diff --git "a/cluster/859.jsonl" "b/cluster/859.jsonl" new file mode 100644--- /dev/null +++ "b/cluster/859.jsonl" @@ -0,0 +1,66 @@ +{"text": "In a case control study of 287 women aged 15-24 years with malignant melanoma and 574 matched controls, findings relating to oral contraceptive use and other hormone use are reported. Ever having used oral contraceptives was not associated with an increased risk of melanoma (relative risk for ever use of the pill = 1.0). Women with melanoma were, however, more likely to have taken oral contraceptives for long periods of time in the past, the relative risk associated with oral contraceptive use for a total duration of 5 years or longer which had begun at least 10 years before the melanoma was diagnosed being 1.5 This elevated risk persisted after controlling for the reported hair and skin colour, frequency of moles on the body, place of birth, and measures of sunlight and fluorescent light exposure. Cases were more likely than controls to have used hormones to regulate their periods, hormonal replacement therapy and be given hormone injections to suppress lactation, the respective relative risks being 1.9, 1.4 and 1.4, but none differed significantly from 1.0. These findings suggest that prolonged oral contraceptive use may, after a lag of 10 years or so, increase the risk of malignant melanoma."} +{"text": "The relationship between female hormone use and primary liver cancer was analysed using data from a case-control study conducted between 1984 and 1992 in Milan on 82 female incident cases with histologically or serologically confirmed hepatocellular carcinoma and 368 controls admitted to hospital for acute non-neoplastic, non-hormone-related diseases. An elevated relative risk (RR) or primary liver cancer was observed in oral contraceptive (OC) users . The RR was directly related to duration of use (RR 1.5 for < or = 5 years and 3.9 for > 5 years) and persisted for longer than 10 years after stopping use . The RR were below unity, although not significantly, for women ever using oestrogen replacement therapy and female hormones for indications other than contraception and menopausal therapy . The long-lasting, association between risk of hepatocellular carcinoma and OC use has potential implications on a public health scale, since primary liver cancer is a relatively rare disease among young women, but much more common at older ages. This study provides limited but reassuring evidence on the possible relationship between oestrogen replacement treatment and subsequent risk of hepatocellular carcinoma."} +{"text": "The relation between the use of combination oral contraceptives (OCs) and the risk of epithelial ovarian cancer was investigated in a case-control study conducted in Milan on 209 women below the age of 60 with histologically confirmed epithelial ovarian cancer, and 418 age-matched controls with a spectrum of acute conditions apparently unrelated to OC use. Combination oral contraceptives were used by 18 (9%) cases, and 59 (14%) controls, giving a relative risk estimate of 0.6 . The risk of ovarian cancer decreased with increasing duration of use and the point estimate remained below unity long after cessation of use. These results were not accounted for by parity, infertility, or other identified potential confounding factors. Thus, the findings of the present study add further support to the evidence emerging from American data of a reduction of approximately 40% in the risk of epithelial ovarian cancer among women who had used oral contraceptives."} +{"text": "Several studies have suggested that prolonged use of oral contraceptives may increase a woman's risk of developing malignant melanoma. In the Royal College of General Practitioners' Oral Contraception Study, 31 cases of malignant melanoma have been reported among ever-users and 27 cases among never-users. The risk ratio (RR) was 0.92 0.55-1.54). There was no significant trend with duration of oral contraceptive use, although those women who had used the pill for at least 10 years had an elevated RR of 1.77 (95% CI 0.80-3.90). The Oxford/Family Planning Association Study has recorded 15 cases among ever-users and 17 cases among never-users; the standardised risk ratio was 0.85 (95% CI 0.42-1.70). None of the rates observed in any duration of use category was materially different from those observed in never-users. The results available so far from the two studies suggest that oral contraceptive use is probably not associated with an increased risk of malignant melanoma."} +{"text": "To assess the relation between oral contraceptive (OC) use and breast cancer, we analysed data from a case-control study conducted in Northern Italy between 1983 and 1991 on 2,309 cases below age 60 and 1,928 controls admitted to hospital for acute diseases unrelated to OC use and to any of the known or potential risk factors for breast cancer. OC use was reported by 16% of cases and 14% of controls. The multivariate relative risk (RR) for ever vs never use of combination OC was 1.2 1.0-1.4). However, there was no trend in risk with duration. The RR was elevated for very short use, but declined to 0.8 (95% CI = 0.5-1.0) for five or more years' use. No noteworthy relationship was found for other major measures of OC use, although RR estimates were above unity for women who had stopped use less than 5 years before , started use less than 10 years before , started when 25 or more years old , or after first birth . No interaction was observed between OC use and family history of breast cancer, parity and age at first birth. A separate analysis of 373 cases and 456 control below age 40 showed no association with ever use ."} +{"text": "The effect of the duration and pattern of oral contraceptive use on breast cancer risk in young women (aged under 36 at diagnosis) has been investigated. Oral contraceptive users were divided into three groups: group 1, continuous users; group 2, interrupted only by pregnancy users; and group 3, intermittent users. There was a clear trend with duration of oral contraceptive use in all three groups of users (P < 0.001 for each category of use) and the relative risks per year of use were similar . The relative risks for intermittent users and for women who had used oral contraceptives except when pregnant were very similar, but the relative risk for users for more than 8 years was highest for continuous users. The results suggest that the relationship between oral contraceptive use and breast cancer risk is dependent upon the total duration of use and is not modified by the pattern of use."} +{"text": "A study among 1960 post-menopausal breast cancer cases and 2258 controls identified through a nation-wide screening program enabled evaluation of effects of oestrogen use on breast cancer risk. Ever use was not associated with increased risk (RR = 1.0), but a significant trend was observed with increasing years of use, with users of 20 or more years being at a 50% excess risk. Elevations associated with long-term use were apparent across all menopause subgroups . Hormones exerted particularly adverse effects in those initiating use subsequent to a diagnosis of benign breast disease, particularly long-term users . There was also some indication that effects predominated among the lower stage tumours, an observation similar to that observed for endometrial cancer. These findings support a role for oestrogens in the aetiology of breast cancer, although risk appears to be enhanced only after extended periods of use, and not to the extent observed for other hormonally-sensitive tumours."} +{"text": "By the end of the follow up in December 1999, there were 226 611 person-years of observation. A total of 1145 malignant tumours were recorded . There was a small excess of breast cancer with 434 observed and 387.69 expected . Among about 3 663 ever users of HRT, there was no increase in overall tumour incidence but a significant excess of breast cancer compared with never users . Breast cancer increased with increasing duration of use and for 48\u2013120 months use the SIR was 1.92 (95% CI 1.32\u20132.70). There was no significant interaction with family history of breast cancer although an independent additive effect was suggested between HRT use and family history. In a Cox regression model time to breast cancer in relation to duration of HRT use was analysed adjusting for age at menarche, age at menopause, age at first full term pregnancy, parity and age at diagnosis. A significantly higher risk was seen for longer duration of HRT use compared with never users. No increased risk is seen in women beyond 5 years after stopping HRT. There was no interaction between previous use of oral contraceptives and later HRT use. \u00a9 2001 Cancer Research Campaign"} +{"text": "The role of aspirin on the risk of cancers of the upper aerodigestive tract was investigated in the combined data of three Italian case\u2013control studies, including 965 cases and 1779 hospital controls. The odds ratio was 0.33 for users of \u2a7e5 years, and 0.51 for \u2a7e5 years since first use. Average frequency of use was 5.7 times per week among cases and 5.6 among controls; 32 cases and 78 controls took aspirin \u2a7e3 times per week, 27 cases and 57 controls every day. Indication of use was analgesia for 10 cases and 38 controls, whereas 27 cases and 49 controls took aspirin for cardiovascular prevention. The multivariate OR for regular use was 0.89 (95% CI 0.56\u20131.43). The OR decreased to 0.33 (95% CI 0.13\u20130.82) for users of \u2a7e5 years, and was 0.51 (95% CI 0.26\u20130.99) for \u2a7e5 years since first use.Table 1A reduced risk with longer duration of aspirin use was observed for all sites considered: the ORs for \u2a7e5 years of use were 0.39 for oral and pharyngeal, 0.80 oesophageal and 0.09 laryngeal cancer. Similarly, the ORs for \u2a7e5 years since first use were 0.26, 0.66 and 0.55 for the three cancer sites, respectively.This study suggests that aspirin may have a beneficial effect on cancers of the upper aerodigestive tract. Although there is evidence of a possible protective effect of aspirin on oesophageal cancer , acts on the arachidonic acid metabolism, blocking the synthesis of thromboxane, prostacyclin and prostaglandins, which in turn can influence cell proliferation, and hence cancer growth . A speciLimitations of our study should be considered that might have introduced a spurious association between aspirin use and the reduced risk of upper aerodigestive tract cancers. It is possible in fact that aspirin use has been affected by early symptoms of the conditions under study. The evidence of an association with longer use is, however, reassuring against this bias. Further, some of the diagnostic categories of the controls may be associated with increased aspirin use. However, the results were similar when cases were compared with each of the major diagnostic categories of controls, thus giving reassurance against potential selection biases. Another limitation of this study is that, although based on a large number of cases, it includes a relatively low number of regular aspirin users, reflecting the pattern of regular aspirin use in Italy. Among the strengths of the study are the similar catchment areas for cases and controls, the almost complete participation rate and the choice of hospitals controls, who are preferable to population ones with reference to reliability and validity of information on drug use, since cases and controls are similarly sensitised towards various aspects of their medical history ("} +{"text": "Use of immunosuppressive drugs post organ transplantation, and prolonged use of glucorticoids for other conditions have been associated with subsequent risk of certain malignancies, that is, skin cancers and lymphoma. There is evidence that the incidence of bladder cancer is also elevated among organ transplant recipients, however, it is unknown whether other groups of patients, that is, those taking oral glucocorticoids, likewise are at an increased risk.In a population-based case\u2013control study in New Hampshire, USA, we compared the use of glucocorticoids in 786 bladder cancer cases and in 1083 controls. We used unconditional logistic regression analysis to compute adjusted odds ratios (ORs) associated with oral glucocorticoid use.In our analysis, the risk of bladder cancer was related to a history of prolonged oral glucocorticoid use . Associations with oral glucocorticoid use were stronger for invasive tumours and tumours with high (3+) p53 staining intensity .Our results raise the possibility of an increased risk of bladder cancer from systemic use of glucocorticoids, and a potential role of immune surveillance in bladder cancer aetiology. With an estimated 68\u2009810 new cases in 2008, bladder cancer is the fifth most commonly diagnosed cancer in the United States of America . There iThrough the New Hampshire State Department of Health and Human Services\u2019 rapid reporting Cancer Registry, we identified newly diagnosed cases of bladder cancer among New Hampshire residents, aged 25\u201374 during the 1 July 1994 to 31 December 2001 period. For efficiency, we shared controls with a study of non-melanoma skin cancer covering a diagnostic period of 1 July 1993 to 30 March 2000, along with additional controls frequency matched to the bladder cancer cases by age and gender , alcohol and other exposures. We requested the original paraffin-embedded tumour specimen for histopathology re-review by the study pathologist who classified tumours according to WHO 1973 and WHO ISUP criteria . Those who responded positively were considered users and were asked the age they were first treated, the condition for which the glucocorticoids were prescribed, the name of the drug, dose and duration of the treatment. Those who responded that they did not use glucocorticoids for at least 1 month were considered non-users. To aid recall, we developed a list of glucocorticoids and other immunosuppressive drugs and a pictorial guide showing the most commonly used drugs grouped by pill colour, size and shape. To minimize potential reporting bias, we did not reveal the specific hypotheses-of-interest to either the interviewer or participant, and we did not inform the interviewers of the case\u2013control status of participants.We computed odds ratios (ORs) and their 95% confidence intervals (CIs) for bladder cancer associated with the use of glucocorticoids before the reference date using unconditional logistic regression, taking into account multiple confounding factors . In addiin situ) and TP53 staining intensity (<3 or 3+) to examine whether glucocorticoid use was associated with tumour aggressiveness.We conducted a combined analysis of all bladder cancers, as well as specifically on pathologically confirmed transitional cell carcinomas. In addition, we carried out a case-only analysis comparing glucocorticoid users to non-users according to subgroups defined by the extent of disease , and from 786 (95%) we obtained data on the use of oral glucocorticoids. A total of 1119 controls took part in the study (70% of those eligible), and from 1083 (97%) we obtained data on the use of oral glucocorticoids . Cases wrcinomas .P for interaction=0.004); however, ORs were elevated in both groups and there were few never smokers.Any glucocorticoid use was associated with an increased risk of bladder cancer, and the association was stronger for oral use than inhaled use . AlthougRestriction to transitional cell carcinomas did not affect our risk estimate for oral glucocorticoid use . Further, using a case\u2013case approach, we found higher ORs associated with more advanced disease stage and grade . Oral glImmunosuppressive therapy, usually a combination of cytotoxic drugs and glucocorticoids, is commonly prescribed to organ transplant recipients, in order to prevent allograft rejection. Glucocorticoids, alone or in combination with other immunosuppressive drugs, are used to help treat many chronic inflammatory conditions, such as rheumatoid arthritis and asthma. Long-term immunosuppressive therapy has been shown to increase the risk of many types of malignancies among organ transplant recipients inhibitors, were twice as likely to develop bladder cancer as the general population or being enrolled in Medicare (those 65 years and older), ensuring our control group was represented within our case group. Our study population was relatively large, although the statistical power diminished in certain categories . We assessed the potential confounding effects of multiple factors and confounding by indication was unlikely, as we did not find systematic differences in the ORs for oral glucocorticoid use by reason for use. A weaker association observed with inhaled steroids is plausible because of the minimal systemic effects of inhaled steroids . Oral gl"} +{"text": "We report the results of a case-control study of oral contraceptive use and breast cancer conducted in London, Oxford and Edinburgh between 1980 and 1984. One thousand one hundred and twenty-five women aged 16-64 years with newly diagnosed breast cancer and a like number of matched controls were interviewed and asked about their past due use of oral contraceptives (OCs). Among women aged 45 years or more at diagnosis there was no evidence of an association between OC use and breast cancer. Among the 351 pairs of women aged under 45 years at diagnosis there was a significantly elevated risk associated with increasing duration of use before first full term pregnancy . Since this result is at variance with the findings in some other studies we have investigated the nature of this association with particular emphasis on possible bias, pill type and a latent effect."} +{"text": "We have evaluated the relation between cancer of the lower urinary tract (\"bladder cancer\") and the use of artificial sweeteners, by means of case-control studies in Manchester, U.K., and Nagoya, Japan, areas where extensive use occurred 30-40 years ago. In each area, a broadly based series of cases was interviewed and a series of controls chosen from the general population. A history of use of sugar substitutes primarily saccharin, was not associated with an elevated risk of bladder cancer in either study area. Risk of bladder cancer did not increase regularly with frequency or duration of use of sugar substitutes. Data on dietetic beverages were not obtained in Nagoya. This exposure was not associated with a greater risk of bladder cancer in Manchester. The results of this study suggest that use of artificial sweeteners confers little or no risk of bladder cancer."} +{"text": "During the interval 1968-74, 17,032 women aged 25-39 years were recruited to the Oxford-Family Planning Association contraceptive study, more than half of whom were using oral contraceptives. These women have been followed up over the years and breast cancer has been diagnosed in 189 of them. We have analysed the available data in two ways. First, we have calculated standardised breast cancer incidence rates in non-users and users of oral contraceptives according to total duration of use, interval since first use, interval since last use, duration of use before first term pregnancy and duration of use before age 25. Secondly, we have conducted case-control within cohort analyses to examine the possible effects of different types of pill and to search for evidence of a latent effect of oral contraceptive use before first term pregnancy on breast cancer risk. We have found no evidence of any adverse effect of oral contraceptive use on the risk of breast cancer in this study. There was, however, little exposure to the pill before first term pregnancy among the participants and virtually no such exposure at a very young age (i.e. below 20 years). Accordingly, the results of this study strengthen the evidence that oral contraceptive use by mature women does not increase breast cancer risk, but add little to the uncertainty about the effects of early use."} +{"text": "Use of phenacetin and other analgesic and non-steroidal anti-inflammatory drugs (NSAIDs) potentially influences bladder cancer incidence, but epidemiologic evidence is limited.We analyzed data from 376 incident bladder cancer cases and 463 controls from a population-based case-control study in New Hampshire on whom regular use of analgesic drugs and NSAIDs was obtained. Odds ratios and 95% confidence intervals were computed using logistic regression with adjustment for potentially confounding factors. Separate models by tumor stage, grade and TP53 status were conducted.>8 years = 3.00, 95% confidence interval (CI) = 1.4\u20136.5). In contrast, use of paracetamol did not relate overall to risk of bladder cancer. We also found that regular use of any NSAID was associated with a statistically significant decrease in bladder cancer risk , and specifically use of aspirin. Further, the association with NSAID use was largely among invasive, high grade and TP53 positive tumors.We found an elevated odds ratio (OR) associated with reported use of phenacetin-containing medications, especially with longer duration of use (OR While these agents have been investigated in several studies, a number of questions remain regarding the effects of analgesic and NSAID use on risk of bladder cancer. We requested the original paraffin-embedded tumor specimen for histopathology re-review by the study pathologist who classified tumors according to WHO ISUP criteria. Of the 342 cases with evaluable pathology, 327 (96%) were deemed cancerous by the study pathologist. Due to the high concordance rates, we classified subjects based on the original pathologist's diagnosis; whereas tumor morphology, extent of disease, and grade were based on the standardized histopathology re-review. Immunohistochemical analysis of the tumors was performed for TP53 and scored for intensity and percent of tumor cells staining positively, as described previously . We obtaSubjects were asked if they used a pain medication at least four times a week for one month or longer prior to the reference date (diagnosis date of the cases and a comparable date randomly assigned to the controls). Those who responded positively were asked the brand names of each medication they took, and for each medication, the age began and stopped, total duration of use and the condition for which the drug was prescribed. Those who responded that they did not use pain medications at the specified frequency were considered non-users.\u00ae were used as the main source of information on drug composition to generate the matrix. Whenever necessary, a variety of other sources were used, such as direct contact with pharmaceutical companies, documents from regulatory agencies and others. Drug categories considered for the analysis were: phenacetin, paracetamol, any NSAID , aspirin, and ibuprofen.Brand names were recoded into active ingredients based on a drug matrix that took into account variations in the composition of the drugs over time, and specifically addressed the exact years of withdrawal/substitution of phenacetin from the formulation. Past editions of the Physician Desk Reference We computed ORs and their 95% confidence intervals for bladder cancer risk for the studied drugs using logistic regression with those who responded that they did not use the pain medications at all or with the specified frequency as the reference category. Risk estimates were adjusted by age in quintiles according to the age distribution among the controls: , sex and smoking status . We further assessed the possibility that education, as a marker of socioeconomic status, could act as a potential confounder but the inclusion of this variable did not appreciably influence our results and therefore was not included in our final models.To distinguish the effects of the specific types of NSAIDs and analgesic drugs, we used two models. One, the \"adjusted model\", simultaneously included each of the drug categories and the other, the \"exclusive use\" model, included subjects that had only used one specific type of pain medication. Duration was classified according to tertiles of control distribution for each drug. Tests for trend were performed by including a single term for categorical exposure variables in logistic regression models using non-users as the reference category.Further, we examined a model in which the drug use variables excluded the year prior to the reference date in order to assess the possibility of drug use consequent to undiagnosed tumor symptoms. In addition to evaluating all bladder cancers combined, we evaluated risks by extent of disease (noninvasive versus invasive), for noninvasive tumors, grade (low grade versus high grade), and TP53 intensity (<3 versus 3+). In the analysis of TP53 intensity, we assessed whether further adjustment by stage or grade affected the results. We did not find evidence of such effects, and thus, did not include stage or grade in the final models of TP53 status. The statistical package SAS v9.1 was used for all the analyses.in situ carcinomas, and 28% were invasive.Of the 472 potentially eligible cases we contacted, 398 (84%) took part in the study, and from 376 (94%) of these, we obtained history of pain medication use. Of the 694 potential controls we contacted, 526 (76%) took part in the study, and from 463 (88%) of these, we obtained history of pain medication use. Subjects were mainly Caucasians and more than half were men Table . More ca>8 yrs = 3.0, 95% CI = 1.4\u20136.5), although even short term users (4 years or less) had an increased risk (P for trend = 0.005). We were unable to fit the exclusive use model for phenacetin because there were no exclusive users. In contrast, regular paracetamol use was not related to any statistically significant association with bladder cancer in the adjusted or exclusive use models and exclusive use model . Exclusion of drugs used in the year prior to the reference date did not materially alter the risk estimates but compromised the statistical power of the analysis (data not shown).We indirectly assessed the effect of dose of aspirin by using the condition for which aspirin was prescribed as a proxy. In general, aspirin is prescribed for coronary heart disease prevention at lower doses than for inflammatory or painful conditions . When we stratified by condition we did not find any differences in the risk estimates i.e., among aspirin exclusive users the odds ratio was 0.5 (95%CI = 0.3\u20130.9) for cardiovascular disease and 0.5 (95%CI = 0.2\u20131.3) for other conditions.When we restricted the analysis of the effect of drugs to transitional cell carcinomas, excluding squamous cell carcinomas (n = 2), small cell carcinoma (n = 2), adenocarcinomas (n = 1), and others (n = 13), results remained unchanged (data not shown). Exclusion of papillary urothelial neoplasia of low malignant potential (n = 84), also yielded similar results for all drug use categories except everusers of phenacetin had a slightly higher bladder cancer risk without these cases.In the analysis stratified by tumor phenotype, we found a stronger inverse association with NSAID use for invasive, high grade non-invasive and TP53 positive tumors Table . There wOur findings suggest that regular use of NSAIDs, in particular aspirin, related to a lower risk of this malignancy. The association appeared strongest for invasive, high grade noninvasive and TP53 positive tumors. Also, our findings provide further evidence of an increased risk of bladder cancer among users of phenacetin-containing drugs, and additionally, suggest that risk is greatest among long term users.Case-control studies like ours have the potential for recall bias. To enhance recall we showed lists of commonly used anti-inflammatory and analgesic medications. One concern is that the prevalence of recalled phenacetin use may be lower for recent studies because the drug was withdrawn from the market nearly two decades ago and recall of past NSAID use decreases with time since last use . ArguingWhile we do not suspect differential misclassification , non-differential misclassification likely exists in our data. We attempted to minimize this by defining drug use as frequent use . By doing this, the \"non-users\" category included subjects who could have used the drugs but did not fulfill our user definition. Such misclassification likely would lead to an underestimate of the effects.We further need to consider the possibility of selection bias, i.e, that non-participants or those on whom we lacked drug use information differed from participants in ways that were not accounted for in the analysis. Overall participants and non-participants were generally similar according to factors we were able to evaluate: age, sex and urban residence (data not shown). Lack of adjustment for occupational history, exposure to water chlorination byproducts or other factors could potentially have biased our results. However, high risk occupational exposures or water chlorination exposure are unlikely to be related to the use of analgesics or NSAIDs, and adjustment for these factors did not affect the relation between medication use and risk of bladder cancer in a case-control study from Spain . In our Knowledge of the effects of phenacetin on urinary cancers largely derives from animal experiments and from studies of renal cancers for which a causal association has been established . RelativParacetamol is a metabolite of phenacetin, but it is unclear whether paracetamol retains the carcinogenic potential of its parent compound. In our study, paracetamol was unrelated to bladder cancer risk. This agrees with the published literature which is generally consistent with no effect ,13-15,19Aspirin and other NSAIDs are COX inhibitors (with varying isoenzyme affinities) and probably have alternative targets of action that could influence cancer occurrence . COX-2 eAmong the commonly prescribed NSAIDs, aspirin and ibuprofen are non-selective COX inhibitors, and diclofenac is COX-2 selective . RandomiRegarding non-aspirin NSAIDs, the southern California study found a decreased bladder cancer risk among acetic acid NSAIDs users, with a suggestion of a dose-response effect . A record linkage study from Denmark with 330 bladder cancer cases, found that prescribed non-aspirin NSAIDs slightly increased the risk of bladder cancer , although no dose-response relationship was observed . MisclasIn conclusion, our data provide evidence that aspirin use may be related to a decreased bladder cancer risk, particularly for more advanced tumors and ones that contain TP53 alterations. They confirm previous findings on an increased risk of bladder cancer among users of phenacetin-containing medications, and additionally suggest that the risk relates to duration of use. Bladder cancer is the most common urologic malignancy in humans, and it typically carries a favorable prognosis, but mortality and morbidity, including a high risk of recurrence, remain important public health concerns. While several studies have investigated the role of NSAIDs and other analgesic medications, many issues remain. In light of the chemopreventive effects of NSAIDs including aspirin for other neoplasms, the possibility that they may reduce bladder cancer incidence warrants further consideration. Studies assessing the effect on bladder cancer prognosis are lacking but would be of great interest.The author(s) declare that they have no competing interests.JF carried out the variable creation, statistical analysis, and drafted the manuscript. MK participated in the drafting of the manuscript and in the analysis plan. MS assisted in the statistical analysis and variable creation. AS performed the histopathology reviews and assessment of p53 immunohistochemistry. AA assisted in the retrieval of pathology materials. JH provided urology expertise. KK leads the somatic alteration component of the project. MRK is principal investigator and participated in all phases of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Literature review of the published evidence on the relation between analgesic and anti-inflammatory drug use and risk of bladder cancer.Click here for file"} +{"text": "Non-steroidal anti-inflammatory drug (NSAID) use has been linked with pancreatic cancer risk; however, findings from epidemiological studies are inconsistent.n=1141) had a diagnosis of primary cancer of the exocrine pancreas between January 1995 and June 2006. Controls (n=7954) were matched with each case on general practice site, sex and year of birth. Conditional logistic regression analyses were used to generate odds ratios (OR) and 95% confidence intervals (CI) associated with NSAID use compared with non-use.A nested case\u2013control study was conducted within the UK General Practice Research Database. Cases (Any use of NSAID in the 5 years before the index date or since entry into the database (excluding the year before diagnosis) was not associated with risk of pancreatic cancer; OR 0.96 and 1.03 (95% CI 0.89\u20131.19), respectively. Exposure to NSAIDs for > 773 days, in the 5 years pre-diagnosis, was associated with a reduced risk of pancreatic cancer OR 0.78 (95%CI 0.62\u20130.97). There was evidence of reduced pancreatic cancer risk with long-term use (5 years or more) of lower doses of NSAIDs OR 0.70 (95% CI 0.49\u20130.99).Long-term exposure to NSAIDs may be associated with a reduction in risk of pancreatic cancer. Pancreatic cancer is rapidly fatal and is the fifth leading cause of cancer mortality in the western world. between January 1995 and June 2006; data were extracted from GPRD in February 2007. Patients aged 85 or older were excluded, as were cases without at least 5 years of UTS data collection before the index date. Controls with no GPRD record of pancreatic cancer were selected using incidence density sampling and matched to cases on year of birth, sex and general practice site. Controls also had at least 5 years of UTS data collection before their index date (date of pancreatic cancer diagnosis in their matched case). Up to seven controls were matched for each case.Pancreatic cancer is difficult to conclusively diagnose without histological or cytological examination of relevant specimens and these specimens are often not available. Pancreatic cancer may therefore be confused with primary malignancies in adjacent organs or with secondary tumours. Histological confirmation was not available from GPRD as there was no routine linkage to cancer registries, at the time the data were obtained. To minimise misclassification, all cancer codes recorded in the medical records of potential cases were reviewed. Only cases with consistently recorded pancreatic cancer codes were included. All other cases, for example patients with codes for both pancreatic cancer and cholangiocarcinoma, or carcinoma of bile duct, gallbladder and so on, and their matched controls were excluded from the dataset; peri-ampullary tumours were also excluded. Cases and controls with a prior history of cancer (at least a year before the index date) were identified by review of cancer codes and flagged as having a previous cancer but were not excluded from the dataset.The primary exposure of interest was NSAID use in the 5 years before the index date, excluding the year before the index date. NSAID use in the year before diagnosis was excluded because early cancer symptoms may have led to NSAID use during this period. NSAID use since entry into GPRD until a year before the index date was also examined. Data were extracted on all NSAIDs prescribed for cases and controls. NSAID use was expressed in units of defined daily dose (DDD), a validated measure of drug consumption maintained by the World Health Organisation (WHO). It is the assumed average maintenance dose per day of a drug used for its main indication in adults. As the DThe number of DDDs for each NSAID prescription issued was calculated by multiplying the dose prescribed by the quantity given and dividing this by the DDD value assigned to that drug. For each time period under study, the total number of DDDs for each NSAID prescribed, for subgroups of NSAIDs , fenamates, propionic acid derivatives, acetic acid derivatives, COX-2 inhibitors, oxicams or others), and for all NSAIDS combined was calculated for each case and control. Cumulative duration of any NSAID use was calculated as the total number of intended days, during the periods under study, for which study subjects were issued any prescription (at any dose) for NSAIDs. Total dose (DDDs) and total duration (days) of any NSAID use, in the periods under study, were categorised into approximate quartiles, on the basis of total NSAID use within the controls.Exposure to any NSAID at any time before the index date, excluding the year before the index date, was further categorised according to both dose and duration of use combined. The median number of DDDs, per day, of NSAID exposure in the controls during this period was one DDD per day, therefore low-dose NSAID exposure was classified as the use of <1.0 DDD of NSAIDs per day and high dose as >1.0 DDDs per day. Categories for duration of low and high NSAID use were subsequently created: no use or use for <1 year (0\u2013365.3 days), use for 1\u20133 years (365.3\u20131095.8 days), use for 3\u20135 years (1095.8\u20131826.3 days) and use for 5 or more years (>1826.3) and 95% percent CIs for the associations between disease status and use of NSAIDs.Models were constructed for ever/never use of all NSAIDs or subgroups of NSAIDs. Dose and duration of NSAID use was examined using continuous variables (output expressed per 200 DDDs and 200 days of use), approximate quartiles of dose and duration of NSAID use and the combined dose and duration variable. NSAID use in the year before diagnosis was excluded in all analyses and models were constructed relating to the 5-year period before diagnosis/index date and since entry into the GPRD.All analyses were adjusted for potential confounders including smoking status , body mass index , alcoholWithin the GPRD, 1361 pancreatic cancer cases met the initial inclusion criteria and these were matched to 9487 controls. After review of cancer codes, 220 cases (16%) and their controls were excluded, leaving 1141 pancreatic cancer cases and 7954 matched controls. Each case had at least one matched control and >90% of cases had seven matched controls. The mean duration of UTS follow-up was 10.6 (s.d. 3.4) years, for both cases and controls. The mean age at database enrolment was also the same for cases and controls at 57.3 (s.d. 9.8) years; over 50% (53.7%) of all subjects were male .Any use of an NSAID in the 5 years before the index date or since entry into the GPRD (excluding the year before diagnosis) was not associated with risk of pancreatic cancer; (OR) 95% CI, 0.96 (0.84\u20131.10) and 1.03(0.89\u20131.19), respectively (Data not shown). Any use of aspirin and its derivatives or high-dose aspirin in the 5 years before the index date was also not associated with pancreatic cancer risk: ORs (95% CI), 0.95 (0.81\u20131.13) and 0.91 (0.61\u20131.35), respectively, and similar results were seen when use since entry into the GPRD was examined . Among sThere was an inverse association between pancreatic cancer risk and duration of NSAID use in the 5 years before the index date. NSAID use for 773 days or more (approximately 2 years) in this period (excluding the year before the index date) was associated with a decrease in risk; adjusted ORs (95% CI), 0.78 (0.62\u20130.97) . No assoIn this large population-based prospective study no significant reductions in risk of pancreatic cancer were seen for ever compared with never use of an NSAID. There was also no overall association between risk and the total dose prescribed and no risk reduction was seen among subjects who used the highest doses of NSAIDs. A 20% reduction in risk was seen among subjects who had been prescribed an NSAID for approximately 2 years or longer in the 5 years before diagnosis and a 30% reduction in subjects who had used lower than average doses of NSAIDs for 5 years (1826.3 days) or more since entry into the GPRD. However, no consistent pattern of reduced risk was seen with increasing dose, duration or combined dose and duration of NSAID use. Modest reductions in long-term users of higher doses of NSAIDs were seen but were not statistically significant. Although chance and residual confounding may explain the latter findings, these data suggest that long-term use (>2 years) of NSAIDs (at regular anti-inflammatory doses) may protect against pancreatic cancer and that duration of use may be more important than dose.Two recent meta-analyses found no association between aspirin/NSAID use and pancreatic cancer. used low-dose aspirin only (<300\u2009mg) and data from cohort studies but statistical significance was not achieved and too few subjects took these preparations to allow a more detailed analysis of use of these drugs. Trials of COX-2 inhibitors in patients with colorectal adenomas or previous colorectal cancer have shown that these drugs can successfully prevent colorectal carcinogenesis . MeloxicThis analysis has several key strengths, being the largest and most detailed of the subject to date, allowing us to stratify the analyses by dose, duration and subgroup of NSAID. The use of prospectively collected prescription data avoids errors of recall and potential recall bias associated with questionnaire-based measures of NSAID use. Moreover, all subjects in our study had at least 5 years of data available before diagnosis and data were available before diagnosis for a mean of over 10 years. We also adjusted for all major confounders and, although data were not available for all subjects, the results of restriction analyses including only those patients who had data on these confounders were not different from the main analyses. However, we cannot rule out residual confounding. We do not believe that confounding by indication is a major problem within the study as indications for which NSAIDs are used are very varied and it is unlikely that each indication would have a common association with pancreatic cancer risk. We attempted to address such confounding by adjusting for a history of rheumatoid arthritis, a common indication of chronic NSAID use; it made no difference to observed ORs.This study has some limitations. Data on prescriptions issued may not reflect actual use of NSAIDs but there is no reason to believe that non-compliance with prescription medication would differ systematically between cases and controls. No information was available on over-the-counter (OTC) NSAID use (including that from non-pharmacy outlets) but there is little evidence of large-scale OTC purchasing of NSAIDs in the UK, especially in the middle aged and elderly. of NSAIDs may protect against pancreatic cancer but replication of these findings is required."} +{"text": "P for trend <0.001; RR for 5-year increment of use=1.25, 95% CI 1.12\u20131.38). Use of estrogen plus progestin was not significantly associated with ovarian cancer risk. Generally, results were similar for serous tumours and slightly stronger for endometrioid tumours . Recency of use was not significantly associated with ovarian cancer risk, but statistical power was limited here.The relationship between postmenopausal hormone use (PMH) and ovarian cancer risk is unclear, particularly for specific hormone formulations, but recent studies suggest that there is a positive association. We conducted a prospective observational study with 82\u2009905 postmenopausal women, including 389 ovarian cancers, in the Nurses' Health Study from 1976 to 2002. Compared with never users of PMH, both current and past users of \u2a7e5 years had a significantly elevated risk of ovarian cancer 1.07\u20131.86 and relative risk (RR)=1.52, 95% CI 1.01\u20132.27, respectively). Examined by hormone type in continuous years, use of unopposed estrogen was associated with a significant increase in the risk of epithelial ovarian cancer ( Ovarian cancer is the fifth most common cause of cancer mortality among women in the United States , and yetWe evaluated the association between PMH use and ovarian cancer in the prospective Nurses' Health Study (NHS) over 26 years of follow-up; we examined duration, recency of use, and PMH type for all ovarian tumours, as well as by histologic tumour type.The NHS began in 1976, when 121\u2009701 female registered nurses in 11 US states completed a self-administered, mailed questionnaire. At enrolment, participants were 30\u201355 years old. Subsequently, follow-up questionnaires were mailed biennially to obtain updated exposure and disease information. Information on deaths was obtained through the post office, relatives and linkages with the National Death Index. Through 2002, these methods yielded a follow-up rate of 93.7% of potential person-years. The study was approved by the Institutional Review Board of Brigham and Women's Hospital.The study population was restricted to postmenopausal women. A validation study in the NHS found self-reported menopause to have high reproducibility (n=209), a bilateral oophorectomy (n=8506), or a diagnosis of cancer other than non-melanoma skin cancer (n=514) before the start of follow-up. Women missing exposure or covariate information (n=1074) were excluded (details below), leaving 14\u2009140 eligible women in the baseline population.In 1976, there were 24\u2009443 postmenopausal women in the NHS. Participants were excluded if they reported radiation as the reason for menopause development of ovarian cancer, (2) report of any cancer other than non-melanoma skin cancer, (3) death, or (4) the end of the study period, 6/1/2002. Person-time with missing exposure or covariate information also was excluded. From 1976 to 2002, 82\u2009905 postmenopausal women accumulated a total of 966\u2009017 person-years.PMH use was assessed in every questionnaire. In 1976, users reported their total duration of use. As 72% of users of a known type reported use of unopposed estrogen in 1978 (when such details were first collected), PMH use in 1976 was classified as unopposed estrogen. This classification probably resulted in a small amount of misclassification of other types of PMH; however, in a sensitivity analysis, we also re-coded PMH use in 1976 to other/unknown type of hormone.Incident cases of ovarian cancer were identified through responses to biennial questionnaires or death certificates and confirmed by medical record review. From 1976 to 2002, there were 760 reported cases of postmenopausal ovarian cancer. We were unable to obtain medical records for 71 (9.3%) and did not confirm the diagnosis upon medical record review for 135 (17.8%) women. Of the 554 confirmed cases, 492 were epithelial tumours (88.8%). After applying exclusion criteria , we were left with 389 cases of primary epithelial ovarian cancer.Histologic type, as coded from pathology reports by a gynecologic pathologist (JLH), had the following distribution: 233 serous/poorly differentiated (hereafter referred to as serous), 60 endometrioid, 35 mucinous, 19 clear-cell and 42 other/unknown subtype. Of the 389 cases, 353 were invasive and 36 were of low malignant potential .Age and time period were used as stratification variables in the Cox proportional hazards models. Based on previous literature, the following covariates were forced into the multivariate models: duration of oral contraceptive use (continuous), parity (continuous), tubal ligation (yes/no), age at natural menopause (continuous) and age at menarche . The complete case method was used, except that women with a hysterectomy were included; the population was restricted to women with natural menopause in secondary analyses.In addition, the following potential confounders were not included in the final models: vigorous physical activity, smoking, alcohol consumption, caffeine intake, lactose/galactose consumption, perineal talc use, breastfeeding, simple hysterectomy, use of non-steroidal anti-inflammatory medications other than aspirin and family history of breast cancer. Data on family history of ovarian cancer, first collected in 1992, was evaluated as a potential confounder by examining its distribution across PMH categories in 1992. Fat intake and body mass index (BMI) were not considered confounders because they were not associated with postmenopausal ovarian cancer in previous NHS analyses , total duration and time since last use. In addition, analyses were performed by type-specific duration in continuous years, simultaneously including all PMH types in the models . Results have not been presented for the \u2018other PMH\u2019 group because it represents a heterogeneous group of hormones, including non-conjugated estrogens, patch hormones, and vaginal hormones, as well as person-time for which hormone type was not reported.n=35) were adjusted only for age, and clear cell tumors (n=19) were not evaluated.Primary analyses included all tumours , but sensitivity analyses restricted cases to invasive tumours. Separate analyses were performed for serous and endometrioid tumour subtypes, but owing to small numbers, those for mucinous tumors , increased risk was observed among both current and past users of five or more years compared with never users . ResultsP for trend <0.001, RR=1.25 for a 5-year increment of use, 95% CI 1.12\u20131.38), whereas continuous years of estrogen plus progestin use were not . ResultsIn analyses restricted to women exclusively using one hormone formulation, unopposed estrogen use of five or more years was associated with an increased risk compared with never use , whereas estrogen plus progestin use of five or more years was not . HoweverTime since last use was not significantly associated with risk. Neither users who quit within the previous three years nor those who stopped over three years ago were at a significantly increased risk of ovarian cancer. Based on the significant association observed with duration, a recency effect would most likely be seen primarily among long-term users. Although limited by small numbers, when we examined the effects of recency and duration together, point estimates suggested an increase in risk among users of five or more years that decreased over time .\u22122) or an intact reproductive system (data not shown). Results were also similar among women reporting natural menopause, although the association with unopposed estrogen use was slightly stronger .No substantial differences were observed when stratifying on BMI or population-based studies , unopposed estrogen use was significantly associated with risk , with similar results for recent and former long-duration users (n=16 cases); although risk was not increased among long-duration past users, there were only four cases in the same model, only duration was statistically significant (data not shown). Overall, the significant increase in risk appeared to be driven largely by duration rather than by status of use. This contrasts with breast cancer PMH findings, where the increased risk is confined to current users (n=655 cases) found that use of estrogen plus sequential progestin was associated with an increased risk, whereas estrogen plus continuous progestin was not (n=32 cases), were consistent with an increase in risk , although not statistically significant (n=13 cases) and continuous estrogen plus progestin use of \u2a7e5 years in women without a hysterectomy and leutinising hormone (LH)), but as the declines associated with PMH use are small, the benefits might be outweighed by estrogen-induced proliferation of ovarian cells . Althougpoptosis .Our analysis has several strengths. The NHS is one of only a few prospective studies of PMH use and ovarian cancer, and associations could be examined by hormone type. Information on exposures and confounders is updated through biennial questions, and follow-up of the cohort is high. The nurses are a relatively homogenous group, with similar education and access to health care, reducing concerns about confounding. Although family history of ovarian cancer was first collected in 1992, this did not vary substantially across exposure. Histologic tumour type was coded by a gynecologic pathologist and was available for most cases.Nurses' Health Study (NHS) participants are not a representative sample of the general population. While it is unlikely that the observed associations would differ in other women, studies covering different race/ethnicity and socioeconomic status are warranted. Generalisability may also be limited by the variations in PMH formulations across countries. We had limited power to look at non-oral formulations of PMH, which are more commonly used outside the US . Small nIn conclusion, we found that use of PMH was positively associated with risk of epithelial ovarian cancer. With other recent studies, our findings suggest that women should be counseled about the potential long-term increase in ovarian cancer risk with extended use of unopposed estrogen. Evidence is insufficient to say whether estrogen plus progestin or very short durations of unopposed estrogen use are associated with risk. Available findings indicate that ovarian cancer is one of several conditions that should be considered by women when weighing the risk and benefits of PMH use."} +{"text": "Melanoma has been increasing in white populations. Incidence rates rise steeply in women until about age 50, suggesting oestrogen as a possible risk factor. Oestrogens can increase melanocyte count and melanin content and cause hyperpigmentation of the skin. We examined prospectively the association between oral contraceptive (OC) use and diagnoses of superficial spreading and nodular melanoma among 183 693 premenopausal white women in the Nurses\u2019 Health Study (NHS) and the Nurses\u2019 Health Study II (NHS II) cohorts. One hundred and forty six cases were confirmed in NHS during follow-up from 1976 to 1994, and 106 cases were confirmed in NHS II from 1989 to 1995. Skin reaction to sun exposure, sunburn history, mole counts, hair colour, family history of melanoma, parity, height and body mass index were also assessed and included in logistic regression models. A significant twofold increase in risk of melanoma (relative risk (RR) = 2.0, 95% confidence interval (CI) 1.2\u20133.4) was observed among current OC users compared to never users. Risk was further increased among current users with 10 or more years of use . Risk did not appear elevated among past OC users, even among those with longer durations of use, and risk did not decline linearly with time since last use. In conclusion, risk of premenopausal melanoma may be increased among women who are current OC users, particularly among those with longer durations of use. Further research is needed to determine whether low-dose oestrogen pills in particular are associated with an increase in risk and to describe possible interactions between OC use and sun exposure or other risk factors for melanoma. \u00a9 1999 Cancer Research Campaign"} +{"text": "We examined ovarian cancer risk in relation to use of phenolphthalein-containing laxatives in 410 epithelial ovarian cancer cases and 713 controls. Compared to women who never used a laxative, ever use of a phenolphthalein-containing laxative was not associated with an increased risk of ovarian cancer . Risk was slightly, but not significantly, higher with more frequent use (OR 1.2 for 75 or more days of use). When women who used non-phenolphthalein containing laxatives was used as the reference group, the associations were slightly, but not significantly larger \u00a9 2000 Cancer Research Campaign"} +{"text": "P for trend <0.0001), and more important than age at start of use or time since first or last use. There was no significant difference between the effects of combined oral contraceptives and progestins-only contraceptives on risk (P=0.98). Similarly, there was no significant difference between the effects of ever use of HC on invasive and borderline ovarian neoplasia (P=0.37). In this cohort, use of HC seems to reduce the risk of epithelial ovarian neoplasia markedly and persistently in relation to the duration of use.The risk of ovarian epithelial neoplasia following use of hormonal contraceptives (HC) was examined in data from the Norwegian\u2013Swedish Women's Lifestyle and Health cohort including 103\u2009551 women aged 30\u201349 years in 1991\u201392. Follow-up through 2000 produced 214 incident cases of histologically confirmed epithelial ovarian neoplasias (135 invasive and 79 borderline cases). Using the Cox proportional hazard models, ever having used HC was associated with a decreased relative risk of epithelial ovarian cancer of 0.6 (95% CI 0.5\u20130.8). The effect of duration of HC use was convincing ( That use of hormonal contraceptives (HC) may reduce the incidence of ovarian cancer is well established from both case\u2013control , as receIn 1991\u201392, the Norwegian\u2013Swedish Women's Lifestyle and Health cohort was established in Norway and Sweden. Using the unique person number, 196\u2009000 women aged 30\u201349 years were sampled at random among all women in their age group from the Central Population Register in Norway and the Swedish Central Population Registry at Statistics Sweden , 15 who were dead or had emigrated before the start of follow-up and 1681 women who reported having an invasive cancer of any type at study enrolment (information obtained from the cancer registries). In addition, 1126 women who with lacked information on use of HC and 463 who had undergone bilateral oophorectomy, and therefore not at risk for ovarian cancer, were excluded from the present analysis, leaving 103\u2009551 women eligible for follow-up.We achieved complete follow-up through linkages between the cohort data set and various population-based registries using the individual unique national registration numbers , and incThe start of follow-up was defined as the date of return of the questionnaire. We obtained information on the dates of death for deceased persons from the death registers, and on the date of emigration from the registers of population migration. The follow-up ended on 31 December 2000, at emigration, death or diagnosis of histologically confirmed primary EON, whichever occurred first. Among the 106\u2009841 women enrolled initially into the cohort, 789 emigrated and 1360 died during the period of follow-up.The information about exposure to HC presented here is based on answers to questions on summary measures as ever having used, current use, total duration of use, age at first use, and use before first full-term pregnancy. We also collected information about each specific period of use. A period was defined as use of a specified HC brand for at least 1 month. Up to 10 different periods of use were reported. For each period, we asked for the age at starting use, duration of use and brand name. We calculated the time since last use as the interval between the end of use and start of the follow-up. \u2018Current use\u2019 was defined as self-reported use of HC at the time of study enrolment, or use within 1 year \u2013 regardless of how many months \u2013 before the start of follow-up. Time since first use was defined as the interval between the start of HC use and the start of follow-up. Information about commercial names or brands in each period of use made it possible to classify HC as combined oral contraceptives (COCs) and progestins-only contraceptives .We considered the use of postmenopausal hormone therapy (ever/never use) among the very few women who had used these at enrolment as either combined estrogen\u2013progestin or estrogen only. At the start of follow-up, only women who reported a natural menopause were considered as postmenopausal . Women reporting use of postmenopausal hormone therapy before natural menopause (2902 women) and those reporting hysterectomy without bilateral oophorectomy (2541 women) were treated as having unknown menopausal status. All other women were considered as premenopausal.The proportional hazards assumptions were met and relative hazards were calculated using the Cox proportional hazard model and SAS We kept the following covariates in the final multivariate model: age at enrolment into the cohort (as a continuous variable), parity (nulliparous/parous women), use of postmenopausal hormone replacement therapy (ever/never users), menopausal status at the start of follow-up , and country of residence (Sweden/Norway). Levels of recreational physical activity, educational status, age at first birth, total duration of breast-feeding, breast cancer in mother/sister, age at menarche, and body mass index were not included in the final multivariate models, because they did not improve the goodness of fit or change risk estimates meaningfully.The characteristics of HC use such as age at first use, time since first use, time since last use, and duration of use are closely related and might therefore be confounded by each other. In the multivariate analyses of age at first use, time since first use, and time since last use, the analyses were performed both with and without duration of use as a covariate. Ordinal ranks were assigned to each category of duration of HC use when calculating the tests for linear trend. The RRs for ever use of COCs and POPs were calculated separately.\u03c72 statistic for differences between log hazards. In this way, we tested the difference in risk for both invasive and borderline epithelial ovarian neoplasms with regard to never use, ever use, current use of HC, use of COCs and POPs, and duration of use of HC, COCs, and POPs.Tests for heterogeneity across subgroups of women were performed by calculating a Wald The responsible Data Inspection Boards and Ethical Committees in both countries approved the study design, and all women provided informed consent to participate in the study.In the study population of 103\u2009551 women aged 30\u201349 years at enrolment in 1991/92, 12% were aged 30\u201334, 31% were aged 35\u201339, 29% were aged 40\u201344, and 28% were aged 45\u201349. During the follow-up through year 2000, 214 cases of primary EON were diagnosed. These were distributed as 135 cases of primary IEON and 79 cases of BEON. The median age at diagnosis was 49 years for IEON and 48 years for BEON; for both, the median year of diagnosis was 1997.We explored the parity, menopausal status, and use of postmenopausal hormone replacement therapy as risk factors for EON, and thus as possible confounders (P=0.98). Similarly, there was no significant difference in risk with regard to ever use (P=0.37) or current use (P=0.70) of HC.The risk of EON was 40% lower among ever users of HC than among never users . The decreased risk of EON was observed both among current and former users of HCs . Ever use of HC was associated with a nonsignificant 20% decrease in risk among these women.P for trend P<0.0001) were statistically significant (data not shown).In our study, we confirm a strong inverse risk association between use of HC and epithelial ovarian neoplasms. Women who used HC for many years were at particularly lower risk as compared to women who were never users. We found no evidence of differential effect of HC use on the development of invasive or borderline tumours. Nor was the impact of use of combined estrogen\u2013progestin contraceptives and progestins-only contraceptives different from each other with regard to cancer risk.The strength of this study is the prospective design, the large size, and the complete follow-up based on linkage to national registers , and may be misclassified over time in our study. The impact of such possible misclassification will only be known once we have updated information on the entire cohort, which is ongoing at the moment. However, due to their ages, we believe that most participating women had probably ended their reproductive life at enrolment, so that changes in parity during follow-up probably have a very modest impact as a confounder on the overall association with HC. It is expected that at least a few women would have undergone hysterectomy and/or tubal ligation during follow-up, but they should not change substantially the association between use of HC and ovarian cancer risk.Our study indicates that the positive effect of HC on ovarian neoplasms is associated with the duration of use, the effect rising with each additional year of use. However, the finding of an important reduced risk in women with very long duration (15 or more years) was based on only one case. After including the duration in the analytical models, no independent effect was observed for age at first use, or for time since the first or last use. Thus, duration, rather than time of use, seems to be the most important in determining a decreasing ovarian cancer risk.invasive and borderline cases). In the analysis of the Nurses' Health Study, based on 260 cases, the exposure was restricted to shorter durations of use than in other studies. The nurses were aged 30\u201355 years in 1976 at enrolment, while the women in our study were 30\u201349 years in 1991. Thus, the opportunity to be ever exposed to HC differed greatly.Few analyses of prospective studies on HC and ovarian neoplasms are available and three out of the four studies were based on small numbers . Only thIn our study, there was no statistically significant difference between the effect of use of HC on risk of invasive or borderline neoplasms, in agreement with most previous studies .Epidemiological evidence from case\u2013control studies on use of COCs and ovarian cancer risk is well defined and consistent : at leasM Kumle and E Weiderpass were responsible for data management in Norway and Sweden, respectively, and for co-ordination of manuscript writing. T Braaten performed the combined Norwegian\u2013Swedish data management and performed the statistical analysis of the data. E Lund and H-O Adami, the principal investigators and initiators of the project in Norway and Sweden, respectively, participated in all phases of the study."} +{"text": "This study was conducted to determine whether use of hormonal contraceptives is associated with cervical dysplasia and cancer in a population where there is widespread use of hormonal contraception and the rates of cervical cancer remain high at 27.5/100,000.A case-control study was conducted among women visiting the colposcopy and gynaelogical clinics at a tertiary referral hospital. Two hundred and thirty six cases CIN I (72), II (59), III (54), cancer (51) and 102 controls, consented and were interviewed on use of contraceptives using a structured questionnaire. Logistic regression was used to determine odds ratios (ORs) and 95% confidence intervals (CIs) associated with use of hormonal contraception in cases and controls and in low and high risk cases. Recruitment was carried out from 2001\u20132002.Contraceptives used were: oral contraceptives \u2013 35%, injections (depot medroxy progesterone acetate (Depo-provera) \u2013 10%, Intrauterine devices \u2013 2%, combinations of these and tubal ligation \u2013 30%. 23% reported use of 'other' methods, barrier contraceptives or no form of contraception. Barrier contraceptive use was not significantly different between cases and controls. Current and/or past exposure to hormonal contraceptives (HC) by use of the pill or injection, alone or in combination with other methods was significantly higher in the cases. In multivariate analysis with age and number of sexual partners as co-variates, use of hormonal contraception was associated both with disease, . When parity and alcohol consumption were added to the model, hormonal contraception was no longer significant. The significant association with high risk disease was retained when the model was controlled for age and number of sexual partners. Depo-provera use was also associated with disease and severity of disease [OR 2.51 p = 0.027]. With parity and alcohol added to this model, depo-provera use retained significance. Exposure to HC > 4 years conferred more risk for disease and severity of disease.Hormonal contraception did confer some risk of dysplasia and women using HC should therefore be encouraged to do regular Pap smear screening. The search for links between the use of hormonal contraception or hormone replacement therapy and the development of some reproductive system cancers in women has yielded conflicting results. Thus there is evidence of a correlation between estrogen and increased risk of breast cancer while on the other hand, it has been suggested that use of oral contraceptives for one year or more is protective against endometrial and ovarian cancers with the protective effect lasting for at least 10 years -8 particThe inconsistent reports of an association between hormonal contraception and cervical dysplasia and cancer may be related, in part, to confounding risk factors that include sexual and lifestyle behaviours ,3. The cSince the 1960s, use of HC for family planning has been actively encouraged in Jamaica. In addition to pills, Depot medroxy progesterone acetate (DMPA) injections and lovonorgestrel implants are available. Data from a Reproductive Health Survey by the National Family Planning Board (2002) showed that in addition to a doubling in the use of condoms, since 1989, there has been a 48% increase in the use of injectables (22.7% vs 33.6%) in women reporting ever use of contraception. Over the period, pill use rose from 47.6% to 56.5% .Given the high incidence of cervical dysplasia and thatEthical approval was obtained from the University Hospital of the West Indies (UHWI) Ethical Review board. Both cases and the comparison group were recruited at the UHWI between April 2001\u2013August 2002. Cases were enrolled from twice weekly conducted colposcopy clinics while women in the comparison group were recruited from weekly gynaecology clinics at the same hospital. Both clinics receive referrals from primary care clinics, hospitals and private practitioners. Consecutive women were invited to participate in the study.Women with abnormal Pap smears who had been referred to the Colposcopy clinic and had a diagnosis that was confirmed by colposcopic biopsy histology served as the sampling frame. Cases were identified from the clinic registry where they were documented according to the degree of cervical intraepithelial neoplasia (CIN) as CIN-I, CIN-II, CIN-III and cervical cancer.Women attending routine gynaecological clinics who had normal Pap smears were regarded to be disease-free and served as the comparison group.The study population consisted of CIN-I (n = 72), CIN-II (n = 59), CIN-III (n = 54), cervical cancer (n = 51) and controls (n = 102). When grouped according to severity of disease using the Bethesda classification, there were 72 with low grade lesions (LGSIL) and 164 with high grade lesions (HGSIL). Informed consent was sought after an explanation of the study and those who agreed were asked to sign a consent form. Confidentiality and anonymity were assured before they were interviewed using a structured questionnaire by a single female interviewer. Data on drinking, smoking and other behavioural variables and sexual characteristics were collected. Participation rates among cases and the comparison group were 72% and 60% respectively.The following persons were excluded from the investigation: women who were pregnant, had hysterectomies or were previously diagnosed with adenocarcinoma.2 as appropriate. Logistic regression was used to determine the association of reproductive and lifestyle factors with disease (presence and severity). Odds Ratios (OR) and confidence intervals (CI) are presented. Analyses were performed using the SPSS software (version 12.0). Statistical significance was achieved when p < 0.05.Differences between cases and the comparison group were examined by the t-test and \u03c7Table Current and/or past exposure to hormonal contraceptives by use of the pill or injection, alone or in combination with other methods was significantly higher in the cases (79.4%) compared to the comparison group (67.4%) p = 0.023. There was also a significant difference between women classified as high risk (HGSIL) (82.9%) and low risk LGSIL (69.8%) (Bethesda classification) p = 0.032 ; p = 0.02] and the severity of the disease, . When parity and alcohol consumption (predictors of disease), were added to the model, HC use was no longer significant . Significant associations were observed for high risk disease when HC, age and number of sexual partners (predictor for severity of disease) were included in the model, . There were also more DMPA users among the HSIL compared to the LSIL . The ORs also shown in Table Of the 236 women who had used HC, 128 (57.2%) reported ever use of DMPA. When these women were compared to those who had never used HC, the percentage of cases who were ever users of DMPA was significantly greater than the controls ] and severity of disease patients suggested that risk of CIS may be confined to long term users .Several studies have contributed to the present understanding of the relationship between the use of HC and disease. The International Agency for Research on Cancer (IARC) pooled data from eight studies of invasive cancer and two of carcinoma in situ in HPV positive cases and found that risk increased significantly with parity . Women wWhereas we did not have data on HPV infection in the comparison group in this study, our findings of high rates of HPV infection among healthy pregnant and non pregnant women in Jamaica , suggestThe data indicates that fewer than 50% of the women underwent regular screening by Pap smear, while in a smaller study of university students only 27% reported screening . Since hUse of HC confers some risk of dysplasia which may be modified/confounded by use of barrier contraceptives, increased number of sexual partners, increased number of biological fathers, parity and alcohol consumption. Given the wide use of HC, regular Pap smear screening should be encouraged.The authors declare that they have no competing interests.NA conceived of the study, carried out statistical analyses and drafted the manuscript. PB was responsible for recruitment and data collection. MJ participated in design and statistical analyses. MS and HF advised on design and recruitment. All authors contributed to the writing of the manuscript and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"} +{"text": "Non-steroidal anti-inflammatory drugs (NSAIDs) prevent the growth of mammary tumours in animal models. Two population-based case-control studies suggest a reduced risk of breast cancer associated with selective cyclooxygenase-2 (sCox-2) inhibitor use, but data regarding the association between breast cancer occurrence and use of non-selective NSAIDs are conflicting.We conducted a population-based case-control study using Danish healthcare databases to examine if use of NSAIDs, including sCox-2 inhibitors, was associated with a reduced risk of breast cancer. We included 8,195 incident breast cancer cases diagnosed in 1991 through 2006 and 81,950 population controls.Overall, we found no reduced breast cancer risk in ever users (>2 prescriptions) of sCox-2 inhibitors (odds ratio (OR) = 1.08, 95% confidence interval (95% CI) = 0.99, 1.18), aspirin , or non-selective NSAIDs OR = 1.04, ). Recent use (>2 prescriptions within two years of index date) of sCox-2 inhibitors, aspirin, or non-selective NSAIDs was likewise not associated with breast cancer risk , 0.96 and 0.99 , respectively). Risk estimates by duration or intensity (low/medium/high) of NSAID use were also close to unity. Regardless of intensity, shorter or long-term NSAID use was not significantly associated with breast cancer risk.Overall, we found no compelling evidence of a reduced risk of breast cancer associated with use of sCox-2 inhibitors, aspirin, or non-selective NSAIDs. Non-steroidal anti-inflammatory drugs (NSAIDs) are inversely associated with the risk of colorectal and other gastrointestinal cancers -5. The pResearch on human cell lines and animal models indicates a role for cyclooxygenase-2 (Cox-2) in breast carcinogenesis , which sResults from epidemiological studies of breast cancer, however, are conflicting ,26. To dTo answer some of the research gaps in the epidemiological literature, we conducted a large population-based case-control study nested within a source population with prospectively collected prescription data to examine the association between use of sCox-2 inhibitors, aspirin, or non-selective NA-NSAIDs and the risk of breast cancer occurrence.This study was approved by the Danish Registry Board, reference #2004-41-4693.We conducted this nested population-based case-control study among the residents of North Jutland and Aarhus counties, Denmark, which together have a total population of 1.15 million inhabitants . The DanAll health-related services are registered to individual patients by use of their civil personal registration (CPR) number, assigned to all Danish citizens since 1968 by the Danish Civil Registration System. This number encodes gender and date of birth and alloth (1977 to 2003) and 10th (1994 on)) [Healthcare data from the two counties have been merged into a research database at Aarhus University -67. This994 on)) . Using tWe used the Civil Registration System ,68 to seAll pharmacies in both counties are equipped with computerised accounting systems that record a customer's CPR number, type and quantity of medication (including tablet and package sizes), and information on the drug according to the Anatomical Therapeutic Classification (ATC) . Healthcare prescription data for refundable drugs have been transferred electronically to a research database at Aarhus University; in North Jutland County since 1989, and in Aarhus County since 1996. We identified prescriptions for the following sCox-2 inhibitors: newer sCox-2 inhibitors , and older sCox-2 inhibitors . We grouped both newer and older sCox-2 inhibitors together. Aspirin use was classified as low dose and high dose (N02BA51 and N02BA01 in tablet size 500 mg). We grouped the other non-selective NA-NSAIDs (remaining codes within M01A) into a separate category. We also retrieved prescriptions for post-menopausal hormone replacement therapy , since use of hormone replacement therapy increases the risk of breast cancer -71 and wWe retrieved information on a hospital history of rheumatoid arthritis and migraine from the research database as proxies for chronic use of over-the-counter NA-NSAIDs and aspirin. Comorbid diseases before the diagnosis/index date were identified using the hospital discharge registries of each county and were summarized using the Charlson Index . The indEver users were defined as women who had more than two prescriptions and never/rare users were women who used less than or equal to two prescriptions. Our data showed that the average length of a prescription was 30 days.We excluded use of all NSAIDs within a year of diagnosis to reduce any potential effect that subclinical disease could have on NSAIDs and sCox-2 inhibitor use . We categorised NSAID use according to the number of prescriptions filled by each patient. ever users into recent and former users. Recent users were those who had three or more prescriptions within two years of index date . Former users were those who had less than three prescriptions within that time period but more than two prescriptions (as defined by ever use) during the entire period of observation.We examined temporality of NSAID use by dividing We examined whether breast cancer risk was associated with the intensity of NSAID use. The intensity of use was defined as low (less than 25%), medium (25 to 50%) or high (greater than 50%), according to the number of days of prescription coverage divided by the total duration of use in days. The duration was defined as the number of days from the date of a first prescription to the date of a last prescription plus the duration of the last prescription. We divided duration of NSAID use into long-term and shorter-term (less than 10 years) use. In most analyses, we considered sCox-2 inhibitors and non-selective NSAIDs separately. However, to examine intensity and duration of NSAID use, we grouped all NSAIDs together and included only women with at least 10 years of prescription history, that is, cases from North Jutland County diagnosed between 1999 and 2006, and from Aarhus County diagnosed in 2006, and their corresponding controls. This approach ensured that women who were long-term users were not misclassified as shorter-term due to a limited prescription history.The cut points for medication exposures were determined based on methods developed by Robertson et al. . RobertsIn all analyses, we used conditional logistic regression to compute odds ratios (ORs) and their associated 95% confidence intervals (95% CI) adjusting for a history of migraine and rheumatoid arthritis, and ever/never hormone replacement therapy use. We completed additional analyses adjusting for the Charlson comorbidity index and separate analyses adjusting for history of heart diseases, but these had no appreciable effect on the risk estimates and were therefore dropped from the final models. In each analysis, we used never or rare use as the reference group. Given the risk set sampling of controls, the ORs were unbiased estimates of the incidence rate ratios (IRRs) in the underlying population. Analyses were performed using SAS version 9.13 .P <0.001). More cases than controls had ever used sCox-2 inhibitors and/or NA-NSAIDs .Characteristics of the 8,195) breast cancer cases and population controls are presented in Table 95 breastOverall, we observed no reduced risk of breast cancer associated with ever versus never/rare use of sCox-2 inhibitors , non-selective NA-NSAIDs , or aspirin , we found no protective effect against breast cancer in shorter-term or long-term users, regardless of the intensity of use Table . We carrIn this population-based case-control study, we found no substantial association between risk of breast cancer and use of NSAIDs, whether aspirin, non-selective or Cox-2 selective NSAIDs.et al. [Our results are inconsistent with those of a case-control study by Sharpe et al. based onet al. and a stet al. investiget al. . Our stuThe only two previous studies to examine the impact of sCox-2 inhibitors on breast cancer risk reported a risk reduction of at least 20% ,77. BothTo overcome potential limitations, we included all available data on use of newer sCox-2 inhibitors, the entire period for which they were available on the Danish market ,79, and Between 5% and 56% of breast cancer patients are estimated to harbour mammogram-detectable carcinomas two to five years before clinical diagnosis . MammogrThe validity of our estimates depends on several factors. First, the use of population-based prescription registries, with a completeness approaching 100% , ensuredOur study also had limitations. We were unable to examine the impact of NSAID use on breast cancer risk by specific breast cancer characteristics such as hormone receptor status. Earlier studies suggest that NSAID use can decrease the risk of oestrogen receptor positive tumours in post-menopausal women . HoweverWe had no information on family history of breast cancer, body mass index, use of oral contraceptives and life-style factors such as alcohol consumption and dietary fat intake . While tOur findings in this large population-based setting do not support an association of prescription NSAID use with the risk of breast cancer.95% CI: 95% Confidence Interval; ATC: Anatomical Therapeutic Classification; Cox-2: cyclooxygenase-2; CPR number: civil personal registration number; ICD: International Classification of Diseases; IRRs: incidence rate ratios; NSAIDs: non-steroidal anti-inflammatory drugs; NA-NSAID: non-aspirin NSAID; OR: odds ratio; sCox-2: selective cyclooxygenase-2.JAB is a consultant to Bayer Pharmaceuticals, Merck Pharmaceuticals and Pozen Incorporated and holds a US patent regarding the chemopreventive efficacy of aspirin in the large bowel. HTS did not report receiving fees, honoraria, grants or consultancies. The Department of Clinical Epidemiology is, however, involved in studies with funding from various companies as research grants to (and administered by) Aarhus University. None of these studies are related to the present study. All other authors declare no competing interests.HTS, DCF, SF and LAP conceived the study idea and designed the study. HTS and LAP collected the data. LAP, DCF, JAB, TLL and HTS planned and performed the analyses. DCF reviewed the literature and drafted the manuscript. DCF, TLL, SF, JAB and HTS edited the manuscript.Tables S1-S3. Table S1: Duration and intensity of non-steroidal anti-inflammatory drug use among women with at least 10 years of prescription history and odds ratio of breast cancer. Table S2: Duration and intensity of non-steroidal anti-inflammatory drug use among women with at least 10 years of prescription history and odds ratio of breast cancer. Table S3: Duration and intensity of aspirin use among women with at least 10 years of prescription history and odds ratio of breast cancer. Click here for file"} +{"text": "Data regarding the effects of oral contraceptive use on women's risk of melanoma have been difficult to resolve. We undertook a pooled analysis of all case\u2013control studies of melanoma in women completed as of July 1994 for which electronic data were available on oral contraceptive use along with other melanoma risk factors such as hair colour, sun sensitivity, family history of melanoma and sun exposure. Using the original data from each investigation , we combined the study-specific odds ratios and standard errors to obtain a pooled estimate that incorporates inter-study heterogeneity. Overall, we observed no excess risk associated with oral contraceptive use for 1 year or longer compared to never use or use for less than 1 year (pooled odds ratio (pOR)=0.86; 95% CI=0.74\u20131.01), and there was no evidence of heterogeneity between studies. We found no relation between melanoma incidence and duration of oral contraceptive use, age began, year of use, years since first use or last use, or specifically current oral contraceptive use. In aggregate, our findings do not suggest a major role of oral contraceptive use on women's risk of melanoma.British Journal of Cancer (2002) 86, 1085\u20131092. DOI: 10.1038/sj/bjc/6600196www.bjcancer.comCancer Research UK\u00a9 2002 Until about age 45 years incidence rates of melanoma in women exceed those in men, after which rates markedly rise in men but level off slightly in women . AdditioWe identified epidemiologic studies of melanoma completed as of July 1, 1994. In addition to an extensive literature review, we contacted an established consortium of melanoma investigators to produce a pooled exposure odds ratio and standard error. The estimator of the pooled exposure effect is a weighted average of the individual study estimators, weighted by the inverse marginal variances; the marginal variance is the sum of the individual study-specific variances and the variance of the random study effects. To assess inter-study variability, we examined the study-specific ORs and tested for heterogeneity using a \u03c7vs 1970 or after). For each, the reference category was \u2018never use\u2019 of oral contraceptives or use of less than 1 year. We also examined whether the effects of duration of use (grouped as <5 or \u2a7e5 years) were modified by number of years since first use (<10 or \u2a7e10 years). Due to small strata in this analysis, we broke the matched pairs and adjusted for age using the same categories used in the frequency matched studies. To further examine the risk of melanoma among current, long-term users, we combined the data from all studies on women less than 50 years of age. We then analysed current use, defined as use in the 2 years prior to diagnosis or interview date, among those with 10 or more years of use compared to our non-user reference group. Stratifying on \u2018study\u2019, we obtained conditional maximum likelihood estimates both for the individual studies and for all studies combined , age began , years since first use , years since last use , and year of first use . We used the classification scheme developed for earlier pooled analyses for eye colour, hair colour and family history of melanoma specifically. There were too few cases of nodular melanomas or lentigo maligna melanomas to perform detailed analyses of these histologic types.A total of 2391 cases and 3199 controls were included in the analysis for the 10 case\u2013control studies . The preTable 22=7.15, P=0.62). The findings generally were similar for superficial spreading melanoma . The risk of melanoma was unrelated to duration of use, age began, time since first and last use of oral contraceptives, or year of first use =0.86; 95% CI=0.74\u20131.01) . Again, none of these odds ratios changed by more than 10% with the additional potentially confounding factors. Results were similar for SSM (data not shown).When we evaluated the effects of duration of use stratified by time since first use, we found that the pORs, for the most part, approached unity .To examine the effects of long-term oral contraceptive use among current oral contraceptive users, we pooled data on women younger than age 50 years. In total, there were 61 cases and 87 controls who were current oral contraceptive users with 10 or more years of use. Combining data into a single conditional logistic model and adjusting for \u2018study\u2019, the odds ratio among current, long-term users compared to non-users was 0.91 (95% CI=0.62\u20131.32). There was no evidence of inter-study heterogeneity (vs population-based), type of interview (in-person vs telephone) and questionnaire format . However, we observed negligible difference in our findings according to these factors.In our combined re-analysis of 10 case\u2013control studies of melanoma in women, we found no overall association between use of oral contraceptives and melanoma risk. Studies included in our analysis represent the largest case\u2013control studies of women completed by 1994 that personally interviewed women about their use of oral contraceptives along with other melanoma risk factors. By applying strict selection criteria, we attempted to minimise inter-study heterogeneity in results that could arise from design characteristics. Still, some aspects varied between studies, such as method of case or control selection in relation to 5 or more years of oral contraceptive use, among those who began use 10 or more years before and NHS II cohorts that involved postal surveys, an elevated risk of melanoma was found in relation to current but not past use of oral contraceptives (Several earlier studies did not collect data on potentially confounding factors such as sun exposure history or pigmentary characteristics or nevi and thus were excluded from our analysis (Increased detection among women who used oral contraceptives is a plausible explanation for any observed association between melanoma and oral contraceptive use. In our analysis, we found little effect on our risk estimates when we adjusted for sociodemographic factors such as level of education that could be related to screening behaviour. Indeed, we extensively evaluated the impact of multiple factors on the odds ratios, but none appreciably altered the results. In conclusion, our pooled epidemiologic data do not suggest a major role of oral contraceptives on women's risk of melanoma."} +{"text": "Epidemiologic studies have identified a number of lifestyle factors, e.g. diet, obesity, and use of certain medications, which affect risk of colon cancer. However, the magnitude and significance of risk factor-disease associations differ among studies. We propose that population trends of changing prevalence of risk factors explains some of the variability between studies when factors that change prevalence also modify the effect of other risk factors. We used data collected from population-based control who were selected as study participants for two time periods, 1991\u20131994 and 1997\u20132000, along with data from the literature, to examine changes in the population prevalence of aspirin and non-steroidal anti-inflammatory medication (NSAID) use, obesity, and hormone replacement therapy (HRT) over time. Data from a population-based colon cancer case-control study were used to estimate effect-measurement modification among these factors. Sizeable changes in aspirin use, HRT use, and the proportion of the population that is obese were observed between the 1980s and 2000. Use of NSAIDs interacted with BMI and HRT; HRT use interacted with body mass index (BMI). We estimate that as the prevalence of NSAIDs use changed from 10% to almost 50%, the colon cancer relative risk associated with BMI >30 would change from 1.3 to 1.9 because of the modifying effect of NSAIDs. Similarly, the relative risk estimated for BMI would increase as the prevalence of use of HRT among post-menopausal women increased. In conclusion, as population characteristics change over time, these changes may have an influence on relative risk estimates for colon cancer for other exposures because of effect-measure modification. The impact of population changes on comparability between epidemiologic studies can be kept to a minimum if investigators assess exposure-disease associations within strata of other exposures, and present results in a manner that allows comparisons across studies. Effect-measure modification is an important component of data analysis that should be evaluated to obtain a complete understanding of disease etiology. Epidemiologic investigations have detected associations between colon cancer and several diet and lifestyle risk factors, but the relative importance of risk factors has changed over time ,2. StudiInconsistencies in results for risk factor-disease associations for colon cancer have been attributed to a variety of non-biological factors. Since earlier results for colon cancer came primarily from case-control studies, while cohort studies contributed more to the recent literature, study design, i.e. the potential biased reporting in case-control studies, has been cited as a potential explanation for differences between results of older and more recent studies. HoweverWe propose that risk factors associated with colon cancer at the population level exist in a shifting context. We hypothesize that the ability of studies to estimate the influence of risk factors for colon cancer depends on the presence of effect-measure modifiers that are changing in the population. We hypothesize that change in the estimated relative risk from a given exposure can occur if prevalence of other exposures that modify its risk are changing. Thus, it is possible that differences in risk factors identified between studies can be attributed, at least in part, to differences in the prevalence of important population characteristics.Changes in U.S. population during the last quarter of the 20th century in diet, physical activity, and weight, characteristics that affect colon cancer risk, have been described based on national surveys . In thisTo test our hypothesis that effect-measure modifiers can influence population level risk of other factors, we consider the inter-relationships of aspirin and other NSAID use, BMI, and HRT in altering the risk of colon cancer. We focus particularly on these factors since their prevalence has changed dramatically over the past decades and they appear to play a major role in modulating colon cancer risk factors.Data from two case-control study populations are included in these analyses,18: a poThe colon cancer study included cases with a first primary of colon cancer diagnosed between October 1, 1991 and September 30, 1994. The rectal cancer study included cases with a first primary tumor in the rectosigmoid junction or rectum diagnosed between May 1997 and May 2001. For both studies, controls were frequency-matched on sex and 5-year age group to the cases. Case eligibility was determined by the Surveillance Epidemiology and End Results (SEER) Cancer Registries in Northern California and in Utah and identified using a rapid-reporting system. At the KPMCP, controls were randomly selected from membership lists. In Utah controls 65 years and older were randomly selected from Health Care Finance Administration (HCFA) lists and controls younger than 65 were randomly selected from driver's license lists. To be eligible for the study, participants had to be between 30 and 79 years of age at time of diagnosis, English speaking, mentally competent to complete the interview, and could not have had previous colorectal cancer. Cases with known (as indicated on the pathology report) familial adenomatous polyposis, ulcerative colitis, or Crohn's disease were excluded.A total of 1346 colon cancer cases and 1544 matched controls were interviewed between January 1991 and December 1994 and 952 cases and 1205 controls were interviewed between October 1997 and January 2002 as part of the rectal cancer study and are included in these analyses. Of participants contacted between 1991 and 1994, 80.8% of cases and 71.8% of controls participated; between 1997 and 2001, 68.8% of controls participated.Data were collected by trained and certified interviewers using laptop computers. For both the colon and rectal cancer studies, data were collected in an identical manner, using the same study questionnaire and the same quality control procedures -21. The 2 was calculated for men and women. Other information of relevance to this analysis included reproductive history, use of aspirin and NSAIDs on a regular basis, and physical activity [For both studies, dietary intake was ascertained using an adaptation of the CARDIA diet history ,22,23. Pactivity . The intTo evaluate differences in population characteristics for the two time periods (i.e. 1991\u20131994 and 1997\u20132001), we compare exposure prevalence among controls after adjusting for possible age and sex differences. Variables assessed included variables thought to be important effect-measure modifiers of other variables. These included, BMI, physical activity, NSAID use (includes use of aspirin), and HRT use among post-menopausal women.In order to describe population changes in exposures to these factors over a longer time period, we used results from national surveys conducted in the U.S., and the published literature, to obtain estimates of exposure prevalence for obesity and NSAID use in the 1970s and 1980s. BMI for men and women was available from the NHANES surveys . We coulFor colon cancer relative risk associations, reported weight for the period two years prior to diagnosis was used to calculate BMI. Using aspirin or non-steroidal anti-inflammatory drugs on a regular basis (defined as at least three times per week for one month) within two years prior to diagnosis was considered positive use, while those not using aspirin or non-steroidal anti-inflammatory drugs during this time period were considered non-users. HRT use was defined as having used HRT within the past two years. Data were categorized into groups based on respondent response to using aspirin/NSAIDs, HRT, and BMI cut-points of 25 and 30. For physical activity levels and western diet, categories were based on distribution of these continuous variables as reported for the colon cancer study.To provide an example of the potential for risk estimates to change due to change in the prevalence of effect-measure modifiers, we estimated odds ratios for the relative risk of colon cancer associated with obesity (BMI \u2265 30) compared to individuals whose BMI was \u2264 23 stratified by HRT users and non-users and by NSAID/aspirin users and nonusers. Unconditional logistic regression models were used to estimate odds ratios associated with effect-modification adjusting for confounding factors that included energy intake, physical activity level, dietary calcium and fiber, HRT (NSAID/aspirin model), and NSAIDs/aspirin (HRT use only model). Statistical testing for interaction was determined by the relative excess risk from interaction (RERI) ,29. The To illustrate the impact of changes in prevalence of use of aspirin and HRT over time on risk estimates for other risk factors, we estimated ORs for BMI (highest quartile vs. lowest) in hypothetical populations with different prevalence of NSAID/aspirin and HRT use. To develop these estimates we used the ORs for obesity or a BMI of 30 or more relative to those with a BMI of 23 or less that were observed in our study population. These risk estimates were calculated within strata of NSAID/aspirin users (risk estimate used was 2.5 from the stratified model) and non-users (risk estimate of 1.2 used from stratified model) and HRT users (risk estimate of 2.1 used for strata of users) and non-users (risk estimate of 0.8 used for strata of non-users). We assumed that risk estimates for BMI within these circumstances would be constant, although variation in exposure to NSAID/aspirin and HRT use in the population would influence the actual BMI detected risk. We calculated the population risk as the variable exposures of NSAIDS/aspirin and HRT using population distributions of 5% users, 95% non-users, then a population with 10% aspirin users, 90% non-users, etc. Additional File Figure We detected a significant interaction between NSAID use and HRT on colon cancer relative risk Table . The intOdds ratios for associations between BMI and colon cancer relative risk differed significantly between subjects who did and did not use NSAIDS and HRT Table . Among tFigure We have demonstrated using data from our studies and the literature that BMI, HRT use, and aspirin use have changed over time. In addition to documenting these changes, we have shown, through assessment of interaction, the potential impact of those changes on ORs for colon cancer relative risk estimates in the population. The presence of exposures such as aspirin and HRT use appear to modify relative risk associated with other the important colon cancer relative risk factors such as obesity. Therefore, changes in prevalence of aspirin and HRT use over time will affect colon cancer relative risk estimates associated with these other factors. The result of multiple risk factors changing overtime further illustrate the complexity of multi-factorial diseases, such as colon cancer, both in terms of risk factor identification and in understanding the disease process itself.As documented here and by others, important diet and lifestyle factors that are associated with colorectal cancer have changed over time ,30-33. OFew studies have reported on changes in aspirin or HRT use over time and different definitions of regular use make comparisons across studies difficult. One of the first studies to detect an association between aspirin use and colon cancer was conducted by Rosenberg and colleagues who reported 7% of the population using aspirin in the late 1970s . In 1976While our study data indicate that HRT use increased during the 1990s, recent research findings regarding potential adverse effects from HRT use are resuThere are several strengths to the study, including a large sample to look at interactions to test our hypothesis, and the ability to look at data collected in the same manner from two population-based control groups, to identify exposure changes overtime. Response rates were slightly lower for the rectal cancer study than for the colon cancer study, although good response rates overall were obtained in both studies. However, because the study in the early 1990s was one of colon cancer and the one in the late 1990s was of rectal, we are unable to directly calculate relative risk estimates for the two time periods at the population level.These findings also have important study design and analysis implications because they suggest that disease associations are complicated when factors that modify risk change. Aspirin, NSAID, and HRT use are examples of these types of effect-measure modifiers. Our results suggest that the risks associated with obesity and HRT is influenced by NSAIDs. In this scenario, case-control studies provide a snapshot of what risk factors are in the population at a given point in time. Studies with sufficiently large sample size can define subsets of the population that have different characteristics. Cohort studies accumulate cases over time; in order to take into account changing population characteristics, changes in risk factors for should be taken into account by incorporating repeated exposure measurements. The importance of identification of effect-measure modification should be stressed since results can have implications for understanding the data and the disease process. Often evaluation of effect-measure modification is secondary and stratified estimates are not presented unless significant effect-measure modification is detected. However, studies are often underpowered to detect statistically significant effect-measure modification . When efIn summary, populations are dynamic and risk factors exist in a shifting context. Of particular importance are factors that modify the risk of other factors. NSAID use, HRT, and obesity are three factors that appear to be changing over time as well as having an impact as effect-measure modifiers of other diet and lifestyle factors. The impact of population changes on the epidemiologic literature can be kept to a minimum if investigators assess exposure-disease associations within strata of other exposures, and present results in a manner that allows comparisons across studies.Numbers used to estimate population risk shown in Figure 2Click here for file"} +{"text": "COX2 influences the risk of advanced prostate cancer. Nine single-nucleotide polymorphisms (SNPs) in COX2 were genotyped among 1012 men in our case\u2013control study of advanced prostate cancer. Gene\u2013environment interactions between COX2 polymorphisms and NSAID use were also evaluated. Information on NSAID use was obtained by questionnaire. Three SNPs demonstrated nominally statistically significant associations with prostate cancer risk, with the most compelling polymorphism (rs2745557) associated with a lower risk of disease (odds ratio (OR) GC vs GG=0.64; 95% confidence interval (CI): 0.49\u20130.84; P=0.002). We estimated through permutation analysis that a similarly strong result would occur by chance 2.7% of the time. Nonsteroidal anti-inflammatory drug use was associated with a lower risk of disease in comparison to no use . No significant statistical interaction between NSAID use and rs2745557 was observed (P=0.12). Our findings suggest that variation in COX2 is associated with prostate cancer risk.Collective evidence suggests that cyclooxygenase 2 (COX2) plays a role in prostate cancer risk. Cyclooxygenase 2 is the major enzyme that converts arachidonic acid to prostaglandins, which are potent mediators of inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the enzymatic activity of COX2 and long-term use of NSAIDs appears to modestly lower the risk of prostate cancer. We investigated whether common genetic variation in COX2 in prostate tumours (COX2 expression has been correlated with higher tumour grade (in vitro as well as prostate tumorigenesis in vivo (Cyclooxygenase 2 (COX2) is an inducible enzyme that converts arachidonic acid to prostaglandins, which are potent mediators of inflammation. Through the production of prostaglandins, COX2 is hypothesised to influence carcinogenesis by promoting cell proliferation, inhibiting apoptosis, stimulating angiogenesis, and mediating immune suppression and +8365 C/T (rs2043), were associated with a reduced risk of disease . Restricting the cases to men diagnosed with advanced disease allows us to focus on the most clinically relevant prostate cancers. To help ensure that the controls were representative of the source population of the cases, controls were men who underwent standard annual medical exams at the collaborating medical institutions. Controls had no diagnosis of prostate cancer or any other non-skin cancers. All controls received a PSA test to detect occult prostate cancer. Controls were frequency matched to cases by age (within 5 years), ethnicity, and medical institution. Detailed information and descriptive characteristics for this case\u2013control study has been reported previously . Advanced prostate cancer cases were confirmed histologically and defined as having either a Gleason score \u2a7e7, or TNM stage \u2a7eT2c, or PSA at diagnosis >10\u2009ng\u2009mlInstitutional Review Board approval was obtained from the participating medical institutions, and informed consent was obtained from all study participants.COX2 by using publicly available genotype data for European populations from the International HapMap project (www.hapmap.org) (http://gvs.gs.washington.edu/GVS/). We assessed the common genetic variation of COX2 >5%) that spanned \u223c2 kilobases (kb) upstream of the transcription start site and \u223c1\u2009kb downstream of the 3\u2032 untranslated (UTR) region. For genetic characterization, seven SNPs (average density=1 SNP every 1.4\u2009kb) and 14 SNPs (average density=1 SNP every 510\u2009bp) were used from the HapMap and Perlegen/Seattle data, respectively. To thoroughly capture the common genetic variation across the locus, we utilised a pairwise tagging approach that reconstructed all SNPs across the locus (r2\u2a7e0.8 between the tag SNP and unmeasured SNP. The Tagger website (http://ww.broad.mit.edu/tagger) and Genome Variation Server (http://gvs.gs.washington.edu/GVS/) were used for tag SNP selection . Because genotyping assays for two SNPs could not be designed and one SNP (rs5272) was monomorphic in our study population, we subsequently used the Perlegen and Seattle SNP data to maintain a comprehensive evaluation of the COX2 locus. Using this data, we selected five tag SNPs exclusive from the tag SNPs identified from HapMap. A genotype assay could not be designed for rs689467.First using the HapMap data, we selected six tag SNPs to capture the common genetic variation across Two common SNPs, +8365 C/T (rs689470) and \u2212899 G/C (rs20417), that were previously associated with prostate cancer (P>0.01 level).Genotyping was performed using the 5\u2032 nuclease Taqman allelic discrimination assay using the manufacturer's predesigned primer/probe sets or custom-designed and manufactured primer/probe sets . All assays were undertaken by individuals blinded to case\u2013control status. For quality control, 2% replicate samples were included. The concordance rate for replicate samples was 100%. The average genotyping success rate was 99.9%. Further details of genotyping methods are described elsewhere . We defined NSAID use as either aspirin or ibuprofen consumption at least twice a week for more than 1 month and no use as less than twice a week use of either of the medications for less than 1 month. For the dose/duration of use, we determined \u2018pill-years\u2019, which is the product of number of pills taken per day and years of drug use, for aspirin and ibuprofen. The summation of pill-years of aspirin use and ibuprofen use is the dose/duration of any NSAID consumption.COX2 genotypes and prostate cancer risk. Odds ratios (ORs) and 95% confidence intervals (CI) were estimated by unconditional logistic regression to examine the association between COX2 SNPs and prostate cancer risk.To investigate the hypothesis that genetic susceptibility to prostate cancer risk is associated with single causal variants, we evaluated the relationship between COX2 haplotypes and prostate risk. Haplotype frequencies among prostate cancer cases and controls were estimated by using genotype data of the tag SNPs as described by COX2 haplotypes and prostate cancer risk.To potentially capture other unmeasured variants that may not be adequately captured by single markers, we evaluated the relationship between common COX2 genotypes and NSAID use, we dichotomized the latter into use and no use and assessed the effect of NSAID use stratified by COX2 genotypes. To test for statistical interaction, the model contained separate terms for the main effect of COX2 genotype, the main effect of NSAID use, and an interaction term for the cross product of COX2 genotype and NSAID use. We also tested the interaction between COX2 genotypes and dose/duration of NSAIDs (categorised into tertiles based on the distribution among controls).To examine the interaction between P-values are two-sided. Permutation testing was conducted to guide interpretation of nominally statistically significant SNP associations. Case\u2013control status within strata of age , racial/ethnic group, and medical institution was randomly permuted 10\u2009000 times for the nine COX2 SNPs.All OR estimates were adjusted for age and medical institution and also for racial/ethnic group in any analysis that combined groups. All reported COX2 SNPs and prostate cancer risk in our study of 506 advanced cases and 506 controls. Three SNPs were nominally statistically significantly associated with prostate cancer risk (P=0.002); specifically, men carrying the GA genotype in comparison to men carrying the GG genotype had a significantly lower risk of disease . An overall consistent pattern of the GA genotype was seen across racial/ethnic groups . In contrast, a higher risk of disease was observed with the AA genotype of rs689470 in comparison to the GG genotype , with this SNP being more common among African Americans than Caucasians . Overall, rs2745557 demonstrated the most compelling association with prostate cancer risk and its permutation P-value was 0.027, indicating that when permuting the data similar levels of significance were observed 2.7% of the time.First, we tested the association between cer risk . The strc groups . SimilarCOX2 haplotypes and prostate cancer risk (P=0.03). We observed a nominally significant association between the AGGGGA haplotype and prostate cancer risk (P=0.009). This haplotype could be defined by the G allele of rs5277, which was associated with risk among Caucasians .Next, we tested the association between cer risk . We idenP=0.12). Among men carrying the GG genotype of rs2745557, a significant reduced risk of prostate cancer was observed with NSAID use . In contrast, among men carrying the GA/AA genotype of rs2745557, there was no significant effect with NSAID use. Similar patterns of associations were observed across racial/ethnic groups. No statistical interaction was observed between rs2745557 and dose/duration of NSAIDs (P=0.97).We examined the potential interactive effects between the rs2745557 SNP and NSAIDs . We prevCOX2, we identified a polymorphism (rs2745557) that was associated with a 36% reduction in risk of prostate cancer (P=0.002). In addition, the major G allele of this polymorphism in combination with NSAID use demonstrated an \u223c40% lower risk of disease in comparison with the G allele and no NSAID use, while the minor A allele was associated with an \u223c50% lower risk and was not influenced by NSAID use. However, a statistically significant interactive effect between rs2745557 and NSAID was not achieved. Our results suggest that inherited variation in COX2 influences prostate cancer susceptibility.In this comprehensive evaluation of vs African Americans: MAF=0.38).In comparison with the previous study that detected an association between the \u2212899 G/C (rs20417) variant and prostate cancer among African Americans . ReasonsP-values and guides the interpretation of results when multiple hypotheses are tested and prostate cancer risk.Our study has several limitations. Because of limited power to conduct African American-specific analyses, we did not characterise the common genetic variation of COX2 may alter the production of inflammatory prostaglandins, which could ultimately influence prostate cancer susceptibility. Furthermore, environmental factors such as use of NSAIDs may modify the biological effects of COX2 variation on disease risk. In particular, the combined effects of rs2745557 and NSAID use are of interest. Among men with the major allele of this polymorphism, those who were NSAID users had a slightly lower risk of prostate cancer than those who were non-NSAID users. While men with the minor allele of this polymorphism appeared to have no additional benefit of NSAID use, it is plausible that NSAID use may actively inhibit COX2 enzymatic activity only among those with the more prevalent major allele, while those with the variant allele may reach a threshold of COX2 inhibition such that NSAID use provides no added protection. To fully explore these potential interactive effects, larger well-powered studies are needed.The functional impact of rs2745557, an intronic variant, on COX2 activity is not yet known. It is possible that either this polymorphism itself or another linked marker may have biological effects on COX2 activity. We hypothesise that genetic variation in LTA C+80A) in lymphotoxin alpha, a proinflammatory cytokine, modified the protective effect of NSAIDs on prostate cancer . Such findings highlight the complex nature of prostate cancer susceptibility, which is likely due to multiple susceptibility alleles acting in tandem with environmental components.We previously reported that a functional variant (COX2 influences the risk of prostate cancer. To date, this is the most comprehensive survey of common genetic variation at the COX2 locus. Our finding supports previous reports showing an association between COX2 variants and prostate cancer risk. Replication is critical in establishing an association between a variant and disease and additional studies either confirming or refuting these findings are needed. Furthermore, our study provides supporting evidence for the overall role of inflammation in prostate cancer susceptibility. By examining how inherited variation in inflammatory genes impacts risk of disease, we will further advance our understanding of prostate carcinogenesis and disease susceptibility.In this study, we demonstrate that common genetic variation in"} +{"text": "Differences between the sexes in time trends of colorectal cancer incidence 1962-87 and mortality 1960-91 in England and Wales are examined in relation to changes in female hormonal factors. There was a trend in the sex ratio of this tumour, particularly marked for the descending colon, whereby the female excess in risk at young ages has almost disappeared but the male excess at older ages has increased. This trend started for cohorts born since the 1920s and coincided with the increase in the use of oral contraceptives and, to a lesser extent, with increases in fertility. The decline has been particularly pronounced for women at young ages born since 1935-39, coinciding with the spread of oral contraceptive use to younger age groups. These results are consistent with the hypothesis that female hormonal factors may play a role in the aetiology of colorectal cancer and with the possibility that oral contraceptive use might exert a protective effect in the descending colon."} +{"text": "There is public concern that use of mobile phones could increase the risk of brain tumours. If such an effect exists, acoustic neuroma would be of particular concern because of the proximity of the acoustic nerve to the handset. We conducted, to a shared protocol, six population-based case\u2013control studies in four Nordic countries and the UK to assess the risk of acoustic neuroma in relation to mobile phone use. Data were collected by personal interview from 678 cases of acoustic neuroma and 3553 controls. The risk of acoustic neuroma in relation to regular mobile phone use in the pooled data set was not raised (odds ratio (OR)=0.9, 95% confidence interval (CI): 0.7\u20131.1). There was no association of risk with duration of use, lifetime cumulative hours of use or number of calls, for phone use overall or for analogue or digital phones separately. Risk of a tumour on the same side of the head as reported phone use was raised for use for 10 years or longer . The study suggests that there is no substantial risk of acoustic neuroma in the first decade after starting mobile phone use. However, an increase in risk after longer term use or after a longer lag period could not be ruled out. More than 1 billion people use mobile phones worldwide, with numbers growing rapidly . There iAcoustic neuroma (vestibular schwannoma) is a nerve sheath tumour of the vestibulocochlear nerve. This tumour is of particular interest in relation to mobile phones because brain deposition of energy from RF fields from a mobile phone is mainly within a small area of the skull close to the handset, which includes the vestibular portion of the eighth cranial nerve where acoustic neuromas develop . Some prWe undertook six population-based case\u2013control studies within the Interphone collaboration to invesSix population-based case\u2013control studies of acoustic neuroma aetiology were conducted in the Nordic countries and the UK. The Nordic studies were conducted in Denmark nationwide, Finland excluding Northern Lapland and \u00c5land, the Southern and middle regions of Norway, and the Stockholm, G\u00f6teborg and Lund regions of Sweden. One UK study was conducted in the Thames regions of Southeast England, and the other in Southern Scotland, the West Midlands, West Yorkshire and the Trent area. The Danish and Swedish studies have been reported previously but withCases were identified through neurosurgery, neuropathology, oncology, neurology and otorhinolaryngology centres in the study areas. Lists of cases were also obtained from the appropriate population-based cancer registries to ensure completeness of ascertainment. Eligible cases were individuals diagnosed with acoustic neuroma between 1 September 1999 and 31 August 2004 (the exact dates within this period vary by centre) at ages 20\u201369 years in the Nordic countries, 18\u201359 in Southeast England, and 18\u201369 in the Northern UK, and resident in the study region at the time of diagnosis.Controls in the Nordic centres were randomly selected from the population register for each study area, frequency matched to cases on age, sex and region. In the UK, where there is no such accessible population register, controls were randomly selected from general practitioners\u2019 practice lists. Controls were subject to the same age and residence criteria as cases and had never been diagnosed with a brain tumour.Subjects were invited by letter to participate in the study. If no reply was received a repeat letter was sent or the subject was contacted by telephone. Each study was approved by the appropriate local ethics committee. Informed consent was obtained from all subjects at interview.Trained interviewers administered a personal interview, which was usually conducted at the subject's home, the hospital or another place convenient for the subject. For almost half the interviews in Norway, however, and a small minority elsewhere, where face-to-face interviews were not possible, interviews were conducted over the telephone. The interview was computer-assisted, with the answers being entered directly into the questionnaire program on a laptop computer, except in Finland, where answers were recorded on a paper copy of the questionnaire and later entered into the program.During the interview, participants were asked about use of mobile phones, and about models and makes of phones they had used. Subjects were shown photographs of the phones to aid their recall of this information.For each phone model, information was collected on the start and end date of use, the average amount of time of use and number of calls. If any substantial changes in use were reported that lasted for more than 6 months, usage information was also collected for these periods. Data were collected on mobile phone network operator, the extent of hands-free use, whether the phone had mainly been used in rural areas, urban areas or both, the side of the head on which the phone was mainly used, and whether the subject was left or right handed.These studies were conducted as part of larger case\u2013control studies on several types of intracranial tumour, with controls recruited for the entire set of cases. To increase statistical power, we used as controls for this analysis all participants interviewed as controls for the study who fitted the frequency matching strata of the acoustic neuroma cases.In the calculation of all exposure indices, except time since first use, any phone use less than 12 months prior to diagnosis was excluded because it was most unlikely to be aetiologically relevant. Time since first use was evaluated up to the diagnosis date. Regular phone use was defined as having used a mobile phone for at least 6 months more than 1 year prior to diagnosis. As controls were not individually matched to cases, we constructed a date equivalent to the cases\u2019 diagnosis date to truncate exposure for controls. We refer to the date of diagnosis or equivalent date for controls as the \u2018reference date\u2019. For controls this was obtained by constructing, for each centre, case-strata by single calendar year of interview and single year interval between diagnosis and interview (\u2018interview lag time\u2019). Controls interviewed in each calendar year were randomly allocated to strata of interview lag time, proportionally to the distribution of the cases in the same calendar year, to obtain a similar distribution of lag time as in the cases. The reference dates for controls were then calculated by subtracting the mean interview lag time in cases in that stratum from the interview dates of the controls.We calculated lifetime cumulative numbers of hours of phone use and numbers of calls by summing the calculated numbers of hours of use and numbers of calls for each usage pattern of each phone model. Risks for cumulative use were analysed with and without modification for reported use of headsets and/or hands-free sets in a vehicle, using methods described elsewhere (P<0.10). The statistical package STATA was used for these analyses were calculated as estimates of relative risk, and were obtained using conditional logistic regression, with strata of centre, region, 5-year age group at reference date and sex, and adjusted for highest educational level and combinations of interview year and interview lag time. Heterogeneity in results between centres was assessed with a log likelihood ratio test comparing a model with an interaction between centre and the exposure with a model with only the main effects. We also conducted parallel analyses using a two-stage random-effects model among cases and 51% (42\u201369%) among controls mailed, and 84% (71\u201393%) and 61% (55\u201376%), respectively, based on individuals definitely receiving the letters. The main reasons for nonparticipation were refusal , inability to contact subjects , no permission from the doctor to approach the subject and illness or death . In total, 684 cases were interviewed, of whom six were excluded because they had neurofibromatosis, leaving 678 cases in the analysis. The number of controls interviewed was 4340, of whom 3553 corresponded to matching strata of the cases and were included in the analysis. The numbers from each centre are shown in P=0.50), or for any of the other results, so centre-specific results are not presented, for brevity. Using a 5-year instead of a 1-year latency period, the relative risk in the pooled data set was 1.0 (95% CI: 0.8\u20131.3) (data not shown).The relative risk of acoustic neuroma for regular mobile phone use was 0.9 (95% CI: 0.7\u20131.1) . Centre-Risk of acoustic neuroma did not increase with increasing time since first regular phone use or lifetime number of years of use . For firThere was no trend in risk with lifetime cumulative hours of phone use or cumulative number of calls . The relRegular use of analogue, or of digital (GSM), phones showed no association with risk. There was no association with time since first use, number of years of use or lifetime hours of use for either type of phone . The relP=0.4), the risk 10 or more years after start of ipsilateral use was 1.5 (P=0.08), and the risk for 10 or more years of cumulative ipsilateral use was 1.8 (P=0.09).The relative risk of a tumour ipsilateral to the side of reported phone use in regular mobile phone users was 0.9 (95% CI: 0.7\u20131.1) using the method described by P trend=0.09). The relative risk for 10 or more years of use in these subjects was 1.5 (95% CI: 0.8\u20132.7) (data not shown).The relative risk of a tumour ipsilateral to handedness was 1.0 (95% CI: 0.8\u20131.3) in regular mobile phone users, and there was some suggestion of a trend in risk with increasing cumulative years of use by ipsilateral handed subjects . Reported side of use among cases was more often contralateral than ipsilateral to the tumour in short-term phone users and the proportion of cases reporting use on both sides was highest in long-term users.P Fisher's exact test=0.34). In regular analogue phone users, the proportion of right-sided tumours was 58.8% (P=0.13) and in regular digital phone users, it was 52.4% (P=0.5). However, there was a nonsignificant deficit of right-sided tumours in long-term phone users overall (48.8%) and in long-term analogue phone users (48.7%) (data not shown).Based on the distribution of preferred side of phone use among controls, if mobile phones cause acoustic neuromas, one might expect a higher proportion of tumours on the right than on the left side of the head among regular phone users. The proportion of right-sided tumours was 53.3% in regular users compared with 49.3% in never or nonregular users , reported bilateral phone use was more common than in short-term users. Secondly, recall bias could distort the findings in the opposite direction: cases might over-report ipsilateral use because they believe it caused their tumour. This bias would be expected not just to raise risks for ipsilateral use but also to reduce apparent risks for contralateral use . ContralWe analysed tumour laterality by handedness as an alternative marker of actual side of use. Handedness has the advantage that it is unlikely to be subject to recall bias, but has the disadvantage that not all people use the phone on their handed side. Risk of acoustic neuroma ipsilateral to side of handedness was nonsignificantly raised after 10 or more cumulative years of phone use \u2013 compatible with, but not giving strong support to, the results on reported side of use.No trend in risk was found in relation to cumulative hours of phone use or number of calls. These measures, however, are subject to substantial misclassification in recall from nonparticipants found a somewhat lower prevalence of phone use in those willing to give such data than in full participants (Confounding by known risks factors is unlikely to explain our results. Neurofibromatosis type II is associated with the tumour, but is rare, and we excluded cases with this condition from the study. High-dose ionising radiation is the only established environmental risk factor (In summary, our findings do not support an increased risk of acoustic neuroma in the first decade after starting mobile phone use. For 10 or more years of use, there was an increased risk of tumours ipsilateral to reported phone use, of uncertain interpretation. There is no consistent biological evidence that exposure to RF fields is implicated in the development of tumours nor has a potential aetiological mechanism been demonstrated . Overall"} +{"text": "Following intracardiac injection of 0.5\u2009mg\u2009kg\u22121 mTHPC, groups of five tumour-bearing animals were used for insitu light-induced fluorescence spectroscopy. Afterwards, the biopsies were taken and snap frozen for fluorescence microscopy. The presence of radioactivity in serum and tissues was determined after chemical digestion in scintillation fluid using a scintillation counter. For each analysed tissue, a good correlation was observed between the three evaluation methods. The highest fluorescence intensity and quantities of mTHPC were observed between 12 and 24\u2009h in liver, kidney, serum, vascular endothelium and advanced neoplasia. The majority of mTHPC was found at around 48\u2009h in smooth muscle and at 96\u2009h in healthy cheek pouch mucosa and early malignant lesions. The lowest level of mTHPC was noted in striated muscle at all times. No selectivity in dye localisation was observed between early squamous cell carcinoma and healthy mucosa. Soon after the injection, a significant selectivity was noted for advanced squamous cell carcinoma as compared to healthy cheek pouch mucosa or striated muscle. A significant difference in mTHPC localisation and quantity was also observed between striated and smooth muscle during the first 48\u2009h following the injection. Finally, this study demonstrated the usefulness of non-invasive in situ spectroscopic measurements to be performed systematically prior to photodynamic therapy as a real-time monitoring for each treated patient in order to individualise and adapt the light dosimetry and avoid over or under treatments.The aim of this study was to evaluate the pharmacokinetics of meta(tetrahydroxyphenyl)chlorin (mTHPC) on different tissues of interest in a hamster tumour model and to confirm our earlier animal studies on semi-quantitative fluorescence microscopy. The results obtained by three different evaluation methods were compared: British Journal of Cancer (2002) 87, 1470\u20131478. doi:10.1038/sj.bjc.6600651www.bjcancer.com\u00a9 2002 Cancer Research UK Photodynamic therapy with meta(tetrahydroxyphenyl)chlorin (mTHPC) has been shown in clinical trials to be a safe and effective treatment of early malignancy in the upper aero-digestive tract, the bronchi and the oesophagus involves the administration of a photosensitiser (PS) and the local illumination of the target with the light of a wavelength corresponding to an absorption peak of the administered drug. The absorption of the light energy by a PS induces a photochemical reaction leading to the damage of the illuminated tissue . After in situ measurements, the animals were sacrificed and the tissue samples were collected and prepared for fluorescence microscopy analysis. The animals that received labelled mTHPC were sacrificed at the same points in time as those for LIFS and FM, and tissues were sampled for quantification of 14C-mTHPC. All experiments were carried out following the recommendation of guidelines for the welfare of animals in experimental neoplasia weighing 140\u2013160\u2009g, housed at room temperature with 12\u2009h light/dark cycle were used in this study. Free access to food and drinking water was allowed throughout the experiments. The animals were divided into two groups (early and advanced SCC) prior to chemically induced carcinogenesis. Early and advanced SCCs were chemically induced by topical application of 5% oily DMBA solution in the hamster's left cheek pouch mucosa three times weekly during 10\u201312 weeks (early SCC) and 14\u201316 weeks (advanced SCC), respectively. The contralateral buccal pouch mucosa, which was not painted with DMBA, served as the healthy mucosa control (in situ (Tis), i.e. intra-epithelial with no invasion of the basement membrane, or micro-invasive (\u03bc-invasive SCC), i.e. with no invasion beyond the lamina propria of the cheek pouch mucosa. Between 6 and 9 weeks from the first DMBA application, different degrees of epithelial dysplasia were observed and invasive SCCs in the upper digestive tract and oesophagus . They were freshly prepared before use by dissolving them in a mixture of 30% (v\u2009v\u22121) polyethylene glycol 400, 20% (v\u2009v\u22121) ethanol and 50% (v\u2009v\u22121) H2O. mTHPC, 14C-labelled in the meso positions, was custom-synthesised by American Radiolabeled Inc. with a specific activity of 74\u2009\u03bcCi mmol\u22121 and a purity at 97%.The mTHPC, now known under the trade name Foscan\u00ae, as well as its \u22121 mTHPC or 14C-mTHPC, groups of five tumour-bearing animals were sacrificed at various points of time ranging from 4\u2009h to 8 days. Before sacrifice, non-invasive in situ LIFS measurements were performed in animals injected with non-labelled mTHPC on various organs such as the liver, kidney, healthy cheek pouch mucosa, early and advanced SCC, striated and smooth muscle (uterus). Then the blood was collected, the serum was separated by centrifugation and LIFS measurements of serum were carried out. Following this step, the various tissue samples were fast frozen and prepared for fluorescence microscopy analysis. The animals injected with 14C-mTHPC were sacrificed at the same times and similar tissues were sampled for 14C-mTHPC quantification.After intracardiac injection of 0.5\u2009mg\u2009kgin vivo LIFS was performed with an optical fibre-based spectrofluorometer described previously (M in water) was used to calibrate the intensity of the tissue fluorescence for changes in excitation intensity and light collection efficiency of the optical system. The absorption peak of mTHPC at 420\u2009nm was chosen as the excitation wavelength in order to be sensitive essentially to the uppermost layers of mucosa. The spectrum of the tissue autofluorescence was subtracted from the recorded spectrum. The height of the emission peak at 652\u2009nm of the mTHPC fluorescence was used as a relative measure for the PS quantification. The fluorescence intensity obtained in this way was reported in the corresponding figures in relative units for in vivo fluorescence (LIFS) and for ex vivo fluorescence microscopy (FM) . For each tissue of interest, we took 5\u201310 spectra by slightly displacing the fibre on the tissue in order to reduce the influence of local inhomogeneities. The results were presented as mean values of these measurements and are reported together with the corresponding standard deviations.Non-invasive The biopsies taken were fast frozen by contact with an isopentane slush and stored at \u221270\u00b0C prior to use. Tissue sections were prepared in the dark to avoid photobleaching. Five consecutive non-stained 5-\u03bcm thick tissue sections mounted on clean glass slides were prepared from each sample. From each frozen section, three images were recorded at three different parts of the slice to avoid photobleaching. The autofluorescence background substraction procedure was standardised on tissue slices from five uninjected animals. The relative fluorescence intensity was analysed using the public domain NIH Image 1.62 program. After recording the fluorescence images, the same slices were carefully removed and stained with haematoxilin and eosin (HE). An HE image was recorded at an identical position. The HE image was compared with the fluorescence image in order to determine the exact histological localisation of the PS fluorescence. We employed an Olympus BH-2 epifluorescence microscope with a filtered 100\u2009W mercury lamp as the excitation light source. Fluorescence images were detected with a cooled slow-scan 16-bit CCD camera . For excitation, an interference band pass filter 420DF30 and a dichroic mirror at 470\u2009nm were used. A long pass filter RG 630 was used to record the fluorescence of mTHPC. An interference band pass filter 560DF40 was used to record the tissue autofluorescence. The CCD camera was operated with an excitation shutter to avoid photobleaching. The system was controlled by an IBM-PC computer using the AT1 software (Wright Instruments), which processes 16-bit images. The localisation and intensity of the dye fluorescence was ascertained by subtracting the autofluorescence from the fluorescence image. Flat field correction was done using a fluorescent reference sample. The fluorescence intensity obtained in this way was reported in relative units (r.u.) in the corresponding figures.\u22121 mTHPC containing 55\u2009\u03bcCi of 14C-labelled mTHPC. Specific activity of 14C-mTHPC was calculated before the experiment. Each animal received 5\u2009\u03bcCi of 14C per 100\u2009g of body weight. Animals were sacrificed at the same points of time, as in the case of LIFS and FM. The blood was collected, and the serum was separated after centrifugation at 4000\u2009g for 10\u2009min. The various healthy tissues of interest and tumours were surgically excised, rinsed in saline, blotted dry and homogenised with scissors. The samples were weighed and digested in 0.2\u2009N NaOH (1\u2009ml per 200\u2009mg of homogenate) by shaking at 50\u00b0C overnight. After digestion, the total radioactive contents of each sample were determined in 4\u2009ml of scintillation liquid using a liquid scintillation counter and calculated as a per cent of total injected dose. Data were expressed in \u03bcg of extracted 14C-labelled mTHPC g\u22121 of tissue. Background radioactivity was measured using corresponding samples from three animals that were administered unlabelled mTHPC. It never exceeded 23\u2009d.p.m. g\u22121 of tissue and was similar to that of scintillation liquid alone.The time-dependent presence of radioactivity in serum and various tissues was determined after administration of 0.5\u2009mg\u2009kgin vivo or ex vivo methods, was determined according to a non-parametric Mann\u2013Whitney U-test (\u03b1\u2a7d0.05). The results were presented as mean values of these measurements and were reported together with the corresponding standard deviations (s.d.). For extraction data, the mean concentration of five samples and their s.d. were calculated for each time point. The data points were fitted with the following function (y=a exp (-bt) (1-exp (-ct))) for visual support.The significance of fluorescence intensities at various points of time, measured either by The relative fluorescence intensities or quantities of mTHPC measured by the three methods showed a heterogeneous PS distribution in different tissues at various times after administration. The autofluorescence level recorded on different tissues using LIFS or FM was fairly comparable between different animals. It ranged from 300 r.u. for striated muscle and 700 r.u. for fibro-elastic and collagen tissue of the lamina propria (data not shown). The mTHPC fluorescence and tissue distribution were within the same order of magnitude at all measured points. Figures 214C-labelled mTHPC between 12 and 24\u2009h after dye administration. The dye amount decreased almost to the background level 96\u2009h after administration A\u2013C.The fluorescence photomicrographs and the corresponding HE stains in A fairly similar kinetic pathway and no significant selectivity in mTHPC fluorescence or quantity between healthy mucosa and early SCC were observed by each of the three methods used . HoweverAll three methods used in this study showed low time-dependent mTHPC fluorescence and a negligible quantity in striated muscle A\u2013C. By c14C-labelled mTHPC gives an overview and confirms the pharmacokinetic data in several tissues of interest relevant for clinical PDT applications. The results presented here are in agreement with the FM study reported previously by our group was observed between advanced SCC and healthy mucosa. Several histopathological features observed in advanced SCC should explain in part this selectivity. As with many human or animal tumours, the DMBA-induced advanced SCC hamster model presents a large number of tumour vessels as compared to healthy mucosa, as well as a more-or-less severe peritumoural inflammatory reaction the tissue's optical properties of the excitation and emission light; (b) the fluorescence quantum yield of the PS used in its environment and (c) the geometry of the measurements. Indeed, if the fluorescence quantum yield of the PS changes with the environment of the latter, then the fluorescence signal and the corresponding PS concentration are no longer related in the same manner. Some of these points have been studied in detail by our group using mTHPC and its PEG-coupled form on a human xenograf colon carcinoma model. A good correlation between LIFS, quantification of radioactivity and improvement of in vivo tumour localisation reported by It is also well known that The measured values obtained by LIFS and FM as well as quantities of extracted mTHPC in different analysed tissues allow us to conclude that there is a highly reproducible \u2018quantitative correlation\u2019 between different tissues of interest. The ratios of nearly 3\u200a:\u200a1 between advanced SCC and normal mucosa and 7:1 between smooth and striated muscle were observed by all three methods.in situ. Our study has shown that any of the three methods should be suitable either when used together or separately for preclinical screening of new generation PS, as long as the therapeutic dose is a function of the mTHPC fluorescence or concentration.One of the major unresolved issues in PDT research is the development of optimal dosimetry tools useful for photosensitiser quantification 14C-labelled mTHPC, and that ex vivo FM measurements are in agreement with in vivo LIFS data. This permits us to develop concept of \u2018validation\u2019 of ex vivo techniques by in vivo LIFS measurements. The observed correlations support the hypothesis that non-invasive in vivo LIFS provide real-time information useful for adapting light dosimetry for each patient treated by PDT, in order to avoid under or over treatment and undesirable side effects. Therefore, LIFS for individualised in vivo dosimetry is a promising in situ technique, which needs to be improved upon by evaluating a large number of therapeutic results.Two main conclusions resulting from this study are that the LIFS measurements are confirmed by absolute concentration of We hope that individualised dosimetry will be relevant for a better understanding of the distribution and kinetics of new photosensitisers and thus will contribute to increasing the efficacy of clinical trials. This assumes that the best therapeutic results will be obtained, side effects will be diminished, and complications will be avoided."} +{"text": "The actions of glyceryl trinitrate (GTN) are the result of its bioconversion into NO; NO increases the intracellular concentration of cyclic guanosine monophosphate (cGMP), which produces pain modulation in the central and peripheral nervous system . In addto investigate the effects of GTN 4 hours after its administration on different experimental pain models in mice.Sixteen Swiss male mice were divided into 2 groups: control group (n=8) and GTN group . Assessment of locomotor activity (activity cage) and nociceptive tests (tail flick-TF and hot plate-HP) were performed before GTN administration and considered as baseline. Four hours after GTN injection, locomotor activity assessment and nociceptive tests were re-evaluated; afterwards, 20 \u00ce\u00bcl of 5% formalin were administrated into the upper right lip in order to assess formalin-induced orofacial pain. The results were compared with paired and unpaired Student\u2019s t test.GTN administration significantly increased HP latencies (p=0.0002) and showed a tendency towards increasing TF (p=0.056). A decrease in the locomotor activity was noted for both vertical movement activity (-78% p =0.001) as well as horizontal movement activity (-87% p=0.0001). GTN had no significant effect in influencing formalin-induced orofacial pain response.In our study GTN administration in mice exerted analgesic effects on acute nociception but had no effect on orofacial formalin pain. In addition, GTN decreased locomotor activity. Taken together, our results demonstrate that trigeminal pain is differently modulated by GTN as compared to nociception in TF and HP."} +{"text": "With recent advances in technology, deep sequencing data will be widely used to further the understanding of genetic influence on traits of interest. Therefore not only common variants but also rare variants need to be better used to exploit the new information provided by deep sequencing data. Recently, statistical approaches for analyzing rare variants in genetic association studies have been proposed, but many of them were designed only for dichotomous outcomes. We compare the type I error and power of several statistical approaches applicable to quantitative traits for collapsing and analyzing rare variant data within a defined gene region. In addition to comparing methods that consider only rare variants, such as indicator, count, and data-adaptive collapsing methods, we also compare methods that incorporate the analysis of common variants along with rare variants, such as CMC and LASSO regression. We find that the three methods used to collapse rare variants perform similarly in this simulation setting where all risk variants were simulated to have effects in the same direction. Further, we find that incorporating common variants is beneficial and using a LASSO regression to choose which common variants to include is most useful when there is are few common risk variants compared to the total number of risk variants. Genome-wide association studies have successfully identified many novel common risk alleles associated with complex traits. Yet these common genetic variants typically have small effect sizes and explain only a small portion of genetic variation for a certain trait. With the advances in whole-genome sequencing technology, data on rare variants have become increasingly available, and many investigators hope that rare variants will enhance our understanding of the biological mechanisms of human diseases and traits. Recently, novel statistical approaches have been proposed to assess the association between traits and rare variants, sometimes including and sometimes excluding nearby common variants. However, many of these methods were developed in a case-control framework and are not applicable to quantitative traits. In this paper, we implement several recently published approaches that can be applied to quantitative traits and compare their type I error and power using the Genetic Analysis Workshop 17 (GAW17) data set.Traditionally, investigators have evaluated genetic trait association by examining a trait\u2019s pattern among the genotypes of single-nucleotide polymorphisms (SNPs), with each SNP being analyzed independently of the other SNPs. Often a regression model is used to account for potential confounding covariates. However, this approach is not adequate for rare variants because the power is directly related to the minor allele frequency (MAF) and is especially decreased when the MAF is low. An alternative to testing each rare variant separately is to combine information across variants in a defined gene region. Intuitively, we can collapse information across different loci for rare variants by dichotomizing the existence of at least one rare variant (indicator method) or by counting the number of minor alleles of the rare variants (count method). These summary measures can then be assessed for association with the trait of interest using a regression or other statistical framework.To analyze rare and common variants simultaneously, Li and Leal proposedOne drawback to the CMC method is that the approach incorporates all common variants in the test statistic. Although this certainly retains any markers that are truly associated, including all common variants likely also retains many falsely associated markers. To balance the inclusion of noise and true signals, we modify the CMC analysis by first using least absolute shrinkage and selection operator (LASSO) regression to selecIn this study, we compare the power and type I error of three methods for collapsing rare variants in a gene region across three strategies to account for the common variants in the gene region.We use the simulated GAW17 data to calculate type I error and power. Genetic data from the 1000 Genomes Project was used to represent exome sequencing in the GAW17 data set. The sample consists of 697 unrelated subjects from seven populations and includes the original sex and age of each subject. Smoking status and traits are simulated across various association scenarios for 200 replicates. We calculate type I error for each method using quantitative trait Q4, a trait with no association to any of the genotypes. We estimate the null distribution for each method by pooling the results for trait Q4 from all gene regions and across all 200 replicates and derive an empirical significance threshold for each method from the empirical null distribution. We then calculate power for the nine genes associated with quantitative trait Q1 using the corresponding empirical significance threshold for each method. We define rare variants as SNPs with a MAF less than 0.005, 0.01, or 0.05. SNPs are assigned to a gene region using the gene assignment in the GAW17 SNP information file.F statistic by comparing the regression model containing genetic variables to the model without genetic variables. In each case, we adjust for sex, age, smoking status, and population group.To evaluate the association between Q1 and each gene region, we calculate an For the indicator method, we assign a dichotomous code to genes with rare variants by creating an indicator variable for each of those genes. The rare variant score is set to 1 if a subject has one or more rare variants within the gene region and 0 if a subject has no rare variants within the gene region.For the count method, we use a count code for each gene with rare variants. Thus for each gene with rare variants and for each subject, we count the total number of minor alleles for each gene region\u2019s rare variants and use the count as the rare variant score.Finally, for the data-adaptive sum test, we implement the method first introduced by Han and Pan . For eacWe use three methods for collapsing rare variants to test the association between the rare variant scores and quantitative traits without using common variants. We also perform analyses including both rare and common variants. For the two methods that include common variants, we apply the three methods for collapsing rare variants and thus arrive at six approaches to jointly test common and rare variants.F statistic as previously described to evaluate the genetic effect.For the CMC method, and for genes with rare variants only, we run a linear regression model using Q1 or Q4 as the outcome and the rare variant score and the previously listed covariates as the predictors. For genes with both rare variants and common variants we include each of the common variants in the model using an additive genetic model. Finally, we calculate the \u03bb) with the minimum CV error. To promote smaller models, we choose the largest \u03bb within 1 unit of standard error from the minimum \u03bb for our final LASSO model. The covariates, rare variant score, and common variants remaining in the model after LASSO selection are then taken to a linear regression model, and genetic association is tested using the F statistic, as previously described.For the LASSO method, we run a LASSO regression using the glmnet package in R ,6 for eaAs shown in Table For the gene regions with causal common variants Figure , includiUnless otherwise indicated, we focus our discussion on the results using the MAF cutoff of 0.01 to define rare variants.FLT1, which was simulated to have 8 out of 25 causal rare variants, but the increase in power is not seen in KDR, which was simulated to have 8 out of 14 causal rare variants, because all methods have 100% power for KDR would perform well when common variants in a gene region explained a relatively moderate to high proportion of the gene region association with the trait compared to rare variants. This is seen most readily with the genes ELAVL4, FLT4, and HIF3A is likely due to moderate linkage disequilibrium between common variants and causal rare variants.Interestingly, incorporating common variants also increased the power for genes that did not contain any causal common variants, albeit to a lesser extent than seen in the genes that did have causal common variants. This slight increase in power seen for ARNT and FLT1 have a ratio less than 1 . For FLT1, the simulated effect is too strong to differentiate between methods . However, in ARNT we do see a slight increase in power for the LASSO regression, as expected. The increase in power for the LASSO method to detect ARNT is small, probably because the ratio of causal common SNPs to total common SNPs is moderate, indicating that not much noise (only two SNPs) can be removed from the regression by using selection on the common SNPs. As the ratio decreases, we would expect to see a sizable increase in power for the LASSO method. Research using different simulation designs is needed to verify this expectation. When the ratio of common variants associated with the outcome to the total number of common variants is large, we expect the CMC and LASSO methods to perform similarly, as seen with the genes HIF1A and KDR, each of which has a ratio of 1.We expected the LASSO method to outperform the CMC method when the ratio of common variants associated with the trait to the total number of common variants was low. Many common variants not associated with the outcome cause noise in the CMC method, whereas LASSO regression is able to filter out some of the noise, leading to a more powerful result. Out of the four risk genes that contain common risk variants, only We also looked at the effect of varying the MAF threshold used to define rare variants on the type I error and power. We found that the type I error stayed consistent for the methods not including common variants and for the CMC method. For LASSO regression, the inflation seen in the type I error appears to decrease as the MAF cutoff increases. This is likely because fewer SNPs are defined as common variants as the MAF cutoff increases, and thus fewer common SNPs are undergoing selection by means of LASSO regression, decreasing the type I error. The changes in power because of the different MAF cutoffs are dependent on the particular characteristics of each gene. The power varies between MAF cutoffs only if the MAF of one or more risk SNPs within the gene is close to the threshold. Because the most appropriate MAF cutoff varies for each gene, a method that uses variable MAF thresholding may be more powerful .A common underlying assumption for the indicator and count methods is that the rare variants within a trait-associated gene have effects in the same direction. An even stronger assumption\u2014that all rare variants within a gene that are associated with a trait have the same effect size\u2014is also required for the count method. Although these assumptions are usually not satisfied, the GAW17 data were simulated to fulfill the first assumption in that all minor alleles are associated with an increase in Q1. The second assumption of equal effect size was not met by most of the simulated risk gene regions. The data-adaptive method also assumes that all causal rare variants within a gene have the same effect size, but unlike the simple count method, the data-adaptive sum test allows the direction of the effect to be different. In real data, the data-adaptive approach requires permutation to retain the appropriate type I error. Using permutation increases the computation expense but is necessary because the method biases the data in favor of association before the analysis. Thus, for the data-adaptive method, there is a trade-off between flexibility and computational expense. As discussed in the Methods section, we use an empirically derived significance threshold calculated from the null phenotype, Q4, to find the power for each method. Given the simulated null distribution, using the empirical significance threshold is computationally fast and efficient compared to permutation. In real data, where a null distribution is not available, permutation is likely the better alternative.In this study, we compared several published approaches and some modifications to analyze rare variants; however, there are other approaches available that we did not use in our comparisons. Capanu et al. applied Here, we have compared approaches that collapse the genotypes for all rare variants within a predefined gene region. An obvious expansion of these methods would be to use existing biological information, such as the functionality of each marker, to choose a subset of rare variants or to weight the rare variants. In addition, other information, such as rare variant transmission through a pedigree or linkage analysis, might also help to guide the weighting and grouping of rare variants within a gene region.The three methods compared here for collapsing rare variants\u2014indicator, count, and data-adaptive methods\u2014performed similarly in this simulation design in which all rare risk alleles were simulated to be in the same direction. Incorporating common variants in order to detect a gene region was useful when there were common causal variants within the gene. And finally, using a selection method, such as LASSO regression, to determine which common variants to include in the analysis was useful when there was more noise than signal as a result of common SNPs in the gene.The authors declare that there are no competing interests.HC carried out the indicator, count, and data-adaptive analyses. AEH carried out the LASSO analysis, and coordinated the study. YC carried out the CMC analysis. LAC and JD helped to conceive the study. CTL conceived the study. HC, AEH, YC, JD, and CTL participated in the design of the study and helped draft the manuscript. All authors read and approved the final manuscript."} +{"text": "FLT1 and the quantitative trait Q1 (P<10\u221230) in a fraction of the computation time of the other methods. The proposed family-based test has inconclusive results. The two methods provide a fast way to analyze simultaneously rare and common variants at the gene level while adjusting for covariates. However, further evaluation of the statistical efficiency of this approach is warranted.Rare variants may help to explain some of the missing heritability of complex diseases. Technological advances in next-generation sequencing give us the opportunity to test this hypothesis. We propose two new methods (one for case-control studies and one for family-based studies) that combine aggregated rare variants and common variants located within a region through principal components analysis and allow for covariate adjustment. We analyzed 200 replicates consisting of 209 case subjects and 488 control subjects and compared the results to weight-based and step-up aggregation methods. The principal components and collapsing method showed an association between the gene With recent technological developments in human genome sequencing, enormous numbers of rare single nucleotide variants can now be detected. This ability to measure rare variants allows researchers to investigate the multiple rare variant/common disease model, which may help to elucidate part of the missing heritability in studies of more common variants. However, the low frequency of these rare variants raises issues about how best to analyze them . In partTo overcome this problem, in this paper we propose two approaches, one for unrelated case and control subjects and one for families. The approaches aggregate rare and common variants into a single variable using a principal components analysis (PCA). These approaches are applied to case-control and nuclear family data sets from the simulations of Genetic Analysis Workshop 17 (GAW17) . Using tTo test jointly the effect of rare and common variants with a case-control sample, we adapted the CMC method )Tij is td ]1/2 has a normal distribution.Then the test statistic Another way to collapse variants is to weight them by allele frequency , a more P-value). Then all combinations of all other variants with P-value is selected as the best combination if its P-value is lower than the P-value of the test of r have a P-value higher than that of the previous rank (r \u2212 1).We also applied a separate data-driven collapsing method (the step-up [SUP] method) proposed by Hoffmann et al. . BrieflyP-value. For the WBC and SUP methods, we used an adaptive permutation procedure to compute the P-value . We allP-values obtained for the top 30 genes ranked by median P-value. All analyses were performed without knowledge of the true simulated model.Association at the gene-level was tested using the three methods on the 200 replicates of 209 case subjects and 488 control subjects and using the family-based PCC method on the 200 replicates of 194 nuclear families derived from the 8 extended families. In the case-control designs, we adjusted for age, sex, smoking, and ethnicity using the corresponding variable provided in the simulations . All analyses were undertaken using nonsynonymous variants only. Variants with a MAF below 5% were considered rare. In the results, we report the distributions of the P = 4 \u00d7 10\u221256).Of the 24,487 variants detected through sequencing of the mini-exome, 21,355 (87%) had a MAF less than 5% and 18,131 (74%) had a MAF less than 1%. A total of 9,433 variants (39%) had a variant allele observed only once among the 697 genotypes . The proportion of nonsynonymous variants significantly increased when MAF decreased: 46.8% of variants with MAF greater than 5%, 50.5% of variants with MAF between 1% and 5%, and 60.2% of variants with MAF less than 1% (FLT1 with the quantitative trait Q1 (median P<10\u221230) Figure and with) Figure . However) Figure . We also4 Figure . The firFLT1 is the gene with the highest median P-value (>10\u22126) and is the only gene that is statistically significant when a stringent Bonferroni correction for multiple testing is applied. In the top 30 list of genes discovered by the three methods using the case-control data sets, 13 genes are common to the three methods, 4 are common to the PCC and WBC methods, and 3 are common to the PCC and SUP methods. Moreover, the WBC and SUP methods have 10 genes in common that do not appear in the top 30 list of the PCC method. The PCC, WBC, and SUP methods ranked in their top 30 lists two, four, and five genes, respectively of the eight genes that had a true effect on Q1. Table P-values over the 200 replicates obtained by the three methods for the nine genes with an effect on Q1.The WBC and SUP methods lead to similar conclusions Figure . FLT1 isFLT1 had a P-value less than 0.0001), the PCC method took 1 s, the WBC method took 170 s, and the SUP method took 6,079 s.We compared the run times of the three approaches used on the case-control data sets. Testing the association of phenotype Q1 with all genes on chromosome 1 (where no markers were significant) took 4 s with the PCC method, 4 s with the WBC method, and 63 s with the SUP method, whereas on chromosome 13 with a moderate or small effect and nine rare variants, each with an odds ratios ranging from 1.17 to 2.9. Some of the other genes controlling Q1 are represented in the top 30 lists obtained with the three methods, but none reaches significance when correction for multiple testing is implemented.The case-control and family-based PCC methods provide a computationally fast method of simultaneously testing rare and common variants and adjusting for covariates. Using a case-control design, the results obtained with the GAW17 simulations were similar to the results obtained using the WBC and SUP methods. The PCC methods are also much faster than the other two permutation-based approaches. Comparing our results to the true model used for simulating the data, we found that genes VEGFA and FLT1) obtained with the family-based association test using the PCC method are in the list of genes controlling Q1, their test statistics are not significant. This can be explained by the simulation procedures used for the GAW17 family data sets, which consisted of eight extended families. Our analysis split the eight pedigrees into 194 nuclear families and assumed that the nuclear families were independent.Although the top two genes . Future In the PCA of each gene, the number of variables included was small (less than five in most cases) because rare variants were aggregated beforehand and only nonsynonymous variants were considered. However, for a larger number of variables, sparse PCA may provide a better alternative .In this paper, we propose a new method to analyze all variants within a predefined region based on the PCA of the collapsed rare variants term and all common variant terms. Applying this method to the simulated data sets of GAW17 provided results similar to two other methods with a greatly reduced computational time. However, evaluation of the statistical efficiency of these approaches is needed on a larger range of models and different family structures.The authors declare that there are no competing interests.RK, TJH and JSW conceived the study. RK and TJH performed the statistical analysis. RK drafted the manuscript. TJH and JSW revised the manuscript. All authors read and approved the final manuscript."} +{"text": "Early methods for estimating divergence times from gene sequence data relied on the assumption of a molecular clock. More sophisticated methods were created to model rate variation and used auto-correlation of rates, local clocks, or the so called \u201cuncorrelated relaxed clock\u201d where substitution rates are assumed to be drawn from a parametric distribution. In the case of Bayesian inference methods the impact of the prior on branching times is not clearly understood, and if the amount of data is limited the posterior could be strongly influenced by the prior.We develop a maximum likelihood method \u2013 Physher \u2013 that uses local or discrete clocks to estimate evolutionary rates and divergence times from heterochronous sequence data. Using two empirical data sets we show that our discrete clock estimates are similar to those obtained by other methods, and that Physher outperformed some methods in the estimation of the root age of an influenza virus data set. A simulation analysis suggests that Physher can outperform a Bayesian method when the real topology contains two long branches below the root node, even when evolution is strongly clock-like.http://code.google.com/p/physher/.These results suggest it is advisable to use a variety of methods to estimate evolutionary rates and divergence times from heterochronous sequence data. Physher and the associated data sets used here are available online at The divergence time of the two lineages is estimated to be 1966 . The same data set was reanalysed using BEAST and the mean substitution rate estimate was 2.1\u2009\u00d7\u200910\u22123 subs/site/year under a constant population size coalescent model and 2.72\u2009\u00d7\u200910\u22123 subs/site/year using the Bayesian skyride tree prior. Similarly the mean root age estimate was 1978 under a constant population size coalescent model and 1998 using the Bayesian skyride method and hence considerably more recent that the \u2018true\u2019 estimate.We analysed the influenza B virus data set using Physher under both non-clock and the discrete clock failed to recover the rate shift pattern that Drummond and Suchard first idNe) scaled by the generation time (\u03c4), and since no assumptions are made about the generation time in our analyses we refer to this composite parameter (\u03b8\u2009=\u2009Ne\u03c4) as the relative genetic diversity. The relative size function can be constant, distributed according to a parametric distribution or any function of time. Importantly, in data sets that contain several lineages that evolved concurrently with incomplete sampling of extant taxa (e.g. influenza B virus data set) these priors can produce spurious results. Using simulations it was shown that relaxed clock models can greatly underestimate the age of the root when lineages exhibit strong rate heterogeneity [With limited data, Bayesian-based inference should perform better than the maximum likelihood method if one can use an appropriate prior on the branching times. Unfortunately, it can produce posterior distributions that are heavily influenced by priors, as is probably the case in our simulation study where no coalescent model might be appropriate. The two main classes of prior that are currently used are coalescent processes and the birth-death process. The coalescent process is a function of the effective population size (ogeneity . In the 2n-2), whereas in the local clock setting the length of each individual is equal to the number of local clocks being investigated. In the influenza A virus data set, we started the GA with ten local clocks. Discrete optimization is notoriously more difficult than its continuous counterpart because functions are generally not convex and because a modification of only one of the variables can significantly change the likelihood of the model.Genetic algorithms appear to be well suited for optimizing the allocation of discrete clocks but their efficiency in the local clock problem is arguable, probably due to the different solution encodings. In the discrete case the individual size is the number of branches and root age (a) were inferred using BEAST for 50 replicates using a birth-death model prior on the phylogeny. Intervals that do not include the true value (blue line) are shown in red.Click here for fileLinear regression estimates of substitution rate and root age in 50 simulated data sets. Point estimates of the nucleotide substitution rate (a) and root age (b) using a root to tip linear regression of the expected number of substitutions per site and sampling dates.Click here for file"} +{"text": "Children get involved in social categorization. Thus, they are able to stigmatize peers as well as to show in-group favoritism theorized by Tajfel and Turner . Moreove According to Tajfel and Turner , social Yee and Brown stated nMoreover, Aboud , 2003 suStigma, like categorization is context-dependent. Therefore, in different cultures and at different times, stigmatization may affect different groups. Today, in our Western society, obesity appears as one of the most stigmatizing and least socially acceptable conditions among children were searched using the following 8 terms \u201cvictimization,\u201d \u201canti-fat bias,\u201d \u201canti-fat attitude,\u201d \u201cstigmatization,\u201d \u201cstigma,\u201d \u201cdiscrimination,\u201d \u201cprejudice,\u201d \u201cstereotype\u201d combined with \u201cobese children\u201d or \u201cobesity AND children.\u201d Databases were investigated limiting the search to psychology and social work but excluding any restrictions about articles' year of publication.Additional manuscripts were derived from the bibliography's analysis of eligible articles.Studies were included in the review if: (1) the age of participants didn't exceed 11 years oldAs described in Figure n = 11), 7 in the USA and 1 in New Zealand.As it can be inferred from Table n = 24 children, Counts et al., n = 1, 861 children, Koroni et al., The papers identified produced a sample size ranging from very small .However, this unexpected result appears mitigated by the conclusions reached by the two studies that added the perceived body size information to the BMI data. Interestingly, both Holub and KornIn particular, Kornilaki observedSimilar conclusions were found by Holub and statThe search revealed 13 papers addressing the relationship between anti-fat stigma and sex . In fact, in the adjective attribution task, both Cramer and Steinwert and KornThree among the possible future developments of this mini-review, which demonstrates the limitation of using a small number of articles written in different time periods, could be: (1) expand the research; (2) consider the effect of the research contexts on the results; (3) more deeply analyze the methodologies used and their impact on the results.RD handled the global structure of the work and the literature selection. LC collaborated in the process of literature selection.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Diabetic foot ulcers are one disabling complication of diabetes mellitus. Pirfenidone (PFD) is a potent modulator of extracellular matrix. Modified diallyl disulfide oxide (M-DDO) is an antimicrobial and antiseptic agent. To evaluate efficacy of topical PFD\u2009+\u2009M-DDO in a randomized, double-blind trial versus ketanserin in the treatment of noninfected chronic DFU. \u03b1, COL-4, KGF, VEGF, ACTA2 (\u03b1-SMA), elastin, fibronectin, TGF-\u03b21, TGF-\u03b23, HIF-1\u03b1, and HIF-1\u03b2. Patients received PFD\u2009+\u2009M-DDO or ketanserin for 6 months. Relative ulcer volume (RUV) was measured every month; biopsies were taken at baseline and months 1 and 2 for histopathology and gene expression analysis for COL-1 Reduction of median RUV in the PFD\u2009+\u2009M-DDO group was 62%, 89.8%, and 99.7% at months 1\u20133 and 100% from months 4 to 6. Ketanserin reduced RUV in 38.4%, 56%, 60.8%, 94%, 94.8%, and 100% from the first to the sixth month, respectively. Healing score improved 4.5 points with PFD\u2009+\u2009M-DDO and 1.5 points with ketanserin compared to basal value. Histology analysis revealed few inflammatory cells and organized/ordered collagen fiber bundles in PFD\u2009+\u2009M-DDO. Expression of most genes was increased with PFD\u2009+\u2009M-DDO; 43.8% of ulcers were resolved using PFD\u2009+\u2009M-DDO and 23.5% with ketanserin. PFD\u2009+\u2009M-DDO was more effective than ketanserin in RUV reduction. Around 415 million of people in the world are living with diabetes mellitus type 2, representing 8.3% of the world population as of 2015 ethyl]-1H-quinazoline-2,4-dione) is a quinazoline derivative, a serotonin antagonist of 5-HTR2, with no agonistic properties .KTS has been used in several clinical trials for treating diabetic foot ulcers. Janssen et al. used 2% KTS to improve wound healing in different kinds of patients, including 6 patients with diabetes. They reported that 36% of ulcers healed in the KTS group as opposed to 15% in the placebo group at 8 weeks . In SwedMore recently in Mexico, Mart\u00ednez-de Jes\u00fas et al. tested 2% topical KTS in diabetic foot ulcers. They reported that an 87% reduction of ulcer area at 12 weeks contrasted with 63% in the placebo group . QuatresNCT02632877 before participant enrollment. All the participants provided their written informed consent.The study was designed as a single-center, randomized, double-blind, active-controlled trial. Patient enrollment took place at the Dr. Valent\u00edn G\u00f3mez Far\u00edas Regional Hospital pertaining to the ISSSTE system in Guadalajara, Mexico, between 2014 and 2015. The clinical trial was approved by The Ethical Review Board of the Dr. Valent\u00edn G\u00f3mez Far\u00edas Regional Hospital, performed in accordance with the Ethical Principles of the Declaration of Helsinki, and took into account the Good Clinical Practice guidelines. The trial was registered at ClinicalTrial.gov under registration ID: Patients with a previous diagnosis of DM2 according to the ADA criteria were enrolled. All of them were under pharmacological treatment for glycemic control, had at least one-foot ulcer classified as A-I following the University of Texas Diabetic Wound Classification (UTDWC), and had at least a 2-month duration. Patients were randomly assigned to receive one of two interventions.2 persisting for a minimum of 2 months.Inclusion criteria are as follows: men and women with diabetes mellitus type two, over 18 years of age, and with a DFU grade A-I following the University of Texas Diabetic Wound Classification\u2009>\u20091cmExclusion criteria are as follows: patients who required either direct (graft) or indirect revascularization procedures during the study, major large-vessel and peripheral arterial disease, grade III insufficiency of the deep venous system assessed by means of the ankle-arm index of 0.9 to 0.7, and autoimmune disease; pregnancy or breast-feeding; inability to attend the monthly evaluations; and patients who, within a period of less than seven days, had applied any topical application to the ulcer, whether pharmacological or not, apart from the water and soap used in the cures.Elimination criteria are as follows: participants with <80% scheduled medical appointments, absence from more than 20% of the visits, with serious side effects or allergic reactions, severe to moderate pain, erythema, edema, and/or necrosis were removed from the study, though all of them were considered for the final statistical analysis . CompliaEligible participants were enrolled and randomly assigned to experimental and active control groups using a random number table. Patients, medical doctors ascribed to the hospital who were in charge of clinical care, the statistical evaluator, and histopathological evaluator were blinded. Concealment was opened at the end of the study.Kitocell-Q\u00ae, a drug in the form of a gel that combines PFD 8% and M-DDO 0.016%, was obtained from Cell Pharma, SA de CV while Sufrexal\u00ae (2% KTS) was acquired from Janssen Pharmaceuticals . Additionally, patients in both groups received integral and conventional care according to the International Consensus on the Diabetic Foot . ConventRelative ulcer volume (RUV) was assigned as a primary outcome. Immediately before the intervention, a physician measured patients' diabetic foot ulcer (DFU) using a flexible sterile ruler. Criteria to measure the size of the wound consisted of assessing the longest, widest, and deepest sides. A photograph was taken later and the assigned sides of DFU were marked on it and kept in the file to serve as a guide to the physician for the following evaluation. Measurements were performed at baseline, 4, 8 12, 16, 20, and 24 weeks, and a photographic record was taken at the same time as a guideline.L\u2009=\u2009the longest extension of DFU in centimeters, W\u2009=\u2009the widest extension of DFU in centimeters, and D\u2009=\u2009the deepest extension of DFU in centimeters.RUV was obtained by multiplying the measure of the longest, widest, and deepest extension of the ulcer, according to the formula:n\u2009=\u2009median of relative ulcer volumes at month 1, 2, 3, 4, 5, or 6 and RUV0\u2009=\u2009median of relative ulcer volumes at baseline.In order to evaluate the reduction percentage of DFU, baseline RUV for each patient was normalized to 100%. Reduction of median RUV was assessed for each month according to the formula:3 were taken from the middle of ulcers at the beginning of interventions and at 1 and 2 months of treatment to accomplish the secondary outcomes. We did not take biopsies beyond three months in order not to reopen healed ulcers and not put patients at risk. Wound healing was defined in this trial as the percentage of healed wounds at the end of the study (24 weeks).Secondary outcomes were wound healing, wound healing histopathological score, molecular assessment of genes involved, and safety and tolerability. Biopsies of approximately 125\u2009mmThe wound healing histopathological score was assessed to evaluate the healing process of DFU. It was assessed according to a previous report in the hA high score represents an improvement of healing and a low score a delay thereof. Parameters assessed in wound healing histopathologic score were as follows: amount of granulation tissue , inflammatory infiltrate , collagen fiber orientation , the collagen pattern , the amount of early collagen , and the amount of mature collagen . The total healing score was calculated by adding the scores of individual criteria, when the score was directly proportional to wound healing.\u03b11), transforming growth factor beta 1 (TGF-\u03b21), transforming growth factor beta 3 (TGF-\u03b23), vascular endothelial growth factor (VEGF), elastin, fibronectin, alpha-smooth muscle actin (\u03b1-SMA), keratinocyte growth factor (KGF), hypoxia-inducible factor 1-alpha (HIF-1\u03b1), and hypoxia-inducible factor 1-beta (HIF-\u03b2) by real-time quantitative reverse transcription (qRT-PCR). Gene expression was assessed based on biopsies and was compared with baseline, as explained below.Molecular assessment was performed to elucidate the effect of treatments on the molecules involved in wound healing over time. Molecular assessment consisted of the evaluation of gene expression for type IV collagen (Col-4), type I collagen alpha 1 was taken using a scalpel blade from the middle of the ulcer. Biopsies were taken at baseline, 1st month (4 weeks), and 2nd month (8 weeks) if the ulcer had not healed. Photographs were taken at all times and relative ulcer volume was measured monthly until complete healing. Relative ulcer volume (RUV) was calculated by measuring the longest, widest, and deepest ulcer side with a sterile flexible graduated ruler, as described previously. Blood samples were obtained at the baseline and at the end of the study to measure biochemical tests and to evaluate any side effects of KTS or PFD\u2009+\u2009M-DDO in biochemical parameters.The ulcer area was washed with aseptic solution and a biopsy (100\u2013150\u2009mm3) were taken using a scalpel blade from the middle of the ulcer at baseline, 1st month, and 2nd month after treatment with either PFD or ketanserin. Anti-human smooth muscle actin (\u03b1-SMA) antibody was obtained from Boehringer , histological-processed wound sections were deparaffinized, and endogenous activity of peroxidase was quenched with a solution 0.03% H2O2 in methanol. The tissue was incubated with a 1/100 dilution of a monoclonal mouse anti-human \u03b1-SMA antibody. Anti-mouse peroxidase-labeled secondary antibody was revealed with diaminobenzidine, and the tissue was counterstained with Harris's hematoxylin. Twenty random fields were evaluated for quantification at 200x magnification. The immunohistochemical positive area was measured with an automated analyzer . Data are expressed as percentage of the \u03b1-SMA stained area.Biopsies . Gene expression was normalized against the housekeeping gene 18S. Relative quantification was achieved by using the 2\u2212\u2206\u2206CT method and KTS 11 (10\u201313) [mean (IQR)], (= 0.621) , Table 2= 0.621) .p = 0.591) and KTS did not show any change 11 (10\u201312.5) (p = 0.648) and no significant difference was observed (p = 0.273) (= 0.273) . Nonethe= 0.273) and abun= 0.273) . Thus, gp = 0.023) in the healing score (p = 0.516) over the first month after KTS treatment, and significant differences were found between treatments (p = 0.050) ] .We then searched for the molecular mechanisms involved in this accelerated wound healing in diabetic foot ulcers induced by PFD\u2009+\u2009M-DDO. The results shown in A significant, in some cases dramatic, increase in the expression of these genes at the first month and the second month in patients from the PFD\u2009+\u2009M-DDO group was observed. Specifically, molecules involved in the synthesis, formation, and organization of granulation tissue or extracellular matrix were overincreased.\u03b1 increased its expression 50-fold at month 1 (p = 0.020) and 69-fold at month 2 (p = 0.050) compared with baseline values and showed a significant difference with the KTS group in both months Additionp = 0.001), which had a statistical difference versus KTS (p = 0.020). TGF-\u03b21 had a significant improvement in the second month (p = 0.036) in the PFD\u2009+\u2009M-DDO group with respect to baseline value, presenting a statistical difference versus the KTS group (p = 0.018). TGF-\u03b23 had an increase of 15.6-fold in the second month (p = 0.013) in the PFD\u2009+\u2009M-DDO group and showed a significant difference against the KTS group (p = 0.022) (COL-4 had a significant increment with PFD\u2009+\u2009M-DDO in the first month (= 0.022) ; this isp = 0.019), which codifies for alpha-smooth muscle actin (\u03b1-SMA), a contractile protein.Remarkably, PFD\u2009+\u2009M-DDO patients showed a 110-fold increase in ACTA2 gene expression and 2 (p = 0.007) and presented a significant difference with KTS (p = 0.031) in month 2.KGF was also overincreased in PFD\u2009+\u2009M-DDO treatment at months 1 , with a statistical difference from the KTS group (p = 0.041); although no differences were found in the expression of HIF-1\u03b2 and VEGF between groups, a clear tendency for increased VEGF was noted.HIF-1Gene expression in the two groups compared with baseline correlates with ulcer resolution. However, it was clear from our observations that patients treated with PFD\u2009+\u2009M-DDO expressed those molecules in a faster and stronger fashion, which is consistent with the fact that 43.8% of ulcers in PFD\u2009+\u2009M-DDO patients fully healed at three months.\u03b1-SMA, a highly contractile protein. PFD\u2009+\u2009M-DDO-treated patients showed a substantial increase of this protein as compared with patients administered with KTS.No serious adverse events were detected in the PFD\u2009+\u2009M-DDO group. 23.7% of the patients reported tolerable pain in the ulcer that was mentioned during the course of treatment, but it was not related to the medication.In the KTS group, 28.2% of the patients reported tolerable pain. Of these, only one patient presented pain probably because of the drug administered. Intolerable pain was detected in one of the patients immediately after receiving the drug, which caused his elimination. The aforesaid pain subsided after ceasing application.All patients included in this clinical trial had similar demographics and biochemical profiles that were not modified after receiving the treatments.KTS has been tried for diabetic foot ulcer treatment with proven effect on decreasing ulcer area between 59 and 60% \u201329, thouIn this same context, due to the anti-inflammatory and antioxidant role and its dynamic effect on matrix extracellular synthesis, pirfenidone is a potential drug for diabetic foot ulcer treatment.Our results in this double-blind, randomized, active-controlled protocol are consistent with the abovementioned Janka-Zires et al. report. They used pirfenidone as a pharmacological treatment of noninfected chronic diabetic ulcer for 16 weeks and reported a 93% reduction of ulcer size when compared to control with conventional treatment of only 21.8% . Our finThese clinical results correlated with the wound healing histopathological score where PFD\u2009+\u2009M-DDO dramatically reduced inflammatory infiltrate, which is consistent with previous findings in different tissues . Further\u03b21 and HIF-1\u03b1 downregulation [\u03b1 and oxidative stress [It is known that neuropathy, peripheral vascular disease with ischemia, coupled with hyperglycemia and infection are associated with a failure to heal and possible amputation . All thegulation , 8, highe stress , delayede stress , and impIn this context, a major amount of healed ulcers from the second month up to six months of treatment in the PFD\u2009+\u2009M-DDO group could be influenced by the guided gene expression of important genes.\u03b23 and TGF-\u03b21 in turn promote fibroblast migration and granulation tissue/extracellular matrix maturation [\u03b1, which eventually will form collagen bundles where fibroblasts will migrate in the proliferation phase [\u03b1-SMA is an indicator of the transformation of fibroblast to myofibroblast since it promotes cell contractility, which would help to close the wound and maintain its structure [\u03b1-SMA in our patients treated with PFD clearly correlates with the expression of its cognate mRNA and further sheds light on the notion that this molecule is acting to \u201creinforce\u201d the resilience of wound closure.The increased expression of KGF in PFD\u2009+\u2009M-DDO treatment could be related to faster reduction of RUV since this powerful mitogen of epithelial cells is very important in wound healing . TGF-\u03b23 turation . PFD\u2009+\u2009Mon phase . \u03b1-SMA itructure . The expOn the other hand, it is critical to consider using an antiseptic agent together with a wound healing \u201cregenerator,\u201d given that infections are a major cause of the occurrence of amputations in diabetic patients , consideThe insights gained from this type of assessment are expected to facilitate the development of novel therapies by stratifying their specific contributions to the wound healing process in time and in a stage-specific manner."} +{"text": "Podoconiosis (mossy foot) is a neglected non-filarial elephantiasis considered to be caused by predisposition to cumulative contact of uncovered feet to irritative red clay soil of volcanic origins in the tropical regions. Data from structured observational studies on occurrence of Podoconiosis and related factors are not available in Kenya.Wuchereria bancrofti microfilaria and (3) determining the concentration of six elements and properties in the soil known to be associated with Podoconiosis. A structured questionnaire was used to identify possible risk factors. Univariable and multivariable Poisson regression analyses were carried out to determine factors associated with Podoconiosis. Thirteen participants were clinically positive for Podoconiosis giving an overall prevalence of 3.4%. The prevalence ranged between 0% and 18.8% across the five villages. Molecular assay for W. bancrofti test turned negative in the 13 samples. The following factors were positively associated with the Podoconiosis prevalence (P<0.1) in the univariable analyses: (i) age, (ii) gender, (iii) education level, (iv) frequency of washing legs, (v) frequency of wearing shoes, (vi) soil pH, and (vii) village. Unexpectedly, the concentration of soil minerals previously thought to be associated with Podoconiosis was found to be negatively associated with the Podoconiosis prevalence (P<0.1). In the multivariable analyses, only frequency of wearing shoes and village turned out significant (P\u22640.05). By modeling the different soil mineral concentrations and pH while adjusting for the variable frequency of wearing shoes, only iron concentration was significant and in the negative dimension (P\u22640.05). However, controlling for Iron, Aluminum concentrations turned significant.To establish the occurrence and aspects associated with Podoconiosis, a cross-sectional survey was implemented in an area located within 30 km from the foot of volcanic Mount Longonot in the Great Rift Valley in Kenya. Five villages and 385 households were selected using multistage and systematic random sampling procedures respectively during the survey. Podoconiosis was determined by triangulating (1) the clinical diagnosis, (2) molecular assaying of sputum samples to rule out This study has pointed to a hitherto unreported occurrence of Podoconiosis cases and has contributed to the baseline knowledge on the occurrence of Podoconiosis in Kenya. Consistent with many studies, wearing shoes remain an important risk factor for the occurrence of the disease. However, our findings are inconsistent with some of the hitherto postulations that associate Podoconiosis prevalence with certain minerals in the soil in other regions in Africa. These findings provide new beginnings for the cross-disciplinary research of Podoconiosis in environmental health, socio-ecology and ecological niche and geo-spatial modeling and prediction. Podoconiosis is a neglected disease in the tropical regions of the world considered to be caused by prolonged contact of uncovered feet to irritant particles found in red clay soil from volcanic origins. The disease presents like filarial elephantiasis. Data from observational studies from Kenya are not available. We conducted a cross-sectional household survey to establish the prevalence and aspects related with Podoconiosis at the foot of Mount Longonot in the Great Rift Valley in Kenya. Podoconiosis was determined by combining results of clinical diagnosis, ruling out filarial elephantiasis in clinically positive Podoconiosis patients using molecular techniques and determining the concentration of elements and properties in the soil known to be associated with Podoconiosis. A structured questionnaire was used to identify possible risk factors. Out of 385 study participants, thirteen were clinically positive for Podoconiosis giving an overall prevalence of 3.4%. Molecular tests for filarial elephantiasis turned negative in the 13 participants. Factors that were associated with Podoconiosis prevalence were age, gender, education level, and frequency of washing legs, frequency of wearing shoes, soil pH and village. The concentration of soil minerals previously thought to be associated with Podoconiosis was found to be negatively associated with the Podoconiosis prevalence. However, the final analyses found frequency of wearing shoes, iron and aluminium as possible predictors of Podoconiosis occurrence in the study area. This is the first structured observational study to report occurrence of Podoconiosis in Kenya. Although some of our findings are inconsistent with some previous reports about the association of Podoconiosis and certain minerals in the soil, this study offers new beginnings for the cross-disciplinary research of Podoconiosis in fields known to influence occurrence of the disease including environmental health, socio-ecology and medical geographical approaches and predictions. Podoconiosis is a neglected geochemical, non-filarial, non-infectious lymphodema of the lower limb . In AfriThe prevalence of the disease is reported to vary considerably from country to country. For instance, reports show an average burden of 1% (range: 0% to 2.1%) in Burundi and 0.6%Numerous structured studies investigating the role of individual-level risk factors have been carried out in Ethiopia. Gender, age, marital status, feet hygiene, level of job skills/employment, education level and house floor type have been associated with risk of Podoconiosis. Being older , female,http://eusoils.jrc.ec.europa.eu/content/soil-map-soil-atlas-africa) in Africa.Podoconiosis has been reported to be prevalent in highlands of tropical Africa, Central America and Northwest India all characterized by certain soil types 21]. Alt. Alt21].Moreover, Podoconiosis is occupational in nature with familial inclination in addition to a deficiency in feet hygiene. Podoconiosis occurs among farmers and other occupational groups whose feet remain uncovered and exposed to clay soil originating from alkaline volcanic rock . Small pPodoconiosis is associated with a host of disease burdens. The quality of life is substantially reduced 27]. Tho. Tho27].Diagnosis of Podoconiosis is not straightforward. To rule in Podoconiosis, geographical location, history, clinical findings and confirmed absence of microfilaria or its antigen on immunological card test are used. Geographically, it has been found that Podoconiosis is prevalent in populations who live at high altitudes (>1000 metres above sea level). Clinically, Podoconiosis is an ascending and commonly bilateral non-filarial elephantiasis though asymmetric and rareThis paper reports a cross-sectional household survey implemented to establish the burden and factors related with Podoconiosis occurrence. This information will be useful to the health administrators and humanitarian agencies responsible for developing and implementing targeted, appropriate and effective public health intervention strategies. This is the first field-based observational survey that has acknowledged the occurrence of Podoconiosis in Kenya.2. Mount Longonot is located within Nakuru County, Kenya which is an agriculturally rich county. It is also a strato-volcano situated southeast of Lake Naivasha in the Great Rift Valley of Kenya in Africa. The county generally has an elevation of 2776m 43].[43].wore.[43].worworedas . VariatiConsistent with published findings, frequency of wearing shoes and soil mineral concentration (particularly iron) were linked with burden of Podoconiosis in this study. These variables show effect after a long period of exposure to reactive alkaline volcanic soils 11]. Min. Min11].However, under univariable analyses, increasing age, female gender, low education level, low frequency of wearing shoes, low frequency of washing legs, high soil pH and some soil elements showed a statistically significant relationship with the prevalence of Podoconiosis.Age, gender and education level are individual-level variables widely reported to be associated with prevalence of Podoconiosis. Increasing age is expectedly associated with occurrence of Podoconiosis most likely because age is a proxy for exposure time (cumulative exposure). Previous work reported that the disease mostly develops in the agriculturally productive ages of 16 to 54 years which could explain the cumulated exposure 32]. Con. Con32].In this study, formal education was associated with decreased risk of Podoconiosis. This shows similarity with research done in Ethiopia which reFoot hygiene practices including low frequency of wearing shoes and low frequency of washing legs were associated with increased prevalence of Podoconiosis. This is consistent with known strategies of Podoconiosis prevention. At early stages, one is capable of managing Podoconiosis and stop further disease development by regular cleaning of feet and consistent wearing of shoes . HoweverIn this study, increasing soil pH was associated with increasing prevalence of Podoconiosis and some soil elements showed a statistically significant relationship with the prevalence of Podoconiosis. Our findings are consistent with observations that the disease occurs as a result of exposure to alkaline clay soils 47]. In . In 47].Diagnosis of Podoconiosis was based on differential diagnosis as done in other studies 20]47].[47].20][.[47].20]This study is not without limitations. The study utilized a cross-sectional study design. These findings therefore need to be interpreted with caution and further and more intensive cross-disciplinary studies are needed to authenticate them. This is because cross-sectional studies are subject to problems of undocumented confounders operating at different scales. Furthermore, the time-relationship between the factors and the disease is not known with such a design. However, in this case, a cross-sectional study design was appropriate as the disease is mostly non-fatal and the associated variables do not have a clear time-onset. Secondly, Podoconiosis is thought to cluster within families/households, partially associated with common environmental exposure or shared genetic susceptibility. This approach could have underestimated the prevalence in the region. However, ours was a cross sectional study in the region with a descriptive purpose with respect to the outcome and a set of risk factors. Additionally, we did not count any additional Podoconiosis suspected cases in the households that were recruited for the study. Future research in the region should investigate the existence of disease clustering at the household and the spatial level to increase the understanding of the disease mechanisms. Distinct study designs particularly case-control studies are also warranted as they are appropriate for studying rare conditions or diseases such as Podoconiosis.This study reports the occurrence of Podoconiosis in the Mt Longonot area in the Rift Valley in Kenya. Soil Iron and Aluminium concentrations and feet hygiene were identified as possible predictors of Podoconiosis occurrence in Kenya. Because of the possible spatial restriction of the exposure, external validity to other areas may not be feasible and, therefore, we restrict our conclusions to the target (source) population. The findings in this study gives the baseline knowledge regarding the occurrence of non-filarial elephantiasis in Kenya and providing a fresh beginning in cross-disciplinary research of Podoconiosis using socio-ecology, environmental health and ecological niche and geo-spatial modeling and prediction.S1 Checklist(DOC)Click here for additional data file."} +{"text": "China launched a health care reform policy due to the aging population and rapid urbanization. However, emergency overcrowding is not improved. We assessed the laboratory efficiency of emergency department (ED) in Shanghai hospitals.We recorded the turn around times for processing laboratory biomarkers to assess laboratory efficiency at 17 EDs in national/regional hospitals. We compared TAT between national and regional hospitals and between central and ED laboratories to analyze the relationship between the laboratory efficiency and the ED overcrowding.P < 0.05). The TAT were longer (65 min (53\u201385 min)) at ED labs than (60 min (42\u201383 min)) at central labs (P < 0.05), independent of the hospital tier and working period. We discovered that only 9% of investigated samples at Tier II EDs and 5% at Tier III EDs were assayed by point\u2010of\u2010care (POC) instruments.All the participating hospitals have an emergency laboratory. The median TAT for c\u2010TNT was 61 min (46\u201376 min) at regional EDs compared with 64 min (46\u201387 min) at national EDs; therefore, the TAT at regional EDs were more efficient (Our TAT level is approaching the recommended international standard. However, the TAT evaluation from ED laboratories demonstrates that their existence does not decrease the waiting time for laboratory reports compared to central laboratory. Thus, they have not yet approached a level to share the burden of the ED overcrowding. Further arrangement should be assigned to separate the function of emergency laboratory and central laboratory. It is worth deploying the POC assay in the ED, which will save twice the TAT level. The idea of evaluating routine laboratory efficiency by TAT at ED is fast, convenient, although it does not represent the general level of laboratory efficiency. The Chinese government launched a reform plan in 2009, which included spending approximately US$125 billion to fund a public health provision In this study, we questioned whether the ED overcrowding is due to the low laboratory efficiency. Although an emergency laboratory has been reconstructed to relieve the pressure of central laboratory and to improve the laboratory efficiency at emergency, we had no idea whether the ED laboratory functions as estimated because there was no statistical prereform report. We then designed this study to evaluate the turnaround time (TAT) of routine biomarkers. We expected to know whether TAT from emergency laboratory is faster than those from central laboratory. We also compared the postreform laboratory efficiency to the international level of TAT. If it is approaching an international level, it will not only suggest an achievement of the reform, but also indicate that laboratory efficiency is not a prominent factor that leads to the particular severe overcrowding in Shanghai.The study was designed to evaluate the laboratory efficiency. Based on the principle of \u201clarge samples and multiple center study,\u201d we selected all tier III hospitals in 10 districts of Shanghai, which is the largest city in China with more than 24 million residents, and one representative tier II hospital in the district. In total, there were 7 tier III hospitals and 10 tier II hospitals in the group. All the enrolled hospitals constructed a lab in their EDs, which were defined as sectional laboratories for ED routine tests. Samples were randomly distributed by laboratory personnel to either a central lab at an inpatient hospital or to an ED lab. Emergency physicians recruited chest pain patients for the study when cardiac biomarkers were required for a diagnosis. A short training session was presented to the enrolled emergency physicians and laboratory personnel after the study was initiated in January 2011. The study lasted 8 months, spanning from April 2011 to December 2011.The TAT was defined as the time point when the cardiac biomarker tests were ordered until the time point when the ED physicians were available to analyze the test results. The emergency physician completed a questionnaire . All the analyses were conducted using SAS software version 8.2 (SAS Institute Inc.).We used the Wilcoxon rank\u2010sum test and Kruskal\u2010Wallis tests to assess the differences between Tier II and Tier III hospitals and between central and ED laboratories if the results of the study failed to satisfy normal distributions (SAS procedures PROC UNIVARIATE and PROC NPAR1WAY). We considered a two\u2010sided The sponsor of this study had no role in the study design, the data collection, the data analysis, the data interpretation, or the writing of the report. The corresponding author had full access to all the data in the study and made the final decision to submit the manuscript for publication. All the funding sources were used for the data collection, the statistical analysis and service fees.1.P > 0.05, see Table\u00a0All 17 participating hospitals we selected have constructed or expanded ED laboratories due to the medical reform policy with the financial support of the health care reform plan. Therefore, we are sure the TAT levels we recorded are not influenced by the limitation of the infrastructure or medical supply. Among these hospitals, 7 were Tier III, and 10 were Tier II. A total of 911 individuals were interviewed and completed questionnaires. Among the 911 questionnaires, 410 were from Tier II hospitals and 501 were from Tier III hospitals. The patient baseline characteristics were similar between groups , which contradicts the purpose of creating an ED lab TAT for c\u2010TNT between central and ED labs were 61 min (47\u201381 min) versus 70 min (60\u2013105 min) in Tier III (P < 0.05), and 51 min (42\u201367 min) versus 65 min (60\u201380 min) in Tier II (P < 0.05) in working period. In night period, the median TAT is 61 min (41\u201390 min) versus 63.5 min (44.5\u201385.5 min) (P < 0.05), and 47 min (34\u201368 min) versus 61 min (48\u201375 min) (p < 0.05). The TAT for c\u2010TNT at the ED labs in Tier II and Tier III hospitals were significantly longer than those at central labs (P < 0.05), regardless of the hours of operation TAT for CK\u2010MB between central and ED labs were 60 min (45\u201392 min) versus 65 min (55\u201383 min) in Tier III and 54 min (43\u201367 min) versus 65 min (55\u201383 min) in Tier II in working period; and there were 61 min (40\u2013100 min) versus 65 min (55\u201383 min), and 47 min (35\u201369 min), versus 65 min (55\u201383 min) in night period, respectively. Moreover, the median TAT for myoglobin were 70.5 min (57\u201390 min) versus 70 min (59\u201387 min) in tier III and 59.5 min (53\u201373 min) versus 70 min (59\u201387 min) in tier II in working period; as well as 70 min (50\u2013105 min) versus 70 min (59\u201387 min) and 52 min (42\u201373 min) versus 70 min (59\u201387 min) in night period. The TAT for CK\u2010MB and myoglobin were also longer at the ED laboratories TAT for c\u2010TNT was 70 min (60\u2013105 min) at Tier III EDs compared with 65 min (60\u201380 min) at Tier II EDs; in addition, there was 61 min (47\u201381 min) compared to 51 min (42\u201367 min) at central labs in working period. In night period, the median TAT is 63.5 min (44.5\u201385.5 min) versus 61 min (48\u201375 min) (p > 0.05), and 61 min (41\u201390 min) versus 47 min (34\u201368 min) (P < 0.05). The TAT for CK\u2010MB and myoglobin were also shorter at Tier II hospitals. The median TAT for CK\u2010MB were 61 min (40\u2013100 min) at Tier III central laboratories versus 47 min (35\u201369 min) at tier II in night period TAT for myoglobin were 70 min (50\u2013105 min) at tier III central labs versus 52 min (42\u201373 min) at Tier II; and 71 min (55\u201391 min) versus 65 min (52.5\u201380.5 min) at ED laboratories in night period cardiac biomarker panels, which are widely used in the United States (US) and Europe, were not well recognised in Shanghai. However, we discovered that only 9% of samples at Tier II EDs and 5% of samples at Tier III EDs from the investigated patients were assayed by POC instruments and Europe. Therefore, we highly advocate improving the spreading usage and standard application of POC assay with the next version of the medical reform policy, which should be based on additional scientific studies and evaluations. These improvements will increase the clinical efficiency and cost\u2010effectiveness of EDs and will enhance Chinese medical reform after the reconstruction of a basic infrastructure.Overall, our study of TAT evaluation demonstrated that ED laboratories have not yet approached a level to share the burden of patient flow at emergency department. Further arrangement should be assigned to separate the function of emergency laboratory and central laboratory. The idea of evaluating routine laboratory efficiency by TAT in the ED is fast, convenient, although it does not represent the general level of laboratory efficiency."} +{"text": "Mycobacterium tuberculosis in a persistent non-replicating dormant state that is associated with tolerance to host defence mechanisms and antibiotics. We have recently reported that vitamin C treatment of M. tuberculosis triggers the rapid development of bacterial dormancy. Temporal genome-wide transcriptome analysis has revealed that vitamin C-induced dormancy is associated with a large-scale modulation of gene expression in M. tuberculosis.Latent tuberculosis infection is attributed in part to the existence of M.tuberculosis (Mtb-TRN) consisting of 178 regulators and 3432 target genes was constructed. The temporal transcriptome data generated in response to vitamin C was overlaid on the Mtb-TRN (vitamin C Mtb-TRN) to derive insights into the transcriptional regulatory features in vitamin C-adapted bacteria. Statistical analysis using Fisher\u2019s exact test predicted that 56 regulators play a central role in modulating genes which are involved in growth, respiration, metabolism and repair functions. Rv0348, DevR, MprA and RegX3 participate in a core temporal regulatory response during 0.25\u2009h to 8\u2009h of vitamin C treatment. Temporal network analysis further revealed Rv0348 to be the most prominent hub regulator with maximum interactions in the vitamin C Mtb-TRN. Experimental analysis revealed that Rv0348 and DevR proteins interact with each other, and this interaction results in an enhanced binding of DevR to its target promoter. These findings, together with the enhanced expression of devR and Rv0348 transcriptional regulators, indicate a second-level regulation of target genes through transcription factor- transcription factor interactions.An updated transcriptional regulatory network of Temporal regulatory analysis of the vitamin C Mtb-TRN revealed that there is involvement of multiple regulators during bacterial adaptation to dormancy. Our findings suggest that Rv0348 is a prominent hub regulator in the vitamin C model and large-scale modulation of gene expression is achieved through interactions of Rv0348 with other transcriptional regulators. Mycobacterium tuberculosis (Mtb) in a state of dormancy/persistence during latent tuberculosis (TB) infection in immunocompetent individuals poses a significant barrier in eradication of this pathogen. Dormant/persistent TB bacteria adapt to a physiologically altered state in response to host-derived stresses and are not easily cleared by the existing TB drugs. Various in-vitro models of dormancy/persistence have provided valuable insights into the signature responses adopted by Mtb for survival under stress conditions [2 depletion, leading to a rapid induction of DevR and its regulon genes [The existence of nditions \u20138. Vitamon genes . The bacon genes .Genome-wide transcriptome studies have provided significant insights into gene regulatory mechanisms and pathways utilised by bacteria during adaptation to dormancy \u201320. Two-Previously, our laboratory reported that vit C-induced dormancy in Mtb is associated with bacterial growth stasis, progression to viable but non-culturable (VBNC) state, loss of acid-fastness and reduction in length, protective response to oxidative stress, lipid metabolism and modulation of anti-tuberculosis drugs . To deciFirstly, we compiled the available data from multiple sources (as described in methods) to generate a Mtb transcriptional regulatory network of the Mtb genome was identified; whereas in the present analysis, we have identified 2543 genes to be differentially regulated (DRGs) at at-least one time point , core repressed (Cluster II) and late time-point induction . Three temporal regulatory patterns were observed: an Early response which comprised of 0.25\u2009h, 0.50\u2009h and 1\u2009h; an Intermediate response which comprised of 2\u2009h, 4\u2009h and 8\u2009h; and a Late response at 24\u2009h are functions related to growth, respiration, metabolism and secretion. Only 1 enriched COG functional class was notably up-regulated (>\u200997% genes), namely \u201cPost-translational modification, protein turnover, and chaperones\u201d , which were prominently regulated by RegX3, Rv0348, FurB, WhiB1, WhiA, SigB, SigE, and SigF that were down-regulated. PhoP, MprA and DevR two-component systems and Rv0348 and CsoR were identified as putative regulators regulating these functions were differentially regulated at 24\u2009h post-vit C treatment and at 24\u2009h was confirmed by RT-qPCR. A comparable trend in expression of Rv0348, devR, regX3, lsr2 and Rv0081 was observed by both techniques , but did not satisfy the FDR correction. Interestingly, no unique DRG targets were present for Rv0348 regulator at any of the time points, rather all of them were also targets of other regulators having 178 regulators with 3448 nodes and 16,040 interactions was constructed using the available literature. We have previously reported that axenic and intracellular Mtb acquire a dormancy phenotype upon treatment with vit C activating a pleiotropic stress response in Mtb , 32. HerNotably, 13 of these regulators were previously shown to be associated with bacterial responses to various stresses such as hypoxia , 56, 57,An important finding that emerged from the analysis of Mtb-TRN was that several physiological functions that were targeted by the enriched regulators identified in this study Fig.\u00a0 were alsA completely different set of regulators was enriched at the Late time point of 24\u2009h. Among these, Lsr2, Rv0081 and Rv0678 had the highest number of connections with the target genes in the network. The number of DRG targets increased steeply from 644 at 8\u2009h to 1777 at 24\u2009h, of which 1416 DRGs were unique to the 24\u2009h time point. The expansion in the expression network at 24\u2009h can be attributed to these Late time point regulators that orchestrate bacterial adaptation response to vit C.Rv0348, which is a prominent hub regulator in the vit C Mtb-TRN, was previously reported as an important regulator during the chronic stage of TB in the murine infection model . This reOverall, our data suggests that the enriched regulators exhibited only a minor or no change in gene expression and false discovery rate (FDR) corrected (Benjamini and Hochberg method) pvalue\u2009\u2264\u20090.05, were considered to be differentially regulated genes (DRGs).Independent triplicate cultures of Mtb H37Rv were exposed to 10\u2009mM vit C for 0.25, 0.5, 1, 2, 4, 8 and 24\u2009h. RNA was isolated from these cultures and microarray was performed. Briefly, Cy3 labeled samples were hybridized to Mtb arrays (Agilent custom 8\u2009\u00d7\u200915\u2009K), scanned and the data were extracted using Feature Extraction Software (GEO accession no: GSE101048 ). This dEscherichia coli (E. coli) and Corynebacterium glutamicum [Firstly, we compiled the available data from multiple sources which included inference from orthology with utamicum , 16, DNAutamicum , 17 and utamicum , 16 intoutamicum , consideutamicum .pvalues obtained were subjected to false discovery rate (FDR) analysis using Benjamini and Hochberg method and a regulator with FDR corrected pvalue of \u22640.05, was considered to be enriched of all the enriched regulators using FET and the genes were classified into the functional sets defined by Cluster of Orthologous Genes (COG) database. A functional class with a Networks were constructed for every time point using Cytoscape software (Version 2.0) which co595 ~\u20090.1\u20130.2 and treated with 10\u2009mM vit C from 0.25\u2009h to 24\u2009h with 0\u2009h untreated cultures as control. RNA was isolated from the treated and untreated cultures using TRIzol based isolation method as described previously [Mtb was cultured in DTA medium as 10\u2009ml cultures in 50\u2009ml tubes with shaking at 220\u2009rpm till an ODRv0348 was amplified from Mtb H37Rv genomic DNA and cloned in E. coli expression vector, pET28a at NcoI and XhoI restriction sites to obtain the C-terminally His-tagged Rv0348. The sequence of primers used for cloning are given in Table E. coli DH5\u03b1 cells. After sequence verification, Rv0348-His6 protein was over-expressed in E. coli C43 (DE3) grown at 37\u2009\u00b0C with shaking at 180\u2009rpm till OD595 of 0.4\u20130.5 in 2x YT media containing Kanamycin (50\u2009\u03bcg/ ml) followed by induction with 1\u2009mM IPTG. Protein was purified by lysing the bacterial inclusion bodies and refolding the protein on the Ni-NTA column as described previously [Rv3134c promoter region (devR operon promoter). The primers used for amplification of probes from genomic DNA are given in Table 6-DevR protein were phosphorylated using 50\u2009mM acetyl phosphate in buffer containing 25\u2009mM Tris -HCl (pH\u20098.0), 0.5\u2009mM EDTA, 20\u2009mM KCl, 6\u2009mM MgCl and 2.5% glycerol for 25 mins at 25\u2009\u00b0C. Phosphorylated DevR (DevR~P) was then incubated with devR operon promoter fragment protein in coating buffer and incubated overnight at 4\u2009\u00b0C in a humidified chamber. Control wells were coated with bovine serum albumin (BSA) or left uncoated. Blocking was carried out for 90 mins at 37\u2009\u00b0C with 4% BSA made in 1x phosphate buffer saline (PBS) and 0.25% Tween-20 following which the plate was washed with 1x PBS. Varying concentrations of the DevR were added to the wells and incubated at 25\u2009\u00b0C for 90 mins. After washing with 1x PBS, polyclonal anti-DevR antibody (generated in rabbit) was then added to each well at a dilution of 1: 5000 for 1\u2009h at 25\u2009\u00b0C. The plate was washed rigorously 5 times with 1x PBS and 0.1% Tween-20 and twice with 1x PBS, followed by addition of goat anti-rabbit IgG-HRP conjugate secondary antibody (1:10000) for 1\u2009h at 25\u2009\u00b0C. The plate was again thoroughly washed and developed with TMB substrate with absorbance measurement at 450\u2009nm.Rv0348-DevR protein interaction was assessed in an Enzyme linked immunosorbent assay\u00a0(ELISA) format\u00a0for assessing protein-protein interaction. Briefly, 96-well microtiter plate was coated with Rv0348-HisAdditional file 1: Table S1.Mycobacterium tuberculosis transcriptional regulatory network (Mtb-TRN). Table S2. Details of the enriched COG functions with the genes and their regulators. Table S3. Temporal network analysis scores obtained using Cytoscape\u2019s NetworkAnalyzer function. Table S4. List of Shared DRG targets of Rv0348 with other enriched regulators at each time point. Table S5. Temporal gene expression data of 56 enriched regulators.Additional file 2: Figure S1. Hierarchical clustering of all differentially regulated genes in the vitamin C Mtb dormancy model. Figure S2. COG functional classification analysis of the differentially regulated gene targets of enriched regulators at 24\u2009h. Figure S3. Temporal expression of selected top scoring nodes (regulatory genes) of network analysis from microarray and RT-qPCR analysis."} +{"text": "Infectious complications are a major cause of morbidity and mortality after heart transplantation (HT). However, the epidemiology and outcomes of these infections in the recent population of adult heart transplant recipients have not been investigated.We conducted a single-center retrospective study on infectious complications occurring within 180\u00a0days following HT on consecutive heart transplant recipients, from January 2011 to June 2015 at Bichat University Hospital in Paris, France. Risk factors for non-viral infections occurring within 8, 30 and 180\u00a0days after HT were investigated using competing risk analysis.n\u2009=\u2009181 episodes) occurred in 80 (71%) patients. The most common bacterial and fungal infections were pneumonia , followed by skin and soft tissue infections . Multi-drug-resistant bacteria were responsible for infections in 21 (19%) patients. Viral infections were diagnosed in 44 (34%) patients, mostly Cytomegalovirus infection . In multivariate subdistribution hazard model, prior cardiac surgery (subdistribution hazard ratio sHR\u2009=\u20092.7 [95% CI 1.5\u20134.6] p\u2009<\u20090.01) and epinephrine or norepinephrine at the time of HT (sHR\u2009=\u20092.3 [95% CI 1.1\u20135.2] p\u2009\u00a0=\u20090.04) were significantly associated with non-viral infections within 8\u00a0days after HT. Prior cardiac surgery (sHR\u2009=\u20092.5 [95% CI 1.4\u20134.4] p\u2009<\u20090.01), recipient age over 60\u00a0years (sHR\u2009=\u20092.0 [95% CI 1.2\u20133.3] p\u2009<\u20090.01) and ECMO following HT (sHR\u2009=\u20091.7 [95% CI 1.0\u20132.8] p\u00a0=\u20090.04) were significantly associated with non-viral infection within 30\u00a0days after HT, as well as within 180\u00a0days after HT.Overall, 113 patients were included. Fifty-eight (51%) HTs were high-priority allocations. Twenty-eight (25%) patients had an extracorporeal membrane oxygenation (ECMO) support at the time of transplantation. Ninety-two (81%) patients developed at least one infection within 180\u00a0days after HT. Bacterial and fungal infections contains supplementary material, which is available to authorized users. Heart transplantation (HT) is the gold standard treatment of end-stage cardiac failure . SubstanThe purpose of this study was to describe the epidemiology and outcomes of infections occurring within 180\u00a0days after HT. Specifically, we aimed to determine risk factors for non-viral infections occurring within 8, 30 and 180\u00a0days following HT.This single-center retrospective study was conducted at Bichat-Claude Bernard Hospital, Paris, France. We included all consecutive adult heart transplant recipients between January 1, 2011, and June 30, 2015. Data on donors and recipients were retrieved from the medical records and the French Transplant Agency database \u201cCristal,\u201d which is a prospectively maintained database designed to store all the donors and recipients\u2019 data. The French Intensive Care Society Ethics Committee approved the study (CE SRLF17-10). Data on intensive care unit (ICU) management and infections were retrospectively collected. In accordance with French law, all the patients or next of kin were informed of the possibility to use their hospitalization\u2019s data for clinical research and gave their consent.Immunosuppressive treatment and antimicrobial prophylaxis for heart transplant recipient are well codified in our transplantation center, in accordance with the International Society of Heart and Lung Transplantation (ISHLT) guidelines . All patStaphylococcus aureus or extended spectrum beta lactamase (ESBL) producing Enterobacteriaceae carriage before HT. Postoperative prophylaxis against infections was initiated between day 3 and day 7 after heart transplantation. Postoperative cotrimoxazole was administered for Pneumocystis jiroveci pneumonia and toxoplasmosis prevention for a duration varying from 1\u00a0year to lifelong, depending on the risk . Oral amphotericin B was given to all patients within 30\u00a0days after HT in the absence of intestinal disease. CMV disease prevention consisted in antiviral prophylaxis with valganciclovir for high-risk patients (donor positive/recipient negative for CMV). For CMV seropositive patients or CMV seronegative donors and recipients, the preemptive strategy based on the level of plasmatic CMV copies was chosen (threshold 4 log10 copies/mL). In case of treated acute rejection, valganciclovir prophylaxis was systematically administered in CMV seropositive recipients, associated with cotrimoxazole prophylaxis in case of interruption. Heart transplant recipients received, if possible, influenza and pneumococcal vaccines before heart transplantation, or within the first year following the transplantation, as recommended by our local protocol.Intraoperative antibiotic prophylaxis was cefazolin except otherwise decided (ongoing infection or multi-drug-resistant (MDR) bacterium colonization). The patients were systematically screened for methicillin-resistant Infections were defined as per the CDC guidelines . The defp values smaller than 0.10 in univariate analysis and adjusted on the sequential organ failure assessment at the time of HT. Subdistribution hazard ratio (sHR) and 95% confidence interval (95% CIs) were estimated for significant risk factors, based on a multivariate analysis. The impact of infectious complications treated as time-dependent events on mortality at 90\u00a0days after HT was determined using Cox regression models. Statistics were performed using SAS 9.4 and R softwares, and a p value of 0.05 or lower was considered significant.Quantitative parameters are reported as median and interquartile range [IQR 25\u201375 percentile] and qualitative parameters as number and percentage. Univariate and multivariate analyses were performed to assess for associations between potential risk factors and non-viral infections within 8, 30 and 180\u00a0days after HT using a Fine and Gray subdistribution hazard model considering death as a competing risk. Factors included in the multivariate model were selected among variables yielding n\u2009=\u200986, 76%), with a median age of 53 years. Main causes of cardiac failure were dilated cardiomyopathy and ischemic cardiomyopathy . Most patients were already hospitalized at the time of HT, including a large number in the ICU . Twenty (18%) patients had VAD as a bridge to transplantation, and 28 (25%) patients were under ECMO support at the time of HT.Overall, 113 patients underwent HT between January 1, 2011, and June 30, 2015, at Bichat-Claude Bernard Hospital, Paris. All those patients were included in the study, with no lost to follow-up within 180\u00a0days after HT. Patient\u2019s baseline characteristics are given in Table\u00a0n\u2009=\u20096), Staphylococci (n\u2009=\u20096), Pseudomonas aeruginosa (n\u2009=\u20093), other Gram-negative bacilli (n\u2009=\u20094) and other pathogens (n\u2009=\u20093). Ninety-two (81%) patients developed at least one infection within 180\u00a0days after HT. There were 181 episodes of bacterial and fungal infections occurring in 80 (71%) patients. Fifty-five (49%) patients had at least one bacterial infection within 8\u00a0days after HT. Although bacteria were the most common identified pathogens in the early postoperative phase, viruses also accounted for a high proportion of infections between 8 and 30\u00a0days following HT , followed by skin and soft tissue infections . We recorded 30 bloodstream infections (BSI) occurring after a median time of 10 [5\u201321] days following heart transplantation. Of these BSIs, 12/30 (40%) were secondary to another infection and 18/30 (60%) were of unknown origin. Six out of eighteen patients with BSI of unknown origin were under ECMO.Twenty 18%) patients of our cohort, including six VAD patients, had an infection within 8\u00a0days before heart transplantation. However, device infection was the main reason for bridging them to transplantation in only two cases. Twenty-two (19%) patients received an antibiotic therapy for an ongoing infection or an alternative prophylaxis other than cefazolin during heart transplantation, preferentially targeting ESBL . Bacterial infections due to Pseudomonas aeruginosa preferentially occurred after 8\u00a0days following HT. Thirty-five (31%) patients developed at least one infection due to Gram-positive cocci, mostly Enterococcus spp. . Twenty-two (19%) patients were colonized by a MDR bacterium before transplantation, and 6 (5%) acquired one during the first 8\u00a0days in the ICU. Nine (8%) colonized patients developed at least one infection due to the same MDR microorganism during their ICU stay. Overall, MDR bacteria were responsible for infections in 21 (19%) patients, mostly ESBL . Sixteen patients presented at least one invasive fungal infection. Aspergillus spp. were responsible for invasive infection in 6 (5%) patients, in all cases over 30\u00a0days following HT. Candida spp. were considered responsible for infection in 4 (4%) patients. Viral infections were diagnosed in 44 (39%) patients, mostly CMV infection , occurring preferentially between 8 and 30\u00a0days after HT patients developed at least one infection caused by Gram-negative bacilli. p\u2009<\u20090.01) and epinephrine or norepinephrine continuous infusion at the time of HT (sHR\u2009=\u20092.3 [95% CI 1.1\u20135.2] p\u2009=\u20090.04) were significantly associated with the occurrence of bacterial infection within 8\u00a0days after HT. Within 30\u00a0days after HT, prior cardiac surgery (VAD excluded) (sHR\u2009=\u20092.5 [95% CI 1.4\u20134.4] p\u2009<\u20090.01), recipient age over 60\u00a0years (sHR\u2009=\u20092.0 [95% CI 1.2\u20133.3] p\u2009<\u20090.01) and ECMO within 24\u00a0h after HT (sHR\u2009=\u20091.7 [95% CI 1.0\u20132.8] p\u2009=\u20090.04) were significantly associated with bacterial and fungal infections. Lastly, within 180\u00a0days after HT, prior cardiac surgery (sHR\u2009=\u20092.3 [95% CI 1.3\u20134.0] p\u2009<\u20090.01), recipient age over 60\u00a0years (sHR\u2009=\u20091.8 [95% CI 1.1\u20133.1] p\u2009=\u20090.02) and ECMO within 24\u00a0h after HT (sHR\u2009=\u20091.6 [95% CI 1.0\u20132.7]) p\u2009=\u20090.05) were significantly associated with non-viral infections patients developed an early severe graft dysfunction and needed an ECMO circulatory support within 24\u00a0h after HT. Data analysis revealed that among those patients, 47 (42%) had a severe primary graft dysfunction and 12 (11%) a secondary graft dysfunction due to acute rejection, hemorrhagic shock or pericardial tamponade. Forty-seven (80%) patients with ECMO were weaned from the circulatory support, while 12 patients (20%) died under support.n\u2009=\u20096, and intra-abdominal infection, n\u2009=\u20092). Bacterial infections in the ICU were associated with a longer stay in the ICU (20\u00a0days [95% CI 13\u201329] versus 12\u00a0days [95% CI 8\u201317] p\u2009<\u20090.001), but not associated with mortality. In an adjusted Cox model, the third non-viral infection was significantly associated with death at 90\u00a0days after HT .The median ICU length of stay after HT was 16 [11\u201323] days, and the median hospital length of stay after HT was 34 [25\u201346] days. Ninety-four (83%) patients were alive 180\u00a0days after HT, and 8 out to 19 (42%) deaths were deemed to be caused by an infectious complication , mostly bacterial or fungal infections . Moreover, we identified four factors associated with non-viral infections depending on the postoperative period: hemodynamic instability at the time of HT, prior cardiac surgery, recipient age over 60\u00a0years and early circulatory support by ECMO following HT. According to the literature, the cumulative incidence of infections following HT varies from 30 to 80%, depending on the definitions of infections and the chosen interval time after HT [Candida infection within 1\u00a0year after HT. Interestingly the population in this study was different from ours as a higher proportion of the patients had a VAD before transplantation (18% vs 30%) [We conducted a large single-center retrospective study to describe the epidemiology, risk factors and outcomes of infections occurring within 180\u00a0days after HT, in the era of high-priority allocations and mechanical support by ECMO and VAD. To our knowledge, it is the largest single-center cohort of recent adult heart transplant recipients analyzed for infections and their impact on prognosis. We found a very high rate of infections within 180\u00a0days after HT . In a re vs 30%) .The most common bacterial and fungal infections in our study were pneumonia (52%) followed by skin and soft tissue infections (14%) in agreement with the literature , 10, 24.We showed that recipient age over 60\u00a0years, prior cardiac surgery (VAD excluded) and ECMO within 24\u00a0h after HT were risk factors associated with non-viral infections within 30 and 180\u00a0days after HT. Interestingly, VAD before HT and national high-priority allocation were not associated with infections following HT. National high-priority allocation criteria depend on the countries, but are everywhere designated for candidates on the waiting list who have the highest priority on the basis of medical urgency. However, in our study, hemodynamic instability at the time of HT, in the context of national high-priority allocation or not, was an independent risk factor for bacterial infection within 8\u00a0days after HT. Besides, recent papers showed that VAD before HT did not increase the risk of post-transplant infection , 11. ECMSpecific risk factors for invasive fungal infections would have been interesting to look for, as ECMO has already been described as an important risk factor in a HT population . As we dCMV infection was the predominant viral infection, occurring in 34% of the population. This rate is in accordance with recent studies focused on CMV , 14, 35.There was no difference in immunosuppressive regimen between patients developing or not infections after HT. However, the high rate of infections in our cohort was also a direct consequence of immunosuppressive therapies. In the current era of HT, those results enlighten the need of personalized immunosuppression, adjusted on patient\u2019s risk factors for acute rejection and early infections , 37.The overall mortality at 6\u00a0months after HT 17%) was in accordance with published data , 33, 38.% was in The first strength of this study was the inclusion of consecutive heart transplant recipients in a transplantation specialized center, in the era of VAD and ECMO mechanical supports and of increased rate of high-priority allocations. The detailed description of epidemiology, risk factors and outcomes of infectious complications in this new population could be a good tool for clinicians taking care of adult heart transplant recipients. We also acknowledge that the present study has several limitations. First, our peculiar population does probably not reflect the reality of all cardiac transplant centers, as the majority of enrolled patients were national high-priority allocations already hospitalized in ICU at the time of HT. It might be difficult to extend these results to all the patients on heart transplant waiting list. Moreover, the sample size is limited, yet it is a recent single-center study. We did not study the donor-derived infections even though it would have been of interest to better characterize early bacterial infections following heart transplantation. As the data collection was retrospective, potential inaccuracies in data reporting could exist. A few infectious episodes were also missing data, most notably the bacterial etiology of infection. Finally, strategies for perioperative management of heart transplant recipients may differ among institutions and may be considered a source of bias.This study confirmed the high rate of infections occurring within 180\u00a0days after HT in adult recipients. Recipient age, prior cardiac surgery, hemodynamic instability at the time of HT and ECMO following HT were independent risk factors for early and late non-viral infections. Thus, the recent pre- and post-transplant recipient characteristics modification could be directly correlated with infections following HT. A prospective study could be of interest to better determine risk factors for infections following HT.Additional file 1: Table S1. Pre-, intra- and postoperative characteristics of heart transplant recipients without non-viral infection and patients developing at least one bacterial or fungal infection within 8\u00a0days, 30\u00a0days and 180\u00a0days after HT"} +{"text": "Analysis of heart rate variability (HRV) can be applied to assess the autonomic nervous system (ANS) sympathetic and parasympathetic activity. Since living systems are non-linear, evaluation of ANS activity is difficult by means of linear methods. We propose to apply the Higuchi fractal dimension (HFD) method for assessment of ANS activity. HFD measures complexity of the HRV signal. We analyzed 45 RR time series of 84 min duration each from nine healthy and five diabetic subjects with clinically confirmed long-term diabetes mellitus type II and with diabetic foot ulcer lasting more than 6 weeks. Based on HRV time series complexity analysis we have shown that HFD: (1) discriminates healthy subjects from patients with diabetes mellitus type II; (2) assesses the impact of percutaneous auricular vagus nerve stimulation (pVNS) on ANS activity in normal and diabetic conditions. Thus, HFD may be used during pVNS treatment, to provide stimulation feedback for on-line regulation of therapy in a fast and robust way. Analysis of HRV represents a common tool for assessment of autonomic cardiac regulation and provides information about pathophysiological changes in various diseases . Based okmax, specifying a maximal distance between the points compared in the time series. As the HFD measures the complexity of the curve that represents the analyzed signal on a plane, it always attains values between 1 and 2. The value of 1 corresponds to a regular time series while for Gaussian-type noise HFD may attain different values: 1.5 for Brownian, 1.8 for pink, and 2.0 for white noise . The study was approved by the local ethics committee of the Medical University of Vienna (no. 1924/2013) and by the Austrian Agency for Health and Food Safety. The RR time series were recalculated from ECG recordings obtained from nine healthy and five diabetic subjects, aged 40\u201380 years, with clinically confirmed long-term diabetes mellitus type II, and diabetic foot ulcer (ulcus cruris) lasting for more than 6 weeks (Table 1). Subject\u2019s exclusion criteria were: participation in another clinical trial over the last 5 weeks before the experiment; addiction to substance abuse; autonomous nervous system dysfunction (except diabetic polyneuropathy); medical treatment with vasoactive substances; history of heart arrhythmia or presence of an active implantable device. Women in childbearing age were not included if pregnant or nursing. All diabetic subjects had a history of diabetes in average(SD) of 14(5) years. The ECGs were acquired by means of a MP36 recording system with a three-lead Einthoven II derivation and a sampling rate of 1 kHz, for further calculation of heart rate and HRV signals. The measurements were obtained between February 24, 2014 and April 3, 2015. The heart rate was calculated using proprietary MATLAB algorithms with manual control . All subjects gave written informed consent in accordance with the Declaration of Helsinki.We retrospectively analyzed 56 RR time series, of 84 min duration each, from an open-label pilot study registered at Each of the healthy and diabetic subjects underwent four sessions of pVNS mediated via four needle electrodes, with one acting as the reference electrode, in vagally innervated regions of the right auricle . Each seEleven out of 56 RR time series were excluded because of significant artifacts. The artifacts were caused by a low quality of the raw data (7 time series) or presence of cardiac arrhythmia in the signal (4 time series). Therefore, further analysis was performed on 45 RR time series .In order to standardize the length of the series, every RR record was linearly resampled with 1 Hz. Low frequency of resampling was used to preserve the original characteristics of the signal. Higher sampling frequencies would change the shape of the original RR curve by introducing additional samples and extending the total length of the signal, subsequently affecting the estimation of real HFD values by introduction of low frequencies.Calculations were performed by means of an in-house implementation of the HFD algorithm in MATLAB R2016b . HRV signals were analyzed within windows of 100 data points displaced by a 50 consecutive samples across the signal, which resulted in 99 HFD values for each of the RR time series. The window of 100 data points reflected approximately 1.5 min windows in RR time series. Consecutively, every phase in each of the time series consisted of the following number of HFD values: B- 11 HFD values; S1- 25 values; P1- 24 values; S2- 25 values; P2- 14 values.kmax parameter, allowing clear differentiation between healthy and diabetic subjects, the HFD was calculated in all of the 45 RR time series, for kmax ranging from 2 to 50 . Calculation for kmax above 50 was not possible due to the window length of 100 samples \u2013 maximal distance between compared samples was less than 1/2 of the window length. Optimal kmax was chosen based on a clear separation of the mean of the aggregated HFD values for healthy and diabetic subjects, and on the minimization of the two-sided Wilcoxon ranksum test p-value for comparison of medians of the HFD values. Subsequent analyses were performed for HFDs calculated with the chosen parameter kmax = 5. In order to find an optimal In each of the RR time series, 99 HFD values were calculated. HFDs aggregated from the time series were tested by Shapiro\u2013Wilk normality test, to confirm non-normal distribution of the values. Afterwards, the aggregated HFD distributions were computed in healthy and diabetics from 2,772 and 1,683 HFD values, respectively. Fifth, 25th, 75th, and 95th percentiles, means, medians, SDs, skewness, and kurtosis were calculated separately in both HFD distributions, for subsequent comparison.t) in healthy and diabetics, for comparison by means of Wilcoxon matched-pairs signed rank test. For visual presentation of the results, the average \u00b195% confidence intervals of the aggregated HFD values from healthy and diabetics were plotted in function of time. Pearson\u2019s linear correlation coefficient was calculated between the average HFDs(t) and the time course of the experiment in both groups. Results were considered significant if correlation exceeded 50% with a p-significance value <0.05.Mean representative vectors of HFDs were calculated in respect to time was applied to reveal significant differences between the average HFDs in healthy and diabetic groups, in respect to the phase of experiment. Bonferroni\u2019s multiple comparison test was used in search of differences between specific phases of the time series. Whiskers-box plots of the aggregated HFDs within the phases were generated for visual representation of the results.To check whether pVNS changes the shape of HFD distribution overtime, 5th, 25th, 75th, and 95th percentiles, means, medians, SDs, skewness and kurtosis were calculated for the HFD aggregated distributions from B, S1, P1, S2, and P2 phases separately, for subsequent comparison. Afterwards, 10 bins-wide histograms of the aggregated HFD distributions were computed within the phases, for healthy and diabetic subjects separately. To objectively assess the magnitude of the changes caused by pVNS, contrast histograms were calculated as a ratio of difference to sum of bins heights between previously computed histograms for S1 and B; P1 and S1, S2 and P1, P2 and S2, and P2 and B phases in both groups.All calculations and statistical analyses were performed by means of MATLAB R2016b.kmax = 5, by means of a two-sided Wilcoxon ranksum test (p < 0.001). Hence, the optimal kmax parameter, allowing clear differentiation between the healthy and diabetes group, was chosen as 5.The aggregated HFD values for healthy and diabetics were found as the most statistically different for W = 0.9745, p < 0.05). Characteristics of HFD aggregated distributions are presented in Table 2.Shapiro\u2013Wilk normality test revealed that HFD values, aggregated jointly from healthy and diabetic subjects, do not form a normal distribution (t) values in healthy and diabetics . The mean(median) difference between the groups was 0.226(0.230). Absolute Pearson\u2019s linear correlation coefficient between the average HFD(t) values and the time course of experiment was larger in diabetics , than in healthy .Wilcoxon matched-pairs signed rank test revealed statistical differences between the average HFD = 60.79, p < 0.0001], and due to the experimental phases . Bonferroni\u2019s multiple comparisons test showed significant differences in mean HFD values between B and P2, S1 and P2, and S2 and P2 phases both in healthy and diabetics . The mean HFD values were significantly different between healthy and diabetics in all phases (largest p < 0.05) except P2 in diabetic subjects, which was not different from B, S1, and P2 in healthy .Two-way ANOVA showed significant differences between the average HFD\u2019s from different experimental phases, in healthy and diabetic subjects. The differences were visible between the healthy and diabetic group [Table 2 presents numerical characteristics of HFD aggregated distributions for experimental phases B, S1, P1, S2, P2 in healthy and diabetic subjects. Figure 4 shows 10 bins-wide histograms of the aggregated HFD distributions for the phases and Figure 5 shows contrast histograms for comparison of the distributions between S1 and B; P1 and S1, S2 and P1, P2 and S2, and P2 and B phases, for healthy and diabetics. The characteristics and the histograms were calculated in total from 4,455 HFD values. Overall mean(median) \u00b1 SD HFD values were found as 1.49(1.48) \u00b1 0.14 and 1.72(1.73) \u00b1 0.17 for healthy and diabetic subjects, respectively. In diabetic subjects, skewness of aggregated HFD distributions was observed to change from negative (\u20130.64) to positive (0.18) between the experimental phases . The opposite, but not monotonic (and not significantly different from zero slope) effect was observable in the healthy group . Moreover, kurtosis of the distributions was found consistently dropping over the experimental phases only in diabetics.Figure 1 and Table 2). Significant differences in mean values of HFD aggregated distributions were found between the B, S1, and P2 phases in both groups. It is also worth to highlight that the mean HFD from P2 in diabetics was found not different from that in B, S1, and P2 phases in healthy . The results indicate no significant influence of pVNS on mean HFD during or directly after the stimulation in both groups. However, the effect seems to be delayed in time and its overall permanence was more explicit in diabetics than in healthy subjects. Moreover, skewness of the aggregated HFD distributions was rising significantly from negative to positive between the phases in diabetics, while a clear trend was not observable in healthy.This study advances knowledge in HRV analysis in healthy and diabetic subjects. By means of our implementation of Higuchi\u2019s method, overall mean \u00b1 SD of aggregated HFD values were found higher for diabetics than for healthy subjects , high frequency power (HF \u2013 parasympathetic activity), low frequency (LF \u2013 mixed sympathetic and parasympathetic activity) power, and the LF/HF ratio in the presented subject population of the HTable 2 and Figure 4). Moreover, a relative increase in HFDs below 1.6 is observable in P2, compared to B in diabetics. The effect is connected with a smaller decrease for HFDs above 1.6 . In contrary, pVNS seems to affect the whole distribution of the HFD values equally in healthy. The shape of the HFD distribution was not changed here substantially over the experiment , however, a slight increase in the higher HFDs associated with a decrease in HFDs of lower range is observable between P2 and B .Our results indicate a slight pVNS-induced shift of HFD values from assembled close to 2 to lower values, in diabetics. The shift was observed mostly for the HFDs above the 50th percentile of distribution and was confirmed by the change in the distribution\u2019s skewness and kurtosis . In both situations the distinction between the healthy and diabetic group might be not possible . Further, with respect to the included subject groups, a significant difference in BMI of healthy and diabetic subjects (Table 1) needs to be considered as a potential co-founding factor when analyzing ANS function. We have performed analysis of HRV time series recalculated from original ECG signals. It was previously shown that HFD may provide similar results when applied to raw data to both diagnostic systems and therapeutic closed-loop pVNS systems.Hence, our results indicate that HFD provides high resolution insight into ANS activity during pVNS based on HRV time series analysis. Simplicity of HFD makes the assessment of ANS activity prospectively possible in on-line systems and may bring accuracy (We have shown that the HFD assesses the ANS activity and differentiates healthy from diabetic subjects, based on HRV signals. Moreover, HFD provides fast and robust distinction between action of parasympathetic and sympathetic ANS activity. Because of its simplicity, HFD may be easily used in pVNS systems to provide direct stimulation feedback for on-line regulation of therapy. Hence, our results have potential implication for patients\u2019 care and technological advancement of pVNS therapy.RG performed the data analysis and wrote the manuscript. SK and FT performed the data analysis and reviewed the manuscript. EK supervised the data analysis and reviewed the manuscript. JS supplied the data and reviewed the manuscript. WK designed the conception of the work, supervised the data analysis, and reviewed the manuscript.JS, EK, and SK own shares and receive honoraria from SzeleSTIM GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors, WK."} +{"text": "Background: The human bocavirus (HBoV) is known to persist latently in the infected host cells and seems to replicate its DNA via the DNA damage response system, which is frequently defect in tumors and correlates with microsatellite instability (MSI). Because HBoV is able to persist in the infected tissues, induces pro-fibrotic and pro- cancerogenic cytokines in vivo and in vitro, and is detected in colorectal and lung tumors, the virus may be involved in cancerogenesis at least as a cofactor. Recently it was shown that the adenotonsillar tissue is an important site of HBoV1 persistence and replication. Considering the background that approximately 60% of oropharyngeal cancers were thought to be attributable to a HPV infection, a co-participation of HBoV in terms of a chronic virus infection might play a role in the cancerogenesis of tonsil tumors.Methods: Formalin-fixed, paraffin-embedded tonsil tumor samples were screened for HBoV and HPV DNA. Positive tissue sections were afterward subjected to fluorescence in situ hybridization (FISH) analysis to identify HBoV and HPV infected cells. By use of an in vitro cell culture model with primary tonsil fibroblasts, keratinocytes, and lymphocytes infected by HBoV we tried to find the target cells of virus replication. MSI testing was based on a previously published protocol using a de-multiplexed PCR followed by fluorescent detection of PCR products in a capillary sequencing device.Results: In total 62 of 103 of the tonsil squamous cell carcinomas tested positive for HBoV DNA and 66 of 103 (66%) samples were identified as HPV positive. The FISH analysis revealed both double infection of HPV and HBoV in the same cells as well as single infections of both viruses within the tumor tissue. Twenty-two of 62 HBoV positive tumors tested HPV negative, 40 of 62 tissue sections were HBoV and HPV positive. We analyzed 21 out of the 62 HBoV positive tumors for MSI. Of those four tonsils displayed MSI in at least 1 of 10 microsatellite markers.Conclusion: Our findings support the hypothesis that human bocavirus infections as a cofactor may have an impact on tumor development in tonsils, although it still remains possible that HBoV solely displays a tumor tropism. Bocaparvovirus genus of the Parvoviridae family, is associated with respiratory tract infections and gastrointestinal infections . This vote was specifically approved for the current study. There is a written informed consent of all patients, whose primary cells were used. Pediatric patients were included exclusively with parental written informed consent. For the retrospective and double blinded part of the study no written consent was required. The study cohort consisted of adult patients (tumors and primary cells) and children (primary cells).n = 11).Hundred and three FFPE tonsillar cancer sections and 20 chronically inflamed tonsil sections were selected from archived samples from our routine clinical laboratory. Primary tonsil cells were gained by routinely performed tonsillectomy in adult patients according to the manufacturer\u2019s protocol. All samples were analyzed with the RespiFinder Smart22 being able to detect 22 respiratory pathogens, including HBoV and Adenovirus. HPV detection was performed by the HPV 3.5 LCD-Array according to the manufacturer recommendations. HBoV real-time PCR was performed as previously described .n = 62) were subjected to FISH analyses for the detection of HBoV1 and HPV 16.All FFPE tonsil tumor samples that tested positive for HBoV by the Respifinder assay following the manufacturers\u2019 recommendations as previously published .4 U/ml each, BioWhittaker/Lonza, Switzerland) at 37\u00b0C. Human embryonic kidney 293 (HEK 293) cells for virus production were obtained from American Type Culture Collection and were cultured according to ATCC\u2019s recommendations. Primary tonsil keratinocytes, fibroblasts, and lymphocytes were obtained from 11 patients suffering from chronic tonsillitis immediately after routine tonsillectomy. Written informed consent was obtained in all cases in agreement with the ethical vote for this study. Resected tonsils were directly placed in 37\u00b0C warm keratinocyte growth medium . Further on lymphatic follicles and epithelial tissues were dissected using a scalpel. Afterward, the epithelial sections of the tonsils were minced in a Petri dish and were then enzymatically dissociated in trypsin for 15 min before being passed through an EASYstrainer cell filter . The lymphocyte section was treated with trypsin for 5 min and was also passed through an EASYstrainer cell filter. While lymphocytes were cultured in six-well plates as suspension cultures in RPMI medium, keratinocytes and fibroblasts were cultured on collagen-coated six-well plates grown in keratinocyte growth medium or epithelial growth medium , respectively. Microscopic analyses and documentation were performed on a Zeiss Primo Vert microscope using the AxioVision 4.8 software .All cultures were performed with 1% v/v Penicillin/Streptomycin .Pearson and the coefficient of determination r2, and the statistical significance were calculated with the one-sided Student\u2019s t-test and a chi-square test.In order to test if a higher tumor stage correlates with the detection of HBoV, Pearson correlation coefficient rFigure 1A). In 66 of 103 (66%) tonsil tumors, but in none (0%) of the 20 chronically inflamed tonsils HPV DNA could be identified . In 40 of 103 (38.8%) tonsil tumors there was a coinfection of HPV and HBoV. In 26 of 103 (25.2%) we could register a single HPV infection and in 19 of 103 (18.4%) a single HBoV infection . All samples were negative for the other 21 airway pathogens tested by the RespiFinder assay.In total, 62 of 103 (60.19%) of tonsil squamous cell carcinomas and 7 of 20 (35%) of chronically inflamed tonsils tested positive for HBoV DNA by the RespiFinder Smart22 PCR . The HPV type 16 FISH assay established for routine diagnostic was in agreement with the results of the HPV PCR, too. In 24 of 40 co-infected tumor sections (60%) the fluorescent signals of HPV 16 and HBoV DNA could be detected in the same cells, implying that the two viruses infected the same host cell. In contrast we found virus persistence in different cells in 13 of 40 co-infected tumor samples (32.5%) which means that FITC signals (indicating HPV DNA) and rhodamine red signals (indicating HBoV 1 DNA) were spread as distinct separated signals all over the tumor sections.To ascertain whether HPV and HBoV DNA are present in the same cells of the tumor tissue an HBoV specific FISH assay was applied (In 3 of 40 tumors (7.5%) we could observe a heterogeneous image with double infected cells and single infections in the same section. In 22 cases (35.4%) of all HBoV positive samples (single and co-infections) the presence of human bocavirus DNA could be determined. In accordance with the PCR based analyses HPV DNA was not detected in the chronic tonsillitis tissue sections. The signals were not always located in the nucleus, but appeared to be in the cytoplasm, as indicated by merging the different color channels. Moreover, multiple signals per cell were registered, indicating that multiple copies of HBoV DNA are present in the cells due to replication or, as a matter of speculation, due to persisting episomes.t-test for two dependent means a trend for the increase in the number of HBoV-positives in higher tumor stages was observed (Table 1A), whereas there was no higher HPV prevalence in higher tumor stages (Table 1B).Furthermore we found a correlation between the presence of human bocavirus and the tonsil tumor stage. We noticed higher bocavirus prevalence in tumor sections with progressed tumor stages. By using the one-tailed Table 2), because only for these tumors a tumor-free control tissue was available to compare the microsatellite status of tumor and otherwise healthy tissue. Of those, only four tonsils displayed microsatellite instabilities in at least 1 of 10 microsatellite markers, as exemplarily shown in Figure 3.In addition, we analyzed 21 out of the 62 HBoV positive tumors (single HBoV infected and HPV coinfected) for MSI . In one infected well of one patient we observed a virus replication after 3 days in accordance with the typical virus growth curve . However, in the remaining three HBoV infected cultures of the other patient there was no virus replication measurable. Virus replication in fibroblasts and lymphocytes was not detectable, indicating that this cell types are not permissive for HBoV.Because it was postulated that HBoV is likely persisting in tonsillar tissue and that this kind of tissue is assumed to be the major replication site in vivo of all tonsillar tumors and in 37% (n = 7) of all chronic tonsillitis tissues. The data presented suggest that there is an association between the presence of HBoV and oropharyngeal squamous cell cancer, especially tonsillar carcinomas. The prevalence of HBoV is statistically significant higher in tonsillar tumor sections than in chronically inflamed tonsils (p = 0.037 by Chi-square test), which not only confirms the hypothesis that HBoV may be a causative pathogen in coinfection, but as well might imply that HBoV may be involved in cancerogenesis. Up to now it is not clearly verified whether HBoV persists in tumors as episomes in form of cccDNA, or whether it is integrated into the human genome but there are several studies concluding that the cccDNA structures, found in patients infected with HBoV may be viral genomes persisting in the cells . For this reason, they thrive in cancerous cells, often giving the false impression of being oncogenic . In addiHowever, we could not detect HBoV in breast, cervical or renal tumors , althougWith the restriction that a single study can only contribute to isolated aspects of carcinogenesis without providing the necessary comprehensive view, these observations lead to the conclusion that human bocavirus appears to be a factor with an unknown role in tonsil squamous cell cancer development. Although it remains to be investigated which cell type within the tonsil acts as a target of HBoV and its replication, it seems possible that the keratinocytes are able to propagate HBoV1. Independently, it turned out that HBoV and HPV occur in the same cells and their possible synergistic interaction with other environmental or inherited factors that eventually lead to malignant progression, will make it difficult if not impossible to classify solely a single agent as the causative factor . Therefore, the present study is a good indicator for the direction of further studies, highly desirable to elucidate the role of HBoV in pathogenesis of oropharyngeal cancer. Finally, the fact that no increased MSI is observed in tonsillar tumors makes it unlikely that the virus interacts directly with the host genome but more likely persists in episomal form.The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.MH and VS performed culturing and infection experiments. MH and OS performed and analyzed FISH experiments. MH and SM were responsible for all clinical procedures. MB was responsible for all pathology-related diagnostics including tumor staging. OS and VS have planned the study. MH, OS, and VS wrote the manuscript. VS supervised the study. IF performed MSI testing and analyses.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Fragility can have a negative effect on health systems and people\u2019s health, and poses considerable challenges for actors implementing health programmes. However, how such programmes, in turn, affect the overall fragility of a context is rarely considered. The Swiss Red Cross has been active in South Sudan and Haiti since 2008 and 2011, respectively, and commissioned a scoping study to shed new light on this issue within the frame of a learning process launched in 2015.The study consisted of a document review, qualitative field research undertaken between June and August 2015 in South Sudan and Haiti, and two data triangulation/validation workshops. Semi-structured key informant interviews and focus group discussions included 49 purposively sampled participants who helped build a deeper understanding of what constitutes and drives fragility in the respective countries. Moreover, interviews and focus group discussions served to grasp positive and negative effects that the Swiss Red Cross\u2019s activities may have had on the overall state of fragility in the given contexts.Qualitative data from the two case studies suggest that the community-based health programmes implemented in South Sudan and Haiti may have influenced certain drivers of fragility. While impacts cannot be measured or quantified in the absence of a baseline , the study nevertheless reveals entry points for designing programmes that are responsive to the overall fragility context and contain more specific elements for navigating a more sustainable pathway out of fragility. There are, however, multiple challenges, especially considering the complexity of fragile and conflict-affected contexts where a multitude of local and international actors with different goals and strategies interfere in a rapidly\u00a0changing setting.Health programmes may not only reach their health objectives but might potentially also contribute towards mitigating overall fragility. However, considerable hurdles remain for aid agencies, especially where scope for action is limited for a single actor and where engagement with state structures is difficult. Thus, cooperation and exchange with other aid and development actors across the spectrum has to be strengthened to increase the coherence of aid policies and interventions of actors both within and across the different aid communities. The provision of healthcare in fragile and conflict-affected states that are home to over two billion people in 2018 remains a significant concern not only to health sector actors but also to the broader state and peace-building community , which lHealth actors agree that fragile contexts pose important challenges in implementing health interventions and programmes. There are strong interlinkages between the dimensions of fragility , health Thus far, the broader implications of such programmes and whether they link to rather top-down peace- and state-building efforts aimed at reducing fragility have not been systematically explored. Indeed, there is a lack of both research and evidence that could guide effective and community-based health interventions in such complex environments , 10, 11.There is continued interest of the donor community to invest in the health sector in fragile contexts \u201314. HencAgainst this background, the Swiss Red Cross (SRC) initiated a learning process in 2015 and commissioned two case study evaluations of community-based health programmes in South Sudan and Haiti. The objectives of the case studies were to better understand the key factors, dynamics and actors driving fragility, considering, beyond the health dimension, the socio-political and economic environment in the given contexts. This, in turn, would allow for further assessment of how the SRC\u2019s interventions interacted with the broader fragility context to ultimately determine whether a sectoral health programme can \u2013 beyond reaching its health targets \u2013 contribute to mitigating fragility in the longer term.The SRC selected South Sudan and Haiti to serve as case studies as they are among the target countries of their long-term investments. Both countries are profoundly affected by myriad aspects of fragility. According to the classification of the Fund for Peace Fragility Index in 2017 to get an overview of the area and the communities because it is the ASEC who live amongst them and know about their various needs. They know the areas much better than the CASEC\u201d (FGD EIC).As regards the Haiti case study, programme activities also attached great value to beneficiary participation and bringing together various stakeholders at the local level as a way of increasing ownership. In this sense, it could be said that the projects also helped achieve promotion of the concept of citizen participation, by providing the beneficiaries with a platform where concerns and ideas can be voiced and shared with the local authorities, albeit only at the local level. Some participants, however, stated that a wider range of local authorities should have been included in the project. While CASECs (executive branch of government) are involved and kept informed about the WASH project implementation, the Assembl\u00e9e de la Section Communale, who officially act as elected representatives of their communities (comparable to a parliamentary structure) \u201cwe will take what we get\u201d attitude and was not necessarily a priority according to the stakeholders interviewed. EIC team leaders involved in the implementation of the WASH 1 and 2 programmes, moreover, stated during a FGD that they would welcome a platform or mechanisms through which community representatives or local authorities could propose or suggest activities to international aid actors that would make sense for the communities.More generally about the inclusion of beneficiaries in decision-making processes within the context of aid programme implementation in Haiti, respondents expressed that projects proposed by international actors were rather guided by their organisations\u2019 specialisations and competencies than the priorities of the communities. The fact that beneficiaries and authorities agreed to have WASH programmes implemented in their communities stemmed, for instance, more from a \u201cThe volunteers helped to build up trust in a fragmented civil society due to tribal conflicts and weak governance [\u2026]\u201d. This may indicate that, by providing services closer to the communities through participatory approaches, building relations of trust and showing sensitivity to ethnic considerations, social cohesion was strengthened throughout the SRC\u2019s programme implementation.While the project in South Sudan had no inbuilt and explicit strategies to tackle the loss of social cohesion through decades of violent conflict, it nonetheless contributed to building trust among various local stakeholders through its activities. Four participants from South Sudan mentioned that as the SRC and SSRC showed sensitivity to ethnic consideration (staff employment and deployment practices and through capacity-building with volunteers) which helped to reconnect people: \u201c[\u2026] When we do door-to-door hygiene promotion activities, we are challenged by the beneficiaries about the fact that we are not distributing hygiene promotion items anymore . They ask us, what is the SRC doing now? This is a big problem\u201d (FGD EIC). Rather harsh reactions of WASH 2 beneficiaries reflected their much more hard-set expectations and frustrations of not receiving the same level of assistance as previous WASH 1 beneficiaries in the neighbouring areas. The SRC\u2019s WASH team in L\u00e9og\u00e2ne stated that there is strong resistance among the population towards doing \u2018unpaid\u2019 work even if the own community or vulnerable members should benefit in the long term. This impression was also confirmed during the FGDs by the beneficiaries, the local authorities and the EIC teams involved in the hygiene promotion activities. Most interviewees said that discontent and tensions over perceived inequalities of aid distribution were a significant contributing factor to frustrations and tensions among and across communities. The failure and inability of the Haitian authorities to play a leading coordination role and to develop standardised approaches for aid programme implementation and inadequate coordination mechanisms among the various aid organisations constituted a great challenge also for the SRC. According to the FGD with WASH 1 beneficiaries, \u201cthese different standards (of the various aid organisations) have created a lot of dissatisfaction and tensions among the beneficiary communities in the area\u201d.The different stakeholders interviewed in L\u00e9og\u00e2ne underlined that the spirit of self-help and mutual support had continually decreased with the arrival of international aid organisations already since the 1970s, especially with the introduction of cash-for-work projects. Social cohesion, moreover, further decreased after the 2010 earthquake following the displacement of a large number of people and in light of a massive increase of vulnerable groups and individuals among Haiti\u2019s population. Changes in attitude were also noticeable during the implementation of the WASH projects in L\u00e9og\u00e2ne according to the members of the EIC teams involved in the implementation of the activities. Particularly during WASH 2, which, marking a transition from relief to a more developmental assistance approach, no longer subsidised the construction of household latrines and discontinued the free distribution of hygiene promotion items, EICs were regularly confronted by the beneficiaries: \u201cThere is an unequal relationship between the Red Cross movement partners. Although initiatives are needs driven, sometimes there is mistrust and confusion about who has what resources for what needs and who collaborates with whom. [\u2026] The imbalance in capacities to cope with the pressure of changing needs in fragile contexts can result in failed partnerships and weak coordination\u201d.As regards SRC health programmes\u2019 potential contributions to decreasing external aid dependency in South Sudan, it needs to be recalled that the context is highly dependent on humanitarian aid as a result of long-term international humanitarian engagement. During the many years of civil war, often exacerbated by natural disasters, people lost their livelihoods and had to live in camps or move from place to place; this has gone on for so long that dependency rates are extremely high. Within the project area, the communities and authorities were often slow to react, as they were used to waiting for others to act rather than taking initiative themselves. Similarly, it was difficult to come up with a common vision for health service provision, as people were more focused on surviving for another day and unused to the concept of long-term planning. Service development depended mostly on foreign aid and on different stakeholders over a long period of time, making it difficult to achieve a coherent approach to health service delivery and impossible to avoid service fragmentation among the various aid actors. As one interviewee noted: \u201cWe have gotten used that things will happen for us, arrive for us. The first important point would be that we become actors, responsible for our own development\u201d. Challenges to reverse aid dependency are, however, huge and individual actors such as the SRC have only little scope for action. Thus, the focus of the SRC programmes in Haiti has, in practice, thus far rather been on strengthening community resilience. Overall, with the sudden drop of aid agencies in Haiti and the lack of proper exit strategies and handover in respect of beneficiaries, local organisations or government counterparts, participants stated that there was a growing concern among the communities: \u201cBeneficiaries are looking to the future with concern, as there are no jobs or livelihood strategies that guarantee them sufficient income after the aid or development programme cease\u201d (FGD WASH1).Several stakeholders in Haiti noted that long-term relief and development aid and the absence of economic reforms and international economic agreements that would benefit the entire population (instead of just the corrupt elite) had turned dependence on international aid into a stark reality. A growing disillusion has become apparent among a large part of the population who have increasingly adopted a \u2018wait-and-see\u2019 mentality, waiting for handouts. As one respondent highlighted: \u201c[\u2026] While the relationship between sectoral programmes and their linkages and possible contributions to state- and peacebuilding are under-researched, there is, unsurprisingly, also a lack of evidence on the potential impacts of health programmes on stability in fragile contexts , 8, 11. With regards to the potential of strengthening the state as a service provider through sectoral health programmes, the findings from both case studies suggest that, while difficult, it is possible and necessary to integrate specific elements in the programme design that help promote state ownership and responsibility to safeguard sustainability in the longer run. Against the backdrop of weak government structures and institutions, apparent both in South Sudan and Haiti, the SRC engagement has focused on community-based health programmes as a means to strengthening community resilience and government institutions from the local (district) up to the national level and thus promoting their role as a service provider Fig.\u00a0. This haIn the frame of the current debate on fragility and how best to operate in fragile contexts , 10, theAs concerns the lack of effective mechanisms to ensure inclusive participation and foster social cohesion in both settings, the two case studies suggest that, when working in a participatory approach with different stakeholders, including government authorities, mutual trust and dialogue may be improved. While it is difficult for organisations such as the SRC to get involved in political reform, the idea and concept of citizen participation can be strengthened vis-\u00e0-vis the various groups involved and put into practice. Another important aspect that emerged in the Haiti case study is that there is a risk that the type of programme implemented in an area is determined rather by the range of services an aid organisation has to offer than by the local needs and priorities. Ideally, there should be mechanisms that enable communities to propose their own specific projects and solutions to be considered by the aid organisations and authorities (demand driven instead of supply driven).Moreover, without coordination and agreement among government and aid actors on types and standards of interventions across the entire country, the opposite may be achieved and social cohesion may be undermined by fuelling tensions across the different beneficiary communities. It is thus vital to conceive projects with a good understanding of other aid actors, including their approaches and quality standards and to help push for coordination with other stakeholders by increasing advocacy. These findings are in line with other studies and reports on the humanitarian response in Haiti , 46, 47.Moreover, it is important to recognise the controversy of attempting to foster social cohesion as part of a development programme, especially when implemented by external actors. Since building social cohesion relies on endogenous processes of building trust and inter-group relations, external actors need to clarify roles and responsibilities with the local stakeholders in a timely and culturally sensitive manner .m\u00e9decins feuille could have been an interesting alternative to explore as a partner for the WASH programmes as they are the go-to person for every Haitian who has health worries. The m\u00e9decins feuille are also represented at the central government level, albeit only recently. One issue that needs further scientific inquiry is the \u2018Build Back Better\u2019 approach, which has become a standard in reconstruction. As seen in the case of Haiti, it is important to note that a vast majority of material used during WASH programmes was bought in Haiti, but was ultimately imported. This is also true for material and goods that were used by other aid organisations that have been active in Haiti for several years. While it is true that local markets could not cope with the increased demand of goods during the influx of hundreds of aid actors, especially during the reconstruction phase, and did not meet international quality standards, some alternative avenues could nevertheless be further explored. For instance, for the construction of latrines, the SRC used, at least partially, high-standard components towards which the local market or the families could not contribute. It should therefore be carefully evaluated whether such high-quality standards as adopted by many, including the SRC, are truly necessary or whether there is some scope for using local materials and technologies that will allow communities and authorities to sustain and replicate activities more easily. Effective coordination and not bypassing but actively involving and strengthening local counterparts are particularly crucial for staying engaged and adapting to changing situations in fragile states, but also for strengthening local and national governance structures.Entry points to help decrease overall dependency on external aid of fragile states are challenging to find for individual NGOs, which often do not have much leverage. To reduce root causes of aid dependency, other forms of engagement are warranted that include international actors from both public and private sectors, and which tackle the issue of the broader asymmetries of political and economic power in the global economic systems. Nonetheless, the scoping study found that, at the level of aid organisations, some measures should be taken to at least avoid further deterioration of the situation. Health programmes should be designed to be firmly anchored in, as far as possible, already existing and established state or community structures to ensure sustained local health service provisions. Developing intimate knowledge about the various actors present in a specific context may allow for new partnerships with less \u2018conventional\u2019 actors. For instance, it was suggested by SRC staff in Haiti that the Engaging in fragile states inevitably brings along a multitude of opportunities and challenges when it comes to adopting strategies to achieve health objectives, while balancing the need not to undermine and contribute to the overall goal of mitigating fragility , 49. In There are several issues from the case studies reported here that need particular attention in designing and implementing future health programmes in fragile and conflict-affected contexts. Indeed, fragile contexts are marked by rapidly changing (conflict) dynamics and vulnerabilities . The traFive specific recommendations emerge from this study for better designing and implementing community-based health programmes in fragile contexts as well as for improved overall strategies and policies. First, systematic and regular training for both international staff and implementing partners on emergency response, and transition to development and preparedness for different phases, should be complemented by conflict-sensitive programme management training. Awareness of the overall fragility context needs to be raised, and a more thorough understanding of the programme\u2019s interactions with the setting should be developed. This will allow project stakeholders to make informed decisions and take actions based on this newly developed and improved understanding.Second, it is crucial to anchor programmes within already existing and established community structures and involve, as much as possible, state structures from the local up to the national levels. This will help in local capacity-building and ensure the handover of responsibility and a sustainable exit strategy and may, furthermore, help smooth the transition between development and humanitarian activities and vice versa.Third, the issue of the sustainability of interventions has been raised at the beginning of project design stages with project partners, stakeholders and beneficiaries. Engaging in community dialogues about the intervention may not only enhance a community\u2019s capacity to integrate interventions into existing practices, but also to address underlying elements, such as socio-cultural/community context, or the organisational settings that can support the sustainability of interventions . ProgramFourth, regular monitoring of the often fast-changing fragility and conflict context along with the development of a good network with local stakeholders can provide crucial information for better and readily adapted project implementation; this will help ensure best possible preparedness for any changes that are not within the control of the project stakeholders.Fifth, advocacy for the establishment and strengthening of adequate coordination mechanisms has to be increased to avoid the creation of inequalities on the ground, which can lead to tensions and further deterioration of social cohesion. Moreover, exchange across the various aid actors is vital for overall more coherent approaches and policies where aid organisations interfering in a context become mutually reinforcing instead of mutually weakening.There are several limitations to this scoping study. First, the retrospective nature of the data collected and the length of the period covered (2008\u20132013 in South Sudan) may have resulted in recall bias. Second, the violent outbreaks in South Sudan shortly before the fact-finding mission may have influenced responses and limited data collection in Mayendit county. As in the case of South Sudan, only primarily programme implementers, donors or employees from SRC were interviewed, and hence the analysis does not include any beneficiary perception; consequently, there may be a bias of the study participants towards the interventions they conducted. Third, due to time constraints, only a limited number of interviews and FGDs were carried out in both countries, and the findings may thus not be representative of the perceptions of all stakeholders involved.The aim of the two case studies was to provide the SRC with an improved understanding of critical issues driving fragility in both Haiti and South Sudan and to assess whether a sectoral health programme can ultimately \u2013 beyond reaching its health targets \u2013 contribute to mitigating state fragility in the longer term. The study of the SRC\u2019s health programmes in South Sudan and Haiti suggests that opportunities and entry points for mitigating some of the identified drivers exist, albeit to varying degrees. Challenges abound, and further investigation and reflection on some of the insights are required to get a better grasp on how far and how best to consolidate immediate health objectives with the state- and peacebuilding goals. Community-based health programmes should consider possible ways of how to promote stability and improved state\u2013society relations. It is critical to improve awareness among health workers about their programmes\u2019 impact on the overall context so that informed decisions can be made. Cross-sectoral collaboration needs to be strengthened, as does coordination among relief, development and peacebuilding actors who often work simultaneously in the same contexts. Ultimately, sustained engagements and strong partnerships with both key stakeholders in the fragile settings and with different international actors are indispensable to support a sustainable transition out of fragility."} +{"text": "Inferior vena cava (IVC) is the most commonly injured abdominal vessel in blunt and penetrating abdominal traumas, and its injury carries a very high rate of mortality. Hemodynamic instability at presentation, poor response to resuscitation, the anatomical level of venacaval injury, low Glasgow Coma Score, and concomitant vascular and visceral injuries are the main factors predicting the outcome of the patient. The primary surgical intervention needed is the control of hemorrhage followed by the repair of IVC defect, which may be done by venorrhaphy, ligation, use of patch or grafts, and other complicated procedures. Each of these techniques carries its own merits and demerits. This case report is of a patient who survived an infrarenal tear of IVC caused by a firearm injury that was repaired by venorrhaphy at a hospital of Pakistan with limited cardiac and endovascular\u00a0facilities. The inferior vena cava (IVC), being retroperitoneal in position and safeguarded by multiple abdominal viscera, has pretty less likelihood of being ruptured in cases of trauma, with the reported incidence of 0.5%-5% in penetrating and 0.6%-1% in blunt abdominal trauma . In the A 28-year-old patient presented to the Accident and Emergency Department of Nishtar Hospital Multan, 30 minutes after he was allegedly shot to the abdomen by armed robbers. Examination revealed that the patient was in hypovolemic shock with the blood pressure of 90/60 mmHg, weak peripheral pulses at the rate of 140 per minute, cold peripheries, and Glasgow Coma Score (GCS) of 13/15. His chest was clear, but his abdomen was tense and diffusely guarded. On removing the pressure dressing done by the rescuing ambulance staff, an actively bleeding entry wound was observed on the left lumbar region roughly 5 cm lateral to the umbilicus and the exit wound on his back at L4 level 4 cm medial to anterior superior iliac spine. Despite immediate resuscitation with two liters of warm crystalloid, the patient remained hemodynamically unstable with minimal urine output. Within the next 20 minutes, the patient was wheeled to the operating room and explorative laparotomy was undertaken via midline incision.Intraoperative findings included a 3x3 cm perforation of anterior wall of the third part of duodenum, multiple perforations in ascending colon, and a zone I retroperitoneal hematoma. Massive hemorrhage ensued when the plane of the hematoma was entered by Kocher maneuver and by medial mobilization of colon. The field immediately got flooded with blood. Hemostasis was obtained by direct gentle pressure by assistant's finger over IVC against the spine above and below the rent. Approximately 3,000 ml of blood was suctioned out to find a 5-cm longitudinal tear in the infrarenal part of IVC, which was repaired by\u00a0direct suturing with prolene (polypropylene) 5/0. Then the duodenal perforation was repaired primarily by vicryl (polyglactin 910) 2/0, and retrocolic gastrojejunostomy was made. Perforated colon was removed, and double barrel colostomy was made.\u00a0He was transfused 2,500 ml of packed cells and 1,000 ml of plasma intra- and postoperatively. For the first three postoperative days, he remained in the intensive care unit, after which he was shifted to the general ward. Ceftriaxone and metronidazole were given as antimicrobial cover and clexane (enoxaparin) 40 mg daily for the first four postoperative days for anticoagulation. No evidence of thrombosis or extension of the defect was seen on the ultrasound done on the sixth postoperative day (Figure His chief postoperative complaint was\u00a0pain at the wound site, which was managed accordingly. The rest of his stay at the hospital remained uneventful and was discharged after 16 days of stay in the hospital. The patient remained stable on successive follow-ups, and colostomy was reversed successfully after 16 weeks.The decrease in mortality rates in patients with abdominal IVC injuries has only been marginal over the past many decades. While 30% to 50% of cases with IVC injury exsanguinate to death before reaching the hospital, another 50% die despite the maximal possible hospital care ,5. NonsuOur patient survived because of his early shifting to the hospital, control of intraabdominal bleed by tamponade effect of the hematoma, and the infrarenal part of IVC being involved. As proven in other studies, these good predictors resulted in the survival of our patient.The primary intervention needed in IVC injuries is the control of hemorrhage as active bleed badly affects prognosis ,8. As inDifferent techniques have been published in the literature for the repair of IVC, including primary repair by direct suturing i.e., venorrhaphy, ligation, grafting, portocaval shunting, atriocaval shunting, splenorenal anastomosis, and venacaval transposition ,9-11. BeIn our patient, grafting or shunting was not possible due to a lack of facilities, required equipment, and specialized vascular surgeons. Ligation could be done as a last resort, but the direct repair was preferred because the infrarenal part was accessible, and lumen diameter looked wide enough. This proved to be a better choice in concordance with other studies, which preferred direct repair to ligation. Our patient also had concomitant visceral injuries needing surgical intervention. This correlates with other studies that report that other viscera are invariably damaged by penetrating injuries reaching IVC and should be operated accordingly ,3.Despite the standardization of resuscitation and operative approaches over the last many decades, IVC injuries carry very high mortality and morbidity, even in specialized trauma units. Delayed transport of patients to the hospital, delay in diagnosis, poor resuscitation, failure control hemorrhage, and inadequate damage control surgery result in dismal outcomes. Radical improvements made in\u00a0these areas will hopefully improve the survival rates of patients with such extensive vascular injuries.In countries like Pakistan, where hospitals are far and wide, have limited facilities, and are enormously overloaded, patients with such injuries rarely survive. There is a need to establish fully equipped trauma units with specialized surgical teams to cope up with such challenging cases. Since surgeons continue to encounter vascular and abdominal trauma, open and endovascular techniques will hopefully evolve constantly, giving us promising results in the coming future."} +{"text": "Plasmodium vertebrate life cycle stages to investigate antimalarials mode of actions and underlying complex host-parasite interactions. Also, we discuss current limitations as well as make several practical suggestions for methodological improvements which could drive metabolomics progress for malaria from a comprehensive perspective.Advances in research over the past few decades have greatly improved metabolomics-based approaches in studying parasite biology and disease etiology. This improves the investigation of varied metabolic requirements during life stages or when following transmission to their hosts, and fulfills the demand for improved diagnostics and precise therapeutics. Therefore, this review highlights the progress of metabolomics in malaria research, including metabolic mapping of Excellent reproducibility alone with non-destructive property for NMR allows all kinds of sample analysis in a short period, making it an ideal candidate in large-scale metabolomics screening. With the growing demand for capturing low-abundance metabolites to meet the requirement of obtaining metabolic patterns as detailed as possible, the MS platform coupled with chromatography could be another powerful and complementary platform for simultaneous analysis of hundreds of compounds in complex biosamples with sufficient resolution, sensitivity, and reproducibility. Until now, the development trend of analytical platforms still mainly focuses on acquiring more metabolic information in a minimized sample, developing high throughput approaches with improved qualitative and quantitative accuracy.Analytical Scope\u2022Untargeted metabolomicsAs a hypothesis generating strategy, it mainly focuses on the global assessment of a broad range of both known and unknown compounds to offer either comprehensive or unbiased approaches to investigate unanticipated perturbations. Using this unbiased strategy, changes of metabolic profiles subjected to different conditions can be revealed, leading to the identification of novel metabolites or metabolic pathways. Then subsequent analysis can be performed to structural or functional characterization of these candidates.\u2022Targeted metabolomicsAs a hypothesis driven strategy, it mainly emphasizes the detection of clearly defined compounds which holds the advantages in obtaining a comprehensive understanding of the kinetic profile in specific pathways.\u2022Functional metabolomicsin vitro and in vivo assays including cell biology and clinical platform. It is able to increase the reliability for metabolomics study and contributes novel knowledge about previous findings.Functional metabolomics, as one kind of emerging concept, spans from the discovery of differential metabolites to investigate the actual function of specific compounds. Based on untargeted and/or targeted approaches, it further characterizes the functional role of selected metabolites as well as the enzymes in related pathways through both Plasmodium-infected erythrocytes. Analysis of metabolic profiles for both intracellular and extracellular metabolomes from different life stages is capable of reflecting the perturbations of specific pathways during culture. As clarified, after infection, the parasite will massively convert host metabolome into essential molecules to maintain survival during the erythrocytic asexual stage, leading to the excretion of products of erythrocyte lysis and parasite metabolism. Actually, this could have a significant effect on the host response, including events at the cellular or molecular level occurring in both host and parasites displayed distinct metabolic patterns, confirming the feasibility for acquiring unique metabolic perturbation rather than non-specific variance. For example, as a cytochrome bc1 complex inhibitor, disrupted de novo pyrimidine synthesis resulted from mitochondrial membrane potential loss has been confirmed by targeting the detection of carbamoyl-L-aspartate and dihydroorotate, the precursor and substrate of affected dihydroorotate dehydrogenase . For example, neither rare homogeneity during Plasmodium research is to be integrated with traditional system biology strategies routinely used in parasitological studies. More system biology approaches will be generate growing interest from genomics, transcriptomics, proteomics, and metabolomics. These combinatorial strategies will allow for better understanding of the biological events from a more global perspective, yielding a detailed view of its intricate networks. Such approaches can undoubtedly inform many aspects of malaria research including, but not limited to, building basic parasite biological models, development of therapeutics, identification of diagnostic biomarkers, and establishment of human pathophysiology models.One current trend toward standardization of varied metabolomics workflows is a practical way to make the technology and the data more accessible. We believe the future of metabolomics in QZ and JC were responsible for providing the idea and framework of the whole work. XY wrote the first draft of the manuscript. GF did critical revision of the article. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs.Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors ( Lung cancer is one of the most genetically complex, aggressive, and lethal solid malignancies . About 2In previous studies, we characterized the tumor suppressor gene HOPX which exerted the tumor growth inhibitory function via RAS-induced senescence ,6,7. In The epithelial membrane proteins (EMP1-3) belong to the peripheral myelin protein 22-kDa (PMP22) gene family containing at least seven members: PMP22, EMP1, EMP2, EMP3, PERP, brain cell membrane protein 1, and MP20 ,9. EMP1 Exosomes are small lipid bilayer membrane vesicles 30\u2013150 nm), containing a variety of active molecules including proteins, mRNA, microRNA, and long noncoding RNA 0 nm, con. For exaCurrently, the tumor biological role of EMP2 and its impact on miRNA expression, particularly exosomal miRNA expression, in NSCLC cells is not yet understood. In this study, we combined EMP2, exosomes, and miRNA into one research frame to investigate the functional role of EMP2, its influence on the expression of exosomal miRNAs, and the effects of EMP2-derived exosomes on tumor cell migratory behavior.We performed real-time RT-PCR to analyze EMP1-3 mRNA expression in ten NSCLC cell lines. Compared to human bronchial epithelial cells (HBEC), EMP1 was significantly downregulated in nine out of ten NSCLC cell lines and decreased expression of EMP2 was found in six out of ten cell lines, while EMP3 was upregulated in more than half (six out of ten) of NSCLC cell lines A.p = 0.004) . Of the lung tumor samples, 52.7% (29 out of 55), 67.3% (37 out of 55), and 45.4% (25 out of 55) exhibited no EMP1, EMP2, and EMP3 expression, respectively. Lower expression of EMP1 was significantly related to tumor size (= 0.004) . The expWe selected NSCLC cell lines with decreased EMP1 and EMP2 mRNA levels for demethylation testing. After treatment with a DNA methyltransferase inhibitor, 5-aza-2\u2032-deoxycytidine (DAC), EMP1 mRNA expression markedly increased in three NSCLC cell lines, including H2170, H23, and A549 , while EBisulfite sequencing (BS) was carried out to analyze the methylation status of EMP1 and EMP2 in the promoter region and exon 1. Partial DNA methylation of EMP1 was found only in the H23 cell line in the region from \u221233 to +220 bp containing nine CpG dinucleotides; no methylation was detected by BS in the EMP2 DNA .To determine the function of EMP2 in NSCLC cells, an expression vector containing the full-length cDNA of EMP2 was transfected into H1299 and H2170 in which no endogenous expression of EMP2 was found A.After stable transfection, overexpression of EMP2 in EMP2-5, EMP2-7, and EMP2-8 (H1299) transfectants, as well as EMP2-1, EMP2-2, and EMP2-3 (H2170), were confirmed by real-time RT-PCR , Westernp < 0.05, p < 0.01, or p < 0.001).As shown in p < 0.001; A colony formation assay showed that EMP2 transfectants formed significantly reduced number of colonies in comparison to mock transfectants , but not invasive ability, compared to control cells . In the cell line H2170, overexpression of EMP2 led to a significantly increased number of cells in the G1 phase (p < 0.01), and a correspondingly reduced number of cells in the S (p < 0.05) and G2/M phases (p < 0.05). The data indicated that EMP2 might be associated with G1 cell cycle arrest in H2170 cells.To determine if EMP2 influences the cell cycle, we performed flow cytometric analysis of propidium iodide-stained nuclei. As shown in p < 0.05, p < 0.01, or p < 0.001; To investigate the molecular changes during cell cycle alteration, we evaluated the mRNA expression of the genes which are involved in the cell cycle regulation such as p53, p21, p27, cyclin D1, and CCNG2, showing that ectopic expression of EMP2 resulted in significant upregulation of all of these genes in both H1299 and H2170 inhibitor PD098059. PD98059 treatment generally led to decreased expression levels of p-ERK1/2 in EMP2 transfectants, cells with EMP2-knockdown, and mock control cells compared to DMSO treatment . In lineAdditionally, altered EMP2 expression was observed to affect EMP1 and EMP3 expression levels. As shown in https://www.proteinatlas.org/ENSG00000135404-CD63/tissue) (accessed on: 13 March 2021). Additionally, transmission electron microscopy (TEM) analysis confirmed the presence of membranous vesicles in the diameter range of 40\u2013100 nm involved in cancer development in both exosomes and cell lysates from the EMP2 transfectants and mock cells was carried out. As shown in As shown above, EMP2 overexpression affected the composition of exosomal miRNA. Therefore, to determine if exosomes could be taken up by H2170 cells and if the uptake of exosomes could influence tumor migratory ability, uptake of PKH67-stained vesicles was visualized by confocal microscopy in H2170 cells incubated with EMP2PKH67 (exosome isolated from EMP2 transfectant stained with PKH67), MOCKPKH67 (exosome isolated from mock cells stained with PKH67), PBSPKH67 (PBS stained with PKH67), or a negative control (NTC) which did not contain PKH67 or exosomes. However, no notable alterations in migratory ability were observed between cells treated with exosomes from EMP2 transfectants and mock cells (data not shown).Epithelial membrane proteins (EMPs) contain four putative transmembrane domain structures and are encoded by the growth arrest-specific 3 (GAS3)/peripheral myelin protein 22 kDa (PMP22) gene family . Accumulhttps://www.proteinatlas.org/ENSG00000213853-EMP2/pathology/lung+cancer#imid_3898442) (accessed on: 13 March 2021).Expression analysis revealed a different mRNA expression pattern of EMPs with significant downregulation of EMP1 (nine out of ten cell lines) and EMP2 (six out of ten cell lines), but significant upregulation of EMP3 in more than half of the cancer cell lines compared to HBEC. During primary lung tumor sample analyses, we found that lower expression of EMP1 were significantly associated with increased tumor size. A study from Sun et al. depicted that overexpression of EMP1 in nasopharyngeal cancer cells led to markedly reduced migration and invasion . In oralEpigenetic modifications, including DNA methylation, are closely associated with cancer initiation and progression . In our Functionally, EMP family members play important roles in the control of cell growth , indicatThe diverse functions of EMPs in human cancer are mediated by numerous signaling pathways, with abnormal activation of these signal pathways shown to influence tumor biological behaviors . EMP2 prThe association between EMP2 and miRNAs in the development of human cancer, particularly in lung cancer, is not yet well elucidated. Recently, analysis of exosomal miRNAs, one of the important exosome cargos, received much attention since they deliver essential information in terms of cell-cell communication . We seleExosomes isolated from oncogenic protein-overexpressing cancer cells were found to be able to promote cell migration ,49. In oLimitations of the study remain. On the one hand, the sample size of the primary lung tumors used for the EMP1-3 protein expression analysis was small, and on the other hand, the function of EMP2 was not assessed in animal models. Additionally, only a handful of tumor pathways were investigated, given the functional diversity of EMP2 in human cancers . These dv/v) fetal bovine serum (FBS) and 1% (w/v) glutamine, and maintained in a humidified atmosphere with 5% CO2 at 37 \u00b0C.Human bronchial epithelial cells (HBEC) were purchased from Clonetics and cultured in BEG media . Ten human non-small cell lung cancer cell lines were purchased from the American Type Culture Collection and the German Collection of Microorganisms and Cell Culture . The cells were cultured in RPMI 1640 medium containing 10% on days 0, 2, and 4. Cells were then harvested for total RNA isolation. Before treatment with the MEK inhibitor PD98059, cells were serum-starved overnight. Afterwards, cells were incubated with 50 \u00b5M of PD98059 for 60 min.For construction of the cell blocks, cell pellets were collected by centrifugation at 1000 g for 3 min as described previously .\u00ae Reverse Transcription Kit . For miRNA expression analysis, reverse transcription with 300 ng of RNA was carried out using a miScript II RT Kit , according to the manufacturer\u2019s instructions.Total RNA was extracted from cell lines and exosomes using Trizol reagent according to the manufacturer\u2019s protocols. For gene expression analysis, reverse transcription with 500 ng of RNA was performed using a QuantiTect\u2212\u0394\u0394Cq method was used for quantification [Real-time RT-PCR was performed in 0.1 mL tubes on the Rotor-Gene Q with the FastStart Universal SYBR Green Master . Twenty-five/fifteen nanograms of total RNA were used for mRNA/miRNA analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for gene expression (mRNA) analysis. RNU6 was applied as an internal control for analysis of miRNA isolated from the cell lysates. For miRNA extracted from the exosomes, a cel-miR-39 Spike-In control was used as the internal control for normalization of the qRT-PCR data . The 2\u2212\u0394fication . The datGenomic DNA isolation, bisulfite modification, and bisulfite sequencing (BS) were performed as described previously . The priA total of 55 primary lung tumor specimens obtained from the University Hospital Jena from 1995 to 2001 were included for the construction of the tissue microarray. None of the patients received adjuvant radiotherapy or chemotherapy before surgery. The study was approved by the local ethical committee of University Hospital Jena (Nr: 3815-07/13).The TMA was constructed using a manual tissue arrayer . Each tumor sample was represented in the TMA by two tissue cylinders of 0.6 mm diameter, as described previously . Immunoh\u00ae 2000 DNA Transfection Reagent Protocol . An empty vector pcDNA3.1 was transfected as a control. Stable transfection was carried out as described previously [A full-length human-EMP2 cDNA purchased from GenScript was transfected into an adenocarcinoma cell line H1299 and a squamous lung cancer cell line H2170 exhibiting no endogenous expression of EMP2, following the instructions of Lipofectamineeviously .4 cells /well) were seeded in 96-well plates and incubated for 1, 3, or 5 days at 37 \u00b0C. Cell proliferation was analyzed by using a BrdU Cell Proliferation ELISA Kit and a Cell Proliferation Reagent WST-1 according to the manufacturers\u2019 instructions. The absorbance at 450/550 (BrdU) or 450/690 nm (WST-1) was measured by using the microplate reader Tecan Infinite 200 PRO for each well. The experiment was carried out in triplicate.For the proliferation assay, cells were seeded in 6-well plates. After 8\u201311 days, colonies were stained with crystal violet and colony numbers were counted.4 cells /well) were resuspended in 500 \u00b5L of RPMI1640 medium and placed in the upper transwell chamber . The upper chambers were placed in a 24-well culture dish containing 700 \u00b5L of medium with 10% (v/v) FBS. Cells were incubated at 37 \u00b0C for 24 h, and nonmigratory cells on upper chamber were removed using a moistening cotton swab. The migratory cells were then fixed with ice-cold methanol and stained with 0.5% crystal violet for 10 min at 37 \u00b0C. For the invasion assay, cells (1 \u00d7 105 cells /well) were harvested after starvation for 24 h using FBS-free medium and seeded in matrigel-coated transwell chambers . Cells were incubated at 37 \u00b0C for 24 h. After removing uninvaded cells, the invaded cells were stained for calculation.For the migration assay, cells (3 \u00d7 105 cells /well) were seeded in 6-well plates and incubated for 72 h at 37 \u00b0C. Cell pellets were collected by centrifugation at 1000 rpm for 5 min, fixed in 2 mL of ice-cold 70% ethanol, and stored at \u221220 \u00b0C overnight. After washing, cells were resuspended in PBS containing 1% glucose, 50 \u03bcg/mL RNase A , and 50 \u03bcg/mL propidium iodide, followed by incubation for 45 min at 4 \u00b0C in the dark. Cell cycle distribution was analyzed using a BD FACSCanto\u2122 II . All experiments were performed in triplicate.For analysis of the cell cylcle, cells (1.5 \u00d7 105 cells/well) and incubated overnight. The cells were transfected with EMP2 siRNA or scrambled siRNA using Lipofectamine 2000 . EMP2 expression was assessed 48 h after transfection by real-time RT-PCR and Western blotting.H1650 cells with endogenous expression of EMP2 were seeded in a 6-well plate (3 \u00d7 10Western blotting was performed as described previously . Thirty-v/v) exosome-depleted FBS (ED-FBS) . After 24 h, the cell media were collected and centrifuged at 2000\u00d7 g for 30 min to remove cellular debris. Ultracentrifugation was performed as described by Th\u00e9ry et al. [g for 30 min, then the supernatants were filtrated through a filter (0.2\u20130.8 \u00b5m pore size), followed by centrifugation at 100,000\u00d7 g for 70 min. Cell pellets were resuspended in PBS and subjected to centrifugation again at 100,000\u00d7 g for 70 min. Exosomes were then dissolved in PBS for characterization using Western blot analysis and electron microscopy.Cells were washed with PBS and cultured in RPMI 1640 medium supplemented with 5% were hydrophilized by glow discharge treatment at low pressure in air. Aliquots of 10 \u03bcL of the exosome solution were adsorbed onto hydrophilic grids for 20 s, washed twice with distilled water, then stained on a drop of 2% uranyl acetate. Samples were analyzed using a Zeiss EM902A electron microscope operated at 80 kV accelerating voltage, and images were acquired with a sharp: eye 2 k \u00d7 2 k CCD-Camera .g for 70 min. H2170 cells were seeded into 6-well plates and incubated with PKH67-labeled exosomes (500 ng/mL) for 24 h in 2 mL RPMI 1640 medium containing ED-FBS. For confocal analysis, 7500 cells were seeded in Ibidi Chambered Coverslips and treated with PKH67-stained exosomes or an equivalent volume of PBS/PKH67 as a control. After 24 h, cells were washed with ice-cold PBS and fixed with 2% formaldehyde for 10 min at RT. The slide was incubated with primary and secondary antibodies (Exosome uptake assay was carried out as previously described . Exosometibodies for 2 h 2) and Fisher\u2019s exact tests were performed to analyze the correlation between EMP1-3 protein expression and clinicopathological parameters. Student\u2019s t-test was carried out to evaluate the differences between EMP2 transfectants and mock transfectant cells. One-way ANOVA with Bonferroni\u2019s multiple comparisons test was performed to analyze exosomal uptake. Two sided p values were calculated, with p values < 0.05 considered to be statistically significant.Statistical analysis was performed using the statistical software package SPSS21 . Two-tailed chi-square (\u03c7"} +{"text": "A three-dimensional (3D)\u00a0cardiovascular magnetic resonance (CMR)\u00a0vessel wall imaging (VWI) technique based on 3D T1 weighted\u00a0(T1w) Sampling Perfection with Application-optimized Contrast using different flip angle Evolutions (SPACE) has recently been used as a promising CMR imaging modality for evaluating extra-cranial and intra-cranial vessel walls. However, this technique is yet to be validated against the current diagnostic imaging standard. We therefore aimed to evaluate the diagnostic performance of 3D CMR VWI in characterizing carotid disease using intra-arterial digital subtraction angiography (DSA) as a reference.Consecutive patients with at least unilateral >\u200950% carotid stenosis on ultrasound were scheduled to undergo interventional therapy were invited to participate. The following metrics were measured using 3D CMR VWI and DSA: lumen diameter of the common carotid artery (CCA) and segments C1\u2013C7, stenosis diameter, reference diameter, lesion length, stenosis degree, and ulceration. We assessed the diagnostic sensitivity, specificity, accuracy, and receiver operating characteristic (ROC) curve of 3D CMR VWI, and used Cohen\u2019s kappa, the intraclass correlation coefficient (ICC), and Bland-Altman analyses to assess the diagnostic agreement between 3D CMR VWI and DSA.The ICC and Bland-Altman plots indicated excellent inter-reader agreement in all individual morphologic measurements by 3D CMR VWI. Excellent agreement in all individual morphologic measurements were also found between 3D CMR VWI and DSA. In addition, 3D CMR VWI had high sensitivity , specificity , and Cohen\u2019s kappa for detecting stenosis >\u200950%, stenosis >\u200970%, ulceration, and total occlusion, respectively, using DSA as the standard. The AUC of 3D CMR VWI for predicting stenosis >\u200950 and\u2009>\u200970% were 0.998 and 0.999, respectively.The 3D CMR VWI technique enables accurate diagnosis and luminal feature assessment of carotid artery atherosclerosis, suggesting that this imaging modality may be useful for routine imaging workups and provide comprehensive information for both the vessel wall and lumen. Carotid atherosclerosis is increasingly recognized as the leading cause of ischemic stroke \u20133. PreviRecently, a three-dimensional\u00a0(3D) CMR vessel wall imaging (VWI) technique based on 3D T1 weighted (T1w)\u00a0Sampling Perfection with Application-optimized Contrast using different flip-angle Evolutions (SPACE) has become technically feasible and gained increasing attention for the diagnosis of atherosclerosis \u201326. ThisThis study protocol was approved by the local Institutional Review Board of Beijing Anzhen Hospital . Written informed consents were obtained from either the patients or their legal representatives prior to study entry.Between July 2015 and June 2018, the study prospectively recruited consecutive patients. The patients had been scheduled for DSA and interventions due to presentation with amaurosis fugax, transient ischemic attack, or suspected recent (<\u200914\u2009days) cerebrovascular ischemia and the diagnosis of \u226550% carotid artery stenosis by ultrasound. All enrolled patients underwent 3D CMR VWI within 7\u2009days before DSA. Patients were excluded if they had metallic or pacemaker implants, severe claustrophobia, renal dysfunction, or if they were allergic to iodinated contrast material. Of note, patients who were unable to tolerate the 20\u2009min CMR imaging protocol were also excluded.3, spatial resolution\u2009=\u20090.63\u2009\u00d7\u20090.63\u2009\u00d7\u20090.63\u2009mm3, matrix size\u2009=\u2009284\u2009\u00d7\u2009320\u2009\u00d7\u2009(208\u2013240) with 7.7\u201325% partition oversampling, number of slices\u2009=\u2009192, number of averages\u2009=\u20091, bandwidth\u2009=\u2009488\u2009Hz/pixel, parallel imaging (GRAPPA) factor\u2009=\u20092, and scan time\u2009=\u20097\u2009min and 3\u2009s. A trailing magnetization flip-down module was used to improve CSF signal attenuation [All examinations were performed on a 3-T whole-body CMR system using an 8-channel phased-array carotid surface coil and a 16-channel head coil . Patients were scanned in a supine, head-first position. After localization, 3D CMR VWI images were obtained using the following imaging parameters: sagittal orientation, echo time (TE)/ repetition time (TR)\u2009=\u200914/900\u2009ms, field of view (FOV)\u2009=\u2009180\u2009\u00d7\u2009200\u2009\u00d7\u2009120\u2009mmenuation . The scaDSA was performed with a digital angiography system operated by an experienced neurointerventionalist. Seldinger\u2019s technique was used to obtain patients\u2019 images of the internal carotid artery (ICA), including the anterior-posterior and lateral projections. A total of 10\u2009mL of contrast medium was injected into the ICA. The parameters were as follows: matrix size\u2009=\u20091024\u2009\u00d7\u20091024, spatial resolution\u2009=\u20092.75 LP/mm, and FOV\u2009=\u200930\u2009cm.One neurointerventionalist with 17\u2009years of experience (T.Z.) reviewed the DSA images and performed morphologic quantification at a workstation . The 3D CMR VWI images was reviewed and measured on a workstation equipped with the multiplanar reconstruction (MPR) functionality.Image quality (IQ) of CMR VWI was first assessed by an experienced reviewer (Y.W.) using a 4-point scale, where 1\u2009=\u2009poor , 2\u2009=\u2009marginal , 3\u2009=\u2009good , and 4\u2009=\u2009excellent . Examples of IQ at different scale levels are provided in Online Figure Each ICA was divided into 7 segments C1-C7) using the Bouthillier classification . Data co-C7 usingP-values <\u20090.05 were considered to indicate statistical significance.Continuous variables are presented as mean\u2009\u00b1\u2009standard deviation (SD), and categorical variables are reported as frequencies (percentages), as appropriate. Kolmogorov-Smirnov tests were used to access whether the measurements were normally distributed. Paired t-tests were used to test for the significant differences between readers or methods. The sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and Cohen\u2019s kappa with 95% confidence intervals of the 3D CMR VWI images were calculated for diagnosing total occlusion, carotid artery stenosis, and ulceration using DSA images as a reference. For continuous variables, the agreement between 3D CMR VWI and DSA, as well as the agreement between two CMR readers were assessed by using the intraclass correlation coefficient (ICC) and Bland-Altman analyses. The level of the agreement was categorized as follows: poor (ICC\u2009=\u20090\u20130.20); fair (ICC\u2009=\u20090.21\u20130.40); moderate (ICC\u2009=\u20090.41\u20130.60); good (ICC\u2009=\u20090.61\u20130.80); and excellent (ICC\u2009=\u20090.81\u20131.00) . Receiven\u2009=\u20091) and severe motion artifacts (n\u2009=\u20091). In total, 65 patients were included in the final analyses. A detailed flow chart of the study design is presented in Fig.\u00a0A total of 67 patients successfully underwent 3D CMR VWI and DSA. The overall image quality for 3D CMR VWI was 3.13\u2009\u00b1\u20090.73. Two patients were excluded from the study due to severe blood flow artifacts at the carotid bifurcation , negative predictive value (NPV), and Cohen\u2019s kappa of 3D CMR VWI for diagnosing total occlusion were 100.0, 98.0, 98.5, 93.5, 100.0% and 0.96, respectively. A case example of the total occlusion assessed by DSA is shown in Fig. Using DSA as the reference, the sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and Cohen\u2019s kappa of 3D CMR VWI for detecting stenosis >\u200950%, stenosis >\u200970%, and total occlusion are presented in Table\u00a0This study confirms the accuracy of the 3D CMR VWI technique based on the 3D T1w SPACE sequence for the diagnosis of carotid atherosclerosis, particularly the assessment of various luminal features, in symptomatic patients with \u226550% carotid artery stenosis. We found that the 3D CMR VWI technique is highly consistent with DSA when measuring the lumen diameter of the CCA and segments C1-C7 segments, as well as the stenosis diameter, stenosis degree, and reference diameter. Additionally, this technique showed excellent accuracy for diagnosing total occlusion, ulcerative lesions, and moderate to severe stenosis. Hence, our results justify the increasing use of 3D CMR VWI as a reliable, accurate, and non-invasive imaging modality for diagnosing carotid artery disease.Analysis of inter-observer reproducibility is an essential requirement for validation of an imaging modality. Zhang et al. previous3, and achieves good flexibility with regard to image visualization, which thereby enables the visualization of plaques located in the tortuous and deep-seated intracranial carotid. Furthermore, dedicated CSF signal suppression is implemented in this technique, which can help to visualize the outer boundaries of the vessel wall [In this study, we evaluated the agreement between 3D CMR VWI and DSA. The morphologic measurements, including the lumen diameter of the CCA and segments C1-C7, and the stenosis diameter, stenosis degree, reference diameter, and lesion length, as measured by 3D CMR VWI, displayed excellent accordance with those measured by DSA (ICC\u2009>\u20090.84). One possible reason for the excellent performance of the 3D CMR VWI technique is that a non-selective excitation radio-frequency pulse has been used to shorten the echo time, which helps improve the SNR of images , 24, 27.sel wall , 27.However, our study showed some systematic differences between CMR VWI and DSA. Firstly, the lumen diameter tended to be underestimated by VWI at larger sized vessel segments. This discrepancy might be caused by the underlying differences in image acquisition and measurement. Specifically, DSA is a projection-based imaging modality and the distance between two parallel vessel boundaries from the imaging perspective is inherently the largest possible diameter of the vessel from that perspective. However, diameter measurement on VWI is performed from reformatted cross-sectional images and derived by averaging two perpendicular hand-drawn straight-line distances. Either of these lines is necessarily aligned with the DSA perspective, and more importantly, each distance may not represent the true diameter at that angle if the straight line misses the lumen center, thus underestimating the diameter. The averaging procedure would further make the measured diameter smaller, particularly at larger vessel segment that are not necessarily round. Secondly, the lesion lengths measured by 3D CMR VWI are about 3\u2009mm longer than those measured by DSA in arteries with >\u200950% stenosis. The potential reason may be the distal portion of a wedge-shaped plaque being regarded as the reference on DSA , 34.In our study, 3D CMR VWI showed a poor ability to identify pseudo-occlusion and small ulcerations (width\u2009<\u20091.5\u2009mm), compared with DSA. Mistaking pseudo-occlusion for total occlusion on 3D CMR VWI might be explained by the fact that DSA has much better spatial and temporal resolution than 3D CMR VWI. This is in line with the findings of a previous study . MissingZhao et al. performeThere were several limitations in this study. Firstly, measurements of 3D CMR VWI were acquired using MPR. Therefore, discrepancy from mismatched slices of the carotid artery lesions between 3D CMR VWI and DSA may have compromised the agreement analysis. Secondly, 3D CMR VWI was still susceptible to motion and residual blood flow artifacts which occurred at certain anatomical locations, including carotid bifurcations and petrous and lacerum segments of ICAs, which can interfere with the correct assessment of the condition . For exa3D CMR VWI can be used to accurately assess carotid atherosclerosis with high spatial resolution and large anatomical coverage in a single scan, suggesting that the imaging modality may serve in routine imaging workup with comprehensive information for both the vessel wall and lumen.Additional file 1: Online Figure 1. Example images of image quality at different scale levels. A, image quality score\u2009=\u20094; B, image quality score\u2009=\u20093; C, image quality score\u2009=\u20092; D, image quality score\u2009=\u20091."} +{"text": "This article has been corrected: Due to errors during figure assembly, the wrong transwell image was used for the lower left panel of 1084-1095. https://doi.org/10.18632/oncotarget.6744Original article: Oncotarget. 2016; 7:1084\u20131095."} +{"text": "ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory\u2010confirmed severe acute respiratory syndrome coronavirus\u20102 (SARS\u2010CoV\u20102), and there has been speculation that SARS\u2010CoV\u20102 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctivas, 12 melanomas, seven squamous cell carcinomas, and seven papilloma samples, were analyzed using high\u2010throughput RNA sequencing to assess messenger RNA (mRNA) expression of the SARS\u2010CoV\u20102 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS\u2010CoV\u20102 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS\u2010CoV\u20102 via these mediators unlikely.SARS\u2010CoV\u20102 is assumed to use angiotensin\u2010converting enzyme 2 ( Patients infected with SARS\u2010CoV\u20102 develop symptoms of fever, cough, and fatigue that can quickly progress to pneumonia. SARS\u2010CoV\u20102 is highly infectious and transmitted mainly by inhaling droplets or aerosols released by an infected individual and possibly by a feco\u2010oral transmission route.ACE2 expression across human tissues, including lung, stomach, ileum, colon, liver, and kidney,ACE2. It was, therefore, speculated that SARS\u2010CoV\u20102 infection may depend on coreceptors or other auxiliary membrane proteins.ENPEP), alanyl aminopeptidase (ANPEP), and dipeptidyl peptidase 4 (DPP4),ACE2 or potential auxiliary proteins and coreceptors such as TMPRSS2, ANPEP, DPP4, and ENPEP.Similar to other coronaviruses, SARS\u2010CoV\u20102 uses angiotensin\u2010converting enzyme 2 (ACE2) protein to gain entry into cells.ACE2 and its potential coreceptors such as ANPEP, DPP4, ENPEP, and TMPRSS2 in transcriptome data of conjunctival samples. We show that ACE2 and its potential coreceptors are not significantly expressed in the human conjunctiva, which suggests a very low probability of SARS\u2010CoV\u20102 propagation in the conjunctiva.In view of the lacking data, this study aims to explore the expression levels of 22.1ACE2 and associated molecules required for cell entry by SARS\u2010CoV\u20102, existing datasets of 38 conjunctival samples from 38 patients were included in this study. The samples comprised twelve healthy conjunctival tissue specimens from twelve subjects who underwent buckle or 20\u2010gauge vitrectomy surgery for retinal detachment as well as twelve conjunctival melanoma, seven conjunctival squamous cell carcinoma and seven conjunctival papilloma specimens that had been treated at the Eye Centre of the University Freiburg from 1996 to 2017. Another eight healthy conjunctival samples from eight subjects undergoing retinal detachment surgery were included for immunohistochemical staining. All specimens contained conjunctival epithelium and subconjunctival connective tissue. All tissue samples were analyzed in an anonymized manner. Institutional Review Board (IRB)/Ethics Committee approval had been obtained for specimen acquisition, use, and data generation.To obtain information on the transcription of 2.2Formalin fixation and paraffin embedding of ocular samples were performed immediately after tissue excision according to routine protocols, as previously described2.3http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ last access on 11/19/2019). Reads were mapped to the human reference genome with RNA STAR Galaxy Version 2.6.0b\u20102ACE2, CD81, LDLR, ANPEP, DPP4, ENPEP, TMPRSS2, KRT19, and KRT13 were extracted from the data and plotted as boxplots using ggplot2.Sequencing data were uploaded to and analyzed on the Galaxy web platform 2.4ACE2 immunohistochemistry was performed as previously described33.1In total, the transcriptome of 38 samples from 38 patients was analyzed in this study. Furthermore, eight healthy conjunctival samples from eight patients were included for immunohistochemistry. A detailed summary of the patient's characteristics is summarized in Table\u00a03.2KRT19 and KRT13 was 4715.6 transcripts per million and 29.6 TPM (IQR: 9.6\u201082.3), indicating significant expression of these markers in our samples . The SARS\u2010CoV\u20102 receptor ACE2, however, showed hardly any expression in conjunctival samples . Of the 38 analyzed conjunctival samples, 35 samples (92.1%) revealed no ACE2 transcripts, and three of them negligible amounts of 0.1, 1.1, and 1.7 TPM, respectively. A subgroup analysis of all conjunctival samples revealed that ACE2 expression was not only insignificant in healthy conjunctival tissue (one sample with 0.1 TPM) but also in altered conjunctival samples such as conjunctival papilloma (one sample with 1.7 TPM), squamous cell carcinoma or melanoma (one sample with 1.1 TPM). In contrast to the above\u2010mentioned data, we found considerable expression of CD81 and LDLR in conjunctival samples was obtained in all 38 conjunctival samples. As a reference, the expression of known conjunctival epithelial markers was analyzed. The median expression for Immunohistochemical staining of eight healthy conjunctival samples confirmed a negligible ACE2 expression in all analyzed samples , ENPEP and TMPRSS2 . Subgroup analysis revealed, that expression of DPP4, ENPEP, TMPRSS2, and ANPEP was comparably low in healthy conjunctiva when compared to diseased conjunctiva . Conjunctival samples do not significantly express the SARS\u2010CoV\u20102 receptor ACE2 but high amounts of the conjunctival markers KRT13 and KRT19. Each dot represents one sampleClick here for additional data file."} +{"text": "Clostridium Difficile Colitis (FCDC), however, occasionally, FCDC is also treated with partial colectomies. The purpose of the study was to identify the outcomes of partial colectomy in FCDC cases.The Total Abdominal Colectomy (TAC) is the recommended procedure for Fulminant P value of\u2009<\u20090.05 is considered as statistically significant.The National Surgical Quality Improvement Program database was accessed and eligible patients from 2012 through 2016 were reviewed. Patients 18\u00a0years and older who were diagnosed with FCDC and who underwent colectomies were included in the study. Patients\u2019 demography, clinical characteristics, comorbidities, mortality, morbidities, length of hospital stay and discharge disposition were compared between the group who underwent partial colectomy and the group who underwent TAC. Univariate analysis followed by propensity matching was performed. A P\u2009>\u20090.99). There were no differences found in the median [95% CI] hospital length of stay (LOS) , post-operative complications , and discharged disposition to home ( 33.8%\u00a0vs. 43.1%) or transfer to rehab between the TAC and partial colectomy groups.Out of 491 patients who qualified for the study, 93 (18.9%) patients underwent partial colectomy. The pair matched analysis showed no significant difference in patients\u2019 characteristics and comorbidities in the two groups. There was no significant difference found in mortality between the two groups (30.1% vs. 30.1%, The overall 30\u00a0days mortality remains very high in FCDC. Partial colectomy did not increase risk of mortality or morbidities and LOS.Level IV.Observational cohort. Clostridium Difficile Colitis (FCDC) is one of the severe conditions of colon that is associated with very high mortality ) for continuous variables, and frequency and percentage for categorical variables). Then, the group that underwent total abdominal colectomy was compared with the group that underwent partial colectomy on patient\u2019s demography, diseases severity, comorbidities, and outcomes. The Wilcoxon Rank Sum test was used for continuous variables, and the Chi-square test was used for the categorical variables.P value was reported for each test. A P value of\u2009<\u20090.05 was considered an indication of statistical significance. Statistical analysis was performed using the R language [The propensity score for total abdominal colectomy was calculated for each patient and the one-to-one matching was performed using the \u201cnearest neighbor\u201d as the matching method to pair a subject who had TAC with the subject who underwent partial colectomy. The propensity score matching was performed using the R package \u201cMatchIt\u201d . The prolanguage .P\u2009=\u20090.03). TAC group presented with higher percentage of life threatening of ASA class and found to have higher percentage\u00a0of patients with history of steroid use . Significantly higher proportion of patients in TAC group mounted severe leukocytosis (\u2265\u200920\u2009\u00d7\u2009109/L) patients underwent total abdominal colectomy. Only 93 (18.9%) patients underwent partial colectomy. Fifty one out of 93 (54.83%) patients underwent right sided colectomy and remaining 42 (45.17%) patients had left sided colectomy. Approximately 84% of patient underwent colectomy for toxic colon and approximately 16% of patients underwent colectomy for perforation. The median [IQR] age of the patient who underwent partial colectomy was 66 [55\u201375], the male and female distribution was almost split equally with slight increase of male dominance,\u2009~\u200953% and about 77% of patients were Caucasians. There were significant differences found between the two groups, TAC and partial colectomy groups, regarding the presence of septic shock prior to surgery \u00a0P values were\u2009>\u20090.05 , septic shock prior to surgery (55.9% vs. 52.7%) and ventilator dependent respiratory failure (37.6% vs. 29%) and comorbidities, all P\u2009>\u20090.99). The median [95% CI] hospital length of stay between the TAC and partial colectomy was . There was no significant difference found between the groups, TAC and partial colectomy, regarding the discharged disposition to home (33.8%\u00a0vs. 43.1%) or transfer to rehab . A recent NSQIP database study included all patients with FCDC who underwent colectomies from 2007 through 2015 [Very few prior studies have examined the comparison of mortality outcome of TAC with partial colectomy. A study examined the surgical mortality of the FCDC found that patients underwent partial colectomy had the worse outcome than the TAC . The majugh 2015 . The stuContrary to above studies, our study included relatively recent NSQIP data set and used propensity-matched analysis, which is better modality for observational study . Our resThe study was done from the NSQIP database; however, the database lacks the detailed information of the some of the patients\u2019 characteristics,\u00a0the timing of the contraction of the clostridium difficile colitis, progression to FCDC and the timing of the colectomy from the time of identification of the FCDC. We used the most recommended analysis method of observational study, the propensity score matching. However, that method does not take into account any unobserved or unmeasured variables that may have influenced the results.The surgical mortality of FCDC remains high. Total abdominal colectomy was the procedure of choice and adapted by majority of surgeons. Partial colectomy did not increase the risk of 30\u00a0days mortality or morbidity. The discharge disposition of patients to home or rehabilitation were same regardless of the patient underwent TAC or partial colectomy.Implications. If the disease pathology limited to one area of the colon, partial colectomy can be an alternative procedure for the FCDC patients."} +{"text": "According to the MNA-SF, only 7.3% of subjects had malnutrition, and 28.2% were at risk of malnutrition. The agreement between the MNA-SF score and the GLIM criteria were observed in only 22.3% of the population. The sensitivity and specificity of MNA-SF against the GLIM criteria were fair . The area under the curve (AUC) was 0.77, indicating the fair ability of MNA-SF to diagnose malnutrition. Based on the present study results, the best solution may be an optional replacement of the screening tool in the first step of the GLIM algorithm with clinical suspicion of malnutrition.Up to 28% of elderly residents in Europe are at risk of malnutrition. As uniform diagnostic criteria for malnutrition have not been formulated, in autumn 2018, the Global Leadership Initiative on Malnutrition (GLIM) presented a consensus on its diagnosis. According to the consensus, the diagnosis of malnutrition requires a positive screening test result for the risk of malnutrition, and the presence of at least one etiologic and one phenotypic criterion. This study aimed to assess the diagnostic performance and accuracy of the Mini Nutritional Assessment\u2014Short Form (MNA-SF) against GLIM criteria. The analysis involved 273 community-dwelling volunteers aged \u2265 60 years. All participants were screened for malnutrition with the MNA-SF questionnaire. Next, the GLIM phenotypic and etiologic criteria were assessed in all subjects. Based on the presence of at least one phenotypic and one etiologic criterion, malnutrition was diagnosed in more than one-third of participants ( The global number of older adults with malnutrition is constantly increasing. The main causes of this phenomenon are demographic changes, the increasing proportion of elderly subjects in society, and the higher risk of poor nutritional status in elderly people compared to younger subjects ,2,3. TheOlder people are at particular risk of malnutrition due to age-related physiologic changes, multimorbidity, psychological and socio-economic problems ,10,11. M2 and the other was unintentional body mass loss >10% in any time or >5% in 3 months, associated with a low BMI or low muscle mass (defined as fat-free mass index (FFMI) < 15 kg/m2 in women and <17 kg/m2 in men) [As uniform diagnostic criteria for malnutrition have not been formulated, in 2015, the European Society for Clinical Nutrition and Metabolism (ESPEN) introduced two proposals on how to diagnose malnutrition. The first one was body mass index (BMI) < 18.5 kg/mThe first step of the diagnostic procedure consists of a screening test with a validated questionnaire, such as Nutritional Risk Screening-2002 (NRS-2002), Mini Nutritional Assessment\u2014Short Form (MNA-SF), Malnutrition Universal Screening Tool (MUST), or Subjective Global Assessment (SGA). In elderly subjects, the MNA-SF is most frequently used to assess the risk of malnutrition ,17,18,19This study aimed to evaluate the diagnostic performance and accuracy of the MNA-SF against phenotypic and etiologic GLIM criteria and to emphasize the importance of clinical suspicion in the diagnostics of malnutrition.n = 178) accounted for 65.2% of the total study population. Each subject provided written informed consent prior to the study conducted under the Declaration of Helsinki. The study protocol was approved by the Bioethical Committee of the Poznan University Medical Sciences, Poland .We performed a cross-sectional study involving 273 community-dwelling volunteers \u226560 years of age (60\u201398 years) living in Poznan, Poland. Women \u2265 7) and the ability to maintain a standing position . Subjects were excluded from the study if they had an artificial cardiac pacemaker, metal implants, or peripheral edemas\u2014conditions that preclude body composition assessment with the bioimpedance method (BIA). Other exclusion criteria were impairment in oral intake and active cancer.In all participants, we performed a screening for malnutrition risk with the MNA-SF. Malnutrition was subsequently assessed with the GLIM criteria.In the present study, we diagnosed malnutrition based on the GLIM criteria in subjects with at least one etiologic criterion and at least one phenotypic criterion, regardless of the MNA-SF score.(1)Reduced food intake was acknowledged in persons who declared at least moderate decrease in the number of meals or amount of food in the past three months;(2)Disease burden/inflammatory condition was acknowledged if at least one chronic disease associated with chronic or periodic inflammation or elevated C-reactive protein levels (>10 mg/L) were identified in the subjects\u2019 medical records.Etiologic criteria:(1)Weight loss was acknowledged in persons who declared unintentional weight loss of at least 1 kg in the past three months;(2)2 in subjects at age <70, and <22 kg/m2 in participants \u226570 years of age;Low body mass index: <20 kg/m(3)2)) derived with the BIA method . The ALM index below cut-off points for the Polish population (5.6 kg/m2 in women and 7.4 kg/m2 in men) were acknowledged low muscle mass [Low muscle mass (LMM). Muscle mass was assessed based on the appendicular lean mass (ALM) index divided by the squared height . The parameters used for further analysis were weight, BMI, skeletal muscle mass, segmental lean mass, fat mass, and percentage of fat mass.According to the GLIM criteria, the severity of malnutrition was graded based on phenotypic criteria .The Mini Nutritional Assessment\u2014Short Form is one of the questionnaires recommended as a screening tool for malnutrition risk by the GLIM experts. Its psychometric properties were assessed in the present study.The MNA-SF contains six questions regarding (1) decrease in food intake, (2) weight loss, (3) mobility, (4) psychological distress or acute disease, (5) neuropsychological problems, and (6) body mass index. The maximum score is 14. A score < 12 indicates a risk of malnutrition, and <7 indicates malnutrition. The cut-off point of 11 was used in the analysis of the MNA-SF psychometric properties against the GLIM criteria.n) and percentages (%). The normality of all quantitative variables were verified with the Shapiro\u2013Wilk test. The equality of the variances was checked with Levene\u2019s test. We used the following tests to assess differences between the groups, Student\u2019s t-test for variables with normal distribution and equal variances, Cochrane\u2013Cox test for variables with normal distribution and lack of homogeneity of variance, and Mann\u2013Whitney U test for data with non-normal distribution. Qualitative variables were compared between groups with a chi-square test with the Yates correction for continuity.Quantitative data are shown as means and standard deviations (SDs), and qualitative variables as numbers , negative predictive value (NPV), as well as the area under the ROC curve (AUC) and the kappa coefficient. Sensitivity reflects the percentage of subjects with malnutrition (based on the GLIM diagnostics criteria) who had a positive result on a screening test . Specificity refers to the percentage of subjects without malnutrition (based on the GLIM criteria) who had negative screening test results . Sensitivity and specificity were classified as good (>80%), fair (50\u201380%), or poor (<50%) . The PPVp-value below 0.05 was considered significant. The statistical analysis was performed with STATISTICA 12.0 software .A The study population consisted of 287 subjects aged \u226560 years (mean age of 72.1 \u00b1 7.7 years). On average, the subjects had three chronic diseases. One-third of the study population was living alone (33.3%), and one out of seven participants was a rural resident (15.4%).p of borderline significance) and had lower MNA-SF scores (p < 0.0001). They had lower body mass (p < 0.0001), a lower BMI (p < 0.0001), and lower muscle mass as assessed with the ALM index (p < 0.0001). They also had lower body fat mass (p < 0.0001), lower muscle mass (p < 0.0001), and lower fat-free mass (p < 0.0001) compared to subjects without malnutrition.The most frequent phenotypic criterion was weight loss, present in more than one out of four subjects . Every fBoth the sensitivity and specificity of the MNA-SF against the GLIM criteria were fair . The AUC was 0.77, indicating a fair diagnostic value of MNA-SF to diagnose malnutrition. Accuracy was 71.4%, and the agreement between the MNA-SF score and the GLIM criteria was fair (kappa coefficient 0.33). The positive predictive value was 62.9%, while NPV was 76.1%.The MNA-SF is a simple and cheap tool widely used to assess nutritional status in elderly subjects . It has According to the recognized standards, a tool of high diagnostic value should have sensitivity and specificity higher than 80% and an AUC above 0.8 . The valn = 32) had malnutrition or risk of malnutrition based on the MNA-SF score. The diagnosis of malnutrition was confirmed with GLIM criteria in 23 subjects (57.5%). Therefore, the sensitivity of MNA-SF was very high (100%), but the GLIM sensitivity was fair (50.0%) [Only two studies investigated the psychometric properties of the MNA-SF against the GLIM criteria in elderly subjects ,24. MatsPsychometric properties against the GLIM criteria of some other screening tools for malnutrition, such as the Malnutrition Screening Tool (MST), MUST, SGA, and NRS-2002, were also investigated in various populations, including elderly subjects . Clark en = 70). The sensitivity and specificity of the screening tools were as follows: MUST, 64.3% and 81.7%; SGA, 95.7% and 14.6%; NRS-2002, 47.1% and 75.6%. The AUC for the three questionnaires was 0.80, 0.77, and 0.69, respectively. Based on their study, Bellanti et al. concluded the MUST questionnaire is the most suitable screening tool for diagnosing malnutrition in hospitalized elderly subjects [The psychometric properties of the MUST, SGA, and NRS-2002 questionnaires as screening tools in the GLIM algorithm were assessed by Bellanti et al. [The limitations of the study are as follows: We utilized information about weight loss and reduced food intake given by the participants in the MNA-SF questionnaire for the phenotypic GLIM criteria. We used the following items: (a) has food intake been declined over the past 3 months due to loss of appetite, digestive problems, chewing, or swallowing difficulties? (answer options: severe decrease in food intake/moderate decrease in food intake/no decrease in food intake); (b) weight loss during the last 3 months? (answer options: weight loss greater than 3 kg/does not know/weight loss between 1 and 3 kg/no weight loss). Many older adults have a problem noticing even short-term changes in body weight, which might have biased their answers.In terms of study strengths, the present study is one of the first attempts to assess the diagnostic performance of the MNA-SF as a screening tool for malnutrition diagnosis according to the GLIM criteria. It was conducted in a large group of community-dwelling elderly subjects. Previous studies included people of various ages or were The GLIM experts recommend assessing malnutrition phenotypic and etiologic criteria exclusively in subjects with a positive screening test result. However, as Da Silva Passos and De-Souza first noticed in their Letter to the Editor in 2019 , the GLI"} +{"text": "Failure of immunologic homeostasis and resultant hepatocyte destruction in autoimmune hepatitis (AIH) is likely the result of environmental triggers within a permissive genetic architecture.We aimed to identify risk factors associated with AIH in a well\u2010phenotyped AIH cohort.We prospectively collected environmental questionnaires from 358 AIH cases and 563 healthy controls. Response frequencies were compared using logistic regression, adjusting for age at recruitment, sex and education.P\u00a0<\u00a0.001) and recurrent UTI (more than 1 per year) compared to controls. Female cases more frequently had ever used oral contraceptives , fewer pregnancies and less often used hormone replacement therapy compared to controls . Current smoking was more prevalent in cases , yet no difference according to historical smoking behaviours was observed. Finally, cases were less likely to have history of mumps and rheumatic fever , but reported higher vaccination frequency to chicken pox (38% vs 28.1%), measles (66.5% vs 39.3%), mumps (58.7% vs 34.6%), rubella (55.3% vs 32.7%), pertussis (59.8% vs 40.1%) and pneumococcus (47.2% VS 39.4%) (P\u00a0<\u00a0.002).AIH cases were more likely to ever have a urinary tract infection (UTI) (53.6% vs 33.9%, Environmental factors are important in AIH pathogenesis. Replication of these findings and prospective examination may provide\u00a0new insight into\u00a0AIH onset and outcomes. AIHAutoimmune \u200bHepatitisGRACEGenetic Repository of Autoimmune Liver Disease and Contributing ExposuresPBCPrimary Biliary CholangitisPSCPrimary Sclerosing CholangitisUCUlcerative Colitis1Broken immune tolerance and failure of immunologic homeostasis underlying autoimmune hepatitis (AIH) pathogenesis are likely the result of environmental triggers in the background of a permissive genetic architecture. Similar to other autoimmune diseases, the Human Leucocyte Antigen (HLA) locusFor other autoimmune diseases, liver diseases, primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), environmental risk assessments have provided a more complete understanding of disease risk, onset and possible modifiable factors.In this study, we aimed to identify associations between AIH and lifetime environmental exposures as well as lifestyles, personal and family histories using the Genetic Repository of Autoimmune Liver Diseases and Contributing Exposures (GRACE) study.22.1The GRACE study contains consecutively recruited AIH patients (cases) from Indiana University.Controls were serially recruited from the Mayo Clinic Division of General Internal Medicine during annual visits for preventive medical examination. Control exclusion criteria included evidence of prior or current liver disease ) or any historical abnormal liver enzyme levels. All participants in the study had informed consent collected by study staff. This study included cohorts that adhere to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Institutional Review Board of Indiana University and Mayo Clinic.2.2The study questionnaire was developed by the\u00a0Mayo\u00a0Clinic\u00a0Survey\u00a0Centre and data acquired via this instrument among other autoimmune liver disease cohorts have been published previously.2.3P\u2010values were calculated using logistic regression models, adjusting for age, gender and education. Statistical analysis was conducted in R version 3.6.2 and a P\u2010value of .05 was considered significant.Information collected by the questionnaires was double entered into a SAS database. Each question was summarized by descriptive statistics for both AIH cases and controls. The 33.1P\u00a0=\u00a0.045), younger at study completion (median =\u00a055 yrs (years) old, (43 (quartile 1(Q1)), 64 (quartile 3(Q3)), P\u00a0<\u00a0.001)), less likely Caucasian and have more college (60.3%) but less professional educational training beyond college than healthy controls. Race remained statistically different between cases and controls after adjustment for age of recruitment, gender and level of education.Demographic comparison of AIH cases and controls is shown in Table\u00a03.2P\u00a0<\u00a0.001) , as was having recurrent UTIs . Other medical conditions were more common in AIH cases as well, including symptomatic gastroesophageal reflux disease , shingles and history of gallstones compared to cases. Frequencies of other assessed medical conditions did not differ the two groups (Table\u00a0History of ever having a urinary tract infection (UTI) was more frequently observed in cases compared to controls compared to controls (Table\u00a0Reproductive history was completed by 284 AIH cases and 414 controls Table\u00a0. Age at ls Table\u00a0.P\u00a0=\u00a0.006) compared to controls, but the duration of OC use between groups was similar (Table\u00a0P\u00a0<\u00a0.001). Among cases with a history of OC use, 93.4% were exposed to OC prior to AIH diagnosis. Ever use of hormone replacement therapy (HRT) was lower in cases compared to controls . Among those that used HRT, AIH cases tended to be younger at HRT start compared to controls vs 49 yrs , P\u00a0=\u00a0.001). Among cases with a history of HRT use, and 91.1% were exposed to HRT prior to AIH diagnosis.AIH cases were more likely to have ever used oral contraceptives (OC) (Table\u00a0P\u00a0<\u00a0.001) but not at home . However, AIH cases were more likely than controls to be current regular smokers . AIH cases were less likely than controls to be current drinkers . Among individuals that ever consumed alcohol, AIH cases had less duration of consumption vs 40 , P\u00a0<\u00a0.001) compared to controls.AIH cases were not any more likely to have every regularly smoked cigarette as controls and rheumatic fever compared to controls (Table\u00a0P\u00a0=\u00a0.005), measles , mumps , rubella , pertussis and pneumococcus , lupus , multiple sclerosis , RA , Sjogren's syndrome , T1DM and colon cancer compared to control FDRs. AIH cases of SDRs were more likely to have RA and T1DM compared to control SDRs.Detailed history of autoimmune diseases, gastrointestinal and liver conditions among both first\u2010degree and second\u2010degree relatives (FDRs and SDRs) was collected in cases and in controls Table\u00a0. The AIH3.7*03:01, worse overall survival and increased development of hepatocellular cancer.Prior disparities among genders in AIH studies have revealed that male patients have higher frequencies of DRB14In this study, patients with AIH compared to controls were more likely to report history of UTI, recurrent UTIs, gallstones, multiple vaccinations , younger age of OC start, ever OC use and current smoking. Moreover, AIH cases were less likely to report a history of working with a smoker, mumps, rheumatic fever, pregnancies and HRT. Finally, patients with AIH often reported concurrent autoimmune diseases as well as FDR with autoimmune diseases as well as SDR .et al reported the only factor that independently increased AIH risk was exposure to antibiotics within 12\u00a0months of diagnosis among 72 AIH cases.et al study could represent an enrichment of drug\u2010induced AIH. Mechanistically, this likely represents a subtype of AIH as immunosuppression removal can be completed in a high proportion of patientsThe GRACE study is the largest cohort of AIH cases to date that has systematically evaluated environmental risk factors associated with the development of disease, but observations in this study should still be interpreted in the context of the study design. Significant differences between case and control responses in this study do not necessarily indicate disease causation, but must be interpreted in the hypothetical web of interactions (environment and genetics) that underscore disease development. Despite a systematic approach in this study, we also realize that there are many unmeasured environmental exposures starting at conception. However, we utilized a questionnaire that has been implemented in other autoimmune liver disease cohorts and collected environmental exposures that have either biologic plausibility in autoimmunity or have been associated with other chronic diseases. To our knowledge, only one other studyP\u2010value =.022) but less likely to ever work with a smoker . These conflicting results may have multiple explanations that could include a dose effect of smoking, variable known/unknown genetic predispositions in our cohort or highlight the tenuous balance between pro\u2010 and anti\u2010inflammatory attributes of smoking among populations. Even despite the relative consistency of smoking associated with PBC in prior studies, published cohorts have shown conflicting assessments.Smoking has not been assessed previously in AIH patients, yet it has been well documented in other autoimmune liver diseases PSC and PBC. In PSC, smoking most recently has been shown to only associate with disease among patients that also had concurrent IBD.Notably, a novel and possibly most important finding in this study was the observation of increased UTI diagnosis (53.6% vs 33.9%) and recurrent UTIs (23.5% vs 15.9%) among AIH cases versus controls Table\u00a0. IncreasGiven female predominance of AIH, we must carefully consider the results of documented exposures that correlate with gender. Our data revealed that AIH cases were more likely to ever use OC, start OC younger (but similar duration), have fewer lifetime pregnancies and less likely to have ever used HRT compared to controls. The observed difference in AIH protection (HRT) and risk (OC use) observed between these oestrogen containing pharmaceuticals may relate to the inherent timing of administration. Infrequent HRT use may be driven by behaviours of cautious providers aware of patients known liver disease ). Similar observations (less HRT use) have been observed in females with PSC,*03:01 in males Historically, only in few, historical and rare cases, has autoimmune pathology been firmly linked with particular vaccines. An observed polyradiculoneuritis associated with the 1976\u20101977 vaccination campaign against swine influenza is one such example.We admit that given the observational approach to our study, there are limitations worth considering. Despite being one of the first systematic approaches to examine environmental risk in well\u2010phenotyped AIH case development, the size of our study remains moderate. We were dependent on patient\u2010reported data for many outcomes of interest which may introduce recall bias. Furthermore, this case\u2010control study does not include a disease control group for direct comparison, yet two other liver disease populations (PBC and PSC) have utilized this questionnaire previously.In summary, we report novel associations between a variety of exogenous risk factors and AIH using an established AIH cohort (GRACE study) and healthy controls. History of UTI, recurrent UTI, ever and earlier start of OC, current smoking and a variety of immunizations were associated with the development of AIH. On the other hand, history of mumps, HRT use, history of working with a smoker were protective against AIH. Despite plausible biologic mechanisms, these associations do not indicate disease causation. Yet, counselling on smoking cessation, screening for other autoimmune illnesses at diagnosis and during follow\u2010up and careful antibiotic stewardship remain important and are currently actionable by clinicians. We advocate for multicentre prospective studies of AIH to better understand modifiable risk factors in the setting of a permissive genetic framework.5This study included cohorts that adhere to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Institutional Review Board of Indiana University and Mayo Clinic.All included authors declare no outside interests that are directly or significantly related to this paper.Craig Lammert: assembly/interpretation of data, drafting and revision of manuscript. Sai Chalasani: assembly of data, review of manuscript. Bryan McCauley: statistical analysis, critical revision of statistics and results. Elizabeth J. Atkinson: statistical analysis, critical revision of statistics and results. Konstantinos N. Lazaridis: Conception of study and design, collection and assembly of data, drafting and approval of final manuscript version.All participants in the study had informed consent collected by study staff.Table S1\u20103Click here for additional data file."} +{"text": "The current wireless communication infrastructure has to face exponential development in mobile traffic size, which demands high data rate, reliability, and low latency. MIMO systems and their variants are the most promising 5G wireless communication systems technology due to their high system throughput and data rate. However, the most significant challenges in MIMO communication are substantial problems in exploiting the multiple-antenna and computational complexity. The recent success of RL and DL introduces novel and powerful tools that mitigate issues in MIMO communication systems. This article focuses on RL and DL techniques for MIMO systems by presenting a comprehensive review on the integration between the two areas. We first briefly provide the necessary background to RL, DL, and MIMO. Second, potential RL and DL applications for different MIMO issues, such as detection, classification, and compression; channel estimation; positioning, sensing, and localization; CSI acquisition and feedback, security, and robustness; mmWave communication and resource allocation, are presented. The main problem with the current wireless communication infrastructure is its dependence on either increasing the spectrum or densifying the cells to obtain the targeted area throughput. Unfortunately, such resources are scarce and are approaching their saturation level in near future. Moreover, increasing the spectrum or densifying the cells increases the hardware price and latency. The spectral efficiency, which can enhance the area throughput, has remained essentially unchanged during the fast development in the wireless systems. Therefore, a wireless access technology must improve the wireless area throughput without increasing the spectrum or densifying the cell to fulfill the essentials requirements of the wireless carriers.MIMO is the most promising and fascinating wireless access technology that is able to deliver the requirements of 5G and beyond networks. The performance of communication systems has been enhanced thanks to the use of MIMO schemes. MIMO utilizes many dimensions that account for multiple antennas, multiple users, and time and frequency resources. Multi-User MIMO (MU-MIMO) is a MIMO system with one BS equipped with many antennas and provides service to more than one downlink user in a one-time slot. Massive MIMO (Ma-MIMO) further extends MIMO systems by using hundreds and thousands of antennas at BS to enhance throughput and spectral efficiency. MIMO technology is integrating bandwidth, radios, and antennas to achieve higher speed as well as capacity for the incoming 5G . The abiRecent advances in AI and ML, specifically the success of RL and DL, In the areas of MIMO systems, recently RL and DL have been applied as an emerging solution to address many challenges efficiently. These technologies have introduced many solutions to different aspects of MIMO communication such as signal detection, classification, and compression; positioning, sensing, and localization; security and robustness; mmWave communications; and resource allocation. AI-enabled MIMO communication is an optimal tool that can give wireless systems the flexibility, intelligence, and efficiency needed to handle the scarce radio resource efficiently and enable top quality of service to the customers. In our work, we are making a research contribution by providing a comprehensive overview of the potential applications of RL, DL, and the mixture of both in different areas of MIMO communication.The remaining of the paper is organized as follows. This section provides a brief introduction to RL, DL, and MIMO systems.RL is a sub-field of Machine Learning where an agent interacts with an environment to achieve a goal, and learning takes place interaction-after-interaction. This section introduces some basic mechanisms and terminology. A detailed presentation of RL can be found in .Agent is an entity that interacts with a given environment by performing actions, and receives a feedback from the environment after any action selected as described in In RL, an Policy is a strategy that indicates to the agent which action to select in every state of the environment. The agent has to learn the optimal policy, that is, the one that maximizes the cumulative reward over the long run.A MDP. A MDP is a tuple S is a set of states; A is a set of actions; a in s at time t), s to state a was taken, respectively; and A RL problem is defined as a RL schemes are normally categorized into two major types: model-free and model-based algorithms. Model-based RL algorithms need a precise description of the dynamics of the environment in terms of the state-transition probability distribution. These methods compute the optimal policy by solving systems of equations. Whereas, model-free RL techniques are adopted when there is not a precise description of the model or its solution would be too complicated. This class of algorithms interacts directly with the environment (or with an emulator of it) using Trial&Error schemes to learn the optimal policy. In inverse RL ,41,42, wDL has revolutionized many research areas with its ability to learn better models from huge volumes of data . Such teThe premises is that the performance of a DNN is generally superior to the one of a classic ANN at the cost of greater training time that, however, can be reduced by using advanced hardware and/or special techniques . The desConvolutional Neural NetworksConvolutional Neural Networks (CNNs) are ANNs with a much higher number of layers and nodes. They are typically adopted for images classification. An CNN needs less preprocessing as compared to other classification schemes. Relevant filters are used in CNNs to capture the temporal and spatial dependencies in the image ,47. The Recurrent Neural NetworksRNN is another important architecture for DL. Differently from CNN, where the layers are sequentially connected, in a RNN there are some nodes whose output is reported back in the input of a previous node. In this way, the network is capable of remembering some information time-related. Recurrent Neural Networks (RNNs), indeed, are massively applied for time-series analysis and prediction in a configuration called LSTM .Generative Adversarial NetworksA Generative Adversarial Network (GAN) consists of two sub-networks\u2014the discriminator and the generator, where the later produces the content and the former validates it. GAN adopts feed-forward and relies typically on CNNs .Deep Belief NetworksDeep Belief Networks (DBNs) are generative neural networks with undirected connections between some layers called Restricted Boltzmann Machines. These layers can be trained using a very fast unsupervised learning algorithm called Contrastive Divergence. In DBNs, hidden patterns are learned globally, while in every layer of other deep nets complex patterns are learned progressively .AutoencodersAutoencoders are applied to reduce the dimension of data and to detect problems. The first layer in an autoencoder is an encoding layer, whereas the transpose of it is used as a decoder. Training is unsupervised and the Regression/Classification problems may be addressed and optimized using Stochastic gradient descent. Input data are translated to a latent space denoted by the encoder, as given below:Input data are reconstructed from the latent space denoted by the decoder as described below.r is the decoded output and it will be identical to input xAutoencoders can be represented essentially by the below equation. Radial Basis Function Neural NetworksRBFNN is a type of ANN that utilizes radial basis functions (RBF) as activation functions. The output of the RBFNN is a linear combination of RBF of the neuron parameters and inputs. RBNN has only one hidden layer which is known as a feature vector. Training in RBNN is faster than in MLP but classification in RBNN takes more time than MLP.MIMO is a wireless technology that employs multiple antennas at transmitters and receivers sides to communicate more data simultaneously as can be seen in MIMO communication uses a multi-path, which is a natural radio-wave phenomenon. Multi-path-transmitted signals may bounce off objects, like ceilings, walls, etc., and arrive at the receiver multiple times at different times and angles. Before the inception of MIMO, interference occur due to multi-path, which can slow down the communication. MIMO technology with multi-path, however, uses smart transmitters and receivers with the addition of spatial dimension that grants enhanced performance and range.MIMO improves signal-capturing of the receiver by empowering antennas to combine signals coming from multiple paths at different times. Smart antennas get the benefit of spatial diversity technique that makes surplus antennas useful. The antennas can increase the range by adding receiver diversity when outnumbered spatial streams.Single User MIMOSingle user MIMO, or multi-antenna MIMO, has more than one antenna both at the transmitter side and receiver side. There are some special variants of MIMO such as \u201cMultiple-input single-output\u201d (MISO) (only one antenna at the receiver side), \u201cSingle-input multiple-output\u201d (SIMO) (single antenna at the transmitter side), and a special scenario when transmitter as well as receiver have one antenna is called SISO .Multi-user MIMOMulti-User MIMO MU-MIMO has been considered in recent WiMAX and 3GPP standards as a candidate technology by various companies such as Freescale, Nokia, Philips, Huawei, TI, Ericsson, Qualcomm, Intel, and Samsung. MU-MIMO systems are more suitable for low-complexity mobile phones with a few receiving antennas, while single-user MIMO\u2019s are more suitable for complex devices having many antennas due to their higher per-user throughput. Moreover, enhanced MU-MIMO uses advanced precoding and decoding techniques.Cooperative MIMO (CO-MIMO)Cooperative MIMO (CO-MIMO) employs multiple surrounding BS to jointly transmit and receive signals to and from users. This prevents inter-cell interference in neighboring BS as may be experienced with traditional MIMO systems.Macrodiversity MIMOMacrodiversity MIMO is a type of space diversity approach that applies many transmit/receive BS for coherent communication with single/multiple users. It is possible that users are distributed in a coverage area that has the same resources of time and frequency ,54,55. TMassive MIMOMassive MIMO Ma-MIMO is a scheme where the number of terminals is inferior to of BS antennas . The maxThis section will review some recent related surveys, their contribution, and limitations as well as the contribution of our work.The list of related survey papers is shown in Another survey paper focused The mmWave communication is discussed in ,63 usingAn encyclopedic overview of the application of DL in wireless and mobile networking is presented in . The autThe work in presentsThe Ma-MIMO systems are highlighted in while prA survey paper on DRL techniques that was proposed to solve emerging problems in communications and networking is presented in . The autAn overview of the DL-based cybersecurity applications for mobile and wireless networks is presented in . The autDifferent cases of 5G wireless communication including cybersecurity, energy efficiency, caching, Ma-MIMO, channel coding, and modulation classification based on AI techniques are discussed in . The autAlthough there are few review papers related to applications of ML, RL, and DL reported in In this paper, we have presented a comprehensive state-of-the-art on the application of RL and DL in different aspects of MIMO communication such as detection, classification, and compression; channel estimation; positioning, sensing, and localization; CSI acquisition and feedback, security and robustness; mmWave communication; and resource allocation. We have also listed the contribution of some survey papers, their limitations for MIMO communication areas, and our contribution in This section presents a comprehensive review of the applications of DL and RL in different areas of MIMO.A growing interest has been developed in recent years to apply DRL techniques to optimize operations of wireless network . IncorpoAn RL-based cognitive BF scheme for co-located MIMO radars is proposed in . The RL-A DNN architecture is presented in in the cA quasi-static flat channel with many antennas is considered for detection of multilevel modulation symbols using DNN in and partThe authors of have stuA likelihood function learning technique for MIMO systems having a one-bit analog-to-digital converter is proposed in using anA letter in considerTwo DNN based detectors, i.e., damped belief propagation (BP) and max-sum by unfolding damped BP and max-A MPD using DNN is introduced in by modifThe performance of a large-scale MIMO receiver is investigated in . The depImplementation methodologies for conventional MIMO transmitters of DL-based signal detection are presented in . The autTwo scenarios of channel information at the receiver using the DL model are considered in for upliThe BP-MMSE-based algorithm is proposed in that iniA DL empowered detector is designed in that aftA likelihood ascent search detection approach based on CNN is given in by usingRL techniques, in particular, the DL method, have proven important tools to improve the accuracy of channel estimation to enhance the performance of Ma-MIMO utilizedDirection-of-arrival and channel estimation using RL techniques for Ma-MIMO systems are discussed in . AuthorsChannel estimation for double directional channels with limited feedback for mmWave Ma-MIMO is done in . The BS Channel estimation with few-bit ADCs in MIMO systems is the focus of the work in . A DNN iA new concept of channel mapping in space and frequency is introduced in . The mapA new channel estimation framework is proposed in with theA DL-enabled method for channel estimation to improve the performance of the Ma-MIMO system is given in . The useThe theoretical analysis on the use of DL in MIMO system channel estimation is discussed in . AuthorsChannel estimation when both low-resolution ADCs and hybrid analog\u2013digital processing in Ma-MIMO systems are utilized is considered in . The autA DL-enabled architecture for channel estimation and joint pilot design of MU-MIMO channels is given in . The pilA decision-directed method for channel estimation using DNN is developed in for the Channel estimation with received SNR feedback is investigated in to estimChannel estimation using DL for MIMO systems for the case of multi-cell and interference-limited is taken into account in . The MIMEffective interference cancellation and reliable channel estimation are necessary for improving the performance of MIMO UAC systems. A single carrier MIMO UAC has been considered in to studyA MIMO receiver with inadequate pilots in a fast fading channel is considered in as well A task-oriented quantization model with scalar ADCs based on DL for MIMO channel estimation is designed in . The proA joint design of channel estimator and pilot signal based on data-driven DL methodology for wideband Ma-MIMO systems are designed in . High-diMIMO systems are meeting the growing need for reliable and faster communications in wireless systems having a large number of terminals. MIMO systems can also be used to estimate the position of a terminal utilizing multipath propagation in multiple antennas. CSI-based user positioning systems using DL technology achieving a high accuracy without any overhead have shown great potential in the MIMO system. These systems are capable of positioning indoor users in both LoS and non-LoS environments with reasonable accuracy. Moreover, with the availability of smart devices and recent development in AI-based wireless systems , localizFingerprinting has been an emerging research area for indoor positioning due to its location-related characteristics and ubiquity . A novelA deterministic \u201cuplink-to-downlink mapping function\u201d is revealed in and a spThe achievable rate of the mmWave Ma-MIMO system can be improved by minimizing training pilots using the DL model for sensing joint channel and hybrid precoding framework . AccordiA channel sounder framework that can measure multi-subcarrier and multiantenna CSI different propagation, antenna geometries, and frequency bands are introduced in . The arcMIMO user positioning using DL techniques that are based on only the OFDM complex channel coefficients is examined in . The proAccuracy of localization using CSI is improved by combing multi-layer perceptron NN and K-nearest neighbors techniques in . Both scIndoor localization based on DL and CSI for 28 MIMO antenna is considered in . The inpDynamic localization using predictive RNN for Ma-MIMO systems is investigated in . The autMIMO communication systems are a major enabler of the excessive throughput requirements NGN, e.g., 5G due to its ability to serve many users at a time with high energy and spectral efficiency. However, the MIMO system requires timely and accurate CSI, which is obtained by a training process including the transmission of a pilot, estimation of CSI, and feedback . The traA DL approach for secure MIMO communications has been employed in by exploTime-varying features have been exploited in using twA CSI feedback mechanism based on bi-directional reciprocal channel properties and limited feedback is introduced in . The Ma-A CS and DL empowered CSI feedback framework for FDD Ma-MIMO communication system is propose in where thThe work in presentsA Bayesian CS-based feedback scheme for MIMO systems is considered in . The wirA compression technique for channel state matrix using DL that is consists of convolutional layers and quantization and entropy coding blocks come after is proposed in . The modCSI reporting which is important for MIMO system transceivers to acquire energy efficiency and high capacity in FDD form is considered in using DLSpatial correlation-based CSI compression feedback for FDD Ma-MIMO systems is considered in and a DLImplicit feedback framework based on DL is applied in to inherA scheme to protect the DL-based CSI feedback process from white-box adversarial is presented in . The autA CNN-based network known as aggregated channel reconstruction model is constructed in to enhanAn uplink-assisted downlink channel acquisition architecture using DL is presented in to minimSuperimposed coding and DL techniques are combined in for CSI A DL-based scheme for the prediction of downlink CSI in Ma-MIMO FDD systems is presented in . The givDeep learning techniques have been utilized recently for interesting and important applications in mmWave and Ma-MIMO systems. DL provides solutions to hard optimization problems due to its powerful capabilities of learning unknown models.A DL-based compressed sensing channel estimation and quantized phase hybrid precoder design scheme is proposed in for the An integration of ML and coordinated BF scheme is employed in to enablA generic dataset for mmWave/Ma-MIMO channels known as the DeepMIMO dataset was introduced in with twoA hybrid processing model for the mmWave Ma-MIMO system is normally employed to minimize cost and complexity. However, channel estimation may be very challenging through this method. The work in presentsBeamforming gains are achievable and high path loss is preventable in mmWave systems by deploying a large number of antennas. However, with a large number of antennas, implementation of digital precoders is difficult due to hardware constraints and, at the same time, analog precoders have performance limitations. Hybrid precoding is an important task in mmWave MIMO systems to reduce the cost and complexity as well as to obtain a sufficient sum rate. Greedy methods or optimization techniques have been used in literature for hybrid precoding. However, these schemes depend on the channel data quality and achieve sub-optimum performance, and also give higher complexity. Therefore, in the next few paragraphs, we will discuss few proposals on the use of DL-based hybrid precoding. Some alternating algorithms for hybrid precoding for mmWave may be seen in .Two schemes, i.e., CNN-based and equivalent channel precoding, are designed in for mmWaA DL empowered hybrid precoding architecture is proposed in that useHybrid precoding for MU-MIMO system is done in using CNFully convolutional denoising (FCD) AMP scheme is introduced in by combiA DL-CS channel estimation method consisting of channel reconstruction and beamspace channel amplitude estimation is given in . The NN Beamforming for mmWave MIMO systems is considered in using thUplink mmWave Ma-MIMO systems are considered in using BaRadio Resource management and userDeep learning has been used in for the A universal DRL-based framework is given in for acceA DQN-based technique is used in for resoThe optimization of sum spectral efficiency for multi-cell Ma-MIMO communication systems is considered in for a diThe optimization of downlink beamforming via DL techniques is for the MISO system is done in ,270. TheA pilot assignment technique using DL for a Ma-MIMO system equipped with a large number of antennas is given in to improAn intelligent algorithm to optimize the performance of the Ma-MIMO beamforming is introduced in . The proThe results of some recent works indicate that deep learning models can learn a form of decoding algorithm, instead of only a classifier. These studies provide that DL architectures can be applied for improving a standard belief propagation decoder, although having large example space . MoreoveMetric normalized validation error is introduced in to invesA practical issue of imperfect successive interference cancellation decoding for real-world NOMA system is considered in . A novelA novel physical layer DL scheme for MIMO system using an autoencoder is developed in by usingA DL-based channel prediction scheme is developed in to enablloped in . The arcChannel characteristics are predicted in using thAn RL actor\u2013critic enabled fault-tolerant control problem is discussed in for MIMOA robust adaptive NN control is discussed in for a geANC for uncertain MIMO nonlinear systems is introduced in when inpAn adaptive backstepping control approach is proposed in uncertaiThis section presents statistics about the surveyed papers and an analysis of their impact.First, we have grouped the literature in four different periods as can be seen in Next, we have considered different categories. Therefore, Finally, as a further detail of data reported in This section discusses the surveyed papers by highlighting the strengths and limitations, along with future directions.It is noted from results of channel estimation that DL frameworks show better results in the large SNR environments, but are outperformed by standard iterative message passing methods. Moreover, adopted DL schemes are suitable for high MIMO dimensions, but converged for comparatively small MIMO dimensions for decoding . TherefoMany problems are associated with 5G Ma-MIMO technology, which can be mitigated through the use of DL. For example, it is difficult to estimate the accuracy of the channel by employing conventional estimation schemes and with a suitable number of pilots. The performance of the low complexity least-squares estimator is not satisfactory. On the other hand, MMSE channel estimation is relatively complex . TherefoIn addition, within linear systems, the DL estimator is near to the linear MMSE estimator, but it outperforms this last one significantly when there is a nonlinear signal model. However, it is sensitive to the training data quality and estimation performance may degrade appreciably when the data in real regimes distribute wider than that of the training data . Both thThe computing capabilities and limited memory of wireless devices may not suite for complex DL algorithms. A considerable amount of time is required for collecting a sufficient number of samples and for training DL models. This can become a critical impediment to implement such algorithms on wireless devices with limited storage and power. Moreover, some MIMO applications need on-fly sampling as well as real-time processing, which makes training difficult. Obtaining more samples and long-time model training results in slow feedback. Therefore, DL models should be designed to acquire optimal accuracy with fewer samples and shorter periods.User privacy is the most important concern of service providers. While deploying DL in wireless systems, one challenge is how the training is enabled on a dataset associated with users without sharing the input data and exposing personal data to risks. It is important to have a security scheme to speed up the integration of DL in MIMO communications.Security of DL networks is another challenge, as NNs are prone to adversarial attacks. These attackers may affect the process of training by inserting fake training data, which can reduce the accuracy of the DL models. This may lead to a wrong design, which can affect the overall performance of the network. Research in the security of RL and DL techniques remains shallow.Multiple antennas are required to mitigate high path loss and to achieve BF gains in mmWave systems. However, it is difficult to employ digital precoders in presence of many antennas due to hardware constraints. Similarly, the performance of analog precoders is limited . TherefoDespite the remarkable progress of DL in communication, still, research efforts are required in many directions to ease the integration of RL and RL. The acceleration of DNN alongside distributed RL systems, cloud computing, faster algorithm, and advanced parallel computing provides an opportunity for 5G to develop the intelligence in its communication systems to provide ultra-low latency and high throughput. Recently, some efforts have been done in DNN acceleration . The accTechniques such as knowledge distillation , projectFurthermore, more exploration of the acceleration of these models may have a significant impact on the adoption of DL to develop intelligence in MIMO systems. Integration of DL and RL in MIMO communication systems can speed up by data collection and subsequent cleansing. With the availability of datasets, researchers can build and test their architectures. Therefore, efforts are required to build systems that can produce datasets.We presented a comprehensive review of the applications of RL and DL to different issues of MIMO communication systems. First, we presented an introduction of both classes of AI methods and MIMO systems. Afterward, we have presented various applications of such AI technologies in MIMO systems. Then, we have analyzed the impact of research papers in the field. Finally, we have outlined open issues, some limitations, and future research directions."} +{"text": "Oncolytic bovine herpesvirus type 1 (BoHV-1) infection induces DNA damage in human lung adenocarcinoma cell line A549. However, the underlying mechanisms are not fully understood. We found that BoHV-1 infection decreased the steady-state protein levels of p53-binding protein 1 (53BP1), which plays a central role in dictating DNA damage repair and maintaining genomic stability. Furthermore, BoHV-1 impaired the formation of 53BP1 foci, suggesting that BoHV-1 inhibits 53BP1-mediated DNA damage repair. Interestingly, BoHV-1 infection redistributed intracellular \u03b2-catenin, and iCRT14 -1H-pyrrol-3-yl]methylene]-3-phenyl-2,4-thiazolidinedione), a \u03b2-catenin-specific inhibitor, enhanced certain viral protein expression, such as the envelope glycoproteins gC and gD, and enhanced virus infection-induced DNA damage. Therefore, for the first time, we provide evidence showing that BoHV-1 infection disrupts 53BP1-mediated DNA damage repair and suggest \u03b2-catenin as a potential host factor restricting both virus replication and DNA damage in A549 cells. Bovine herpesvirus type 1 (BoHV-1) and herpes simplex virus-1(HSV-1) are members of the family Herpesviridae and the subfamily Alphaherpesvirinae . AlthougDNA damage poses a constant threat to cells. If left unrepaired, it may give rise to deleterious mutations and genome instability, consequently leading to cell death or disease . Double-Accumulating studies have indicated that tumor suppressor p53-binding protein 1 (53BP1) plays an essential role in the maintenance of genome integrity and stability by orchestrating the repair of double-strand breaks (DSBs) . For DSB\u03b2-catenin is involved in the regulation of multiple cellular functions, such as adhesion and gene transcription. Mutations and overexpression of \u03b2-catenin are closely associated with many cancers, including lung cancers . Thus, \u03b2In this report, we demonstrated that BoHV-1 infection decreased 53BP1 expression and reduced 53BP1 foci formation. Moreover, for the first time, we found that \u03b2-catenin is involved in the restriction of viral protein expression and virus-induced DNA damage in A549 cells.In response to DSBs induced by many toxic elements, such as X-rays, \u03b3-radiation, and UV-light irradiation, \u03b3H2AX levels increase and accumulate in the DSBs to develop specific foci under fluorescence microscopy, which are generally considered molecular markers of DNA damage . NotablyThe expression of viral glycoprotein gC was detected at 24 and 36 hpi D. In supCounting the number of \u03b3H2AX foci by fluorescence microscopy is a useful method to assess the extent of DSBs quantitatively . Thus, aTaking these data together, the assessment of \u03b3H2AX foci under fluorescent microscopy can be used for the examination of DNA damage induced by BoHV-1 infection in A549 cells.53BP1-mediated NHEJ events play a critical role in the repair of DSBs . RecruitWe then detected the steady-state protein expression of 53BP1 by Western blot analysis. At 24 and 36 hpi, 53BP1 protein levels were consistently reduced. Similar to mock-infected cells, 53BP1 protein levels were approximately reduced to 34.1% and 52.6% at 24 and 36 hpi, respectively C. Since Accumulating studies have indicated that \u03b2-catenin signaling had effects on DNA damage response and thatIFA assay indicated that, in the mock-infected cells, intracellular \u03b2-catenin was evenly distributed in the cytosol and the nucleus and highly expressed on the plasma membrane . HoweverOur data indicated that the increased formation of \u03b3H2AX foci could be used to assess DNA damage induced by BoHV-1 infection. iCRT14 is a known \u03b2-catenin-specific inhibitor that inhibits \u03b2-catenin-dependent transcription . To undeThese data suggested that \u03b2-catenin has the capacity to restrict DNA damage, and the \u03b2-catenin-specific inhibitor iCRT14 promotes DNA damage elicited by virus infection.To understand why iCRT14 promotes DNA damage elicited by virus infection, we investigated whether it had effects on viral gene expression. For this aim, A549 cells infected with BoHV-1 were treated with either DMSO control or iCRT14, and the viral proteins gC and gD were detected. At 24 and 36 h after infection, a gC-specific band was detected in virus-infected cells, which was enhanced when treated with iCRT14 A,B. QuanExposure to various genotoxic stresses, such as UV treatment, leads to a sharp peak of \u03b3H2AX and accumulation of \u03b3H2AX at the sites of damaged DNA that forms highlighted foci with IFA assay. \u03b3H2AX foci or \u03b3H2AX protein levels are widely detected to measure DNA damage . MechaniInterestingly, we found that a subset of \u03b3H2AX foci was also located at the cytoplasm B. ThoughWe found that virus infection led to the depletion of 53BP1 and reduced 53BP1 foci , which m\u03b2-catenin stimulates BoHV-1 productive infection in CRIB (BVDV resistant MDBK cells) cells, and the \u03b2-catenin-specific inhibitor iCRT14 significantly inhibits productive viral infection in cell cultures . We alsoMoreover, we found that the \u03b2-catenin-specific inhibitor iCRT14 enhanced DNA damage , which iOf note, \u03b2-catenin is overexpressed in diverse tumors, including lung cancers, and it plays an important role in the stimulation of tumorigenesis, invasion, metastasis, and chemotherapy resistance, and, therefore, is regarded as a novel target of cancer therapy. Unexpectedly, our data indicated that BoHV-1 oncolytic activity was not relying on the suppression of \u03b2-catenin signaling. In contrast, it seems that \u03b2-catenin had limiting effects on viral gene expression. However, our data indicated that a \u03b2-catenin-specific inhibitor, such as iCRT14, could efficiently enhance virus infection-induced DNA damage and viral gene expression, raising a possibility that combination therapy with both BoHV-1 and iCRT14 might achieve synergism of antitumor efficacy via the induction of DNA damage, which needs extensive studies in vivo in the future.In summary, in this study, for the first time, we provided insight into how \u03b2-catenin was involved in BoHV-1 productive infection and virus-infection induced DNA damage in human tumor cells. We also provided the first evidence that the \u03b2-catenin inhibitor, iCRT14 promoted viral gene expression (gC and gD). Therefore, \u03b2-catenin may restrict virus replication and inhibit DNA damage in human lung carcinoma A549 cells. A549 cells and MDBK cells were both purchased from the Chinese Model Culture Preservation Center . They were maintained in DMEM medium supplemented with 10% fetal bovine serum . BoHV-1 was pro\u00ae-conjugated goat anti-rabbit IgG (H + L) .The antibodies used in this study were as follows: 53BP1 polyclonal antibody (pAb) , phosporylated-H2A.X (\u03b3H2A.X) monoclonal antibody (mAb) , \u03b2-catenin mAb , BoHV-1 gC mAb , BoHV-1 gD mAb , \u03b2-Actin mAb , HRP (horseradish peroxidase)-labeled goat anti-mouse IgG , HRP-labeled goat anti-rabbit IgG , and Alexa Fluor 488A549 cells in 60 mm dishes were mock-infected or infected with BoHV-1 at an MOI of 1 for 24, 36, and 48 h. Cells were lysed with an RIPA buffer that was supplemented with protease inhibitor cocktail . Cell lyThe intensity of the detected protein bands was quantitatively analyzed with free ImageJ software. \u03b2-Actin was tested, along with individual proteins for protein loading control. The band intensity was initially normalized to \u03b2-Actin, and the fold change after infection was calculated. Protein levels in mock-infected cells were set to 1.\u00ae-conjugated goat anti-rabbit IgG (H + L) for 1\u2009h in the dark. After three washings, nuclei were stained with DAPI . The cells were covered with coverslips using an antifade mounting medium . Images were captured using a confocal microscope (Leica Camera).A549 cells seeded into 2-well chamber slides were mock-infected or infected with BoHV-1 (MOI = 0.1) for 36\u2009h. Cells were fixed with 4% paraformaldehyde prepared in PBS (pH\u20097.4) for 10\u2009min at room temperature, permeabilized with 0.25% Triton X-100 in PBS (pH\u20097.4) for 10\u2009min at room temperature, and blocked with 1% BSA in PBST (PBS+ 0.1% Tween-20) for 1\u2009h followed by incubation with the antibodies against \u03b3H2A.X (1\u2009:\u2009800 dilution), \u03b2-catenin (1\u2009:\u2009800 dilution), and 53BP1 (1\u2009:\u2009500 dilution) in 1% BSA in PBST for 12\u2009h at 4 \u00b0C. The cells were washed three times with PBST and incubated with Alexa Fluor 488DNA damage was evaluated through an alkaline comet assay (single-cell gel electrophoresis) according to the method described elsewhere with modification . In brie"} +{"text": "Financial toxicity, the material and psychological burden of the cost of treatment, affects 30\u201350% of people with cancer, even those with health insurance. The burden of treatment cost can affect treatment adherence and, ultimately, mortality. Financial toxicity is a health equity issue, disproportionately affecting patients who are racial/ethnic minorities, have lower incomes, and are <\u200965\u2009years old. Patient education about treatment cost and patient-oncologist cost discussions are recommended as ways to address financial toxicity; however, research shows cost discussions occur infrequently . Our overall goal is to address the burden of financial toxicity and work toward health equity through a tailorable education and communication intervention, the DISCO App. The aim of this longitudinal randomized controlled trial is to test the effectiveness of the DISCO App on the outcomes in a population of economically and racially/ethnically diverse cancer patients from all age groups.Patients diagnosed with breast, lung, colorectal, or prostate cancer at a NCI-designated comprehensive cancer center in Detroit, MI, will be randomized to one of three study arms: one usual care arm (arm 1) and two intervention arms (arms 2 and 3). All intervention patients (arms 2 and 3) will receive the DISCO App before the second interaction with their oncologist, and patients in arm 3 will receive an intervention booster. The DISCO App, presented on an iPad, includes an educational video about treatment costs, ways to manage them, and the importance of discussing them with oncologists. Patients enter socio-demographic information and indicate their financial concerns. They then receive a tailored list of questions to consider asking their oncologist. All patients will have up to two interactions with their oncologist video recorded and complete measures at baseline, after the recorded interactions and at 1, 3, 6, and 12\u2009months after the second interaction. Outcome measures will assess discussions of cost, communication quality, knowledge of treatment costs, self-efficacy for treatment cost management, referrals for support, short- and longer-term financial toxicity, and treatment adherence.If effective, this intervention will improve awareness of and discussions of treatment cost and alleviate the burden of financial toxicity. It may be especially helpful to groups disproportionately affected by financial toxicity, helping to achieve health equity.ClinicalTrials.gov NCT04766190. Registered on February 23, 2021 Financial toxicity, the severe material and psychological burden of the cost of cancer treatment, affects an estimated 30\u201350% of patients \u20135. As caThe burden of financial toxicity is a health equity issue, disproportionately affecting patients who are racial/ethnic minorities , 21\u201324, Cancer treatment costs and related material and psychological burden influence treatment recommendations , treatmeHealth insurance, whether public or private, does not protect patients against financial toxicity , 4. The n = 103), we found tl et al. .In an attempt to increase patient awareness and communication about cost, the American Society of Clinical Oncology (ASCO) developed tools, including ASCO Answers: Managing the Cost of Cancer Care, ASCO\u2019s VIncreasing patient active participation during oncology interactions has the potential to improve the frequency and quality of patient-oncologist treatment cost discussions , 54. ResThere is a great need for tools to improve patient treatment cost education and to prompt patient-oncologist treatment cost discussions. Question prompt lists (QPLs) are communication tools designed to enhance patient active participation in interactions with physicians. QPLs are lists of questions that patients might consider asking their healthcare provider during a clinical interaction , 69\u201372 aFinancial toxicity comprised psychological and material economic burden, both of which can contribute to poor treatment adherence and mortality. Our overall goal is to address the burden of financial toxicity and work toward health equity through a tailorable education and communication intervention. Our conceptual model Fig. illustraPatient Patient-oncologist interaction Healthcare utilization Aim 1: Determine the effectiveness of the Discussions of Cost App (DISCO App) on short-term outcomes at three levels:We hypothesize that:H1a: The DISCO App will significantly improve the outcomes for all intervention patients as compared to usual care patients.H1b1: Black patients will experience significantly greater improvement than White patients.H1b2: Lower-income patients will experience significantly greater improvement than higher-income patients.H1b3: Younger patients will experience significantly greater improvement than older patients.H1b: The DISCO App will significantly improve the outcomes for patient groups suffering the disproportionate burden of disparities in the financial consequences of cancer care, specifically:2.Aim 2: Determine the effectiveness of the DISCO App on longer-term outcomes . We will compare longer-term outcomes across arms.The DISCO App is desigH2a: Patients receiving the DISCO App + booster will experience the greatest improvement in the outcomes, followed by patients receiving the DISCO App, and last, usual care patients.H2b: Patient groups suffering the disproportionate burden of disparities in the financial consequences of cancer care will experience the greatest improvement in outcomes, specifically:H2b1: Black patients will experience significantly greater improvement than White patients.H2b2: Lower-income patients will experience significantly greater improvement than higher-income patients.H2b3: Younger patients will experience significantly greater improvement than older patients.3.H3: We hypothesize that the DISCO App will increase the frequency and quality of patient-initiated cost discussions, which will increase social work/financial navigation referrals, social work/financial navigation referral uptake, and patient self-efficacy for managing treatment cost, which in turn will reduce financial toxicity and improve adherence.H4: We hypothesize that this relationship will be moderated by patient socio-demographic characteristics.Aim 3: Test potential mediators and moderators of the relationship between the short-term and longer-term outcomes of the DISCO App.We hypothesize that:This is a clinical trial involving a behavioral intervention focused on patients (with and without an intervention booster), which will be evaluated with a longitudinal between-subjects randomized controlled trial in which patients will be randomized to intervention or usual care groups, and outcomes are compared between the groups. All study procedures have been approved by Wayne State University\u2019s (WSU) IRB (IRB-20-2836). SPIRIT guidelines were used to report this protocol .This trial will be conducted at WSU/Karmanos Cancer Institute (KCI), a NCI-designated comprehensive cancer center located in Detroit, MI, USA, which serves a highly diverse population.We will recruit up to 15 medical oncologists and medical oncology fellows at the beginning of data collection, prior to patient recruitment. Medical oncologists are eligible to participate if they treat patients with breast, prostate, lung, or colorectal cancers at KCI. We focus on these cancers because they are the leading sites of cancer cases and deaths in the USA and because, as solid-tumor cancers, their treatment protocols are similar . MedicalTo recruit oncologists, research staff will explain the study at clinic program meetings and meet with interested oncologists individually to answer questions and obtain consent. Oncologists who consent will agree to (1) complete a baseline survey, (2) inform their eligible new patients about the study prior to their initial appointment to discuss treatment, (3) have interactions with participating patients video recorded, and (4) complete a brief survey following interactions with participating patients. Upon recruitment, oncologists will receive a \u201ctip sheet\u201d to help prepare them for treatment cost discussions with patients Fig. . Oncolog>\u200918\u2009years of age, identify as either Black or White, are able to read and write in English, have an email account, and are newly diagnosed with breast, prostate, lung, or colorectal cancer (stages I\u2013IV) for which systemic therapy is a likely recommended treatment. Strata will be created to ensure study arms are balanced by patient race, income, age, and sex. The majority of patients treated at KCI and living in the Detroit Metropolitan Area identify as either Black or White, and therefore, only the members of these two populations will be recruited.We will recruit up to 240 White and Black patients from various socio-economic statuses and ages. Patients are eligible if they are Eligible patients will be identified by research staff who will review the participating oncologists\u2019 schedules weekly. Patients will be contacted via phone call, face-to-face, or email from the participating oncologist\u2019s clinical staff to inform them of the study and assess interest. If interested, patients will be contacted by research staff via a phone call or in person in the clinic prior to a scheduled appointment. The research staff member will explain the study and obtain consent and collect baseline data. Patient participants will be (1) asked to arrive 30\u2009min early to their next scheduled appointment with their oncologist, (2) randomized to one of the study arms, (3) asked to complete a brief survey following the video-recorded interactions with a participating oncologist, (4) asked to have up to two of their clinical interactions video recorded, and (5) asked to complete up to four follow-up surveys at 1, 3, 6, and 12\u2009months after their last video-recorded interaction. All patients will provide baseline socio-demographic information at the time of consent. We will ask the patients for their preferred method of contact for follow-up surveys.Participating physicians and patients can stop their participation at any time. If a participating physician leaves the institution or stops their practice, they are no longer eligible, and if patients receive their care at another institution, they are no longer eligible.Just prior to the second patient-oncologist visit to finalize treatment plans, but before treatment begins, a research assistant will use data collection software to randomize patients into one of three study arms (1:1:1). Patients in arm 1 will receive usual care, patients in arm 2 will receive the intervention, and patients in arm 3 will receive the intervention and an intervention booster. Patients in arms 2 and 3 will receive the DISCO App. Patients in arm 3 will receive an intervention booster provided 2 months after receiving the DISCO App. For all patients, we will video record the second patient-oncologist interactions using our established, unobtrusive video recording system , 86\u201388.Just before the second patient-oncologist interaction, patients in arms 2 and 3 will access the DISCO App on an iPAll patients will be contacted via their preferred method of contact to complete follow-up measures including questions about their disease and treatment status, whether they received a referral for SW/FN; if they followed up on that referral, self-efficacy managing treatment cost; and perceived short-term financial toxicity in the first follow-up, actual financial toxicity in the remaining follow-ups, and treatment adherence. Follow-up will occur at 1, 3, 6, and 12\u2009months after the second recorded interaction. Patients who receive the intervention booster will also complete booster assessments .The DISCO App Fig. is displFinancial toxicity is multifaceted and long-term. We expect the DISCO App to influence short-term outcomes, but we expect it may need reinforcement to influence longer-term outcomes . Thus, we will explore the effects of a booster to reinforce the effects of the DISCO App. Patients in arm 3 will receive the booster 2 months after receiving the DISCO App. The booster will be a tailored email or text message reminding patients of (1) the content in the educational video, (2) the questions they selected, and (3) that treatment costs are something they can discuss with their oncologist.During DISCO App acceptabWe designed the tip sheet to be a two-sided, tri-fold document that fits in physicians\u2019 white lab coats.Two months after receiving the DISCO App, patients in arm 3 will receive an intervention booster. The booster will be an email or text message (depending on patient preference) to remind patients (1) of the content in the education video, (2) the questions they selected from the DISCO App, and (3) that treatment costs are something they can discuss with their oncologist or other providers. The email or text message will include a \u201cread receipt\u201d so we can track whether the patients view the email or text message.Data include patient and oncologist self-report, video-recorded patient-oncologist treatment discussions, and medical chart data. Video recording allows us to use our validated coding systems , 53, 92 Most of the measures in this study have been used with cancer patients, including in the DISCO App\u2019s feasibility pilot, with high completion rates and few complaints about burden. However, the first ten patients who complete all measures will be specifically queried about the burden. Measures will be adjusted if necessary.It is difficult for me to live on my total household income right now) [\u03b1 = .91; e.g., How confident are you in your ability to know what questions to ask your doctor?), patient-practitioner orientation , their self-efficacy in managing the cost of treatment , [I am concerned about how much my cancer treatment will cost me), and their anticipated material hardship due to their cancer treatment [: After providing consent, oncologists will complete a one-time assessment of their socio-demographic and professional information, including race/ethnicity, gender, age, and years in practice. Oncologists will also complete measures of patient-practitioner orientation , their perceptions of the oncologists\u2019 role in treatment cost discussions , and their self-efficacy with discussing treatment cost.Baseline measures from patients and oncologists will be used as moderators and covariates in analyses of the intervention\u2019s effects. Patients: After providing consent, patients will provide socio-demographics including age, race/ethnicity, gender, education, marital/personal status, income, employment, and financial situation . They wiatment), their leeatment) . OncologWe will use the patients\u2019 medical records to abstract information on cancer diagnosis, co-morbidities, and their zip code.Cancer treatment may cost me in the following ways) and ways to manage those costs ; perceived financial toxicity, comprising treatment cost distress ; and perceived material hardship [The DISCO App helped me ask my doctor my cost questions) [Immediately after the video-recorded interactions, patients will complete the following measures: self-efficacy in patient-physician interactions; self-effatment?) . Interveestions) .\u03b1 = .90; e.g., My out-of-pocket medical expenses are more than I thought they would be) [The reminder email or text message was helpful with my cost questions and concerns).At the 1-, 3-, 6-, and 12-month follow-ups, patients will be contacted via their preferred method by research staff to complete measures on their disease and treatment status to help account for any differences observed in the outcome measures. Patients will also complete measures assessing self-efficacy in patient-physician interactions, self-effould be) . At the Did you and your oncologist (patient) discuss the cost of your (his/her) cancer treatment today?) and satisfaction with treatment cost discussion(s) and I discussed treatment cost today).Immediately after the video-recorded interactions, patients and oncologists will complete measures of the perceived presence of treatment cost discussion(s) , who initiated the cost discussion , and what topics were discussed [the patient asked a lot of questions) [\u03b1 = .75; e.g., the doctor encouraged the patient to express concerns and worries) [Trained research assistants (RAs), blind to research questions, will observe and rate video-recorded interactions using our established procedures to ensure acceptable inter-rater reliability , 96\u201398. n, etc.) . RAs wilestions) and oncoworries) . Anotherworries) and inteworries) .Immediately after the video-recorded interactions, patients will complete measures of whether they wanted and/or received a SW/FN referral, and if so, if they followed up on the referral.Oncologists will complete measures on whether they made a SW/FN referral for the patient.I had a hard time doing what the doctor suggested I do for treating my cancer) and treatment cost-related adherence ; [At the 1-, 3-, 6-, and 12-month follow-ups, patients will be contacted by their preferred method of contact by research staff to complete measures, including whether they wanted a SW/FN referral, and if so, whether they followed up on that referral; treatment adherence ; and clinUsing medical records, we will assess whether the oncologist made a SW/FN referral, and if so, if the patient followed up on the referral, treatment adherence, and clinic appointment adherence.For the first primary objective ; income (high vs. low); and age (<\u200965 vs. \u2265 65)) . The primary analyses will be based on complete data without missing values. As a sensitivity analysis, we will also perform hypothesis testing after multiple imputation. For time-independent variables , multiple imputation will be performed using chained equations (MICE) , and forThis research is highly significant in several ways. First, if successful, reducing the material and psychological burden of financial toxicity will improve the quality of cancer care. Second, the intervention can easily be adapted to other cancers, where expensive treatments are emerging. Third, this research will provide empirical data regarding the mechanisms through which treatment cost discussions and other aspects of clinical communication improve patient outcomes related to financial toxicity. Finally, this research directly addresses disparities in cancer care by improving communication quality for patient groups suffering the disproportionate burden of disparities in the financial consequences of cancer care.This study is not without potential limitations. One of these is the focus on oncologists, rather than or in addition to other providers, such as nurses and social workers, who are instrumental to helping patients navigate the financial issues related to treatment and survivorship. However, we focus on oncologists because they, with their patients, make the final treatment decisions that have the greatest influence on financial consequences. Also, patients consider physicians to be their primary and preferred source of information. Second, it is possible that some patients may not be comfortable using iPads. However, our feasibility testing and the prevalence of smartphones and app usage in this population lead us to believe this will be of limited concern , 89, 107Our research team is well-positioned to disseminate our preliminary and final findings. We have ongoing collaborations with several community-based cancer education and advocacy groups through KCI\u2019s Office of Cancer Health Equity and Community Engagement (OCHECE). These groups, called Cancer Action Councils (CAC), are located throughout Metro Detroit and CAC members served as key informants at the earliest stages of the DISCO App\u2019s development. Dr. Hamel (first author/PI) is also a member of the Michigan Cancer Consortium (MCC), a statewide, broad-based partnership of public and private organizations that provides a forum for collaboration to reduce the burden of cancer among the residents of Michigan. Dr. Hamel is also an active member of several professional organizations including ASCO, the American Academy of Communication in Healthcare/European Academy of Communication in Healthcare (AACH/EACH), and the Society of Behavioral Medicine (SBM).If we demonstrate the DISCO App\u2019s effectiveness, we can disseminate our findings to community-, state-, and nationally focused organizations, in addition to presenting our interim and final findings to academic conferences and high-impact scientific journals. Furthermore, the DISCO App is a product, and if found to be effective, we are in a position to disseminate the DISCO App in KCI\u2019s out-patient clinics across the state of Michigan, thus contributing an evidence-based tool to reduce financial toxicity in diverse populations.All research activities will occur at KCI/WSU, which will also serve as the study\u2019s coordinating center. Dr. Hamel (PI) is based at KCI/WSU, and she will oversee all scientific and administrative aspects of the recruitment of physicians and patients, implementation of the intervention, data collection and analysis, and preparation of reports and manuscripts.A data monitoring committee (DMC) comprising the principal investigator and co-investigators will monitor all self-report, video, observational, and medical record data throughout the duration of the study. The DMC will operate independently from the funder. The biostatistician on this study will conduct interim audit analyses quarterly. Following the WSU IRB rules and definitions, adverse and expected events will be reported to the WSU IRB. The principal investigator will be responsible for all aspects of the trial and will make the final decision when to end the trial.All data files will be stored on a network server at the KCI/WSU in the Department of Oncology. Only the WSU staff listed in the application will have access to the files and at no time will data files be shared with collaborators outside the institution. The KCI/WSU network server utilizes hardware-based encryption at the level of the hard drives. Approved domain users are granted project-specific permission on the server folders.The server is backed up to an off-site software-encrypted disk-based backup solution. Dr. Hamel\u2019s computer and network server are protected under the same CISCO firewall. The recordings will be kept in a locked file cabinet in KCI\u2019s Behavioral and Field Research Core\u2019s editing suite . Patient medical record numbers will be assigned a study ID number in a master key, and study IDs will be used on all research documents. Only the principal investigator, co-investigators, and data manager will have access to the master key, which will be locked in password-protected computers as described. We assure that any publications and presentations of the data will not allow for the identification of patients, hospitals, or physicians.Protocol # 2020-117 was approved on October 21, 2020, by Karmanos Cancer Institute\u2019s Protocol Review and Monitoring Committee and approved on December 17, 2020, by Wayne State University\u2019s Institutional Review Board (# IRB-20-10-2836-B3).Recruitment began on March 10, 2021, and will continue until approximately September 30, 2023."} +{"text": "Neurovascular system plays a vital role in controlling the blood flow into brain parenchymal tissues. Additionally, it also facilitates the metabolism in neuronal biological activities. Cerebral microvascular endothelial cells (MECs) are involved in mediating progression of the diseases related to cerebral vessels, including stroke. Arachidonic acid can be transformed into epoxyeicosatrienoic acids (EETs) under the catalysis by cytochrome P450 epoxygenase. We have reported that EETs could protect neuronal function. In our research, the further role of 14,15-EET in the protective effects of cerebral MECs and the potential mechanisms involved in oxygen glucose deprivation and reperfusion (OGD/R) were elucidated. In our study, we intervened the SIRT1/FOXO3a pathway and established a TSPO knock down model by using RNA interference technique to explore the cytoprotective role of 14,15-EET in OGD/R injury. Cerebral MECs viability was remarkably reduced after OGD/R treatment, however, 14,15-EET could reverse this effect. To further confirm whether 14,15-EET was mediated by SIRT1/FOXO3a signaling pathway and translocator protein (TSPO) protein, we also detected autophagy-related proteins, mitochondrial membrane potential, apoptosis indicators, oxygen free radicals, etc. It was found that 14,15-EET could regulate the mitophagy induced by OGD/R. SIRT1/FOXO3a signaling pathway and TSPO regulation were related to the protective role of 14,15-EET in cerebral MECs. Moreover, we also explored the potential relationship between SIRT1/FOXO3a signaling pathway and TSPO protein. Our study revealed the protective role and the potential mechanisms of 14,15-EET in cerebral MECs under OGD/R condition. Stroke is a common cerebrovascular disease, which may cause ischemic and hypoxic damage to neurons. Although emergency thrombolytic therapy can save the lives of some patients, they are still bothered with the dysfunction brought by this treatment. During ischemic stroke, the perfusion in neuronal tissues is decreased, resulting in a lack of glucose and oxygen in the injured site, which consequently causes dysfunction in energy-dependent activities for cell survival. Additionally, it causes disruption of mitochondrial membrane integrity, exacerbates the depletion of cellular energy and aggravates cellular apoptosis . To explvia cytochrome P450 pathway. EETs have the functions of anti-inflammation, promoting angiogenesis, and reducing apoptosis. EETs mainly includes four isomers: 14,15-EET, 5,6-EET, 8,9-EET, and 11,12-EET. 14,15-EET and 11,12-EET are significantly expressed in brain tissues. Our previous research demonstrated that 14,15-EET could promote the survival of cerebral microvascular smooth muscle cells through PI3K/Akt and JNK signaling pathway and protect against oxygen glucose deprivation and reperfusion (OGD/R)-induced apoptosis. However, it is unclear whether 14,15-EET exerts a neuroprotective effect through other molecular mechanisms.Epoxyeicosatrienoic acids can be generated from arachidonic acid Mitochondrial energy metabolism has been a new research direction of stroke injury in recent years. During ischemia, due to the lack of nutrients and oxygen, the production of mitochondrial ATP decreases and reactive oxygen species are produced, resulting in cell damage and apoptosis. In the reperfusion stage, MPTP opens due to mitochondrial calcium overload, and excessive ROS is produced, resulting in a series of damages . AutophaIn the cardiac tissue, it was found that 14,15-EET could enhance the SIRT1 activity of HL-1 cardiomyocytes under starvation and promote mitochondrial biogenesis . In the in vitro OGD/R model was established in our study.Our research aimed to explore the role of 14,15-EET in cerebral MECs and reveal the potential mechanisms. It was proved that 14,15-EET played a protective role through modulating SIRT1/FOXO3a pathways and TSPO protein expression. An 14,15-EET was obtained from Cayman Chemical Corporation (United States). Antibodies for SIRT1 (#8469), TSPO (#70358), LC3 (# 12741), P62 (# 23214) and Foxo3a (#12829) were purchased from CST. GAPDH (Ab8245) was purchased from Abcam (United States). Anti-rabbit polyclonal antibodies and anti-mouse polyclonal antibodies were provided by abmart (United States). Cell culture dishes were obtained from Icell Biotechnology Co. The experiments abided by the ethical rules of Harbin Medical University.2, 37\u00b0C). After centrifugation for 10 min, cell pellets were prepared and suspended.Human cerebral MECs lines, HCMEC/D3 were obtained from Icell Biotechnology Co. The cells were cultured in Endothelial Cell Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and glutamine (1%) in an incubator with adequate humidity the control group; (2) OGD/R group; (3) OGD/R + 14,15-EET group, in which the cells were treated by 14,15-EET (1 \u03bcM) for 30 min followed by OGD/R exposure; (4) OGD/R + 14,15-EET + 3-MA group, in which the cells were treated by 14,15-EET (1 \u03bcM) and 3-methyladenine (3-MA) (5 mM) for 2 h before OGD/R; (5) OGD/R + 14,15-EET + siRNA group, in which the cells were transfected with siRNA (SIRT1-siRNA and TSPO-siRNA) for 6 h before OGD/R treatment; and (6) OGD/R + 14,15-EET + siRNA + 3-MA group, in which the doses of 14,15-EET 3-MA and siRNA were chosen based on recent studies.2, 95% N2). The cells in the experimental group were incubated with DMEM/F12 medium free of serum and glucose for 4 h. After the cells underwent hypoxia for 4 h, reoxygenation was carried out for 24 h by placing the cells in normal culture environment. SiRNA or 14,15-EET was used to treat the cells before OGD/R treatment. Cells in the control groups were treated with the same procedure except for OGD/R exposure. Western Blot analysis, flow cytometry and CCK8 assay were used to detect corresponding indicators.The plate in the experimental group was placed in a thirty-seven centigrade degree anaerobic incubator with adequate humidity supplemented with mixed gas and FITC (fluorescein isothiocyanate)-Annexin V (5 \u03bcl) were added into the cells (100 5 \u03bcl) followed by incubation for a quarter in darkness at room temperature. The cells were immediately analyzed after the aforementioned treatment by using the flow cytometer. FACS Calibur cell sorter (BD FASAria Cell Sorter) was used in this assay. Three independent experiments were conducted and triplicated samples were used.14,15-EET was incubated with the cells for 30 min prior to OGD/R treatment. CCK-8 assay was used to detect the survival of cerebral MECs. After 24 h of OGD/R treatment, CCK-8 (10 \u03bcl/well) was added into the cells for incubation for 2 h in a thirty-seven centigrade degree environment. The Microplate spectrophotometer was used to detect the OD at a wavelength of 480 nm. Cell viability was presented as the proportion of the cells without treatment.The content of MDA and SOD in each group of HCMECs was detected by using the ELISA kit . Supernatant samples (50 \u03bcl) were added into each well in the test plate, which was incubated for 45 min at thirty-seven centigrade degree. The dried wells were then washed by buffer for five times (10 s for each wash). HRP-conjugate reagent (100 \u03bcl/well) was added, following by incubation for 30 min at thirty-seven centigrade degree. The wells were then washed for five times. Substrate A and B solutions (90 \u03bcl for each solution) were mixed well for 15 min at thirty-seven centigrade degree. Afterward, stop solution (50 \u03bcl) was added to each single well. Finally, a spectrophotometer was used to detect the light absorbance.JC-1 fluorescence mitochondrial imaging was used to detect mitochondrial membrane potential. JC-1 solution was added into HCMECs for incubation in a thirty-seven centigrade degree environment for 15 min. After centrifugation for 5 min at 1,200 rpm, the cells were collected and washed for two times by using JC-1 buffer. Subsequently, culture medium was added into each well. The fluorescence microscope (Leica) was used to obtain the images. Red/green fluorescence ratio reflected the mitochondrial membrane potential. The ratio of fluorescent intensity of the red to green was analyzed by Image J software, and represented \u0394\u03a8m level .A series of concentrations of 14,15-EET were prepared and mixed with culture medium for 30 min. Human cerebral MECs were harvested. PBS was used to wash the cells, which were then lysed on ice for a quarter. Cells were then centrifuged for 10 min at 12,000 rpm. The supernatants were collected and stored in a \u221220\u00b0C refrigerator for further experiments. Protein (15 \u03bcg) was added into SDS-PAGE (10%) gel and transferred onto nitrocellulose membranes. Bovine serum albumin was used to block the membranes for 1 h. Primary antibodies against sirt1, foxo3a, TSPO, LC3, and P62 were used to incubate with the membranes. The enhanced chemiluminescence detection system was used to conduct the densitometric analysis.1 a database of known and predicted PPI, to evaluate PPI ,uate PPI . For eacuate PPI , and theuate PPI , and theuate PPI , 2013.t-test were used to evaluate the differences among different groups. We applied GraphPad Prism version 9.0 for graphing and analysis. P-value < 0.05 indicated that the results were statistically significant.The results were described as average value \u00b1 SD of at least three independent experiments. ANOVA and Student\u2019 s 2). Cerebral MECs were treated with 14,15-EET (1 \u03bcM) before OGD/R treatment for 30 min. CCK8 and flow cytometry were used to measure the cell function. Viability of cerebral MECs was detected by CCK8 method. Cell viability was reduced after treated by OGD/R in comparison with the cells in the control group. 14,15-EET could remarkably ameliorate the decrease in cell viability resulting from OGD/R treatment was utilized to construct PPI network of SIRT1 and TSPO. The confidence score in STRING database was 0.4. Cytoscape version 3.9.1 software was used to visualize PPI network, which was shown as graphs containing nodes (proteins) and edges (related interactions). After PPI analysis using STRING software, 99 edges and 22 nodes were identified . The keyGene ontology and pathways enrichment analyses were performed on these nine genes. 33 pathways were classified into 4 clusters , which wFour clusters including \u201cregulation of endogenous apoptosis signaling pathway by a p53 class mediator,\u201d \u201csmooth muscle cell apoptotic process,\u201d \u201cnegative regulation of post-transcriptional gene silencing\u201d and \u201cresponse to metformin\u201d contain 24 terms or 73% all biological terms. It seems that these four clusters of biological terms are the core of terms related to the central nodes. As it is appeared among \u201csmooth muscle cell apoptotic process,\u201d \u201cnegative regulation of post-transcriptional gene silencing\u201d and \u201cresponse to metformin\u201d are concerned by PPARG genes. And in the \u201cregulation of endogenous apoptosis signaling pathway by a p53 class mediator,\u201d it contain the MDM2 gene. PPARG and MDM2 genes may be the main genes involved in the interaction between SIRT1 and TSPO .We have previously demonstrated the protective role of 14,15-EET in smooth muscle cells in brain under the condition of OGD/R , 2017. HStroke is a common cerebrovascular disease with high morbidity and mortality. The therapeutic effect window of the neuroprotective agents that play a pivotal role in reperfusion is longer compared with the agents which play a role in the ischemic cascade reaction in the early stage . TherefoOur previous studies have shown that 14,15-EET could reduce the apoptosis of cerebrovascular smooth muscle cells in rats under glucose and oxygen deprivation through PI3K/AKT and MTOR signal pathways and alleviate hypoxic-ischemic brain injury . HoweverBecause of the focal cerebral ischemia, especially when reperfusion occurs, reactive oxygen radicals are produced during enzyme transformation. Oxidative stress plays an essential part in cellular apoptosis in ischemic stroke. In addition, reoxygenation of ischemic brain injury leads to a worse injury to the brain tissue compared with ischemia alone. Ischemia-reperfusion injury can induce the production of ROS, increase MDA, decrease SOD, and lead to apoptosis . SuperoxMitochondrial autophagy is a form of autophagy . The mPTIn addition, human CMECs autophagy was observed after OGD/R, and the effect of 14,15-EET on autophagy was investigated. LC3 is a well-known autophagosome marker and the concentration of LC3-II indicates the count of autophagy-associated structures and autophagosomes. Nutrient starvation (the culture medium free of serum and/or amino acids) can increase the generation of autophagosomes and the concentration of LC3-II . It was via regulating autophagy. Mitochondrial quality control is an important mechanism for maintaining cell survival state through multi-dimensional regulation of mitochondrial morphology, quantity and quality. Combined with the results of cell activity, apoptosis and oxygen free radical detection in our study, It is suggested that 14,15-EET may achieve mitochondrial quality control by regulating the process of mitochondrial autophagy, thus playing a protective role in cells. It was hypothesized that this protective function might be related to activated autophagy-related or regulatory proteins.Taken together, our results revealed that 14,15-EET could exert a protective role during starvation Sirtuins is a conservative family containing ADP ribosyltransferase and nicotinamide adenine dinucleotide (NAD +)-dependent deacetylase. SIRT1 is located in both the cytoplasm and nuclei and plays a role through the deacetylation of the substrate, which is a regulator of metabolism and senescence . SIRT1/2via regulating TSPO expression in brain ischemia. It was found that after treatment with 14,15-EET, the expression level of TSPO protein was increased, which indicated that 14,15-EET could regulate the expression of TSPO under the condition of OGD/R. Then, we used siRNA to inhibit the expression of TSPO. According to the results of flow cytometry and CCK-8 assay, TSPO-siRNA suppressed the protective role of 14,15-EET compared with the OGD/R group. In addition, with TSPO-siRNA treatment, the LC3 II/LC3 I ratio was decreased and the expression level of P62 was enhanced as compared with the 14,15-EET + OGD/R group. The inhibition of TSPO protein greatly suppressed the protective role of 14,15-EET. Our study first revealed that TSPO protein regulation may be an important factor in the neuroprotective effect of EETs.Translocator protein (18 kDa), mainly located at the outer membrane of mitochondria, is a transmembrane protein. TSPO is well acknowledged as a biomarker for the activation of microglia cells. It was first recognized as a binding site for diazepam. Most research has focused on the main function of TSPO in immunomodulatory, mitochondrial activities and cellular bioenergetics . TSPO liThe expression of TSPO has also been shown to be altered by different stress conditions . Some reThe cross-talk or connection between the SIRT1/FOXO3a pathway and TSPO protein is largely unknown. In our research, in order to explore the relationship between SIRT1/FOXO3a pathway and TSPO protein, we used the STRING database and converted the results visually by using Cytoscape software. In these identified proteins, TP53, EP300, SIRT1, MDM2, PPARG, PPARG C1A, MYOD1, FOXO1, and SMAD2 were identified as the common hubs. PPARG, encodes a member of the peroxisome-activated receptor subfamily of nuclear receptors. PPARG-gamma was encoded by PPARG . PPARG-gvia modulating SIRT1/FOXO3a pathway and TSPO protein. The in vitro experiments further supported that 14,15-EET may regulate mitophagy in human cerebral MECs elicited by oxygen glucose deprivation. Moreover, we also explored the potential relationship between SIRT1/FOXO3a signaling pathway and TSPO protein. A thorough investigation of the mechanisms involved in the protective role of 14,15-EET may provide new insights on the treatment of cerebral ischemic injury.In conclusion, 14,15-EET could protect human cerebral MECs against OGD/R damage The data that support the findings of this study are available from the corresponding author upon reasonable request.YQ and YZ: conceptualization and funding acquisition. JC: methodology and data curation. SW, YL, and DW: investigation. YZ: resources and writing\u2014review and editing. YQ: writing\u2014original draft preparation. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "The interaction between teachers and students is vital for promoting teaching quality. Online learning spaces have various features that can support teacher-student interaction in online learning contexts. In this study, a preliminary model was developed by analyzing the principles underlying the interaction between teachers and students and the support features of online learning spaces. Then, the interaction model was refined and validated in three rounds of teaching practice involving 31 college students. A real-time dynamic artificial intelligence analysis system was used to analyze the teacher-student interaction during three rounds of design-based research. The results showed that the model significantly fostered students\u2019 engagement during the interaction. Moreover, students significantly improved their final exam scores and their innovative problem-solving ability after the intervention. Today, higher education is under great pressure and faces considerable challenges. Teachers must not only help students acquire existing knowledge and skills, but also and more importantly, they must enable students to apply and generate new ideas. Fostering an effective teacher-student interaction can positively impact learning in the higher education setting Zhang, . The teaThe use of information technologies may help to foster active teacher-student interactions more effectively Zhang, . ExploreTo explore how to better support teacher-student interaction and effectively gain knowledge in online learning spaces, an in-depth theoretical and practical research on the teacher-student interaction model was conducted. A set of theoretical frameworks for the teacher-student interaction was established, and the principles of teacher-student interactions and the functional support of online learning spaces for teacher-student interaction were clarified. Then, the teacher-student interaction model in online learning spaces was constructed, and the interaction mechanism, interactive behaviors, tools support, and implementation strategies of the model were further improved through a design-based study.S plane analysis model show that the average level of the final examination scores of the subjects was high.The effectiveness of the teacher-student interaction model was examined from three aspects based on online learning spaces: the teacher-student interaction, students' ability to solve innovative problems, and learning performances. Firstly, the S-T curve of teacher-student interaction shows that the degree of teacher-student interaction increased. Secondly, the independent sample T test results of the innovation problem solving ability scale identified a significant difference between pre-test and post-test periods. Thirdly, the results of the\u203eX-Therefore, the teacher-student interaction model in the online learning spaces can effectively enhance the teacher-student interaction, and help students to improve their ability to solve problems innovatively, achieve higher cognitive attainment, and improve their learning performance.Teachers and students develop a bilateral and interactive relationship in the education process via interaction and mutual influence analysis system A real-time dynamic AI analysis system for teaching and learning was used to analyze classroom interaction behaviors. This system was also used to both collect and analyze classroom discourses as well as various teaching data (such as the percentage of teacher-student speech and response rate) generated in the classroom through AI analysis and computational models. Analysis reports were also formed with the same system. System-generated S-T curves and the data of Flanders analysis were used to examine whether the teacher-student interaction has improved.The Scale of Creative Problem-solving Skills (CPS) Based on the CPS model values were similar in both rounds of the design-based study, indicating that the teacher-student interaction remained relatively stable. \u2461 Compared with the first round, the second round specified the content and form of teacher-student interaction. Additionally, the percentage of teacher talk (TT) decreased, while the percentage of pupil talk (PT) and pupil steady state ratio (PSSR) increased. This result indicates that the teacher paid more attention to the students' learning experience and returned the initiative to the students. \u2462 The teacher response rate (TRR) increased, indicating that teachers coordinated the cooperative learning status of each group and were able to make targeted suggestions more efficiently.In summary, the results of the Flanders interaction analysis showed that, after three iterations of refinement, the TT value decreased, PT and PSSR increased, and TRR improved. This indicates that the teacher-student interaction model in online learning spaces could effectively improve teacher-student interactions and enhance the quality of teacher-student interactions.Pre-tests and post-tests on students\u2019 innovative problem-solving ability were compared before and after the three-iteration design-based study using paired samples t-tests conducted via SPSS 26.0 (see Table t (30)\u2009=\u2009\u2013\u20092.74, p\u2009=\u20090.010). The mean score of problem-solving ability in the pre-test was 3.53 and the mean score in the post-test was 3.84. The problem-solving ability of experimental subjects was significantly improved after the application of the model (t (30)\u2009=\u2009\u2013\u20092.71, p\u2009=\u20090.011). The mean score of innovation ability of the experimental subjects was 3.56 in the pre-test and 3.84 in the post-test. The innovation ability of the experimental subjects was also significantly improved after the application of the model (t (30)\u2009=\u2009\u2013\u20092.39, p\u2009=\u20090.023).As shown in Table In conclusion, the teacher-student interaction model in the online learning spaces significantly improved students' innovative problem-solving ability.The average score of the final exam reflects the concentration trend of the overall class level distribution, and the standard deviation reflects its degree of dispersion. By forming a two-dimensional plane with the mean and standard deviation of the final exam, the overall characteristics of the grades can be comprehensively and visually presented. The descriptive statistics of the course final exam results are shown in Table X\u2009=\u200975, S\u2009=\u200910) as reference standard. The two parameters S are used to determine in which quadrant of the plane the achievement samples fall into. Different quadrants represent groups of achievement samples with different statistical characteristics.The As shown in Fig.\u00a0X\u2009=\u200987.58 and the standard deviation is S\u2009=\u20098.036; is located in the fourth quadrant of the The mean score of the course final exam in this study is Through theoretical and practical research, a set of theoretical frameworks of teacher-student interaction was established based on online learning spaces. Further, the principles of teacher-student interaction and the functional support of online learning spaces for teacher-student interaction were clarified, and a teacher-student interaction model in the online learning spaces was constructed. Moreover, the interaction mechanism, behavior and tool support, and implementation strategies about the model were further improved through design-based research. In addition, the effectiveness of the model was verified, teacher-student interaction was improved, the activity of teacher-student interaction was increased, and students' innovative problem-solving skills were enhanced.Song et al. developeCertain aspects of this study still need to be improved. For example, in teaching practice, different teaching contents may impose different requirements on teacher-student interaction; therefore, the effective interaction between teachers and students could be explored according to the requirements of different teaching contents to further improve the integrity of the study. Effect evaluation showed that the ability to reflect on problems and the ability to communicate and express could not be greatly improved within a short period of time. Thus, the improvement effect between before and after the application of the model was not significant. Consequently, further research is needed that addresses how the model can be improved continuously. Considering these limitations, a follow-up study will develop the theoretical research and practical application further based on the construction of the teacher-student interaction model in online learning spaces.The engaged interaction between teachers and students is central for promoting teaching quality. In this study, literature research and design-based research were adopted to build and test a teacher-student interaction model in online learning spaces. First, a preliminary model was developed by analyzing the principles behind the interaction between teachers and students and the support features of online learning spaces. Additionally, the interaction model was refined during three rounds of teaching practices. The results of three teacher-student interaction analyses showed that the model significantly promoted students' engagement during the interaction and was also able to significantly improve students' final exam scores and innovation problem-solving ability. This model provides new ideas and methods for the integration and innovative application of online learning spaces in the information era."} +{"text": "Objectives: Sunlight exposure is an important environmental factor in the pathogenesis of skin cutaneous melanoma (SKCM). Ultraviolet (UV) from sunlight can cause excessive intracellular production of reactive oxygen species (ROS), resulting in damage from oxidative stress to cells. As a major iron-rich and ROS-producing organelle, mitochondria are considered as an important place for cell ferroptosis. Thus, the pathology and potential biological process of UV exposure-induced ferroptosis in the development of SKCM has aroused our strong interest.Methods: Gene expression profile datasets of melanoma cell line datasets (GSE31909) and UV-irradiated mitochondria dataset (GSE3632) were downloaded from the Gene Expression Omnibus (GEO) database, and ferroptosis-related genes were obtained from the FerrDb v2 database. After identifying the common differentially expressed genes (DEGs), comprehensive analyzes were performed, including functional annotation, protein-protein interaction (PPI) network construction, hub gene identification, and gene and tissue protein expression levels, survival analysis, and immune cell infiltration analysis.Results: A total of 14 common DEGs was identified for subsequent analyses. Seven DEGs, including PSMB4, CRELD2, CDKN2A, TIMP1, NDRG1, ATF3 and JUND, have consistent performance in mRNA and protein expression in normal skin and SKCM tissues can be regarded as a good biomarker with SKCM diagnostic effectiveness. Functional enrichment analysis results indicate that HIF-1 signaling pathway and angiogenesis involved in the pathogenesis and development of SKCM. Induction of ferroptosis in tumor cells by enhancing the function of CD8+ T cells is expected to be an effective intervention to promote tumor therapy.Conclusion: Our study reveals the pathogenesis and potential biological processes of UV exposure-induced ferroptosis in the development of SKCM, which may provide potential immunotherapy targets for SKCM treatment via tumor cell ferroptosis mechanisms. Skin cutaneous melanoma (SKCM) is the most common malignant tumor of skin cancer with the highest mortality rate. It originates from melanocytes and is highly malignant and prone to metastasis . MelanocThe UV of sunlight can cause an excessive intracellular production of reactive oxygen species (ROS), resulting in damage by oxidative stress to cells . MitochoExploration of cotranscriptional signatures help to understand important links between cell injury and disease. In this study, intrinsic associations with ferroptosis-related genes in a melanoma cell lines datasets (GSE31909) and UV-irradiated mitochondria dataset (GSE3632) were analyzed. The physiological mechanisms of ferroptosis-related DEGs and their important role in the expression, pathological stage, and prognosis of SKCM were explored through a comprehensive bioinformatics analysis. Subsequently, the connection between ferroptosis and tumor immune microenvironment of primary SKCM was explored to provide new therapeutic strategies for ferroptosis-based tumor immunotherapy.https://www.ncbi.nlm.nih.gov/geo) (http://www.zhounan.org/ferrdb/current/).Gene expression profiles on melanoma cell lines and UV-irradiated mitochondria were downloaded from GEO database (gov/geo) . The melp-value < 0.05, and a base-2 logarithm with a fold change value greater than \u00b11. For cDNA expression data set of UV-irradiated mitochondria, the threshold was set to a p-value < 0.05. The common DEGs were screened by an online Venn diagram tool.The gene expression profile data were read by GEOquery packages, and the differential expression fold change was calculated by Limma R packages. The probe without corresponding genes was deleted, and multiple probe sets with the same gene were averaged. For the mRNA expression data set of the melanoma cell line, the threshold of DEGs was set to a https://david.ncifcrf.gov/) is currently the most widely used biological annotation database, enabling rapid enrichment annotation of pathways in the biology of genes. GO annotation enrichment analysis, including molecular function (MF), biological process (BP), and cell component (CC), and KEGG pathway enrichment analysis was performed using DAVID. The enrichment analysis based on the expression of each gene was analyzed and visualized by the \u201cclusterProfiler\u201d R package. In addition, the gene-disease enrichment analysis was performed by using Enrichr database (https://maayanlab.cloud/Enrichr/).DAVID database (https://cn.string-db.org/) was used to search for the regulatory network of protein-protein interactions (PPI), and the GeneMANIA (http://genemania.org/) online database was used to explore the coexpression network. The connectivity degree of each gene in PPI network is calculated using the degree algorithm in cytohubba plug-in, and the gene with the highest node degree is defined as the hub gene. The top 10 hub genes PPI network was visualized with Cytoscape (version 3.7.2) software.The STRING database and disease-free survival (DFS) . The HumTo investigate whether the genes can be used to predict the occurrence of SKCM, the expression data of SKCM specimens in TCGA database was extracted using \u201cggplot\u201d R package. Then, a least absolute shrinkage and selection operator (LASSO) logistic regression model, Cox regression prediction model and ROC analysis prediction model were established by \u201cglmnet\u201d package, \u201cpROC\u201d package and \u201csurvival\u201d package, respectively, and used to verify genes effectiveness.+ T cells, CD8+ T cells, B cells, neutrophils, macrophages, and dendritic cells, and the correlation of endothelial cell infiltration abundance and cancer-associated fibroblasts.The tumor immune microenvironment is an important factor in the onset and development of tumors. CIBERSORTx analytical tool calculate the enrichment status of 22 immune cells based on the gene expression profile of the specimen, LM22 (22 immune cell types) and 1000 permutations as selection parameters. The TIMER (version 2.0) database was used to explore the correlations of hub gene expression with six types of immune cell infiltration abundance, including CD4p-value < 0.05 was considered significant of comparison of two groups.All data of two sets comparisons were standardization and analyzed by Z-score algorithm and Student\u2019s t-test, respectively. In this study, A A total of 271 ferroptosis-related genes were downloaded from FerrDb v2 database. The gene expression profiles data were standardized, DEGs (5028 in GSE3632 and 1352 in GSE31909) were identified . After tp = 1.02E-04), positive regulation of angiogenesis (p = 1.63E-04), cytosol (p = 8.78E-03) and the binding of enzymes (p = 2.17E-03). For the analysis of the enrichment of the KEGG pathway (p = 2.60E-04), hepatocellular carcinoma (p = 9.26E-04) and cancer pathways (p = 2.72E-03). In terms of gene-disease enrichment analysis (p = 3.74E-07), stage 0 skin melanoma (p = 5.34E-07) and secondary malignant neoplasm of prostate (p = 4.19E-05). These results indicate that these common DEGs are strongly involved in the onset and development of melanoma.GO annotation, KEGG pathway and gene-disease enrichment analysis were performed to understand the biological functions and pathways of common DEGs. For the enrichment analysis of the GO annotation , these g pathway , these ganalysis , these gThe PPI network of the common DEGs was constructed and analyzed using the STRING database . Then, tp = 3.95E-05), regulation of transcription of the RNA polymerase II promoter in response to oxidative stress (p = 4.00E-05) and cellular response to hypoxia (p = 8.22E-05). In terms of CC (p = 5.42E-04), RNA polymerase II transcription factor complex (p = 9.65E-04) and the cytosol (p = 2.12E-03). In term of CC (p = 2.28E-04), mitogen-activated protein kinase kinase binding (p = 2.30E-04) and transcription factor binding (p = 4.71E-04). The KEGG enrichment analysis result (p = 2.04E-05) and pathways in cancer (p = 1.06E-03) were the most enrichment pathway.The genes in the co-expression network were also performed to enrichment analysis. In terms of BP , the genms of CC , the genrm of CC , the gens result showed tThe mRNA expression levels of genes in the coexpression network were explored using GEPIA. FADS1, MAP3K11, SLC39A14, PSMB4, CRELD2, CDKN2A and TIMP1 were higher in SKCM tissues than normal skin tissues, and NDRG1, ATF3, IL1R1, JUND and MMP3 had an opposite trend . The expp < 0.001) and NDRG1 have significant correlation with SKCM. For the results of the multivariate analysis, TIMP1 has a significant correlation with SKCM. Based on the TCGA database, ROC analysis was used to validate the diagnostic effectiveness of these DEGs for SKCM can be used to predict the appearance of SKCM, the expression profile of these DEGs from the TCGA database was extracted to construct the LASSO model, and it was found that the genes can be identified with nonzero regression coefficients . Univarifor SKCM . The valp < 0.05), and MMP3 was highly correlated with stage II skin melanoma . For the multifaceted prognostic value index (OS and DFS) in patients with SKCM , and IL1B, CDKN2A, NDRG1, IL1R1 and HMOX1 mRNA levels were significantly associated with DFS . Patients with low IL1B, NDRG1, and IL1R1 mRNA levels were significantly associated with low OS and DFS.The relationship between these co-expression genes and tumor pathological stage and multifaceted prognostic value were explored. For tumor pathological stage , the resith SKCM , the resp = 0.012) in the enrichment of immune cells between the two samples , and negatively correlates with CD 4+ T cell and neutrophils infiltration. PTEN is positively correlated with infiltration of CD 8+ T cells and neutrophils infiltration. IL1B positively correlates with macrophages , neutrophils and endothelial cells infiltration. PRKCA is positively correlated with neutrophils . HMOX1 positively correlates with macrophages and dendritic cells infiltration. ATF3 is positively correlated with neutrophil infiltration infiltration. BRAF is positively correlated with CD 8+ T cell infiltration and neutrophils infiltration. MMP3 is positively correlated with endothelial cell infiltration and cancer-associated fibroblasts infiltration. CDKN2A is negatively correlated with macrophage infiltration and neutrophils infiltration. TIMP1 is positively correlated with CD 4+ T cell and macrophage infiltration.Subsequently, the association of the expression of hub genes with the infiltration of immune cells from the tumor microenvironment in SKCM was analyzed by the TIMER database . HIF1A pSKCM is a highly malignant and insidious type of skin tumors, and its incidence is increasing rapidly. Currently, it is the leading tumor type for skin cancer mortality . SurgicaVarious biological therapies such as cellular immunotherapy, cytokine, and monoclonal antibody therapies have been widely used in the treatment of SKCM, and in-depth research on the pathogenesis of SKCM and the discovery of new therapeutic targets is still the current focus . NumerouFerroptosis has recently been identified as a natural tumor suppressor mechanism, and mitochondria are important organelles regulating ferroptosis. The pathology and potential biological process of UV-exposure-induced mitochondrial ferroptosis in the development of SKCM have aroused our strong interest. In this study, we explored the cotranscriptional signatures of UV exposure-induced mitochondria and ferroptosis to reveal the intrinsic mechanisms of SKCM. Based on the enrichment results of the common DEGs, these genes are mainly enriched in cellular response to hypoxia, positive regulation of angiogenesis, and the HIF-1 signaling pathway. Hypoxia and intratumoral angiogenesis are common features of solid tumors and contribute to cancer progression and drug resistance. The process of neoangiogenesis has been shown to be critical for melanoma proliferation and is believed to be so primarily in the metastatic process . In partPPI coexpression networks contribute to increasing understanding of functional connections between proteins under disease conditions. We obtained the top 10 genes with the most nodal degree in the PPI coexpression network by using the degree algorithm. HIF1A, PTEN, and IL1B were found to be the main hub gene for the coexpression network. The high expression of HIFA in melanoma samples is consistent with the characteristics of hypoxia and angiogenesis of SKCM tissue . PTEN, aUnderstanding the expression pattern of genes and proteins in tissues with melanoma can effectively improve the early diagnosis and prognosis of SKCM. We examined the mRNA and protein expression levels of genes in the co-expression network in SKCM. For the level of mRNA expression in the TCGA database and melanoma cell lines, SLC39A14, PSMB4, CRELD2, CDKN2A and TIMP1 were higher in SKCM tissues than in normal skin tissues, and NDRG1, ATF3, and JUND had an opposite trend. For protein expression level in HPA database, except for SLC39A14, the protein expression of the other seven genes was consistent with the mRNA expression in SKCM samples. Then, to investigate whether these DEGs can be used to predict the occurrence of SKCM, the expression profile of these DEGs from the TCGA database was extracted to construct the LASSO model, Cox proportional risk regression model and ROC analysis prediction model, and found that PSMB4, CRELD2, CDKN2A, TIMP1, NDRG1, ATF3 and JUND have expression consistency and prediction stability, which can be used as a molecular marker for early diagnosis of SKCM , 5. Intep = 4.85E-06). in vivo studies that MMP3 was an important factor promoting melanoma tumor growth and lung metastasis. The prognostic correlation analysis of SKCM with molecular markers found that patients with low mRNA levels of IL1B, NDRG1, and IL1R1 were significantly associated with low OS and DFS. Notably, IL1B, NDRG1 and TIMP1 act as drivers of ferroptosis, and their low expression in patients with poor prognosis implies a correlation between tumor cell resistance to ferroptosis mechanisms and poor prognosis. This result indicated that the promotion of tumor cell ferroptosis is expected to be an effective means of improving the prognosis of patients with SKCM.Then, the relationship between these co-expression genes and tumor pathological stage and multifaceted prognostic value were explored. MMP3 was highly correlated with stage II skin melanoma and a low mRNA level of PTEN (a ferroptosis driver) positively correlate with CD8+ T cell infiltration, implying that a ferroptosis resistance mechanism exists in the tumor microenvironment of SKCM, and it is expected that the induction of ferroptosis in tumor cells by enhancing the function of CD8+ T cells is expected to be an effective intervention to promote tumor therapy.Growing evidence suggests that tumor immune microenvironment is an important factor affecting tumor progression, and tumor immunotherapy is expected to be an effective means to delay tumor progression . Therefost cells . Wang etIn summary, this study provides a comprehensive analysis of the mechanisms of ferroptosis induced by Sun exposure on the pathology and potential biological processes of primary melanoma and explored the impact of ferroptosis-related genes on the immune microenvironment of the tumor . Our fin"} +{"text": "Escherichia coli O157:H7, Salmonella Typhimurium, aerobic mesophilic counts, and molds and yeasts, respectively, as compared with traditional US-assisted methods. Sensory properties, color index, total soluble solids, titratable acidity, and weight loss were not negatively affected by any of the treatments. Firmness was slightly reduced after all treatments; however, the firmness of the samples was maintained during storage, in contrast with the decreased firmness observed in the control. Phenolic content and antioxidant activity significantly increased after all treatments. Further analysis of two key enzymes involved in phenolic synthesis showed that their levels significantly increased following all treatments, leading to an increase in phenolic content and antioxidant activity. This result also indicated that US-assisted washing could act as an abiotic elicitor to increase nutritional content. Overall, US-PAA\u00a0+\u00a01%AA treatment served as an effective method for disinfecting produce during washing and for controlling microbial growth after washing without prolonging the processing time, which is an advantage over traditional US-assisted washing.Traditional ultrasound (US)-assisted disinfection is only effective during washing. Coating is an effective method to control microbial growth after washing; however, cross-contamination can occur during immersion in the coating aqueous solution. Tap water (TW) rinsing is generally used to remove sanitizer residues after US-assisted washing; however, the Food and Drug Administration stated that rinsing is unnecessary when the peracetic acid (PAA) concentration does not exceed 80\u00a0ppm. In this study, we proposed a novel US-assisted hurdle technology of 80\u00a0ppm PAA combined with low-frequency US (25\u00a0kHz) during washing, followed by US-assisted aerosolization processing (nonimmersion coating). Ascorbic acid (AA), a safe and low-cost agent, was selected as the aerosolization solution. Cherry tomatoes were selected as the model, and the proposed method was compared with traditional US-assisted disinfection methods (US-10\u00a0ppm free chlorine washing\u00a0+\u00a0TW rinsing and US-5\u00a0ppm chlorine dioxide washing\u00a0+\u00a0TW rinsing) to analyze the disinfection efficacy and quality changes. During storage, US-PAA\u00a0+\u00a01%AA facilitated additional 0.7\u20130.9, 0.6\u20130.8, 0.7\u20131.0, and 0.5\u20131.0 log CFU/g reductions in the counts of Typhimurium, in which the youngest infected person is less than one year old E. coli OH157:H7 caused 15 illnesses and 4 hospitalizations from October 13, 2021 to November 8, 2021 Fresh produce is an important source of daily vitamins, minerals, and fiber. Due to an accelerated lifestyle, the demand for ready-to-eat produce is increasing. Since fresh produce is not thermally treated, consumption is accompanied by food safety hazards caused by foodborne pathogens. Among these pathogens, Salmonella on iceberg lettuce, which is consistent with free chlorine treatment; however, survival counts in washing water after US treatment were 5.70 log CFU/g, which is similar to that of tap water washing, in contrast to the undetectable counts after FC treatment Pseudomonas fluorescens in lettuce compared to US alone Salmonella and E. coli O157:H7 was consistent with that in tap water washing; however, cross-contamination was completely prevented after US\u00a0+\u00a0FC. Therefore, US should be combined with effective disinfectants in washing fresh produce. Among all disinfectants, peracetic acid (PAA), FC, and chlorine dioxide (CD) are commonly used owing to their low cost, excellent disinfection efficacy, and ability to prevent cross-contamination Salmonella present on strawberries compared to US alone Ultrasound (US) is a non-thermal processing technology that can generate shear force and shock waves in an aqueous solution to detach and kill microorganisms on the surface of the produce US-assisted disinfection was only effective during washing; thus, procedures to control microbial growth after washing should be developed. If a combination with this method extends the processing time as compared with the traditional method, the production efficiency will be reduced, thus reducing the acceptance by industry US-assisted nebulizers have been successfully used in indoor air disinfection and disease treatment. For fresh produce, aerosol droplets can adhere to the surface and continuously effective after treatment. Owing to the characteristics of ready-to-eat produce, the aqueous solution to produce aerosols should be safe, is low-cost, and do not affect sensory quality. Ascorbic acid (AA), a low-cost food additive, has been used for food preservation and against browning 22.1S.\u00a0Typhimurium (ATCC14028) recommended by the FDA in food safety testing and non-toxic E. coli O157:H7 (NCTC12900) used for fresh produce disinfection experiments were selected in this study 9 CFU/mL cell concentration. Then, 10 cherry tomatoes and adjusted bacterial suspension were added into sterile stomacher bags at a ratio of 1:8 (w/v) and manually massaged for 15\u00a0min. The samples were transferred into a biosafety cabinet for air drying for 3\u00a0h. Finally, the inoculated samples were stored at 4\u00a0\u00b0C for 24\u00a0h to allow sufficient bacterial attachment.Cherry tomatoes were purchased from a local market on the day of the experiment, and samples without rotting and apparent bruises were selected for the experiment. 2.22.2.1The washing water used for fresh-cut vegetables is recycled and the soluble matter from the produce can lead to a high chemical oxygen demand (COD) in the washing water, consuming oxidizing sanitizer. Thus, the use of produce homogenate in preparing the wash water with a certain COD value was recommended in previous studies 2.2.2The processing periods for washing and rinsing were 5 and 1\u00a0min, respectively, based on previous studies US-FC, US-CD, and US-PAA washing were performed as follows: 20 samples were placed into a stainless steel cage (18\u00a0cm\u00a0\u00d7\u00a015\u00a0cm\u00a0\u00d7\u00a05\u00a0cm) and into the US washer containing 10 L of wash water. A submersible pump was placed at the bottom of the washer to generate water flow.The rinsing process was performed using a spray system consisting of a bucket, a self-priming pump, and a spray nozzle. After US-FC and US-CD washing, the sample was placed on a shaker at 120\u00a0rpm , and the spray nozzle was set at 70\u00a0cm above the sample. The pump was immersed in the bucket to initiate rinsing at a rate of 0.75\u00a0L/min After the above treatments, the samples were transferred to a polyethylene terephthalate box and packaged using a polyvinyl chloride cling film 2.3E. coli O157:H7 and S. Typhimurium, respectively. For naturally-present microbes, 1\u00a0mL of the bacterial suspension was pour-plated in plate count agar (Hopebio) and incubated at 37\u00a0\u00b0C for 2 d to obtain the AMC, and 1\u00a0mL was pour-plated in rose bengal agar (Hopebio) and incubated at 28\u00a0\u00b0C for 5\u00a0days to quantify the M&Y.Eight samples were randomly selected from the package, transferred to a sterile stomacher bag, diluted 1:9 (w/v) in sterile 0.85% NaCl solution, and homogenized in a stomacher for 2\u00a0min. Then, 1\u00a0mL of the diluted bacterial suspension was spread-plated on modified sorbitol MacConkey agar (Hopebio) and xylose lysine deoxycholate agar (Hopebio) and incubated for 24\u00a0h at 37\u00a0\u00b0C to analyze 2.42.4.1\u00a0wtWeight loss in the sample was calculated as follows:2.4.2The values of L*, a*, and b* were determined using a colorimeter . Five samples were randomly selected from each package, and each sample was analyzed four times for a total of 20 readings per replicate. The color index was calculated using the formula described previously 2.4.3Firmness was determined using TA. XT Plus Texture Analyzer equipped with a cylindrical probe with a diameter of 3\u00a0mm. Five samples were randomly selected from each package, and the firmness of each sample was determined using the following parameters: pretest speed, 2\u00a0mm/s; test speed, 1\u00a0mm/s; post-test speed, 5\u00a0mm/s; auto trigger force, 5\u00a0g; and travel distance of the probe, 5\u00a0mm.2.4.4Eight panels were invited to evaluate the sensory color, flavor, and firmness of the samples. A 3-point scale method was used for evaluation, in which 0 indicated very poor, 5 indicated acceptability threshold, and 10 indicated liking very much 2.4.5Five samples were randomly selected from each package and rinsed for 1\u00a0min using TW to remove the AA present on the sample surface. After air-drying, the sample was soaked in liquid nitrogen for 30\u00a0s and then transferred to an IKA analytical mill for grinding. The resulting powder was used for analysis, as described in 2.4.6The ground powder (0.5\u00a0g) was mixed with distilled water at a ratio of 1:5 and analyzed using a hand-held refractometer to determine the TSS content. TA analysis was performed according to GB/T 12293-1990.2.4.7The ground powder (0.5\u00a0g) was mixed with 80% methanol at a ratio of 1:10 and then allowed to stand for 10\u00a0min. After centrifugation at 11,000 g for 10\u00a0min, the supernatant (50\u00a0\u03bcL) was mixed with 250\u00a0\u03bcL Folin reagent (Sinopharm) and 3\u00a0mL distilled water. After reaction for 6 mins, 750\u00a0\u03bcL of 20% sodium carbonate was added and incubated for 90\u00a0min in the dark. The absorbance was recorded at 765\u00a0nm, and the results were defined as gallic acid equivalents (mg/100 g) expressed on a fresh weight basis.The 1,1-diphenyl-2-picrylhydrazyl (DPPH) method was used for the antioxidant analysis. Briefly, 24\u00a0mg DPPH was dissolved in 100\u00a0mL methanol to prepare a stock solution and stored at \u221220\u00a0\u00b0C until use. Before each measurement, a working solution with an absorbance of 1.1\u00a0\u00b1\u00a00.02 at 515\u00a0nm by mixing a 10\u00a0mL stock solution with 45\u00a0mL methanol. The supernatant (150\u00a0\u03bcL) was mixed with 2850\u00a0\u03bcL working solution and reacted for 8\u00a0h. The absorbance was recorded at 515\u00a0nm, and the results were defined as Trolox equivalent (\u03bcM/g) expressed on a fresh weight basis.2.4.8Phenylalanine ammonia-lyase (PAL) was analyzed following the protocol of Zheng et al. 2, 0.15\u00a0mL 50\u00a0mM ATP, 0.15\u00a0mL 1\u00a0mM CoA, and 0.15\u00a0mL 5\u00a0mM p-coumarate), and incubated at 40\u00a0\u00b0C for 10\u00a0min. The reaction was stopped by adding 0.1\u00a0mL 6\u00a0M HCl. The absorbance was determined at 333\u00a0nm, and 4CL activity (U) was defined as the amount of enzyme that caused an increase in absorbance of 0.1 at 333\u00a0nm per minute.The method reported by Liu et al. 2.5All data were analyzed using the SPSS v.20. Differences between the means of the groups were evaluated using one-way analysis of variance and post hoc Duncan\u2019s multiple range test. Statistical significance was set at p\u00a0<\u00a00.05. Each experiment was independently performed three times, and the samples were analyzed on days 0, 3, and 5. Samples without any treatment were used as controls.33.1E. coli O157:H7, S.\u00a0Typhimurium, AMC, and M&Y were reduced by 1.95\u20132.11, 1.85\u20131.99, 1.44\u20131.48, and 1.12\u20131.22 log CFU/g, respectively (E. coli O157:H7 and 2.60 (day 3) and 2.47 (day 5) log CFU/g for S.\u00a0Typhimurium, which are significantly higher than those in traditional US-assisted washing method and US-PAA\u00a0+\u00a00.5%AA , ectively . Meanwhi+\u00a00.5%AA A, B. Sim+\u00a00.5%AA D. Moreov3.2The control group exhibited 0.92% weight loss at day 3, and the four combinations showed similar values ranging from 0.86 to 1.16%, which were not significantly different from the control group A. After Sensory quality was analyzed during the five-day period, and the results are shown in 3.3An increasing trend of polyphenolic content in the control group was observed from days 0\u20135. At day 5, the polyphenolic content was significantly improved from 20.79 (day 0) to 25.29\u00a0mg/100\u00a0g A. For th4E. coli than either of the individual treatments. Furthermore, a recent study indicated that the antibacterial mechanism of CD, another chlorine-based disinfectant, does not primarily involve the cell membrane and instead harms cells by damaging intracellular components, a mechanism that is significantly different from that of FC FC damages the cell membrane, leading to intracellular component leakage Similar to FC, PAA damages the cell membrane, and the combination of US and PAA accelerates membrane damage E. coli O157:H7. However, AA has been shown to have strong antibacterial activity during food storage. Recently, AA was successfully employed to disinfect Salmonella in soft cheese during storage E. coli O157:H7 present on AA-coated tomatoes was undetectable, whereas samples coated with carvacrol, citric acid, curcumin, and riboflavin showed 2.01, 1.46, 2.60, and 2.65 log CFU/g reductions in E. coli O157:H7 counts, respectively In general, hurdle technology combines different disinfection methods with different antibacterial mechanisms to further reduce microbial contamination Listeria monocytogenes on lettuce treated with 0.5% citric acid, propionic acid, and acetic acid were even higher than those of the control at the end of storage and that L. monocytogenes was significantly inactivated during storage when the concentration was increased to 1% L. monocytogenes, leading to an imbalance in the microbial composition and resulting in the rapid growth of L. monocytogenes. However, the organic acid concentration of 1% exceeds the upper limit of L. monocytogenes. This could also explain why US-PAA\u00a0+\u00a00.5% AA treatment did not lead to a greater reduction in E. coli O157:H7 and S.\u00a0Typhimurium counts during storage compared with the US-FC and US-CD treatments. A significantly greater reduction was observed as the AA concentration increased to 1%.Organic acids have a higher disinfection efficacy against M&Y than against AMC Changes in the sensory quality following acid treatment should also be considered. Vijayakumar and Wolfhall When plants are subjected to external stimuli, particularly abiotic stresses, the phenylpropanoid metabolic pathway is activated by accelerating secondary metabolite synthesis, mainly that of phenolic compounds 5E. coli O157:H7, S.\u00a0Typhimurium, AMC, and M&Y) on day 0; however, a significantly higher reduction was observed during storage. Second, US-PAA\u00a0+\u00a01%AA treatment did not lead to additional quality loss as compared with the control. Third, US treatment induced the upregulation of PAL and 4CL, leading to an increase in the polyphenolic content and antioxidant activity of cherry tomatoes.In this study, PAA was combined with US during the washing stage, and a US-assisted AA aerosolization approach was used to achieve microbial control during storage. There were three main findings of this study. First, compared with the traditional US-assisted disinfection method (US-FC\u00a0+\u00a0TW and US-CD\u00a0+\u00a0TW), the proposed method (US-PAA\u00a0+\u00a01%AA) did not lead to further microbial reduction (in terms of Although AA did not affect the sensory flavor of cherry tomatoes, its effects on sweet fruits should be explored in the future. Natural products are emerging as green and safe antibacterial treatments, and the combination of US-PAA with an aerosolized mixture containing AA and natural products should be evaluated in future studies. In addition, the mechanisms underlying the enhancement of phenolic contents have not been elucidated at the molecular level; therefore, such molecular analyses using multi-omics techniques should be undertaken in future studies.Jiayi Wang: Conceptualization, Supervision, Funding acquisition, Writing \u2013 original draft, Writing \u2013 review & editing. Zhaoxia Wu: Data curation, Writing \u2013 original draft, Writing \u2013 review & editing. Hongbin Wang: Data curation.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."} +{"text": "Despite existing observations of religious delusions in epilepsy in classical psychiatric literature, such clinical cases are rare in current practice.To reveal features of disease progression, interference of combined mental pathology, treatment specifics, markers of possible harmful behavior.Psychopathological, Multichannel eyes closed resting EEG in interictal period.Patient N, 39 years old, manifested her illness at age 13 with affective bipolar disorder; phases lasted several months each. From age 19, rare recurrent generalized convulsive paroxysms preceded by an aura; non-convulsive paroxysms were observed. The patient was uncritical of paroxysms and discontinued anticonvulsive therapy. At age 29 and 30 she suffered two psychotic attacks with sensory delusions of meaning, staging, persecution, megalomaniacal ideas of apocalyptic content . Delusional behavior . Anticonvulsive therapy accompanied by antipsychotic medications. Schizoaffective disorder and epilepsy diagnosed. From age 35, acute psychotic attacks with apocalyptic delirium preceded by the same aura lasted maximum one day, followed by partial amnesia. Epileptiform polyspikes (up to 150 \u03bcV) registered in the right temporal-central EEG leads.Presence of religious delusion in combined schizoaffective disorder and epilepsy, requires special approach: combination of anticonvulsants and antipsychotics. Religiosity of patient should be taken into account as well.No significant relationships."} +{"text": "Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4.in vitro and their biodistribution in vivo.We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vivo tumour uptake and retention of exendin(9-39).Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo imaging of GLP1R.We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based The online version contains supplementary material available at 10.1007/s00259-022-06041-y. Currently, several exendin formulations are clinically used to help normalise glucose levels in people with type 2 diabetes In-DTPA-exendin(9-39) in subcutaneous INS-1 tumours In-DTPA-exendin-4 (referred to as exendin-4), 12 mice received [111In]In-DTPA-exendin-4-Pen (referred to as exendin-4-Pen), 11 mice received [111In]In-DTPA-exendin(9-39) (referred to as exendin(9-39) and 11 mice received [111In]In-DTPA-exendin(9-39)-Pen (referred to as exendin(9-39)-Pen). To determine non-specific uptake, an excess of unlabelled peptide was injected (blocking group). A 100-fold excess of unlabelled DTPA-exendin-4 or DTPA-exendin(9-39) was co-injected (5 or 6 mice per group). Blocking groups were only included at the 4 h timepoint, to reduce the number of mice used as a control. After 1 h and 4 h after injection, mice were euthanized by CO2/O2 asphyxiation.All mice were intravenously injected with 20 pmol/mouse (0.37 MBq/mouse) of the corresponding radiolabelled exendin variant in approximately 200 \u03bcL PBS/0.5% BSA. 12 mice received In-exendin-4, [111In]In-exendin-4-Pen, [111In]In-exendin(9-39) and [111In]In-exendin(9-39)-Pen could be labelled with a high molar activity , reaching a radiochemical purity of > 99%. HPLC profiles showed single peaks at the expected elution times, confirming the purity of the radiolabelled exendin variants -Pen showed increased binding and internalisation compared to [111In]In-exendin(9-39) at 30 minutes and 4 hours incubation are reliable and easy to handle cell lines for in vitro testing of receptor specificity, we considered INS-1 cells preferable for in vivo studies because our group previously demonstrated that results obtained with INS-1 xenografts are more representative of the physiological situation In-exendin-4 (17.2 \u00b1 4.3 %ID/g after 1 h and 19.5 \u00b1 6.5 %ID/g after 4 h), and lower for [111In]In-exendin-4-Pen (8.8 \u00b1 1.7 %ID/g after 1 h and 6.5 \u00b1 0.8 %ID/g after 4 h). For the antagonist, addition of penetratin had no effect on pancreatic uptake. Both antagonistic variants showed very low uptake (1.7 \u00b1 0.5 %ID/g after 1 h and 2.2 \u00b1 0.3 %ID/g after 4 h for [111In]In-exendin(9-39) and 2.2 \u00b1 0.3 %ID/g after 1 h and 1.2 \u00b1 0.2 %ID/g after 4 h for [111In]In-exendin(9-39)-Pen) Fig. , B.Both [111In]In-exendin-4-Pen and [111In]In-exendin(9-39)-Pen showed unspecific accumulation in the liver and a small increase in spleen accumulation in comparison to the unconjugated peptides as well.in vitro and specific tumour uptake in vivo. Our results provide proof of concept that CPP conjugation can be used to turn a non-internalising antagonist into an internalizing tracer molecule for molecular imaging and theranostics.The objective of this study was to investigate the effect of the CPP penetratin on the cell and tissue interactions of exendin-4 and its antagonistic analogue exendin(9-39). We were especially interested in the opportunity to improve exendin(9-39) retention in GLP1R-expressing tissues. We found that penetratin increases binding and internalisation of exendin(9-39) In vitro, penetratin led to increased binding and uptake of [111In]In-exendin(9-39) in GLP1R-expressing cells. In contrast to binding, internalisation could not be completely blocked by an excess of unlabelled exendin(9-39). This observation is consistent with the presence of receptor unspecific internalisation triggered by the CPP. However, uptake of the exendin-CPP conjugates was low in cells that do not express GLP1R , conjugation of exendin-4 to penetratin did not lead to an increase in internalisation. A slight increase in binding was observed, which is probably due to interaction of the CPP with the cell membrane. Exendin-4 internalisation was very efficient, as has been reported before In-exendin(9-39)-Pen reached a higher tumour uptake in comparison to [111In]In-exendin(9-39) -Pen is likely due to internalization of the tracer and subsequent intracellular trapping of the residualizing complex [111In]In-DTPA. This confirms our hypothesis that CPP-mediated internalisation leads to higher tissue accumulation of the antagonist.The results 39) Fig. . Importa39) Fig. . In cont[111In]In-exendin-4 showed considerable uptake into the pancreas which, however, was lower for the [111In]In-exendin-4-Pen conjugate. This difference in uptake may be explained by sequestration of the CPP conjugate in the liver. In contrast, neither [111In]In-exendin(9-39) nor [111In]In-exendin(9-39)-Pen showed increased pancreatic uptake. However, one must be cautious in drawing conclusions from mouse pancreatic uptake. The mouse exocrine pancreas takes up exendin in a GLP1R unspecific manner, which does not reflect the human situation. Rats are a more suitable model for pancreatic uptake, as our group previously reported .Both [111In]In-exendin-4-Pen and [111In]In-exendin(9-39)-Pen showed unspecific liver uptake, which was not observed for the exendin analogues without penetratin. For [111In]In-exendin-4, the increased hepatic sequestration of the penetratin conjugate correlated with a decreased distribution to the pancreas and tumours. By comparison, for the [111In]In-exendin(9-39)-Pen conjugate this was not the case. Liver sequestration is a common phenomenon among CPPs , 28, andA tracer with the characteristics of exendin(9-39)-Pen is likely to quickly find a way to application. Our group provided proof of concept that exendin can be used for image-guided surgery (IGS) and targeted photodynamic therapy (tPDT) , 37. TheIn this study, we restricted ourselves to a proof-of-concept of increased antagonist internalisation and increased tumour retention. When aiming at clinical applications, further work will have to address the potential toxicity of the conjugated antagonist. We consider this risk low as for free penetratin cellular toxicity is only observed at concentrations higher than 10 \u03bcM.in vitro to in vivo. Previously, non-arginine was shown to increase the in vitro uptake of a peptide conjugate consisting of bombesin and an endosome-disrupting peptide, aiming at the cytosolic delivery of plasmid DNA [in vivo data have been presented. The previous investigation that most resembled our approach was the N- and C-terminal conjugation of several CPPs to the agonistic peptide PTH(1-34), derived from the parathyroid hormone (PTH) [50 values and in vitro epithelial permeability were assessed, but not cellular internalisation or biodistribution. Interestingly, that study showed that C- and N-terminal conjugation of the CPP changed the properties of the PTH(1-34) conjugate, in a different way for each CPP [Importantly, this is the first study that investigated the impact of CPP conjugation for an antagonistic G-protein-coupled receptor peptide ligand from smid DNA . Howeverne (PTH) . The IC5each CPP . This tiin vivo. This result opens the door to further unleashing the great potential of exendin, as a research tool and as a theranostic agent. Future research into CPP conjugates should be extended to other non-internalising peptide antagonists.In conclusion, we showed for the first time that a CPP efficiently causes cellular internalisation of an antagonistic, non-internalising peptide ligand, thereby increasing tumour retention of the tracer ESM 1(DOCX 1404 kb)"} +{"text": "In addition, the integration between the SGgel network and ABG moieties within a nano-scale level enabled the hybrid hydrogel to form adhesion to tissue, maintain the durable osteogenesis and accelerate bone regeneration. Therefore, a robust approach to the simultaneously satisfying tough adhesion onto the tissue defects and high efficiency for bone regeneration on a mouse skull was achieved, which may represent a promising strategy to design therapeutic scaffolds for tissue engineering in clinical applications.The significant efforts being made towards the utilization of artificial soft materials holds considerable promise for developing tissue engineering scaffolds for bone-related diseases in clinics. However, most of these biomaterials cannot simultaneously satisfy the multiple requirements of high mechanics, good compatibility, and biological osteogenesis. In this study, an osteogenic hybrid hydrogel between the amine-functionalized bioactive glass (ABG) and 4-armed poly(ethylene glycol) succinimidyl glutarate-gelatin network (SGgel) is introduced to flexibly adhere onto the defective tissue and to subsequently guide bone regeneration. Relying on the rapid ammonolysis reaction between amine groups (-NH Bone tissue possesses unique self-repair and regeneration capacities for minor injuries, however, severe bone fractures and defects, as well as trauma and osteosarcoma, are still harmful diseases in orthopedics ,2,3. AltHydrogels comprising of 3D crosslinking polymer networks with multifariously physical structures and chemical properties have been widely explored as biological scaffolds to mimic the natural ECM environment when properly designed, thus facilitating the viability, growth, proliferation, and differentiation of the stem cells for tissue repair ,24,25,26Using high performance hydrogels as biological materials for bone tissue repair has led to great progress in enhancing the robustness of bone tissue in recent years. In general, the incorporation of bioactive components is an effective method to improve the biological performance of bone constructs ,36,37,38In this work, in order to fabricate an advanced osteogenic scaffold that could simultaneously satisfy the multiple requirements of strong adhesion onto tissue defects, good compatibility, high mechanics and promote the sustainable osteogenesis effect, a class of highly integrated inorganic-inorganic hydrogel scaffold (SGgel@ABG) was developed via the rapid ammonolysis reaction between NHS-activated tetra-PEG-SG polymer and gelatin in the presence of an amine-terminated bioactive glass (ABG) particle in the one-pot strategy . On acco1H NMR spectrum could be found and belonged to its explicit molecular structure and the clear integration peak ratio (a/b/c/d = 2:1:1:1), as shown in The schematic illustration of the SGgel@ABG hybrid hydrogel was shown in 4 kPa), which is larger than G\u2033, indicated an elastic state and rigid backbone, which is indicative of its potential values for the tissue regenerations.The SEM images showed the morphologies of the hydrogel scaffolds, as can be seen in w/v of ABG, was an optimized formula with optimal structures and well-balanced properties. Therefore, with facile injection, tough adhesion and biological properties, this hydrogel adhesive may spark innovative clinical treatments to guide bone regeneration.4 cells/mL on day 1, (1.74 \u00b1 0.08) \u00d7 104 cells/mL on day 3 and (3.51 \u00b1 0.1) \u00d7 104 cells/mL on day 5, higher than those of the SGgel hydrogel and control group, indicating the important roles of ABG substrate on improving cell proliferation after prolonged incubation.Biomaterial compatibility is a prominent prerequisite for high-qualified cell carriers. The FDA-approved PEG and biocompatible gelatin are widely used in biomedical applications, such as in neural, articular cartilage tissue, bladder and bone repair. Therefore, cytotoxicity was first conducted to assess the safety of the SGgel and SGgel@ABG hydrogel scaffolds, by the BMSCs, using a live/dead assay. To explore whether this hybrid hydrogel could support cell activity, growth, and proliferation, we performed the proliferation experiment and measured the OD values at 450 nm after the in vitro culture on the hydrogel scaffolds using a CCK8 assay. After the co-culture of the SGgel@ABG hydrogel, the cell proliferation rate was slightly improved initially, and significantly increased at days 3 and 5 of in vitro incubation C, revealIn addition to the requirement good biocompatibility, ideal engineered scaffolds should also meet the needs of boosting progenitor cell\u2019s osteogenic differentiation. ALP staining, ARS staining and qPCR were applied to assess the osteoinductivity of the SGgel@ABG hydrogel scaffold. As shown in Inorganic BG is a series of bioactive, biodegradable and osteoconductive materials for bone tissue engineering, but its application in bone tissue engineering is limited due to its easy removal from the target site. In regard to the SGgel@ABG composite hydrogel with good mechanical, compatible and biological properties in vitro, we performed in vivo bone-defect experiments of this bioactive scaffold to demonstrate its strong bonding capacity, excellent cell ingrowth and regenerative bioactivity. A circular bone piece was fixed within freshly formed cranial defects and treated with the SGgel and SGgel@ABG hydrogels. The stability and bone regeneration of the circular bone plates were evaluated eight weeks after surgery. The micro-CT images exhibited the dislocation of the circular bone piece in the control group. This was the result of its lack of fracture fixation, sustainability and regenerative ability A. No simA histologic analysis experiment was carried out to differentiate the bone and soft tissue within the defect after eight weeks E\u2013G. ThisThe repair mechanism of the SGgel@ABG composite hydrogel may be attributed to the following factors: (1) the component of osteoid and bone tissues. Biocompatible natural gelatin was favored for the chondrogenesis to improve proteoglycan/collagen secretion, and polymeric tetra-PEG-SG provided a dense crosslinking junction and sufficient adhesion to the tissue surface to maintain the durable mechanical support and long-lasting stability; (2) the SGgel@ABG composite hydrogel could offer a moisture environment to ensure the surrounding cells\u2019 survival, growth and proliferation, which played a role in the initial stage of bone regeneration; (3) in addition to the hydrogel degradation in vivo, ABG could be exposed-contacted into, and subsequently bound onto, the tissues to facilitate angiogenesis, which was of vital importance to enhance bone regeneration; (4) on account of the main biological components of silicon, calcium and phosphorus elements within the ABG, the gradual degradation and element release could be absorbed to promote bone mineralization. Consequently, in comparison to the previous attempts to fabricate tissue engineered hydrogel scaffolds for tissue regeneration, the current attempt presents a distinctive formula to offer a potential clinic alternative with good biocompatibility, facile injectability, usage flexibility and long-lasting osteogenic capacity.In summary, we designed a novel SGgel@ABG hybrid hydrogel adhesive for local bone regeneration. The porous structure and favorable mechanics of the SGgel@ABG scaffold could enhance cell activity, growth, proliferation, and differentiation, while its strong adhesive strength and durable stability on bone tissue was essential for clinical bone repair. In addition, the amine-functionalized ABG moieties not only rationally provided the physical-chemical crosslinking junction on the improving the mechanical properties, but also significantly contributed to the bioactive osteogenic activity, which was found to effectively guide the osteogenesis, maintain the perdurability of bone formation, and accelerate the bone repair both in vitro and in vivo. This study demonstrated an adaptive strategy for producing bone scaffolds with a rational design and fabrication of advanced biomaterial hydrogels for clinical treatments.w = 20 kDa, Mw/Mn = 1.03, SINOPEG), glutaric anhydride , N-hydroxysuccinimide , 3-aminopropyltriethoxysilane , gelatin , and bioactive glass was purchased from Shanghai Aladdin Bio-chem Technology Co., Ltd. . All other chemical and biological compounds were purchased from Beijing Chemical Works and used without any further treatments and purification steps.Four-arm poly(ethylene glycol) with a velocity of 2 mm min\u22121. The adhesion testing was also performed using Instron 3365 at room temperature. One side of fresh squared porcine skins (diameter: 16 mm \u00d7 16 mm) was adhered to a Perspex cylinder using an \u03b1-cyanoacrylate glue, while the other side was adhered using the hydrogel adhesive at room temperature. When the attaching porcine skins were torn apart under the driven force, the shear strength was measured with a force sensor of 100 N.The nuclear magnetic resonance spectrum was obtained on a Bruker DRX-400 spectrometer. Scanning electron microscopy (SEM) images were measured at an acceleration voltage of 5 kV, on a JSM-6700F microscope. The sample was sputter-coated with a thin layer of Pt for 120 s before the measurement. The rheological measurements were carried out on a Thermo Haake Rheometer, equipped with cone-parallel plate geometry (diameter: 35 mm), at a gap of 0.5. The sample was spread on a parallel plate, followed by sealing with the oil to prevent water evaporation during the experiment, which was conducted at 25 \u00b0C with a constant strain of 0.05%, in the frequency range of 100\u20130.1 rad s2Cl2. After stirring for 24 h, the solution was washed with a hydrochloric acid aqueous solution, saturated sodium chloride aqueous solution and DI-water, several times over, and dried over anhydrous MgSO4. Next, the obtained product was further precipitated into diethyl ether for twice to obtain the tetra-arm PEG-glutaric acid polymer. Then, tetra-PEG-COOH , NHS and EDCI were dissolved in 25 mL of anhydrous CH2Cl2. After stirring for 24 h, the solution was washed with a hydrochloric acid aqueous solution, saturated sodium chloride aqueous solution and DI-water, three times, and dried over anhydrous MgSO4 to yield the white solid of tetra-PEG-SG.First, tetra-PEG-OH , DMAP and glutaric anhydride were dissolved in 50 mL of anhydrous CHThe modification method used to prepare ABG particle was carried out by adding 5 mL APTES into 100 mL of hexane, containing 0.3 g of BG. After stirring for 24 h at 60 \u00b0C, the amine-terminated ABG was facilely yielded after being washed with the ethanol and DI water several times.The SGgel adhesive was prepared by mixing the same volume of solution A, containing 20 wt% of gelatin, and solution B, containing 15% of tetra-PEG-SG polymer. For the SGgel@ABG, 5 wt% of ABG was incorporated into solution A with stable sonication, 10 min before mixing solution A and B.4 cells/100 \u00b5L were incubated for 24 h in a 5% CO2 humidified incubator. The hydrogel was treated in fresh cell medium for another 24 h in order to obtain the extracts. Then, the treated cell medium was used to replace the fresh cell medium to obtain further cell cultures. The cells that were cultured in the fresh medium formed the control group. The cell viability was calculated via the equation:A CCK-8 assay was used to evaluate the cytotoxicity of the hydrogel scaffold. After suspending it into a fresh cell culture medium and seeding it into a 48-well plate, the BMSCs with a density of 1 \u00d7 10The cell biocompatibilities were evaluated using a Live/Dead Viability Kit. The BMSCs on either a blank plate or hydrogels were stained with live/dead staining working solution , and the stained cells were directly observed under the inverted optical microscope . Calcein-AM generates a green fluorescence signal in living cells and PI only reaches the nuclei of dead cells to emit red fluorescence. The number of live cells was quantitatively analyzed using Image J software .Cell proliferation measurement was also carried out using a CCK-8 assay. Firstly, the BMSCs were incubated in a growth medium for 1 day. Next, the hydrogel samples were added into the medium and further incubated for another 1 day. After 5 days of cell incubation, 100 \u00b5L of fresh culture medium was used to replace the cell culture medium and added into 10 \u00b5L of CCK-8 for another 4 h. Then, the absorbance was measured at 450 nm and recorded, using a microplate reader to calculate the cell proliferation.6 cells/well in osteogenic induction medium. On differentiation days 14 and 21, the cells were washed several times, fixed with 4% paraformaldehyde for 15 min, and stained for 30 min using the ALP staining kit (day 14) and ARS solution (day 21). An inverted optical microscope was used to capture the images. The areas of ALP positive colony forming unit (Cfu-ALP) and ARS positive colony forming unit (Cfu-ARS) were calculated using Image J software. The stained cells were lysed with 10% hexadecylpyridinium chloride monohydrate for 15 min and the absorbance of the collected supernatant at 540 nm was measured using a spectrophotometer. The relative ALP and ARS activities were normalized to total protein content, which was determined using a BCA kit.The BMSCs were seeded on the blank plate or the hydrogels pre-coated plate, with a density of 1 \u00d7 10The total mRNA extraction was performed using TRIzol and was then subjected to reverse transcription by PrimeScript RT Master Mix. The qRT-PCR was conducted using the ABI 7500 Real-time PCR System . The expression of the target genes was measured using TB Green Premix Ex Taq and the expression of \u03b2-actin was used as an endogenous control.In order to study the osteogenesis effects of hydrogel scaffolds in vivo, the animal experiments were carried out on 8-week-old male Sprague\u2212Dawley (SD) rats using the calvarial defect model. These rats were anesthetized by an intraperitoneal injection of pentobarbital , and the sagittal incisions were made on the skin and the muscle to the cranial roof. After elevating the skin-muscle-periosteum flap, a 5 mm thick circular defect was created in the center of each parietal bone with a ring drill of a corresponding external diameter. Next, the implanted hydrogel samples were injected into the rat defects and the incisions were closed with the resorbable sutures. The rats returned to normal function after surgery. A total of 30 rats were randomly divided into the graft study groups: (1) negative control group (defect only), (2) SGgel/BMSCs group, and (3) SGgel@ABG/BMSCs group. All surgical interventions and postoperative animal care procedures were approved by the Animal Research Committee of Peking University (LA2020465).To assess the quality of the new bone formation, skull bones possessing a defect region were extracted 8 weeks after the implantation. Micro-CT was performed to scan the skull defects using the SkyScan 1076 System , under a standard condition . DataViewer was used to reconstruct 3D images of micro-CT. A diameter of 5 mm, covering the bone defect region in the reconstructed 3D image, was defined as the region of interest (ROI), and the bone morphometric parameters, including bone volume (BV), bone volume/tissue volume (BV/TV) and bone mineral density (BMD), were analyzed in ROI.The skull samples were fixed in 4% paraformaldehyde for 2 d and subsequently decalcified with a daily change of 15% tetrasodium EDTA for 28 d. From the center of the defect region in the coronal plane, a decalcified skull sample was divided into 8 mm thick slices. Next, the samples were dehydrated in an ethanol series before being embedded in paraffin. From the center of the defect region in the coronal plane, decalcified skull samples were sliced into 5 \u00b5m thick sections for the histological examination and morphological analysis with Hematoxylin-Eosin (H&E) staining and Masson\u2019s trichrome staining.p < 0.05 was considered statistically significant.All the results were expressed in the form of mean and standard deviation and there were at least 3 independent experiments. For multi-group comparisons, the one-way ANOVA was assessed, followed by either the Student-Newman-Keuls or Dunnett\u2019s test, wherever appropriate;"} +{"text": "Relying on the dense integration between the TSG-PEI network and ABG moieties on a nano-scale level, this hydrogel expressed powerful adhesion to tissue as well as durable stability for the engineered scaffolds. Therefore, its self-endowed biocompatibility, high adhesive strength, compressive modulus, and osteogenic potency enabled the prominent capacities on modulation of bone marrow mesenchymal stem cell (BMSCs) proliferation and differentiation, which may propose a potential strategy on the simultaneous scaffold fixation and bone regeneration promotion for the tissue engineering fields.Osteogenic scaffolds reproducing the natural bone composition, structures, and properties have represented the possible frontier of artificially orthopedic implants with the great potential to revolutionize surgical strategies against the bone-related diseases. However, it is difficult to achieve an all-in-one formula with the simultaneous requirement of favorable biocompatibility, flexible adhesion, high mechanical strength, and osteogenic effects. Here in this work, an osteogenic hydrogel scaffold fabricated by inorganic-in-organic integration between amine-modified bioactive glass (ABG) nanoparticles and poly(ethylene glycol) succinimidyl glutarate-polyethyleneimine (TSG-PEI) network was introduced as an all-in-one tool to flexibly adhere onto the defective tissue and subsequently accelerate the bone formation. Since the N-hydroxysuccinimide (NHS)-ester of tetra-PEG-SG polymer could quickly react with the NH Bone tissue has an inborn and unique ability of self-regeneration for many variously minor injuries, but some serious diseases like bone fracture defects, trauma, and even osteosarcoma are still dangerous in orthopedics ,2,3. AltThe rapid development of hydrogel scaffolds with preferred biocompatibility, sufficient mechanical strength, matching degradation rate, and promotional osteogenic activity has become a hot spot in bone tissue engineering research ,18,19,20Branched polymers with dendritic structures and abundant terminal groups have been an ideal crosslink to design the complex networks and advanced functions for the topological polymers and hydrogels ,30,31,32Bioactive glass (BG) comprised of silicon, calcium, and phosphorus elements has advanced the ability to induce bone mineralization to repair bone defects. Especially under physiological conditions, BG can produce a hydroxyapatite layer through chemical combination with bone tissue, similar to the stage of forming bone mineral, and provides a favorable environment for the activity, growth, proliferation, and differentiation of osteoblast-related cells ,39. HoweHerein, we designed and prepared an inorganic\u2013organic TSG-PEI@ABG hydrogel via the highly efficient ammonolysis reaction between (NHS)-ester of tetra-PEG-SG and branched PEI polymers in the presence of ABG particles in one-pot step . Covalenw = 20 kDa) was purchased from SINOPEG, China. Glutaric anhydride (98%), dimethylamino-pyridine , and N-hydroxysuccinimide were purchased from Energy Chemical Co., Ltd., Shanghai, China. 3-aminopropyltriethoxysilane was purchased from J&K, Beijing, China. Branched polyethyleneimine was purchased from Alfa Aesar. Bioactive glass was purchased from Aladdin. All other chemical reagents were purchased from Energy Chemical Co., Ltd., Shanghai, China, and used as received without further purification.Tetra-arm poly(ethylene glycol) polymer. Then, tetra-PEG-COOH , EDCI , and NHS were dissolved in 25 mL of anhydrous DCM. After continuously stirring for 24 h, the DCM solution was washed with 2 M HCl solution, saturated NaCl solution, and DI water three times and was followed by drying over the anhydrous MgSO4 to afford the white solid of tetra-PEG-SG polymer under the vacuum.First, tetra-PEG-OH , glutaric anhydride , and DMAP were dissolved in 50 mL of anhydrous DCM. After continuously stirring for 24 h, the DCM solution was washed with 2 M HCl solution, saturated NaCl solution, and DI water three times, followed by drying over the anhydrous MgSOThe modification method to prepare ABG particle was performed according to the previous works ,42. BrieTSG-PEI hydrogel was easily prepared after mixing the 15 wt% of tetra-PEG-SG and 1 mg/mL of PEI solutions at 37 \u00b0C without adding any other additive crosslinkers. As for the preparation of TSG-PEI@ABG hydrogel, the ABG was added into the PEI solutions in advance with continuous sonication for 30 min, and then mixed with tetra-PEG-SG solution via vortexing at 37 \u00b0C to achieve the gelation.1H NMR and 13C NMR) spectra were obtained on a Bruker DRX-400 spectrometer using the deuterated chloroform as the solvent and tetramethyl-silane (\u03b4 = 0 ppm) as the internal standard.Nuclear magnetic resonance (The network architecture of hydrogels was observed using scanning electron microscopy (SEM). Firstly, the hydrogels were freeze-dried to obtain the dry samples, and then a thin gold layer of Pt was sputter-coated onto the sample surface for 90 s to make them conductive. After that, the hydrogel networks could be observed via a JEOL JSM-6700F microscope with an acceleration voltage of 10 kV.The compressive strength of hydrogels was measured using a universal tensile machine at a compressive speed rate of 2 mm/min. The hydrogel samples were cut into cylinders (diameter of 15 mm and height of 8 mm) for the measurement.The rheology behavior of hydrogels was conducted using a rheometer . The measured hydrogel samples were spread on a parallel plate (25 mm) and sealed with silicone oil. A dynamic frequency scan in the range from 0.1 to 100 rad/s was used to record the storage and loss moduli (G\u2032 and G\u2033). The stress amplitude was set as 0.1% and the temperature was set at 25 \u00b0C.Adhesion measurement was performed using the lap shear test by injecting 1 mL of gels onto the porcine skins, and commercially available fibrin glue was used as the control group. Briefly, standard lap shear test was performed on porcine skins that were adhered using the TSG-PEI, TSG-PEI@ABG, and fibrin glue for 30 s of pressing under a 200 g weight at room temperature before the tests, respectively. The porcine skins were cut into slices with a length of 60 mm, thickness of 10 mm, and width of 20 mm for the usage. All tests were performed with the universal testing machine at a constant rate of 10 mm/min. The experiments were carried out at room temperature with a humidity of 45%, and each sample was tested five times to obtain the average values.2. Isolation, culture, trilineage differentiation potential assay, and immunophenotypic identification of BMSCs were verified as previously described in the literature [The bone marrow mesenchymal stem cells (BMSCs) were isolated from 3-month-old New Zealand white rabbits. The original generation of BMSCs was expanded up to passage 3 for experiments with the culture medium. The cells needed to be cultured in the medium and refreshed every 2 days in an environment of 37 \u00b0C and 5% COterature .4 cells/100 \u00b5L and incubated for 24 h at 37 \u00b0C in a 5% CO2 humidified incubator. The culture medium was then replaced with the hydrogel extract and incubated for another 24 h. Cells cultured in fresh medium were set as the control group. The cell viability (%) was calculated via the following equation:The cytotoxicity was performed using CCK-8 assay. The hydrogel extracts were prepared by placing the hydrogel into Dulbecco\u2019s Modified Eagle\u2019s medium (DMEM) at 37 \u00b0C for 24 h and then sterilizing them by filtration with a 0.22 \u03bcm filter. BMSCs were suspended in cell culture medium and seeded into 48-well plates with a density of 1 \u00d7 10Cell proliferation was also measured using CCK-8 assay. Firstly, the BMSCs were incubated in growth medium for 1 day, and then the hydrogel extracts were added into the medium and further incubated for another several days. After 7 days of incubation, cell culture medium was replaced with 100 \u00b5L of fresh culture medium and 10 \u00b5L of CCK-8 were added for another 4 h. Then, the absorbance at 450 nm was recorded on a microplate reader . Cell number was correlated with OD value for calculating the cell proliferation.Cell biocompatibilities were evaluated using a live/dead viability kit. The BMSCs after culture with hydrogel extracts was stained with live/dead staining working solution according to the manufacturer\u2019s protocol. After culturing for 1 day, the stained cells were directly observed under the inverted optical microscope . Calcein-AM generates a green fluorescence signal in living cells and PI dye only affects the nuclei of dead cells to emit a red fluorescence light. The number of live cells was quantitatively analyzed by Image J 1.8.0 software .6 cells/well in osteogenic differentiation medium including \u03b1-MEM supplemented with 10% of FBS, 1% of antibiotics, 50 \u03bcM of ascorbic acid, 10 mM of \u03b2-glycerol phosphate, and 0.1 \u03bcM of dexamethasone. After the osteogenic incubation for 7 and 14 days, the cells were washed three times, fixed with 4% of paraformaldehyde for 15 min, and stained for 30 min using the ARS staining kits at room temperature. The stained BMSCs were dried, and image J 1.8.0 software was utilized to calculate the stained areas for semi-quantitative analysis.BMSCs were seeded on the hydrogel extracts with a density of 1 \u00d7 10The total RNA was extracted using the typical TRIzol Reagent and cDNA was synthesized from 200 ng of total RNA by RevertAidTM H Minus First Strand cDNA Synthesis Kit . Template PCRs were performed after 33 cycles of amplification with the adjustment annealing temperature. The primer sequences were listed as follows in p < 0.05 was considered to be significant.All the results were presented as the mean and standard deviation of 3\u20136 independent experiments. The statistics were analyzed by SPSS 22.0 software . One-way ANOVA was used for more than two groups. 1H NMR spectrum shows the explicit attribute of the characteristic peaks of tetra-PEG-SG with a very close integration ratio (a:b:c:d = 2:1:1:1) in 13C NMR spectrum also clearly verified the structural integration and further proved the successful preparation of targeted polymer. Taking into consideration the abundant amine groups in the branched PEI polymers and ABG particles, the NHS-reactive tetra-PEG-SG polymer can be quickly reacted to form the TSG-PEI or TSG-PEI@ABG hydrogel networks after mixing them together, within seconds. It was mentioned that the ABG particles should be mixed with PEI solution under the sonication in advance to achieve the uniform arrangement as far as possible between the inorganic phases and organic materials. As shown in The scheme illustration of preparation process of TSG-PEI@ABG hydrogel is shown in Compared to the TSG-PEI hydrogel, the mechanical performances of TSG-PEI@ABG hydrogel were significantly improved in For assessing one of the key factors to endow the engineered scaffold with high stability and durability during the practical application, the adhesive property of the hydrogel was quantitatively characterized on porcine tissue by a lap shear test C. PrevioIn view of its favorable structures, pores, mechanics, and adhesion force of the TSG-PEI@ABG hydrogel, it is necessary to assess the cell biocompatibility for this engineered scaffold. Therefore, we co-cultured the BMSCs with the hydrogel extract to evaluate the in vitro cytocompatibility by CCK-8 and live/dead staining assays. Quantitatively, the TSG-PEI hydrogels had capacities on maintaining a high cell survival level over the times with or without being laden with ABG. The cell viability ratio at day 3 was obviously higher than that of day 1, indicating the low cytotoxicity and high cell proliferation behaviors A. In addTo reveal the effect of hydrogel scaffolds on the osteogenic differentiation in vitro, we seeded BMSCs cells on the control, TSG-PEI, and TSG-PEI@ABG hydrogel scaffolds. ARS staining and qPCR assay were used to examine the osteoinduction capacity. As shown in In addition, the mRNA levels of expression of the osteogenesis-related marker genes of OCN, Osterix, ALP, and Runx 2 were investigated using the control, TSG-PEI, and TSG-PEI@ABG hydrogels, as shown in 2-modified ABG introduction not only contributed to the hybrid crosslinking junction to enhance the cohesive strength and structural stability, but also provided the crucial osteogenic components to guide the osteogenesis. Consequently, compared to previous attempts at preparing the engineered hydrogel scaffolds for osteogenesis, the current attempt has put forward an adaptive hybridization strategy, offering a potential candidate with feasible usage availability and robust osteogenic potency.In summary, we constructed a kind of hybrid hydrogel adhesive via the efficient NHS\u2013amine chemistry due to the availability and affordability of the polymeric components of NHS-activated tetra-PEG-SG polymer and the amine groups of branched PEI polymer and ABG particles. Under the high efficiency of the crosslinking reaction, the obtained TSG-PEI@ABG hydrogel displayed suitable porous architectures, sufficient compressive and adhesive strength, and facilitated a moisture environment for the stem cell survival, growth, and proliferation. It was particularly stressed that NH"} +{"text": "Background: Case-based surveillance of antimicrobial resistance (AMR) provides more actionable data than isolate- or sample-based surveillance. We developed A Clinically Oriented antimicrobial Resistance surveillance Network (ACORN) as a lightweight but comprehensive platform, in which we combine clinical data collection with diagnostic stewardship, microbiological data collection and visualisation of the linked clinical-microbiology dataset. Data are compatible with WHO GLASS surveillance and can be stratified by syndrome and other metadata. Summary metrics can be visualised and fed back directly for clinical decision-making and to inform local treatment guidelines and national policy.Methods: An ACORN pilot was implemented in three hospitals in Southeast Asia to collect clinical and microbiological data from patients with community- or hospital-acquired pneumonia, sepsis, or meningitis. The implementation package included tools to capture site and laboratory capacity information, guidelines on diagnostic stewardship, and a web-based data visualisation and analysis platform.Results: Between December 2019 and October 2020, 2294 patients were enrolled with 2464 discrete infection episodes . Overall, 28-day mortality was 8.7%. Third generation cephalosporin resistance was identified in 54.2% (39/72) ofE. coli and 38.7% (12/31) ofK. pneumoniae isolates. Almost a quarter ofS. aureus isolates were methicillin resistant . 290/2464 episodes could be linked to a pathogen, highlighting the level of enrolment required to achieve an acceptable volume of isolate data. However, the combination with clinical metadata allowed for more nuanced interpretation and immediate feedback of results.Conclusions: ACORN was technically feasible to implement and acceptable at site level. With minor changes from lessons learned during the pilot ACORN is now being scaled up and implemented in 15 hospitals in 9 low- and middle-income countries to generate sufficient case-based data to determine incidence, outcomes, and susceptibility of target pathogens among patients with infectious syndromes. The benefits of case-based versus isolate-based surveillance of antimicrobial resistance have recently been discussed in peer-reviewed literature. In a recent ACORN protocol manuscript, correspondence on ACORN and a review on surveillance strategies the three purposes of surveillance of antimicrobial resistance (AMR) were listed as \u201c(i) to inform empiric treatment guidelines and clinical decision-making; (ii) to characterize trends in space and time including outbreak detection; and (iii) to provide a benchmark to measure the impact of interventions provides a platform for standardised data collection and submission allowing comparison of data between countries and regions. Sixty-six of 82 enrolled countries (as per 31st July 2019) have submitted data. Most countries submitted isolate or sample-based data, with low- and middle-income countries (LMICs) relatively underrepresented . The neeIsolate and sample-based surveillance data without clinical denominators may have various biases favouring resistant organisms due to lack of diagnostic stewardship, underuse of diagnostic microbiology resources and a sampling preference for more severe cases or treatment failures, especially in LMICs .Other authors summarised the benefits of case-based surveillance in an opinion piece as: \u201c(i) to allow linking of AMR profiles with patients at risk; (ii) to better inform treatment guidelines; (iii) to identify high-risk populations; (iv) to provide reliable data streams for analyses of effectiveness of interventions; and (v) to study the linkage of AMR phenotypes and burden\u201d collection of 28-day mortality data, and (v) direct feedback of local data, visualised using an interactive dashboard. The main objective of ACORN is to generate data in LMICs that can be directly used to inform local treatment guidelines and national policy, in addition to inclusion in international pathogen-focused AMR surveillance systems and burden of disease studies.Drawing from these issues and benefits, we have developed ACORN: A Clinically-Oriented antimicrobial Resistance surveillance Network. ACORN aims to be a routine hospital activity of collecting and combining clinical and microbiology data for AMR surveillance with direct feedback and visualisation for end-users. To achieve this, five components are added to a GLASS compatible backbone of microbiology data: (i) active case-finding and provision of diagnostic stewardship materials to support correct use of microbiology diagnostics, (ii) bedside clinical data collection using a short Open Data Kit (ODK) e-questionnaire on a tablet computer, (iii) software solutions to link collected data to existingHere we describe the results of a pilot in three central hospitals collaborating with the University of Oxford Tropical Network in Southeast Asia: Mahosot Hospital in Vientiane, Laos; Angkor Hospital for Children (AHC) in Siem Reap, Cambodia and the National Hospital for Tropical Diseases (NHTD) in Hanoi, Vietnam. The surveillance protocol was published previously . PatientClinical data was captured using password protected Android smartphones / tablets via Open Data Kit (ODK) Collect. Clinical records and electronic hospital information systems of enrolled patients were reviewed by study doctors daily to capture ICD10 codes for infection episode (if recorded), final classification of surveillance diagnosis , hospitalisation outcome and date and number of days admitted to an intensive care unit. The participant was contacted by telephone on day 28 post enrolment to determine health status and date of death, if appropriate. The protocol implementation package included tools to capture site and laboratory capacity information, guidelines on diagnostic stewardship, and a web-based data visualisation and analysis platform to support standardisation of clinician diagnosis and specimen collection (see1 and 2 ). SurveiExtended data).Clinical variable selection was informed by a stakeholder workshop, with representation from WHO GLASS, WHONET, University of Oxford Tropical Network, International Vaccine Institute (IVI), Foundation for Innovative Diagnostics (FIND), Fleming Fund, Centre for Disease Dynamics, Economics & Policy (CDDEP), held in Bangkok in May 2019. Clinical variables in addition to age, sex, ward and date of admission included co-morbidities, date of original admission if transferred from another healthcare facility, hospitalisation and/or surgery in last three months, severity features, presence of medical devices, diagnostic blood culture taken within 24 hours of admission or symptom onset (HAI), systemic antibiotics received in 24 hours before diagnostic blood culture, and empiric antibiotic treatment received. Patients admitted with CAI were re-classified as having healthcare associated infections (HCAI) if they had hospitalisation and/or surgery in last three months. Patients admitted with active infection on transfer from another hospital were considered as a subset of CAI, given that the majority of patients were transferred early and site investigators felt that most transfers were likely escalation of care CLSI and EUCAST guidelines or WHONET software, was matched to ACORN clinical data in andelines .Ethical approval was obtained through the Oxford Tropical Research Ethics Committee , Cambodia National Ethics Committee for Health Research , Laos Ministry of Health \u2013 University of Health Sciences Ethics Committee , and NHTD Institutional Review Board, . All committees agreed to waive the need for written individual informed consent as this surveillance was defined as a minimal/negligible risk activity, consisting of implementation of accepted quality improvement tools (diagnostic stewardship) and collection and use of limited clinical data that is expected to be collected as part of standard of care. Patients were instead given an information sheet explaining the surveillance project and given the opportunity to opt out on screening and enrolment. No patient samples were collected other than for clinical diagnostic purposes. All patient identifiable data were removed during the clinical-laboratory data linkage procedure, rendering an anonymised dataset for analysis and onward sharing.Preliminary data on key infection syndromes and associated pathogens was collected prior to study start. Challenges and potential solutions to implementation of the surveillance protocol were identified using a baseline AMR knowledge, attitudes, and practices (KAP) survey of clinicians working on the surveillance wards, an open call for feedback, augmented by formal requests for feedback to be presented at investigator meetings (data not shown).R version 4.1.0 .Patients were enrolled between December 2019 and May 2020, with an extension until end October 2020 after an investigator meeting because of coronavirus disease 2019 (COVID-19) delays and quarantine of the Vietnam site and 518 classified as HCAI (21.0%). Weekly point-prevalence surveys identified 160 HAI (6.5%). CAI accounted for 66.3%, 88.9% and 62.2% of cases at AHC, Mahosot and NHTD, respectively. At presentation, 253 episodes were clinically diagnosed as meningitis, 677 as pneumonia and 1534 were sepsis. On review at hospital discharge 39.9% meningitis diagnoses, 23.9% pneumonia diagnoses and 25.7% sepsis diagnoses were rejected based on clinical course / diagnostic test results by treating physicians. During the pilot, alternate final diagnoses were not recorded, and thus further results are presented here based on presentation diagnosis.The discharge outcome of 83/2408 (3.5%) admissions was death, with an additional 93 (3.9%) patients discharged moribund. Seven patients had no discharge status recorded. Day-28 follow-up occurred post-discharge for 1995 admissions, at a median of 25 days after discharge . An additional 126 deaths were identified . MortaliExtended data).Restricting to 2287 first infection episodes where discharge status was available , 2114 (92.4%) patients were discharged alive and expected to recover with 92 patients (4.0%) discharged moribund and 81 (3.5%) whose hospital outcome was death. There were no clear differences between hospital outcome by clinical syndrome. However, a higher proportion of patients with HAI were discharged moribund compared to those with CAI or HCAI of infection episodes in total and in 87.7% (1215), 74.8% (552) and 68.0% (232) of episodes at AHC, Mahosot and NHTD, respectively. Collected blood cultures are summarised by syndrome and origin of infection inBlood cultures were reported to contain contaminants in 70/1310 (5.3%) and 31/569 (5.4%) specimens at AHC and Mahosot, respectively. At NHTD these numbers were not available as the laboratory does not report growth of these contaminants back to the wards, often after checking with treating physicians.E. coli, 19S. aureus, 18K. pneumoniae, 12A. baumannii , 14Salmonella spp. (including 2S. Typhi and 1S. Paratyphi A), and 8S. pneumoniae. At least one target organism was cultured from 110/2123 (5.2%) blood cultures. In addition,Burkholderia pseudomallei, a regionally important pathogen,was isolated from 22 blood cultures (Extended data).In total, 192 potentially pathogenic organisms were isolated from 2123 blood cultures from 1999 infection episodes. Six out of seven of the most frequently isolated organisms were target organisms (115/192): 44Extended data).Among 1999 blood-cultured infection episodes, the incidence of blood stream infections (BSI) caused by any target organism was 51 per 1000 episodes : 18 (95% CI 5 \u2013 47) for meningitis, 25 (95% CI 12 \u2013 44) for pneumonia and 66 (95% CI 53 \u2013 81) for sepsis. Breakdowns of BSI incidence by target organism, age group and infection timing are shown in Supplementary Tables 3 \u2013 4 and to a lesser extent to the number of \u201cSepsis 6\u201d severity indicators in children . Both amExtended data). Lower respiratory tract specimen data was captured in 6% of pneumonia episodes in total and in 4% (14), 6% (3) and 19% (20) of episodes at AHC, Mahosot and NHTD, respectively. Overall, 41 lower respiratory tract specimens were captured from patients with clinically suspected pneumonia, more frequently from hospital-acquired than community-acquired or healthcare-associated pneumonia episodes. Of these, 13 were culture positive and 26 potentially pathogenic isolates were recovered . The range of non-blood culture specimen and organism data are summarized in Supplementary Table 7 and Supplementary Figure 5 (Extended data).Data from 1306 non-blood culture specimens was captured. Cerebrospinal fluid (CSF) specimen data was captured in 58% of meningitis episodes in total and in 58% (76), 56% (33) and 58% (37) of episodes at AHC, Mahosot and NHTD, respectively. A total of 147 CSF specimens were captured from patients with clinically suspected meningitis, 14 were culture positive yielding a single potentially pathogenic isolate in each case . After deduplication to the first isolate per species per patient, there were >30 isolates forE. coli, K. pneumoniae, andS. aureus. Third generation cephalosporin resistance was identified in 54.2% (39/72) ofE. coli and 38.7% (12/31) ofK. pneumoniae isolates. Almost a quarter ofS. aureus isolates were methicillin resistant .Due to the small numbers of isolates generated in the pilot, detailed exploration of the data was considered inappropriate. However, examples of AST profiles were generated for the two most prevalent organisms,online or offline dashboard. ACORN was piloted in three hospitals in Southeast Asia between December 2019 and October 2020.To overcome the biases of isolate-based surveillance and improve direct feedback of AMR surveillance data to hospitals and prescribing doctors we developed ACORN. ACORN starts with a patient rather than a pathogen, and consists of bedside collection of clinical metadata that is subsequently linked to clinical microbiology laboratory data and displayed on anWe show objectives of the pilot with predefined outcome measures and how we met those inDenominators of generated data and antibiograms in this pilot were too small for immediate use in clinical guidelines and definitive analyses of AMR impacts. However, the framework developed, and iterated as described below, will permit such usage during longer periods of surveillance.Data from 2464 clinical infection episodes were collected and matched with 192 potentially pathogenic isolates from blood culture and an additional 229 potentially pathogenic isolates from other specimens. In total, 290 infection episodes (11.8%) were matched to a pathogen. Receiving antibiotics when blood culture was taken was associated with a lower proportion of positive culture results (3.6 vs. 5.8%).In total, 83 patients died in hospital and an additional 126 deaths occurred following hospital discharge . Of these, 93 patients were discharged moribund to die at home rather than the hospital, which is often preferred by patients and families in the cultural contexts of these three countries . Low numbers of lower respiratory tract samples were collected for patients with a clinical diagnosis of pneumonia, both among children and adults. Hospital-acquired pneumonia cases were sampled more frequently than community- or healthcare-associated pneumonia cases and this perhaps reflects views on clinical utility and the fact that around 50% of hospital-acquired pneumonia patients were mechanically ventilated. High quality sputum specimens from community-acquired pneumonia cases are notoriously hard to generate, resulting in culture results that frequently represent upper respiratory tract colonisation.These results show that case-based surveillance of AMR requires collection of data from a considerably larger number of patients to achieve the same amount of isolate based information compared to strictly isolate based surveillance. However, the clinical data collected is richer and allows for stratification by syndrome, severity, origin of infection and other metadata allowing for rapid actionable feedback to clinicians and hospitals. The results from this pilot also show that ACORN is feasible to deliver, but at this stage and in these settings required dedicated clinical and data staff and therefore funding was a prerequisite for delivery. During further roll-out we will assess to what extent ACORN surveillance can become part of routine work on site and self-sustainable.Salmonella andB. pseudomallei infections were labelled as HAI/HCAI according to protocol, which probably does not reflect their true origin of infection. Similarly, the proportion of patients with blood culture taken per site and syndrome and the proportions of rejected admission diagnoses were heterogeneous between sites, showing rigorous on-site diagnostic stewardship and clear guidance regarding case definitions is important. Clinical feedback during the pilot highlighted that several patients with a pathogenic organism grown from blood were not included in surveillance due to lack of a clearly documented clinical diagnosis. Alternative enrolment strategies were thus considered and a two-week audit of patient numbers on surveillance wards was undertaken (data not shown). We concluded that enrolment could be improved by selection of patients based on clinician antibiotic treatment choices, rather than written diagnosis.Categorisation between CAI, HCAI and HAI was heterogeneous between three sites, reflecting differences in patient populations. Pragmatically, we disregarded transfers from lower-level hospitals because most were hospitalised <48h before transfer and this was considered escalation of care and transferred cases were classified as CAI. This may have led to some HAIs being misclassified, especially at NHTD which receives >70% of its patients from peripheral sites. Detecting HAI with weekly point-prevalence surveys but without case definitions may not have caught all infections. Conversely, somePhase 2 of ACORN is currently being implemented, with a wider roll-out in 15-18 sites in 9 countries in Africa and Asia. After having reviewed the pilot experiences with study staff from hospitals and research units, the following changes to the protocol were made:Pilot enrolment criteria were based on clinical diagnosis without requirement for specific criteria to be checked. Diagnoses could be confirmed or rejected at discharge. For ACORN2 the only requirement for patient enrolment will be the presence of a clinically suspected acute infection and the intention to commence / receipt of intravenous antibiotics on screening for eligibility. Patients will be categorised subsequently by syndrome on discharge.An adapted pragmatic clinical syndrome list modified from the Global PPS protocol and the WHO attributable mortality protocol will be used . Study sWe will convene meetings with surveillance experts to discuss pragmatic syndromic definitions of community and hospital acquired infections for use in future generations of ACORN.Data capture on day 28 will be more nuanced and expanded to allow for more detailed capture of ongoing morbidity and health economics analyses.E. coli andS. aureus bloodstream infections with the WHO attributable mortality protocol , influenced by the insights from this initial study (which can be seen as the pilot for ACORN II). Due to this, I find myself more at ease with this version of the manuscript.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Clinical Microbiology, Computational Microbiology,\u00a0 Clinical Bioinformatics (Genomics)I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The study by van Doorn et al. describes the result of the pilot phase of the ACORN project, which has the objective to provide case-based AMR surveillance. Using the inpatient setting, the project matches clinical information, laboratory data, and outcome data from individual patients admitted at an emergency ward for suspected infections. Case-based AMR surveillance should overcome the inherent bias seen in conventional isolate- or sample-based AMR surveillance, and as such contribute better to guidance of antibiotic therapy. The authors conclude that the implementation of such a surveillance strategy is feasible and decided to scale up the project to 15-18 hospitals in the region. The manuscript provides detailed descriptive information on the entire process from enrolment to assessing outcome data. By doing so, the authors show that the approach can lead to a rich dataset that provides insight in diagnostic stewardship, clinical record keeping, and follow-up procedures. As such, the manuscript delivers clearly on its objectivesMain comment. There is no doubt that current surveillance is severely hampered by the approach taken. The WHO GLASS initiative is welcome but can only provide a limited view of the AMR-situation, given its sample-based approach. Case-based surveillance is urgently needed, something acknowledged by WHO, and being addressed by the ACORN project. It is therefore somewhat surprising that the Discussion section does not further elaborate how case-based AMR\u00a0 surveillance could be designed. Adding clinical data to samples submitted to the laboratory is one option, especially if this is done through active case-finding and point-prevalence surveys as in ACORN. But does this inform antibiotic use in those large number of patients not being admitted to an emergency ward? The majority of antibiotic prescription is done in an outpatient setting.\u00a0The discussion section would be strengthened if the potential limitation of the inpatient setting of ACORN is discussed. Furthermore, there are other approaches to case-based AMR surveillance being discussed in the literature. Some of these try to overcome one of the limitations of ACORN (as mentioned in the Discussion section): the need for the large number of patients to be able to decipher AMR-profiles with a reasonable level of precision. Without these profiles, guidance on antibiotic treatment will be impossible.Minor comments In Table 6, the authors mention the coverage of the objective of cost assessment. I did not see this in the main text, nor in the supplemental material. In Figure 5 middle bar chart, the Y-axis is labelled in steps of 0.5. As it refers to the number of isolates, I suggest to change this to make assessment easier. As direct visualisation is a major objective of the project, it would be great to see an example. The shiny-app does not show anything as no data are uploaded. Could there be a screen-shot of how an actual visualisation on the dashboard would look like?Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Infectious disease epidemiologist working in the field of antimicrobial resistance surveillanceI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard."} +{"text": "Background: Antimicrobial resistance surveillance is essential for empiric antibiotic prescribing, infection prevention and control policies and to drive novel antibiotic discovery. However, most existing surveillance systems are isolate-based without supporting patient-based clinical data, and not widely implemented especially in low- and middle-income countries (LMICs).Methods: A Clinically-Oriented Antimicrobial Resistance Surveillance Network (ACORN) II is a large-scale multicentre protocol which builds on the WHO Global Antimicrobial Resistance and Use Surveillance System to estimate syndromic and pathogen outcomes along with associated health economic costs. ACORN-healthcare associated infection (ACORN-HAI) is an extension study which focuses on healthcare-associated bloodstream infections and ventilator-associated pneumonia. Our main aim is to implement an efficient clinically-oriented antimicrobial resistance surveillance system, which can be incorporated as part of routine workflow in hospitals in LMICs. These surveillance systems include hospitalised patients of any age with clinically compatible acute community-acquired or healthcare-associated bacterial infection syndromes, and who were prescribed parenteral antibiotics. Diagnostic stewardship activities will be implemented to optimise microbiology culture specimen collection practices. Basic patient characteristics, clinician diagnosis, empiric treatment, infection severity and risk factors for HAI are recorded on enrolment and during 28-day follow-up. An R Shiny application can be used offline and online for merging clinical and microbiology data, and generating collated reports to inform local antibiotic stewardship and infection control policies.Discussion: ACORN II is a comprehensive antimicrobial resistance surveillance activity which advocates pragmatic implementation and prioritises improving local diagnostic and antibiotic prescribing practices through patient-centred data collection. These data can be rapidly communicated to local physicians and infection prevention and control teams. Relative ease of data collection promotes sustainability and maximises participation and scalability. With ACORN-HAI as an example, ACORN II has the capacity to accommodate extensions to investigate further specific questions of interest. Surveillance for the emergence and spread of antimicrobial resistance is a cornerstone of our response strategies. It is essential both at patient-level for informing empiric antibiotic prescribing and treatment guidelines, and at system-level for designing and evaluating treatment, infection prevention and control policies. Antimicrobial resistance surveillance involves systematic data collection, analysis and communication. The aim is to describe disease burden and epidemiology in a timely and unbiased manner that is relevant to the community and various stakeholders, including patients, physicians, researchers and policy makers.Antimicrobial resistance is declared as 1 of the top 10 global public health threats facing humanity. This is mainly because assembling comprehensive surveillance data which incorporate both clinical and microbiological records is challenging. Infection episodes are defined using clinical criteria which involves sourcing individual-level data from medical notes, microbiological reports and antibiotic prescription charts. Existing population-level surveillance databases, such as the European Centre for Disease Prevention and Control Surveillance Atlas, The Center For Disease Dynamics, Economics & Policy Resistance Map, and the World Health Organisation (WHO) Global Antimicrobial Resistance and Use Surveillance System (GLASS), are isolate-based and rely mainly on microbiology data alone.Despite a global emphasis on antimicrobial resistance in recent decades, effective surveillance networks and systems remain sparsedue to orwith a resistant bacteria. In addition, quantifying morbidity and mortality impacts of drug-resistant infections requires longitudinal follow-up and cannot be reliably determined from death certificates. Such high quality surveillance data is especially lacking in LMICs where antimicrobial resistance burden is the highest.These isolate-based data present geospatial and temporal trends of resistant organisms but are prone to biases introduced by local characteristics such as differential access to antibiotics in the community versus hospital settings, antibiotic prescription practices, availability of laboratory resources, and practices of microbiological culture collection and reporting. Particularly in low- and middle-income countries (LMICs), microbiology cultures may only be performed after multiple courses of empirical antibiotics have failed or in situations where diagnostics are affordable. Importantly, interpreting microbiology cultures without considering clinical syndromes makes it impossible to differentiate innocent \u2018bystander\u2019 colonising versus true pathogens, i.e. if a patient became ill or died. It aims to implement an efficient clinically-oriented antimicrobial resistance surveillance system, incorporated as part of routine workflow in hospitals in LMICs. In addition, ACORN II is designed to accommodate extension studies with specific focuses on infection syndrome, pathogens or patient population.A Clinically-Oriented Antimicrobial Resistance Surveillance Network (ACORN) is a large-scale multicentre study which captures essential data on patient clinical features, management, and outcomes. ACORN adds value to isolate-based antimicrobial resistance surveillance ), using patient rather than pathogen denominators, syndromic and pathogen outcomes, along with associated health economic costs. Specific target pathogens includedStreptococcus pneumoniae, Staphylococcus aureus, Salmonella spp., Klebsiella pneumoniae, Escherichia coli, andAcinetobacter baumannii. The current ACORN II study protocol refines the methodology ACORN I, and expands the scope to include all infection syndromes and bacterial pathogens. To illustrate the ability of ACORN design to accommodate extension studies, we describe the ACORN-healthcare associated infection (ACORN-HAI) extension study which focuses on healthcare-associated bloodstream infections and ventilator-associated pneumonia. Study procedures including site set up, diagnostic stewardship, data collection and rationale behind their design features are detailed below. An initial pilot phase (ACORN I) was completed in 2020, focusing on patients with clinically suspected meningitis, pneumonia, or sepsisAn overarching approval for ACORN II and ACORN-HAI was given by the Oxford Tropical Research Ethics Committee . Local ethics approvals are sought prior to enrolment. Written informed consent is required for participation in all study sites except the following, where anonymised data may be collected without individual consent: Angkor Hospital for Children in Siem Reap (Cambodia), Sarawak General Hospital , Queen Elizabeth II Hospital , Sabah Women and Children's Hospital , University Malaya Medical Centra , National Hospital of Tropical Diseases, Hanoi (Vietnam).The aim of ACORN II is to provide actionable data to local institutions, national and international surveillance systems, researchers and policy makers via an efficient clinically orientated antimicrobial resistance surveillance system (extended data 2). It is designed to be implemented alongside routine clinical care in hospitals, especially in LMIC settings. The data collected are fully compatible with WHO GLASS and are further expanded to enable classification of infection syndromes and outcomes. These data will be used to estimate syndromic and pathogen outcomes along with associated costs.Escherichia coli and methicillin resistantStaphylococcus aureus bloodstream infection using the WHO attributable mortality protocol); iv) determine the major indications for prescribing parenteral antibiotics by patient group, timing of prescription, and location; and, v) to determine the major empiric antibiotics used by clinical syndrome, place of acquisition, patient group, and location .The specific objectives are to i) implement and assess a hospital-based system for patient-centred surveillance of drug resistant infections; ii) characterise drug-resistant infections by clinical syndrome, place of acquisition, patient group, and location; iii) quantify burden of drug-resistant infections in terms of attributable mortality and excess length of hospital stay ; ii) set up infrastructure such as computers, data collection software and laboratory capacity; iii) form communication channels between the research investigators, clinicians and laboratory teams for collaborations and data sharing; and iv) familiarise themselves with local ethics and regulatory bodies.Participating sites will i) undergo basic research training such as \u201cGood Clinical Practice (GCP)\u201d surveys:Laboratory assessmenthttps://tinyurl.com/ACORNlabsurvey), the Site Laboratory Assessment tool (extended data 3), document review and virtual audits. The assessment tools allow the assessors to gather information on the current capacity of site laboratories including what routine specimen processing the laboratory already carries out, the current level of quality management and quality control in the laboratory, safety procedures and specimen reporting. Following completion of the laboratory assessment continual laboratory quality monitoring is performed using the Site Laboratory Quality Monitoring tool (extended data 3).The purpose of laboratory assessment is to make a baseline evaluation of the laboratories that will be used for processing samples and submitting data for antimicrobial resistance (AMR) surveillance. Laboratory assessments will be performed using the Laboratory Pre-Assessment online survey . The endpoint of diagnostic stewardship is to ensure that the right patients have the right tests at the right time and that results are used to ensure that they receive the right treatment. Systematic testing of patients with suspected infection will result in data that can be used to inform local treatment guidelines as well as be used for AMR surveillance activities. It is important that ACORN II stewardship is aligned with existing specimen collection, processing, and feedback procedures. A diagnostic stewardship checklist is available to determine the extent of existing diagnostic stewardship activities / materials at the site. Negative answers to any of the questions should prompt development of that particular item / activity in the form of recommendations for standardised investigations for suspected infection, at least covering when to collect a blood culture and / or sterile site fluid culture. Examples of appropriate guidelines are included in the ACORN diagnostic stewardship guideline and can be adapted to the local situation. Monitoring of culture rates, by patient group, clinical location, clinical syndrome, via the ACORN project app described will permit assessment of diagnostic stewardship activity success.Clinician Knowledge, Attitudes and Practices surveyhttps://tinyurl.com/ACORNclinicianKAP). The survey gathers information on current knowledge, attitudes and practices around diagnostic microbiology and AMR surveillance. The data will be used primarily to inform and iterate site-specific surveillance training and implementation.The purpose of the clinician knowledge, attitudes and practices survey is to make a baseline assessment of the clinical staff working on ACORN II surveillance wards , in whom the decision to start intravenous antibiotic treatment has been made, and are willing to participate in the surveillance. This includesPatients transferred directly from another facility with an acute infection;Patients admitted to a non-surveillance ward initially but transferred to a surveillance ward within 48 hours of admission;Patients investigated and treated for suspected CI in the Emergency Department (Emergency Room / Accident and Emergency Department) with delayed transfer to the surveillance ward for any reasons, e.g. bed shortages, COVID-19 screening procedures, or other local operational challenges.For HI cases, all admitted patients in the surveillance wards will be reviewed on a single day per week via a point prevalence survey (PPS). The HI inclusion criteria include:Clinical suspicion of bacterial infection and prescription / commencement of a new intravenous antibiotic ; andOnset of infection syndrome on or after day 3 of admission (day 1 refers to the day of admission); andInfection syndrome was not active during the previous weekly review, i.e. onset at least one day following the most recent previous healthcare-associated infection point prevalence surveyAn overarching approval for ACORN II to enrol without the need for individual consent was given by the Oxford Tropical Research Ethics Committee . The requirement for informed consent will be catered to local ethics committees\u2019 advice. In participating sites where the need for explicit informed consent can be waived for minimal/negligible risk studies, potential participants in participating wards will be given a patient information sheet with details about the study. At least one ACORN II information poster will be on display in each surveillance ward. Potential study participants will be asked to confirm verbally that they have read the surveillance information material and agree to participate. Any patient who requests not to be included in the study will be recorded accordingly in the screening logbook. In sites where informed consent cannot be waived, a typical consent taking process will be in place.Further infection episodes may be identified during the weekly PPS. The final follow up is 28 days after the final infection episode. This can be done by telephone if the participant has been discharged. If the patient is readmitted before the 28 day follow up, the 28 day follow up should be completed as scheduled if no further infection episodes are diagnosed during the readmission.On enrolment, baseline clinical data will be extracted from the patient clinical records/electronic hospital information systems :DemographicsDate of admission and ward locationPrimary reason for admissionCo-morbidity status (modified Charlson comorbidity index)Healthcare exposure in the three months before current admissionThe following data will be collected about the infection episode on enrolment and when a new infection episode is detected in the weekly ward PPS:Ward detailsInfection syndromeClinical severity signs at symptom onsetqSOFA score for adults, \u226518 yearsSepsis six recognition features for children, <18 yearsGeneral WHO severity signs for neonates, <28 daysPresence of medical devices / surgical proceduresMicrobiology and antibiotic susceptibilityEmpiric antibiotic treatment prescribedE. coli orS. aureus BSI, additional clinical data are captured to fulfil the requirements of the WHO GLASS attributable mortality protocol. The Pitt BSI score is calculated and details of immunosuppression are collected, along with complete details of antibiotic treatment for the infection and likely source of infection.For patients with laboratory provenClinical data entry can be done either directly using the Open Data Kit (ODK) Collect app on an Android tablet or via paper case report forms (CRFs) with subsequent entry into the surveillance REDCap database using a laptop and web browser. All clinical data entered will be stored centrally in the ACORN REDCap database, where error checking and correction is performed regularly. All data stored in the central database are anonymised and securely stored.https://github.com/acornamr/acorn-dashboard) (https://moru.shinyapps.io/acornamr/). The application allows for real-time monitoring of surveillance and can be used for data visualisation and reporting. If it is not possible to access existing laboratory LIMS or WHONET data extracts, microbiology data can be directly entered into a WHONET database specifically for the project.Microbiology laboratory data, either extracted from an existing laboratory information management system (LIMS)/WHONET file or entered into WHONET software specifically for surveillance, is linked to clinical data using the ACORN project RShiny application (hboard) . This daACORN-HAI follows the ACORN II main study methodologies and procedures closely, with a specific focus on healthcare-associated bloodstream infection and ventilator-associated pneumonia (extended data 4 and 5). These two infection syndromes are chosen due to their association with drug resistant organisms and high mortality. In ACORN-HAI, healthcare-associated bloodstream infections encompass hospital-acquired bloodstream infections, i.e. any bloodstream infections taking place either after two days of admission or within 3 months of significant healthcare exposures, can be enrolled into the study.ACORN-HAI is complementary to ACORN II as it is designed to capture more cases of HAI and in more clinical detail for each enrolled case than ACORN II. ACORN II identifies HAI via weekly point prevalence surveys and daily ward screening (case-initiated), which will provide estimates for incidence and prevalence. However, the absolute number of HAI cases enrolled is likely to be lower than the actual incidence. ACORN-HAI aims to enrol all healthcare-associated bloodstream infection and ventilator-associated pneumonia in the participating wards through screening via positive blood or sputum cultures (isolate-initiated). Additional variables describing admission progression, antibiotic prescription and functional outcomes are collected with an addendum CRF .A participating site may participate in both or either ACORN II and ACORN-HAI study. In sites which implement both ACORN II and ACORN-HAI, only study participants who are enrolled into ACORN II will be enrolled into ACORN-HAI. In sites or wards which participate only in ACORN-HAI, similar clinical, microbiology and ward-level data as the ACORN II study will be collected such that the two datasets can be merged and analysed in a coherent manner .Enrolment criteria for ACORN-HAI are as follows:Bloodstream infectionInclusion criteria:1) Prescription/commencement of an intravenous antibiotic;Candida spp. pathogen(s) identified from blood specimen(s) either taken on or after day 3 of admission (Day 1 refers to the day of admission), or within 3 months of significant healthcare exposures; and 2) Growth of bacterial or3) Pathogen(s) in the blood specimen(s) satisfies either of the following:i.\u00a0 bacterial orCandida spp. pathogen(s) identified from 1 or more blood specimens obtained by a culture; or1 or more non-common commensalii.\u00a0; andthe same common commensal bacterial pathogen identified from 2 or more blood specimens collected on separate occasions4) The same pathogen(s) in the blood specimen(s) was/were not present in the blood specimen(s) taken during the first 2 days of admission among those without significant healthcare exposures in the past 3 months.Exclusion criteria:Patient who has positive growth of organisms belonging to the following genera which are typically causes of community-associated infections and are rarely or are not known to be causes of healthcare-associated infections, or associated with severe immune suppression:a) Burkholderia pseudomallei,b) Brucella spp., including but not limited to,B. melitensis, B. abortus, B. suis, B. canis,c) Campylobacter, Salmonella, Shigella, Listeria, Vibrio and Yersinia.Ventilator-associated pneumonia1) Prescription/commencement of an intravenous antibiotic;; and 2) Clinical suspicion of ventilator-associated pnuemonia taken on or after day 3 of ventilation.3) Growth of bacterial pathogen(s) identified from respiratory specimen(s)Exclusion criteria:Patient who has positive growth of organisms belonging to the following genera which are typically causes of community-associated infections and / or are rarely or are not known to be causes of healthcare-associated infections:a) Streptococcus pneumoniae, Streptococcus suis, Haemophilus influenzae,b) Burkholderia pseudomallei,c) Coagulase-negative staphylococci,d) Talaromyces marneffei,e) Mycobacterium tuberculosis,f) M. mucogenicum, M. fortuitum, M. abscessus, M. chelonae, M. neoaurumRapidly growing Mycobacteria, including but not limited to,Acinetobacter spp.,Pseudomonas spp., Enterobacterales,Staphylococcus aureus,Enterococcus spp. and Candida (only from positive blood cultures), grown from routine clinical cultures will be collected from the microbiology laboratory and stored. The isolates will undergo whole genome sequencing at accredited laboratories.Bacterial isolates includingThere is no formal sample size calculation. This surveillance will enrol all eligible and consenting patients admitted to the surveillance wards during the surveillance period.All participating sites will develop an internal data monitoring plan to ensure adherence to ethics requirements, completeness and accuracy of data entry, and if applicable, isolate collection and storage. The local and overall data manager will conduct regular audits of the data entered and raise queries when necessary. Collated reports for monitoring progress and quality of data collected will be generated and presented to participating sites regularly.ACORN II site teams will interact with their local data in real time using the project app / dashboard. Via this dashboard, site investigators will have access to enrolment frequencies and patient demographics, follow-up on clinical outcome and day 28 status, weekly active HI point prevalence, microbiology summaries, and antimicrobial susceptibility and resistance patterns.The secondary outcomes are to: i) characterise multidrug resistant infections by clinical syndrome, place of acquisition , patient age group , sample type, and location , ii) quantify burden of multidrug-resistant infections in terms of attributable mortality and excess length of hospital stay, and iii) determine major parenteral antibiotic prescription indications by clinical syndrome, patient group , timing of prescription , and location . Analyses for these secondary outcomes will be performed after at least two years from study commencement (extended data 6).In general, data will be summarised in tables and graphs using descriptive statistics. For each selected key pathogen, the proportions of cases will be calculated, using the total number of participants from whom any pathogen was isolated as the denominator. Summaries will include the proportions of isolates resistant to key antibiotics, as defined by WHO GLASS or categorised as multidrug resistant, using standard definitions. Univariable and multivariable logistic regression models will be fitted to explore whether any clinical or microbiological variables are associated with the outcomes of resistance, mortality and discharged moribund. The unit of analysis will be admissions, with patient and site fitted as random effects.. At each site, observed crude case fatality rates will be compared between cohorts: patients with multidrug resistant infection for selected pathogen-antimicrobial combination (cohort 1), and patient with non-multidrug resistant infection for selected pathogen-antimicrobial combination (cohort 2). Patients from cohort 1 and 2 will be matched 1:1 retrospectively. Ideally, matching will be by age category, admission ward, month of infection, and clinical syndrome at enrolment, and by the time from admission to infection. The effect of antibiotic resistance on vital status will be estimated using cause-specific Cox proportional hazards models, assessing the competing events of mortality and discharge alive, from the time of infection. Admissions, rather than patients, will be used as the unit of analysis and only the first relevant infection episode will be considered. A composite all-cause end- of-stay endpoint will also be assessed which may be interpreted as an indication of the daily hazard of a patient\u2019s admission ending. All models will include adjustment for the time between admission and infection. Proportional hazards assumptions will be checked using Schoenfeld residuals and visual inspection of log-log plots. Non-proportional hazards will be corrected using stratification.For attributable mortality analyses, the survival model approach outlined in the WHO GLASS protocol will be followed. Concordance (i.e. cultured isolate was susceptible) or discordance (i.e. cultured isolate was resistant) with microbiology test results will be determined. To assess the impact of initial treatment on mortality another set of Cox proportional hazards models will be run, but antibiotic resistance will be replaced by receipt of active initial therapy as the exposure of interest.Excess length of stay in days will be calculated using multistate models. The difference in expected length of stay will be estimated between the multidrug resistant and susceptible states. For empiric antibiotic analyses, drugs prescribed on the day of admission or symptom onset will be classified according to the WHO AWaRe criteria.To examine trends in infection rates over time, we will first use plots to visually inspect the trends to determine the most appropriate model/approach for analysis, while anticipating that random effects modelling with hospital clusters will be likely. Monthly incidence will be modeled controlling for hospital characteristics including the country\u2019s income status, type of hospital (tertiary versus community), and proportion of patients in specific age rangesIn the ACORN-HAI study, all clinical isolates will be processed for genomic sequencing. These genomic data will inform geospatial distribution of pathogen species and phenotypic resistance patterns and trends over the study period.Both ACORN II and ACORN-HAI are being implemented in study sites across Asia and Africa. ACORN II commenced in September 2021 in Asia and Africa and is actively enrolling in 19 hospitals. ACORN-HAI started enrolment in September 2022 and is fully implemented in 25 hospitals across Asia including Singapore, Malaysia, Vietnam, Thailand, Nepal, India, and Pakistan . ACORN II is a comprehensive antimicrobial resistance surveillance activity which advocates pragmatic implementation, and prioritises improving local diagnostic and antibiotic prescribing practices through patient-centred data collection and rapid feedback to physicians. It uses a systematic methodology to collect clinical, microbiology, and antibiotic use data. These data can be rapidly communicated to local physicians and infection prevention and control teams via an interactive web application to guide empiric antibiotic prescription and stewardship. Relative ease of data collection promotes sustainability and maximise participation and scalability.. In addition, prospectively collected data is less prone to information bias as relevant data are collected at baseline (without knowledge on outcomes) using standardised methods. Selection bias is minimised by implementing this surveillance tool in multiple antimicrobial resistance \u2018hotspot\u2019 regions to achieve a representative cohort of patients with multidrug-resistant infections.A prospective cohort design has strengths with regards to less risk of bias and confounding when compared to cross-sectional and retrospective study designs, albeit at the expense of higher cost. Important confounders in evaluating burden of drug resistant infections, e.g. attributable mortality, include comorbidities, initial disease severity at symptom onset, prior antibiotic exposure and time from admission to infectionACORN II has the potential to fill important gaps in our understanding of antimicrobial resistance epidemiology. Firstly, most existing antimicrobial resistance surveillance systems are passive, and based on routine antimicrobial susceptibility testing results generated by clinical microbiology laboratories alone. While such designs are useful to give broad pictures on the types of resistance and bacteria prevalent in a population, they cannot be used to relate the effect of resistance with patient outcomes. These proportions of resistance per pathogen antimicrobial combination lack relevant clinical metadata to be informative for local clinicians in their decision making or guideline development. Secondly, ACORN II focuses on LMICs where there is a mismatch in terms of antimicrobial resistance burden and surveillance capacity. ACORN II presents opportunities for participating sites to adopt and integrate a systematic surveillance programme into their routine workflows.Acinetobacter baumannii, carbapenem-resistantPseudomonas aeruginosa, carbapenem-resistant Enterobacterales and extended-spectrum beta-lactamase-producing Gram-negative bacteria. These antibiotic-resistant pathogens have been identified by the WHO as of the highest concern. This is because treatment options for carbapenem-resistant organisms are severely limited, associated with high toxicity, and often ineffective. ACORN-HAI, led by the ADVANCE-ID network, will form the groundwork for subsequent interventional clinical trials targeting drug resistant infections. This reflects ADVANCE-ID\u2019s main mission, which is to promote transnational research collaboration for a sustainable pipeline to conduct large-scale clinical trials in infectious diseases, particularly tackling antimicrobial resistance in LMICs where the need is the most urgent. With ACORN-HAI as an example, we foresee such extensions based on the ACORN II protocol in the future as hospital and research networks identify specific questions of interest.By focusing on severe healthcare-associated infections, ACORN-HAI builds on the main ACORN II protocol by collecting more detailed data on disease presentation, progression and treatment. The commonest pathogens causing healthcare-associated bloodstream infections and ventilator-associated pneumonia in LMICs are carbapenem-resistantWe acknowledge several potential limitations. Firstly, though ACORN is designed to be as efficient as possible such that it can be incorporated as part of routine workflow, additional staff and resources are required to maintain the surveillance system. This is a barrier to sustainability beyond the research funding period, and will necessitate further development and adaptation of enrolment and data collection procedures for sustainable scale-up going forward. Secondly, surveillance for antimicrobial resistance serves to inform interventions, guide treatment decisions, and identify emerging threats. Hence, ACORN may be less beneficial for sites that have limited familiarity and resources in infection prevention and control, as well as antibiotic stewardship. However, implementing an antimicrobial resistance surveillance system is an important first step to stimulate the initiation of such activities. Thirdly, differences in study implementation procedures across study sites, such as waiver of consent and choice of surveillance wards, may bias the demographics and disease severity of patients who enrol into the study. Lastly, identification of patients through clinical suspicion of infection syndromes, though pragmatic, may be sensitive to local physician practices and result in over- or under-estimation of certain infection syndromes such as ventilator-associated pneumonia.Implementation of a robust surveillance network for drug resistant infections is the necessary first step towards improved empirical antibiotic prescription, designing infection prevention and control policies, guiding resource allocation, and motivating novel treatment therapy clinical trials. A focus on severe drug resistant infections with the highest health and economic consequences will align the interests and expectations from various stakeholders including patients, clinicians, health ministries and pharmaceutical industry. Such surveillance data will engage all parties to direct a concerted effort to tackle the urgent issues in antimicrobial resistance. In response to my prior feedback, the authors have provided thoughtful revisions to the manuscript. I appreciate their clarification on the evolution of the ACORN study, detailing the transition from ACORN I (pilot) to ACORN II, and further to the extension study, ACORN-HAI. Their emphasis on the synergy between ACORN II and ACORN-HAI, especially in the context of enrolling an increased number of patients with HAI and capturing in-depth clinical data, is well-articulated. Furthermore, the inclusion of key study limitations in the discussion adds depth and transparency to the paper. The improvements notably address the initial concerns and enhance the manuscript's overall clarity and contribution.Is the study design appropriate for the research question?PartlyIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Microbiology,\u00a0 AMR, infectious diseasesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have thoroughly reviewed the current version provided by the authors, and I am pleased to note that the majority of my concerns have been effectively addressed. I extend my appreciation to the authors for their diligent efforts and commendable work. I wish them all the best moving forward.Is the study design appropriate for the research question?PartlyIs the rationale for, and objectives of, the study clearly described?PartlyAre sufficient details of the methods provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Clinical Microbiology, Computational Microbiology,\u00a0 Clinical Bioinformatics (Genomics)I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The article presents the protocol for ACORN II, a surveillance network aimed at monitoring antimicrobial resistance (AMR) in a clinically oriented manner. The study focuses on patients with severe bacterial infections in low- and middle-income countries (LMICs), where the burden of AMR is high and surveillance capacity is limited. The ACORN II surveillance network aims to provide a comprehensive picture of AMR, focusing on both community-acquired and healthcare-associated infections. The study includes patients with clinically compatible acute bacterial infection syndromes and collects data on patient characteristics, infection syndromes, microbiology, and antibiotic use. The data is then rapidly communicated to local physicians and infection prevention and control teams via an interactive web application to guide empiric antibiotic prescription and stewardship. The study also includes an extension, ACORN-HAI, which focuses on healthcare-associated bloodstream infection and ventilator-associated pneumonia due to their association with drug-resistant organisms and high mortality. The ACORN-HAI study follows similar methodologies and procedures as the main ACORN II study but collects more detailed data on disease presentation, progression, and treatment. Constructive Feedback: Clarify the Objectives/methods: The authors should provide a clearer link between the specific objectives of the study and the methods used to achieve them. This would help readers understand how the study design and data collection methods will help achieve the stated objectives. Clarify the Role of ACORN-HAI: The authors introduce ACORN-HAI as an extension of ACORN II, but it's not clear how these two studies are related. The authors should provide more information on the role of ACORN-HAI and how it fits into the overall ACORN II study. Limitations: Every study has limitations, and it's important for the authors to acknowledge these. For instance, are there potential biases in the study design? How generalizable are the findings likely to be, given that the study is being conducted in LMICs? Figures: The content of Figures 3A and 3B is not easily readable, which limits their usefulness in the context of the study. Overall, the article presents a valuable protocol for monitoring antimicrobial resistance in LMICs. With some improvements in clarity and detail, it could serve as a useful guide for other researchers in this field.Is the study design appropriate for the research question?PartlyIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Microbiology,\u00a0 AMR, infectious diseasesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I applaud the team for their brilliant work. The authors' aim to incorporate patient- or clinically-oriented AMR surveillance alongside isolate-based AMR surveillance is important because it acknowledges the multifaceted nature of AMR and can enable a comprehensive understanding of the problem. This approach can enhance the development of effective strategies to combat AMR and promote better patient outcomes through targeted interventions and improved antimicrobial stewardship. Upon evaluating this protocol, I have the following observations: The authors describe ACORN-II and ACORN-HAI in this protocol. In the title, they present this as an ACORN-II protocol. They describe a surveillance system that includes hospitalized patients\u2026 with clinically compatible acute community-acquired or healthcare associated bacterial infection syndromes\u2026 They also mention diagnostic activities that will be implemented to optimize culture specimen collection practices and later state that patient characteristics are already being recorded on enrolment and after 28 days of follow up... My concern with the authors\u2019 approach is that they mix various elements in this protocol, making it somewhat unclear. Moreover, the fact that the protocol is already being implemented adds complexity to evaluating its suitability. I would have preferred the authors to follow a more systematic flow in their writing. They should have summarized ACORN-I since it presumably serves as the baseline for ACORN-II. Additionally, they should have described the advancements or updates in ACORN-II compared to ACORN-I, such as novel approaches, methodologies, or updated protocols etc. For instance, ACORN-I (the pilot study) focused on three infection syndromes, and it would be helpful to clarify which of these ACORN-II focuses on and why? Currently, the authors seem to present ACORN-I as GLASS, which is somewhat perplexing. Since they state that ACORN-II builds on the WHO-GLASS, to measure... I think they need to base ACORN-II more on the pilot study (ACORN-I) and less on GLASS. The authors briefly mention a surveillance system but fail to provide a comprehensive description of its components. Is it unclear whether this system encompasses ACORN in general, including both ACORN-I and ACORN-II, or if it specifically refers to ACORN-II and its extensions, such as ACORN-HAI? Although the authors introduce ACORN-HAI as an extension of ACORN-II, they should consider describing it fully in this protocol since it appears to be an independent study on its own. It would be helpful for the authors to clearly distinguish between ACORN-II and ACORN-HAI and explain the rationale behind the extension (ACORN-HAI). The authors mention ongoing activities while also stating that some activities are not yet underway (based on their writing tenses). This makes it challenging to provide feedback on the protocol if certain study activities are already underway. I would appreciate clarification from the authors to better understand what has been done, what is in progress, and what remains to be done. Regarding the study design versus the specific objectives, the authors state that ACORN-II is a prospective observational study . This implementation phase makes it difficult to provide comments on a protocol that is already being executed. However, I am concerned that the general design may not effectively achieve the specific objectives outlined by the authors (i to v). I recommend that the authors provide further clarification in this regard. Lastly, I suggest that the authors clearly define the methods that they intend to use to achieve each specific objective in a systematic manner. This would facilitate a more thorough evaluation and allow for feedback on the proposed approaches.Is the study design appropriate for the research question?PartlyIs the rationale for, and objectives of, the study clearly described?PartlyAre sufficient details of the methods provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Clinical Microbiology, Computational Microbiology,\u00a0 Clinical Bioinformatics (Genomics)I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above."} +{"text": "Non-communicable diseases (NCDs) such as cancer, diabetes, heart disease, mental disorder and chronic lung conditions are the leading cause of death and disability in Indonesia. Adolescence is when risks for NCDs emerge and it is also an important life stage for intervention, yet young people are often at the margins of NCD policy and actions. This study aimed to understand how policies and actions should address NCD risks for adolescents in Indonesia, and how young people can be meaningfully involved.Qualitative in-depth interviews over videoconference (n\u00a0=\u00a021) were conducted in English or Bahasa with stakeholders in Indonesia. Participants included policymakers, implementation partners, and advocates who were focused on adolescent health or NCDs. Interviews were recorded, transcribed, translated, and thematically analysed using NVivo12. Findings were disseminated to participants for validation and feedback. Youth participants (n\u00a0=\u00a07) attended an additional workshop and considered recommendations and actions arising from this research.Participants identified that government and non-government organisations are acting on NCDs in Indonesia, but few of the existing initiatives target adolescents, and adolescent services rarely addressed NCD risks. Participants also felt that policies to protect adolescents from NCD risks were not always enforced. For programs or initiatives focused on adolescent health, those that had engaged adolescents as co-creators and leaders were perceived to be more successful. As such, participants recommended more meaningful engagement of young people, including young people's leadership of initiatives. Additional recommendations included the need for intersectoral engagement and a \u2018whole-of-government\u2019 approach to prevention given the complex determinants of NCD risks, and the need for evidence-based actions that are underpinned by quality data to enable monitoring of progress.There is a recognised need to strengthen policies and actions to address NCD risks amongst adolescents in Indonesia. Meaningful youth engagement that allows young people to take the lead, intersectoral actions, and evidence-based data driven responses were key strategies identified.UNICEF East Asia and Pacific Regional Office. We searched the literature for articles published from 2012 to August 2022 using terms \u201cnon-communicable diseases\u201d, \u201cprevention\u201d, \u201crisks\u201d, and \u201cIndonesia\u201d; we kept search terms purposefully broad and then searched within these publications for adolescent focused studies. Our search of Ovid MEDLINE, and PubMed returned 413 titles after duplicates were removed; title, abstract and relevant full text screening identified 20 relevant publications. Searching the grey literature and reference lists revealed 5 additional publications which were relevant to our search.We did not find any studies which explored the perceptions and experiences of stakeholders around NCD prevention. Publications were largely focused on adults with only 3 that specifically focused on adolescents. The first was an intervention in high school students to increase understanding of NCD risks using peer educators and general practitioners in collaboration with the community NCD screening program (Posbindu PTM), which was found to be successful. The investigators partnered with adolescents to develop study materials and incorporated youth empowerment as part of the intervention. A second study, from the grey literature, was our own earlier work that defined a reporting framework for NCDs and risk factors in Indonesia. That study mapped disease burden, policy, and data to highlight key NCDs of interest across the life course, and used available data to examine prevalence, geographic inequality, and data gaps. We found that greater focus on NCDs as they occur in children and adolescents, and also embedding objective measures for these age groups within existing data collection systems, would help advance actions on NCDs in Indonesia. This study had input from young people aged 18\u201325-years within the stakeholder consultation process. A third publication, also from the grey literature, was a research report that highlighted the need to address NCDs, with a focus on 10\u201324-year-olds. The authors assessed progress on NCDs in 10 countries, including Indonesia. The report recommended involving young people in the policy making process, that more data are needed including greater focus on the social determinants of health and the importance of multisectoral approaches to NCD prevention. The authors also noted that political will can be more important than a country's financial resources in its orientation to NCDs.This paper is the first to explore what existing strategies are in place to reduce NCD risks for adolescents in Indonesia, how young people are involved, and what is needed to advance actions in this space. A strength of this study is the way young people were prioritised throughout, with a place on the research team, participating in interviews, and formulating recommendations for action. The participants came from a broad range of stakeholder organisations and government, and were engaged in health services, program delivery, policy and strategic planning, youth advocacy, communications, monitoring and evaluation, and education. Notwithstanding the breadth of experience and backgrounds represented, clear themes emerged from the data: we report on the perceptions and experiences of those currently engaged in NCD prevention and adolescent health in Indonesia; what they identified as the strengths and limitations of programs and policies; and whether or not there was a clear focus on adolescents and young people within current programs or policies. We also provide clear recommendations for action, based on thematic analysis of the in-depth interviews, and refined by young people through a consultation workshop.Investment and actions to address NCDs has been fragmented and inadequate not only in Indonesia, but globally, and there is a need to rethink how we address these issues from a public health perspective. Our study is distinct in identifying how adolescents and young adults can play an important role in accelerating progress on NCDs. The key recommendations developed in consultation with participants included more meaningful engagement of young people; support for young people to take the lead; greater focus on intersectoral engagement and \u2018whole-of-government\u2019 approaches to prevention; and ensuring that evidence-based actions are underpinned by quality data.Several aspects of our findings echo the findings in the literature. NCDs remain a significant challenge; barriers to action such as uneven policy and program implementation, poor service quality and coverage, and a lack of data continue to stymie prevention and control efforts. We also found that participants\u2019 experiences partnering with adolescents and young people were overwhelmingly positive, with evidence of greater involvement of young people in NCD prevention in Indonesia than is reflected in the literature. This indicates the need for thorough evaluation and scaling up of implementation for those successful local partnerships between government, NGOs, and grass roots organisations.Our findings and recommendations highlight the importance of young peoples\u2019 leadership in driving actions on NCDs. We present clear actionable steps to improve youth engagement and to improve the current approach to addressing NCD risks in adolescents. Targeted preventative actions in this stage of life are essential to mitigate the upward trend of NCD morbidity and mortality in Indonesia.Routinely collected health data in the Republic of Indonesia (Indonesia) show a rapidly changing burden of disease.,,,,,,Effective prevention of NCDs requires actions across the life-course, including the developmental stage of adolescence.We have an issue that adolescents are often seen as a healthy group; from ten onwards we often forget about health and address it when people are in their 30s, 40s and 50s and already suffering from NCDs or one or more risk factors.\u201d(p.9) There also appears to be limited adolescent engagement in research and implementation despite evidence that young people have the desire and the skills to be involved in issues which impact them.,Globally, adolescents mostly remain at the margins of policy and prevention focused on NCDs. Dr. Marie Hauerslev (NCD Alliance board member and past-Chair of NCD Child) noted that \u201cThis study seeks to understand what is required to address NCD risks for adolescents in Indonesia. We explored three inter-related questions: What are stakeholders\u2019 perspectives on existing strategies for the prevention of NCDs in Indonesia; how are adolescents currently included in these strategies; and how can adolescents be better involved to advance actions on NCDs?This qualitative study used semi-structured interviews to explore stakeholders\u2019 perspectives on NCD prevention in Indonesia. Indonesia was selected by the UNICEF East Asia and Pacific regional office due to the increasing burden of NCDs, planned investments to address NCDs, and the identified need to understand how young people can be involved in these efforts.We engaged young people in this research in three ways: we engaged an Indonesian young person as a co-researcher within the research team from the beginning of the project (ANT) to ensure the overall project and data collection was appropriately framed and \u2018youth friendly\u2019; we purposively sampled a diverse range of young people during the enquiry phase and aimed to have half of participants aged 18\u201325 years; and we invited young people to an additional forum to discuss the findings and co-define key recommendations.We developed a question guide for semi-structured interviews based on the research aims. It was then translated to Bahasa Indonesia for Bahasa language interviews agreed to take part and none withdrew post consent.Due to the COVID-19 pandemic, interviews were conducted online using Zoom videoconferencing between 6th April 2021 and 25th May 2021. Participants were advised to join the videoconference from a private space of their choosing. Interviews were led by one of two female post-graduate qualified researchers, who were either English-speaking (KC) or Bahasa-speaking (NW). Six of the Bahasa-language interviews were also attended by a young Indonesian graduate researcher (ANT), who participated in planning the interviews, note-taking, asking questions from the interview guide, translation, and transcribing. Videoconference software provided separate audio and video recordings of the interviews; video recordings were deleted and audio recordings were retained for transcribing and translation . Both workshops had a short presentation of findings and draft recommendations which supplemented a written report that had been previously circulated. In the UNICEF and research team workshop, feedback was given, and the findings discussed. In the second workshop seven young people attended , with three researchers, one of whom provided translation between English and Bahasa. The young people who attended represented Indonesian NGOs, International NGOs, the UN sector, and youth-focused organisations. Following the presentation participants were asked to consider three questions to guide the discussion see . The youThis study received ethical approval from the Alfred Hospital Ethics Committee, Melbourne, Australia and the Health Research Ethics Committee of the Faculty of Public Health, Universitas Hasanuddin (1305/UN4.14.1/TP.02.02/2021).This study was funded by UNICEF East Asia and Pacific Regional Office (EAPRO). The overarching aims of the study were guided by the funding brief from UNICEF EAPRO, but UNICEF was not involved in data collection, analysis or drafting the key recommendations. UNICEF Indonesia staff were consulted on the draft recommendations, and their feedback was considered by the lead investigators and young people in framing the final recommendations. None of the interviewers or interviewees were employees or volunteers at UNICEF. All authors had access to the data in the study, approved the final manuscript and were responsible for the decision to submit the manuscript.We interviewed 21 stakeholders . All incNCDs and risk factors were considered important health issues impacting all ages and populations across Indonesia. Participants described the link between individual behaviours and NCDs. Social determinants as underlying drivers of risks were acknowledged, as was the influence of social norms, gender norms, and societal expectations.Participants identified strategies that targeted education, youth empowerment, health promotion, and NCD screening, that were developed by government , NGOs, and grassroots or youth-led organisations. Strategies relevant to adolescents are outlined in \u201cWhen it comes to adolescents in terms of health the focus is always SRH or infectious disease \u2013 it\u2019s not tobacco or obesity.\u201d(Public sector)Participants reported that existing NCD focused screening and health promotion programs were adult focused. For example, Posbindu PTM, the government's NCD screening program, although open to adolescents from age 15, are not orientated towards this age group. Participants felt that current adolescent health initiatives did not sufficiently focus on NCDs.\u201cPosyandu Remaja is actually one of the success stories of youth engagement at the grassroots level \u2013 it is a bottom-up program \u2013 initiated by communities in East Java which has already spread to many provinces, yet the quality of the implementation varies\u201d(Youth-focused org)However, Posyandu Remaja and Pelayanan Kesehatan Peduli Remaja were both identified as adolescent services which had some NCD focused activities. Examples of these activities included an adolescent smoking cessation program at PKPR, co-led with a local youth-focused organisation, and NCD screening events held at Posyandu Remaja organised and facilitated by local adolescents, to identify those at risk .Participants expressed that these two services had great potential to meet the needs of adolescents and fill an important gap in prevention activities. They provided examples of partnerships between Posbindu and a local university, and PKPR with local NGOs, which they described as successful. Importantly, participants felt that the success of these examples was due to adolescents\u2019 active involvement and meaningful engagement in these activities. Some believed that Posbindu activities should be extended to a younger age, while others felt that PKPR and Posyandu Remaja were the better forum for adolescent focused activities. However, regardless of which service participants felt should be extended, they highlighted the need for better monitoring and evaluation of implementation to ensure an evidence-based approach to service provision. While program-level evaluation was regularly built into local services, participants said it was not possible to evaluate outcomes against national targets and indicators.Participants identified various government policies and regulations which they felt were designed to control NCDs. However, they felt that efforts were hampered in two important ways: lack of enforcement of key regulations, and lack of meaningful youth engagement in the policy-making process.\u201c...Food labelling is often covered by promotional messages.\u201d(Indonesian NGO)\u201cI am aware of the PP 109 (tobacco control). I think it says do not sell the cigarettes to children, but in reality, the shops are selling cigarettes to children or adolescents. There is no punishment or sanction if they are doing this.\u201d\u201cKawasan Tanpa Rokok : However, the implementation is not going well. Even though there are plenty of signs in public to not smoke in the specific designated areas but there are still people smoke in there. For sanctions on that regulation, it still has to be strengthened.\u201d(Public Sector)Participants noted that regulations around food advertisement and labelling, tobacco advertising and sponsorship, tobacco packaging and labelling, and the enforcement of smoke-free places were not well implemented.\u201cWe want more for youth than just come and talking to the Minister, we want to stay involved in the policy setting and in policy publications as well. We don't have enough of that kind of platform\u2026 voicing their needs and making the regulations. As I mentioned before a lot of regulations are not designed to protect our youth...\u201d(Indonesian NGO)\u201cWe were told we (the youth) were being invited to discuss about some policies\u2026 at the event it was already drafted, and we were just revising and reading them\u2026 it was pre-made by the government and then it was given to the youth to just review and that was really disappointing - we really thought that we were going to be a part of this policy.\u201d\u201cI think one of the most important things to consider is to involve young people from early\u2026 if the government or UNICEF would like to initiate new activities that are targeted to young people, they should listen to young people\u201dWhile issues such as sanctions are key to enforcement, we also found that participants wanted to support greater involvement of adolescents in the policy-making process. In the past, participants felt this work had been largely tokenistic.\u201cListen to them! They have brilliant ideas\u2026 young people are underestimated.\u201d\u201cThey [young people] have a style we don't understand, but the best part is that they can communicate effectively with their age group when we can\u2019t.\u201d(Indonesian NGO)Participants reported many examples of adolescents on the front-line of NCD prevention initiatives in Indonesia: in peer-to-peer programs, advocacy, education, and community actions, where they were reported to be leaders, project coordinators, program and evaluation officers, health professionals, national coordinators, and student volunteers. Participants felt that when adolescents were involved, they contributed unique ideas and energy to programs and initiatives, they were skilled in communicating with and engaging other adolescents, and very aware of the issues impacting their cohort.\u201cA positive with youth led orgs and UN agencies - they are really interested in what we are saying. With government organisations its harder to do - it\u2019s harder to convey our opinions to them.\u201d\u201cI prefer to work with the research division to be honest, because they work in partnership with the Universities, who are well informed\u2026 So, they really listen to us, about what kind of questions we want\u00a0to address, as patients, as young people, and on how to deliver the program and disseminate the research results.\u201d(NCD and youth-focused org)\u201cOur journey doing advocacy wasn't easy - the government is not always cooperative working with young people - as we know there's a social hierarchy in our society.\u201dYouth engagement was seen by many participants as both the strength of existing strategies and the best opportunity for driving further actions. However, participants described varying experiences of collaborating with government and other organisations. It appeared that government departments were more difficult to engage with than non-government organisations.\u201cThere is no accountability for what we tell them... how are young people's opinions being heard in the government? We can reach out and speak to them, but we don't know what they do with the information.\u201d\u201cWe often are not equal... We don\u2019t get feedback from them. We need a clear framework from the beginning.\u201dYoung participants also described a lack of follow-up on issues raised and an absence of transparency on how their engagement was applied, leaving many with the perception that their work was tokenistic.Box 21.Meaningfully engage young people as equal partners in the response to NCDs.2.Identify and support areas where young people can take the lead.3.Prioritise intersectoral engagement for action on NCDs.4.Facilitate evidence-based actions and policy, based on sound data.Draft recommendations were based on the findings from analysing 21 stakeholder interviews. The final key recommendations were the\u201cA priority action is to create a roadmap for youth engagement with the government, a framework \u2026 what do they envision that youth will be engaged in? I don't think that kind of discussion has happened internally in government.\u201d\u201cLink youth organisations with (government), make safeguarding principles together\u2026 have an open and transparent way of doing things, like deciding who gets to be involved in what.\u201d\u201cSo, before we include more people the government needs to improve how they engage.\u201dMost participants saw meaningful youth engagement as crucial for driving actions on NCDs in Indonesia. However, for youth engagement to avoid the pitfalls of tokenism, many participants cited the need for a framework (a set of priorities and guidelines) to govern youth engagement with mutually agreed expectations and outcomes. Participants also felt that there was a need to develop the capacity of policy makers and stakeholders to work more effectively with adolescents. Additionally, they urged steps be taken to ensure that engagement was representative of the diversity of adolescents in Indonesia.\u201cFirst thing is that we made our own guidelines of youth involvement and engagement. So that we as the members of that council knew our rights\u2026 Even before you ask the youth of their opinions, start with what kind of expectations you have, or what do you think is your right to be heard, because it will really affect any other activities after that\u2026 but make sure that everyone understands how to meaningfully engage young people.\u201dThose who had developed such frameworks previously found they had facilitated quality engagement with youth.\u201cBut even in Indonesia that claims it's country as a very diverse country, but yet we do not have such representation\u2026 LGBTQI, indigenous, ethnic minorities. I'm trying to tell them that young people... we aren't only people who live in Java and, you know, a certain religion and ethnicity background, no\u2026 it\u2019s also us who are not included in (those) categories.\u201d(NCD and youth-focused org)\u201cWe need to offer routes for young people to get involved who are usually marginalised\u2026 there is a risk when a big international organisation is involved, that group (of young people) you\u2019re talking to becomes elite or exclusive, just an extension of the bureaucracy.\u201dParticipants felt that another step towards meaningful engagement was to ensure that youth engagement is representative of diversity , noting that such efforts must be adequately resourced and explicitly defined. Some felt that there was a risk of bias if only particular groups were consulted.1.Young people can raise awareness of NCDs and their risk factors within their families, peer groups and communities.2.Young people can support other young people and amplify their voices.3.Young people can lead campaigns for the inclusion of youth in policy.4.Young people can advocate for the transparent and accountable implementation of laws, regulations, and policies.Participants identified four specific ways that young people can take the lead with the support of other stakeholders.\u201cWe try to foster collaboration between initiatives\u2026 Without connections they (young people) are trying to solve problems on their own.\u201d\u201cI hope we could receive more supports that are maybe in\u00a0the form of networks or connections to the right people.\u201dParticipants felt a crucial part of allowing young people to take the lead is providing support structures to enable them. One action suggested by several participants was to promote connections between youth initiatives and leaders (government or non-government): creating intergenerational partnerships. There was a common interest from young participants to be more connected to both mentors and other stakeholder networks.\u201cSome people that I know, they make the effort to give me a seat at the table\u201d\u201cI thought I was just studying a bachelor and didn't have enough knowledge or experience in this field, but my co-workers (adults) always encouraged us to speak up. For example, giving us opportunities in the meetings. I have overcome it and I have now become more confident talking in public including to stakeholders from the government.\u201dParticipants shared some examples of how these intergenerational partnerships had specifically helped them to engage in meaningful projects.\u201cYoung people have been pressured by industry after working with us\u2026 young people being bullied on social media and other platforms; we need groups of young people that support each other.\u201dIt was also acknowledged that those young people who advocate in public forums need additional support, as this kind of advocacy was understood to carry an element of risk.\u201cThe difficulty is a siloed approach when we talk about young people. Ministry of Finance, Ministry of Industry, other ministries need to be involved - they can't all be pretending nothing is happening or that they're not involved.\u201d(Public sector)Participants identified effective intersectoral actions as central to progress in Indonesia. This included facilitating interactions between different ministries within government, across the various levels of government, and government funded service providers.Stakeholders felt that better intersectoral engagement was needed to improve enforcement, regulation, and implementation of laws, particularly those focused on tobacco control and advertising, and food labelling and advertising. To improve, participants felt that intersectoral coordination needed to extend beyond government and across communities, schools, businesses, the arts, agriculture, and policy makers.\u201cWe don't even have any data on... inequality, on gaps, illness gaps between different ethnic groups, or sexual orientation - we don't have such data, we don't have such research.\u201d(NCD and youth-focused org)Participants identified the need for high quality data to enable transparency and accountability in three areas: firstly, for priority setting \u2013 to understand the health needs of the adolescent population; secondly, for advocacy \u2013 to drive changes in legislation, programming, and funding; and thirdly, for monitoring and evaluation. Participants emphasised the role of data for better monitoring and evaluation, both for their own work and to build accountability and trust in government-funded programs. Participants were clear that they felt they did not have access to adequately disaggregated epidemiological data for the local communities their programs were focused on.Several participants acknowledged the existence of current data systems in Indonesia which have some relevant coverage for adolescents and NCDs, such as RISKESDAS survey data, and data generated from screening activities in Posbindu and Posyandu Remaja. However, they were unsure how to access the data or receive the support to meaningfully use it.,,,,We found that almost all existing national NCD strategies target adults. Furthermore, most current health services and programs for adolescents do not routinely address NCD risks. The few that have both an adolescent and NCD focus are either smaller targeted programs or have insufficient quality data to monitor and evaluate their effectiveness. This includes those programs that were perceived to be the most successful, such as Posyandu Remaja. We found that efforts to address NCDs are beginning to be embedded in data systems (in surveillance and outcome monitoring), and in policies and regulations with an NCD focus.,Our study is distinct in identifying that adolescents and young adults are a major resource to drive progress on NCDs in Indonesia. We have shown that stakeholder representatives from a variety of backgrounds and roles identified meaningful youth engagement as both vital to current achievements and the best opportunity for future success in NCD prevention in Indonesia. The themes which arose as priority actions to ensure meaningful youth engagement resonate with those from other studies. Actions such as developing standards and guidelines for engagement, transparency in monitoring, evaluation, and dissemination, and supporting and strengthening partnerships involving young people, are consistent with a recent global stakeholder consultation on how to improve youth engagement in health research.,,Another theme underpinning our findings was the need for greater transparency and accountability in NCD prevention and control. Improved accountability in implementation, monitoring, youth engagement, communication, and governance is likely to improve priority setting, contribute to better opportunities for intergenerational partnerships, and ultimately accelerate progress. This need is not new to global health or the NCD agenda,The Indonesian Ministry of Health's current action plan for the directorate for prevention and disease control (2020\u20132024) has only one NCD target that includes adolescents, the \u2018early detection screening for NCD risks including people aged 15 and above\u2019, despite the focus on prevention within the action plan generally. The absence of policy targets highlights that young people are not being adequately considered in the context of the significant NCD burden in Indonesia. The reported success of grassroots services like Posyandu Remaja is promising, given the lack of relevant policy focus. With strengthened implementation and evaluation there is a real potential to meet adolescent needs for screening and early intervention of NCD risks. Posbindu PTM is the government's NCD screening program which is mandated to screen for NCDs in people over 15, however due to resourcing and access issues it has not been well utilised by adolescents.,,The Indonesian government has invested heavily in health data collection, for example, Posbindu PTM (screening for NCDs), the sample registration system, and the national health survey.,,,A strength of this study is that young people's voices were prioritised throughout. We had a female Indonesian young person on the research team, young people participated in the interviews, and young people also helped to define the recommended actions. The young participants were all working in some aspect of NCD prevention, adolescent health, or youth advocacy which is not only a mechanism from which the recommendations are contextually grounded, but also helps ensure that there is alignment with the needs of those who wish to translate this research into action. Another strength of this study is the breadth of expertise represented in the interviewees. We spoke to people engaged in health services, program delivery, policy, strategic-planning, youth advocacy, communications, evaluation, and education. What we gained in breadth of experience was lost in depth of detail from specific sectors, which could be viewed as a limitation of the study. However, despite the variety of roles and responsibilities of our participants, clear themes emerged across perspectives that ranged from student volunteers through to national directors. This commonality indicates that the recommendations to drive action in partnership with young people could\u00a0be broadly useful. Another potential limitation to the study is that the findings may not be widely generalisable beyond Indonesia. However, many of the issues and recommendations discussed by our participants are consistent with other global and regional studies.In conclusion, our study found universal support amongst a diverse group of stakeholder representatives for greater youth engagement in the prevention and control of NCDs in Indonesia. In fact, young people's involvement in policy and implementation was seen as the key to current and future success, and young participants in our study articulated that further steps are needed to ensure meaningful youth engagement. Arguably, finding pathways to amplify adolescent voices and allowing them to take the lead will help to give greater priority to NCD prevention within government and non-government organisations alike, improve intersectoral engagement, and help address critical data gaps.KC and PA conceptualised this study. KC, PA, and AA developed the study protocol. KC, PA, and NW developed the data collection tools, with expert feedback from AA and ANT. KC, PA, NW, AA, ANT, AC, GS supported implementation of the study, with interviews led by KC, and NW, and assisted by ANT and AA, and validation workshops held by KC, AA, and PA. NW & ANT handled all translation of transcripts in Bahasa Indonesia to English. KC, IA, LL, and DAP completed the literature review. KC, PA, and NW led the analysis. Specific expertise for this study was sought from SMP, AC, SAE, GS, AA, ANT, DAP (the Indonesian context); ANT, IA, LL, DAP (youth perspective and engagement); KF (data availability and interpretation), DD, SS, GP , and all authors contributed to the interpretation and drafting of the manuscript. All authors approved the final manuscript and were responsible for the decision to submit the manuscript.Qualitative transcripts are not available for sharing due to the risk of identifying individuals. De-identified data may be shared on a case-by-case basis upon requests to the corresponding author.Researchers from Murdoch Children's Research Institute are supported by the Victorian Government's Operational Infrastructure Support Program.KC is supported by a postgraduate scholarship from Australia's National Health and Medical Research Council (NHMRC) GNT1190911. KC & DD are supported by The Centre of Research Excellence in Driving Global Investment in Adolescent Health funded by NHMRC GNT1171981. DD is supported by\u00a0an NHMRC\u00a0Early Career Fellowship\u00a0GNT1162166 (2019-2022), and by an ARC Discovery Early Career Award DE230101174 (2023-2025). PSA is supported by a NHMRC Investigator grant GNT 2008574.The authors declare no conflict of interests."} +{"text": "Biopolymers represent a great resource for the development and utilization of new functional materials due to their particular advantages such as biocompatibility, biodegradability and non-toxicity. \u201cIntelligent gels\u201d sensitive to different stimuli have different applications in many industries . This review summarizes the research efforts presented in the patent and non-patent literature. A discussion was conducted regarding biopolymer-based hydrogels such as natural proteins and polysaccharides . In this analysis, the latest advances in the modification and characterization of advanced biopolymeric formulations and their state-of-the-art administration in drug delivery, wound healing, tissue engineering and regenerative medicine were addressed. Nowadays, there is great research motivation regarding materials based on biopolymers due to the desire to replace the use of traditional polymers and monomers originated from petroleum, the resources of which are limited. The other problem with synthetic polymers is the removal of accumulated waste plastics and their hazardous and toxic residues after decomposition in the environment . AdditioBy definition, hydrogels are known as three-dimensional insoluble supramolecular or covalent polymer networks that can hold a great quantity of water or fluids due to the balance in the osmotic pressure forces and the elastic forces of the crosslinked macromolecular chains. Usually, they are formed by physical or covalent interactions of hydrophilic macromolecules. Biopolymer-based hydrogels can be built from biopolymers soluble in physiological fluids, involving natural proteins and natural polysaccharides by the crosslinking process . HydrogeBiopolymer-based hydrogels are applied in drug delivery systems as formulations or carriers that allow for the encapsulation of therapeutic active substances and the control of transport through biological membranes to the site of action for the treatment of diseases with improved efficiency and safety . They caThis review aims to present the latest achievements in this field, comprising an analysis of patent documents and published scientific works in the field of biopolymer-based hydrogels. In addition to information contained in scientific journals, the information contained in patent documents is also very interesting as a source of significant technical information. The technical knowledge which can be found there has not yet been published anywhere else. Patent documentation databases can serve as both problem-solving resources and inspiration for future studies. After the examination procedure, a patent is granted for an invention that is new , includes an inventive step and finally, is industrially applicable . A patent gives its holder the exclusive right to use the protected invention in the territory of the country that recognized it, as well as the right to prevent others from using that invention for commercial purposes ,12. The A review was carried out using worldwide Espacenet and ScopProteins are compounds of high molecular weight consisting of amino acids interconnected by peptide bonds. They are the main structural components of the human organism . PolypepFibrin is an insoluble, non-globular protein in the form of long fibrous chains, formed in the process of soluble protein fibrinogen conversion via protease thrombin enzymes in the process of blood coagulation. Fibrin was discovered by Marcello Malpighi in 1666 . EventuaIn the worldwide Espacenet database for fibrThe novelty of registered patent ES2527800B1 is its photothermal composition suitable for generating hyperthermia in biological tissues in which this composition is implanted . This paThe subject of registered patent US11371021B2 is the production of tissues and cell cultures from stem cells (pluripotent and undifferentiated) by PEG-fibrinogen hydrogels as three-dimensional biomimetic materials . T. T146]. In the worldwide Espacenet database, 7151 patent applications and granted patents were found, filtered according to title, abstract and claims for alginate biopolymer hydrogels in the period from 1953 to 27 May 2023 . ObtaineThe subjects of patent US9090868B2 are alginate hydrogel fibers and related materials with their preparation methods . A threePatent EP2688397B1 protects a hydrogel which could be applied to entrap or encapsulate live cells, as well as transporting methods for live cells (entrapped or encapsulated into hydrogels) . The patPatent EP2916881B1 discloses a method for providing embedded mammalian cells, which includes a phase in which an aqueous solution of sulfated alginate is provided; a gelation phase of its reaction to build a hydrogel; and, in the last phase, the embedding of the precursor cell in the sulfated alginate hydrogel. In such a way, a sulfated alginate hydrogel-embedded cell was yielded . The patPatent EP3086822B1 protects pharmaceutical formulations based on a homogeneous hydrogel consisting of Fibroblast Growth Factor 18, formed in situ . The hydThe subject of granted patent TWI472536B is the molecular structure modification of the alginate monomer and hydroxyl groups to form carbonyl groups . metal cPatent EP3433282B1 protects a semi-permeable hydrogel composition based on an alginate matrix covalently crosslinked in the periphery with 3\u201310 polymer arms . The perPatent CN110548214B proposed a method for preparing a miniature smart calcium alginate hydrogel, monolayer film terminal and micro-manipulator based on different micro-electrodes to realize different functions . This me+ to obtain meta synthesizing acrylic alginate, mixing methacrylic alginate, an acrylic monomer and a photoinitiator, and photocrosslinking by irradiating UV light.The object of patent KR102521317B1 is a hydrogel based on an alginate, a methacrylate bonded to a hydroxyl group, an acrylic monomer, and a photoinitiator, characterized by an elastic modulus which increases as the number of uronic acid residues bonded to the methacrylate increases . ProduciA hydrogel spraying device and a method for obtaining calcium carbonate solution and alginate solution were disclosed in patent TWI776389B . HydrogeA hydrogel based on a copolymer of oxidized sodium alginate/polyethylene glycol amine was prepared to solve the problem of the poor biocompatibility of the existing hydrogel in 3D cell culture and the lack of corresponding stimulating factors that promote cell proliferation . Patent Alginate was initially used as an ingredient and additive in the food industry. Due to its properties , it has been extensively used as a significant biomaterial in many advanced applications in the medical, pharmaceutical and cosmetics industries. Similar to many other natural polymers, alginate has some disadvantages including low mechanical characteristics, poor biocompatibility and dimensional stability. Future investigations into alginate should be directed towards examinations, improvements and the development of new products to overcome its weaknesses. Some of those possibilities have been analyzed in this review. Future development of these biomaterials include technology transfer, innovated production processes, safety and regulatory requirements and users\u2019 acceptability. For the use of alginate in medical or pharmaceutical practices, its consequences for human usage have to be rigorously analyzed in clinical trials.Rhodophyceae. It is an anionic, linear polymer consisting of 1,3\u03b1-1,4\u03b2-galactans repeating units with one (\u03ba-), two (\u03b9-) or three (\u03bb-) sulfates per disaccharide unit [Carrageenan is a high-molecular-weight sulfated mucopolysaccharide, originating from red seaweed of the algae class ide unit ,158. Thride unit . Lambda-ide unit ,161,162 ide unit ,164, conide unit , cartilaide unit thanks tA total of 1525 patent applications and granted patents were found in the Espacenet database, filtered according to title, abstract and claims for carrageenan in the period from 1968 to 27 May 2023 . The seaThe aim of patent JP6689052B2 is to provide a carrageenan with excellent compatibility with other ingredients in pharmaceutical or food preparations. The divalent cation content of carrageenan can be significantly reduced by carrying out the divalent cation reducing step using potassium citrate or sodium citrate and removing the citrate solution from the mixed carrageenan and citric acid solution at the time of carrageenan production . Using tPatent KR101354180B1relates to a method for producing a butter cake using a butter cream with enhanced cohesiveness of carrageenan, wherein the shape of the cake is not deformed by an external impact or then temperature due to an improved thickening effect and a coagulation force .Patent CN104921964B solves the technical problem of \u03ba-carrageenan having poor solubility, a large molecular weight and easy gelation. Because of this, its application in antioxidant activity is limited . The patGranted patent CN104894100B protects a method for preparing immobilized \u03ba-carrageenase . The optThe development of a novel gel based on a mixture of \u012d- and \u03ba- types carrageenan, which is moderately soft, hardy and flexible under conditions of low concentrations of \u012d-type and \u03ba-type carrageenans, was the main subject of patent CN105778122B, which also provides a preparation method by means of swelling and cooling . The \u012d-tPatent CN106556654B, belonging to the agricultural product quality and safety testing field, relates to a method for kappa-carrageenan detecting in livestock meat, especially t application of a liquid chromatography\u2013tandem mass spectrometry method .Patent CN112430290B discloses a \u03ba-carrageenan-based high-strength double physical crosslinked hydrogel and an obtaining method thereof . The \u03ba-cThe aim of the patent CN112220046B is to provide a compound nutritional fortifier containing selenized carrageenan which can be absorbed, synergized and applied as a supplement with beneficial elements, such as selenium and iron, for improving the human body\u2019s immunity . The macA carrageenan sulfatase, along with the enzyme, is used to degrade \u03ba-carrageenan CN112522235B . By remoCarrageenan is an ancient, natural, plant-based food ingredient. Carrageenan-based hydrogels have great advantages and applications in the food industry, and they are also promising natural polysaccharides for biomedical applications, especially for controlled and sustained drug delivery, tissue engineering and wound healing. The abundance of functional groups is very convenient for further chemical modification for enhancement of the physicochemical properties of these hydrogels, e.g., the hydrophilic surface could be activated by hydrophobic modification. Similar to previously noted approaches, scientists make additional structural modifications for innovative products and processes with the aim of enhancing their applications and ensuring their safe usage based on clinical trials. Many therapeutic applications of carrageenan-based hydrogels are in the experimental stages; we await validation of their effectiveness and objective capability.Cellulose is the most abundant polysaccharide worldwide. It is produced in nature as a structural polymer in plant life, and it is also formed by some algae, fungi and bacteria. Cellulose is a linear, hydrophobic homopolymer derived from a dimer of glucose as repeating cellobiose units, especially two \u03b2-D-glucopyranose units connected by \u03b2-(1\u21924) glycosidic bonds which form a ribbon structure, stabilized by many strong intramolecular hydrogen bonds ,176. TheCellulose, as one of the renewable and easily accessible materials, is extensively used in the food , paper , textileWater-soluble 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose was obtained from bamboo-dissolving pulp . In the In the worldwide Espacenet database, 8514 patent documents were found, filtered according to title, abstract and claims for biopolymer hydrogels based on cellulose in the period from 1915 to 27 May 2023 . The seaBacillus subtilis) and Gram-negative (Escherichia coli) bacteria were tested.Patent US10207252B2 discloses pristine cellulose nanocrystals with functionalized surfaces, incorporated in hydrogel beads applied as adsorbents for pollutants in water treatment . These hPatent US9650742B2 provides a production method for value-added hemicellulose, extracted from a cellulosic pulp and paper industry byproducts, and for commercial uses . The patPatents EP3335695B1 and EP332)n-S(O)m-L1-N3, where 1 < n < 10; m is 0 or 1; L1 is a linker), which is attached to a carbon atom of glucosyl units (one or more) of azido-modified nanofibrillar cellulose, and which, accordingly, forms an ether bond with carbon atom [The nanofibrillar cellulose hydrogel disclosed by patent EP3572434B1 may consist of azido-modified nanofibrillar cellulose with a substituent and water . The hydEscherichia coli and Staphylococcus aureus). The water retention of the composite hydrogel increases with the decrease of potato flour content and keeps moisture up to 48 h at 40 \u00b0C, which can provide sufficient nutrients for the soil after dehydration and can also be applied in agriculture.The preparation method of an antibacterial wheat straw cellulose composite hydrogel, disclosed in a patent CN112724422B, comprises the following steps: wheat straw pretreatment, potato pretreatment and the preparation of wheat straw cellulose composite hydrogel . The obtPatent CN113150319B discloses a high-efficiency self-healing hydrogel which is reinforced by cellulose nanocrystals, together with a hydrazide-modified hydrogel matrix material with a disulfide bond and a crosslinked network structure formed therewith . The obtPatent CN114230719B provides a double crosslinked cellulose-based hydrogel prepared by cellulose from dried pineapple pomace, with added acrylic acid and 2-acrylamide-2-methylpropanesulfonic acid in the cellulose solution, initiated by cold plasma . The meaN,N\u2032-methylene bisacrylamide and polycarboxylic acids or irradiations). Some of the study outcomes include lower crystallinity degree and thermostability. Marking a difference from many analyzed biomaterials, major advancement has been achieved in improving the features of cellulose-based hydrogels in various fields of application. This includes adopted technologies for cellulose-based hydrogels production and the optimized process conditions. New physicochemical approaches to simultaneously control the gelation process and interactions between the obtained hydrogel and native tissues could additionally improve their applications in tissue engineering and drug delivery.Cellulose is a low-cost, available, biodegradable natural polysaccharide with very good functional properties and ease of preparation. Its main feature is its swelling ability when in contact with water or aqueous fluids. Cellulose-based hydrogels are extensively applied as biosensors, functional food for delivering nutrients, as active/smart food packaging, as adsorbers for heavy metals, as dyes in wastewater treatment, etc. The chemically crosslinked cellulose-based hydrogels have satisfactory levels of stability and strength. However, the main weakness of the chemical crosslinking method is the toxicity of the residual crosslinkers. Many studies found alternative approaches, e.g., the use of low toxic crosslinkers in the latest published papers. A patentability analysis of selected documents based on natural hydrogels based on proteins and polysaccharides is provided.Nowadays, information is kept secret or usually protected by patent rights, especially in industry. Globalization has demanded that universities also adapt to such a trend and open up for business and international cooperation. This includes effective protection, management of research results and cooperation with industry. As a result of the presented patent search, it was noticed that a large number of patents originate from universities, as well as from industry\u2013academic cooperations and foundations . AdditioBiopolymer hydrogels are suitable starting materials for biomaterials, which is obvious by their significant scientific and technological benefits. Because of the characteristics of biopolymer-based materials, such as biocompatibility, particular interactions with living tissue, degradability, responsivity and regrowing resources, biopolymers are very useful. Biopolymers\u00b4 usability for biomedical applications, especially as carriers for the controlled delivery of drugs, proteins and nucleic acids, cell binding and proliferation, requires particular modifications and the synthetic designing of the desired properties. Major progress in recent times presents the functional groups\u2019 introduction to allowing controlled crosslinking density and elastic moduli to design better mechanical properties. Novel biopolymer hydrogels ensure better bioactivity, enable physical interactions with bioactive molecules and ensure control of the diffusion rates of incorporated active substances, as well as the conformation, properties and functions of obtained biopolymer materials. Therefore, they have shown great potential.Despite the numerous investigations into new, innovative biopolymer-based hydrogels, their production processes and the development of new, inventive applications, the biological interactions between cells, tissues and biopolymers still remains under-researched. The summarized properties of each kind of hydrogel analyzed in this review are presented in The degradation processes of biopolymer-based hydrogels, and the metabolic processes and immunological body responses to biopolymers, as well as their degradation, are hard to predict and are not sufficiently recognized and understood. They are of most importance in biomedicine, with the aim of controlling the delivering time in vivo, the dosage of released drugs and the produced biological effects. Interdisciplinary collaboration for the integration of knowledge from different disciplines\u2014e.g., bioengineering, medicine, pharmacology\u2014is required in order to put the new biopolymer-based hydrogels into clinical application. Considering the many advantages of biopolymer-based hydrogels, it should be expected that their new applications will advance clinical practice.This review could be useful in future studies of biopolymer-based hydrogels, and it may serve as a starting point from which to plan and design future research in this particular field."} +{"text": "Both auditory and vestibular primary afferent neurons can be activated by sound and vibration. This review relates the differences between them to the different receptor/synaptic mechanisms of the two systems, as shown by indicators of peripheral function\u2014cochlear and vestibular compound action potentials (cCAPs and vCAPs)\u2014to click stimulation as recorded in animal studies. Sound- and vibration-sensitive type 1 receptors at the striola of the utricular macula are enveloped by the unique calyx afferent ending, which has three modes of synaptic transmission. Glutamate is the transmitter for both cochlear and vestibular primary afferents; however, blocking glutamate transmission has very little effect on vCAPs but greatly reduces cCAPs. We suggest that the ultrafast non-quantal synaptic mechanism called resistive coupling is the cause of the short latency vestibular afferent responses and related results\u2014failure of transmitter blockade, masking, and temporal precision. This \u201cultrafast\u201d non-quantal transmission is effectively electrical coupling that is dependent on the membrane potentials of the calyx and the type 1 receptor. The major clinical implication is that decreasing stimulus rise time increases vCAP response, corresponding to the increased VEMP response in human subjects. Short rise times are optimal in human clinical VEMP testing, whereas long rise times are mandatory for audiometric threshold testing. The classical methods of testing human otolith function using linear acceleration stimuli have now been complemented in the clinic by the use of air-conducted sound (ACS) and bone-conducted vibration (BCV) as vestibular, and specifically otolithic, stimuli ,2,3. SouACS or BCV stimuli can activate vestibular receptors, resulting in small myogenic potentials called vestibular evoked myogenic potentials (VEMPs). VEMPs are due to synchronized action potentials of vestibular afferents activated at the onset of the stimulus since, as we explain below, prolonging the stimulus rise time degrades the synchronization of action potentials and degrades VEMPs . VEMPs aThe use of VEMPs as indicators of vestibular function rests on physiological evidence that vestibular receptors and neurons are activated by sound and vibration. Mostly, this has come from recording the response of single identified otolithic afferent neurons to sound or vibration in guinea pigs and other species . As we In anesthetized guinea pigs, we have measured VIII nerve activation ) in response to transient ACS and BCV stimuli ,13,14. CThe vestibular and cochlear divisions of the labyrinth share the same fluids and have receptor cells of superficially similar appearance, so the vestibular and cochlear divisions appear to be interdependent. That appearance is false . It has (a)use stimuli that are accepted as otolithic\u2014transient linear accelerations\u2014and interpret the results (such as the VsEP) as being due to vestibular rather than cochlear activation There are two approaches to differentiating vestibular from cochlear responses in animal experimental studies. One can either(b)use transient BCV or ACS clicks, which can activate all labyrinthine sensory regions but use various controls, such as disabling or even removing the cochlea, to identify the vestibular component of the total CAP nerve response ,26.orThe problem with using linear acceleration is that the presumably specific otolithic stimulus most likely has aspects that affect the cochlea\u2014it is difficult to deliver a transient linear acceleration without sound or vibration\u2014so it is wise to use broadband masking noise to minimize any possible cochlear contribution . As we sInner hair cells have a fairly uniform appearance throughout the cochlea, although stereocilia height varies along the basilar membrane channels on the stereocilia to open and depolarize the hair cell and therefore generate a receptor potential\u2014the cochlear microphonic (CM)\u2014and action potentials in individual afferents , but thvestibular since it is present after total cochlear ablation and disappears after vestibular endorgan ablation ;The level of potassium in the synaptic cleft;Resistive coupling between the type I receptor and the enveloping calyx .Contini et al. have shown that synaptic transmission at the type I\u2013calyx synapse is very unusual ,64. Cont(a)Glutamate release is apparently similar to that in the cochlea; vestibular type I receptors contain ribbon synapses, which release glutamate probably in response to relatively slow (sustained) stimuli. This is quantal glutamate transmission. The activation of the post-synaptic neuron by glutamatergic transmission is relatively slow .(b)Vestibular stimulation causes the deflection of the stereocilia and therefore opens the mechanoelectrical transduction (MET) channels on the stereocilia of type I receptors, so potassium enters the type I receptor from the potassium-rich endolymph and is released by the receptor into the narrow (femtolitre) synaptic cleft between the type I receptor and its enveloping calyx. Potassium levels in the narrow synaptic cleft modulate the membrane potential of the receptor and the calyx ,67,68.(c)Most importantly, the simultaneous dual patch clamp recording of a type I receptor and its enveloping calyx afferent conclusively demonstrated non-quantal transmission experimentally. This is a form of synaptic transmission that is not dependent on glutamate reception on the post-synaptic membrane, and one key component of this non-quantal transmission is called resistive coupling, which is essentially electrical transmission that is dependent on membrane potential. This is a form of ultrafast electrical coupling between the type I receptor and calyx afferent; channels on the type I receptor membrane and the facing membrane of its calyx afferent are both open near resting potential, enabling the ultrafast depolarization of the irregular primary afferent ,66. We cEach of these factors is addressed below:One consequence of blocking glutamate transmission via CNQX is that after CNQX blockade, a stimulus will still open the MET channels on the stereocilia of the type I receptors and will still cause receptor membrane depolarization (and therefore vestibular microphonics). Receptor membrane depolarization enables ultrafast resistive coupling between the type I receptor and its calyx to take place independently of the (relatively slow) quantal glutamatergic synaptic transmission ,70. ThisBy using the peaks of the cochlear and vestibular microphonics as the benchmark for comparing the latencies of the vCAP and the cCAP, we have shown that the vCAP has a shorter latency than the cCAP see . This reIn response to sinusoidal stimuli, individual cochlear and vestibular afferents with irregular resting activity show precise phase locking; action potentials are generated at a particular phase angle (or a narrow band of phase angles) of the stimulus with high precision ,35,53,71Further evidence of the temporal precision of irregular vestibular afferents can be found by examining the frequency limits of phase locking . The uppIn light of the above evidence, we consider how the cochlear and vestibular systems respond to various stimulus manipulations of relevance for clinical vestibular testing. In some experiments, these manipulations are carried out using the kind of procedures outlined above to eliminate or minimize cochlear contributions to the measured vestibular response.onset of the stimulus that is the key parameter in the generation of VEMPs.The importance of stimulus onset in VEMP testing on human subjects was shown in a study in which the total duration of a 500 Hz tone burst stimulus was systematically decreased from 10 ms to 2 ms, leading to the oVEMP n10 for a 2 ms stimulus having the same amplitude as the response amplitude to a stimulus of 10 ms duration . We haveThe systematic variation in the rise time of short 500 Hz BCV tone bursts shows the importance of stimulus rise time in generating VEMPs in human subjects; increasing the rise time of 7 ms 500 Hz tone bursts decreased VEMP amplitude , with thThese clinical data correspond to the results of the primary vestibular afferents. We have shown that as the rise time of a click increases, the amplitude of the vCAP decreases. This is expected because increasing rise time will decrease the synchronization of action potentials across neurons see Fig. The optexactly opposite\u00a0to that in the audiometric testing of pure tone thresholds: for VEMPs, the most effective VEMP stimulus is one with a very short rise time, which synchronizes vestibular action potentials in primary afferent neurons. Unfortunately, some devices intended for use in VEMP testing do not allow for rise times less than 2 ms, and some clinicians do not realize how important it is to minimize the rise time for VEMP testing as opposed to auditory threshold testing.Evidence from single neuron recordings shows the excellent temporal precision of irregular vestibular afferents, which respond with short latencies to transient stimuli and phasThe amplitude of the cCAP to ACS or BCV is reduced by masking (both simultaneous broadband masking or forward masking ), whereaReducing the interval between successive pulses reduces the cCAP to the second pulse, with reductions in cCAP amplitude beginning at an interstimulus interval of about 50 ms and reaching a 50% response suppression at an interstimulus interval of ~10 ms ,77 see . HoweverVestibular afferents are resistant to masking, whereas with cochlea afferents, it is relatively easy to mask their response. We contend that the differences in synaptic transmission (cochlea vs. vestibular) are responsible for this differential effect regarding stimuli masking.The vestibular evoked potential at the scalp (VsEP);The vestibular summating potential ;Auditory Nerve Neurophonic (ANN) ;Vestibular Nerve Neurophonic (VNN).For both auditory and cochlear systems, there are other components and variants of these labyrinthine electrophysiological potentials to stimuli other than transients (see ), includOne outcome of measuring vCAPs is that they allow us to evaluate which particular stimulus parameters at the level of otolithic receptors are likely optimal for clinical VEMP testing. So, for example, the results have confirmed the importance of short rise times in optimizing VEMP stimuli. A simple way of reducing cochlear contributions to vestibular responses is to use simultaneous masking noise, as confirmed by the physiological results presented in this paper. Another preliminary result is that the CE chirp stimulus (500\u20134000 Hz sweeps), also called narrow-band chirps, are particularly clinically effective stimuli for oVEMPs ,80. Our It is otolithic receptors and their afferents with irregular resting discharge originating from the striola which are activated by sound and vibration .exactly opposite to that in the audiometric testing of pure tone thresholds; effective VEMP stimuli have a very short rise time, synchronizing vestibular action potentials in primary afferent neurons. In contrast, for the audiometric testing of thresholds, a long rise time is mandatory. Unfortunately, some stimulus generators do not allow for rise times less than 2 ms, and some clinicians who measure VEMPs do not realize how important it is to minimize the rise time for VEMP testing as opposed to auditory threshold testing. Our recordings of the vCAP in response to chirp stimuli confirm that the chirp stimulus is particularly effective in generating vCAPs at the level of primary afferents in comparison to simple clicks.In clinical VEMP testing, the situation is"} +{"text": "Clinical trials demonstrated that co-targeting angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF-A) with faricimab controls anatomic outcomes and maintains vision improvements, with strong durability, through 2 years in patients with neovascular age-related macular degeneration and diabetic macular edema. The mechanism(s) underlying these findings is incompletely understood and the specific role that Ang-2 inhibition plays requires further investigation.We examined the effects of single and dual Ang-2/VEGF-A inhibition in diseased vasculatures of JR5558 mice with spontaneous choroidal neovascularization (CNV) and in mice with retinal ischemia/reperfusion (I/R) injuries.In JR5558 mice, Ang-2, VEGF-A, and dual Ang-2/VEGF-A inhibition reduced CNV area after 1 week; only dual Ang-2/VEGF-A inhibition decreased neovascular leakage. Only Ang-2 and dual Ang-2/VEGF-A inhibition maintained reductions after 5 weeks. Dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 1 week. Both Ang-2 and dual Ang-2/VEGF-A inhibition reduced macrophage/microglia accumulation around lesions after 5 weeks. In the retinal I/R injury model, dual Ang-2/VEGF-A inhibition was statistically significantly more effective than Ang-2 or VEGF-A inhibition alone in preventing retinal vascular leakage and neurodegeneration.These data highlight the role of Ang-2 in dual Ang-2/VEGF-A inhibition and indicate that dual inhibition has complementary anti-inflammatory and neuroprotective effects, suggesting a mechanism for the durability and efficacy of faricimab in clinical trials. Retinal and choroidal vascular diseases, such as diabetic retinopathy, diabetic macular edema (DME), retinal vein occlusion, and neovascular age-related macular degeneration (nAMD), are among the leading causes of blindness and visual impairment globally . A key dTissue ischemia leading to hypoxia and oxidative stress in nAMD, coupled with chronic hyperglycemia in diabetic retinopathy, mediates increased expression of vascular endothelial growth factor (VEGF)-A and pro-inflammatory cytokines, which stimulate increased vascular permeability and pathological neovascularization . VEGF-A The angiopoietin (Ang)/tyrosine kinase with immunoglobulin (Ig) and epidermal growth factor homology domains (Tie) pathway plays an important role in vascular development and homeostasis. While Ang-1 activates the Tie2 receptor and promotes cell survival and vascular stability during development , Ang-2 a+ microglia/macrophages around CNV lesions, was statistically significantly reduced with dual Ang-2/VEGF-A inhibition compared with inhibition of Ang-2 or VEGF-A alone in which mice develop subretinal neovascularization and fibrotic lesions resembling human nAMD . One wee-A alone . The red-A alone . These p\u00ae) is a bi-specific antibody designed using Roche\u2019s CrossMAb technology, which simultaneously neutralizes both Ang-2 and VEGF-A to 12- (5 weeks post treatment) week-old male and female animals. Studies on retinal I/R injury were performed using 10- to 11-week-old C57BL/6J male mice.Mice were housed in temperature- (22 \u00b1 1\u00b0C) and humidity- (57%) controlled rooms (automatic 12-h light/dark cycle) under pathogen-free conditions in groups of up to five in ventilated cages. Standard laboratory chow and tap water were supplied in vivo experimentations.The JR5558 genetic model was discin vivo imaging. The 10 mg/kg anti\u2013Ang-2/anti\u2013VEGF-A antibody dose was selected based on our previously reported (in vivo by fluorescein angiography (FA) at baseline (day [D] 0) and at 1 week (D15) or 5 weeks (D43) after treatment to assess immediate and long-term treatment effects on CNV lesion activity, respectively. Blood-retinal barrier permeability was quantified by fluorophotometry 1 week after the last antibody dose. At the end of the study (D15 or D43), neovascularization and subretinal inflammatory cell infiltration were evaluated by immunohistochemistry on retinal pigment epithelium (RPE)/choroid flatmounts. Antibody treatment administration and the qualitative and quantitative analyses of results were made on a blinded basis.The JR5558 experiments were designed as previously described , but werreported anti-angFluorescein fundus angiography was carried out at baseline (D0) to equally assign JR5558 mice across experimental groups by sCNV lesion leakage area before treatment and 1 week (D15) or 5 weeks (D43) after treatment. Before fluorescein administration, mice were anesthetized by subcutaneous (SC) injection of a fentanyl (0.05 mg/kg), medetomidine (0.5 mg/kg), and midazolam (5 mg/kg) mixture, and eyes were dilated with 0.5% tropicamide. Once pupils were dilated, 3.2-mm plano contact lenses were placed on the eyes, and 2% fluorescein sodium salt (10 mL/kg) was injected intraperitoneally. Mice were then placed in front of the Heidelberg Spectralis ophthalmic imaging system for infrared (IR) and FA image acquisition. Both eyes underwent a complete analysis, with an initial central FA image captured 5 min after fluorescein injection and a subsequent six images to cover all sections of the eye. Following IR and FA imaging, anesthesia reversal was achieved by subcutaneous injection of a buprenorphine (0.2 mg/kg), atipamezole (2.5 mg/kg), and flumazenil (0.5 mg/kg) mixture. FA images from each individual eye were manually exported as JPEG files for analysis using Adobe Photoshop software . The number of lesion leakage areas per eye at baseline was counted using the Photoshop Count tool. For quantification of the lesion leakage area after treatment, areas of fluorescein leakage were selected for each eye with the Photoshop Lasso tool and expressed as pixel numbers for statistical analysis.In vivo quantitation of blood-retinal barrier permeability was performed by fluorophotometry 1 week (D15) after treatment. As per the FA procedure, JR5558 mice were anesthetized by subcutaneous injection of a fentanyl, medetomidine, and midazolam mixture. Eyes were then dilated using 0.5% tropicamide and contact lenses (Cantor & Nissel Ltd.) were placed to prevent eye drying and protect against cataract formation. For measurements of fluorescein concentration profiles in the vitreous ocular compartment and in the plasma, 1% fluorescein sodium salt (10 mL/kg) was administered by subcutaneous injection. Mice were then placed on a temperature-controlled (37.0 \u00b1 1\u00b0C) stage, and their position was adjusted so that the eye being analyzed was aligned precisely and in parallel to the optic device. Data acquisition was started 45 min after fluorescein injection using 450- to 490-nm excitation and 520- to 600-nm emission for fluorescein detection. After scanning both eyes, tails were punctured for blood sampling (25 \u03bcL), and plasma was extracted by centrifugation to correct for circulating fluorescein and, thus, account for potential administration differences. Plasma was then diluted 1:100 with phosphate-buffered saline (PBS) in a microcuvette, and fluorescein levels were measured using a cuvette holder provided with the fluorophotometer. Raw data for the vitreous compartment and plasma fluorophotometric measurements were analyzed and quantified using Microsoft Excel by calculating the average value from the five highest steps corresponding to the graphical peak for fluorescein concentration in the vitreous and plasma. Average fluorescein levels in the vitreous of JR5558 mice were corrected by the maximum plasma fluorescein values and expressed as a ratio on the final bar/scatter dot blot graph.Bandeiraea simplicifolia, 1:100, #L2140; Sigma-Aldrich, St. Louis, MO, United States), goat anti-Iba1 , rat anti-CD45 , or rat anti-CD11b . The following day, the tissue was washed for 6 \u00d7 5 min with 0.3% Triton X-100 in PBS, followed by incubation for 2 h at RT with the following secondary antibodies in 0.3% Triton X-100 with 5% donkey serum in PBS: 1:100 fluorescein isothiocyanate (FITC)-conjugated DyLight 488 Streptavidin , donkey anti-goat IgG , and anti-rat IgG . RPE/choroid tissues were then washed for 6 \u00d7 5 min in 0.3% Triton X-100 in PBS at RT before being flatmounted on Superfrost glass slides (Thermo Fisher Scientific) using Dako fluorescent mounting medium . Following RPE/choroid tissue immunostaining, isolectin B4, CD45, and CD11b signals were detected and acquired using an Olympus VS120 scanner equipped with a XM10 camera and a UPLSAPO 10 \u00d7 /0.40 objective . Specific isolectin B4, Iba1, CD45, and CD11b signal areas were selected with the Lasso tool in Adobe Photoshop and areas of isolectin B4 signal or Iba1-, CD45-, and CD11b-positive cells number per RPE/choroid flatmount were quantified using ImageJ.Subretinal inflammatory cell infiltration was detected by Iba1, CD45, and CD11b immunostaining and was evaluated by flatmounted RPE/choroid histology at 1 and 5 weeks after antibody treatment. Enucleated eyes were fixed with 4% paraformaldehyde solution for 2 h at room temperature (RT) and then transferred to PBS. The RPE/choroid complex was carefully separated from the retina and permeabilized for 2 h at RT in 3% Triton X-100 solution in PBS. After permeabilization, RPE/choroids were incubated overnight with the following primary antibodies in 0.3% Triton X-100 with 5% donkey serum in PBS: biotinylated isolectin B4 , followed by natural reperfusion, as previously described of IgG control antibody (3 \u03bcg/\u03bcL) or anti\u2013VEGF-A (anti\u2013VEGF-A at 1.5 \u03bcg/\u03bcL and IgG control at 1.5 \u03bcg/\u03bcL), anti\u2013Ang-2 (anti\u2013Ang-2 at 1.5 \u03bcg/\u03bcL and IgG control at 1.5 \u03bcg/\u03bcL), or bi-specific anti\u2013Ang-2/anti\u2013VEGF-A (3 \u03bcg/\u03bcL). Two days after treatment, retinal ischemia was transiently induced by injection of saline into the anterior chamber to raise the intraocular pressure to 85\u201395 mmHg was measured at 48 h after I/R injury using a previously described method . BrieflyA DNA fragmentation assay was used to evaluate ongoing retinal cell death 48 h after I/R injury using a previously described method . BrieflyAll personnel involved in image acquisition, image analysis, and histology were blinded to treatment until raw data were fully processed. Only personnel involved in drug administration had information about the identity of the treatments the animals received.P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical analyses were performed using Prism 7 .Data were compared between groups using one-way analysis of variance followed by Tukey\u2019s multiple comparison test. Statistical significance was indicated by *Animal maintenance and experimental procedures were approved by the Federal Food Safety and Veterinary Office of Switzerland (reference BS-2734) and were conducted in strict adherence to the Swiss federal ordinance on animal protection and welfare, as well as according to the rules of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research guidelines (European Directive 86/609/EEC) and the Roche Ethics Committee on Animal Welfare (JR5558 sCNV mouse model) or with approval of the University of Michigan Institutional Animal Care and Use Committee .in vivo by FA and fluorophotometry, respectively, at 1 week after treatment. The extent of neovascularization was analyzed by immunohistochemistry using isolectin B4 as a vascular marker on RPE/choroid flatmounts to assess immediate treatment effects compared with IgG control\u2013treated mice . There wThese results suggest that intravitreal delivery of antibodies affording dual Ang-2/VEGF-A inhibition is more effective than antibodies causing Ang-2 or VEGF-A inhibition alone in preventing both vascular permeability and neurodegeneration following retinal I/R injury.Phase 3 clinical trials in patients with DME or nAMD have demonstrated that dual Ang-2 and VEGF-A pathway inhibition with faricimab improves and maintains vision, with strong durability, and improves anatomic outcomes compared with VEGF-A pathway inhibition alone. However, the underlying individual contributions of Ang-2 and VEGF-A in mediating these outcomes is unclear. Here, we have described the results of preclinical studies that aimed to delineate the longer-term (5-week) effects of Ang-2 and Ang-2/VEGF-A inhibition on vascular permeability, inflammation, and neurodegeneration in mouse models of retinal neovascularization and I/R. Of note, our findings highlight that Ang-2 plays an important underlying role in the extended efficacy and durability effects observed here.+, CD11b+, and CD45+) accumulation at the lesions, whereas only Ang-2 and dual Ang-2/VEGF-A inhibition, but not VEGF-A inhibition, reduced immune cells accumulation after 5 weeks. Taken together, these findings not only suggest that dual Ang-2/VEGF-A inhibition may promote vascular stability by decreasing neovascular leakage and neovascularization, but also that Ang-2 inhibition plays an important underlying role, particularly when considering the more prolonged (5-week) effects. Further, these findings suggest that Ang-2 inhibition may play an important role in mediating the strong durability and anatomic improvement observed with faricimab in clinical trials and were conducted in strict adherence to the Swiss federal ordinance on animal protection and welfare, as well as according to the rules of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research Guidelines (European Directive 86/609/EEC) and the Roche Ethics Committee on Animal Welfare (JR5558 sCNV mouse model) or with approval of the University of Michigan Institutional Animal Care and Use Committee .All authors had full access to all the data in the study, took responsibility for the integrity of the data and the accuracy of the data analysis, and contributed to the planning, conduct, and reporting of the work described in the article."}