chemotherapy started and during the course of the treatment, and only these patients qualified . The control group was chosen in such a way that the following criteria matched the patient group: eating habits, age, body weight, sex, and smoking status . The study was approved by the medical ethics committee of the collegium medicum nicolaus copernicus university bydgoszcz, poland, (in accordance with good clinical practice, warsaw 1998), and all the patients gave informed consent . Venous blood samples (18 ml) from the patient and volunteer groups were collected . The blood was carefully applied on top of histopaque 1119 solution (sigma - aldrich inc . ; st . Louis, mo) and leukocytes were isolated by centrifugation according to the procedure specified by the manufacturer . Determination of 8-oxodg by the mean of hplc / ec technique was as described previously . Urine sample preparation, hplc purification and gc / ms analysis were conducted as described earlier . Mann - whitney testing for independent groups with abnormal distribution was performed . For normal distribution, analysis of daily excretion of 8-oxogua and 8-oxodg with urine was done in a study group consisting of 222 patients with malignant cancer (iii and iv degree of clinical stage). Leukocytes from peripheral blood samples for analysis of 8-oxodg level were obtained from 179 patients from among the study group . Control peripheral blood samples for analysis of 8-oxodg in leukocytes were obtained from 134 healthy volunteers . From this group, 85 urine samples were taken for the measurement of daily excretion of 8-oxogua and 8-oxodg in urine . The patients had various malignant tumors:, head and neck cancer (n=45), breast cancer (n=32), colon cancer (n=25), lung cancer (n=37), uterine cancer (n=15), ovarian cancer (n=39), testicular cancer (n=7), prostate cancer (n=11), gastrointestinal cancer (n=11). The patients were asked to abstain from vitamin supplementation for at least 1 month before the chemotherapy started and during the course of the treatment, and only these patients qualified . The control group was chosen in such a way that the following criteria matched the patient group: eating habits, age, body weight, sex, and smoking status . The study was approved by the medical ethics committee of the collegium medicum nicolaus copernicus university bydgoszcz, poland, (in accordance with good clinical practice, warsaw 1998), and all the patients gave informed consent . Venous blood samples (18 ml) from the patient and volunteer groups were collected . The blood was carefully applied on top of histopaque 1119 solution (sigma - aldrich inc . ; st . Louis, mo) and leukocytes were isolated by centrifugation according to the procedure specified by the manufacturer . Determination of 8-oxodg by the mean of hplc / ec technique was as described previously . Urine sample preparation, hplc purification and gc / ms analysis were conducted as described earlier . Mann - whitney testing for independent groups with abnormal distribution was performed . For normal distribution, significantly elevated levels of 8-oxogua and 8-oxodg excreted in urine daily as well as 8-oxodg in dna of leucocytes in venous blood were observed in cancer patients as compared with healthy subjects, with considerable statistic significance (table 1). For the whole patient population (n=222), the median values of 8-oxogua and 8-oxodg in urine samples were 12.44 (interquartile range: 8.1420.33) [nmol/24 hr] and 6.05 (3.1215.38) [nmol/24 hr], respectively . The median values of 8-oxogua and 8-oxodg in urine samples of the control group (n=85) were 7.7 (4.6510.15) [nmol/24 hr] and 2.2 (1.72.8) [nmol/24 hr], respectively . The level of 8-oxodg in dna isolated from leukocytes in the patient group (n=179) and of the control group (n=134) was 4.93 (3.469.27) per 10 dg and 4.46 (3.825.31) per 10 dg, respectively . Certain amounts of oxidatively modified bases are present in every cell, reflecting the balance between ros attacking dna in the course of many metabolic processes and damage repair of these molecules by specific enzymes repairing dna . It is not known at present what is the endogenous level of these potentially mutagenic damages . According to the reports of authors applying different analytical techniques, the values range from 0.2 to several modifications/10 pairs of bases for healthy cells . They are removed in the process of repair and excreted in urine in unchanged state . A balance between producing ros which induce oxidative dna damages and removing these damages was observed in a cell (background level). According to some authors, the levels of these modifications it is possible to estimate the extent of repair on the level of the whole organism while analyzing the amount of oxidative dna damages in urine . High values of oxidative damages of dna excreted in urine indicate an intensified level of oxidative stress, but they may also reflect high efficiency of repair systems of these damages (oxidative stress may be high, yet repair mechanisms remove its effects). However, combining the background level specific for every patient and analyzing 8-oxogua and 8-oxodg in excreted urine may clearly reflect information about dna repair mechanisms . The results presented in the present study comparing amounts of 8-oxogua and 8-oxodg excreted in urine daily as well as amounts of 8-oxodg in venous blood dna leucocytes in patients with diagnosed cancer when compared with healthy subjects demonstrate higher levels of these modifications in cancer patients (figures 13). Significantly elevated levels of analyzed markers of oxidative damage dna may reflect the oxidative stress situation which accompanies cancer . Several studies have analyzed 8-oxogua and 8-oxodg in urine in different types of cancer patients and control groups, reporting elevated level of 8-oxogua in urine of cancer patients and in a control group of smoking subjects, and the level of 8-oxodg in dna isolated from venous blood leucocytes was higher in the patient group . These findings suggest that in cancer patients repair mechanisms of oxidative damage to dna are less efficient (the concentration of modified nucleoside / base in urine reflects dna damages of the whole organism, and the level of 8-oxodg in cellular dna reflects balance between processes generating damages and the ability of their repair). Compared a group of cancer patients and healthy volunteers with regard to the amount of 8-oxogua and 8-oxodg excreted in urine, demonstrating elevated levels of oxidatively modified base in patients when compared with healthy subjects by 50% . Our work indicates the levels of these derivatives elevated to such an extent cannot be related to its increase in cancer cells exclusively . The results suggest that oxidative stress in patients with cancer, demonstrated by elevated levels of these modifications in urine, may be typical not only of affected tissue but also of other tissues and even the whole organism . From practical point of view, a test that would enable determination of background levels of basic markers of oxidative stress (8-oxogua and 8-oxodg in urine and 8-oxodg in dna in leukocytes), might be applied as an additional and helpful marker for early detection of the development of cancer.
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Lumbar degenerative spondylolisthesis (lds) is an acquired slippage of one lumbar vertebra on the lower one as the result of degenerative instability, in the absence of a defect in the pars interarticularis . The disease is frequently seen in middle and old aged female and patients may have no clinical symptoms . Most of the time, the symptomatic patients respond well to non - surgical treatments such as lifestyle modification (reducing environmental pain generators), medication, physical therapy, weight reduction, multidisciplinary pain clinics or epidural injection . In refractory cases with intolerable symptoms (a dramatic decline in quality of life, unresponsive to a reasonable trial of> 3 months conservative treatment, rest pain, progressive neurologic deficit, or sphincter disturbances) surgery, 70 - 80% of the surgically treated patients have a satisfactory outcome, but due to the continuing degenerative process the results get worse over time . Poor prognostic factors commonly quoted for the patients with surgical treatment, include age> 65 years, chronicity of symptoms> 24 months, instrumentation> 4 levels, inability to restore sagittal balance, comorbidities> 4, preoperative back pain more than leg pain, posterolateral fusion versus 360 degrees, intermittent claudication more than several hundred meters, previous surgery, andinability to fuse . These include indirect reduction alone, decompression alone, decompression plus lumbar fusion with or without instrumentation, decompression and slip reduction plus instrumented fusion . In this study, we aimed to evaluate the surgical outcome of degenerative spondylolisthesis with neural decompression, pedicular screw fixation, and posterolateral fusion . After local institutional review board approval (code number 88194) and based on standard error calculation, this study was carried out on 45 surgically operated patients with refractory lds from august 2008 to january 2011 . Our inclusion criteria were lds unresponsive to more than three months aggressive conservative treatment, progressive neurologic deficit (especially motor deficit) and a careful clinical and radiological evaluation that confirmed patients complains were due to the lds . We excluded those patients with associated significant comorbidities (like psychoneurotic disorders, advanced diabetes mellitus, severe untreated hip, or knee osteoarthritis, etc . ), previous lumbar spine surgery, underlying lumbar congenital or traumatic lesion, and a follow - up period of less than two years . Preoperatively, routine standing anteroposterior and lateral views of the lumbosacral spine and magnetic resonance imaging scan were obtained from all patients . This method describes the degree of slip as a percentage of the anteroposterior diameter of the top of the lower vertebra . One of the advantages of this method is its percentage expression; therefore, differences in radiological magnification and patient s body size do not distort the results . Correction rate was calculated as below: slip correction rate (%) = ((preoperative postoperative slip percentage)/preoperative slip percentage)100). The patients pain and disability were assessed by a 0 - 10 numerical rating scale (vas and odi) questionnaire version 2.1 . The surgical procedure was explained in simple terms to patients and they signed the informed consents . All the surgical procedures were performed by a single surgical team and in the same manner . Initially, efficient neural decompression, pedicular screw insertion, and posterolateral spondylodesis were carried out as routinely performed and then vertebral reduction tried . Throughout these years, we used a mixture of local bone graft besides matchstick allograft (10 pieces 5535 millimeters of freeze dried cortical cancellous matchstick, tissue regeneration corporation (trc), kish, iran) to achieve posterolateral fusion . At the time of screw insertion, depending on the amount of replacement needed, therefore, with tightening of the proximal screws, the slipped vertebra will come back to the original place . To do this, we first tightened both distal screws (usually l5 vertebral pedicular screws) to the longitudinal rods . Then, we applied mild distracting force between proximal and distal screws to facilitate the reduction maneuver and later we tightened the proximal screws to draw back the slipped vertebra (figure 1). 49-year - old man with l4-l5 lds presented with debilitating claudication and pain . One or two days after surgery, the patients began to walk with a rigid lumbosacral orthosis and slip percentages were calculated again . Postoperatively, they are followed - up by x - ray and physical examination at 6 weeks and 3, 6, 12 months, and then annually . At annual visits, the questionnaires were completed again . To confirm osseous union, we relied on observing the bony bridge between transverse processes on plain anteroposterior and ferguson radiographs . Statistical package for the social sciences (spss) software version 16 was used for statistical analysis, data management, and documentation . The pearson correlation coefficient was used to measure the linear dependence between the amount of vertebral reduction and clinical (odi) improvement . Initially, efficient neural decompression, pedicular screw insertion, and posterolateral spondylodesis were carried out as routinely performed and then vertebral reduction tried . Throughout these years, we used a mixture of local bone graft besides matchstick allograft (10 pieces 5535 millimeters of freeze dried cortical cancellous matchstick, tissue regeneration corporation (trc), kish, iran) to achieve posterolateral fusion . At the time of screw insertion, depending on the amount of replacement needed, therefore, with tightening of the proximal screws, the slipped vertebra will come back to the original place . To do this, we first tightened both distal screws (usually l5 vertebral pedicular screws) to the longitudinal rods . Then, we applied mild distracting force between proximal and distal screws to facilitate the reduction maneuver and later we tightened the proximal screws to draw back the slipped vertebra (figure 1). 49-year - old man with l4-l5 lds presented with debilitating claudication and pain . One or two days after surgery, the patients began to walk with a rigid lumbosacral orthosis and slip percentages were calculated again . Postoperatively, they are followed - up by x - ray and physical examination at 6 weeks and 3, 6, 12 months, and then annually . At annual visits, the questionnaires were completed again . To confirm osseous union, we relied on observing the bony bridge between transverse processes on plain anteroposterior and ferguson radiographs . Statistical package for the social sciences (spss) software version 16 was used for statistical analysis, data management, and documentation . The pearson correlation coefficient was used to measure the linear dependence between the amount of vertebral reduction and clinical (odi) improvement . We evaluated 45 patients that included 37 (82.2%) women and 8 (17.8%) men . The mean age of the patients at surgery and the mean follow - up period were 58.33.5 years (ranged 43 - 76) and 31.24.8 months (ranged 24 - 52), respectively . The distribution of the spondylolisthesis levels in our patients was as follows: l4 - 5 (35 cases, 75.5%), l5-s1 (3 cases, 6.7%), l3 - 4 (3 cases, 6.7%), l3-l4-l5 (3 cases, 6.7%), l2-l3 (1 case, 2.2%), and l4-l5-s1 (1 case, 2.2%). The mean pre- and postoperative clinical and radiological indices (at the last follow - up visit) are shown in table 1 . The mean slip correction rate was 52.211.6% (ranged 21 - 95%) with a mean loss of correction of 4.81.1% (ranged 0 - 11%). Slip reduction of more than 5% was obtained in 39 cases (86.7%), ranged 0 to 31% . Pre- and postoperative radiological and clinical characteristics of our patients odi: oswestry disability index, vas: visual analogue scale, comparison based on paired t - test pearson correlation between the amount of vertebral reduction and odi improvement was not statistically significant (r=0.15, p=0.322). Although regarding to positive r, one may initially conclude that with more obtaining of vertebral reduction, the more improvement in odi can be achieved; this correlation was not significant statistically . We did not encounter any significant intraoperative complications, but we had two superficial postoperative wound infections (4.4%), both in diabetic patients and both responded efficiently to local wound care, antibiotics, and blood sugar control . Postoperative implant failure (screw breakage at the distal level) was seen in only one patient due to asymptomatic pseudoarthrosis . None of the patients required reoperation . Our findings confirm vertebral reduction, neural decompression, posterolateral fusion, and instrumentation with pedicular screws and rods in surgical treatment of patients with refractory lds has resulted in a significant improvement in pain and disability . Although vertebral reduction was associated with improved function, we cannot find a significant correlation between these two indices statistically . We had carried out a relatively similar study on 23 patients with lds in 2008 and followed them up for an average period of 29 months . In that study, we treated them without any attempt on vertebral reduction . Other details of the research were completely alike and odi improved from preoperative 72.2% to 14.4% at the last postoperative visit . In comparison with that study, in the present research (the same surgical techniques, but with the accomplishment of vertebral reduction) odi improved from 71.6% to 28.7% . It seems that reduction maneuver in the present study could not lead to significantly better functional outcomes . In another retrospective study conducted by han et al ., they evaluated the clinical outcomes and loss of correction in 37 patients with low grade lds with a mean follow - up period of 36.4 months . For assessing pain and disability improvement, they also used vas and odi, but they also used computerized tomography beside a plain radiograph to appraise fusion status and loss of correction . In their study, they achieved a postoperative reduction rate of 76.4%, although in 34 cases (91.9%), 5.8% of correction was lost . At the last follow - up visit, odi and their complications included superimposed degenerative scoliosis (2 cases), upper adjacent segment retrolisthesis (1 case), screw breakage (1 case), and postoperative infection (1 case). In contrast to our study, they could gain more reduction rate, but their loss of correction was also higher . These authors finally recommended reduction, decompression, and posterolateral instrumented fusion, although limited loss of correction commonly occurs . He believed that this indirect force facilitated vertebral reduction . In a study on 32 patients, he and his colleagues momentarily instrumented the upper adjacent vertebra to apply distraction on the instrumented vertebrae and then lumbar interbody fusion was also performed . At the last follow - up visit (averaged 32 months), they reported 81% reduction rate, 100% bony union, and significant improvement in quality of life (assessed by short form 36 questionnaire). Bednar in 2002 reported the surgical outcome of lds in 56 patients treated by instrumented fusion and slip reduction without associated laminectomy . They attained clinical and imaging outcomes comparable to previous outcomes published in the literature had been achieved by in situ fusion and laminectomy . The author concluded that laminectomy may not be required in surgical treatment of lds . In the present study, laminectomy was performed routinely, but we also could not find a significant advantage for slip reduction in lds surgery . In 2006, all the patients were treated by decompression, posterolateral fusion, and stabilization and followed up for more than two years . Functional outcomes of surgery between the patients with bony union and pseudoarthrotic cases were similar and they recommended instrumented spondylodesis in the surgical treatment of these patients . There are many controversies about vertebral reduction, spinal sagittal rebalancing, and their relationship with surgical outcome of the patients . Overall, most authors seem to agree with re - establishment of the normal anatomy . In this regard, kawakami et al . Conducted a study about the effects of lumbar sagittal balance on clinical outcome of surgery in lds . They used l1 axis s1 distance as a radiologic index for evaluating the sagittal balance . They retrospectively assessed 47 patients with a mean follow - up period of 3.6 years and they finally concluded that in the patients with a l1 axis s1 distance more than 35 mm, a reduction of the vertebral slip may advance functional outcome of surgery . In the study we conducted, we could not gain a strong relationship between vertebral reduction and surgical results, although we did not have any neural injury as well . We did not use postoperative computerized tomography for careful evaluation of the fusion mass, although the osseous union is not meant to cure . It would have been better that we could compare two separate groups with and without slip reduction and follow them up for a longer period . The strengths of our study were its acceptable number of patients and follow - up assessment . In the future, it is strongly recommended that a randomized control trial study should be conducted . In conclusion, although spinal decompression with fusion and posterior instrumentation in surgical treatment of the patients with lds result in satisfactory outcome, vertebral reduction cannot significantly enhance the clinical improvement.
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Outcomes and quality of life (qol) assessment instruments are important for assessing the results of surgical and radiosurgical treatment, as well as comparing results across studies . Many of these instruments assess qol but not in a manner that addresses the cns symptoms / signs of specific tumor sites . This becomes increasingly relevant when dealing with tumors that tend to arise in specific intracranial locations which can lead to distinct symptom complexes with unique effects on qol that can be overlooked by less specific qol instruments . Recognizing these location - specific symptom complexes allow us the opportunity to personalize the qol assessment towards distinct patient groups . Accounting for some 20% of intracranial tumors, they are generally histologically benign and slow growing, but may become locally invasive . Due to the risk of tumor growth and development of focal neurological deficits from mass effect, standard treatment usually consists of surgical resection or stereotactic radiosurgery . Qol assessment provides information regarding efficacy of surgical / radiosurgical treatment and may serve as an important decision - making tool for patients and physicians who are weighing the risks and benefits of brain surgery, especially if their meningioma is of the benign variety . While there are a variety of outcome assessment tools currently available, they vary widely in terms of scope (post - surgery qol, post - cancer therapy qol, post - brain surgery qol, etc . ), and none are currently location - specific . Currently, the instrument most accurately targeted at assessing qol following brain tumor resection is the validated functional assessment of cancer therapy - brain (fact - br) [22, 23, 40]. The location - specific nature of clinical symptoms and signs of intracranial meningiomas provides an opportunity for an even more targeted qol assessment; one that takes into account site - specific symptoms . This would allow for a more detailed examination of an individual patient s qol based on tumor site and pre / post - surgery signs and symptoms . With these goals in mind, an important part of developing such an instrument was making it available freely in a web based platform so that patients could access the questionnaire on regular set intervals without the need for direct physician contact or instruction . The visiontree optimal care (vtoc) secure web - based portal allows for patients to enter the site through self - registration and complete the fact - mng electronically . Upon registration, the patient automatically receives an email with their secure login information providing ongoing access to the survey . Vtoc includes full reporting / tracking capabilities to query the data which can be exported to excel for additional analysis . Initial data was gathered by way of reviewing relevant literature (via pubmed) which discussed and examined intracranial meningioma surgical therapy as well as subsequent outcomes . With 38 papers [115, 1722, 2430, 3241] in hand meeting our search criteria, we reviewed the assessment instruments utilized in each . While all of the papers included outcomes, when it came to qol assessment, 2 of the 38 used sf36 [3, 19, 39] others used fact - br [5, 22, 40], and 8 used a more basic patient functionality - oriented karnofsky performance status (kps) comparison [10, 13, 28, 32, 34, 36, 37], and less than half (15 of 38 papers) utilized any qol instrument at all (fact - br, sf36, kps scale, or some other instrument). To address the issue of site specificity, we created fact - mng (functional assessment of cancer therapy - meningioma), an amalgamation of fact - br, sf36, and newly formulated questions addressing tumor site - specific signs and symptoms . Specifically, we started with fact - br as our base and added questions from sf36 that were more detailed in ascertaining the patient s physical capabilities (e.g., ability to perform light, mild, or strenuous activity without difficulty) (table 1). Our most significant modification was the creation and inclusion of questions that were relevant to the signs and symptoms that characterize one of 11 tumor sites: olfactory groove, tuberculum sella, optic nerve sheath, sphenoid wing, cavernous sinus, parasagittal, tentorium cerebelli, petroclival region, cerebellopontine angle, cerebellar convexity, and foramen magnum (table 2, 3).table 1sf-36 questions incorporated into fact - mngnot at alla little bitsomewhatquite a bitvery muchi am limited in performing vigorous activities, such as running, lifting heavy objects, participating in strenuous sports01234i am limited in performing moderate activities, such as moving a table, pushing a vacuum cleaner, bowling, or playing golf01234i have difficulty climbing several flights of stairs01234i have difficulty walking several blocks01234i have difficulty bathing or dressing myself01234table 2site - specific questions incorporated into fact - mngnot at alla little bitsomewhatquite a bitvery mucholfactory groove meningioma my sense of smell is altered01234 my sense of taste is altered01234 my vision is altered01234 my short term memory is worse01234tuberculum sellae meningioma my vision is altered01234 compared to before my surgery, my vision is improved01234 my short term memory is worse01234optic nerve sheath meningioma my vision is altered01234 compared with prior to treatment, my vision is improved01234sphenoid wing meningioma my vision is altered01234 compared with prior to treatment, my vision is improved01234 i have numbness on my forehead01234 i have numbness on my cheek01234 i have double vision01234 my short term memory is worse01234cavernous sinus meningioma my vision is altered01234 i have double vision01234 i have numbness on my forehead01234 i have numbness on my cheek01234 i have numbness on my chin01234parasagittal / falx meningioma my short term memory is worse01234 my leg is weak01234 my leg is numb01234 my arm is weak01234 my arm is numb01234 i have a blind spot in my vision01234tentorial meningioma my vision is altered01234 i have double vision01234 i have numbness on my face01234 my short term memory is worse01234 i have a blind spot in my vision01234petroclival meningioma my vision is altered01234 i have double vision01234 i have numbness on my face01234 my short term memory is worse01234 my coordination is affected01234 my arm is uncoordinated01234 my leg is uncoordinated01234 my hearing is worse than prior to treatment01234 my balance is worse than prior to treatment01234cerebellopontine angle (cpa) meningioma i have numbness on my face01234 i have pain in my face01234 my hearing is worse than prior to treatment01234 i have ringing in my ear on the side of surgery01234 i have weakness of my face01234 i have problems with dizziness01234 my balance is worse than prior to treatment01234 my coordination is affected01234 my arm is uncoordinated01234 my leg is uncoordinated01234cerebellar convexity meningioma my coordination is affected01234 my arm is uncoordinated01234 my leg is uncoordinated01234 my balance is worse than prior to treatment01234 my arm is weak01234 my leg is weak01234 my arm is numb01234 my leg is numb01234foramen magnum meningioma i have neck pain01234 my speech is slurred01234 i have trouble swallowing01234 my voice is hoarse01234 my balance is worse than prior to treatment01234 my walking is worse than prior to treatment01234 i have shoulder pain01234table 3fact - mngnot at alla little bitsomewhatquite a bitvery muchphysical well - being i have a lack of energy01234 i have nausea01234 because of my physical condition, i have trouble meeting the needs of my family01234 i have pain01234 i am bothered by side effects of treatment01234 i feel ill01234 i am forced to spend time in bed01234 i am limited in performing vigorous activities, such as running, lifting heavy objects, participaing in strenuous sports01234 i am limited in performing moderate activities, such as moving a table, pushing a vacuum cleaner, bowling, or playing golf01234 i have difficulty climbing several flights of stairs01234 i have difficulty walking several blocks01234 i have difficulty bathing or dressing myself01234social / family well - being i feel close to my friends01234 i get emotional support from my family01234 i get support from my friends01234 my family has accepted my illness01234 i am satisfied with family communication about my illness01234 i feel close to my partner (or the person who is my main support)01234regardless of your current level of sexual activity, please answer the following question . If you prefer not to answer it, please check this box and go to the next section i am satisfied with my sex life01234emotional well - being i feel sad01234 i am satisfied with how i am coping with my illness01234 i am losing hope in the fight against my illness01234 i feel nervous01234 i worry about dying01234 i worry that my condition will get worse01234functional well - being i am able to work (include work at home)01234 my work (include work at home) is fulfilling01234 i am able to enjoy life01234 i have accepted my illness01234 i am sleeping well01234 i am enjoying things i usually do for fun01234 i am content with the quality of my life right now01234additional concerns i am able to concentrate01234 i have had headaches01234 i have had seizure convulsions01234 i can remember new things01234 i get frustrated that i cannot do the things i used to01234 i am afraid of having a seizure (convulsion)01234 i have trouble with my eyesight01234 i feel independent01234 i have trouble hearing01234 i am able to find the right word(s) to say what i mean01234 i have difficulty expressing my thoughts01234 i am bothered by the change in my personality01234 i am able to make decisions and take responsibility01234 i am bothered by the drop in my contribution to the family01234 i am able to put my hands together01234 i need help caring for myself (bathing, dressing, eating, etc. )01234 i am able to my thoughts into action01234 i am able to read like i m used to01234 i am able to write like i m used to01234 i am able to drive a vehicle (my car, truck, etc. )01234tumor site - specific questionsolfactory groove meningioma my sense of smell is altered01234 my sense of taste is altered01234 my vision is altered01234 my short term memory is worse01234tuberculum sellae meningioma my vision is altered01234 compared to before my surgery, my vision is improved01234 my short term memory is worse01234optic nerve sheath meningioma my vision is altered01234 compared with prior to treatment, my vision is improved01234sphenoid wing meningioma my vision is altered01234 compared with prior to treatment, my vision is improved01234 i have numbness on my forehead01234 i have numbness on my cheek i have double vision01234 my short term memory is worse01234cavernous sinus meningioma my vision is altered01234 i have double vision01234 i have numbness on my forehead01234 i have numbness on my cheek01234 i have numbness on my chin01234parasagittal / falx meningioma my short term memory is worse01234 my leg is weak01234 my leg is numb01234 my arm is weak01234 my arm is numb01234 i have a blind spot in my vision01234tentorial meningioma my vision is altered01234 i have double vision01234 i have numbness on my face01234 my short term memory is worse01234 i have a blind spot in my vision01234petroclival meningioma my vision is altered01234 i have double vision01234 i have numbness on my face01234 my short term memory is worse01234 my coordination is affected01234 my arm is uncoordinated01234 my leg is uncoordinated01234 my hearing is worse than prior to treatment01234 my balance is worse than prior to treatment01234cerebellopontine angle (cpa) meningioma i have numbness on my face01234 i have pain in my face01234 my hearing is worse than prior to treatment01234 i have ringing in my ear on the side of surgery01234 i have weakness of my face01234 i have problems with dizziness01234 my balance is worse than prior to treatment01234 my coordination is affected01234 my arm is uncoordinated01234 my leg is uncoordinated01234cerebellar convexity meningioma my coordination is affected01234 my arm is uncoordinated01234 my leg is uncoordinated01234 my balance is worse than prior to treatment01234 my arm is weak01234 my leg is weak01234 my arm is numb01234 my leg is numb01234foramen magnum meningioma i have neck pain01234 my speech is slurred01234 i have trouble swallowing01234 my voice is hoarse01234 my balance is worse than prior to treatment01234 my walking is worse than prior to treatment01234 i have shoulder pain01234 sf-36 questions incorporated into fact - mng site - specific questions incorporated into fact - mng following its creation, fact - mng was converted into a web - based format (currently available for patients / physicians to download for free at https://www.optimalcare.com . In addition to the benefits of offering fact - mng digitally (streamlined data collection, storage, and analysis), the dynamic delivery offered by a web - based application allows patients to only answer questions pertinent to their tumor s site . At ucsf we plan to introduce patients to the web site and questionnaire with a small business card containing the web site url and suggested time interval for assessment post - operatively at 3, 6, 12, 18, 24, 36, 48, 60, and 72 months . Any additional intervention will re - start the assessment clock for the initial intervention and begin a new follow - up session . The vtoc system can be configured to release the fact - mng into the patient s portal at the specific time points noted above . An email alert is automatically sent to remind the patient to login and complete the survey at each time interval . The process is fully automated within vtoc using templates and calendar reminders to ensure accuracy and compliance . A subset of patients may not be proficient with or have access to computers, either due to educational differences, socioeconomic status, or as a result of their disease process and/or treatment . With qol assessment becoming increasingly important as an information, education and outcomes assessment tool for both physicians and patients, relying on an outcome instrument too general in scope may leave more detailed, but nonetheless pertinent, issues unexplored and unmeasured . While fact - br has been validated as an assessment tool pertaining to brain tumors in general, those tumors which associated with characteristic site - specific signs and symptoms readily lend themselves towards qol assessment with an even further refined scope . Intracranial meningiomas offer one such opportunity and, on account of their comprising almost one - fifth of all intracranial tumors, warrant the development of an outcome instrument with a narrower scope . Further examination of fact - mng should revolve around qualifying its validity as compared to the outcome assessment provided by other qol instruments (most notably fact - br) following meningioma surgical therapy . The authors of this paper plan to investigate the use of this instrument in a prospective fashion on their patient population and report on the results at a future date.
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First performed in 1989, laparoscopic cholecystectomy quickly became the gold standard for surgical extirpation of the gallbladder . Soon after, reports began to emerge of an increased incidence of significant bile duct injuries . Reported injury rates varied from 0.1% to 2.2% compared with 0.1% for open cholecystectomy . The increased rate of bile duct injury has been attributed to a steep learning curve for laparoscopic cholecystectomy . Subsequent referral to tertiary centers for repair of transected common bile ducts resulted in published series that outlined the mechanisms of injury . Risk factors for iatrogenic injury to the common duct appear to be the surgeon's inexperience, patient obesity, scarring and acute inflammation . A common factor in the reported cases of bile duct injury is the misidentification of common duct for cystic duct, resulting in resection of a portion of the common bile and hepatic ducts and a right hepatic artery injury . Precautions suggested to decrease the possibility of bile duct injury have included: routine cystic duct cholangiography, cholecystocholangiography, confirmed identification of the junction of the cystic duct with the common bile and hepatic ducts and identification of the junction of the cystic artery with the right hepatic artery . Our experience with laparoscopic cholecystectomy supports the importance of anatomic identification in the safe performance of the procedure and also suggests the presence of a useful and reproducible anatomic landmark which can help define a zone of safe dissection within the triangle of calot . This study is derived from a single surgeon's experience with more than 300 laparoscopic cholecystectomies performed since 1991 . All patients with acute or chronic gallbladder disease were considered for the laparoscopic approach with the exception of those with suspected gallbladder masses or neoplasm . The anatomy of the biliary tree was defined prior to division of the cystic duct and artery . Cholangiograms were abandoned only if the lumen of the cystic duct would not accommodate the epidural catheter used for this purpose . During 1996, patients were chosen in one of three representative categories as they randomly presented in the course of our practice . These were chronic disease, acutely inflamed gallbladder and intra - operatively identified aberrant anatomy . I.m ., a 67 year - old - female with biliary colic systems and ultrasound proven cholecystolithiasis, underwent outpatient laparoscopic cholecystectomy . Two 1 mm veins were demonstrated within the triangle of calot between the cystic duct and cystic artery (figure 1). Case i: acute chdeaystititis and multiple cystic veins m.b ., a 56-year - old female with biliary colic symptoms and cholecystolithiasis on ultrasound, underwent outpatient laparoscopic cholecystectomy . A small venous structure was found to be crossing it and extending to the gallbladder (figure 2). Further dissection showed the artery to be an aberrant right hepatic artery with a short cystic artery identified later ., a 49-year - old female, was admitted with acute abdominal pain, fever, leukocytosis and thickened gallbladder containing stones on ultrasound . Dissection within the triangle of calot demonstrated venous structures between the later confirmed cystic duct and artery (figure 3). Early in our experience with laparoscopic cholecystectomy, the presence of small tubular structures within the triangle of calot became evident as being necessarily divided to proceed with the dissection . Among these was a structure that presented as a bridge between the cystic artery and duct; the division of which, unless controlled, resulted in insignificant but annoying bleeding . It quickly became a standard part of the procedure to clip or cauterize this ves sel during the dissection . Its color and non - pulsatile bleeding when divided, indicated that this was a vein, ostensibly unnamed . It was subsequently determined that these venous channels were named as the cystic veins and their courses documented in early textbooks of anatomy . I.m ., a 67 year - old - female with biliary colic systems and ultrasound proven cholecystolithiasis, underwent outpatient laparoscopic cholecystectomy . Two 1 mm veins were demonstrated within the triangle of calot between the cystic duct and cystic artery (figure 1). M.b ., a 56-year - old female with biliary colic symptoms and cholecystolithiasis on ultrasound, underwent outpatient laparoscopic cholecystectomy . A small venous structure was found to be crossing it and extending to the gallbladder (figure 2). Further dissection showed the artery to be an aberrant right hepatic artery with a short cystic artery identified later . N.m ., a 49-year - old female, was admitted with acute abdominal pain, fever, leukocytosis and thickened gallbladder containing stones on ultrasound . Dissection within the triangle of calot demonstrated venous structures between the later confirmed cystic duct and artery (figure 3). Early in our experience with laparoscopic cholecystectomy, the presence of small tubular structures within the triangle of calot became evident as being necessarily divided to proceed with the dissection . Among these was a structure that presented as a bridge between the cystic artery and duct; the division of which, unless controlled, resulted in insignificant but annoying bleeding . It quickly became a standard part of the procedure to clip or cauterize this ves sel during the dissection . Its color and non - pulsatile bleeding when divided, indicated that this was a vein, ostensibly unnamed . It was subsequently determined that these venous channels were named as the cystic veins and their courses documented in early textbooks of anatomy . A consistent anatomic feature during laparoscopic cholecystectomy has been the presence of venous twigs that run parallel to the cystic duct and perpendicular to the common bile duct, bridging the gap between cystic artery and cystic duct . As the veins do not cross the common hepatic duct, their presence can serve as an anatomic feature of a safe dissection space between the important structures of the triangle of calot . During surgical residency a common question intended to " trip up " junior residents during division of the cystic artery, has been to ask them to define the cystic vein . It is usually pointed out to the less assured that there is no cystic vein per se, but that the venous drainage is through the bed of the liver . In contradistinction to this, however, grant's anatomy, in illustrating the veins of the extrahepatic bile passages and gallbladder (figure 4), clearly demonstrates venous drainage directly into the liver, and describes venous twigs which clinging to the passages .... join branches of the portal vein . Gray's anatomy of the human body likewise describes cystic veins which join at the neck of the gallbladder to form either single or double cystic veins which flow along the cystic duct and upward along the hepatic ducts . Textbooks of surgery, however, do not assign much importance to the venous drainage of the gallbladder . The textbook of surgery edited by sabiston reports only that the venous drainage of the gallbladder and extrahepatic ducts is into the portal vein . Schwartz's principles of surgery describes the venous drainage as variable and generally does not run parallel with the arteries . A prominent surgical atlas similarly omits all veins from it's anatomic diagrams and does not describe operative management of these structures during the procedure . One of the differences between laparoscopic and open surgery is that magnification of the anatomy gives a new perspective to the dissection process . The magnified field of view demands meticulous attention to operative detail lest bleeding from a source not significant during open cholecystectomy, completely obscures the field during laparoscopic cholecystectomy . This fractal * anatomic view uncovered the presence of venous structures within the triangle of calot, which, if not clipped or cauterized prior to division, would obscure the operative field . Only with experience did we find the presence of the cystic veins to be constant . During difficult cases, the cystic veins can serve as a landmark for that space between cystic duct, cystic artery and common bile duct, that allows for safe handling and isolation of these vital structures . A review of anatomy texts has shown that the cystic veins were known to classical anatomists . It was only later that, without apparent clinical relevance, a description of the cystic veins was omitted from surgical textbooks and their very existence devolved into a trick question for unsuspecting house staff . I do not doubt that experienced laparoscopic surgeons have encountered these venous structures and are dealing with them on a regular basis, but that they exist as a reliable anatomic feature of the region is not well appreciated . Its importance, however, may lie in the fact that the cystic vein can serve as a landmark during a difficult laparoscopic cholecystectomy to decrease the risk of inadvertent bile duct injury.
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Endoscopic endonasal transsphenoidal surgery (etss) has become a more common procedure in the treatment of midline skull - based lesions such as pituitary tumors [16]. Etss offers several technical advantages, including wider, multidirectional views of the operative field [7, 8]. In spite of these advantages, there is significant risk of injury to the surrounding vital structures, particularly when the tumors have extensively invaded into the sphenoidal sinus and when the bony components of the skull base have been destroyed . In such cases, it is critical to identify and monitor the vital structures by both preoperative simulation and intraoperative real - time detection . To date, we have reconstructed 3d - ct models from 2d - ct images to allow more accurate visualization of the anatomical orientation of the nasal cavity and paranasal sinuses . With these models, we can precisely locate vital structures, such as the internal carotid arteries (icas) and optic nerves, as well as the anatomical relationships between these structures and the sellar floor . However, in cases where the tumor occupies the paranasal sinus, where normal bony structures have been destroyed, or where the icas are buried in the tumor, only 3d - ct images are insufficient for identifying the exact location of the icas . In other cases, where the tumor has extensively infiltrated the cavernous sinus and largely extends into the suprasellar region, it becomes very difficult to identify the icas and the normal pituitary gland . To overcome these problems, we created a fusion model of 3d - ct and mri, which can demonstrate the positional relationship between the tumor and the sellar structures, including the icas . Recently, fluorescence angiography using an indocyanine green (icg) microscope has been utilized to confirm real - time blood flow [911]. Furthermore, angiography with an icg endoscope has been shown to be useful in verifying the patency of vessels hidden from microscopic view during etss [1216]. In the present study, we investigate the usefulness of a fusion model of 3d - ct and mri along with an icg endoscope as a multimodal assistant system in etss, particularly in cases requiring identification of the icas for safe and maximum resection of the tumor . All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . Thirty - five patients with sellar or intrasphenoidal sinus tumors were treated with etss at ehime university hospital from april 2014 to march 2015 . They included patients with pituitary macroadenoma (tumor size 10 mm), craniopharyngioma, rathke's cleft cyst, chordoma, and giant cell tumor . Neurological and endocrinological examinations were assessed preoperatively as well as immediately after surgery and at 3 months later . All patients had visual examinations including visual acuity and visual field assessed by goldmann perimeter . The diagnosis of acromegaly was based on clinical symptoms and the endocrinological requirements, consisting of an elevated level of insulin - like growth factor 1 (igf-1) compared to the age- and gender - matched value and insufficient suppression of growth hormone (gh) after loading 75 g oral glucose . The patient with prolactin (prl) producing adenoma was involved in the study because she could not take a dopamine agonist due to her myocardial disturbance . The radiologist created a fusion model from the reconstructed preoperative 3d - ct and mri of all patients . In addition, from january 2015, icg endoscope was introduced in our department . By the subsequent operation, during etss, icg was administered intravenously to the remaining 10 patients, and vital structures, including the icas, were visualized via the icg endoscope . Informed consent was obtained from all individual participants enrolled in the study, including the surgical procedure and potential risks of etss . Contrast - enhanced ct was performed immediately after injection of 70 ml of intravenous iodinated contrast medium (omnipaque injection, 350 mg i / ml; daiichi sankyo company, tokyo, japan) using a 64-multidetector row ct (brilliance 64; philips healthcare, andover, ma, usa and best, netherlands). The collimation was 64 0.625 mm and the gantry rotation time was 0.4 s. scan parameters were as follows: voltage, 120 kv; tube current, 300 mas (228 ma); field of view (fov), 250 mm; matrix, 512 512; and brain smooth (uv) filter . Mri studies were performed using a 3.0-t whole - body mr scanner (gyroscan achieva; philips medical systems, best, netherlands) and an eight - channel phased - array head coil . For each patient, a high - resolution anatomic data set was scanned using 3d spoiled gradient - recalled echo sequences (repetition time, 15 ms; echo time, 2.3 ms; flip angle, 10; matrix, 256 320; fov, 230 mm; thickness, 0.9 mm) with gadopentetate dimeglumine . Image processing of the acquired ct and mri was performed using a 3d advantage workstation volume share 4 (ge healthcare, waukesha, wi, usa). Step 2: set the fov to 18 cm and adjust the pituitary gland to the center of the monitor with other images (either axial, coronal, or sagittal). Step 3: set position and select the volume rendering front cut function (squarely whittle at image) and then cut from the tip of the nose to the end of virus ring . Step 4: confirm position and scope and then save . Step 5: overlay the ct volume - rendered (vr) image (cta image) obtained from the above process onto the mra image and the vr image obtained from contrast - enhanced mri . Step 6: for overlaying the image, perform (1) autofusion (overlay an image with automatic adjustment of fov and position information) or (2) manual fusion (overlay an image while checking the image and adjusting the triaxial x - y - z). Fusion image model data from mri and 3d - ct were stored in digital imaging and communications in medicine (dicom) format . A slide presentation file was prepared from digital snapshots taken at different phases of the simulated operation, and this file was used as a visual reference during the actual surgery . Based on images from the fusion model, we evaluated the structure of the sphenoidal sinus and location of the vital structures, including the bony prominences of the icas and optic canals buried under the invasive tumor, and made comparisons with the actual endoscopic views observed intraoperatively . The system has both optical type and magnetic field type and it is possible to choose a system according to an operative situation . For etss in the present study, the magnetic sensor was fixed to a mouthpiece that was made to order for each patient . Preoperative ct and mr images were taken for the navigation system and these data were transferred to the workstation of the navigation system . The icg compound (25 mg), purchased from daiichi - sankyo, was dissolved in 10 ml of sterile water . In 10 patients, 5 ml of the solution (12.5 mg of icg) was injected into a peripheral vein as a bolus during surgery, which was followed by flushing with 10 ml of saline . The maximum absorption and emission wavelengths of icg in water are 780 nm and 805 nm, respectively; in plasma, they are 800 nm and 825 nm, respectively . One was a rigid 0, 30, and 70 endoscope (4 mm in diameter, 11 cm in length; olympus, japan) that was used for etss with or without endoarm assistance . The other was an icg endoscope with the following features: a straightforward 0 telescope (5.8 mm in diameter, 19 cm in length), a tricam pdd 3-chip camera head, a tricam slii 3-chip camera control unit, and a cold light fountain d - light p (all from karl storz). The light source could be converted from white light to near - infrared light with a foot switch . A tricam pdd system was calibrated by white balancing under white light with no filter . We placed the icg endoscope in a suitable position under white light and then switched to near - infrared light, whereupon the fluorescent signals from the icg flow in the icas became visible . Statistical analysis was performed using fisher's exact test for categorical variables and analysis of variance for continuous variables . Two - tailed tests were performed for each scenario, and the significance level was set at p <0.05 . All analyses were performed using office excel 2013 software (microsoft, redmond, wa, usa). 35 patients in the present study included those with pituitary adenomas (n = 27), craniopharyngioma (n = 3), rathke's cleft cyst (n = 3), chordoma (n = 1), and giant cell tumor (n = 1) (table 1). The hormonal types of the patients with pituitary adenoma were nonfunctioning adenoma (n = 22), gh producing adenoma (n = 4), and prl producing adenoma (n = 1). Mean age at the time of surgery was 55.5 years (range, 1684 years). Subjects comprised 17 women (48.6%) and 18 men (51.4%). No significant difference in age was evident between male and female patients (p> 0.05). Preoperatively, visual impairment was seen in 14 patients of pituitary adenomas, one of craniopharyngioma, and one of rathke's cleft cyst . Two patients of pituitary adenomas, two of rathke's cleft cyst, and all three of craniopharyngiomas showed deficiency of anterior pituitary hormones (table 2). One patient showed cerebrospinal rhinorrhea that required repair surgery, decreased visual function was seen in one, and one needed hormone replacement therapy . Histological examination showed gh expression in four gh producing adenoma, prl expression in one prl producing adenoma, and no expression of all pituitary hormones in 22 nonfunctioning adenoma . Histopathology of craniopharyngioma and rathke's cleft cyst revealed a cytokeratin - positive adamantinomatous type and a feature of columnar epithelium, respectively . Postoperative mri showed sufficient resection of the tumors with no or little evidence of residual tumor tissue in 33 patients except for the patients with chordoma and giant cell tumor . Three patients of four gh producing adenomas showed normalization of gh secretion, and in the patient with prl producing adenoma the level of prl decreased to 33.5 ng / ml . Visual disturbance was immediately improved after surgery in eleven patients, but only one patient with pituitary adenoma showed deteriorated visual function after surgery (table 2). One patient with craniopharyngioma was in need of all pituitary hormones including antidiuretic hormone after surgery . We successfully created a fusion model of 3d - ct and mri in all patients . In the model, critical structures such as the icas, optic canals, the pituitary gland, and the tumor were completely reconstructed, and their spatial relationships were better visualized by successively deleting adjacent bony structure images . The intraoperative anatomy was consistent with the preoperative simulation that had been made based on the fusion models (figures 1(a) and 1(b)). However, two major issues can complicate visualization of the anatomical structures of the sphenoidal sinus: the presence of multiple onodi (sphenoethmoidal) cells and an invasive tumor mass occupying the sphenoidal sinus . The former was observed in 6 patients (17.1%), and the latter was observed in 7 patients (20.0%). The models revealed the entire course (c3c5) of the bilateral icas and the intracranial arteries by successively eliminating images of the sphenoidal bony structures from anterior to posterior, even if the icas were involved in the tumor . The fusion model of 3d - ct and mri was possible to be viewed in the operating room during surgery as multislice presentations on a computer monitor . The images of ct / mri fusion model were not created to be utilized for neuronavigation system . Compared to 2d images and 3d - ct models alone, the fusion model had the advantage of presenting images that much more clearly delineated the anatomical relationships between the sella, the surrounding vital structures such as the icas and optic canals, and the extensive infiltrating tumor . Icg was prepared by the anesthesiologist and administered on demand of the surgeon . In all patients, good identification with respect to the icas, cavernous sinus (cs), intercavernous sinus (ics), and normal pituitary gland was obtained . Intravenous icg (12.5 mg) was administered via bolus injection and followed by flushing with 10 ml of saline . At 10 to 15 seconds (s) after flushing, the bilateral icas were visualized as a strong fluorescent signal through the thin bone of the carotid prominence (figures 1(c) and 1(e)). A few seconds later, the cs was also visualized . At 30 to 40 s after flushing, the fluorescent signal of icg from the normal pituitary gland that had been extracapsularly dissected from the tumor came into view (figures 1(d) and 1(f)). We also used an electromagnetic navigation system to localize the anatomical structures, but icg endoscopic visualization was far superior to the navigation system, particularly just before opening the sellar floor and in the case of a little bleeding from tumors, because the icg endoscope allowed for real - time monitoring of the vital structures and displayed the anatomical relationships between the tumor and surrounding structures . A 49-year - old woman was diagnosed with a growth hormone producing pituitary adenoma 3 years before and she had been treated with an intramuscular injection of octreotide . Gadolinium- (gd-) enhanced mri showed a macroadenoma with inferior extension into the sphenoidal sinus . The sellar floor had been destroyed by the invasive tumor and the bilateral icas from the c3 to the c5 portions were completely encased by the tumor (figure 2(a)). We created the fusion model to make a preoperative plan for how to resect the tumor effectively and safely because it was difficult to identify the exact locations of the icas that were buried in the tumor with the 3d - ct model alone . Successive elimination of bony structures on the fusion image of 3d - ct and mri clearly revealed the location of the bilateral icas surrounded by infiltrating tumor (figure 2(b)). The intraoperative endoscopic view correlated with the images on the fusion model, but the exact location of the icas could not be identified due to the widely protruding tumor mass (figure 2(c)). Thus, the electromagnetic navigation system was used to identify the sellar structures, including the icas (figure 2(d)). Finally, we were able to resect most of the tumor that had extended through the destroyed sellar floor (figure 2(e)). Gd - enhanced mri showed a cystic lesion in the sella (figure 3(a)). A reconstructed 3d - ct and mri fusion model allowed localization of the icas, optic nerves, and the normal pituitary gland, which were observed through the thinning bone structures (figure 3(b)). Under the guidance of the 3d - ct and mri fusion model, we made a preoperative plan and decided how best to open the sellar floor, particularly to expose the upper part of the dura mater because the normal pituitary located in the sellar floor and the anterior - superior part of the sella was determined to be the appropriate site for opening the cyst wall . Introduction of the icg endoscope allowed visualization of the bilateral icas 10 s after icg flushing; the ics could be seen 20 s later, and the normal pituitary gland could be seen 25 s later (figures 3(d), 3(e), and 3(f)). The cyst contents were removed, and a part of the cyst wall was removed and used for histological examination . Postoperatively, the patient's visual function improved, although he still required hormone replacement therapy, including hydrocortisone and levothyroxine . A 59-year - old man visited our department after demonstrating gradually worsening visual field deficits . Coronal and sagittal gd - enhanced mri showed a macroadenoma involving bilateral icas with invasion into the sphenoidal sinus and destruction of the sellar floor (figures 4(a) and 4(b)). Reconstructed 3d - ct and mri fusion model images revealed the location of the optic canals and bilateral icas (figure 4(c)). However, during the actual etss procedure, the extensive tumor made it difficult to identify the exact location of the icas that were buried in the tumor . The endoscopic view after decompression of the tumor within the safety zone demonstrated residual tumor bilaterally, with the descending diaphragma sellae situated centrally in the surgical field (figure 4(d)). To confirm the exact location of the right ica and the normal pituitary gland, we utilized icg endoscopic visualization . The right ica was visualized after icg administration under the residual tumor (figure 4(e)), and the fluorescent signal persisted until visualization of the normal pituitary gland, which was observed at the posterior part of the diaphragma sellae (figure 4(f)). As a result, the tumor was removed as completely as possible without any complications, and the postoperative course was uneventful . In the current study, we successfully performed etss for 35 patients with pituitary tumors and intrasphenoidal sinus tumors using multimodal assistant systems, consisting of a fusion model of 3d - ct and mri and icg fluorescence endoscopy . These assistant systems enabled simulating the surgical procedure as a competent guide and visualizing the vital structures such as ica involved in tumors in real time, resulting in maximum tumor resection without serious complications . Etss is highly effective in the resection of pituitary tumors with suprasellar and/or infrasellar extension including midline skull base lesions [16]. Introduction of an endoscope in transsphenoidal surgery provides surgeons with a wide panoramic view and the use of multiangled endoscopes provides multidirectional views, facilitating much clearer visualization of the anatomic relationships between the sella and surrounding vital structures, such as the icas and the optic nerves . However, their anatomic landmarks are sometimes hidden by bony structures and extensive tumors, which can result in spatial disorientation during etss . To avoid any injury to the icas, to date, we have adopted a 3d - ct model to help clarify the anatomy of the nasal cavity and paranasal sinuses . With this model, we obtained information on the locations of the icas and optic nerves and the anatomic relationship between these structures and the sellar floor . However, in case of tumors infiltrated into paranasal sinus extensively, normal bony structures were destroyed and icas were involved in the tumor . In addition, the dura mater and the normal pituitary gland may also be indistinguishable from the invasive tumor . In those cases, we also applied a magnetic navigation system to monitor the exact location of the icas in both the cavernous sinus and intracranial subarachnoid space . While a neuronavigation system can direct the field of view to the exact site of interest, the usefulness of the system is limited because some vessels cannot be detected on preoperative mri scans . Thus, to perform etss safely, surgeons must be highly skilled, and multidisciplinary assistant systems for etss are required . The 3d - ct and mri fusion model in this study facilitates identification of the exact location of important structures, including the icas, optic nerves, and onodi cells, when the surgeon is preparing to perform etss . If preoperative planning is performed conventionally with 2d images or a 3d - ct model alone, a considerable amount of effort is required to obtain imaginary images that are as clear as those acquired via 3d reconstruction, in particular, when the tumors have grown extensively in the paranasal sinuses . The 3d - ct and mri fusion model provides the surgeon with a clearer method of orientation, which is critical for performing etss . Furthermore, the model can be optimized for preoperative surgical planning according to the individual anatomy of the patient . However, there are some cases in which this 3d - ct and mri fusion model may be less effective . For example, when the sphenoidal sinus is occupied by a downward extending tumor, bony structures, including the carotid prominence, cannot be identified . In such cases, it is very important to accurately identify the icas in the early stage of the surgery in order to resect the tumor without injury to the icas . To handle this situation, we have applied other methods in combination with this model, such as fluorescence detection of the icas by icg endoscopy . In recent years, endoscope - integrated icg video angiography has been introduced to assist with confirmation of the patency of vessels hidden from microscopic and plain endoscopic views [12, 13, 15]. Icg is an ideal agent for imaging vessels as it is tightly bound to plasma albumin, has a short half - life, and maintains an acceptable safety profile [14, 18]. In previous reports, the icas and the patent cs were clearly detected with icg endoscopy in real - time and at high resolution . Researchers reported that the icas appeared at 10 to 15 s after flushing with icg; the ics and cs were seen at 3 to 5 s after ica detection, and the normal pituitary was seen at 30 s after ica detection [1315]. In addition, litvack et al . Reported that adenomas were less icg - fluorescent than the normal pituitary gland . In the current study, we demonstrated that intraoperative use of icg endoscopy provides visualization of the exact location of the ica, cs, and ics in etss . Additionally, we demonstrated that icg endoscopy enables differentiation between normal pituitary gland tissue and tumor . On the other hand, it is limited in that icg fluorescent signals persist in solid tissues, so background signals might be high . Therefore, it is important to know what structures are to be inspected and how to interpret the findings . By combining icg endoscopy with our reconstructed 3d - ct and mri fusion models, we can operate on sellar and midline skull - based tumors safely by etss . Preoperative 3d - ct and mri fusion models along with icg endoscopy allow for distinct visualization of vital structures, such as the icas, in the case of tumors that have extensively infiltrated the sphenoidal sinus . The fusion images are useful for preoperative planning and for clear guidance during etss . In addition, the icg endoscope is a very useful tool for real - time monitoring during etss . These multimodal assistant systems may improve the efficiency of etss and facilitate maximum tumor resection without complications.
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T - cells of the human immune system recognize antigens as short peptide fragments (t - cell epitopes) derived from the degradation of proteins . Major histocompatibility complex (mhc) proteins play a vital role in the initiation and regulation of immune responses (14). Their primary function is to bind and subsequently present antigenic peptides on the cell surface for recognition by t - cells of the immune system . The recognition of t - cell epitopes is critical for the immune response to infectious, autoimmune, allergic and neoplastic disease . T - cell epitopes are important for the development of peptide - based vaccines (5). There is a great diversity of human leukocyte antigens (hlas; human mhc) genes with some 2000 known variants characterized to date (6). Hla proteins share 3d structure with main differences observed in residues that form the peptide - binding groove . Hla proteins that have small differences in their peptide - binding grooves and share similar peptide - binding specificities are grouped into hla supertypes (7,8). Promiscuous peptides those that bind more than one hla variant are prime targets for vaccine and immunotherapy development because they are relevant to higher proportions of the human population . Because of the large number of hla proteins, experimental approaches for identifying t - cell epitopes (from overlapping peptides that span the length of protein antigens) are time - consuming and costly, and thus not applicable for large - scale screening . Computer modeling methods can help to simulate the biological process of antigen presentation, minimize the number of experiments required, enable a systematic scanning for candidate mhc - binding peptides and thus speed up vaccine development (9). Multipred is a web - based system for the prediction of peptides that bind multiple hla alleles . Current implementation can predict peptides that bind hla proteins belonging to supertypes a2 and a3 (hla class i) as well as dr (hla class ii) and in future will be extended to other supertypes . The predictive engines implemented in multipred are hidden markov models (hmms) and artificial neural networks (anns). A novel data representation method enables multipred to predict peptides that bind to multiple hla alleles belonging to one hla supertype by a single prediction model per supertype . The predominant length of peptides that bind hla - a2 and - a3 (class i) proteins is nine amino acids (10). Hla - dr (class ii) proteins bind longer peptides through the core binding region, which is nine amino acids long (11). The training data comprise 3050 9mer peptide sequences (664 binders and 2386 non - binders) related to 15 variants of the hla - a2 supertype, 2216 9mer peptide sequences (680 binders and 1536 non - binders) related to eight variants of the hla - a3 supertype and 2396 9mer peptides (448 binders and 1948 non - binders) related to six hla - dr variants . These data are mainly from three sources, the mhcpep database (12), published articles and a set of hla non - binding peptides (v. brusic, unpublished data). For both training and prediction the data representation includes both the peptide and its binding environment (hla contact residues). This virtual peptide representation comprises both peptide residues and the environment for each residue of the 9mer peptides (13,14). To simplify the data representation and eliminate redundant information, for each hla supertype, we considered only those contact residues that vary across various hla variants and discarded the residues, which are conserved . In multipred, a three - layer backpropagation network with sigmoid activation functions was built for hla - a2 and -a3 supertype and a four - layer backpropagation network with a hyperbolic tangent sigmoid activation function between the two hidden layers and a sigmoid activation function between the second hidden layer and the output for hla - dr supertype . Various techniques, including optimization of ann architecture and balancing datasets, were explored to improve the prediction accuracy of the ann models (14). Multipred also has a first - order hmm as an alternative prediction engine (13). The user can select either the ann or the hmm model for prediction both methods have been optimized and show similar performance . The aroc is> 0.8 in all cases, indicating good prediction capability [see (13,14) for details on hla - a2 models, (15) for hla -a3 models, and v. brusic, a. sette, g. l. zhang, k. n. srinivasan, j. t. august and v. brusic, manuscript in preparation for hla - dr models . In addition to individual 9mer predictions, multipred also predicts immunological hotspots (regions of high concentration of 9mer promiscuous binders). We have developed two scoring schemes to identify immunological hotspots within antigens for hla classes i and ii supertype . The scheme for hla class i supertype is based on high - scoring individual 9mers within a window of 30 amino acids (15) and the scheme for hla class ii supertype is based on average scores of individual 9mers within a window of 15 amino acids . The selection of window lengths was based on a trial - and - error process . Window lengths of 15, 20, 25 and 30, were explored and the results were compared with the representative experimental results . The window length 30 was found to suit class i predictions and window length 15 to class ii predictions . The lengths outside these ranges are considered too short or too long as targets for experimental validation . The prediction performance of multipred for hla - a2 and -a3 hotspots was validated using experimental results from a systematic study of human papillomavirus type 16 e6 (p03126) and e7 (p03129) proteins (16). The prediction performance of multipred for hla - dr hotspots was validated using experimental results from systematic binding studies of overlapping peptides from myelin oligodendrocyte glycoprotein (mog) (caa88109), bee venom protein (1poc) and hepatitis c virus 1b protein (aab00216). The web interface of multipred uses a set of graphical user interface forms with a combination of perl, cgi and c background programs . The functions provided by multipred include (i) running predictions, (ii) model building, (iii) prediction accuracy evaluation and (iv) identifying consensus predictions among up to three sets of predictions on the same input protein sequence . Run prediction. The required input is the selection of supertype and prediction method (pre - defined ann or hmm). Alternatively, users can select a pre - defined model (built by model building function). By selecting the submit button users get to a sequence input page where the required input is a protein sequence and its name . The length of the input sequence must be between 9 and 2000 amino acids . If the input sequence contains symbols other than amino acids (space and carriage returns are allowed) or if the sequence is outside the length limits, an error message will be displayed . Protein sequence, which means the input sequence is treated as one single protein sequence and carriage returns are ignored . If users changed the sequence type to a list of peptide sequences, then sequences divided by carriage returns are treated as separate peptides . The detailed description on processing steps involved when the input sequence is a protein sequence or a list of peptides are available at and, respectively . The 9mer binding scores range from 1 to 9 (figure 1a), with scores 49 referring to predicted binders (8 or 9 referring to high, 6 or 7 to moderate, and 4 or 5 to low confidence of peptide binding). Multipred saves the prediction result and the users may note down the i d number of the saved jobs for the comparison of prediction results generated by different prediction models (figure 1a). Two scoring schemes to identify immunological hotspots within antigens were developed for hla classes i and ii supertype . The scheme for hla class i supertypes is based on high - scoring individual 9mers within a window of 30 amino acids (15). In the result table (figure 1a), sum is the sum total of the individual binding scores of a peptide to the mhc proteins, score 1 is the top 1 sum in a 30mer window (a 30mer window comprises 22 consecutive 9mer peptides). Score 2 is the average of the top 2 sum in a 30mer window . Score 5 are the average of the top 3, 4 and 5 sum, respectively, in a 30mer window . To show the user a clear view of the binding capacity of an input protein, scores 15 of all 30mer peptides of the input protein can be displayed as graphs, in which x - axis represents the starting position of a 30mer window and the y - axis represents score 1 (2/3/4/5) of the 30mer window . For example, in figure 1b, which is the graph of score 4 of the protein e6, the first three 30mer windows (starting at positions 1, 2 or 3) are 36.82 and the next two windows (starting at positions 4 or 5) have scores 39.50 . The following 13 30mer windows (starting at positions 619) have scores> 42, the recommended threshold for score 4 for hla - a2 ann models (figure 1a), indicating a predicted hotspot, which corresponds to an experimentally determined hla - a2 hotspot in e6 protein (16). To locate the individual 9mers with top binding scores in each 30mer window, the align function can be used . Figure 1c shows an example of the alignment view of the top four 9mers in each 30mer window . The default values on the web page are the recommended thresholds for score 4 . In a hla - dr prediction result table, average was calculated as the average of the sum within a 15mer window (seven consecutive 9mers make a 15mer window). When users select the input sequence as a list of peptide sequences, the input sequences separated by carriage returns or line breaks are treated as different peptides . All overlapping 9mers in each peptide are submitted for prediction . In the result tables, predicted binding scores are represented by the highest individual binding score of each input peptide . The predicted binding scores of individual 9mers in each peptide in the list are data not shown (figure 2a). To display the input peptides in the order of their binding scores, the user can use the function sort the result. In the result page (figure 2b), the input peptides are listed in descending order of their binding scores . To display the predicted 9mer binders from each input peptide, the user can use the function alignment view. In the result page (figure 2c), the 9mers with binding scores 4 are aligned with the input peptides . The predicted 9mer binders are displayed with the names of the hla alleles, which produced binding scores above the selected threshold . If the user has 9mer peptides with known binding affinities to proteins belonging to hla - a2, -a3 or -dr supertypes and wants to build his own prediction models, the user can use the model build function in multipred . The users have the option to use their data only, or combine their data with the existing multipred data and build the model on the server . Currently, users can expect to train an hmm model within 1 min while training of ann models may take up to 50 min (depending on the size of the training dataset)there are actually four anns trained in the background . The ann models trained by the same dataset are usually slightly different because the initial weights of networks are assigned randomly (14). To make the trained models more stable, the training is repeated four times, and four sets of weights are trained the predictions are the averages of these four predictions . When the model building request is submitted, an intermediate page (figure 3) will be displayed providing the result url can be bookmarked for later model retrieval . If the user has 9mer peptides with known binding affinities and would like to evaluate the prediction accuracy of a model with these peptides, the user can use the accuracy evaluation function of multipred . The system predicts the binding affinities of the input 9mers and calculates aroc of the predictions . For each supertype the comparison of predictions helps identify the most promising peptides picked up as predicted binders by multiple models . The user needs to input the individual result ids (up to three) to the system . The result ids must be predictions of the same protein and to the same hla supertype, if the result ids belong to predictions on different proteins, an error message will be displayed . The user can select the analysis of top 5 or 10% of the predicted binders . In the output table, top 5 or 10% predictions are displayed in the descending order of their binding scores . Several web - based systems have been developed and widely used for the prediction of mhc binders, such as syfpeithi (17), bimas (18), smm (19), mhcpred (20), rankpep (21), tepitope (22), netmhc (23) and svmhc (24). Although multipred is similar to them in its overall goal of predicting mhc - binding peptides, there are significant differences in both functionality and methodology . Bimas, mhcpred, rankpep and tepitope use quantitative matrices, and smm is based on an improved matrix - based algorithm called stabilized matrix method . Each of these methods uses one prediction model per mhc proteins, making them difficult to maintain and assess accuracy . Tepitope allows prediction of peptides to many different class ii proteins (using multiple prediction models), but it is not available through the web . Since hla proteins are highly polymorphic, promiscuous peptides that bind more than one hla protein are prime targets for vaccine and immunotherapy development because they are relevant to higher proportions of the human population . T - cell epitope hotspots are highly promising regions as targets of t - cell immune responses, which are of interest for experimental validation as potential vaccine targets . In addition, multipred provides several functions which are not available in other prediction systems, such as model building by user function, accuracy evaluation function and consensus prediction function . The pathway from epitopes to vaccine development is lengthy and cost - intensive, involving exhaustive experiments . The main utility of multipred is in the selection of key antigenic regions to minimize the number of experiments required for mapping of promiscuous t - cell epitopes and t - cell epitope hotspots . An example of the output pages of multipred when the input is a single protein sequence . The input protein sequence is a human papillomavirus type 16 e6, the prediction method used is ann and the hla supertype of interest is hla - a2 . (a) the main result page . The input sequence is truncated into overlapping 9mers for the prediction of binding scores to multiple hla - a2 variants, 0201, 0202, 0203, 0204, 0205, 0206, 0207 and 0209 . An example of the output pages of multipred when input is a list of peptides . The input protein peptides are from hepatitis c virus, the prediction method used is ann and the hla supertype of interest is hla - a3 . (as can be seen here, the input sequence is truncated into overlapping 9mers for the prediction of binding scores to multiple hla - a3 variants, 0301, 0302, 1101, 1102, 3101, 3301 and 6801 . When the model building request is submitted, an intermediate page will be displayed providing the result url that can be bookmarked for later model retrieval.
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Constrictive pericarditis is a progressive condition that is characterized by pericardial fibrosis with or without calcification, and impairs ventricular filling . The most common cause of constrictive pericarditis is previous cardiac surgery, followed by thoracic radiation, previous myocardial infarction, idiopathic disease, and infection . In the past, the development of constrictive pericarditis was believed to be irreversible, but recent studies have reported the resolution of a transient form of constrictive pericarditis without surgical intervention . Moreover, approximately 60% of patients with constrictive pericarditis caused by cardiac surgery have been shown to have constrictive physiology on postoperative echocardiography . Therefore, we aimed to assess whether constrictive physiology based on early postoperative echocardiography after off - pump coronary artery bypass graft (opcab) may require medical or surgical treatment to prevent constrictive pericarditis and whether it would have an effect on early mortality and morbidity of patients undergoing opcab . Between january 2008 and july 2011, 903 patients underwent isolated opcab at yonsei cardiovascular hospital and were enrolled in this single center, randomized, retrospective study . Group a consisted of patients who showed constrictive physiology on postoperative echocardiography, and group b was the control group that consisted of the patients without evidence of constrictive physiology . Mortality, 30-day major adverse cardiac and cerebrovascular events (macces), surgical characteristics, and other complications were compared between the two groups . All the patients underwent surgical bypass using the off - pump method and median sternotomy . Systemic heparinization was administered to maintain an activated clotting time of 250 seconds during the operation . A commercially available cardiac stabilizer and an apex vacuum device were used for cardiac displacement and stabilization . The internal mammary artery (i m a) was harvested using a semi - skeletonized technique . In most cases, the left i m a was anastomosed to the left anterior descending artery . For the other left coronary artery regions, a radial artery or saphenous vein graft was used as a composite graft . For right coronary artery bypass, the radial artery or a saphenous vein free graft attached to the left i m a was used in the majority of the cases depending on the degree of native coronary artery stenosis and the degree of aortic atherosclerosis . Statins were routinely used during the study period, and a small dose of diuretics was administrated until the patients were discharge according to their conditions . All of the patients' charts were reviewed, and data were collected retrospectively from the reviewed charts . The patients' preoperative baseline characteristics, surgical record, postoperative complications including 30-day macces, renal failure, respiratory complications, early postoperative echocardiography, intensive care unit (icu) stay, and hospital stay were reviewed . Constrictive physiology was defined as septal bouncing and flattening of the left ventricular posterior wall during diastole, respiratory variation in ventricular size (inferior vena cava plethora), respiratory variation of 25% or more in the mitral inflow e velocity, increased diastolic flow reversal with expiration in the hepatic vein, e' 7 - 8 cm / sec with respiratory variation of e', and absence of pericardial thickness and calcification . A macce was defined as the occurrence of death by any cause, nonfatal myocardial infarction, a stroke, or the need for target vessel revascularization . Myocardial infarction was defined as creatine kinase muscle - brain elevation with the appearance of a new q - wave or s - t segment elevation greater than 2 mm on an electrocardiogram . The chronic renal failure patients were defined as those who required hemodialysis, peritoneal dialysis, or whose preoperative serum creatinine level exceeded 2.0 mg / dl . Statistical analyses were performed using statistical software spss ver . 13.0 (spss inc ., the values for continuous variables were displayed as meanstandard deviation . For the comparison of two variables, between january 2008 and july 2011, 903 patients underwent isolated opcab at yonsei cardiovascular hospital and were enrolled in this single center, randomized, retrospective study . Group a consisted of patients who showed constrictive physiology on postoperative echocardiography, and group b was the control group that consisted of the patients without evidence of constrictive physiology . Mortality, 30-day major adverse cardiac and cerebrovascular events (macces), surgical characteristics, and other complications were compared between the two groups . All the patients underwent surgical bypass using the off - pump method and median sternotomy . Systemic heparinization was administered to maintain an activated clotting time of 250 seconds during the operation . A commercially available cardiac stabilizer and an apex vacuum device were used for cardiac displacement and stabilization . The internal mammary artery (i m a) was harvested using a semi - skeletonized technique . In most cases, the left i m a was anastomosed to the left anterior descending artery . For the other left coronary artery regions, a radial artery or saphenous vein graft was used as a composite graft . For right coronary artery bypass, the radial artery or a saphenous vein free graft attached to the left i m a was used in the majority of the cases depending on the degree of native coronary artery stenosis and the degree of aortic atherosclerosis . Statins were routinely used during the study period, and a small dose of diuretics was administrated until the patients were discharge according to their conditions . All of the patients' charts were reviewed, and data were collected retrospectively from the reviewed charts . The patients' preoperative baseline characteristics, surgical record, postoperative complications including 30-day macces, renal failure, respiratory complications, early postoperative echocardiography, intensive care unit (icu) stay, and hospital stay were reviewed . Constrictive physiology was defined as septal bouncing and flattening of the left ventricular posterior wall during diastole, respiratory variation in ventricular size (inferior vena cava plethora), respiratory variation of 25% or more in the mitral inflow e velocity, increased diastolic flow reversal with expiration in the hepatic vein, e' 7 - 8 cm / sec with respiratory variation of e', and absence of pericardial thickness and calcification . A macce was defined as the occurrence of death by any cause, nonfatal myocardial infarction, a stroke, or the need for target vessel revascularization . Myocardial infarction was defined as creatine kinase muscle - brain elevation with the appearance of a new q - wave or s - t segment elevation greater than 2 mm on an electrocardiogram . The chronic renal failure patients were defined as those who required hemodialysis, peritoneal dialysis, or whose preoperative serum creatinine level exceeded 2.0 mg / dl . Statistical analyses were performed using statistical software spss ver . 13.0 (spss inc ., chicago, il, usa). The values for continuous variables were displayed as meanstandard deviation . For the comparison of two variables, among the 903 patients included in this study, 153 (17%) were diagnosed with constrictive physiology (group a). They were not given specific treatment for constrictive physiology, and 1-year follow - up echocardiography showed complete resolution of the constrictive physiology in all 153 patients . Group a showed a low representation of women (p=0.04), a low incidence of hypertension (p=0.03), and low ejection fraction (p=0.05) compared to group b. other reviewed data showed no significant differences between the two groups . There were no significant differences between the two groups in the number of distal anastomosis and in the operative time (table 2). Increased blood loss and transfusion volumes during the first postoperative 24 hours were observed in group a compared to group b, but these differences were not statistically significant (p=0.20). Other postoperative comparisons such as hospital stay and icu stay showed no significant differences between the two groups (table 3). Thirty - day macces including death by any cause, nonfatal myocardial infarction, and stroke were not significantly different between the two groups . There were also no significant differences in respiratory complications (such as pneumonia, prolonged ventilator care, or acute respiratory distress syndrome) and renal failure (such as the need for renal replacement treatment). Admissions for more than 12 days and prolonged hospitalizations were also not significantly different between the two groups . Among the 903 patients included in this study, 153 (17%) were diagnosed with constrictive physiology (group a). They were not given specific treatment for constrictive physiology, and 1-year follow - up echocardiography showed complete resolution of the constrictive physiology in all 153 patients . Group a showed a low representation of women (p=0.04), a low incidence of hypertension (p=0.03), and low ejection fraction (p=0.05) compared to group b. other reviewed data showed no significant differences between the two groups . There were no significant differences between the two groups in the number of distal anastomosis and in the operative time (table 2). Increased blood loss and transfusion volumes during the first postoperative 24 hours were observed in group a compared to group b, but these differences were not statistically significant (p=0.20). Other postoperative comparisons such as hospital stay and icu stay showed no significant differences between the two groups (table 3). Thirty - day macces including death by any cause, nonfatal myocardial infarction, and stroke were not significantly different between the two groups . There were also no significant differences in respiratory complications (such as pneumonia, prolonged ventilator care, or acute respiratory distress syndrome) and renal failure (such as the need for renal replacement treatment). Admissions for more than 12 days and prolonged hospitalizations were also not significantly different between the two groups . Constrictive pericarditis, a compressed ventricle, and impaired ventricular filling have been shown to occur because of tuberculosis, uremia, previous thoracic irradiation, and past chest trauma; however, recently the most common cause of constrictive pericarditis has been shown to be previous cardiac surgery . Pericardiotomy with concurrent cardiac surgery leads to a transiently thickened and inelastic pericardium, resulting from edema, fibrin deposition, or inflammation . Approximately 1 to 2 weeks later, constrictive physiology develops . In the last phase, pericardial effusion resolves or decreases in size, and the hemodynamics normalize with no evidence of constriction recurrence in most patients . However, in some patients, pericardial inflammation continues and pericardial fibrosis and calcification subsequently develop, leading to chronic constrictive pericarditis [3 - 5]. The common symptoms of constrictive pericarditis have been dyspnea on exertion, chest pain, fever and a sign of right heart failure consistent with edema, abdominal swelling, and jugular venous distension . As mentioned above, a transient form of constrictive pericarditis can be resolved with medical treatments including a nonsteroidal anti - inflammatory drug (nsaid), steroids, and diuretics without surgical intervention . Approximately 60% of the patients with constrictive pericarditis caused by cardiac surgery have shown constrictive physiology on postoperative echocardiography . Specifically, constrictive pericarditis after coronary artery bypass graft (cabg) has been reported in a few studies where symptoms developed from myocardial ischemia because of graft occlusion derived from diastolic dysfunction of constrictive pericarditis . These cases had required repeat cabg including extensive decortication of the left ventricle, right atrium, and right ventricle . Constrictive pericarditis following coronary bypass surgery is an unusual complication with an occurrence rate of 0.2% to 0.3%, but as mentioned earlier, serious negative results can develop . In the same way, postoperative constrictive pericarditis can have an unfavorable effect on the prognosis of patients undergoing cardiac surgery, especially cabg . Therefore, we analyzed whether constrictive physiology seen on early postoperative echocardiography after opcab may require treatment to prevent constrictive pericarditis and whether it would have an effect on early mortality and morbidity of patients undergoing opcab . In our study, constrictive physiology on postoperative echocardiography was observed in 153 (17%) patients with isolated opcab . No patient displayed any typical symptoms of pericarditis, and no patient received preventive medication or specific treatments for constrictive pericarditis . After 1 year, follow - up echocardiography showed complete resolution of constrictive physiology in all the patients . There were no significant differences in the postoperative mortality and morbidity rates between the two groups . Therefore, we suggest that if a patient showing constrictive physiology on early postoperative echocardiography is hemodynamically stable and has no symptoms, specific medication is not needed and a follow - up echocardiography should be considered . Transient constrictive pericarditis was originally described in the english literature by sagrista - sauleda et al . In 1987 . They reported the development of objective evidence for constrictive physiology occurring in 16 of 177 patients (9%) with effusive acute idiopathic pericarditis, which subsequently resolved with conservative treatment . The interval of time until the first echocardiogram showing pericardial effusion ranged from 5 to 30 days . The time to normalization of the noninvasive recordings for all the patients ranged from 7 days to 58 months . At a mean follow - up of 31 months, none of the patients had presented with a recurrence of constriction . The authors suggested that the mechanism responsible for the findings in these patients was a transiently thickened and inelastic pericardium resulting from edema, fibrin deposition, or inflammation . Similarly, we thought that constrictive physiology does not accompany pericardial fibrosis, calcification rarely progresses to constrictive pericarditis, and resolution occurs with conservative treatment . In the cases of constrictive physiology followed by pericardiotomy, therefore, we assumed that postoperative bleeding, tube drainage volumes, and icu stay duration that disturb ambulation are risk factors for constrictive physiology . In our study, group a had more blood loss and a longer icu stay, but these were not statistically significant . Reported the clinical characteristics of 11 patients that developed postoperative constrictive pericarditis among a total of 463 cabg patients . Ten patients (91%) had evidence of pericardial effusion, which was the primary risk factor for postoperative constrictive pericarditis . In addition, left ventricular ejection fraction and warfarin administration were risk factors for postoperative constrictive pericarditis based on multivariate analysis . Effler reported that residual blood elements within the pericardial sac, especially in the areas of major involvement, were found at the time of cardiac surgery in most of the patients with chronic constrictive pericarditis of various etiologies . Cohen and greenberg also explained that organized hematomas, loculated clots, and unclotted viscous blood within the pericardial space were the most important causes of constrictive pericarditis after cardiac surgery . Most of the studies until now, and those cited above, are studies of on - pump cabg, and a study on constrictive pericarditis after opcab can only be found in one case report . Elena reported that a prolonged cardiopulmonary bypass time is an independent risk factor for pericardial effusion after cardiac surgery because of the systemic inflammatory response . Although no study has compared on - pump cabg with opcab, the on - pump cabg can be a risk factor for constrictive pericarditis compared with opcab because on - pump cabg has a risk for pericardial effusion caused by a systemic inflammatory response . Pericardial effusion is the most important factor in constrictive physiology, but we were unable to find the appropriate factors that represent the effectiveness and factors disturbing tube drainage that affect pericardial effusion . Because our follow - up duration was limited to the immediate postoperative period, the clinical symptoms were mixed, with postoperative chest pain, fever, and other symptoms . Lastly, because our follow - up period was short, a study with a long term follow - up period and repeated echocardiographic data is necessary to confirm our conclusions . The results of this study suggest that patients with early postoperative constrictive physiology do not need medical or surgical treatment, and that conservative care is sufficient.
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Data on all campylobacter isolates obtained from fecal or lower gastrointestinal tract samples, reported in england and wales from 1990 through 2007, were extracted from the national laboratory database (labbase) and stored in a microsoft access (microsoft corp ., cases were assigned to 10-year age groups and to geographic areas on the basis of laboratory location (northern, mid - country, southern). The season was assigned on the basis of the earliest available specimen date (spring, march may; summer, june august; autumn, september data on cases of cryptosporidiosis and nontyphoidal salmonellosis for the same period were extracted, grouped, and categorized and manipulated as above for comparative purposes . Denominator data for england and wales for the same period were obtained from the office for national statistics . Data were analyzed in microsoft excel 2003 and stata version 10 (statacorp, college station, tx, usa). Estimates of incidence per 100,000 population were calculated throughout; relative risks (rr) and 95% confidence intervals (cis) were calculated as required . From 1990 through 2007 in england and wales, 838,436 cases of campylobacter infection were reported; patient age was available for 810,632 case - patients (96.7%). From 1990 through 1999, incidence increased in all age groups (figure 1), but the increase was proportionate to increasing age (09 years of age rr 1.07 [95% ci 1.031.10]; 1019 years rr 1.47 [95% ci 1.411.55]; 2059 years rr 1.78 [95% ci 1.751.81];> 60 years rr 2.51 [95% ci 2.412.61]). From 2000 through 2004, incidence declined in all age groups . However, although the degree of decline was similar for those 09 years of age (rr 0.77 [95% ci 0.740.8]), 1019 years (rr 0.73 [95% ci 0.700.76]), and 2059 years (rr 0.75 [95% ci 0.740.76]), for patients> 60 years of age, the degree of decline was significantly lower (rr 0.88 [95% ci 0.860.91]; p<0.001). Finally, although the incidence increased only moderately among those 1019 years of age (rr 1.02 [95% ci 0.981.07]) and 2059 years (rr 1.04 [95% ci 1.031.06]) from 2005 through 2007, greater increases were observed for those 09 years (rr 1.12 [95% ci 1.081.17]) and those> 60 years (rr 1.33 [95% ci 1.291.36]). During the surveillance period, therefore, the incidence among patients> 60 years of age, compared with the incidence among younger patients, increased markedly (rr 0.45 [95% ci 0.440.47] in 1990 to rr 1.17 [95% ci 1.151.19] in 2007). This effect was observed for campylobacteriosis in both sexes, in 3 geographic areas of england and wales, and in all 4 seasons, but was not observed for nontyphoidal salmonellosis or cryptosporidiosis (figure 2; technical appendix). Incidence of laboratory - reported campylobacteriosis, england and wales, by age group, 19902007 . Relative incidence of campylobacteriosis by sex, region, and season, compared with rates of salmonellosis and cryptosporidiosis, among patients> 60 years of age, england and wales, 19912007 . Northern, northwest, northeast, as well as yorkshire and the humber regions; mid - country, wales, west midlands, east midlands, and east of england regions; southern, london as well as southeast and southwest regions . Salmonellosis includes nontyphoidal salmonellae, with age data available for 356,270 of 380,915 case - patients (94%); cryptosporidiosis includes age data for 76,462 of 79,808 case - patients (96%). We report a striking change in the population at risk for campylobacteriosis in england and wales, which is independent of gender, geography, or season . The absence of a similar change in the age distribution of laboratory - reported salmonellosis or cryptosporidiosis from the same population suggests that this is unlikely to be a surveillance artifact . Campylobacter infections are rarely typed beyond the genus level in england and wales, so infections are routinely reported as campylobacter species . Therefore, changes in the incidence of the various species that constitute this broad case definition could possibly explain some of the altered disease pattern reported . For example, infections caused by certain species (e.g., c. fetus) are more often associated with coexisting conditions that might occur more frequently in the elderly . First, isolation methods used in england and wales favor the growth of c. jejuni and c. coli (4,5) over that of other species, including c. fetus . Furthermore, c. fetus normally causes systemic infections detected through blood culture, and the proportion of blood to fecal isolations of campylobacter species in patients> 60 years of age reported in england and wales remained constant from 1990 through 1999 (275/58,139 fecal isolations; 0.47%), from 2000 through 2004 (185/44,349; 0.42%), and from 2005 through 2007 (135/31,637; 0.42%) (health protection agency, unpub . Data). When disease incidence was ranked according to specific population group, children 09 years of age had the highest ranking in 1990, but by 2007, incidence for this age group ranked seventh of 9 age groups . This finding led to the hypothesis that the introduction and increased utilization of the national health service s nhs direct (a 24-hour telephone, online, and interactive digital tv service, which provided health advice and information) at this time had a triage effect on those seeking care for children in this age group . The 2 events are correlated in time (initial nhs direct pilot sites began taking calls in march 1998; by april 1999, 40% of the population of england had access, and by november 2000, the service was available throughout england and wales), nhs direct has had a demonstrable negative effect on the use of general practice (7), and infants and young children are overrepresented among calls to nhs direct about gastrointestinal conditions (8). The second infectious intestinal disease study in england, currently under way (9), will provide further information upon which to assess this hypothesis, which is not readily testable by using laboratory data . By far the most striking finding of this study is the emergence of older persons as the population most at risk for campylobacteriosis in england and wales . Although the elderly were not the only group at risk in 2007 (because of increasing incidence), the overall trend singles them out as the main emerging at - risk group (the increase in other age groups requires continued monitoring, however). The pattern of infection in older patients is perhaps predictable, given the similar pattern for incidence of listeriosis in england and wales since 2001 (10). As life expectancy increases in the united kingdom, the number of persons living with chronic conditions is likely to increase; these factors suggest that the incidence of campylobacteriosis in older persons will continue to increase in the future . Therefore, risk factors for campylobacter infection specific to older uk residents must be identified . Relative incidence of campylobacter infection in each year compared with that in 1990 among patients> 60 years of age infected with selected gastrointestinal pathogens, england and wales, 1991 - 2007
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Sciatic hernias are one of the rarest types of hernia and often pose diagnostic difficulty to clinicians . Imaging is often required to confirm the diagnosis and usually involves computed tomography (ct) or magnetic resonance imaging (mri). To our knowledge, we report the 115th case of sciatic hernia in the literature who had a falsely negative ct but had the diagnosis confirmed using ultrasonography . An 80-year - old lady was referred to the on - call surgical team by her gp with a 3-week history of a right - sided swelling of the buttock . She had a past medical history of hypertension, osteoarthritis and pemphigus vulgaris and a past surgical history of a perianal abscess requiring incision and drainage . She was allergic to penicillin and took regular oral betamethasone, xylometazoline nasal spray and topical aqueous cream . She did not consume alcohol, was a non - smoker and lived in warden - controlled accommodation . On examination, her cardiac, respiratory and abdominal examinations were normal . Digital rectal examination revealed a non - tender stool - filled rectum with no palpable masses . On standing, a swelling on the medial aspect of the right buttock became apparent which was easily reducible, had audible bowel sounds and a positive cough impulse . She was reviewed by the on - call consultant who discharged her with a working diagnosis of possible obturator hernia, with a plan for an outpatient ct of her pelvis and follow - up in clinic . Ct scan of the pelvis did not identify a cause for the swelling (fig . 1). Due to the positional nature of the swelling, a gluteal ultrasound was organized, which revealed a large colonic sciatic hernia (fig . 2). As the patient had minimal symptoms and was not keen for surgical intervention, a plan for conservative management was agreed and the patient was discharged from clinic . Figure 1:ct of the patient's pelvis demonstrating a normal right sciatic foramen (arrowed). Us hip rt: confirms reducible herniation of colon in the right sciatic region into the buttock.) Ct of the patient's pelvis demonstrating a normal right sciatic foramen (arrowed). Hip rt: confirms reducible herniation of colon in the right sciatic region into the buttock.) Sciatic hernias are one of the rarest types of hernia and often pose diagnostic difficulty to the clinician . A sciatic hernia is defined as herniation of intraperitoneal contents through either the greater or lesser sciatic foraminae (fig . The majority of sciatic hernias are found in women (77%) with more than one - third of these being aged 60 or over . The contents are variable and hernias containing ovaries, ureters, bladder, small and large intestine, omentum and dermoid cysts have been reported [16]. Half of patients report non - specific abdominal or pelvic pain and one third have a mass on clinical examination . Sciatica, intestinal obstruction, urinary sepsis and hydronephrosis have also been described [15]. Diagnostic laparoscopy or laparotomy is often required to fully evaluate the sac contents and to repair the defect [2, 3, 5]. In symptomatic patients, surgical repair is indicated due to the high risk of bowel strangulation [1, 3]. Figure 3:pelvis demonstrating the greater (red) and lesser (green) sciatic foraminae . Diagnosis by clinical examination alone is possible in only the minority of cases and imaging is frequently used to confirm the suspicion of sciatic hernia . Commonly used imaging modalities are computerized tomography (ct) [25] and mri [3, 4], particularly if the sciatic nerve is thought to be involved . Currently, the most commonly used imaging modality of choice is a ct scan with the patient supine . However, if the hernia is only apparent in a dependent position, this can produce a false - negative result, as in our case . The benefit of ultrasound is that it allows for real - time positional assessment of the region of interest, to reduce the risk of missing this rare but significant hernia . Due to their rarity, sciatic hernias are often not considered in the differential diagnosis . The authors recommend that all patients presenting with non - specific abdominal or pelvic pain associated with a gluteal swelling should have sciatic hernia considered amongst their differential diagnosis . For patients in which there is a high degree of clinical suspicion for a sciatic hernia and a negative ct, the use of ultrasonography for positional defects may be a useful aid to confirming the suspected diagnosis.
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The growing use of high - throughput screening (hts) as a discovery tool in academic translational centers has resulted in the pursuit of assay artifacts, promiscuous bioactive compounds, and screening actives with major absorption, distribution, metabolism, excretion, and toxicological (admet) liabilities . A similar situation may exist in industry, and this observation may simply be a reflection of academic pressures to publish . In either case, the follow - up of such compounds can significantly burden the post - hts triage and hit - to - lead stages of the discovery process . Therefore, chasing assay artifacts and promiscuous screening compounds can waste both time and other valuable resources, and failure to triage these compounds has led to many artifacts and frequent hitters making their way into the scientific literature, patent applications, and research funding applications . As an example, pan - assay interference compounds (pains) can display apparent bioactivity and/or interfere with assay readouts across unrelated biological targets and testing methods . Multiple sources for promiscuous behavior or assay interference have been described, including: chemical aggregation, chelation, singlet oxygen production, compound fluorescence effects, redox activity, sample impurities, membrane disruption, cysteine oxidation, and nonselective compound reactivity with proteins . Reporter interference as the most likely source of biological assay readouts in a compound that has progressed to human clinical trials . An important point with these luciferase experiments is that confounding readouts are not isolated to cell - free assays . Cell - based assays with perturbations in cell proliferation may be particularly susceptible to assay interference or off - target and confounding effects . Misleading readouts can have clinical relevance, as a recent study suggests the pharmacological activity of acamprosate (an fda - approved drug for relapse prevention in alcoholism) may be due to the calcium cation component of its formulation rather than the long - presumed bioactive ingredient, n - acetylhomotaurinate . Despite the risks associated with pursuing these types of undesirable compounds, their identities and the chemical mechanisms by which they can mislead even seasoned researchers often go uncharacterized in the pursuit of identifying lead compounds . Unfortunately, this leaves open the possibility for other groups to fall into the same scientific maelstrom that most often results in costly failure . In an effort to alert the uninitiated, we describe herein the structure interference relationships (sir) in five series of problematic compounds we encountered in a recent hts campaign . Epigenetic enzymes, such as histone deacetylases, methyltransferases, and histone acetyltransferases (hats), are an important emerging class of therapeutic targets . Epigenetic chemical probes and enzymatic modulators are sought for a variety of human diseases including cancers . Our group and others have focused on a series of enzymes unique to fungi, rtt109 hats, that are critical for dna replication - coupled nucleosome assembly and genomic stability and therefore may represent a novel antifungal therapeutic approach . Antibody - based approaches can probe for specific histone modifications such as methylation and acetylation . Another more indirect approach probes for reaction byproducts via chemical probes or reporter enzymes . One chemical probe, n-[4-(7-diethylamino-4-methylcoumarin-3-yl)phenyl]maleimide (cpm), readily reacts with free thiols to form highly fluorescent adducts . Several cpm - based assays and screens have been reported for multiple biological targets, including some epigenetic enzymes . Recently, our group screened approximately 225k small molecules for their ability to inhibit rtt109-catalyzed histone acetylation using a cell - free cpm - based hts . Pains were computationally filtered at the beginning of our triage and were not initially evaluated in our post - hts counter - screens . Post - hts triage of approximately 1.5k primary screening hits demonstrated only a few confirmed actives . In retrospect, this indicated a significant portion of the screening hits were either false positives or assay artifacts resulting from fluorescence quenching, compound reagent interference, and/or other mechanisms . On the basis of the chemical structures of the triaged compounds, we speculated many chemotypes from the primary hts campaign were reacting with the coa byproduct to produce an interfering assay readout mimicking enzymatic inhibition . We also speculated many of these thiol - reactive compounds, including several series of pains we had previously triaged, could also inhibit enzymatic activity by reacting with protein cysteines, a recognized source of promiscuous enzyme inhibition and metabolic liability . We observed chemotypes that were enriched among the actives that appeared chemically similar to many of the published pains substructures but were not flagged by our cheminformatic pains filters . There is a growing interest in both assay interference and promiscuous enzymatic inhibition, including nonspecific thiol reactivity . Therefore, identifying thiol - reactive chemotypes in compound screening libraries is important for enhancing library design and post - hts decision - making . Additionally, the characterization of the chemical mechanisms of thiol reactivity may also be useful for reactivity prediction, compound optimization, and the avoidance of follow - up on compounds that may have metabolic or selectivity liabilities further downstream in the drug discovery pipeline . The observation that some pains - like compounds may escape cheminformatics filters may have significant consequences for screening centers without experienced hts triage personnel, especially if those performing hts triage are overly reliant on cheminformatics filters . Herein, we report the identification of multiple chemotypes (chemical structural motifs) that showed publication - quality ic50 values in our primary assay but through a series of orthogonal assays and counter - screens showed thiol - reactive assay interference and also promiscuous enzymatic inhibition . Using sir and structure activity relationships (sar) from our rtt109 post - hts triage, along with multiple analytical techniques, we provide evidence supporting several chemical mechanisms of assay interference relating to thiol reactivity . We show these chemotypes can form covalent adducts with other biologically relevant thiols such as glutathione (gsh) and cysteine residues on multiple structurally unrelated proteins . The chemical mechanisms we propose contributing to assay promiscuity include addition elimination reactions, nucleophlic aromatic substitution, buffer instability, disulfide bond formation, and h2o2 production . Our findings may be more broadly applicable, as several compounds containing these chemotypes formed covalent adducts with the la antigen (alarm nmr), demonstrating potentially broad - spectrum thiol reactivity . Furthermore, compounds with these chemotypes showed evidence of assay promiscuity in analyses of pubchem and the hts database of a major pharmaceutical company . Despite these red flags, several such compounds have been reported in the patent literature and reputable scientific journals with varying claims of target specificity and utilities as either chemical probes or therapeutic leads . It is hoped that the identification and detailed characterization of these thiol - reactive chemotypes can accelerate post - hts triage, enhance lead identification, and prevent follow - up on unpromising chemical matter by other researchers . We previously reported the use of a cpm - based method to screen approximately 225k compounds for their abilities to inhibit rtt109-catalyzed histone acetylation in vitro . In a hat reaction, an acetyl group is enzymatically transferred from acetyl - coa to the -amino group of a histone lysine side chain, resulting in the production of an acetylated lysine and a coa byproduct . The free thiol on coa can then react with suitable substrates, such as maleimide - based probes like cpm, to form highly fluorescent adducts that can indirectly assay hat activity (figure 1a). In practice, we and others have found this method to be low - cost and relatively robust . We were also aware that this method is subject to assay interference by thiol - containing compounds . However, we soon learned that this assay is highly susceptible to other mechanisms of reactive compound interference especially when testing potentially heterogeneous chemical matter like hts libraries . In principle, compounds can interfere with the cpm fluorescence intensity by fluorescence quenching . Also, compounds with nucleophilic or electrophilic reactivity can react with either the cpm probe or the coa reaction product, respectively . (a) assay schematic for the cpm - based hts used in this study . The assay measures the hat activity of the rtt109vps75 complex, which catalyzes the transfer of an acetyl moiety from acetyl - coa to specific lysine residues on the asf1dh3h4 substrate complex to produce acetylated histone residues and coenzyme a (coa). Addition of the thiol - scavenging probe cpm leads to a highly fluorescent adduct by reacting with the coa byproduct, which is used to quantify hat activity via fluorescence intensity measurement . Despite identifying 1.5k actives in the primary screen, a post - hts triage consisting of computational filtering, experimental counter - screens, and orthogonal assays demonstrated only three compounds that could inhibit rtt109-catalyzed histone acetylation . Therefore, significant portions of this collection of experimental and computationally filtered (filtrand) compounds were either false positives or assay artifacts . In addition, many of the computationally filtered actives were flagged as pains, which we believed could inhibit hat activity by nontherapeutically useful mechanisms . In all, several prominent chemotypes were identified among the primary actives during the course of the post - hts triage (figure 1b). On the basis of their chemical structures, it appeared likely that each chemotype could interfere with our hts assay readout through different and, perhaps, multiple thiol - trapping mechanisms . Many of these compounds were flagged as pains, but still many were not verified as bad actors until relatively late in our triage process . To further confound matters, these electrophilic compounds could conceivably inhibit enzymatic activity by nonspecific reactivity with the protein components in the assay, further scrambling the assay readout . To better understand the chemical mechanism(s) behind this form of assay behavior and to assess whether this interference could have implications beyond our hts, we examined five prominent subclasses of compounds in more detail . Several classes of compounds demonstrated low micromolar ic50 values in the cpm - based hts method (figure 2). 2) have been associated with cooperativity and anomalous binding behaviors, such as chemical aggregation . Most of the compounds showed slightly elevated hill slopes, but this was not an immediate concern, as we had included a detergent (triton x-100) in our assay buffers to mitigate micelle formation . Dose responses of select screening compounds in the rtt109 hts and an assay interference counter - screen . Shown are representative examples from chemotypes 1, 2, 3, 4, and 6, which displayed promising low micromolar ic50 values by the primary hts assay (solid lines). A counter - screen that replaced the acetyl - coa substrate with the coa reaction product produced similar dose reaction aliquots from the active compounds (1a, 2a, 3a, 4a, 6a, and 6b) all showed decreases in histone acetylation at 125 m when they were analyzed by an orthogonal slot blot assay that uses h3k56ac- and h3k27ac - specific antibodies to probe for the acetylated histone lysine product rather than the coa byproduct (tables 15). We examined both histone modifications because the rtt109vps75 complex is capable of acetylating multiple histone h3 residues . Overall, reaction aliquots showed similar levels of histone acetylation, regardless of whether h3k27ac or h3k56ac was examined, strongly suggesting that any observed enzymatic inhibition was not specific to one particular histone modification . This assay also detected decreases in histone acetylation with garcinol, a natural product previously shown to inhibit rtt109 activity and other hats in vitro at low micromolar compound concentrations . When the same assay was used to examine reaction aliquots at lower compound concentrations (8 m), we observed more discrepancies between the hts and slot blot readouts (data not shown). Overall, these observations showed that representatives of these five compound classes could inhibit acetylation activity at high concentrations, but their hts assay readouts were confounded by assay interference near their apparent ic50 values . To further examine the mechanisms underlying this assay interference, we first established a fluorescence - quenching counter - screen to assess for fluorescence interference . This assay accurately identified the fluorescence quencher bhq-1, a positive control (supporting information, figure s1). However, none of these compounds (1a, 2a, 3a, 4a, and 6b) showed evidence of fluorescence quenching, intrinsic fluorescence or the generation of fluorescent adducts with either coa or cpm in a set of assays mimicking our hts procedures (tables 15). When the acetyl - coa reactant was replaced by the coa reaction byproduct, these compounds provided assay readouts that were strikingly similar to those obtained under the hts assay conditions (figure 2). The degree of assay signal reduction was also dependent on the levels of coa present (data not shown). Similar assay behavior was observed for a variety of chemical analogues bearing these chemotypes (tables 15). Therefore, the body of evidence strongly implicated a thiol - trapping mechanism of assay interference . Next, we sought to understand the chemical basis of this interference and potential sources of enzymatic inhibition . Coa adducts, we incubated the compounds with coa under hts - like conditions . We also tested for adducts with reduced l - glutathione (gsh), another important biological thiol, to assess if this presumed thiol reactivity was unique to coa . Ms and lc hrms analyses showed that the test compounds (1a, 2a, 3a and 4a) form adducts with both coa and gsh (figure 3 and supporting information, figure s2). Ms for multiple other representative compounds from each chemotype (data not shown). While the p - hydroxyarylsulfonamides 6a6e yielded the expected compound - gsh adducts (6a6e; figures 4a, b), we also observed that 6a6e were really intermediates that converted to a common adduct in situ after only 15 min under the hts conditions (figure 4b, c). (a) selected interference compounds were incubated with meoh (black traces), hts buffer (blue traces), or hts buffer plus gsh (red traces) and analyzed by uplc ms . Shown selected mass spectra are also shown for a select sample in meoh (black spectrum) and selected adducts (red spectra). Numbers in parentheses represent the predominant ion molecular weight (denotes negative ion mode). (b) simplified schematics of the proposed reaction mechanisms to generate the observed adducts . Labile adducts between p - hydroxyarylsulfonamides (6) and gsh detected by qualitative uplc (a) simplified scheme of adduct formation between biological thiols and chemotype 6 . (b) uplc ms analyses of compound 6a mixed with gsh in hts buffer . 6a was treated with gsh after varying lengths of incubation in hts buffer (5, 15, 30 min). Trace (iv) shows the same sample from trace (i) analyzed 15 min later . A common breakdown product 6 was detected for all five sulfonamides tested (rt = 3.28 min, m / z = 446). See supporting information, figures s5, s7, and s11, for additional stability studies with chemotype 6 . A = compound incubated in hts buffer for 5 min, then gsh added, then analyzed by uplc - ms 5 min later; b = same sample from a, but analyzed by uplc - ms 15 min later . Together, this data is consistent with a thiol - trapping mechanism as a major contributor to the cpm - based assay signal reduction in the compound classes studied, as the tested compounds reacted with both coa and gsh . Gsh adducts is an important consideration for certain cell - based assays, or for in situ or in vivo assays, where xenobiotic the selected compounds interfere with the hts assay readout and form thiol adducts by a variety of chemical mechanisms (figures 3b and 4a). On the basis of the uplc ms and chemical principles, we propose the following chemical mechanisms of thiol reactivity for chemotypes 1, 2, 3, 4, and 6 (figure 1): benzo[b]thiophene 1,1-dioxides 1 (benzothiophenes) interfere via a straightforward michael addition elimination reaction at the electrophilic c3-position through thiolate nucleophilic attack . The compounds with chemotype 1 most likely to interfere and form thiol adducts in our experimental conditions were those with s - linked heteroaromatic substituents (table 1). The uplc ms experiments using 1a confirmed the presence of the adduct 1a and the leaving group 1a (figure 3a). This proposed mechanism is also supported by the observations that several 2,3-dihydro analogues did not show appreciable levels of apparent rtt109 inhibition or interference in the coa the level of assay interference is consistent with the leaving group ability of the c3 substituent, as compounds with n-, o-, or c - linked groups at this c3-position did not show as significant levels of interference or apparent inhibition (supporting information, figure s3). Most of the compounds with chemotype 1 showed only partial decreases in histone acetylation at high compound concentrations (table 1), demonstrating these compounds can weakly inhibit rtt109 activity in our hts, most likely by nonspecific thiol reactivity (table 1). Hts refers to ic50 values calculated from the cpm - based rtt109 hts method, coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen . Hts reaction aliquots from compounds tested at 125 m final concentrations; yes (y), no (n), or partial (p). Compounds flagged as quenchers if greater than 20% assay signal reduction at 10 m final concentrations . Compounds flagged if fluorescence intensity greater than 20% assay signal at 125 m final concentrations; compounds tested in either hts buffer (buffer), hts buffer plus 20 m cpm (+ cpm), or hts buffer plus 7.5 m coa (+ coa). The benzothiadiazole / benzofurazan scaffold 2 likely forms thiol adducts via nucleophilic aromatic substitution through a meisenheimer complex intermediate between the nucleophilic thiol and the strongly electrophilic heteroaromatic core . The benzofurazan core has been previously associated with promiscuous thiol reactivity, while some related benzothiadiazoles have been reported as pains (e.g., substructures diazox_b and diazox_sulfon_a). The related compound 4-chloro-7-nitrobenzofurazan (nbd - cl) and its derivatives are widely used as probes for studying thiols in biological systems . Ms analysis of 2a demonstrates that the parent compound is stable to the hts buffer but that the addition of a thiol source leads to near - complete conversion to the thiol adduct 2a with the thiopurine serving as the leaving group (figure 3a). The compounds that showed the strongest apparent enzyme inhibition and interference contained electron - withdrawing substituents such as nitro groups and halogens, although there was no apparent reactivity difference between benzothiadiazoles and benzofurazans . Another important feature for interference was the presence of an s - linked aryl substituent, which serves as the leaving group even when there are other electron - withdrawing groups present (table 2). Benzothiadiazoles without these features did not show significant levels of apparent enzyme inhibition or assay interference (supporting information, figure s4). While flagged as pains, many sulfoxide - substituted analogues tested in our system were inactive and noninterfering, an observation we attribute to the absence of an additional strong electron - withdrawing group (e.g., nitro). Many compounds with chemotype 2 were capable of completely inhibiting rtt109-catalyzed histone acetylation in our hts (table 2). Hts refers to ic50 values calculated from the cpm - based rtt109 hts method, coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen . Hts reaction aliquots from compounds tested at 125 m final concentrations; yes (y), no (n) or partial (p). Compounds flagged as quenchers if greater than 20% assay signal reduction at 10 m final concentrations . Compounds flagged if fluorescence intensity greater than 20% assay signal at 125 m final concentrations; compounds tested in either hts buffer (buffer), hts buffer plus 20 m cpm (+ cpm). Or hts buffer plus 7.5 m coa (+ coa). While the core 1,2,4-thiadiazole heterocycle 3 may appear benign, many such compounds can react quite readily with thiols but not other functional groups like alcohols or amines . The 1,2,4-thiadiazole core can be susceptible to ring - opening reactions, recyclization side products, and nonenzymatic reductions . Pains substructure, although it differs by resonance and substitution at the n2-position, meaning this chemotype could bypass some pains filters depending on its structural representation and certain chemoinformatic parameters . This speculation aside, the likely chemical mechanism of interference in our assay is sulfhydryl - scavenging by the 1,2,4-thiadiazole core at the s1-position, specifically a ring - opening reaction that generates a disulfide that can then be reduced by another thiol or electron source in situ to form the corresponding thiourea . Indeed, we first observed the formation of the thiourea form (3a), as evidenced by a major shift in the uplc retention time upon the addition of thiols (figure 3a). The parent ions for this entity (i.e. Ms, and notably we did not observe any coeluting gsh ions, suggesting this peak was not the 3a form with an attached gsh moiety . To gain a further structural understanding of the 3a adducts, we synthesized it under hts - like conditions and characterized its identity and structure in situ by lc gsh 3a adduct (supporting information), which is consistent with a previous report on this chemotype . These data, combined with our findings that compounds 3 are strongly reactive in our thiol - trapping interference screen, suggests the 3gsh adduct forms (3) are not stable to our characterization procedures and/or our lc - ms conditions . Examination of close analogues showed the assay interference strongly correlates with additional alkylation at the core n2-position to generate a partially cationic nitrogen, which presumably activates the s1n2 bond for thiol - mediated cleavage . Compounds lacking these substituents on the n2-position were inactive and showed minimal interference (supporting information, figure s5). Of note, het_5_inium, which bears resemblance to the charged 1,2,4-thiadiazoles in this chemotype . Neither the nature of the r r substituents nor the particular salt composition appeared to have significant effects on thiol - trapping (table 3). Consistent with this mechanism, many 1,2,4-oxadiazole analogues showed minimal assay activity and interference (supporting information, figure s5). We observed that many of these 1,2,4-thiadiazoles inhibited rtt109-catalyzed histone acetylation quite effectively (table 3), suggesting these compounds cannot only interfere with the hts assay but can also inhibit enzymatic activity, again presumably by nonspecific reactivity with protein thiols . Hts refers to ic50 values calculated from the cpm - based rtt109 hts method, coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen . Hts reaction aliquots from compounds tested at 125 m final concentrations; yes (y), no (n) or partial (p). Compounds flagged as quenchers if greater than 20% assay signal reduction at 10 m final concentrations . Compounds flagged if fluorescence intensity greater than 20% assay signal at 125 m final concentrations; compounds tested in either hts buffer (buffer), hts buffer plus 20 m cpm (+ cpm), or hts buffer plus 7.5 m coa (+ coa). Our data is consistent with an elimination event followed by a michael addition of a free thiol to the resulting maleimide . This is the same sulfhydryl - sensitive group present in the cpm probe used in our hts . Elimination is likely, given the slightly alkaline ph of the assay buffer (ph 8.0). This same proposed leaving group was not detected when 4a was incubated in neat meoh (figure 3a). First, the proposed elimination product maleimides 5 showed nearly identical ic50 values in both the hts and interference counter - screens compared to their parent succinimides (table 4). Second, the apparent enzymatic inhibition and counter - screen ic50 values correlate well with the presumed leaving - group ability of the succinimide substituent . For instance, succinimides with s - linked aryl groups showed significant assay interference (table 4), while nonaryl, s - linked leaving groups showed no significant activity and interference (supporting information, figure s6). Third, succinimides with n - linked substituents did not appear to inhibit rtt109 in our hts or show interference in our counter - screens (supporting information, figure s6). The substituents on the resulting maleimides did not have a noticeable effect on either the hts or counter - screen ic50 values (table 4). Most of the interfering succinimides 4 could only partially inhibit rtt109 activity at higher compound concentrations in our hts, which may be a reflection of the kinetics of the succinimide - to - maleimide conversion in buffer . Hts refers to ic50 values calculated from the cpm - based rtt109 hts method, coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen hts reaction aliquots from compounds tested at 125 m final concentrations; yes (y), no (n) or partial (p). Compounds flagged as quenchers if greater than 20% assay signal reduction at 10 m final concentrations . Compounds flagged if fluorescence intensity greater than 20% assay signal at 125 m final concentrations; compounds tested in either hts buffer (buffer), hts buffer plus 20 m cpm (+ cpm), or hts buffer plus 7.5 m coa (+ coa). Others have shown this scaffold to be redox - active as well as subject to addition elimination at the substituted c3-position . During our post - hts triage, we also observed that many of these compounds produced h2o2 in our assay buffer, both in the presence and absence of the reducing agent dtt using a horseradish peroxidase phenol red assay (hrp - pr; supporting information, table s1, and data not shown). Several of these compounds did not produce detectable levels of h2o2 in our assay, however . This may be related to compound stability in assay buffer, as discussed below . Given these results, we suspected that h2o2 production might be another source of assay interference for this chemotype (by oxidizing the free thiol on coa to sulfenic and sulfinic acids). However, we found that even relatively high levels of h2o2 (1 mm final concentrations) did not interfere with the assay readout in the coa cpm counter - screen when compared to control reactions (p = 0.61, n = 8). Even h2o2 present in levels greater than those observed in our hrp - pr redox assay did not appreciably inhibit the hat activity of rtt109vps75 or other hats either in the presence or absence of dtt (supporting information, table s2), suggesting these particular proteins are not overly susceptible to h2o2-mediated inactivation under our experimental conditions . Additionally, none of the other prototype compounds used in this report showed evidence of redox activity when tested (supporting information, table s1). Together, these data suggest h2o2 release by these redox - active compounds is not the primary factor behind their compound - mediated reductions in hts signal or enzymatic activity . Ms experiments provided important insights into the complex nature of this triple - threat chemotype . Elimination of a thiol on the parent compounds at the c3-position (figure 4b, c). Importantly, these sulfonamides and the aforementioned adducts were not stable to our assay conditions . To our surprise, compounds 6a6e (table 5) and adducts 6a6e showed a time - dependent degradation in our hts buffer when monitored by uplc (figure 4b, c and supporting information, figure s7). These data are consistent with and extends a previous report examining a similar screening compound . On the basis of this data and plausible chemical mechanisms, we speculate that the degradation products are arylsulfonamides and naphthoquinones resulting from imine hydrolysis and perhaps other as yet unidentified intermediates . Evidence supporting the complex and subversive reactivity of this class of compounds includes the observation that treatment of 6a6e with gsh led to a common compound adduct (6) with an m / z of 446 . We propose that this gsh adduct is formed by loss of the arylsulfonamide and water, perhaps by imine hydrolysis of 6a6e at the c hts refers to ic50 values calculated from the cpm - based rtt109 hts method, coa - cpm refers to ic50 values calculated from the coa - based hts counter - screen hts reaction aliquots from compounds tested at 125 m final concentrations; yes (y), no (n) or partial (p). Compounds flagged as quenchers if greater than 20% assay signal reduction at 10 m final concentrations . Compounds flagged if fluorescence intensity greater than 20% assay signal at 125 m final concentrations; compounds tested in either hts buffer (buffer), hts buffer plus 20 m cpm (+ cpm), or hts buffer plus 7.5 m coa (+ coa). Ic50 values shown are means sd for three replicates . In the aqueous and slightly alkaline hts conditions, it is likely chemotype 6 can also undergo imine hydrolysis to generate a reactive naphthoquinone in situ, although we were unable to observe this compound directly by our uplc ms setup . Naphthoquinone formation is consistent with the production of h2o2 and our observation of a common compound glutathione adduct . It is likely the thiols could react with a resulting naphthoquinone via michael addition elimination . Interestingly, compounds with a quinone moiety 7 in place of a naphthoquinone generally showed less interference (supporting information, figure s8) and none of these compounds were active in the slot blot or showed signs of redox activity (supporting information, table s1). Taken together, these data suggest the napthoquinone moiety is an important structural factor for both redox activity and thiol reactivity, at least under our experimental conditions . Many compounds with the chemotype 6 inhibited rtt109-catalyzed histone acetylation as determined by slot blot (table 2), suggesting these compounds can inhibit enzymatic activity either by reacting with proteins and/or other nonspecific mechanism(s). We also examined the ability of the presumed aromatic leaving groups formed from these substrates (e.g., 1a) to interfere with the assay readout . Many of these leaving groups did not reduce the hts or coa - based counter - screen readouts, especially at the same low micromolar compound concentrations used for the prototype compounds (supporting information, figure s9), and none inhibited enzymatic activity in the slot blot orthogonal assay . The severity of interference, however, appears to increase when they were allowed to incubate longer with coa (unpublished observations). Despite containing a thiol group, none of these leaving groups formed fluorescent adducts with cpm, suggesting they are not sufficiently nucleophilic to react with the maleimide probe under the conditions tested . Interestingly, the only leaving groups that formed fluorescent adducts with cpm were some p - hydroxyarylsulfonamides (e.g., 6a) with thioglycolic or 3-mercaptopropionic acid substituents, as these compounds showed profiles consistent with false - negative enzymatic inhibition (table 5). Although compounds with chemotypes 1, 2, 3, 4, and 6 interfere with the assay readout by trapping coa, several of these same compounds were shown to inhibit rtt109-catalyzed histone acetylation at high compound concentrations by slot blot assay (tables 15). We confirmed this inhibition for several compounds using a second, lower - throughput orthogonal hat assay that utilized [h]-acetyl - coa . We found that most of these compounds inhibited rtt109-catalyzed histone acetylation in the low micromolar range, particularly scaffolds 2, 3, and 6 (table 6). This shows compounds with these chemotypes can inhibit rtt109 enzymatic activity in vitro, but this is most likely via nonspecific protein reactivity, given the ability of these compounds to form thiol adducts . As expected for compounds with nonspecific thiol reactivity, these same compounds also inhibited the human hat p300 and the yeast gcn5ada2ada3 hat complex at similar concentrations (table 6). This inhibition was profoundly attenuated by the inclusion of dtt (table 6), which is consistent with these chemotypes being thiol - reactive agents . In parentheses compounds tested identically in the presence of 1 mm dtt; results similar versus rtt109-vps75, p300, and gcn5; typically <20% inhibition was observed at 125 m final compound concentrations . Previously published value . To further examine the thiol reactivity of these problematic compounds, we performed protein mass spectrometry (lc ms / ms) using tryptic digestions of samples containing select prototype compounds incubated with the protein components of the hts assay . As expected for potent thiol - trapping compounds, we observed several ionized peptides with accurate mass measurements corresponding to covalently modified cysteine residues on rtt109 (figure 5 and supporting information, table s3). Detectable adducts were also observed with select cysteine residues on vps75 and asf1 (figure 5 and supporting information, table s3). These compounds did not form detectable adducts with all the cysteines in the hts proteins under our experimental conditions . We speculate this may be because some of the adducts were particularly labile under the experimental conditions or were not amenable to ionization or because sterically inaccessible and/or chemically inactivated cysteines (via sulfur oxidation) were not subject to reactivity . Prototype compounds were incubated with purified proteins from the rtt109 hts, and then samples were subjected to lc - ms / ms analyses after in - gel proteolysis . Shown are peptide ms / ms spectra with assigned y- and b - type fragments . (b) compound 6a forms a detectable adduct with mono - oxidized c21 on yeast vps75 . Shown in each spectra are the sequences for the precursor peptide and a simplified reaction scheme for the adduct formation . See supporting information, table s3, for additional examples of compound peptide adducts detected by peptide mass spectrometry . Overall, these data demonstrate the prototype in each of the chemical classes can covalently modify the protein components of our hat assays in a promiscuous fashion . Given this data, and the strong attenuation of enzymatic inhibition by the inclusion of dtt in our radiolabeled hat assays (table 6), the most likely mechanism of enzymatic inhibition is nonspecific thiol reactivity . Because rtt109 does not have a known catalytic cysteine residue, it is most likely the case that this thiol modification alters protein structure and dynamics rather than directly inhibiting the catalytic mechanism . The fact that several of the protein components included in the hts method were modified (and that hat inhibition can be significantly attenuated with dtt) further suggests these compounds react with thiols indiscriminately and may therefore show promiscuous bioactivity . It is known that the reactivity of thiols in a proteinaceous microenvironment may be different than their reactivity with small - molecule thiols like gsh (and presumably coa). To complete our study, we investigated whether these interfering compounds could react with protein cysteines from a completely unrelated protein, the la antigen, using alarm nmr . Importantly, this assay utilizes a completely orthogonal detection method, that is, not based on fluorescence, mass spectrometry, antibodies, or radioactive substrates . We tested the prototype compounds (1a, 2a, 3a, 4a, 6a, and 6b) as well as positive and negative control compounds 2-chloro-1,4-naphthoquinone and fluconazole, respectively (figure 6a). Consistent with the previous findings, all of these prototype compounds induced peak shifts in the regions of interest in the absence of dtt . These effects could be prevented by the inclusion of dtt in the assay buffer, the addition of which does not lead to peak shifts or signal attenuation (figure 6a and supporting information, figure s10). Together, these results indicate these prototype compounds (1a, 2a, 3a, 4a, 6a, and 6b) covalently modify cysteines located on the la antigen . In the case of the arylsulfonamides 6a and 6b, it appears the protein conformation is strongly perturbed (denatured) without the inclusion of dtt . Of possible relevance, related compounds thiol reactivity of select screening compounds with the la protein as measured by alarm nmr . C hmqc spectra of selected c - labeled methyl groups for the selected compounds 1a, 2a, 3a, 4a, 6a, and 6b as tested by alarm nmr for protein reactivity . These methyl groups have been shown to undergo peak shifts and intensity decreases in the presence of many compounds that covalently react with neighboring cysteine residues . Compounds were incubated with the la protein in either the presence or absence of 20 mm dtt . (b) summary of the additional compounds tested by alarm nmr, including several negative compound controls that were inactive in the rtt109 hts and thiol - reactive counter - screen . To further show the utility of this method and that the results were not exclusive to a select subset, we also tested several other analogues of these prototype compounds, including some negative controls comprised of structural analogues that did not show interference in our hts counter - screens nor inhibition of rtt109-catalyzed histone acetylation in the slot blot assay . As expected, all of the prototype analogues, but not the negative controls, were alarm nmr - positive (figure 6b). As before, including dtt in the sample buffer prevented the alarm nmr reactivity . This demonstrates by a non - ms - based method that the interfering chemotypes are also susceptible to reactions with protein cysteines, a known source of nonspecific enzymatic inhibition and bioassay promiscuity . As there is considerable chemical overlap in many academic screening libraries (unpublished observations), due in part to shared commercial vendors and the combiphilic nature (i.e., amenable to synthesis by combinatorial schemes) of many screening scaffolds, we examined the scientific literature and the pubchem database to gauge whether our findings may be more broadly applicable to other biological systems and assay formats . Not surprisingly, compounds with the interfering scaffolds and some closely related derivatives have been reported in the context of many biological systems with varying degrees of biological activity and claims of utility . Several compounds bearing the scaffolds described in this report also showed patterns of bioassay promiscuity in a simple search of pubchem bioassay records (figure 7). On the basis of our findings, it is likely much of this bioassay promiscuity is due to nonspecific thiol reactivity . Select examples of compounds containing thiol - reactive chemotypes that demonstrate promiscuous pubchem bioassay profiles . Shown are conspicuous examples of compounds containing chemotypes 1, 2, 3, 4, and 6 that have promiscuous bioassay profiles according to a pubchem substructure search (accessed 1 march 2014). Accompanying each structure is the pubchem cid followed by the ratio (number of bioassays where the compound was classified as active / number of bioassays that the compound was tested). Finally, we analyzed hts records from a major pharmaceutical company for evidence of frequent - hitter behavior across the chemotypes that we have described above . It is commonly understood that academic and corporate libraries vary in size, composition, and chemical diversity, and therefore it is not immediately obvious that the trends seen in academic data would apply outside of this domain . For the purpose of comparison, we derived frequent - hitter scores for a large subset (> 1 m compounds) of astrazeneca s corporate compound collection . The frequent - hitter scores are based on the body of historical hts screening data for these compounds, typically, compounds in the corporate screening deck will have been tested in several tens to hundreds of hts campaigns . The frequent - hitter score we derive takes into account the anticipated incidence of activity for an average compound, with high scores suggesting a higher - than - expected level of activity . A score cutoff is defined to identify those compounds with an unexpectedly high level of activity, thereby designating frequent hitters empirically . Details of the derivation of the frequent - hitter score (pbsf) have been described previously . It should be noted that the corporate data set used to identify frequent hitters covers a wide range of assay types, and does not solely encompass assays like those described earlier in this publication . We examined the incidence of frequent hitters across various categories of nuisance chemotypes in the astrazeneca collection (table 7 and supporting information, table s4). It is clear that some of the nuisance chemotypes derived from academic data display an elevated incidence of frequent - hitter behavior in the corporate data as well, although not all chemotypes showed the same degree of promiscuity . Chemotypes 1 (benzothiophene dioxides), 2 (benzothiadiazole / benzofurazans), and 6 (p - hydroxysulfonamides) exhibit high levels of promiscuous behavior in the astrazeneca screening deck, suggesting their indiscriminate and deleterious influence is present in a wide range of assay technologies . For the p - hydoxysulfonamides 6, the observation that they may also cause protein denaturation in the alarm nmr assay in this study suggests another mode of action along these lines (that is, in addition to their other liabilities of redox activity and thiol reactivity). The astrazeneca corporate data showed high levels of assay promiscuity for chemotypes 6 and 7, which suggests the inactivity and weaker interference of chemotype 7 in our systems may be an assay - specific observation . That is, chemotype 7 may still be relatively promiscuous under other assays conditions, a speculation that may be pursued in future investigations . Nonsalt forms of the 1,2,4-thiadiazoles 3 show only slightly elevated levels of promiscuous behavior in the corporate data set (supporting information, table s4), while the salt forms were relatively promiscuous (table 7). The succinimide chemotype 4 did not exhibit high promiscuity in the astrazeneca screening deck, but this may be an indication of problematic behavior under specific assay conditions such as alkaline assay buffers, which we expect would be needed to generate the reactive maleimides 5 . We note that some of the assays used for generating the frequent - hitter scores have been stabilized with additions of dtt, which has the potential to mitigate the effects of reactive behavior depending on assay specifics . Therefore, the bioassay promiscuity emerging from this set of data may also be indicative of interference cause by mechanisms other than thiol reactivity . Overall, the observations derived from the larger set of corporate data corroborate the evidence derived from the academic data in this publication . Structure annotations: a, any atom; ns, number of substituents (e.g., 2s); nr, number of connected ring bonds (e.g., 2r); x, halogen . Ndata designates the subset of compounds for which a pbsf score had been derived . Expected incidence of anomalous binders is 6% (averaged over all compounds). Observed fractions of frequent hitters for structural classes (chemotypes). Note only biochemical assay data, and not cell - based assay data, were used to derive the frequent hitter score . In this article, we characterized the chemical basis of assay interference for five problematic chemotypes (1, 2, 3, 4, and 6) identified during the course of a recent triage of a sulfhydryl - scavenging hts for inhibitors of rtt109-catalyzed histone acetylation . These chemotypes were flagged as pains or have close chemical structural similarities to certain pains substructures . We first showed that while compounds containing any of these five scaffolds are capable of inhibiting rtt109-catalyzed histone acetylation, this inhibition was confounded by the ability of these compounds to interfere with the hts assay readout by reacting with free coa in the rtt109 hts . Ms and lc hrms that these compounds can form adducts with other biological thiols such as gsh, and in another orthogonal enzymatic assay, can inhibit several different hats in vitro only when dtt is absent in the reaction mixture . Protein mass spectrometry confirmed several of these compounds could covalently modify multiple cysteines in the hts . Using alarm nmr, yet another orthogonal detection method, we showed that the majority of these compounds can covalently modify cysteines on a completely unrelated protein system . The findings described herein strongly suggest investigators (and reviewers) flag these problematic compounds and avoid their follow up . It is particularly troubling that many of these compound classes are still being reported in the patent literature and reputable scientific journals, some with dubious claims of biological utility (see supporting information). Perhaps not coincidentally, these compound types were active in other bioassays according to pubchem queries . The propagation of these nuisance compounds in reputable journals suggests that many academicians and reviewers alike are not fully aware of nuisance compounds such as pains and perhaps not appreciative of their potential to sidetrack early drug discovery projects . On the basis of our studies, we highly recommend the problematic chemotypes described in this report be pursued as chemical leads with high levels of skepticism and that investigators currently working with these compounds carefully re - evaluate the interpretation of their results when there are claims of biological utility, including apparent enzymatic and cell - based selectivity, bioactivity, and mechanism - of - action studies . For instance, we posit that much of the selectivity observed for these chemotypes (a common defense for those publishing pains as bioactive compounds) is due to different susceptibilities of assay components (e.g., enzymes, cell lines) to thiol - reactive compounds or other nonspecific mechanisms and that observed bioactivity is likely attributable to off - target effects . With regards to mechanistic studies, it is likely some key component is missing from the experimental design (e.g., assessing the effect of dtt or rigorously testing for irreversibility). A recommended list of assays for evaluating the potential for compound we recommend that knowledge - based methods be supplemented by more than one of the experimental - based methods . Triage of active compounds from hts (real or virtual) should always include knowledge - based methods to flag potential reactive entities . Flagged compounds should then either be removed from consideration or investigated more rigorously using two or more of the experimental - based methods described above . Notes: several of these methods have been described in the text and elsewhere . The use of frontier molecular orbital (fmo) calculations has been reported as a gross method of flagging frequent - hitters . Certain cysteine proteases (e.g., caspase-1, -8) have been used as probes for reactivity including cysteines oxidation by redox - active compounds . The emergence of epigenetic targets such as hats has led to the development of several types of hts assays to study epigenetic modifications like acetylation . Hat activity can be probed with antibody - based methods (e.g., western blots, amplified luminescent proximity homogeneous assays, tr - fret) or sulfhydryl - scavenging methods (e.g., fluorescent probes or coupled - enzyme reporters). Other methods such as radiolabeled substrates, mass spectrometry, and electrophoretic mobility, have been used to assay the status of protein acetylation, including cell - based adaptations . While each method has distinct advantages and disadvantages, each is still susceptible to false positives, assay artifacts, and identifying promiscuous the maleimide - based screens are subject to several mechanisms of chemical interference . We determined thiol - trapping compounds represented a significant source of assay artifacts in our cpm - based rtt109 hts, especially chemotypes 1, 2, 3, 4, and 6 . These compounds interfered with our hts readout by forming covalent adducts with the coa produced by the hat reaction, creating a convoluted readout of enzymatic inhibition . Nucleophilic screening compounds can also form adducts with cpm that can create either a false - positive or false - negative readout pattern, depending on the fluorescent characteristics of the adduct . Another artifact source is fluorescence quenching, although we did not encounter many examples of fluorescence quenchers in our post - hts triage (data not shown). On the basis of the chemotypes in this report, along with the other triaged compounds, the majority of assay artifacts from our cpm - based hts resulted from thiol - trapping rather than compound, a major (though not insurmountable) disadvantage of this screening method is the high levels of assay interference and the time and resources needed in the post - hts phase to triage these artifacts . In fairness, it is worth noting that this method is capable of identifying compounds that inhibit enzymatic activity and has distinct advantages such as low cost and robustness . To prevent follow - up on bad chemical matter discovered by maleimide - based screens, first, we strongly recommend having a validated, robust orthogonal assay in place prior to conducting an hts with this method . Relying solely on the cpm - based method could lead to the selection of thiol - trapping compounds, and if used for the basis of compound optimization could lead to the unfortunate case of optimizing for thiol reactivity rather than the desired enzymatic inhibition . This could conceivably happen if one were to view the apparent enzymatic inhibition data in tables 15 as evidence of a preliminary sar, when in fact it would be more appropriately called sir (i.e., structure interference relationship). As the nature of the cpm - based format contraindicates the use of dtt and other biological reducing agents, it would be advisible to have an orthogonal assay that can test candidate compounds in both the presence and absence of dtt or similar reducing agent to further rule out thiol reactivity (e.g., table 6). This assay can identify assay artifacts, and if used in parallel with an orthogonal assay, can identify potentially problematic thiol - reactive enzymatic inhibitors . Third, the ratio of acetyl - coa to test compound should be kept as high as possible, although this must be balanced with other important factors such as the acetyl - coa km . If fluorescence quenching is a concern, we recommend the facile counter - screen used in this manuscript, as it should not be easily susceptible to interference from thiol - trapping compounds . A deeper understanding and appreciation for the chemical mechanisms of assay interference and thiol reactivity can have important implications for early analogue selection and screening library design (see figure 1 for the general chemotypes discussed here). Elimination reactions, and on the basis of our results, we recommend a leaving group analysis for such compounds . Therefore, selecting and/or testing analogues with weaker leaving groups or a reduction at the c2c3 position (supporting information, figure s2) may be a potential strategy to overcome thiol reactivity in this chemotype . The former strategy may be useful for certain succinimides 4 with good leaving groups (e.g., s - linked heteroaromatics; supporting information, table s4). Certain 1,2,4-thiadiazoles 3 are susceptible to attack by thiol nucleophiles at the s1-position, specifically when the n2-position is positively charged . We note our findings with this chemotype are consistent with other previous mechanistic work on these compounds . Should investigators choose to pursue compounds bearing chemotype 3, it may be useful to assess the effect of switching to 1,2,4-oxadiazole analogues, as well as testing the nonsalt forms of 1,2,4-thiadiazoles . The benzothiadiazoles / benzofurazans 2 interfere by nucleophilic aromatic substitution, and this interference correlated with the apparent strength of the presumed leaving group . As with chemotypes 1, 4, and 6, a strategy for navigating away from this problematic chemotype would be to select analogues with weaker leaving groups or with less electron - withdrawing functional groups on the heteroaromatic core . Substructure to include additional strong electron - withdrawing moieties such as nitro groups (table 2). The sar / sir of these chemotypes also raises important questions about the ability of certain pains to be converted to non - pains . It is interesting to note that even in the cases where incidence of anomalous behavior is high, presence of the offending substructure does not predispose all compounds to anomalous behavior . This suggests that it may be possible to design out such behavior if the activity seen in the assay is true after all . Nonetheless, presence of the nuisance chemotype does suggest that there is a very significant risk of failure in attempting such optimization, as chances are high the active is acting via a therapeutically uninteresting mechanism, thereby rendering such hits unattractive start points for hts follow - up . Both the succinimides 4 and the p - hydroxyarylsulfonamides 6 illustrate the susceptibility of screening compounds to undergo chemical transformations under certain assay conditions . The decomposition of chemotype 6 was unexpected, and it will be interesting to examine the conditions critical for this conversion as well as more detailed characterization of this decomposition process . For instance, it appears several of these problematic arylsulfonamides were also unstable in alarm nmr buffer, suggesting this scaffold is likely unstable in many other biologically relevant aqueous buffers and not an isolated phenomenon (supporting information, figure s11). Ms / ms, further attesting to the instability of this scaffold in our assay conditions (supporting information, table s3). Given the instability of scaffolds 4 and 6, we recommend assessing the stability of any promising compounds in assay buffer by analytical techniques to verify the structure of the active chemical entity in the biological context . This is a rather straightforward experiment, and in light of our findings, it may be an important confirmatory experiment to perform before proceeding to more extensive experiments, such as molecular modeling, that are based on correct structure identification and integrity . There has been a recent resurgence of interest in covalent drugs (e.g., ibrutinib and dimethyl fumarate). This renewed interest has been used as a line of defense in the reporting of known reactive compounds, including pains, as viable drug leads . We have shown in this manuscript that all interference compounds are not created equal and that they can exhibit a distinct sir . However, we suggest it is highly unlikely that compounds that show indiscriminate protein reactivity, as do pains, will ever be useful drug or probe leads . While there may be exceptions, we expect that most of the recently developed covalent drugs have either been purposefully designed as such or have undergone extensive mechanistic studies and medicinal chemistry optimization . They are usually not the outcome of the optimization of nonselective, reactive, and promiscuous compounds that sometimes are reported from hts . Therefore, we recommend that the scientific community apply an extremely high standard of rigor to the review and publication of manuscripts that claim any drug- or probe - like potential for these types of compounds . Additionally, we caution researchers that commercially available probes that feature known thiol - reactive moieties, including but not limited to those chemotypes discussed herein, may be less selective versus the proteome than their probe - like status suggests (supporting information, table s5). Our findings highlight the importance of taking a chemocentric approach to hts triage and hit prioritization and highlight the need for carefully planned counter - screens and orthogonal assays in a well - validated cascade of hit - triaging assays (figure 8). We believe our investigation also demonstrates the importance of partnering with medicinal chemists in the post - hts triage process and should serve as caution for lead selection based primarily on initial potency and sar data without confirmation of activity by orthogonal methods . The continued growth of cheminformatics and the incorporation of pains filters into both commercial software suites (e.g., sybyl, schrodinger canvas) and freeware is undoubtedly a positive advancement for the field . However, many compounds with chemotypes 14 were not flagged by our cheminformatics pains filters . This raises important concerns about the potential for overreliance on cheminformatics filters . For instance, unseasoned researchers (and reviewers) may fall into the trap that because pains were removed by substructure filters, that they no longer have to consider any related nuisance compounds . Strategies to mitigate this risk are to (1) take a chemocentric approach to hts triage using a well - validated cascade of deconvoluting assays, (2) encourage more mechanistic studies of nuisance compounds to further the understanding of their behavior, (3) periodically update pains filters as more data is made available, and (4) mine the ever - increasing amount of hts data for insights into pains substructures (e.g., along the lines shown in a recent report). Such undertakings also raise important follow - up questions for those in hts triage about what exactly should constitute a pains and what criteria should form the basis for classifying a compound as promiscuous and/or pan - assay . Likely, these definitions will have to be dependent on the screening context, at least in part, and guided by those with sufficient expertise in hts triage . The following reagents were obtained from sigma - aldrich: dmso, cpm, coa (sodium salt hydrate), acetyl - coa (sodium salt), bovine serum albumin (bsa), h2o2, and triton x-100 . Compounds tested in post - hts assays were repurchased as solid powders from standard chemical vendors (e.g., emolecules). In a quality - control sampling of a random 5% of the chemical library samples used in this report, greater than 90% of the tested commercial samples had acceptable purities (> 90%) by uplc the cpm - based rtt109 assays have been detailed in a previous report with minor modifications . Briefly, all compounds studied in this report were rescreened in assay buffer containing freshly prepared 0.01% triton x-100 (v / v) and enzyme concentrations of 50 nm rtt109vps75 complex . For ic50 experiments, compounds were tested in triplicate at eight compound concentrations ranging from 200 nm to 125 m final compound concentrations . Slot blots were performed on reaction aliquots using standard techniques with a bio - rad bio - dot sf microfiltration apparatus . Membranes were imaged with a li - cor odyssey and analyzed using image studio (li - cor biosciences). Z factors for each plate were calculated using eq 1:1where and represent the standard deviation and mean of the positive (c) and negative (c) plate controls, respectively . Response data to the sigmoidal dose response variable slope four - parameter equation in graphpad prism 6.0 . Compounds interfering with the cpm - based assay readout were identified as previously described with minor modifications . Compounds were tested in triplicate at eight concentrations ranging from 200 nm to 125 m final compound concentrations using an adaption of the rtt109 hts assay format . Proteins and assay buffer were dispensed to assay plates analogously to the hts procedure, then the acetyl - coa substrate was replaced with coa in concentrations titered to match the fluorescence intensity observed for the uninhibited enzyme reaction in the hts assay (approximately 5 m coa). Compounds were incubated with coa and allowed to react with cpm under conditions identical to the hts procedure . Assay interference was quantified by comparing the background - corrected (compound + proteins + coa + cpm) fluorescence intensities to the (dmso + proteins + coa + cpm) controls . To further investigate their fluorescence behavior under the hts conditions, select compounds were also incubated with assay reagents (buffer - only, buffer + coa, buffer + cpm) and their fluorescence intensity measured . The overall plate layout, controls, protocols, and assay readouts were unchanged from the aforementioned compound coa compounds were tested for evidence of fluorescence quenching using a modification of our published procedure . Briefly, cpm and coa (20 and 5 m final concentrations, respectively) were allowed to react to completion in assay buffer . Completion was defined as a stable signal plateau, usually after 5 min reaction time . The cpm coa adduct solution (20 l per well) was then dispensed into assay plates preplated with dmso and test compounds . Microplates were shaken for 5 min and allowed to equilibrate for another 5 min at room temperature . Fluorescence intensity was measured, and the data was analyzed as percent signal reduction compared to dmso controls . Selected compounds (1 equiv) and either coa or reduced l - glutathione (2 equiv) were incubated under hts - like conditions, except with 5% dmso (v / v) and no detergent in the assay buffer . Compounds were also tested in hts buffer or methanol minus the addition of biological thiols . . Samples with visible precipitates were passed through 0.25 m syringe filters to remove particulates . Sample injections were typically 1.0 l in volume performed by an autosampler and were analyzed on a waters uplc system using a beh c18 2.1 mm 50 mm column . The flow rate was 0.250 ml / min with a standard gradient starting at 95% solution a (950 ml h2o, 50 ml mecn, 1 ml formic acid) and ending with 100% solution b (1000 ml mecn plus 1 ml formic acid) over 6.5 min . The samples were monitored simultaneously using an els detector, a diode array detector (214, 220, 244, and 254 nm), and a zq mass spectrometer (esi positive and negative modes). Selected compounds were assessed for redox activity using published protocols . Freshly prepared 100 m h2o2 (sigma) was included as a positive plate control, while nsc-663284 and 4-amino-1-naphthol were used as positive redox - active controls for dtt and dtt - free assay conditions, respectively . Compounds were tested in triplicate at eight final concentrations (200 nm to 125 m via 2.5-fold dilutions) in either the presence or absence of 1 mm dtt final concentration . All active compounds did not interfere with the assay readout at a610 (data not shown). For selected compounds, inhibition of hat activity was also checked with an orthogonal in vitro radiolabeled substrate assay . Rtt109 inhibition was tested at eight compound concentrations (200 nm to 125 m final compound concentrations via 2.5-fold dilutions) in an adaptation of a previous procedure . Briefly, reactions were performed in standard volume 384-well microplates using 45 l total reaction volumes containing the following in final concentrations: 50 mm tris hcl, ph 8.0, 50 mm kcl, 0.1 mm edta, 1 mm dtt, 0.01% triton x-100 (v / v), 50 ng/l bsa, and 2.5 m [h]-acetyl - coa (perkinelmer). Purified recombinant yeast rtt109vps75 was tested at approximately 5 nm final concentrations, while purified recombinant asf1dh3h4 (approximately 250 nm) was used as acetylation substrate . Compounds and dmso were plated with a multichannel pipet, followed by a similar addition of a solution containing enzyme and histone substrate (36 l). Test compounds were allowed to equilibrate with enzyme and histone substrate for 10 min at 30 c in an incubator . The hat reaction was initiated by adding [h]-acetyl - coa solution (7.5 l). Dmso content was kept constant across all reactions at 3% (v / v). After 5 min, the reactions were quenched by multichannel pipet transfer of reaction aliquots (35 l) to adjacent microplate wells each containing 35 l of 2-propanol . Aliquots (35 l) of the quenched solutions were carefully spotted onto whatman p-81 phosphocellulose paper filters (ge healthcare) and air - dried . Filter papers were washed five times for 5 min per cycle with 50 mm nahco3, ph 9.0, then rinsed with acetone and then allowed to air - dry for 30 min . [h]-acetate incorporation was then measured by an ls6500 liquid scintillation counter (beckman coulter). Testing versus p300bhc and the gcn5ada2ada3 complex were performed similarly, except that the final enzyme concentrations were approximately 500 pm and the substrate was purified recombinant dh3h4 tetramers . Compounds and proteins were incubated together at 30 c for 60 min at 100 m and 10 m final concentrations, respectively . Reaction mixtures were denatured with gentle heating and then further resolved by sds - page . Peptide extracts were dried in vacuo and reconstituted in 98:2:0.1 h2o: acetonitrile: tfa; approximately 0.2 g of each gel band was analyzed by capillary lc ms on a velos orbitrap mass spectrometer (thermo fisher) with higher energy collision induced dissociation activation . Peaks studio 6.0 build 20120620 (bioinformatics solutions) software package was used for interpretation of tandem ms and protein inference . Search parameters for rtt109, vps75, and asf1 proteins were uniprot database (sacharomyces cerevisiae strain atcc 204508/s288c, taxonomy i d 559292, accessed 19 may 2014) concatenated with the common lab contaminant proteins (www.thegpm.org); parent mass error tolerance = 20.0 ppm; fragment mass error tolerance = 0.1 da; precursor mass search type = monoisotopic; enzyme trypsin with max missed cleavages = 2 and nonspecific trypsin cleavage; variable modifications = methionine oxidation and dioxidation, cysteine oxidation, and dioxidation, and suspected compound adducts; maximum variable modifications per peptide = 5; false discovery rate calculation = on; spectra merge options = 0.2 min within 10.0 ppm mass window; charge correction = on for charge states 28; spectral filter quality> 0.65 . Support for the detection of peptides plus adducts from each supporting tandem ms data was based on: (1) high confidence peaks peptide score (minimum 10 log p 35), (2) a minimum of five consecutive b- or y - type peptide fragment ions, (3) high precursor mass accuracy (<7 ppm), and (4) supporting signature ion peaks for the site localization of the pertinent cysteine modification on one or more peptide fragments . Incidence of frequent - hitting behavior was checked in the astrazeneca corporate screening deck by mining the historical screening data . We calculate a descriptor, pbsf, for each compound to determine whether it is more active than expected . The pbsf score is the negative logarithm of the probability that the observed pattern of activity and inactivity is observed by chance, given the known average behavior across all compounds in the screening deck and across the historical set of screening campaigns they have been measured in . If the likelihood of seeing the pattern at hand is high, the compound is likely not a frequent hitter and all is fine . However, if the probability of seeing the pattern is low, the resulting pbsf score will be high and the pattern should be regarded as anomalous . A cutoff of pbsf> 2 was used to designate compounds exhibiting suspicious binding behavior . To check the incidence of frequent - hitting behavior, we searched the corporate collection using substructures (with in - house tools), collated pbsf scores for the set, and counted the number of frequent - hitting compounds using the pbsf threshold stated in the above . For reference, the average fraction of compounds displaying frequent - hitting behavior across the collection of compounds with historical hts data is 6% . The number of hts data points is variable for each compound, as it depends on the number of times a compound has been screened . The median number of data points per compound in the data set is approximately 200, with only 10% of the compounds having less than 50 data points . Only biochemical assay data, and not cell - based assay data, were used to derive the frequent hitter score alarm nmr was performed as previously described with minor modifications . The gene encoding amino acids 100324 of the human la antigen was cloned into pet-28b+ vector (novagen) such that it contained both an n- and c - terminal his tag . The plasmid was freshly transformed into escherichia coli rosetta cells (novagen) and cultured in m9 minimal media supplemented with nh4cl (cil) in an adaption of published procedures . The la antigen was enriched with c at the -methyl groups of leucine, the -methyl group of isoleucine, and the -methyl groups of valine by the addition [3-c]--ketobutyrate and [3,3-c]--ketoisovalerate (sodium salts, cil) to the culture medium 30 min before inducing in the presence of 1 mm iptg for 8 h at 25 c (od600 was approximately 0.8 at time of induction). Harvested cells were lysed by french press in ice - cold lysis buffer consisting of 50 mm tris, ph 7.6, 300 mm nacl, 10% glycerol (v / v), 5 mm -mercaptoethanol (bme), 5 mm imidazole, 2 mm mgcl2, benzonase (sigma), and protease inhibitor cocktail . This solution containing the lysed cells was sonicated briefly (3 15 s pulse sequence) on ice, then loaded onto a prewashed ni - bead column (ge healthcare) kept at 4 c . Proteins were eluted from the beads with an elution buffer consisting of 50 mm tris, ph 7.6, 300 mm nacl, 10% glycerol (v / v), 5 mm bme, and an imidazole gradient ranging from 5 mm to 0.5 m. pooled elution fractions containing the la antigen were dialyzed overnight (25 mm sodium phosphate, ph 7.0, 5 mm dtt), flash - frozen in liquid n2, and stored at 80 c until further use . Prior to use, aliquots of 500 m protein was incubated in the presence of 20 mm dtt at 37 c for 1 h, then dialyzed versus 2 2 l of 25 mm sodium phosphate buffer, ph 7.0 (no dtt) at 4 c with constant n2 bubbling . The h / c - hmqc spectra were acquired in 25 mm sodium phosphate buffer, ph 7.0, 10% d2o (v / v; cil) 200 m test compounds delivered from 10 mm dmso stock solutions, and 20 mm dtt . Compounds were incubated with proteins at 37 c for 1 h and then 30 c for 15 h prior to data collection . Data were recorded at 25 c on a bruker 700 mhz nmr spectrometer equipped with a cryoprobe (bruker) and autosampler . Samples were loaded into bruker 1.7 mm samplejet tubes with 40 l total sample volumes and stored at 4 c while in queue . The alarm nmr samples were tested at 50 m protein concentrations using 16 scans, 2048 complex points in f2, and 80 points in f1 using standard protein hmqc and water suppression pulse sequences . Nonreactive compounds were identified by the absence of chemical shifts (c - methyl) 20 mm dtt . Reactive compounds induced chemical shifts in certain diagnostic peaks in the absence of dtt, and this effect was significantly attenuated when 20 mm dtt was included in an otherwise identical sample . As an additional precaution against trace reactive contaminants, compounds tested by alarm nmr were repurified in - house by standard hplc procedures using mass - directed collection . Detailed adduct synthetic procedures and chemical characterization can be found in the supporting information . The gene encoding amino acids 100324 of the human la antigen was cloned into pet-28b+ vector (novagen) such that it contained both an n- and c - terminal his tag . The plasmid was freshly transformed into escherichia coli rosetta cells (novagen) and cultured in m9 minimal media supplemented with nh4cl (cil) in an adaption of published procedures . The la antigen was enriched with c at the -methyl groups of leucine, the -methyl group of isoleucine, and the -methyl groups of valine by the addition [3-c]--ketobutyrate and [3,3-c]--ketoisovalerate (sodium salts, cil) to the culture medium 30 min before inducing in the presence of 1 mm iptg for 8 h at 25 c (od600 was approximately 0.8 at time of induction). Harvested cells were lysed by french press in ice - cold lysis buffer consisting of 50 mm tris, ph 7.6, 300 mm nacl, 10% glycerol (v / v), 5 mm -mercaptoethanol (bme), 5 mm imidazole, 2 mm mgcl2, benzonase (sigma), and protease inhibitor cocktail . This solution containing the lysed cells was sonicated briefly (3 15 s pulse sequence) on ice, then loaded onto a prewashed ni - bead column (ge healthcare) kept at 4 c . Proteins were eluted from the beads with an elution buffer consisting of 50 mm tris, ph 7.6, 300 mm nacl, 10% glycerol (v / v), 5 mm bme, and an imidazole gradient ranging from 5 mm to 0.5 m. pooled elution fractions containing the la antigen were dialyzed overnight (25 mm sodium phosphate, ph 7.0, 5 mm dtt), flash - frozen in liquid n2, and stored at 80 c until further use . Prior to use, aliquots of 500 m protein was incubated in the presence of 20 mm dtt at 37 c for 1 h, then dialyzed versus 2 2 l of 25 mm sodium phosphate buffer, ph 7.0 (no dtt) at 4 c with constant n2 bubbling . The h / c - hmqc spectra were acquired in 25 mm sodium phosphate buffer, ph 7.0, 10% d2o (v / v; cil) 200 m test compounds delivered from 10 mm dmso stock solutions, and 20 mm dtt . Compounds were incubated with proteins at 37 c for 1 h and then 30 c for 15 h prior to data collection . Data were recorded at 25 c on a bruker 700 mhz nmr spectrometer equipped with a cryoprobe (bruker) and autosampler . Samples were loaded into bruker 1.7 mm samplejet tubes with 40 l total sample volumes and stored at 4 c while in queue . The alarm nmr samples were tested at 50 m protein concentrations using 16 scans, 2048 complex points in f2, and 80 points in f1 using standard protein hmqc and water suppression pulse sequences . Nonreactive compounds were identified by the absence of chemical shifts (c - methyl) 20 mm dtt . Reactive compounds induced chemical shifts in certain diagnostic peaks in the absence of dtt, and this effect was significantly attenuated when 20 mm dtt was included in an otherwise identical sample . As an additional precaution against trace reactive contaminants, compounds tested by alarm nmr were repurified in - house by standard hplc procedures using mass - directed collection . Detailed adduct synthetic procedures and chemical characterization can be found in the supporting information.
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Of the various behaviors and adaptations that have evolved in marine environments, bioluminescence stands out as one of the particular importance, given the fact that it has evolved independently more than 40 times (haddock et al . 2010). While it does not necessarily give specific advantage to species in cold environments, the long periods of continuous darkness that characterize winters at high latitudes create an environment, at least with respect to light, that is similar to the deep - sea . Depending on which taxa these include defensive functions such as the counter - illumination, the burglar alarm and offensive mechanisms such as prey attraction and intraspecific communication other adaptations have evolved in both phytoplankton and zooplankton to survive in these harsh conditions that relate more to metabolic rates and the actual vertical space occupied in the water column within which a species spends the winter months . One such strategy is to enter a dormant state and overwinter at depth, seen for the copepods calanus glacialis and c. hyperboreus which are commonly reported to enter a state of diapause at depth during winter months (fortier et al . They, then, return to shallower water in the summer to take advantage of the high productivity rates (ashjian et al . 2009) for the first time provide acoustic evidence of active vertical migrations of zooplankton throughout the polar night in the high arctic . More recently, this phenomenon was corroborated in the southern hemisphere where diel vertical migration (dvm) was shown to continue through the austral winter in the lazarev sea, but ceased during the austral summer (cisewski et al . The goal of the current study was to characterize plankton abundance and distribution patterns during a time of year that has rarely been studied by means of vertical net tows and autonomous underwater vehicle (auv) surveys . Fitted on the auv were adcps, a ctd and a bathyphotometer designed to register bioluminescence potential in the water column . Data were collected off the coast of ny lesund in kongsfjord, svalbard (7857 n, 1156 e) in ~120 m of water from january 19 to 22, 2010 . Spatial and temporal dynamics of acoustic backscatter, salinity, temperature and bioluminescence were measured using a remus-100 auv (moline et al . 2005). The vehicle was equipped with upward and downward facing rd instruments 1,200-khz workhorse navigator acoustic doppler current profilers (adcp), configured in this study to provide relative acoustic backscatter as an estimate of scattering volume, rather than current velocities, a neil - brown ctd and a bioluminescence bathyphotometer (bp; moline et al . The bp utilizes an impeller to continuously draw a measured volume of water into a chamber through the front of the nosecone where bioluminescence is measured by a photomultiplier tube at 60 hz [see herren et al . (2005) for details]. For statistical purposes, we restricted our analysis to the non - zero observations . The auv was deployed at 10:27, 12:25 and at 13:40 (local time 19th of january) at depths of 15, 45 and 75 m, respectively . The vehicle was also deployed at 21:30 (19th of january) along the same transect as during the day but ran one mission surveying the transect at 15, 45 and 75 m successively without breaks between each depth . Adcp data were processed to remove noise and calculate relative backscatter coefficient (sv) according to deines (1999) for the data collected between 0.5 and 5.0 m away from either side of the vehicle . Following the auv deployments, continuous bp observations were made from 15:00 on january 21, 2010 until 09:00 on january 22, 2010 at 1 m depth at the ny lesund harbor about 2 km from the study site . For logistical reasons (power supply, stable holdfast for the instrument and weather conditions), it was not possible to carry out the continuous bp observations in the fjord at the same place as the auv deployments . However, given the short distance between the two locations and the absence of any physical barriers, the two sites are regarded as comparable for the examination of circadian rhythm (see also " discussion "). Concurrent with the remus deployments, vertical net hauls were conducted using a wp2 plankton net, 180-m mesh size with a 0.25 m opening . In order to collect vertical net hauls from the same depths surveyed by the remus adcp, two replicates from each depth were taken from 300 m, 600 m and from 900 m between 10:3013:00 lt . During the nighttime auv deployment, replicate hauls were taken between 300 m, but inclement weather resulted in only one sample from 600 m was collected and none between 900 m. to determine species composition and abundance for specific depth intervals of 6030 m and 9060 m, the results from 300 m were subtracted from those of 600 m, and results from 600 m from those of 900 m, respectively . Additionally, the remus bp was equipped with cylindrical plankton nets (20-m mesh size), and these nets were fitted to each of the two exhausts of the bp (moline et al . 2009), which hence provided two replicate samples from each of the depths (15, 45 and 75 m) sampled using the auv . For all plankton samples, samples were later enumerated and identified to the lowest possible taxonomic unit using a leica stereomicroscope with 6.340 magnification . The temperature, salinity and density profiles were similar between night and day during the sample period (fig . 1) and did hence not reveal any major advection events that would otherwise influence the measurements and results presented below . Based upon willis et al . (2006), physical parameters indicated that the water mass in kongsfjord was of artic water origin . Bioluminescence was detected throughout the water column both night and day, with higher bioluminescence at depth during the day and increased surface bioluminscence at night (fig . Comparison of the bioluminescence at the specific depths also provided that this result with significantly greater bioluminescence intensity per flash was observed at 75 m during the day and at 15 m at night (table 1), suggesting that the organisms with more intense flashes, i.e., larger zooplankton (see moline et al . 1vertical profiles of a temperature (c), b salinity (ppt), c density (kg m) and d bioluminescence (photons / l) as a function of depth (m) taken from the remus auv during the final ascents from 75 m during the day and night deployments . Daytime observations represented in gray and nighttime in black . For this profile, bioluminescence for the upper water column (<45 m) was significantly less than that for the lower water column during the day (mann whitney, p = 0.009, n = 102) and higher bioluminescence in the upper water column at nighttable 1mean bioluminescence intensity per flash (se) surveyed by the auv at the three different depths during the daytime and nighttime deployments (lt is local time)depth (m)mean intensity / flash (10) daytime (10:3014: 25 lt)mean intensity / flash (10) daytime (21:3022:30 lt)mean s v difference (day night)159 2 * 160 142 16457 28 3357518 6 * 4 1 17significant differences were found between depths using mann whitney (* p = 0.016, n = 1,030; p = 0.025, n = 286; p = 0.022, n = 126), and additionally, the difference in the mean acoustic backscatter coefficients (s v) between day and night is shown for each depth . The differences in s v between day and night were significant (mann whitney, p <0.001) at all depths vertical profiles of a temperature (c), b salinity (ppt), c density (kg m) and d bioluminescence (photons / l) as a function of depth (m) taken from the remus auv during the final ascents from 75 m during the day and night deployments . Daytime observations represented in gray and nighttime in black . For this profile, bioluminescence for the upper water column (<45 m) was significantly less than that for the lower water column during the day (mann whitney, p = 0.009, n = 102) and higher bioluminescence in the upper water column at night mean bioluminescence intensity per flash (se) surveyed by the auv at the three different depths during the daytime and nighttime deployments (lt is local time) significant differences were found between depths using mann whitney (* p = 0.016, n = 1,030; p = 0.025, n = 286; p = 0.022, n = 126), and additionally, the difference in the mean acoustic backscatter coefficients (s v) between day and night is shown for each depth . The differences in s v between day and night were significant (mann whitney, p <0.001) at all depths it is important when interpreting changes in bioluminescence signals that the circadian rhythms in the bioluminescence potential of planktonic organisms be taken into account (batchelder et al . The continuous bp observations at the ny lesund harbor showed no evidence of a circadian rhythm in the bioluminescence signal (fig . 2) in a location (sheltered by the pier and with a max depth of 5 m) where vertical migration of zooplankton would be restricted . Organisms collected by net hauls next to the moored bp and by nets connected to the bp during this period using identical methods described above for the auv showed> 80% similarity to the study transects (data not shown), thus making these results applicable to the observations made by the auv.fig . 2hourly means of log bioluminescence potential (solid black line) with standard deviations (dotted black lines) collected at 1 m depth from 15:00 lt on january 21 to 09:00 lt on january 22, 2010, (n = 9,545, non - zero observations). Flashes per unit time were also found to be consistent throughout the time series hourly means of log bioluminescence potential (solid black line) with standard deviations (dotted black lines) collected at 1 m depth from 15:00 lt on january 21 to 09:00 lt on january 22, 2010, (n = 9,545, non - zero observations). Flashes per unit time were also found to be consistent throughout the time series estimates of relative backscatter coefficient as a relative measure of zooplankton biomass in a 10-m swath around the prescribed vehicle depths showed significantly higher intensity between 70 and 80 m during the day, and between 10 and 20 m and 4050 m at night (table 1). In combination with the changes in bioluminescence intensity, these data demonstrated a coordinated movement of biomass indicative of dvm . Plankton enumerations from wp2 vertical net hauls show an increase above 60 m in the majority of the most abundant zooplankton taxa at night, including pseudocalanus spp . These genera have been reported to present throughout the year in this regions (lischka and hagen 2005). Table 2 provides specific species classification and shows that this increase above 60 m at night is also apparent in the enumerations of other less abundant taxa including calanus finmarchicus, acartia longiremis, oncaea borealis and eukrohnia hamata . Of these, metridia lucens, metridia longa, oncaea borealis, thysanoessa inermis and thysanoessa longicaudata most likely account for the increase in high - intensity bioluminescent flashes at 15 and 45 m during the night (table 1).table 2concentrations of the plankton captured by the 180 m wp2 plankton net during the day and at night for depth intervals 300, 6030 and 9060 mtaxadepth (m)ind./m (day)ind./m (night)taxadepth (m)ind./m (day)ind./m (night) calanus finmarchicus 030 5082 heterorhabdus norvegicus*030 <1<13060 411483060 <166090 896090 4 calanus glacialis 030 410 harpacticus chelifer 030 9503060 31313060 543060901476090 <1 calanus hyperboreus 030 <11harpacticoida spp.030 2<13060 <1<13060 11<16090 46090 <1 pseudocalanus spp.030 3,10010,300 oithona atlantica 030 3887253060 <116,8203060 <15556090 7756090 <1 microcalanus spp.030 5632,300 oithona similis 030 4008253060 1631,9003060 <11,3356090 636090 131 metridia lucens*030 317appendicularia*030 17153060 4183060 336090 66090 96 metridia longa*030 <1<1 limacina helicina 030 2<13060 <133060 <116090 116090 12 acartia longiremis 030 175375 sagitta elegans 030 <123060 <13853060 5116090 <16090 12 paraeuchaeta norvegica 030 <1<1 eukrohnia hamata 030 4303060 1<13060 2216090 <16090 11 diastylis lucifera * 030 <1<1 thysanoessa longicaudata*030 <123060 1<13060 376090 <16090 2 bradyidius similis 030 <11 thysanoessa inermis*030 <1<13060 <133060 <1226090 66090 <1 oncaea borealis*030 12<13060 512006090 <1asterisks indicate organisms known to be bioluminescent concentrations of the plankton captured by the 180 m wp2 plankton net during the day and at night for depth intervals 300, 6030 and 9060 m asterisks indicate organisms known to be bioluminescent plankton enumerated from the> 20 m net collection of the bp exhaust suggests that during the day, the greatest biomass occurred at 45 m and was dominated by copepod nauplii, copepod eggs and the tintinnid acantostomella norvegica (table 3). The same three groups of organisms dominated the biomass at 15 and 75 m. other major contributors at each of these three depths were ceratium and protoperidinium spp . Microcalanus spp ., oithona similis and oithona atlantica (table 3), consistent with the wp2 nets samples (table 2).table 3concentrations of the plankton captured by the 20 m plankton nets covering the remus bp exhaust for daytime deployments at 15, 45 and 75 mtaxadepth (m)ind / m taxadepth (m)ind / m ceratium arcticum 15229copepod nauplii*151,40945352452,72675246751,403 ceratium fusus * 159.6copepod eggs159034591.3451,6317580.3751,372 ceratium furca 15<1 oncaea borealis * 15545134552754754 protoperidinium spp. *15211 harpacticoida spp . Microsetella norvegica 1574539145775387754diatom spp.151015545594577580754 acantostomella norvegica 151,252 oithona atlantica 1567452,02345117758277559 salpingella acuminata 15241 oithona similis 157245163451117512275108 heliocostomella subulata 152 eukrohnia hamata 15<1451345175<175<1 parafavella denticulata 157gastropoda larvae15234533453375457511 calanus finmarchicus 158appendicularia*1511458453975117521 paraeuchaeta norvegica ciii 15<1membranipora larvae1519451452075<17521 acartia longiremis 1529bivalve larvae15<1457457751875<1 pseudocalanus spp.15205 limacina helicina 15<14521545<175154754 microcalanus spp.15137 thysanoessa longicaudata * 15<14543745<175140751asterisks indicate organisms known to be bioluminescent concentrations of the plankton captured by the 20 m plankton nets covering the remus bp exhaust for daytime deployments at 15, 45 and 75 m asterisks indicate organisms known to be bioluminescent evident from organisms collected from both net sampling approaches is that the day / night changes in the vertical distributions of bioluminescence and acoustic scattering resulted from the larger zooplankton . And appendicularia) collected by the wp2 vertical net tows showed decreased abundance at the surface (upper two depth layers) during the day than during the night and the highest abundance of this size class was found at depth during the day (table 2). The smaller size class (pseudocalanus spp, microcalanus spp . Histograms of the bioluminescence intensity (data not shown) in conjunction with plankton enumerations from each depth suggest that an underlying low - to - intermediate - intensity bioluminescence was consistent between day and night and likely attributed to the dinoflagellates, from protoperidinium that occurred throughout the water column and to a lesser degree, ceratium furca and c.fusus that were present in significantly lower abundances (table 3). Though ubiquitous in the world s oceans and important from an ecological and evolutionary perspective (for review see haddock et al . 2010), few studies have described bioluminescent communities and their distributions in the arctic, particularly during the winter darkness . Buskey (1992), and lapota et al . (1989, 1992) examined bioluminescence distributions and community structure with the goal of developing methodology to use bioluminescence as a way to measure total biomass and light budgets of a given water mass during the spring in the greenland sea, during the fall in the beaufort sea and in summer in a norwegian fjord, respectively . In contrast, this study quantified the bioluminescent community during the polar night and demonstrated the absence of circadian rhythm in bioluminescence . Furthermore, both the diurnal distribution of bioluminescence intensity and concurrent changes in acoustic backscattering provide independent evidence for an active dvm of the larger bioluminescent zooplankton (and likely non - bioluminescent zooplankton) within the upper 75 m of the water column . Histograms of intensities showed the major differences between day and night occurring at the highest intensities, which is consistent with larger zooplankton (lapota et al . 1992; moline et al . 2009) and with the enumerations in this study . Bioluminscence and acoustic backscatting may in fact not be directly linked, but the circumstantial evidence provided herein suggests that the diurnal signal in bioluminescence is in fact caused by vertically migrating organisms . Numerous studies have examined proximal triggers for dvm (forward 1988; ringelberg 1995; ringelberg and van gool 2003; benoit - bird et al . 2009), and many others have looked at triggers for the inhibition of bioluminescence as related to its circadian rhythm (batchelder et al . 1992; kelly and katona 1966; raymond and devries 1976; swift et al . 1995). Interestingly all studies have in one way or another implicated a relative or absolute change in irradiance intensity, angle or daylength as a means of regulation for both dvm and the circadian rhythm of bioluminescence . Although changes in light during the time of this study are not visible to the human eye, it is possible that they were sufficient to initiate dvm in the organisms present at the time of the study as was suggested in the study by berge et al . (2009). However, while external light cues may play a role in regulating dvm, it might not be the only factor relevant to consider for understanding this behavior . It has been well established for photosynthetic dinoflagellates and for heterotrophic dinoflagellates of the protoperidinium genus that the bioluminescent inhibition occurs when light intensities are greater than the intensity of bioluminescence of the organisms themselves (sweeney et al . This phenomenon could explain why no circadian rhythm existed in bioluminescence during december and january in antarctica (raymond and devries 1976) and may also be related to the absence of a circadian rhythm in bioluminescence in the current study . Previous studies have found that protoperidinium spp contributed between 20 and 90% of the total light budget from the surface to a depth of 100 m in the beaufort sea (lapota et al . 1992) and that dinoflagellates were estimated to account for 96% of the total light budget in vestfjord, norway (lapota et al . 1989), so it is reasonable to assume that they were significant contributors to the overall light budget during this study . Wherein the lack of sunlight facilitated an environment where the intensity of bioluminescence was not inhibited by light greater than the bioluminescence of the organisms themselves . While this regulatory factor has been established for dinoflagellates, it is possible that it plays a role in other bioluminescent taxa as well, such as copepods, appendicularians and arctic krill as in the case of this study . The most notable finding in this study is the detection of bioluminescent activity among zooplankton during the polar night, which may be an important ecological feature . While the ultimate and proximate explanations for both the bioluminescence and the dvm behavior detected during the campaign fall outside the data collected during this study, these results provide evidence for both endogenous and exogenous control of poorly understood or previously unknown processes . Also, during this expedition, which took place during the darkest period of the polar night, we observed five different species of seabirds actively foraging at sea; little auk (alle alle), black - legged kittiwake (rissa tridactyla), northern fulmar (fulmarus glacialis), black guillemot (cepphus grylle) and brnnich s guillemot (uria lomvia). These seabirds have, to the best of our knowledge, not been reported to overwinter at these latitudes . Whether bioluminescence and/or dvm are playing roles in the foraging behavior of these visual predators is an exciting possibility, although still an open question . Despite the limited scope of this study, results open new lines of enquiry regarding the function and process during a time of year when classical paradigms of arctic ecosystems postulate that organisms are predominately in a state of hibernation (see berge et al . Ultimately, these questions also have implications for human activities (i.e., oil exploration) in the high arctic, which up until to now has been considered without life during the polar night.
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Some of the strongest predictors of type 2 diabetes mellitus (t2 dm) are obesity and family history of diabetes [1, 2]. However, lifestyle factors are also important in the etiology of the disease, and several studies have shown that t2 dm can be prevented through modifications to a healthier lifestyle, such as dietary modification [35] or increased physical activity [46]. Recent meta - analyses have concluded that there is either a u - shaped [7, 8], j - shaped, or inverse association between t2 dm incidence and alcohol consumption . Furthermore, one prospective study reported that increased alcohol consumption over time was associated with lower risk of t2 dm among initially rare and light drinkers . Few studies have stratified the analyses of risk of t2 dm and alcohol intake by body mass index (bmi). A u.s . Study reported a u - shaped association between alcohol consumption and risk of t2 dm in women with a body mass index (bmi) <25 kg / m but an inverse association in the overweight and the obese . We have previously reported that individuals with newly diagnosed t2 dm were inversely associated with income and were higher in maori and pacific adults compared to europeans . We assessed whether low to moderate alcohol consumption was associated with risk of t2 dm both overall and separately in low-, middle- and high - bmi individuals in a cross - sectional study of middle - aged new zealand men and women . Between may, 1988, and april, 1990, 5,672 adult workers (4,103 men, 1569 women) were interviewed at 41 work sites in auckland and five work sites in tokoroa (response rate 67 percent). Participants comprised 78.8% european, 7.7% maori, 11.7% pacific, and 1.8% asian workers . Pacific participants were 53.7% samoan, 10.7% tongan, 6.8% niuean, 26.5% from cook islands, and 2.3% from other islands . Approval for the study was obtained from the ethical committee of the university of auckland, and all participants gave informed consent . The median number of staff interviewed at each worksite was 72 (range 10567). Management of 10 worksites refused giving permission for their staff to be interviewed (response rate for worksites was 82%). All participants completed a self - administered questionnaire about sociodemographic factors, occupation, smoking habit, and current medication use . The new zealand socioeconomic index (nzsei) was calculated based on the occupation of the worker and spouse and a participant was assigned the higher of the two . All participants were asked to fast from 10 pm the evening before their interview . On the morning of the interview, participants were given a 75 g polycose (abbott laboratory) after the collection of fasting blood samples . Glucose tolerance status was evaluated by 2006 who criteria and 2011 ada alternative criteria (using fasting glucose 7.0 mmol / l or 2 h post glucose load of 11.1 mmol / l for diabetes, fasting glucose <7.0 mmol / l, and 2 h glucose between 7.8 and 11.0 mmol / l for igt) [14, 15]. As all new cases of diabetes were diagnosed after 39 years of age, all workers provided fasting blood specimens for glucose, cholesterol, hdl - cholesterol, and triglyceride estimations, and a blood sample two hours after a 75 g polycose load . Interbatch coefficients of variation for normal control material, in percentages, were as follows: glucose, 3.9; cholesterol, 3.6; hdl - cholesterol, 3.4; and triglycerides, 4.7; and from abnormal control sera: glucose, 2.1; cholesterol, 3.6; and triglycerides, 5.4 . Leisure exercise was assessed using a three - month physical activity recall questionnaire that has been validated . Vigorous activities were defined as those that made participant's breathe hard and moderate activities involved movement such as brisk walking . After a rest of 15 minutes or more, blood pressure was measured twice in the sitting position with a hawksley random zero sphygmomanometer . Hypertension was defined as systolic blood pressure 140 mm hg, and/or diastolic blood pressure 90 mm hg, or current use of blood pressure lowering medications . Shoes and heavy clothes were removed for measurement of weight to the nearest 0.2 kg, and height to the nearest 0.5 cm . Body mass index was calculated as weight (kg) divided by the square of height (m). Food intake over the previous 3 months was estimated by a 142-item food frequency questionnaire, which was filled in by participants at their home, and checked for errors and omissions at their interview the following morning . Natural serving sizes, such as the can of beer or serving of spirits, were used or published serve sizes . The comprehensive version of the food composition tables was used to calculate nutrient intakes . We have previously reported that alcohol intake from this food frequency questionnaire was valid (r = 0.84 in europeans and r = 0.68 in polynesians) and reproducible (r = 0.88 in europeans and r = 0.83 in polynesians). A generalised linear model was used to calculate adjusted means by body mass index and alcohol consumption groups . Relative risks and 95% confidence intervals were estimated using poisson regression models, adjusted for potential confounders . An overall model was fitted first to examine the interaction between alcohol intake and body mass index, and then separate models were fitted for three subgroups of participants defined as normal weight (bmi <25 kg / m), overweight (25 bmi <30 kg / m), and obese (bmi> 30 no alcohol consumption was taken as a reference group to estimate the relative risks for alcohol consumption in each bmi group . Alcohol consumption was also categorized into no alcohol consumption during the past three months, <5 g / day, <20 g / day and 20 g / day . 103 (1.8%) were excluded due to a previous history of diabetes mellitus, 53 (0.9%) who did not complete the food frequency questionnaire, and 4 (0.1%) with missing body mass index, leaving 5,512 (%). The median daily alcohol consumption was 4.8 g / day . The majority (75%) drank <13 g / day of alcohol and 95% drank <37 g / day . The mean age of participants was 48.8 years (range 4078 years) and 87.6% consumed alcohol during the previous three months . Only 3.1% of individuals were diagnosed as new cases of t2 dm and 4.5% had impaired glucose tolerance . There were 34.4% individuals who were classified as normal weight, 43.7% as overweight, and 21.9% as obese (table 1). Among normal weight individuals, the proportion of males was lower than overweight and obese individuals, as was the proportion of new cases of t2 dm and impaired glucose tolerance . The group of obese individuals had the highest proportions of those with hypertension, current smokers, igt, and new cases of t2 dm . Mean fasting and 2-hour glucose levels, total cholesterol, triglycerides, and systolic and diastolic blood pressure increased with increasing bmi level (all p <0.001). In contrast, hdl - cholesterol levels and nzsei reduced with increasing bmi level (p <0.001). Mean physical activity was significantly lower (p <0.001) in the obese group compared to normal and overweight individuals . There appeared to be a u - shaped relationship between alcohol consumption categories and new cases of t2 dm, impaired glucose tolerance, and hypertension . Compared to the no alcohol consumption group, those consuming alcohol had lower 2-hour post - polycose glucose levels and nzsei levels, and mean hdl - cholesterol and triglycerides levels increased over the alcohol consumption categories . Mean fasting glucose levels were lower in those who drank more than zero and less than 5 g / day, and geometric mean exercise levels were higher in those consuming 5 g / day or more . Participants consuming 20 g / day had higher mean total cholesterol and systolic and diastolic blood pressure levels . Lower mean body mass index levels were seen in those consuming 5 to less than 20 g / day . Compared to the group with no alcohol consumption and after adjusting for age, sex, and ethnicity, the group consuming some alcohol during the past three months had the following relative risks of t2 dm: normal weight individuals0.22 (95% ci: 0.08, 0.64); overweight individuals0.39 (0.18, 0.84); and 1.10 (0.65, 1.87) in obese individuals . After further adjusting for total cholesterol, hdl - cholesterol, triglycerides, smoking habit, physical activity, socioeconomic status, body mass index, and hypertension, the relative risks of t2 dm were 0.16 (0.05, 0.50) in normal weight individuals, 0.43 (0.19, 0.97) in overweight individuals, and 0.92 (0.52, 1.60) in overweight individuals . The p value for the interaction term between bmi categories and alcohol consumption was <0.0001 . The relative risks comparing no alcohol consumption to the various alcohol consumption groups are shown in table 3 (column headed overall). After adjusting for age, gender, and ethnicity, the relative risk for t2 dm overall was 0.53 (95% ci: 0.34, 0.82) in those drinking more than zero to <5 g / day, and 0.60 (0.37, 0.96) after also adjusting for current smoking habit, body mass index, total cholesterol, hdl - cholesterol, triglycerides, physical activity, socioeconomic status, and hypertension (referred to hereafter as all potential confounders). Figure 1 shows the relative risks and 95% confidence intervals for normal, overweight, and obese adults and overall, by alcohol consumption group . Relative risks associated with risk of t2 dm by alcohol consumption and bmi groups are also shown in table 3 . There were lower relative risks for t2 dm in those consuming more than zero to <5 g / day in both normal weight and overweight individuals, but not in obese individuals, after adjusting for age, gender, and ethnicity . After further adjusting for all potential confounders, similar relative risks were observed; however, the relative risk of type 2 diabetes in those consuming 5 to <20 g / day in normal weight individuals was also significantly lower (rr 0.23; 0.06, 0.83). In the overweight group, the relative risks for t2 dm in the group consuming 20 g / day was 0.36 (0.13, 1.01), but this just failed to be statistically significant at the 5% level of significance . There was no significant association between alcohol consumption and igt after adjusting for all potential confounders with relative risks of 0.92 (0.37, 2.30) in normal weight individuals, 1.14 (0.56, 2.34) in overweight individuals, and 0.62 (0.38, 1.02) in obese individuals . In this study, low to moderate alcohol consumption over the past three months was associated with reduced risk of t2 dm among normal weight and overweight individuals . However, there was no significant association between alcohol consumption and risk of t2 dm in obese individuals . In the full model looking at no alcohol versus alcohol consumption these associations persisted after adjustment for body mass index and other known and probable risk factors for t2 dm . However, in analyses that categorised alcohol into various groups, there appeared to be an approximate u - shaped relationship between alcohol consumption group and risk of t2 dm in the full model and in the normal and overweight groups but not the obese group . It is possible that this is, in part, due to a lack of statistical power, or to the cut - off points chosen to classify alcohol intake . Our findings of a possible u - shaped association between alcohol consumption and risk of t2 dm in normal weight and overweight individuals have been described previously, but our finding of no association in the obese is not consistent with some other studies . A study of 84,941 female nurses reported a u - shaped association between alcohol consumption and risk of t2 dm over 16 years of followup in those with a body mass index (bmi) <25 kg / m but an inverse association in the overweight and the obese . However, in the same study carried out 6 years earlier in 109,690 nurses aged 25 to 42 years they reported an inverse relationship between alcohol intake and incidence of t2 dm in both the obese (bmi 30 kg / m) and nonobese . In contrast to the current study where diabetes status was determined from a glucose tolerance test, diabetes status was by self - report in initially employed women in these latter two studies [4, 22]. Moderate alcohol consumption was also associated with an inverse association with risk of self - reported t2 dm in 16,154 european men and women which was stronger in the overweight and obese particularly at higher levels of alcohol intake . However, the findings from a large study conducted over 12.1 years among us physicians showed no such interaction . In that study there was an inverse association between alcohol consumption among individuals both above and below the median bmi of 24.4 kg / m . A japanese study of 5,636 adults reported an increased risk of self - reported t2 dm among low bmi individuals (bmi 22 kg / m) and a decreased risk of t2 dm among middle - bmi individuals (22.124.9 kg / m) but no significant difference in high - bmi individuals (25 kg / m). These findings in the overweight and obese are consistent with the current study; however, we could not assess the effect of bmi 22 another japanese study of employed men found that alcohol intake was associated with an increased risk of glucose tolerance test diagnosed t2 dm in the lean but a protective effect in the overweight . In contrast, another japanese study of 28,893 men reported a positive association between self - reported diabetes incidence over 10 years of followup and moderate to high alcohol (23 g / day) intake in men with bmi 22 kg / m but no significant association at> 22 kg / m . Alcohol consumption was also a positive risk factor for the development of fasting glucose diagnosed t2 dm in overweight (bmi 23<25 kg / m) and obese (bmi 2538.7 kg / m) 2,500 young korean males . Differences in drinking patterns or quality of the alcohol (e.g., drinking beer and wine versus drinking vodka and other spirits) may have contributed to these inconsistencies, in addition to incomplete adjustment for confounders or measurement error in alcohol assessment or the differences in the measurement of alcohol consumption levels (e.g., usual alcohol consumption versus recent alcohol). In agreement with the findings in the current study, a study of over 8700 subjects from micronesian and mauritian populations, alcohol intake had no association with prevalence of impaired glucose tolerance (igt), nor was there a significant association in 6,362 japanese men . In contrast, a cross - sectional study of 3,128 swedish men aged 35 to 56 years reported a reduced prevalence of igt with moderate alcohol consumption levels, and another study reported that alcohol consumption was a positive risk factor for the development of igt in overweight (bmi 23<25 kg / m) and obese (bmi 2538.7 kg / m) young korean males . T2 dm is caused by both insulin resistance and -cell dysfunction [15, 31]. Several factors may explain the inverse or u - shaped association between moderate alcohol consumption and reduced risk of t2 dm including increased insulin sensitivity with lower plasma insulin concentrations [32, 33], increased hdl - cholesterol or due to the anti - inflammatory effect of alcohol [35, 36]. At higher alcohol consumption levels, body weight, blood pressure, and triglyceride concentrations increase [3739]. Both moderate and acute alcohol consumption have also been to be reported to inhibit lipolysis with a reduction in free fatty acid levels [40, 41]. Such studies provide a possible physiological explanation for the inverse or u - shaped association between alcohol consumption and t2 dm . It has also been hypothesized that regular moderate alcohol consumption promotes insulin sensitivity of skeletal muscle, resulting in a protective effect for risk of t2 dm . The possibility of differential reporting of alcohol consumption according to disease status was minimized as alcohol consumption data was collected prior to diagnosis of t2 dm; therefore, drinking habits and recall of alcohol intake could not have been influenced by disease status . We relied on self - reported alcohol consumption data in this study, as did most other alcohol - related epidemiological studies, since it has been reported that other approaches are not practical in large studies . Furthermore, the usefulness of simple self - administered questionnaires to reliably estimate alcohol consumption in these participants has been demonstrated and was indirectly validated by the significant positive correlation with hdl - cholesterol . It is unlikely that misclassification accounted for the observed associations, since measurement error in observational studies tends to reduce or obscure a true association . A further limitation is that cross - sectional data cannot differentiate between cause and effect . The key strength of the current study was that diabetes status was ascertained by oral glucose tolerance test rather than self - reported diabetes as has been used in the majority of previous studies . In conclusion, results from the current study support the hypothesis that there is an inverse association between light to moderate alcohol consumption and the risk of t2 dm in normal weight and overweight individuals but not in the obese . These results are limited to healthy working individuals consuming light to moderate amounts of alcohol and suggest that light to moderate alcohol consumption in addition to other known risk factors can modify the incidence of t2 dm.
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