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+{"text": "PLoS Medicine addressed the issue of ensuring equity in distributing AIDS medications. Reis and Capron discuss the study's implicationsA study by Wilson and Blower in the February issue of  Of the roughly 40 million people living with HIV , an estiGiven this gap between what can be done and what needs to be done, the people who set policies and administer programs to provide ART in high-burden countries are faced with difficult questions of distributive justice. Decisions regarding the pricing of ARTs and other care for patients with HIV/AIDS, the distribution of treatment centers, and potential measures to overcome barriers for vulnerable populations will determine who will get access to treatment and who will die. In order to deal with these crucial issues, decision-makers need guidance on how to design policies on equitable access to ART that respect human rights norms and ethical standards.PLoS Medicine [A new study by Wilson and Blower, published in the February issue of Medicine , addressMedicine . Wilson While the authors suggest that their method could be adapted to take other objectives into account, here they have taken an exclusively egalitarian approach to equity. Although this notion of equity is broadly accepted, other important approaches could have been taken into consideration. Simple equality in access can actually produce inequities (because a fair approach would differentiate among groups in the population according to their different needs); further, under some theories, those who are least advantaged generally should receive a disproportionate share of newly distributed benefits (the maximin principle) . In geogIn determining equitable spatial accessibility for the application of their model to KwaZulu\u2013Natal, the authors used a rather rough estimation of HIV prevalence . As prevalence greatly varies between specific communities, future studies would certainly benefit from using more disaggregated data where available . SimilaPlace matters, but spatial accessibility is only one factor to be overcome in ensuring equitable access to health services. Studies show that even when services are available at a near distance, factors such as temporal accessibility, disease perception, stigmatization, and outright discrimination heavily influence \u201ceffective demand\u201d . MoreoveWilson and Blower have developed a mathematical model to determine the fair geographical distribution of ART treatment sites and have applied it to the specific setting of KwaZulu\u2013Natal. Despite some methodological and data limitations, such studies can inform policy-makers' decisions regarding the location of HIV services. Since distance to a treatment center is strongly determinant of patients' ability to access care, WHO is developing a service availability mapping tool to monitor relative equity between districts and identify major gaps in service availability, for example, availability of ART and prevention of mother-to-child transmission programs.Not only is further research needed to refine the spatial accessibility model presented by the authors but careful attention must be paid to other factors that affect access to HIV services and to the underlying assumptions as to what would constitute fair distribution. In a recent guidance document, WHO and UNAIDS recommended that ART programs include special measures to ensure access of vulnerable and marginalized populations and women to ART . The dec"}
+{"text": "Recently, a global commitment has been made to expand access to antiretrovirals (ARVs) in the developing world. However, in many resource-constrained countries the number of individuals infected with HIV in need of treatment will far exceed the supply of ARVs, and only a limited number of health-care facilities (HCFs) will be available for ARV distribution. Deciding how to allocate the limited supply of ARVs among HCFs will be extremely difficult. Resource allocation decisions can be made on the basis of many epidemiological, ethical, or preferential treatment priority criteria.Here we use operations research techniques, and we show how to determine the optimal strategy for allocating ARVs among HCFs in order to satisfy the equitable criterion that each individual infected with HIV has an equal chance of receiving ARVs. We present a novel spatial mathematical model that includes heterogeneity in treatment accessibility. We show how to use our theoretical framework, in conjunction with an equity objective function, to determine an optimal equitable allocation strategy (OEAS) for ARVs in resource-constrained regions. Our equity objective function enables us to apply the egalitarian principle of equity with respect to access to health care. We use data from the detailed ARV rollout plan designed by the government of South Africa to determine an OEAS for the province of KwaZulu\u2013Natal. We determine the OEAS for KwaZulu\u2013Natal, and we then compare this OEAS with two other ARV allocation strategies: (i) allocating ARVs only to Durban  and (ii) allocating ARVs equally to all available HCFs. In addition, we compare the OEAS to the current allocation plan of the South African government . We show that our OEAS significantly improves equity in treatment accessibility in comparison with these three ARV allocation strategies. We also quantify how the size of the catchment region surrounding each HCF, and the number of HCFs utilized for ARV distribution, alters the OEAS and the probability of achieving equity in treatment accessibility. We calculate that in order to achieve the greatest degree of treatment equity for individuals with HIV in KwaZulu\u2013Natal, the ARVs should be allocated to 54 HCFs and each HCF should serve a catchment region of 40 to 60 km.Our OEAS would substantially improve equality in treatment accessibility in comparison with other allocation strategies. Furthermore, our OEAS is extremely different from the currently planned strategy. We suggest that our novel methodology be used to design optimal ARV allocation strategies for resource-constrained countries. A mathematical model that allows to determine a strategy for distribution of limited medical resources  such that each individual in need will have an equal chance of receiving treatment The HIV/AIDS epidemic is having a devastating impact in sub-Saharan Africa and other resource-constrained regions. Recently, the World Health Organization and other organizations have committed to expand access to antiretrovirals (ARVs) in the developing world, the United States government has pledged to provide $15 billion for AIDS in Africa and the Carribean, and drug prices have fallen . It has Here, we use operations research to address this important resource allocation problem and to design ARV allocation strategies that are rational and equitable. The allocation decisions that we make here are based on ethical criteria, and not on epidemiological or preferential treatment priority criteria. Specifically, we determine the optimal allocation strategy that would ensure that each individual with HIV has an equal chance of receiving ARVs. We present a novel spatial mathematical model of treatment accessibility that we use in conjunction with an equity objective function to determine an optimal equitable allocation strategy (OEAS) for ARVs in a resource-constrained region. We quantify how changing the size of the catchment region surrounding each HCF, and the number of HCFs utilized for ARV distribution, alters the OEAS. Specifically, we use data from the detailed ARV rollout plan designed by the government of South Africa to determine an OEAS (based upon a variety of assumptions) for the province of KwaZulu\u2013Natal. We also discuss how our proposed ARV allocation strategy differs from the currently proposed plan.Our current analysis is applied to the South African province of KwaZulu\u2013Natal, although our methodology could be applied to any resource-constrained setting. KwaZulu\u2013Natal is the largest province in South Africa with a population of approximately 9.4 million and has more people infected with HIV than any other province  as a weighting function that determines the treatment accessibility to a HCF based upon distance i,j,d and iI as the number of people with HIV in community i. The distance, ij,d between community i and HCF j is based on the longitude (lon) and latitude (lat) of each location and is determined byWe assume that the number of people with HIV who will travel to a specific HCF is directly proportional to the number of individuals with HIV in that particular community, but that the probability of an individual traveling to receive ARVs  decreases with distance from the HCF. We define R is the radius of the earth, taken to be 6,371 km, and the angles are in radian measure. We calculate the \u201ceffective demand\u201d of community i on HCF j to be the number of people with HIV in community i that will travel to HCF j for ARV regimes, namely, fiI. Thus, demand on HCFs for ARVs is reduced by the treatment accessibility function. Our model is conceptually similar to the \u201cgravity\u201d models that have been used to predict retail travel [f(d) = exp(\u2212kd2), where k is a dispersal length scale parameter determining the radius of the catchment region. The size of the actual catchment regions is unknown, but based upon distances from communities to HCFs in KwaZulu\u2013Natal (see k = 0.003786). We vary the catchment region by considering a 20-km radius (k = 0.0151) and a 60-km radius (k = 0.00168). The different catchment regions that we simulate  for each HCF are illustrated in where l travel , plan lal travel , and detl travel . Howeveratal see A we assu= exp\u2212kd, where kj,j, D to beTo determine how many ARVs should be allocated to each HCF, we first calculate how a given supply of ARVs will be distributed from each HCF to the surrounding communities in the catchment region. We calculate the \u201ceffective demand\u201d on HCF j (where there are m communities). Then, we model the distribution of ARVs from a HCF to each community within the catchment region as the proportion of the \u201ceffective demand\u201d on HCF j that is contributed by the respective community. Accordingly, ARVs will be distributed from HCF, j, to each community as the ratiowhich sums the \u201ceffective demand\u201d of all communities on HCF i by the drug supply allocated to HCF j isTherefore, the number of people treated in community jS is the number of regimes allocated to HCF j. Hence, the total number of people with HIV treated in community i,i,T summing over all n HCFs iswhere A ARV regimes for a total of We establish an equity objective function to determine the optimal equitable allocation of ARVs to each HCF so that all individuals with HIV have an equal chance of receiving treatment. To obtain the same fraction of treated individuals in each community, given that there are E, by solving for the number of ARVs to be allocated to each HCF , whilst enforcing the following three constraints: (i) ensure that the total number of ARVs available is equal to the sum of the supply allocated to all HCFs,Our goal is to minimize jS \u2265 0, j = 1\u2026n); and (iii) ensure that the number of people treated in each community is not greater than the number of people with HIV in the community . We note that if a different objective is required, then all of our preceding analysis still holds and only the functional form of the objective function needs to be altered. To solve the problem, and determine the OEAS, we used successive linear programming operations research techniques [(ii) ensure that only a positive number of ARVs are allocated to each HCF (chniques .k = 0.003786). The proportion of infected individuals that are treated at each location is displayed graphically in E = 0.27 for our OEAS, compared with (i) E = 0.50 and (ii) E = 133.88 for the comparison allocation strategies. There is large diversity in the fraction of individuals with HIV treated per community when equal quantities of ARVs are given to each HCF, evidenced by an inter-quartile range of 0.025%\u201341.746% compared with inter-quartile ranges of 0%\u20130% and 0.011%\u20139.982% for the first comparison strategy and our OEAS, respectively. Therefore, equal access is not obtained if equal quantities of ARVs are allocated to each HCF. Obviously, allocating to only one HCF (the first comparison strategy) could also be considered unequal because although the inter-quartile range is minimal, effectively only one community (Durban) receives ARVs. Our OEAS, while not perfect, achieves the best equality possible given the accessibility constraints and limited ARV supply.The OEAS of ARVs in KwaZulu\u2013Natal that we determined is complex see A and 3B.The catchment region for HCFs is a factor of large uncertainty. We considered three catchment region sizes: radii of 20 km, 40 km, and 60 km. We also simulated two additional cases with increased numbers and locations of HCFs  should be used. The strategy that we are advising may be fairly easy to accomplish at the policy level because the health-care infrastructure  already exists, although consideration must be made for issues such as the training and transportation that is necessary, which may be costly. In contrast, increasing the size of catchment regions may be very difficult. Obviously, increasing both the number of HCFs and the size of the catchment region each services would substantially increase equality of access to health care in KwaZulu\u2013Natal.Future modeling studies could extend our work by not making the simplifying assumption that all patients have similar ease of travel over the same distance and by including weighting functions on distance impedance for different communities  . Here, we have shown how to calculate optimal ARV allocation strategies based upon the principle of equity. Future research is necessary to compare ARV allocation strategies based upon the principle of efficiency  in order to determine whether utilizing different principles for optimization would result in similar (or different) allocation strategies.The World Health Organization and the Joint United Nations Programme on HIV/AIDS have identified three core principles that should underlie the effort to fairly distribute ARVs, namely: urgency, equity, and sustainability . They stAlthough we have focused on one equitable strategy, there are many other ARV allocation strategies that are ethical. Uneven access to HIV treatment has the very real potential to fracture social and political structures and could lead to intrastate and/or interstate conflict . GovernmTreatment priority decisions for individuals could be based on many different criteria, including disease progression , socioeconomic status, ethnicity, and who is thought to have the greatest risk of transmitting infections . Although it could be argued that behavioral core groups should be targeted to receive ARVs because this may have the greatest epidemiological impact, such an allocation strategy would be neither feasible nor practical to implement. For example, sex workers are an obvious behavioral core group, but many women would likely claim to be sex workers if they knew that ARVs were only available to sex workers. Additionally, the ethics of targeting such groups in favor of other societal groups must be questioned. It could also be argued that, to maximize the preventative effect of ARV therapy, ARVs should be concentrated in virological core groups  ,26; thisOur model has been applied to the South African province of KwaZulu\u2013Natal, but it can be applied by government health officials in any resource-constrained country. In many of the countries worst affected by the HIV pandemic, scarcity of resources will mean that not everyone that could potentially benefit from ARVs will be able to access them. Many of the decisions that must be made to develop an effective response to the HIV/AIDS epidemic are inevitably underpinned by ethical considerations. Leadership in most resource-constrained regions cannot avoid these decisions. Whilst there has been considerable attention given to South Africa, many other countries worldwide either have plans in place  or are in the process of developing national programs for ARV distribution through the public health system  . LegitimAntiretroviral drugs can change the lives of patients with HIV/AIDS. Their high price, however, means that many poor countries do not have enough of these drugs to treat all the people who need them. The decision of who will get treatment is very difficult, and different ways to come up with ethical solutions to the problem have been proposed.One of the approaches is to try to make sure that every infected person has the same chance to get antiretroviral drugs. David Wilson and Sally Blower, the authors of this study, wanted to find a scientific strategy to achieve this goal of equal access.They used mathematical models to calculate how to distribute available drugs among hospitals and doctor's offices so that each patient in a particular area had an equal chance to get treated.When they used their approach on a real example, the South African province of KwaZulu\u2013Natal, they found that making some changes to the current plans for drug distribution would lead to more equal access among all of the individuals with HIV in the province. Instead of only 17 out of the 54 health care facilities in KwaZulu\u2013Natal distributing the drugs (which is the current plan of the South African government), Wilson and Blower calculate that it would be fairer if all 54 facilities distributed the medicines.Mathematical models like the one used here are always based on assumptions and simplifications. As a consequence, they are never perfect matches for a real-life situation, but they can help to guide complicated decisions. This article suggests that the approach Wilson and Blower developed could help to determine strategies for equitable allocation of limited HIV treatment resources.The authors hope that the tools they developed will be used by policy makers in resource-poor countries to guide their strategies. They are keen to work with these policy makers to adapt and optimize the method to local settings and priorities.http://www.who.int/hiv/pub/advocacy/en/ethicsmeetingreport_e.pdfReport by the World Health Organization and the Joint United Nations Programme on HIV/AIDS on ethics and equitable access to HIV/AIDS treatment: http://www.who.int/ethics/en/background-macklin.pdfRuth Macklin's report on ethics and equity in access to HIV treatment: http://www.who.int/ethics/en/background-pro-poor3.pdfThe Pro-Poor Health Policy Team's report on priority in HIV/AIDS treatment: http://www.who.int/bulletin/volumes/82/1/en/news.pdfNews article from the World Health Organization Bulletin on the South African HIV/AIDS treatment program:"}
+{"text": "Antiretroviral drugs change the lives of patients with HIV/AIDS\u2014if they have access to them. Most patients in resource-poor countries cannot afford the drugs. Major initiatives are under way to expand access to antiretrovirals in developing countries, but the number of individuals in need of the drugs currently vastly exceeds the supply, and will continue to do so for the foreseeable future. These circumstances make for difficult decisions about treatment allocation. David Wilson and Sally Blower have shown how it is possible to design an equitable antiretroviral allocation strategy, that is, to come up with a plan that would give each individual with HIV an equal chance of receiving antiretrovirals. Their novel spatial model enables them to model the \u201cspatial diffusion\u201d of antiretrovirals in a resource-constrained country.Based on the premise that only a limited number of drugs will be available and only a limited number of health-care facilities can be used for drug distribution (each of them serving the population in a specific area), they determine an optimal equitable allocation strategy. They then apply this approach to a practical example\u2014the equitable allocation of antiretrovirals to patients with HIV/AIDS in the South African province of KwaZulu\u2013Natal. Using data from a detailed rollout plan for antiretrovirals designed by the South African government, they come up with an allocation strategy that differs substantially from the current governmental plan for the province.KwaZulu\u2013Natal has a total of 54 health-care facilities, of which 17 are assigned to allocate antiretrovirals under the current plan. It is the largest province in South Africa, with a population of about 9.4 million, and it has more people with HIV than any other province . Wilson and Blower assume that the available amount of antiretrovirals can treat 10% of the individuals with HIV in KwaZulu\u2013Natal. Modeling the 17 health-care facilities and the 51 communities of individuals with HIV, they determine the amount of drugs to allocate to each facility to achieve equitable access by patients throughout the province. They then extend the analysis assuming that additional health-care facilities could be made available to distribute drugs. They conclude that in order to achieve the greatest degree of treatment equality, all 54 health-care facilities should be used, and they should, on average, each serve the population within a radius of 50 km.Wilson and Blower discuss how their model can be adjusted and therefore used by policy makers in resource-constrained countries to determine a scientifically based allocation strategy for limited resources based on a number of specific objectives. They also recognize that there are other considerations that influence ethical treatment allocation besides equity, for example, the desire to maximize epidemic reduction, or the imperative to give priority to the least advantaged individuals They believe that their model can be adjusted and therefore \u201cused by policy makers to determine an optimal scientifically based allocation strategy\u201d for a number of specific objectives. Another possibility would be to apply the equity strategy to allocate drugs to particular health-care facilities , and then take additional ethical considerations into account at the community level."}
+{"text": "For many people in the developed world, a diagnosis of HIV/AIDS is no longer a death sentence. Since the introduction in the 1990s of effective antiretroviral therapy (ART)\u2014combinations of three or four drugs that interfere with different stages of the HIV life cycle\u2014HIV/AIDS mortality rates have dropped by 50%\u201370% in affluent countries. By contrast, in developing countries, at least 6 million people need immediate access to ART but fewer than 10% of them get it. In 2005, 3 million people in poor countries died from HIV/AIDS. To improve this situation, the World Health Organization (WHO), the Joint United Nations Programme on HIV/AIDS (UNAIDS), and other international bodies are working toward providing universal access to ART for all those who need it by 2010. In addition to reducing AIDS morbidity and mortality, the hope is that this strategy will also reduce HIV/AIDS prevalence, because sexual transmission of HIV is more likely if the partner who is HIV-positive has a high viral load.Many challenges need to be overcome to achieve universal access to ART, not least of which is determining how to maximize the benefits of ART to patients and their communities in resource-poor settings. Regional differences in health-care facilities, local changes in sexual behavior in response to treatment, and many other factors can alter how ART affects both HIV transmission rates and HIV/AIDS mortality. Ideally, the best strategy for each setting would be determined through large-scale randomized trials of different approaches\u2014for example, the time at which treatment is initiated relative to the time of infection\u2014with HIV prevalence and HIV/AIDS\u2013related mortality as primary endpoints. However, such trials are lengthy and costly, so researchers and policy makers are also using mathematical models to explore the impacts of different treatment and monitoring strategies. Rebecca Baggaley and colleagues now describe a new approach to modeling the impact of ART in resource-poor settings. Their model predicts that HIV epidemics in sub-Saharan Africa will not be controlled through ART alone, even if universal access is achieved. Additional prevention methods such as counseling patients and their communities about safe sex are essential. Without them, their results suggest, access to ART is likely to increase HIV/AIDS prevalence.The researchers' deterministic model of HIV transmission incorporates ART and stratifies infection progression into four different stages , each of which is associated with a different degree of infectiousness. In effect, the model is a complex flowchart through which patients move inexorably as they become infected and receive treatment\u2014which can fail (virologic failure) or succeed \u2014or from which they can withdraw. Sexual behavior and changes in sexual behavior in response to HIV/AIDS and ART is also plugged into the model\u2014people treated with antiretrovirals often become more sexually active as they begin to feel better. Effective counseling, on the other hand, can increase safe-sex practices. To turn this flowchart into predictions of how HIV epidemics in sub-Saharan Africa might develop over time given different ART strategies and, for example, the availability of diagnostic laboratories to monitor the immune status and viral load of individuals with HIV, the researchers used published estimates of relevant parameters such as the fraction of patients that drop out at each stage of treatment and the transmission probability per sexual partnership for patients in whom ART failed.Baggaley and her colleagues make several predictions. They suggest, for example, that unlimited ART provision initiated once patients have developed AIDS will increase the prevalence of infection , a worrying result given that one aim of the universal access initiative is to reduce HIV infection rates. Furthermore, although different coverage levels in this scenario will not affect the years of life gained per person-year of treatment, increased coverage will increase the emergence and spread of drug resistance. If pre-AIDS patients are treated as well, the researchers predict that additional infections will be averted per person-year of treatment, but the effect will be small and highly dependent on how pre-AIDS patients change their sexual behavior in response to ART.As with all modeling exercises, this new model includes many assumptions that may limit its applicability in the \u201creal world.\u201d For example, it includes only first-line triple-therapy ART and does not allow for second-line therapy if one drug regimen fails; it does not consider the sexual behavior of people who don't know they are infected with HIV; and it does not allow for a reduction in the quality of ART programs as coverage increases, a likely problem in countries with limited resources. Nevertheless, the model's predictions sound a warning: ART is not likely to function as a direct method for transmission prevention even when coverage is high. Counseling of patients and their communities to promote safe sexual practices must accompany ART provision. Difficult decisions regarding the allocation of finite resources will have to be made as ART is rolled out in resource-poor countries, conclude the researchers, decisions that can best be made by combining mathematical modeling with data from early programs as they become available."}
+{"text": "PLoS Medicine study that models the impact of antiretroviral drugs upon HIV transmission in developing countries.Bertozzi and Bautista-Arredondo discuss the implications of a new Ideally, evaluations of strategies related to the use of antiretroviral agents would use large-scale randomized trials with HIV-related mortality as the primary endpoint. In reality, however, such studies are rarely conducted, given the long follow-up needed and the substantial costs. Furthermore, people currently living with HIV/AIDS need treatment now, not at the conclusion of long-term prospective trials.Model-based methods are a powerful and practical means of performing formal comparisons of health interventions when the ideal is not possible. Model-based analyses synthesize data from multiple sources, permitting decision makers to understand the likely impact of different strategies and to set priorities for clinical trials . Models Probably the most important contribution of mathematical models relies not in the specific point estimates they generate, but rather, in the insights they provide regarding the likely impact of changes in different variables\u2014such as access to treatment, sexual behavior, natural history of the disease, quality of care and adherence\u2014on life expectancy and epidemic dynamics.Mathematical models have been used to assess the impact of antiretroviral programs on HIV transmission . Even thModel-based methods are a powerful and practical means of performing formal comparisons of health interventions when the ideal is not possible.PLoS Medicine, Rebecca Baggaley and colleagues constructed a model that describes the impact of ART on HIV transmission in developing countries, and they chose Malawi to apply their model [In a study inr model . The autThe authors' model predicted that the unlimited provision of ART, started at late-stage infection (AIDS), would lead to an increase in HIV prevalence. The effect of additionally treating patients with earlier infection (pre-AIDS) would depend on whether treated patients changed their sexual behavior. The authors concluded that one cannot expect the provision of ART, at least in high-prevalence African epidemics, to have important effects on HIV transmission, a finding that is consistent with the previous studies summarized above.The authors' modeling was restricted to considering changes in risk behavior by those persons with HIV infection who initiated ART. They did not consider how availability of therapy may lead to dramatic changes in risk behavior (in either direction) among the far larger population of people who are not infected, infected but don't know their status, or infected but not eligible for therapy.prevention-enhancing. To accomplish the latter, programs must evaluate different strategies to maximize the potential positive impact on prevention and minimize the potential for disinhibition.The authors' decision not to consider these effects is understandable given their focus on comparing different initiation/discontinuation strategies for ART, and given the lamentable lack of data on population-level changes in risk behavior following introduction of ART. However, as the work by Mead Over and colleagues in India suggests, small changes in population-level risk behaviors indirectly caused by ART are likely to be far more important determinants of the epidemic's course than are changes among those receiving therapy . Thus, wThe other important limitation of Baggaley and colleagues' study is suggested by the authors in their conclusion: \u201c\u2026in reality, scaling up programmes is likely to compromise quality, meaning higher dropout rates, mortality and treatment failure, negating the beneficial impacts of ART, and increasing the rate of drug resistance emergence.\u201d Not only does the model not capture likely declines in quality with increasing scale, it also does not appear to adequately capture the likely population heterogeneity with respect to quality of care and patient adherence to treatment.distribution of adherence in the population is an important determinant of average effectiveness, not just the mean adherence. Our concern about this issue is heightened because of our experience in Mexico, where we have documented very high levels of provider and patient heterogeneity [While the model considers four different classes of sexual risk behavior , it appears to treat quality and adherence as parameters that are constant across patients and providers. While the amount of bias introduced by this simplification will clearly depend on the true degree of heterogeneity in the population, the highly nonlinear relationship between adherence and viral suppression suggests that thegeneity . In poorBaggaley and colleagues suggest that policy makers, in their quest to maximize the efficiency of treatment, need to consider both the likelihood that a patient will benefit from ART  and the possibility of reallocating treatment from someone in virological failure to someone who has not yet been able to initiate therapy. If our hypotheses are correct, then it is likely to be even more important, in order to maximize the efficiency of treatment, to consider the probability that a patient will receive care of adequate quality, will have consistent access to drugs, and will be both willing and able to maintain high levels of adherence. The next generation of models should seek to incorporate a more nuanced treatment of these issues\u2014but their ability to do so will also depend on whether data are collected in programs currently being scaled up on prescription patterns, consistency of drug supply, adherence, and duration of viral suppression.We have argued that mathematical modeling can be very useful for informing policy choices, but it is important that such an argument not be construed to mean that mathematical modeling can replace data on effectiveness of interventions. On the contrary, for future modeling exercises to usefully inform policy, better data are needed. For reasons that are not entirely apparent, the scientific community, nongovernmental organizations, and government officials have not insisted that sufficiently rigorous data be generated on effectiveness of HIV/AIDS interventions as they are scaled up. Without even considering prevention interventions, the amount of information known about how to achieve high rates of ART adherence in developing countries, to take just one example, is nowhere near commensurate with either the size of the problem or the resources being committed to address the problem. If we don't take advantage of the current scale-up of interventions to collect data on which approaches work and which ones don't (thus reducing the depth of our ignorance), it will be impossible to convince people to continue to fund such programs. And if we don't figure out how to implement ART in a way that greatly potentiates the effectiveness of prevention, the costs of care in the future will likely outstrip even the most optimistic projections of resource availability."}
+{"text": "Much emphasis is put on providing evidence to assist policymakers in priority setting and investment decisions. Assessing the cost-effectiveness of interventions is one technique used by policymakers in their decisions around the allocation of scarce resources. However, even where such evidence is available, other considerations may also be taken into account, and even over-ride technical evidence. Antiretroviral therapy (ART) is the most effective intervention to reduce HIV-related morbidity and prolong mortality. However, treatment provision in the developing world has been hindered by the high costs of services and drugs, casting doubts on its cost-effectiveness. This paper looks at Thailand's publicly-funded antiretroviral initiative which was first introduced in 1992, and explores the extent to which cost-effectiveness evidence influenced policy.This article reviews the development of the national ART programme in Thailand between 1992 and 2004. It examines the roles of cost-effectiveness information in treatment policy decisions. Qualitative approaches including document analysis and interview of key informants were employed.Two significant policy shifts have been observed in government-organised ART provision. In 1996, service-based therapy for a few was replaced by a research network to support clinical assessments of antiretroviral medication in public hospitals. This decision was taken after a domestic study illustrated the unaffordable fiscal burden and inefficient use of resources in provision of ART. The numbers of treatment recipients was maintained at 2,000 per year throughout the 1990s. It was not until 2001 that a new government pledged to extend the numbers receiving the service, as part of its commitment to universal coverage. Several elements played a role in this decision: new groups of dominant actors, drug price reductions, a pro-active civil society movement, lessons from experience on treatment benefits, and global treatment advocacy. Unlike previous policy discourse, human rights, ethics and equity notions were explicitly raised to support therapy extension.In the early decision, moving from a relatively limited ART service to a research network was clearly influenced by cost-effectiveness data. But in the 2001 decision to include ART in the universal coverage package, cost-effectiveness arguments were over-ruled by other considerations. Thai ART policy was shaped by many factors, and was not a simple rational process which relied on evidence. While human immune-deficiency virus (HIV) infection is incurable, use of antiretrovirals (ARVs) is the most effective intervention to prolong patients' lives. Combination antiretroviral therapy (ART), widely introduced in developed countries since the mid-1990s, has resulted in a dramatic decline in opportunistic diseases and therefore HIV-related mortality . FurtherDespite potentially desirable outcomes, ART provision in developing countries is limited: by the end of 2005 only 20% of the global population in need had accessed to therapy . Scarce While policy decisions in the developing world's health systems have been enhanced by the increasing availability of findings from research studies , these aThis paper describes the development of the publicly-subsidised ART programme in Thailand, and examines the role of cost-effectiveness information and other sorts of evidence in policy making. It draws on a larger research study undertaken by the first author as a PhD thesis . DocumenAlthough ART had been adopted as standard care for AIDS patients in industrialised societies in the late 1990s, it remained inaccessible among most PLWHA in poor settings. In 1996 Brazil became the first developing country which provided universal coverage for ARV-based medication -13. DespGlobal agencies were key players in improving access to HIV treatment and care including ART, for example the establishment of the Joint United Nations Programme on HIV/AIDS (UNAIDS)' Drug Access Initiative in 1997; and Accelerating Access Initiative in 2000 ,16. FurtThe scale up of ARV medication in poor settings was facilitated after the instigation of the Global Fund to Fight AIDS, Tuberculosis, and Malaria (GFATM) in 2002, and the launch of WHO's policy in 2003 to get 3 million PLWHA on treatment by 2005 \u2013 the so-called 3 by 5 initiative . During HIV in Thailand moved rapidly from a concentrated epidemic in the early 1980s among high-risk groups such as male homosexuals, intravenous drug users and commercial sex workers, to a generalised epidemic in the beginning of 1990s . Owing tThe national response to the disease evolved over time, and initial efforts focused on prevention. But in 1992, a limited, public-sector ART programme was implemented, offering zidovudine (AZT) monotherapy to people living with HIV/AIDS (PLWHA) in low-income groups . The numA dramatic policy shift took place in late-2001 as the newly elected government pledged to provide free treatment to all clinically eligible people living with HIV/AIDS (PLWHA) through the recently instigated Universal Health Coverage (UC) plan . ThereafIn the following section, we analyse how far cost-effectiveness information and other contributing factors influenced policy over two periods: the replacement of limited ARV monotherapy service with the collaborative Clinical Research Network in 1996, and the adoption of universal ART policy in 2001.Within four years after AZT was introduced, evidence from developed countries suggested limited clinical benefits from single-drug regimens. By the mid 1990s, demand for therapy in Thailand had risen sharply . Policy Senior health officials noted that, in response to the study's findings, the Health Ministry had two options: to terminate ARV supply of AZT, didanosine and zalcitabine to hospitals or to improve the efficiency of the treatment programme . The shiIn this shift in policy empirical information derived from programme reviews, epidemiological projections and cost-effectiveness analyses ,25 had a'In the first years of fully subsidised ARV supply, physicians and hospital administrators were interested in HIV/AIDS care. One of the reasons was they received ARVs without any need to negotiate for more budget. ... From 1994, the MoPH's AIDS Division reached its full capacity of supply because of overwhelming requests.' [20:431]It is noteworthy that in 1994 the coverage of ART offered through the public initiative was only 6% of the population in need. Projecting the fiscal burden of providing universal access to treatment was thus compelling evidence of the unaffordability of programme costs . On the In 2001 the new government introduced, as part of pre-election promises, a reform of health care financing, instigating the universal health coverage plan (UC), which aimed to ensure equitable access to essential health services among Thai people . Given twere subsidised by the UC plan, since they anticipated that ART administration would be more cost-effective [Between February and October 2001, there were intense campaigns run by ART advocate coalitions, which included, for example 53 PLWHA organizations under the Thai Network of People Living with HIV/AIDS; the National AIDS NGO Network; Drug Study Group; the Thai AIDS Society; the Thai Lawyers Association; individual scientists from the Government Pharmaceutical Organization; experts on intellectual property laws; and HIV clinicians from medical institutes . While Nffective . DespiteAn analysis of ART policy process in this crucial period between March and November 2001 suggests that apart from the intense domestic treatment advocacy by civil society organisations, programme expansion was facilitated by several other factors discussed in greater detail in Tantivess . Among t'Cost-effectiveness is only one consideration in allocating resources to specific diseases and interventions; epidemiological, medical, political, ethical, cultural, and budgetary factors also affect such decisions.'Policy making in HIV is complex because the causes, manifestations and implications of the epidemic are multi-dimensional, involving socioeconomic, political and health factors. The policy environment has also been extremely dynamic, as knowledge has advanced since the early 1980s. Since most HIV-afflicted people are marginalised and vulnerable to deprivation, the extent to which they can access proper care and treatment largely relies on public services . To addrEconomic evaluations suggest that ART in poor countries does not offer value for money since therapy is less cost-effective than HIV prevention interventions ,33. FollOverall, it seems that policies to roll out ART in poor countries have been propelled by factors other than cost effectiveness considerations, and in spite of perceptions that treatment is often not cost effective. It is probably the case that in many low-income countries with high HIV prevalence, ART roll-out has been strongly influenced by the global discourse on expanding access to treatment, as well as significantly increased financial and technical support through different international initiatives .In this paper we suggest that the information on cost-effectiveness played a role in the first shift in policy \u2013 with the result of limiting, rather than expanding ART. However, it could be argued that the termination of service-based therapy programme was guided by the projected financial burden of the programme, rather than the cost-effectiveness information. Indeed, the, findings from economic evaluations can be used to inform resource allocation in several ways. Although the study commissioned by the MoPH in 1995 suggested that ART for adults was less cost-effective than most HIV prevention interventions, at 33,300 baht per QALY gained, the medication could have been justified. This is because when the cost per QALY gained is compared with the national ability to pay, the benchmark usually used is the cost per QALY gained should be less than three times of gross national product . Given tIn the analysis of policy, caveats are in order. This paper is based on an analysis of the policy process in the public sector, and not specifically on the attitudes of Thai policy makers towards particular techniques of resource allocation and prioritisation. Moreover, the paper focuses on ARV therapy \u2013 an intervention with unique characteristics \u2013 so the conclusions reached may not be generalisable to other policies. The medicines prescribed in ART have changed over time and while prices have fallen they remain expensive; ART is demanded by large numbers of patients and is used for an incurable disease for which prevention measures are obviously more cost-effective; ART is complicated to administer; and may cause both desirable and undesirable externalities. Finally there has been global commitment to expanding access to ARVs. These features shaped the decisions on publicly-subsidised service provision in Thailand, and may not be comparable with decision making on other health interventions. Furthermore, this study emphasises the extension of treatment delivery under the national programme organised by the Health Ministry. It excludes the adoption of ARV medication in the private sector or in the country's health benefit plans.It is noteworthy that although the pledge to include ART in the UC benefit package was made in 2001, treatment costs were only formally covered by the Plan in 2005. This was because negotiations were necessary between the MoPH's Disease Control Department, which had been responsible for treatment provision since 1992, and the UC plan managers . The traIn conclusion, the allocation of public resources to subsidise ART in Thailand has always been driven by policy considerations of resource availability and affordability. However, at certain points in the policy process, other motivations, specifically over human rights and equity, were extremely important in providing the impetus for radical change. The sustainability of ART programme in Thailand remains uncertain. The drug costs may escalate dramatically if viral resistance to the current first-line regimens develops among large proportions of the treated population, resulting in the rising demand for expensive, imported original ARV products. International and bilateral regulations on trade-related intellectual property rights protection are other threats. While the production of many new generic ARVs including indinavir, ritonavir and saquinavir, as well as other drugs in new dosage forms and fix-dose combinations  has expaThe author(s) declare that they have no competing interests."}
+{"text": "The South African Government has outlined detailed plans for antiretroviral (ART) rollout in KwaZulu-Natal Province, but has not created a plan to address treatment accessibility in rural areas in KwaZulu-Natal. Here, we calculate the distance that People Living With HIV/AIDS (PLWHA) in rural areas in KwaZulu-Natal would have to travel to receive ART. Specifically, we address the health policy question 'How far will we need to go to reach PLWHA in rural KwaZulu-Natal?'.We developed a model to quantify treatment accessibility in rural areas; the model incorporates heterogeneity in spatial location of HCFs and patient population. We defined treatment accessibility in terms of the number of PLWHA that have access to an HCF. We modeled the treatment-accessibility region (i.e. catchment area) around an HCF by using a two-dimensional function, and assumed that treatment accessibility decreases as distance from an HCF increases. Specifically, we used a distance-discounting measure of ART accessibility based upon a modified form of a two-dimensional gravity-type model. We calculated the effect on treatment accessibility of: (1) distance from an HCF, and (2) the number of HCFs.In rural areas in KwaZulu-Natal even substantially increasing the size of a small catchment area (e.g. from 1 km to 20 km) around an HCF would have a negligible impact (~2%) on increasing treatment accessibility. The percentage of PLWHA who can receive ART in rural areas in this province could be as low as ~16%. Even if individuals were willing (and able) to travel 50 km to receive ART, only ~50% of those in need would be able to access treatment. Surprisingly, we show that increasing the number of available HCFs for ART distribution ~ threefold does not lead to a threefold increase in treatment accessibility in rural KwaZulu-Natal.Our results show that many PLWHA in rural KwaZulu-Natal are unlikely to have access to ART, and that the impact of an additional 37 HCFs on treatment accessibility in rural areas would be less substantial than might be expected. There is a great length to go before we will be able to reach many PLWHA in rural areas in South Africa, and specifically in KwaZulu-Natal. Accessibility to all types of healthcare is generally inadequate in rural settings of resource-constrained countries . AntiretImplementing HIV/AIDS treatment programs in rural regions of resource-constrained countries is a realistic goal as several pilot programs have shown -5. In ruIn rural South Africa there is an overall lack of health care infrastructure, reliable statistics and adequate resources . This grFor most PLWHA living in rural areas who do not have local clinic-based ART programs, the maximum distance that they would be able (or willing) to travel for ART is not clear, but it is the primary determinant of health service utilization . Road anThe South African government has outlined detailed operational plans  for ART To quantify treatment accessibility, we first determined the total number of PLWHA that live in a catchment area of a specific size around each of the HCF (i.e. the 'demand'). We then calculated the 'effective demand' by weighting the 'demand' according to the distance that PLWHA that reside in the catchment area have to travel to reach the HCF. We assumed that accessibility to ART decreases as the distance from the HCF increases; therefore we calculated the 'effective demand' around each HCF by using a distance-discounting measure of ART accessibility based upon a modified form of a gravity-type model -16. Simif(d) = exp (-kd2), where d is distance and k is an access-scaling parameter that quantifies treatment accessibility. The access-scaling parameter is used to delineate the circumference of the catchment area; 'k' is defined such that accessibility at the circumference is only 1% of accessibility at the HCF. We used this Gaussian distribution function to calculate the probability that a PLWHA at any given distance from the HCF has access to ART relative to a PLWHA living extremely close to the HCF. The total number of PLWHA in any rural community i that has access to any of the (n)available HCFs is then calculated by di,j specifies the distance between the rural community i and the specified HCF j, and Ii specifies the number of PLWHA in community i .Our calculations show that in rural areas of KwaZulu-Natal, there is a nonlinear relationship between treatment accessibility (i.e. the percentage of PLWHA with access to ART) and the size of the catchment area. This nonlinear relationship is most apparent if 17 HCFS are used, but is also evident if all 54 HCFS are used Figure . In ruraSubstantially increasing the size of small catchment areas around HCFs in rural areas is unlikely to 'capture' a much larger population of PLWHA, because of the large distances between rural communities. Catchment areas around HCFs in KwaZulu-Natal could be as small as 5 km . Thus the percentage of PLWHA who can receive ART in rural areas in this province, if 17 HCFs are used, could be as low as ~16% (~18000/110000) Figure . Even ifThe number of HCFs that are utilized (17 versus 54) obviously also affects treatment accessibility Figure . HoweverOur results show that many PLWHA in rural KwaZulu-Natal are unlikely to have access to ART. We highly recommend that studies collect data on the distance that PLWHA are able (or willing) to travel for treatment. This will commence the facilitation of discussion and decisions on ART allocation, and would help focus goals towards enabling PLWHA to access ART. A mobile clinic that travels between remote communities to take healthcare workers and resources to locations of demand in Nigeria is an initiative that other regions could implement. If new HCFs were to be constructed to increase ART access and other basic health care needs, optimization techniques could be used to determine the most appropriate location . FollowiAlthough there is no single solution regarding how best to introduce ART into resource-constrained settings, obviously more drugs, healthcare personnel and HCFs are needed in these countries ,3, partiThe author(s) declare that they have no competing interests.DPW and SMB developed the concept and study design, and wrote and edited the manuscript. DPW conducted mathematical analyses. SMB also supervised the project.The pre-publication history for this paper can be accessed here:"}
+{"text": "The implementation of the comprehensive plan for the care, management, and treatment of HIV and AIDS in South Africa  needs toAn equitable allocation of HCFs is necessary to ensure that each individual with HIV will have an equal chance of receiving antiretroviral drugs (ARVs). We have applied their method to determine the number of ART HCFs per district in KwaZulu\u2013Natal.We first set out to assemble basic details about the KwaZulu\u2013Natal health districts, namely, population and number of hospitals and fixed and mobile clinics. Secondly, by using population data as reported in the KwaZulu\u2013Natal Department of Health 2004 annual report , and basThe national target for access to HCFs is 10,000 habitants per one fixed primary health-care (PHC) facility . A PHC fWith regard to ART, the 54 HCFs proposed by Wilson and Blower translate to 18,076 people with HIV per facility . This nuIn terms of equity, it could be argued, for instance, that the two facilities in the eThekwini district cannot be expected to provide ARVs to the estimated 319,994 individuals with HIV. In comparison, the Umziyathi, Amajuba, Umkhanyakude, Uthungulu, and Ugu districts currently have the same number of ART HCFs as eThekwini, but serve smaller populations . This reFrom our calculations, it seems that in order to achieve treatment equity for individuals with HIV in KwaZulu\u2013Natal, more ARV HCFs should be established as follows: 15 in eThekwini, four each in Umgungundlovu and Zululand, three each in Uthukela and Uthungulu, two each in Ugu and iLembe, and one each in Amajuba, Sisonke, Umzinyathi, and Umkhanyakude.As a recommendation, future rollout of ART should take into consideration the principle of equity. This will ensure that all people with HIV have equal access to ARVs from their nearest HCF. We show that by applying the Wilson\u2013Blower method, it is possible to determine the number of health-care facilities where ARVs would be equitably provided.This correspondence letter was peer reviewed."}
+{"text": "In response to our commentary  on theirOn a factual level, we believe Wilson and Blower's results were appropriately described for the purposes of our commentary. As their letter points out, they applied the operations research methods to model the allocation of antiretrovirals (ARVs) among 17 health-care centers in KwaZulu\u2013Natal, based on a hypothetical distribution of HIV/AIDS among the communities in that province. Their article characterizes this as \u201can elegant and simple theoretical framework,\u201d but they object to our concluding that it could \u201cinform policy-makers' decisions regarding the location of HIV services,\u201d since they took the treatment sites as given. Yet their article compared the alternatives of using all 54 centers in the province, at one extreme, and of using only a single treatment site (in Durban), at the other extreme; in each case, the possibility of allocating to a larger number of centers is equivalent to the creation of additional centers closer to remote groups of patients.Wilson and Blower write that geographic accessibility is improved if the number of health-care facilities is increased, and they calculated that it would be optimal if all 54 facilities in the province of KwaZulu\u2013Natal distributed the medicines, instead of just 17. We took this result to confirm the need to reach out and build capacity. We are sorry if we were mistaken in assuming that Wilson and Blower would want to see their stated objective of ensuring fair distribution applied in the real-world context of many poor countries with a high HIV burden and where fairness in ARV care cannot be achieved solely by allocating resources among the existing sites.A wider gap in perception can be seen in Wilson and Blower's repeated conflation of \u201coptimal,\u201d \u201cequal,\u201d and \u201cequitable,\u201d combined with their suggestion that decision makers who fail to apply their model must be following an \u201cad hoc approach.\u201d The central point of our commentary was that various ethical theories reach very different conclusions about what result would be optimal, and that even among those aiming to achieve the greatest equity (rather than some other optimum), many would not take equality as the measure of equity. Wilson and Blower themselves recognize that apparent equality of access (in terms of distance to treatment) needs further study to determine whether patients can in fact access treatment. We need to know whether some distances are simply too far for patients to travel for chronic care, and when distances of equal length affect access very differently because of the characteristics of particular patient populations, transportation systems, and so forth.Wilson and Blower seem unwilling to accept the notion that, in the furtherance of a rational strategy to achieve equity, some health authorities might decide, for example, to allocate a disproportionate share of ARVs to traditionally disadvantaged populations. Wilson and Blower's model could still be useful in allocating resources among the centers chosen (or established) to reach the target population, but the calculation would have to take account of more information about the centers and the population, lest assumptions about catchment areas produce a formal equality that does not translate into actual equality in access, much less into equitable access in light of all relevant factors.Plainly, we share Wilson and Blower's aim of optimizing countries' responses to the tragedy of treatable, but untreated, HIV/AIDS. Any tools that are useful to that end are welcome. But besides using models to distribute ARVs in a way that optimizes spatial equality, governments that want to achieve equity will need also to overcome nongeographic barriers to accessing treatment. These include ignorance, stigma, discrimination, and outright criminalization of vulnerable groups, as well as fees at point of service that are prohibitive for the poor. All of these are given attention within the context of the \u201c3 by 5\u201d program of the World Health Organization and the United Nations Joint Programme on HIV/AIDS, including in the guidance document on equitable access to ARV treatment cited in our commentary ."}
+{"text": "Limited literacy is common among patients with chronic conditions and is associated with poor health outcomes. We sought to determine the association between literacy and blood pressure in primary care patients with hypertension and to determine if this relationship was consistent across distinct systems of healthcare delivery.We conducted a cross-sectional study of 1224 patients with hypertension utilizing baseline data from two separate, but similar randomized controlled trials. Patients were enrolled from primary care clinics in the Veterans Affairs healthcare system (VAHS) and a university healthcare system (UHS) in Durham, North Carolina. We compared the association between literacy and the primary outcome systolic blood pressure (SBP) and secondary outcomes of diastolic blood pressure (DBP) and blood pressure (BP) control across the two different healthcare systems.th grade level comprised 38.4% of patients in the VAHS and 27.5% of the patients in the UHS. There was a significant interaction between literacy and healthcare system for SBP. In adjusted analyses, SBP for patients with limited literacy was 1.2 mmHg lower than patients with adequate literacy in the VAHS , but 6.1 mmHg higher than patients with adequate literacy in the UHS ; (p = 0.003 for test of interaction). This literacy by healthcare system interaction was not statistically significant for DBP or BP control.Patients who read below a 9The relationship between patient literacy and systolic blood pressure varied significantly across different models of healthcare delivery. The attributes of the healthcare delivery system may influence the relationship between literacy and health outcomes. Over 90 million adult Americans lack the literacy skills to effectively function in the current healthcare environment  \u2013 a numbWe examined the relationship between literacy and blood pressure (BP) in primary care patients with hypertension. Hypertension affects approximately 65 million individuals in the United States ,12 and iAlthough a great deal of research has focused on defining and understanding the relationship between literacy and health, it is less clear how to improve outcomes for patients with limited literacy. If adequate literacy is required for a patient to successfully navigate encounters with a healthcare delivery system, the impact of low literacy may be exacerbated or mitigated by the features of that system. This is supported by the finding that patients with limited literacy particularly benefit from disease management interventions that address the increased challenges of limited literacy and improve patient self-management skills ,18. HoweTo evaluate this, we examined a racially and economically diverse sample of patients who received primary care for hypertension in one of two healthcare systems in Durham, North Carolina. Patients received care in either the Veterans Affairs health system (VAHS), an integrated delivery system with affordable prescription medications for all of its enrollees , or a unWe pooled data from patient interviews performed at the time of enrollment for two separate randomized controlled trials to improve BP control. All patients provided written informed consent and both studies were approved by their respective institutional review boards. The patients who comprised our VAHS sample were all enrolled in the Veteran Study to Improve the Control of Hypertension (V-STITCH), which was conducted in three Durham VA Medical Center primary care clinics . This trUsing similar recruitment strategies and inclusion/exclusion criteria, a second sample of hypertensive individuals was enrolled in the Take Control of Your Blood pressure (TCYB) study , and comReasons for exclusion were similar between the two studies and included: spouse participating in the study; not living in a surrounding eight county catchment area; receiving kidney dialysis; recipient of an organ transplant; planning a pregnancy; hospitalization for stroke, myocardial infarction (MI), coronary artery revascularization in the prior 3 months; diagnosis of metastatic cancer or dementia; residence in a nursing home or receiving home healthcare; difficulty speaking or understanding English; or severe hearing or speech impairment. All measures reported were obtained in the baseline face-to-face interview with the research assistant except for blood pressure and insurance status which are described below. With the exception of the literacy assessment, all questions were read aloud to patients by the research assistant.Blood pressure readings were abstracted from the individuals' medical record at the time of study entry. For both studies, clinic nurses using standard automated devices systematically obtained the patient's resting seated BP prior to their visit with the primary care provider. Only 8.6% of patients had more than one BP reading available from the day of enrollment; in these cases we used the minimum systolic and diastolic for the baseline BP as we believed this would most likely be the BP reading used by the primary care provider for decision making. Our results for the adjusted analyses were unchanged when using the mean SBP rather than the minimum SBP. There was no formal review of the clinic BP measurements by study personnel for quality control. We chose to use SBP as the primary outcome for the adjusted analysis because it is both a more important risk factor for cardiovascular disease and more difficult to control than diastolic BP . BP contThe Rapid Estimate of Adult Literacy in Medicine (REALM) was used to measure literacy . PatientThe following patient demographic information was collected in both healthcare systems by patient self-report at the time of enrollment: age, gender, race, marital status, education level, presence of diabetes, and financial situation. Race was dichotomized as white or non-white; Hispanic patients were categorized as non-white. Patients were asked to categorize their highest level of education according to the following categories: 0\u20139th grade, 10\u201312th grade, some college or vocational school, or college graduate. Financial situation was assessed by asking patients to describe their current finances by one of the following four categories: 1.) enough money after paying bills for special things; 2.) enough to pay the bills, but not purchase extra things; 3.) enough money to pay bills by cutting back on things; or 4.) difficulty paying bills no matter what was done. Patients who reported either of the last two answers were categorized as inadequate income .Self-reported medication adherence was assessed using a four-item measure based on the Morisky scale . PatientHealth insurance information was collected for patients in the UHS from the Duke University Health System billing database. Patients' insurance was categorized as Medicare, Medicaid, commercial, or uninsured. Because non-VA insurance coverage has little effect on out of pocket healthcare expenses for patients in the VA , no addiDescriptive statistics were calculated for each variable and are presented by healthcare system. To test for differences between healthcare systems we used chi-square for categorical variables and Wilcoxon rank-sum for continuous variables. Multiple linear regression was performed to determine the relationship between literacy and healthcare system with the primary outcome SBP after controlling for potential confounders. We included the interaction term of literacy and health system in the model to test the hypothesis that the association between literacy and SBP may differ across healthcare systems. We adjusted for the following demographic factors that we hypothesized could be related to SBP: age, race, marital status, education level, and adequacy of income. Gender was excluded from the analysis because it was confounded with health system; 98% of the VA population was male. We included diabetic status in the model because this variable may influence the treatment goals for patients with hypertension. Medication adherence, smoking, and exercise were included based on their potential relationship with SBP. Because of the many different physician providers, we included participatory decision making score in the model as an attempt to adjust for physician level differences.In addition to the primary outcome of SBP, we evaluated the relationship between literacy and DBP using methods identical to those described above for SBP. We also examined the relationship between literacy and healthcare system on the outcome BP control using logistic regression models with and without adjustment for the covariates included in the linear regression models described above. Two sided p-values were used for all analyses with alpha set at 0.05 for rejecting the null hypothesis. All statistical analyses were performed using SAS software, version 9.1 .th grade reading level) was present in 38.4% of the VAHS sample (n = 226 patients) and 27.6% (n = 175 patients) of the UHS sample.The combined study sample consisted of 1224 patients from the VAHS (n = 588) and UHS (n = 636). Participants' mean age was 62.3 years . The sample was evenly balanced between white (52.5%) and non-white (47.2%) patients. The majority (94.5%) of non-white patients identified their race as black. Limited literacy  and study team, thereby reducing the chance of bias from differential measurement in the two healthcare systems.An alternative explanation for our findings is that there were systematic differences between the patients in the two healthcare systems that confounded our results. Gender was heavily imbalanced between the two healthcare systems and we were unable to adjust for this variable in the combined model. Also, veterans may adopt different health behaviors as a result of their training in the military that were not captured in our available patient measurements. Although we adjusted for several patient variables that may be associated with systolic blood pressure, we did not include measures of patient knowledge, health beliefs, or health status, which have previously been associated with literacy and may differ between the two patient populations ,7. In adFinally, although the relationship between literacy and blood pressure significantly differed between these two healthcare systems, further work including a larger number of representative healthcare systems would provide more definitive evidence that literacy's impact on disease outcomes varies across different systems of healthcare delivery. Further work should include more explicit measurement of the financial and organizational characteristics of healthcare delivery. Within a healthcare delivery system, there may be many factors that interact with patient literacy to influence health outcomes. Future studies with more detailed measurement of organizational characteristics are needed to both validate our findings and provide greater information about the factors that may mediate the interaction between literacy and healthcare delivery systems.Limited literacy is common in all healthcare delivery systems. This study adds to the existing literature by showing that the relationship between literacy and systolic blood pressure is complex and may differ significantly across different systems of healthcare delivery. This finding is consistent with clinical trials showing that changes in the system of care for chronic disease may influence the relationship between literacy and health outcomes ,18.Future studies should include more explicit measurement of the financial and organizational characteristics of healthcare systems to identify the features that may directly interact with literacy to influence health outcomes. Our findings should also be verified in other patient samples as well as with other diseases as the impact of literacy may change as the self-management responsibilities of the patient increase. As we move forward to improve healthcare for our most vulnerable patients, we believe that patients' reading ability and comprehension is a difficult variable to modify. Therefore, a better understanding of the circumstances under which literacy is (or more importantly is not) a contributor to poor health outcomes may be the best way to overcome its impact.The authors declare that they have no competing interests.BJP was responsible for the overall study design and manuscript draft. MKO conducted the statistical analysis and ensured data accuracy. CTT assisted in the analysis approach as well as drafting and editing the manuscript. EZO and HBB were involved in the design and implementation of the two separate clinical trials included in the analysis. Both also provided oversight and direction in the analysis, interpretation, and writing of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "A complete renin-angiotensin system (RAS) is locally expressed in the brain and fulfills important functions. Angiotensin II, the major biologically active peptide of the RAS, acts via binding to two main receptor subtypes designated AT1 and AT2. The present paper focuses on AT2 receptors, which have been reported to have neuroprotective effects on stroke, degenerative diseases, and cognitive functions. Our group has identified a family of AT2 receptor interacting proteins (ATIPs) comprising three major members  with different intracellular localization. Of interest, all ATIP members are expressed in brain tissues and carry a conserved domain able to interact with the AT2 receptor intracellular tail, suggesting a role in AT2-mediated brain functions. We summarize here current knowledge on the ATIP family of proteins, and we present new experimental evidence showing interaction defects between ATIP1 and two mutant forms of the AT2 receptor identified in cases of mental retardation. These studies point to a functional role of the AT2/ATIP1 axis in cognition. The renin-angiotensin system (RAS), a major regulator of blood pressure and cardiovascular functions, is now fully recognized as playing important roles in the brain \u20136. Amongin vivo studies tend to indicate protective effects of the AT2 receptor against stroke, Alzheimer disease, and cognitive impairment [In contrast to the ubiquitous AT1 receptor, the AT2 subtype is predominantly expressed during embryonic development and is restricted to few sites in the adult , 17, 18.pairment , 41\u201345. pairment \u201348. Howepairment \u201352, and pairment  followinpairment \u201356, shouMTUS1. This gene contains 17 coding exons encompassing more than 112 kilobases and localizes at chromosomal position 8p22 [A family of AT2 receptor-interacting proteins (ATIPs) has been identified by a two-hybrid system cloning strategy using as a bait the 52 carboxy-terminal residues of the AT2 receptor , 15. Thrion 8p22 . All thrion 8p22 , 16. Thuion 8p22 , 15, 34.ATIP1  is the first characterized member of the ATIP protein family , 15, 58.Real-time PCR analysis of ATIP transcripts in human tissues has revealed that ATIP1 is ubiquitous and the most abundant ATIP mRNA species expressed in the brain  Figure . HoweverATIP1 interacts with the C-terminal intracellular portion of the AT2 receptor , 15. Intin vitro and tumor growth in vivo, in line with tumor suppressor effects of ATIP3 reported in breast cancer [As mentioned before, ATIP3 is identical to ATIP1 in the carboxy-terminal region carrying the AT2-interacting domain; however whether this isoform indeed interacts with the AT2 receptor in living cells remains to be determined. QPCR analyses revealed that ATIP3 transcripts are expressed in all human tissues including in the central nervous system . Howevert cancer . In the brain, microtubules play essential roles by regulating neuronal differentiation, neurite outgrowth, and cell migration , 62. Altin silico observations, it is tempting to speculate that ATIP4 might be structurally organized as a transmembrane protein with a short (36 residues) N-terminal extracellular domain and an intracellular region (456 residues) able to interact with the AT2 receptor (The cDNA cloning and functional characterization of the ATIP4 isoform have not been undertaken to date. ATIP4 presents two interesting features that make it a good candidate for mediating AT2 functions in the brain. First, expression of the ATIP4 mRNA is restricted to the brain and remains undetectable in peripheral tissues  Figure . Of notereceptor . Future receptor  may suggAGTR2 mutations identified in cases of mental retardation. These data relaunch the debate on the implication of AT2 receptors in mental retardation and point to MTUS1 as an attractive target gene in human brain pathologies.Since the discovery of a new family of AT2 receptor interacting proteins in 2004, the question of whether these polypeptides may play a role in normal and/or pathological brain functions has not been addressed. Notably, all ATIP members are abundantly expressed in the brain and share the same C-terminal domain able to interact with the AT2 receptor, suggesting that each ATIP member may contribute to brain AT2 receptor functions. A functional AT2/ATIP1 axis has been previously reported to be involved in rat fetal neuron differentiation. We present here evidence that AT2/ATIP1 interactions are altered by"}
+{"text": "In Germany, clinical trials and comparative effectiveness studies in primary care are still very rare, while their usefulness has been recognised in many other countries. A network of researchers from German academic general practice has explored the reasons for this discrepancy.Based on a comprehensive literature review and expert group discussions, problem analyses as well as structural and procedural prerequisites for a better implementation of clinical trials in German primary care are presented.In Germany, basic biomedical science and technology is more reputed than clinical or health services research. Clinical trials are funded by industry or a single national programme, which is highly competitive, specialist-dominated, exclusive of pilot studies, and usually favours innovation rather than comparative effectiveness studies. Academic general practice is still not fully implemented, and existing departments are small. Most general practitioners (GPs) work in a market-based, competitive setting of small private practices, with a high case load. They have no protected time or funding for research, and mostly no research training or experience. Good Clinical Practice (GCP) training is compulsory for participation in clinical trials. The group defined three work packages to be addressed regarding clinical trials in German general practice: (1) problem analysis, and definition of (2) structural prerequisites and (3) procedural prerequisites. Structural prerequisites comprise specific support facilities for general practice-based research networks that could provide practices with a point of contact. Procedural prerequisites consist, for example, of a summary of specific relevant key measures, for example on a web platform. The platform should contain standard operating procedures (SOPs), templates, checklists and other supporting materials for researchers.All in all, our problem analyses revealed that a substantial number of barriers contribute to the low implementation of clinical research in German general practice. Some issues are deeply rooted in Germany\u2019s market-based healthcare and academic systems and traditions. However, new developments may facilitate change: recent developments in the German research landscape are encouraging. Research in general practice is fundamental to ensure patient safety and efficient patient care. Recently, there has been increasing awareness of the need for clinical trials in primary care settings, or extension of translational research projects into everyday primary care, which can provide evidence with significant impact on public health. Translating research \u2018from the ivory tower to the village green\u2019 (Chris van Weel) is essential to make research findings useful for the population, but requires sufficient infrastructure and funding opportunities -5. In thIn Germany, fundamental biomedical research and technological development are the strongholds of medical research. By comparison, clinical trials are still under-represented. This need has been recognised by stakeholders in research policy. To address this, a single national funding programme has been launched . All cliIn Germany, clinical research has been almost exclusively based in university or tertiary care hospitals to date; clinical studies in general practice remain the exception ,17-23.In 2009, a group of researchers from various academic departments of general practice interested and involved in clinical research applied to the German Research Foundation to fund a network. Their long-term aim is to facilitate the implementation of clinical trials in primary care in Germany, increasing the numbers of successful grant applications and execution of high-quality clinical trials. The network group defined three work packages to be addressed regarding clinical trials in German general practice: (1) problem analysis, and definition of (2) structural prerequisites and (3) procedural prerequisites. This paper presents the main results of the problem analysis and highlights the next steps for the network and future development of clinical research in German general practice generally.According to German Research Foundation rules, network size is limited to a maximum of 15 persons . Funding is intended to enable meetings of the group and to invite experts . Structuhttp://info.scopus.com) and by hand searching publication lists of general practice departments, details are given in [Participation in the network group was based on personal experience and expertise. The group has met face to face six times so far. In addition to the network members, external national or international experts  were involved in some meetings , or consulted individually by a member. The work packages (1) problem analysis, and definition of (2) structural prerequisites and (3) procedural prerequisites were addressed in an iterative discussion process based on the group\u2019s own expertise and knowledge as well as a comprehensive review of published and German grey literature. The group identified problem areas, which were then looked at in more detail by small working groups. Research papers published by German general practice researchers were sought in Scopus  training for any trial physician/participating general practitioner (GP) . Funding problems may be particularly acute for pragmatic studies or comparative effectiveness studies: the vast majority of clinical trials are funded by the pharmaceutical industry, either as commissioned contract research, or as investigator-initiated trials attractive to the industrial partner. They are usually run by highly specialised hospital-based clinicians. In contrast, research questions from a primary care perspective usually carry no substantial financial interest for industry partners as they often focus on the non-inferiority of less invasive and/or inexpensive interventions or comparison of established, off-patent therapies . ExampleGermany lacks a long-standing tradition of primary care research ,27. AcadAn overview of barriers for clinical trials initiated by academic general practice , identified within the structure and tradition of German universities and medical faculties, is shown below.Problems and barriers at the university level in German general practice:\u2022 Medical faculties\u2022 Research profiles of medical faculties expected to focus on a few specific research fields: little awareness and little compatibility of general practice principles and research agenda with most universities\u2019 current research priorities .\u2022 University hospitals are aiming to be profitable, opening new \u2018business areas\u2019 in a market-oriented health care system. Primary care is not perceived as a profitable research field.\u2022 University hospitals  compete with general practitioners (GPs) for some aspects of (out-) patient care. Some are aiming to establish their own primary care facilities in competition with traditional practices, resulting in a conflict of interest for academic and collaborating GPs.\u2022 Coordination centres for clinical trials need to fund themselves (after a publicly funded starting period of a few years). They usually have no experience with (research in) primary care. Good Clinical Practice (GCP) training is run as a self-funded  activity, focused on hospital-based research.\u2022 Universities/academic departments of general practice or the German College of General Practice/Family Medicine are not formally involved in vocational training of GPs, which is under the auspices of the regional chambers of physicians. Thus, research is not a part of vocational training, and awareness of primary care research as well as evidence-based medicine remains quite low . One exc\u2022 Academic departments of general practice\u2022 Usually small, mostly \u2018first generation\u2019, without a long-standing research tradition\u2022 Few trained general practice researchers \u2022 Limited experience in clinical trials\u2022 No funding or (stable) support structure for research practices/practice-based research networksMost academic departments collaborate with a number of general practices in their area, which are contracted to receive and teach undergraduate students. These teaching GPs are not considered university staff; they receive a small service-based fee for teaching but not for train-the-trainer activities. Training and supervision is usually provided by the local academic department of general practice , and the German College of General Practitioners and Family Physicians. While many of these teaching GPs are willing to participate in research, there is no financial or organisational support for general practice-based research networks.The small size of the academic departments makes clinical trials particularly challenging: due to a two-stage reviewing procedure in the federal funding programme, the time lag between submission of the initial and main proposals and the receipt of funding is at least a year. Study centre recruitment must be successfully completed before a proposal is considered in the second review round. This is very challenging in primary care, where study centres represent participating general practices run by individually motivated GPs. For most general practice-based studies, a relatively high number of practices/centres must be recruited to accommodate the relatively low incidence/prevalence of most conditions in a non-selected practice population. In order to conduct a clinical drug trial, all research staff as well as all participating GPs must be formally GCP trained by an accredited centre. Depending on the region of Germany, up to 16 hours of compulsory formal GCP training is required, constituting a considerable commitment for busy GPs. Another barrier is the high cost of the training, which adds to the trial costs.Other structural barriers relate to the German health care system: there are no formal practice lists (as patients do not need to register with a practice), and GPs are not gatekeepers to specialist care  German GFurther to addressing the obstacles described above, facilitating clinical trials in German general practice will also require some fundamental change. The funding environment to support trials needs to be improved and should include provision for pragmatic or comparative effectiveness trials. This is a political goal, which cannot be achieved by a small group or within a short time.Further implementation and upgrading of academic departments of general practice would enable the recruitment, training and retention of qualified researchers and research staff beyond single projects. This would facilitate proposals for and the conduction of clinical trials in primary care. Some structural or organisational support for general practice-based research networks could provide practices with a point of contact, information, training facilities as well as support with recruitment and inclusion phase of a trial. GPs and practice staff will need professional recognition and financial compensation for their training and the time invested in research. A very recent survey on attitudes of German GPs towards participation in clinical trials has been published elsewhere .A lot of support facilities and technology (including information technology), operating instructions and predefined standard procedures exist for clinical trials in hospital settings or specialised trial clinics. However, most are setting-specific and cannot be conveyed to the challenge of running a clinical trial in primary care simultaneously with patient care in a busy general practice . Peer suOur problem analysis revealed that a substantial number of barriers contribute to the low implementation of clinical research in German general practice. Some issues are deeply rooted in Germany\u2019s health care and academic systems and traditions. However, new developments may facilitate change: the 2010 modification to German drug law emphasises the need for comparative effectiveness studies: within three months the majority of newly licensed drugs must be assessed for superiority compared to the current standard treatment for the particular condition ,38. PricNevertheless, interest in general practice and general practice research is growing , fuelledSome academic departments of general practice and participants of the network have published clinical studies that have received international attention ,19,21,43This problem analysis results from the work of a small network group with very limited, temporary funding. Our review of the international literature was conducted comprehensively and cannot be considered systematic, as relevant papers are not always easily identified due to often inconsistent or incomplete MeSH labeling . HoweverThe first requirement of establishing inventories, providing materials, engaging practices interested in research and networking, and providing information and peer support has the potential to improve the quality and success of grant applications. The network group aims to further stimulate the development of additional research capacity and research strategies for clinical trials in general practice, and to be considered a research group eligible for  funding by the German Research Foundation.Ultimately, the network group aims to underpin cooperation with coordination centres and funding bodies by, for example, defining accreditation standards and rewards for participating practices. It could also act as a \u2018clearing house\u2019, reviewing study proposals and protocols, providing access to practices and facilitating bottom-up communication of research needs in primary care to funders and policy-making bodies .Successful conduction of clinical trials in general practice settings can be hampered by characteristics of a market-based health care system, insufficient research capacity and funding, and unfulfilled need for structural support and established facilitative procedures. Though we identified barriers related to the low implementation of clinical trials in Germany, many of these, as well as the prerequisites, may apply to other countries. Increasing recognition of the importance of primary care and its role in comparative effectiveness studies or translational research may facilitate new developments. In Germany, recent developments in the research landscape are encouraging.CRFs: case report forms; GCP: Good Clinical Practice; GPs: general practitioners; PBRNs: practice-based research networks; SOPs: standard operating procedures; US: United States of America; UK: United Kingdom.The authors declare that they have no competing interests.EH-P and JB initiated the network. EH-P is the network\u2019s speaker, she wrote the article, with input from all other authors. JB wrote the funding proposal , she is the network manager. JB, GS, JFC and MS revised the draft on several occasions, AA, AB, and SJ contributed to the paper in several working sessions and correction rounds. All network members participated in the meetings and group discussions which provided the content for article. EH-P, JB and MS determined the final structure of the paper, all authors agreed to this version.EHP, AA, JFC, MS, are professors of general practice and heads of the departments of general practice at their respective universities . AB is professor of general practice in Marburg. GS, JB, and SJ are general practitioners and senior research fellows. Further members of the network are Angela Buchholz , Ildik\u00f3 G\u00e1gyor , J\u00f6rg Haasenritter  Frank Peters-Klimm , Michael M Kochen , Antonius Schneider  and Wilhelm Niebling ."}
+{"text": "Limb injuries comprise 50-60% of U.S. Service member\u2019s casualties of wars in Afghanistan and Iraq. Combat-related vascular injuries are present in 12% of this cohort, a rate 5 times higher than in prior wars. Improvements in medical and surgical trauma care, including initial in-theatre limb salvage approaches (IILS) have resulted in improved survival and fewer amputations, however, the long-term outcomes such as morbidity, functional decline, and risk for late amputation of salvaged limbs using current process of care have not been studied. The long-term care of these injured warfighters poses a significant challenge to the Department of Defense (DoD) and Department of Veterans Affairs (VA).The VA Vascular Injury Study (VAVIS): VA-DoD Extremity Injury Outcomes Collaborative, funded by the VA, Health Services Research and Development Service, is a longitudinal cohort study of Veterans with vascular extremity injuries. Enrollment will begin April, 2015 and continue for 3\u00a0years. Individuals with a validated extremity vascular injury in the Department of Defense Trauma Registry will be contacted and will complete a set of validated demographic, social, behavioral, and functional status measures during interview and online/ mailed survey. Primary outcome measures will: 1) Compare injury, demographic and geospatial characteristics of patients with IILS and identify late vascular surgery related limb complications and health care utilization in Veterans receiving VA vs. non-VA care, 2) Characterize the preventive services received by individuals with vascular repair and related outcomes, and 3) Describe patient-reported functional outcomes in Veterans with traumatic vascular limb injuries.This study will provide key information about the current process of care for Active Duty Service members and Veterans with polytrauma/vascular injuries at risk for persistent morbidity and late amputation. The results of this study will be the first step for clinicians in VA and military settings to generate evidence-based treatment and care approaches to these injuries. It will identify areas where rehabilitation medicine and vascular specialty care or telehealth options are needed to allow for better planning, resource utilization, and improved DoD-to-VA care transitions. Hughes in Korea ) studies in addition to interview and survey data collected during the course of this study.Our denominator includes all individuals verified as having undergone initial in-theatre limb salvage (IILS) during the GWOT VI study. Inclusion criteria for IILS included: 1) received a vascular repair on the affected limb(s) and 2) transferred to the post-anesthesia unit and to the ICU after surgery. Individuals with amputations performed as the first, in-theatre operation are excluded.To date, DoD chart abstractions using the Department of Defense Trauma Registry (DoDTR) have identified 1,363 individuals with confirmed extremity vascular injury and initial limb salvage; approximately 1,266 of these have separated from active service , linking files from each source using the unique identifier. Once VA patients in the confirmed IILS cohort are identified, we will conduct medical chart abstraction of VA electronic medical records, and conduct a patient interview and survey to obtain patient reported outcomes. The study will start on April 1, 2015 and will be open for three years.Data abstracted from the DoD medical chart in the GWOT VI study will be used in this study as control variables, or characteristics for stratification based on expert consensus of our clinical team. Table\u00a0We will access the electronic medical record for any VA patient with IILS from the time of VA entry to the time of abstraction, with special attention to functional outcomes including physical therapy, prosthetic care, and care for vascular injuries or conditions such as skin ulcers that may result from graft failure or complications of the initial injury. After development of the electronic data collection template, database, and training, the PI and the chart abstractors will independently review 10 charts and meet together to identify inconsistencies, errors, or disagreements and resolve any by consensus. This will continue until there is 90% agreement on all items abstracted.After obtaining the appropriate IRB approvals, HIPAA, and OMB waivers we will proceed with the interview and survey component using a modification of the Dillman method . During For the online/mail survey, one week after the interview, the research team will send a letter thanking those who have responded and remind non-respondents to reply, followed 2\u00a0weeks later by a second mailed invitation to non-respondents to complete the on-line survey (or mailed back-up). We will provide a gift card to participants who complete the mailed surveys.Short Musculoskeletal Function Assessment. Additional instruments include the Paffenbarger Physical Activity Questionnaire, Chronic Pain Grade Scale, and the Military to Civilian Questionnaire MC2-Q (a measure of community re-integration). Short Musculoskeletal Function Assessment (SMFA) is a validated, 46-item self-reported health status questionnaire on extremity functional status. The SMFA contains two parts: the dysfunction index and the quality-of-life limitation index. The SMFA questionnaire has excellent internal consistency and stability, with most values greater than 0.90. Content validity for the dysfunction and bother indexes was supported with few ceiling effects (less than 5 percent), and no floor effects. Moreover, significant correlations were found between the SMFA dysfunction and other indexes and the physicians\u2019 ratings of patient function  and clinical measures including grip strength and walking speed [rho\u2009\u2265\u20090.40] [The primary survey outcome measure for this study is one used for the GWOT VI: the \u2009\u2265\u20090.40] .The Paffenbarger Physical Activity Questionnaire). We will calculate descriptive statistics for all IILS patients\u2019 demographic characteristics, long-term clinical outcomes , and healthcare utilization (note that long-term use of opioid therapy will be assessed only among VA patients). We will also compare these descriptive statistics between those who received VA vs. non-VA care using chi-square tests for categorical variables and t-tests for continuous variables (with appropriate Box-Cox power transformation if necessary).To address the first aim, we will compare injury, demographic and geospatial characteristics of patients IILS and identify late vascular surgery related limb complications and health care utilization in Veterans receiving VA vs. non-VA care using merged VA\u2009+\u2009VA-Medicare and DoD study data. We will identify vascular procedures performed, limb salvage survival time, and indicators of persistent morbidity over time , and the percent patency each year for individuals without amputation. We will then examine the relationship between percent patency and preventive care using random effects analyses that models the vascular repair patency as a linear function of the indicator of receiving antiplatelet agents, statins, duplex ultrasound , 52, andWe will also conduct generalizability analysis using the inverse propensity probability weight technique. , 54 The Based on preliminary data from the TRACC study, we anticipate that the proportion of OEF/OIF/OND VA patients with ILLS will receive antiplatelet agents/statins/or yearly duplex ultrasound will be no greater than 44.7%. We also assume that the mean vascular repair patency falls between 50%-60%, and that the increased patency due to receiving antiplatelet agents/statins/or yearly duplex ultrasound is no less than 12% (conservatively). Under these conditions, the study will have \u22650.84 power to determine if Vascular Preventive Care processes are associated with positive patient outcomes.To address the third aim  we will begin by examining descriptive statistics associated with SMFA, PPFA, CPG scale, SF-8\u2122, and M2C-Q). We will compare scores on these instrument among IILS patients with and without indicators of persistent morbidity, controlling for possible confounders associated with having indicators of persistent morbidity (from Aim 1) including CES-D, and PCL-M. We will then conduct separate generalized estimation equation analyses for each outcome, modeling the mean of each outcome as a linear function of the indicator of having one or more indicators of persistent morbidity and other covariates .Secondary analyses will stratify individuals by initial injury severity type  to further control for initial conditions that may be more difficult to control for in multivariable analyses. We will also examine the role of various prosthetic devices, including the extent to which the technology of the device, the number of different prosthetic devices per limb, etc. mediate the relationship between injury severity, persistent morbidity and functional outcomes. Furthermore, we will conduct generalizability analysis (described above) to examine whether the outcome model derived from the VA patients can be generalized to the non-VA patients.Our power analysis for the third aim is based on the primary outcome: SMFA. Based on preliminary data, we assume that the proportion of patients with persistent morbidity falls between 10% and 50%. We also assume that the effect size associated with persistent morbidity on SMFA, PPFA, CPG scale, SF-8\u2122, and M2C-Q no less than 0.435. Under those conditions, the study will have >0.80 power to identify a true difference in outcomes among those with and without persistent morbidity.This study will provide key information about the current process of care for OEF/OIF/OND Veterans with polytrauma/vascular injuries at risk for persistent morbidity and late amputation. The results of this study will be the first step for clinicians in VA and military settings to generate evidence-based treatment and care approaches to these injuries. It will identify areas where rehabilitation medicine and vascular specialty care or telehealth options are needed allowing for better planning, resource utilization, and improved DoD-to-VA care transitions. Using the DoDTR to identify injured Service members and a DoD-VA research team to determine the long term outcome of traumatic vascular injury repairs provides an unprecedented opportunity to define current processes of care and improve the evidence base for further treatment. In addition, methods used in VAVIS can be applied to other health care problems encountered in combat casualty care to determine long term outcomes. These studies have the potential to improve battlefield care, transitions of care between the DoD and VA, as well as to determine care teams within the VA to optimally treat these complex patients."}
+{"text": "Foodborne-associated bacteria with increased incidence of antimicrobial drug resistance were the most common cause. Salmonella spp., Shigella/enteroinvasive Escherichia coli, and enterotoxigenic E. coli were the main pathogens associated with severe symptoms. We identified genes associated with resistance to third-generation cephalosporins \u224840% of Salmonella- and E. coli\u2013positive samples. Hajj-associated foodborne infections pose a major public health risk through the emergence and transmission of antimicrobial drug\u2013resistant bacteria.Hajj, the annual Muslim pilgrimage to Mecca, Saudi Arabia, is a unique mass gathering event that raises public health concerns in the host country and globally. Although gastroenteritis and diarrhea are common among Hajj pilgrims, the microbial etiologies of these infections are unknown. We collected 544 fecal samples from pilgrims with medically attended diarrheal illness from 40 countries during the 2011\u20132013 Hajj seasons and screened the samples for 16 pathogens commonly associated with diarrheal infections. Bacteria were the main agents detected, in 82.9% of the 228 positive samples, followed by viral (6.1%) and parasitic (5.3%) agents.  Hajj, the annual pilgrimage by Muslims to Mecca, Saudi Arabia, is a unique mass gathering event in terms of scale , diversity of the pilgrims, nature of the activities performed, and regularity. Approximately 2 million pilgrims from 185 countries, in addition to hundreds of thousands of residents of Saudi Arabia, travel to holy sites in Mecca each year ; viruses ; and parasites  . The Institutional Biosafety and Ethics Committee of King Abdullah University of Science and Technology also approved the study in 2013.We conducted the study for 3 successive Hajj seasons, starting in 2011. Fecal samples from pilgrims having medically attended diarrhea while performing Hajj were collected. Healthcare facilities distributed along the Hajj sites were enrolled in the study.>3 unformed stools in a 24-hour period or passing stool more frequently than normal for the patient, accompanied with >1 other gastrointestinal symptom . Patients who had unformed stool with visible blood were defined as having cases of dysentery. Patients with increased body temperature were categorized as having either mild (>37.5\u00b0C and <39\u00b0C) or severe (>39\u00b0C) fever.We included patients with symptoms who were seeking medical care for diarrhea or who were admitted to hospitals or primary care centers established in the holy sites during the 7\u201310 day Hajj period. We defined diarrhea as the occurrence of >6 unformed stools per day; diarrhea requiring hospitalization; or diarrhea accompanied by fever, dehydration, or bloody or mucoid stools. We classified patients with diarrhea not fulfilling the criteria for severe symptoms as having mild cases. We screened all the samples molecularly, antigenically, or both for a panel of 16 infectious agents commonly associated with diarrheal infection.We categorized the patients into 2 groups according to degree of symptom severity. We defined severe diarrhea as Giardia/Cryptosporidium Quik Chek test  for the detection and differentiation of Cryptosporidium oocyst antigen and Giardia cyst antigen.We used qualitative enzyme immunoassays for the initial detection of viral agents in the fecal samples according to manufacturers\u2019 instructions. We used the IDEIA Norovirus test  to detect norovirus genogroups 1 and 2 and ProSpecT tests (Oxoid) to detect of group A rotaviruses, adenoviruses, and astroviruses. For parasitic agents, we used the We used the QIAsymphony SP , an automated high-throughput platform, for the isolation and purification of total DNA from the collected fecal samples. We used the QIAsymphony DNA 800 complex kit (QIAGEN) to extract DNA from 800 \u03bcL of pretreated diluted samples according to the manufacturer\u2019s instructions.eae and bfpA (enteropathogenic E. coli), aggR (enteroaggregative E. coli) and Vero cytotoxin (enterohemorrhagic E. coli). The M2 multiplex PCR used primers targeting the genes elt and st (enterotoxigenic E. coli [ETEC]), daaE (diffusely adherent E. coli), and virF and ipaH (Shigella spp./enteroinvasive E. coli [EIEC]). The M3 multiplex PCR used primers targeting the hipO gene (Campylobacter jejuni), internal transcribed spacer region , Yersinia stable toxin gene (Yersinia enterocolitica) and rtxA gene (Vibrio cholerae). Primer details and the expected PCR fragment sizes are provided  in parallel to detect the bacterial pathogens commonly associated with diarrheal infections (provided Table 1.http://www.mbio.ncsu.edu/bioedit/page2.html) to trim and align bidirectional sequence reads and used the consensus sequences to identify the viral genotype. We identified rotavirus genotypes by using RotaC version 2.0 software  to extract viral RNA from antigenically positive samples for rotavirus, norovirus, and astrovirus according to the manufacturer\u2019s instruction. We performed reverse transcription by using the SuperScript III First-Strand Synthesis System  and PCR amplification by using Platinum Taq DNA Polymerase High Fidelity  and previously described primers for the detection of rotavirus Table 2.Enterobacteriaceae species for the detection of \u03b2-lactamase genes  as previously described  patient age was 40.17 (+13.17) years. By Hajj season, median age was 40 (+12.25) years in 2011, 40 (+13.25) in 2012, and 40.5 (+14) years in 2013 (During 3 consecutive Hajj seasons 2011\u20132013), we collected 544 fecal samples from pilgrims who had diarrhea while performing Hajj and who sought treatment at healthcare facilities , 2. Thes011\u20132013, in 2013 . Most pa in 2013 . We summarized the distribution of the clinical features among the patients during the 3 Hajj seasons . Most pa>1 of the pathogens screened for in 41.91% (n = 228) of the samples. We observed no significant difference between the numbers of positive samples during the 3 seasons . The percentages of positive samples detected were 43.22% (n = 51) for 2011, 40.40% (n = 120) for 2012, and 44.19% (n = 57) for 2013. Bacterial pathogens were the predominant infectious agents detected for the 3 Hajj seasons and the agents identified in 34.74% (n = 189) of the total samples, followed by viral  and parasitic  agents. Thirteen patients  had samples testing positive for >1 pathogen. We observed no significant difference in the distribution of infectious agents across the 3 seasons .We screened the 544 fecal samples collected from the patients during the 2011\u20132013 Hajj seasons for 16 infectious agents, including bacteria, viruses, and parasites commonly associated with diarrheal infections. We calculated the number of the samples tested and the number and percentage of the positive samples from each season . We dete2\u00a0= 8.59; p = 0.2).We calculated the distribution of patients by age group and the enteric pathogens identified  of the bacteria-positive samples. Of the serovars tested, ETEC was the most common, detected in 25.4% (n = 48) of the positive samples, followed by enteropathogenic E. coli , enteroaggregative E. coli , diffusely adherent E. coli , and enterohemorrhagic E. coli . We detected Salmonella spp. in 32.80% (n = 62) and Shigella/EIEC in 21.69% (n = 41) of the bacteria-positive samples. We observed significant differences in the distribution of bacterial pathogens across the 3 Hajj seasons  and among the different age groups .We also calculated the distribution of the bacterial agents associated with the diarrheal patients during 2011\u20132013 Hajj seasons by age group , panel CWe calculated the distribution of the viral and parasitic agents associated with diarrheal infections of pilgrims during the 2011\u20132013 Hajj seasons . ScreeniGiardia spp. was the most common parasitic agent, identified in 83.33% (n = 10) of the parasite-positive samples, followed by Cryptosporidium spp. in 16.66% (n = 2) of the samples. We isolated Giardia spp. from patients originating from 10 countries: 4 from Pakistan, 3 from Nigeria, 2 from Bangladesh, and 1 each from Ethiopia, Somalia, Egypt, Jordan, Niger, India, and Afghanistan. We identified Cryptosporidium spp. in 2 children (<5 years of age) from Saudi Arabia and 1 older pilgrim (65 years of age) from Chad  and ETEC (n = 16/48). This finding suggests that 40.32% of Salmonella infections and 33.33% ETEC infections associated with the Hajj might be resistant to at least some third-generation cephalosporins, and this number might be growing with successive seasons.We calculated the distribution of \u03b2-lactamase genes among the identified bacterial samples . blaCTX-Salmonella spp., , noroviruses (3.5%), astroviruses (1.9%), and adenoviruses (1.4%)  have all been identified by the World Health Organization as being among the top 9 bacteria likely to have a serious impact on global public health (blaCTX-M-15) and carbapenemase  genes in \u224840% of Salmonella spp. and E. coli\u2013positive samples collected.The 3 most commonly identified bacteria in our study (Enterobacteriaceae (ESBL-PE) but not carbapenemase-producing Enterobacteriaceae (CPE), with the rate of acquisition varying by destination , and understand the dynamics of disease transmission. In addition, active surveillance for enteric diseases is needed to define the potential impact of Hajj on the baseline status of enteric infections in residents of Saudi Arabia and to investigate foodborne outbreaks of disease in a timely manner.Description of primers used and additional characteristics of enteric infections associated with travel for Hajj, 2011\u20132013."}
+{"text": "However, progress in this direction is hindered by a lack of control of the kinetics and microstructure due to inherent complexity arising from competing energy and topology. We have studied thermal and electrokinetic effects on disclinations in a three-dimensional nonabsorbing nematic material with a positive and negative sign of the dielectric anisotropy. The electric flux lines are highly nonuniform in uniaxial media after an electric field below the Fr\u00e9edericksz threshold is switched on, and the kinetics of the disclination lines is slowed down. In biaxial media, depending on the sign of the dielectric anisotropy, apart from the slowing down of the disclination kinetics, a nonuniform electric field filters out disclinations of different topology by inducing a kinetic asymmetry. These results enhance the current understanding of forced disclination networks and establish the presented method, which we call fluctuating electronematics, as a potentially useful tool for designing materials with novel properties  NLC phases display rich birefringence under a polarizing microscope during phase ordering from a disordered state after a rapid quench in pressure or temperature, resulting in the formation of disclinations with integer and fractional topological charge. These singularities proliferate after nucleation and form contractile loops after intercommutation12. Unlike dislocations, disclinations possess intricate kinetics, microstructure, and equivalence with an electric charge. Strings are charge neutral with either topological charge \u00b11 or \u00b11/2 residing at the two segments or end points of the string to form topological dipoles. Higher multipoles and integer charged dipoles also nucleate within the charge neutral strings at the early stage of kinetics. Subsequently, these structures rupture into fractionally charged dipolar strings. Similar to electrodynamics, like topological charges repel and unlike charges attract and annihilate in pairs while monopoles are nonexistent to retain charge neutrality unless created by symmetry-breaking boundaries, an inclusion of impurity or external drive with a laser beam13.Topological singularities such as points, lines and walls are ubiquitous in phases with broken symmetry. Canonical examples include dislocations in solids12. Strings in uniaxial NLC displayed in Fig.\u00a0\u03c01 in the projective plane 12. After a theoretical proposal14, \u03c0 solitons have been seen in fluorescence confocal polarized-light microscopy of pentylcyanobiphenyl (5CB) NLC7, molecular simulations15 and field theoretic computations16. Likewise, biaxial disclinations displayed in Fig.\u00a0Cx,y,z. In monolayered thin films, the simultaneous and pairwise coexistence of fractionally charged point dipoles of either class Cx,y, Cy,z or Cx,z is predicted17 and observed in field theoretic computations18. Albeit topologically proscribed in three dimensions, strings of disparate topology do not entangle but annihilate pairwise within the respective class16.Existence, classification and recombination rules of disclinations in equilibrium, which play an important role in the material design, is governed by the energy landscape as well as the geometry (topology) of the order parameter space13, and, how the anisotropy of the nematic orientation embedded in the dielectric tensor leads to nonuniformity in the local electric field. For example, nematic regions at the top of a colloidal inclusion are generated due to the asymmetric distribution of the field intensity19. Such control is hard to characterize in experiments, impossible in nanoscale molecular simulations and limited in field theoretic calculations due to numerical complexity, as ref.19. mentions, \u201cmolecular alignment in the inhomogeneous electric field has not yet been well studied as it is not easy to solve the Poisson equation with an inhomogeneous dielectric constant to calculate the local electric field\u201d. Attributing to the scale invariant property of the Ginzburg-Landau-de Gennes (GLdG) field theory, relaxational kinetics of the orientation tensor has quantitatively reproduced experiments in silico from mesoscale20 to nanoscale21. State of the art grand challenge is attributed to the nonavailability of a robust numerical scheme22 which is, in descending order of complexity, (a) free from numerical artifacts of the traditional methods23, accounts for (b) local nonuniformity in electric field and (c) equilibrium thermal fluctuations by respecting physical laws, (d) guarantees zero-trace property of the orientation tensor and (e) incorporates anisotropic elasticity to probe beyond the single diffusion (one elastic constant) approximation23. Recent advances in fluctuating hydrodynamics of isotropic suspensions24 incorporating point (a) demand a natural, yet challenging, extension for anisotropic suspensions25 while on the other hand, numerical achievement of points (c\u2013e) is fairly recent26.Depending on the anisotropic elastic constants of the medium, soft disclinations are vulnerable to thermal fluctuations and external stimulus like an electromagnetic field. Regulated by the sign of the dielectric anisotropy constant of the material, an electric field at the Fr\u00e9edericksz threshold can orient the nematic director along or perpendicular to the field direction. It is particularly interesting to examine whether locally uniform and nonuniform electric field can lead to a time-dilated kinetics of the disclination network27, in this article, we have developed a fluctuating electronematics method based on the thermal description of the GLdG theory, with the physical control over the role of each forcing to accurately describe thermal and electrokinetic phenomena in three dimensional NLC media. Using simple analytical argument, we provide an understanding of the underlying mechanism responsible for the outcome in both uniaxial and biaxial media in the free draining limit at moderate to small electric field intensity, where the advective flow of the anisotropic media can be neglected. The interdependency between the topology in the orientational order of the media and morphology of the external field is elucidated through the measurement of disclination kinetics and morphology. It turns out that small magnitude of nonuniform electric field can significantly dilate the coarsening kinetics of disclination network and can eradicate certain topological class of disclinations in biaxial media.By investigating beyond the uniform field assumptionkBT, E) on the kinetics and microstructure of the string disclination assembly.Here we systematically probe on the role played by the elasticity of the medium and various external forcing  we show a portion of the three-dimensional volume that distinguishes the disclination kinetics between the athermal and thermal scenario. In the athermal scenario, three different regime with marked exponents emerges out in the evolution process, that had been previously quantified as diffusive regime\u2009\u2192\u2009Porod\u2019s law regime\u2009\u2192\u2009diffusive regime16. The early diffusive regime corresponds to domain coarsening before nucleation of disclinations (t\u2009=\u20090.84\u2009ms), while Porod\u2019s law scale designates the defect annihilation kinetics (t = 1.4\u2009ms) and finally, the late stage diffusion is attributed to contraction of isolated loops (t\u2009=\u200911\u2009ms). Clearly, thermal fluctuations tend to increase the disclination surface density without affecting the scaling laws, that is important for materials (e.g. PAA) having transition temperature way above the room temperature. Fluid viscosity \u03b7 can be obtained from the Stokes-Einstein relation kBT/K\u03b7\u2009=\u2009constant. However, frame c shows that disclination density in the athermal media not only increases for an increase in \u03ba, but also stretches the Porod\u2019s law regime. The slope changes from 0.8 to 1 as elastic anisotropy is increased. While the slope of unity is also obtained in experiments with 5CB29, we re-establish our earlier claim20 about the crucial contribution of the anisotropic elasticity of the medium. Intuitively, anisotropic elastic constant results into asymmetric diffusion constants in Cartesian directions and thus brings asymmetry in the speed of \u00b11/2 integer point defects30. The loss of area in forming a contractile loop is greatly reduced for higher anisotropy.The disclination kinetics is immensely influenced by external agents like thermal and electric forces, anisotropic elastic effects or shear. To illustrate the role of thermal fluctuations, we estimate the decay of disclination density per unit area in the degenerate elastic constant approximation without an electric field (equation ). Using t, the disclination surface density \u03c1 is found7 to scale as \u03c1 \u221d t\u22121. An identical scaling law is obtained for a planar disclination when equating the rate of change of the line tension per unit volume with the energy density loss rate1. Our method accurately reproduces the slope of 1\u2009\u00b1\u200910\u22123 within the errorbar shown in Fig.\u00a0CB. Thermal fluctuations and elastic anisotropy lead to an increment in the disclination surface density at a given time and the period of disclination annihilation kinetics is extended without affecting the physical laws.Before we embark on discussing coarsening in the presence of an electric field, we revisit the coarsening kinetics when no electric field is present. As seen in Supplementary Movie\u00a0ton when only disclinations with dipolar charge \u00b11/2 are present, and, the medium is free of \u00b11 dipolar strings or point charges. The switch-off time of the field is set at an instant toff when the electric flux lines within the medium do not change substantially. For a shallow quench below the supercooling line, for both signs of the dielectric anisotropy constant and below the Fr\u00e9edericksz threshold of the electric field, the orientational order is comparable in magnitude with the non-dimensionalized electric potential. Thus within the medium, the flux lines are very much distorted as seen in Fig.\u00a06 and have a little contribution in distorting the flux lines. This is attributed to the weak coupling of scalar order to the electric field, unlike the director that strongly couples to the electric field. In no switch-off scenario (toff \u2192 \u221e), the nonuniformity of the flux lines is retained in the electrically forced nematic phase devoid of disclinations around t\u223c35\u2009ms (not shown). The flux lines in Fig.\u00a0\u03c0 solitons under an intense field above the Fr\u00e9edericksz threshold, the lack of backflow in our method cannot reproduce a ceasing motility of disclinations6. Rather, they annihilate at a faster rate due to the uncompensated electric drag force. Electrokinetic effects under intense forcing, such as electroconvection or rheochaos, can be quantitatively captured only if backflow is systematically included. Unlike in colloidal suspensions24, the question of the existence of correlations between fluctuations in orientation and velocity has to be answered from experiments31 before attempting a numerical study in which backflow effects are included.Next, we elaborate on the thermal and electrokinetic effects on coarsening in uniaxial NLC after the onset and cessation of an unidirectional voltage pulse. The electric field is applied at an instant CB while twist constant is much smaller than splay or bend constants20. Also deep within the uniaxial phase where the director is aligned uniformly, Frank constant K can sufficiently define the medium elasticity26. In such situations, planar disclination energy per unit length is f\u2009=\u2009k\u03d5 + c, with 0 \u2264 \u03d5 \u2264 2\u03c0, k\u2009=\u2009\u00b11/2 being the topological charge, c a constant, defines a planar defect configuration n\u2009=\u2009. Performing the surface integral, the reduced disclination energy per unit length is\u03b7\u2202t\u03be per unit length yields contributions from (i) the equilibrium kinetics \u03bd\u2009=\u2009\u03c0\u03b50\u03b5aE2/8k\u03b7. While \u03bd is independent of the sign of k or \u03b5a, \u03bd > 0 implies of a temporal reduction of the loop extinction kinetics. Physically this can be interpreted as a reduction in speed of approach between \u00b11/2-charged topological dipole within a charge-neutral loop due to the external forcing. As observed in Fig.\u00a0We qualitatively argue about the slowing down of disclination kinetics for a planar isolated disclination loop. An in-plane estimate is valid for 5\u03b5a > 0, in Fig.\u00a0S around a planar defect for different field intensity and compare with the equilibrium scenario. The fluctuation amplitude at Sueq is reduced due to the application of an electric field, resulting in a reduction of the disclination surface density  on the kinetics and microstructure of the string disclination assembly of different homotopy class. We do not find excitingly different outcome when investigating the role of anisotropic elasticity and, thus, here we restrict ourselves in reporting results in \u03ba\u2009=\u20090 limit, in par with other investigations32.Next, we examine the role played by the isotropic elasticity of the medium and various external forcing  in morphology  >0 qualitatively explains the slowing down. For both signs of the dielectric anisotropy constant of the material, flux lines are massively distorted in the presence of disclinations . After an interval of ~5\u2009ms, a clear asymmetry between the disclination kinetics of different topology becomes evident. As shown in frames b,c,e,f,g, this results in long-lived disclinations of either class with uniform electric field lines. This is attributed to an increment (decrement) of the total free energy with a positive (negative) value of the dielectric anisotropy constant (frame h). Thus, the dielectric energy has a strong influence in selecting disclinations of the desired class as the bulk and elastic energies increase negligibly from the no field scenario. Physically, the acceleration (or retardation) in the loop extinction kinetics at the late stage can be interpreted as effective acceleration (or retardation) in speed of approach between \u00b11/2-charged topological dipole within a charge-neutral loop of different class due to the electric force. Consistent evidence of class selection is also obtained, however on a much longer timescale, for values of the electric field magnitude much smaller than the Fr\u00e9edericksz threshold value. However, the decay kinetics of disclinations of a particular homotopy class is accelerated in the presence of an intense electric field due to the absence of backflow in our model to compensate the electric drag. We expect new phenomena in experiments on thermotropic biaxial media under an intense electric field, perhaps similar to the behavior of uniaxial disclinations under an intense electric field6.To conclude, we examine the thermal and electrokinetic effects on the coarsening of biaxial NLC when the sample is rapidly cooled from a disordered phase in the presence of a steady voltage pulse. Figure\u00a037 or without23 particulate inclusion, under an intense electric field have established the coupling of orientation tensor with hydrodynamics and uniformity in the electric field, though the effect of nonuniformity in the electric field40 and the effect of thermal fluctuations are less explored. The effect of hydrodynamics is assumed to be negligible under moderate to weak electric field intensity41. We have examined the role of thermal fluctuations and nonuniformity of electric field in this limit and have shown that fluctuating electronematics is a robust tool to mimic laboratory experiments13in silico for anisotropic NLC in three dimensions. From the structure of the orientation tensor, we present a simple way to identify and classify the line defects and to compute physical quantities from the geometry of disclinations.Electrorheology of line defects, withWe have shown how the spatial uniformity in electric field is gained in approaching the Fr\u00e9edericksz limit. Apart from modifying the kinetic pathway of the coarsening of athermal disclination network, the external stimuli in terms of temperature fluctuations and local electric field essentially probe two emergent length scales: (i) interfacial correlation length between isotropic and nematic phase and (ii) radius of curvature of disclination loop. Neglecting any local heating effects due to the variation of temperature, any change in the correlation length is attributed to the disclination core size as well its geometric position within the three-dimensional volume. Although the evolution is temporally dilated compared to the no-field scenario, the external field cannot sufficiently modify the radius of curvature of isotropic disclinations - thus the lines are not stretched along the direction of the electric field, rather they retain their shape even when the director gets aligned along or perpendicular to the field direction depending on the sign of material\u2019s dielectric anisotropy. The inhomogeneity of the nematic orientation is manifest in the inherent nonuniformity of the local electric field - resulting in the highly nonuniform electric flux lines within the sample. The electric field induces a memory to the material that exhibit an elastic response and also induces a kinetic asymmetry within disclinations of the different class. On the other hand, increase in thermal fluctuations tends to increase the disclination surface density.42, metadevices43 and photonic applications44. The electric field induced kinetic asymmetry leading to the class selection of biaxial disclinations can develop into novel materials in topologically similar systems. Other than NLC, the presented work has resemblance with line defects in passive9 and active45 soft matter including conducting microwires46 and self-assembled resonators47, and thus has the potential to bring exciting applications in diverse systems.This complex interaction can be intelligently engineered to yield a fascinating outcome in a more complex scenario, for example, fractal nematic colloids1l with order m, \u2329\u22c5\u232at denotes the ensemble average at that instant t and symmetric traceless is symbolized withB2 cease to null as S attains its maximum, other moments R2 generated from the planar projection of {n, l} is also exercised to define biaxiality33. B2 is numerically less expensive in considering one less degree of freedom, nevertheless, R2 plays an important role in understanding the field-induced switching kinetics48.Instantaneous orientational order, that distinguishes between the disordered liquid state and partially ordered nematic state, is characterized by a symmetric traceless second rank tensorA,B} control the system temperature and size disparity, C > 0 preserves the boundedness and E\u2032 \u2260 0 brings the notion of biaxiality1. Higher order expansions are not required while32 (TrQ2)3/6 \u2265 (TrQ3)2. Order in equilibrium is obtained from Sueq =\u2212B/6C + (B2/36C2 \u22122A/3C)1/2; (B2)ueq\u2009=\u20090, while for a biaxial media, Sbeq is omitted for brevity.The ground state free energy including the excitations due to the spatial distortions and dielectric coupling are represented in the phenomenological Ginzburg-Landau-de Gennes (GLdG) free energy functional\u03ba\u2009=\u2009L2/L1 and \u0398\u2009=\u2009L3/L1 of the elastic constants can be mapped to the Frank constants splay, bend and twist49. For MBBA,  \u2248 1 while for 5CB, \u03ba \u2248 40,\u0398 \u2248 1 which designate nearly equal splay and bend constants for both materials (\u0398 \u2260 0) but the twist constant is an order smaller than splay or bend. This results into nucleation of integer topological charged nematic droplets in the metastable isotropic medium of 5CB, while topologically uncharged nematic droplets nucleate in the metastable medium of MBBA50.Inhomogeneities due to the excitations above the ground state is concealed within the symmetry allowed lowest order terms in \u03b5\u2009=\u2009\u03b5s\u03b4 + \u03b5aQ, where \u03b4 is the Kronecker delta, \u03b5s\u2009=\u2009Tr\u03b5/3 and \u03b5s\u2009=\u20090.74\u03b5a, where for 5CB \u03b5a\u2009=\u20095.8 at T\u2009=\u200933.65\u2009\u00b0C and \u03b5a\u2009=\u2009\u22120.7 for MBBA at T\u2009=\u200925\u2009\u00b0C52. Application of a spatially varying electric field E=\u2212\u2202\u03a8 leads to an electric displacement D\u2009=\u2009\u03b5 \u22c5 E and therefore to a dielectric (free) energy term \u03b50 being the vacuum permittivity and \u03a8 the electric potential. We characterize the intensity of the electric field with respect to the order53 and thermal energy by the nondimensional ratios \u03b51\u2009=\u2009(\u03b50\u03b5aE2/8\u03c0ASueq)1/2 and \u03b52\u2009=\u2009(\u03b50\u03b5aE2/8\u03c0kBT)1/2. We estimate the Fr\u00e9edericksz threshold to orient a director in a uniformly oriented nematic state along  an electric field in a twist geometry, calculated by minimizing the free energy for director distortion and field coupling, to be54Lx is the spacing between the electrodes.The optical dielectric permittivity tensor is related to the orientation tensor when separated into symmetric and antisymmetric part e.g. centrosymmetry breaking geometric restriction at the boundary by coverslips, patterned or chemically active walls, curvature induced polarization and the presence of free ions are not considered. For bent-core molecules, curvature induced polarization can be incorporated by adding Pf\u2009=\u2009c1\u2202 \u22c5 Q\u2009+\u2009c2\u2202\u2009\u22c5\u2009(Q\u00b7Q)\u2009+\u2009c3\u2202(TrQ2)\u2009+\u2009c4[Q\u2009\u22c5\u2009(\u2202 Q)\u2009\u2212\u2009(Q\u2009\u22c5\u2009\u2202)\u2009\u22c5\u2009Q] to the electric displacement D, where c1,\u2026,4 are coefficients55. Free ions can also be neglected by retaining n is the free ion density and e the electric charge19.In the present study, confinement effects \u0393\u2009=\u2009\u0393[\u03b4ik\u03b4jl + \u03b4il\u03b4jk\u2009\u2212\u20092\u03b4ij\u03b4kl/3] is a 4th rank tensor that maintains the symmetric-traceless property on the right-hand side of second equation \u232a\u2009=\u20090, \u2329\u03be\u03be\u232a\u2009=\u20092kBT\u0393\u03b4(x\u2009\u2212\u2009x\u2032)\u03b4(t\u2009\u2212\u2009t\u2032) and is constructed as a summation of Wiener process to keep discrete fluctuation dissipation (FDT) spectrum intact over all Fourier modes and thus to sample Gibbs distribution in thermal equilibrium26. We stress at this point that the functional derivative of D38. This legitimate the imposition of Maxwell\u2019s equation along with the Q-tensor equation. Unlike ref.40, we neglect cross-coupling between terms proportional to tQ equation and t\u03c8 equation for simplicity.When the medium is sufficiently dry so that the long ranged hydrodynamic interaction produced by the motile disclinations do not interfere the kinetics and fluid inertia plays no role - thus restricting to an overdamped relaxational kinetics without convection of momentum, the Langevin equation displaying the time evolution of the electric potential together with the orientation tensor can be written asBy substituting equation  in equatT < T*, the orientational order is small and is affected by a moderate to small magnitude of the electric force. Conversely, under an intense electric field Q-tensor equation (A significant departure from uniformity in electric flux lines is expected when \u03ba\u2009=\u2009\u0398\u2009=\u20090) to examine electrokinetic effects of point defects in switching experiments56. However, an impeccable role of backflow and elastic anisotropy is found to decipher asymmetric speed of \u00b11/2 integer defects under intense electric field30, that also leads to ceasing motility of disclinations due to the backflow6. A quantitative measure in three-dimensional media under intense electric field, albeit experimentally posed for single disclination in deep uniaxial state6, is yet to be sought by including Beris-Edwards model41 for fluid flow to the presented fluctuating electronematics model. Here instead, we focus on moderate to the small magnitude of electric forces including thermal fluctuations at temperature close and below T* where advective effects can be neglected for simplicity.The deterministic part of the equation  has been\u03bcm\u2009\u00d7\u2009160\u2009\u03bcm base and 80\u2009\u03bcm height. At equilibrium, periodic boundaries in three Cartesian directions are retained, that can be realized as a free standing anisotropic thick film from a groove. The electric potential at x\u2009=\u20090 is fixed at zero and at x\u2009=\u2009Lx is held according to the desired field magnitude with Dirichlet boundary condition and periodicity is maintained in the yz-directions. This is mimic by suspending the thick slab within two planar laser beams kept at a different potential or placing within an electrode without the notion of an easy axis.We consider a thick rectangular slab of insulating thermotropic NLC material in thermal equilibrium, with an 80\u2009Q, \u03be} tensor is by projection on a basis of five 3 \u00d7 3 matrices as57 Q\u2009=\u2009\u2211lalTl;\u03be\u2009=\u2009\u2211l\u03b6lTl , so that the kinetics is projected into the basis coefficients al. The fluctuating force in the projected basis has the property, \u2329\u03b6l\u232a\u2009=\u20090,\u2329\u03b6l\u03b6m\u232a\u2009=\u20092kBT\u0393\u03b4lm\u03b4(x \u2212 x\u2032)\u03b4(t \u2212 t\u2032). After projection, equation  as \u03be\u2009=\u2009[\u222bd3qSq2/\u222bd3qS] \u22121/2, where S\u2009=\u2009QijQij/\u222bd3qQijQij with the orientation tensor defined as 62. At equilibrium, the Ornstein-Zernicke form of the spatial correlation \u2329Q(0)Q(x)\u232a \u221d \u0393e\u03b6\u2212|x|//|x| defines the coherence length \u03b6\u2009=\u2009[{1 + 2(\u03ba + \u0398)/3}L1/A]1/2. By definition, \u03b6 determines the core size of a disclination which is distinct from the length scale \u03be that denotes the mutual separation between two strings and also the separation between \u00b11/2 integer dipoles within a shrinking disclination loop (or the radius of curvature). Thus, the length scale obtained from the disclination surface density 16. In our numerics, time, length, and energy scales are resolved by non-dimensionalizing equations (20 and strictly retaining (i) Courant-Friedrichs-Lewy condition for timestep to avoid stiffness63, (ii) \u2202S < S/\u03b6 for the validity of the GLdG method and, (iv) 16. With the chosen values of {\u03b50, \u03b5s, \u03b5a}, electric field relaxation is rapid compared to the orientational kinetics, so as a steady electric field is obtained for each step of Q-evolution. Grid size independence, numerical accuracy, and validity of physical tests were confirmed for each presented graphics and for both equations 66. Also, the finite volume method (FVM) applied to Smoluchowski equation for the orientational probability density function is yet at a preliminary stage67. On the other hand, after the advent of large scale computation, numerical solution of the athermal orientation tensor equation retaining all independent degrees of freedom is exercised through cell-dynamic scheme (CDS)18, finite difference methods (FDM)68 as well as the method of lines (MOL)59 approach to the GLdG theory. Attempts to include hydrodynamics and thermal fluctuations to the FDM via fluid particle dynamics (FPD)35 were replaced with the Lattice-Boltzmann method (LBM)69, while inclusion of thermal fluctuations retaining second-order numerical accuracy is extended as a stochastic generalization of the method of lines (SMOL)26. In this manuscript, SMOL complements the inclusion of Maxwell\u2019s equation to the existing formulation and motivates the association of hydrodynamics24 for intense electric field studies and Fourier\u2019s law of latent heat conduction70 for confined NLC systems.We finally discuss difference of our method with existing methodology in connection with the electrorheology of disclinations. Within Leslie-Ericksen (LE) theory in one dimension using free energy minimization technique, both the effect of rheologyS,B2,n,l} from the basis coefficients ai on each space point by a similarity transformation. The scalar values are colour rendered according to the indicated bars in Fig.\u00a0\u03b3 displayed in Fig.\u00a018. The angular shift of {n,l} is measured while traversing one complete loop and the charge and sign of defects are estimated using the hodograph method1. If u\u2009=\u2009 is directionally equivalent with \u2212u\u2009=\u2009 where  are polar and azimuthal angle in an arbitrary frame, then following transformation retains the centrosymmetry,Disclinations of different homotopy class are obtained after extracting {\u03b3, n rotates by \u00b1\u2009\u03c0. In biaxial NLC, homotopy classes are identified using the following recipe:18 (i) for Cx class of disclinations, n rotates by \u00b1\u03c0 but l does not rotate, (ii) for Cy class of disclinations, n does not rotate but l rotates by\u2009\u00b1\u2009\u03c0 and, (iii) for Cz class of disclinations, both {n, l} rotates by \u00b1\u03c0. We do not find any Cx class of disclinations, which is consistent with the analytic prediction on two-dimensional nonabelian vortices17 and numerical computations18. This algorithm not only supersedes the traditional defect classifying approaches using vector field, tensor glyph or hyperstreamline seeding through Mueller and Westin matrices72 but can uniquely determine disclinations from the structure of orientation tensor rather than rely on the vectorial information.For \u00b11/2 integer disclinations in a slice plane of uniaxial NLC shown in frame 1c, while traversing Supplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6"}
+{"text": "STAT3 promoter regions and verified their contribution to bovine body size traits. We first estimated the degree of conservation in STAT3, followed by measurements of its mRNA expression during fetal and adult stages of Qinchuan cattle. We then sequenced the STAT3 promoter region to determine genetic variants and evaluate their association with body size traits. From fetus to adult, STAT3 expression increased significantly in muscle, fat, heart, liver, and spleen tissues (p < 0.01), but decreased in the intestine, lung, and rumen (p < 0.01). We identified and named five single nucleotide polymorphisms (SNPs): SNP1-304A>C, SNP2-285G>A, SNP3-209A>C, SNP4-203A>G, and SNP5-188T>C. These five mutations fell significantly outside the Hardy\u2013Weinberg equilibrium (HWE)  and significantly associated with body size traits (p < 0.05). Individuals with haplotype H3H3 (CC-GG-CC-GG-CC) were larger in body size than other haplotypes. Therefore, variations in the STAT3 gene promoter regions, most notably haplotype H3H3, may benefit marker-assisted breeding of Qinchuan cattle.Signal transducer and activator of transcription 3 (STAT3) plays a critical role in leptin-mediated regulation of energy metabolism. This study investigated genetic variation in  SIRT2, MTNR1A, SIX4, MC4R and FTO, identified in cattle, pigs, and other livestock [Body size is a pleiotropic suite of traits essential to livestock genetic breeding programs. Successfully applying marker-assisted selection (MAS) in livestock depends on the identification of relevant genes or tightly linked markers . Growth ivestock ,4,5,6.STAT3 gene exhibited increased appetites, obesity, partial leptin resistance, and glucose intolerance [STAT3 regulates differentiation of brown adipose tissues (BAT), involved primarily in burning energy [STAT3 in cattle directly influence body size and carcass quality traits [Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous cytoplasmic protein expressed in multiple metabolic tissues. It is a member of the STAT protein family, characterized by the presence of Src homology domain 2 (SH2), Src homology domain 3 (SH3), and a tyrosine phosphorylation site at the carboxyl-terminal region. Various cytokines and growth factors phosphorylate STAT3 and translocate the activated protein to the cell nucleus, where it functions as a transcription factor ,8. Acetyolerance . Interesg energy . Geneticy traits .Qinchuan cattle have been popular farming and meat breeds in China for thousands of years , due to SIRT3 promoter region influences intramuscular fat deposition in beef cattle [GPAT3 promoter region are associated with pig body-size traits and promoter activity [STAT3 promoter regions should be investigated and their contribution to Qinchuan body size verified.Because STAT3 is important to almost every aspect of energy metabolism, its variants should predispose carriers to specific body-size traits. To the best of our knowledge, few studies have investigated this possibility in cattle, with most research focusing on humans and laboratory animals ,25,26,27f cattle , while Sactivity , and so STAT3 gene function. We then detected STAT3 mRNA expression at the fetal and adult stages of Qinchuan cattle. Next, we sequenced the STAT3 promoter region in 420 Qinchuan cattle to analyzed genetic variation. Finally, we tested for associations between SNPs and haplotype combinations with body size traits of Qinchuan cattle. Our results should greatly benefit MAS breeding programs.In the present study, we used bioinformatics techniques to predict We performed multiple sequence alignment on STAT3 from seven species, including common ruminants , monogastric animals , and humans . As the STAT3 mRNA expression in nine different tissues from fetal (FQC) and adult (AQC) Qinchuan cattle. Relative expression varied in all examined tissues (We determined  tissues . Among Fp < 0.01). In contrast, intestinal, lung, and ruminal STAT3 expression decreased dramatically among AQC (p < 0.01). Finally, renal STAT3 expression did not differ between FQC and AQC.We observed a significant increase in STAT3 expression of heart, liver, spleen, muscle, and fat from the fetal to adult stage . Additionally, PIC classification indicated that the five SNPs were moderately polymorphic (0.25 < PIC < 0.50).We identified five SNPs in STAT3 promoter regions: SNP1-304A>C, SNP2-285G>A, SNP3-209A>C, SNP4-203A>G, and SNP5-188T>C. Sequencing each SNP respectively yielded genotypes AA, AC, CC; GG, AG, AA; AA, AC, CC; GG, AG, AA; and TT, CT, CC . GenotypIn silico analysis indicated that alternative alleles may generate gains or losses of transcription factor binding sites. The substitution of A with C in SNP1 and SNP3, as well as the substitution of T with C in SNP 5 produced a putative gain of binding sites Sp1, MyoD, and SRF, respectively . Substitr2. The latter index is a pairwise measure of LD and less sensitive to allele frequencies than D\u2019 [r2 range was 0.052\u20130.452  , all happ < 0.05). However, body length, wither height, hip height, hip width, and chest circumference did not differ between genotypes (p > 0.05). At the SNP2 locus, AG and AA individuals had greater wither height than GG individuals (p < 0.01). At the SNP3/4 locus, genotypes AA/AA and AC/AG were significantly related to wither height and chest depth (p < 0.01). Additionally, chest depth differed significantly between these two genotypes (p < 0.05), whereas they were not associated with other body-size parameters (p > 0.05). At the SNP5 locus, TT individuals had greater hip width, hip height (p < 0.01) and chest depth (p < 0.05) than CC individuals. p < 0.01).We examined relationships between the five SNPs and body size traits in 420 Qinchuan cattle . At the STAT3 gene (24 exons) is located on chromosome 19 and is involved in leptin-mediated regulation of energy metabolism [STAT3 deletion interferes with normal body weight homeostasis and glucose metabolism, leading to obesity, diabetes, and thermal dysregulation [STAT3 polymorphisms significantly affected body size traits in Xinong Saanen dairy goats and Hainan black goats [STAT3 promoter regions are associated with body size traits in Qinchuan cattle. Thus, here we successfully identified five SNPs in bovine STAT3 promoter regions. Chi-squared tests revealed that the five SNPs were not in HWE, possibly due to artificial selection from draft cattle to beef production, or small sample size [The bovine tabolism ,15,16,17tabolism . In vitrgulation ,15. Furtgulation . Among lck goats . Identifck goats ,15,38,39ple size .p < 0.05), while SNP2 genotypes AG and AA were associated with improved wither height (p < 0.01). The SNP3/4 genotypes AA/GG and AC/AG also affected wither height and chest depth (p < 0.05), while genotype CC/AA resulted in the widest hips (p < 0.01). At SNP5, genotype TT increased hip height, hip width, wither height, and chest depth over genotype CC (p < 0.05). Our investigation of associations between haplotype combinations and body size traits revealed that H3H3 individuals differed significantly from other haplotypes (p < 0.01). These results suggested that H3H3 could see potential use as a molecular marker in future breeding programs to increase Qinchuan cattle growth rates. In our future studies, we aim to investigate how SNPs alter STAT3 promoter activity to influence body size traits.Promoters can alter QTL expression through regulating mRNA isoforms . In the In complex signaling cascades, transcription factors activate the target gene via directly binding to DNA, or else control gene expression through altering chromatin configuration . Thus, wSTAT3 can influence relevant traits through altering gene activity, leading to consequences such as increasing the risk of gastric cancer [STAT3 promoter SNPs and body size traits of Qinchuan cattle. The underlying mechanism of this link may be related to methylation-induced promoter activation. However, further research is necessary before we can fully understand how promoter methylation influences the association between STAT3 and traits of interest.Methylation of c cancer . In turnc cancer , glioma c cancer , and denc cancer . In thisBos taurus NP_001012689.2, Homo sapiens NP_644805.1, Rattus norvegicus NP_036879.1, Mus musculus NP_998824.1, Capra hircus NP_001301207.1, Ovis aries XP_014954273.1, and Sus scrofa NP_001038045.1). Multiple sequence alignment was performed in MUSCLE (MUltiple Sequence Comparison by Log-Expectation), while a neighbor-joining phylogenetic tree was constructed in MEGA version 7.0.26  [Amino acid sequences of STAT3 were acquired from NCBI for seven species  ,51.ad libitum, following based on the Nutrient Requirement of Beef Cattle .The Experiment Farm of the National Beef Cattle Improvement Center  supplied Qinchuan cattle for this study. The experiment used 420 adult females . All procedures were performed in accordance with the guidelines of the China Council on Animal Care. Protocols were also approved by the Experimental Animal Management Committee (EAMC) of Northwest A & F University . The same care protocol and the same environment were employed for cattle rearing. Subjects were fed a diet of 25% concentrate and 75% roughage (corn silage and dry straw) on a total mixed ration (TMR) basis and provided water Three fetal Qinchuan cattle  and three adult Qinchuan cattle  were randomly selected . The adults and fetuses were unrelated within the last three generations. The FQC embryos (cattle gestation: 280 days) were placed in sterile physiological saline immediately after removal from the reproductive tract of slaughtered cattle at a local abattoir. In addition, FQC age was estimated following published research .To detect transcriptome-level STAT3 expression, we collected nine tissues  at both FQC and AQC. Samples were immediately frozen in liquid nitrogen upon collection from the carcasses before being transferred to the laboratory for RNA extraction.\u03b2-actin and GAPDH were used as internal controls. Ct\u2212\u0394\u0394 method [The RNAprep Pure Tissue kit  and reverse transcription kit  were used for RNA extraction and cDNA synthesis, respectively. Real-time quantitative PCR (RT-qPCR) was performed in an Applied Biosystems thermocycler , using the SYBR Premix Ex Taq kit . Bovine t method .Blood samples for DNA extraction were collected from 420 Qinchuan cattle, aged 24\u201330 months, following published protocols . Body si2, and 0.5 U Taq DNA polymerase . Amplicons were sequenced in Sangon  to screen for variants. Thermocycling conditions were as follows: 95 \u00b0C for 5 min; 94 \u00b0C for 30 s, 63.1 \u00b0C for 35 s, and 72 \u00b0C for 40 s; 35 cycles from 94 \u00b0C to 72 \u00b0C; followed by a final extension at 72 \u00b0C for 10 min. Sequences were identified in SeqMan . Promoter positions were numbered by designating the first nucleotide of the first exon as +1 and the nucleotide immediately upstream as \u22121.Promoter regions were PCR-amplified  sequenced. The reaction mixture (20 \u03bcL) contained 50 ng DNA, 10 pM of each primer, 0.20 mM dNTP, 2.5 mM MgClhttp://gene-regulation.com/pub/databases.html).Genotypic and allelic frequencies were directly calculated for all five SNPs. The Hardy-Weinberg equilibrium (HWE) was estimated with a Chi-squared test in PopGene version 3.2  . Populatp values.Linkage disequilibrium (LD) and haplotypes were analyzed using SHEsis . The BonSTAT3 promoter regions. We conclude that this genotype could be used as a molecular marker in future breeding programs that aim to select for body size traits in Qinchuan cattle.In this study, we determined that the combined genotype H3H3 (CC-GG-CC-AA-CC) had the strongest effect on body size traits among all identified SNP variants of"}
+{"text": "To correlate acne severity with elevated androgen levels and to compare androgen levels between cases and controls.This case-control study was carried out in the Department of Dermatology, Mayo Hospital, Lahore from March 2016 \u2013 March 2017. Two hundred and seventy patients and eighty age and gender-matched controls were recruited after ethical approval and informed consent and categorized into mild, moderate and severe acne. Severity was correlated with serum Testosterone, Dihydrotestoststerone and Dihydroepiandrosterone Sulphate levels. Quantitative variables were expressed as median and percentiles, comparisons done by Mann-Whitney and correlations by Spearman correlation. P value of < 0.05 was considered statistically significant.There were 142 (41%) males and 208 (59%) females. Ninety-Seven patients had mild, 108 moderate and 65 had severe disease. Median hormonal levels were 3.5ng/ml, 184pg/ml and 0.82ug/dl for Testosterone, Dihydrotestosterone and Dihydroepiandrosterone Sulphate respectively which differed significantly between cases and controls. There was no correlation with severity but the levels differed significantly between the different grades in case of Testosterone and DHEAS.Androgens are not directly correlated with acne severity, but affect acne severity as seen in difference between their levels in different grades of acne. Anti-androgens may be initiated early in acne resistant to conventional therapy. Acne is one of the commonest dermatological disorders with a prevalence of 22%Testosterone (T) and dihydrotestosterone (DHT) are synthesized in the skin and bind to the androgen receptor.DHEA-S was found to be in the highest concentration in both sexes, which is converted by sebocytes and dermal papilla into testosterone and DHT. Androgen receptor (AR) malfunction is associated with acne.8Acne may be associated with endocrinopathies, PCOs and ovarian tumours, if associated with hyperandrogenism. It is important to standardize the hormonal profile and control relapses in breakouts of acne. Treatment includes androgen receptor blocker, lowering androgen production by adrenals and ovaries.9The objectives of this study were to correlate acne severity with elevated androgen levels and to compare androgen levels between cases and controls.Two hundred and seventy acne patients and eighty age and gender matched controls presenting to the outpatient department of Mayo Hospital, Lahore were included. Patients were studied for hyperandrogenism. Patients having features of hyperandrogenism clinically (e.g. hirsutism) were excluded as well as those patients having a known cause of hyperandrogenism like PCOS or oral contraceptives in case of females or Cushing\u2019s syndrome (as tested on Ultrasonography and fasting serum cortisol levels). Patients presenting for the first time were selected. After approval from the ethical committee and informed consent, after ruling out polycystic ovaries on ultrasound and after basal serum cortisol, fasting androgen levels were recorded. Levels were measured by ELISA using kits by Diasource Belgium and tests performed on Diamate 310. Levels of androgens were compared between cases and controls and correlated with acne severity. All data was entered into SPSS 20. Since the distribution was non-normal, quantitative data was expressed in median and percentiles. Non-parametric tests like Mann-Whitney were done for comparison between two groups. Correlation was measured by Spearman Correlation. A P value of < 0.05 was considered statistically significant.There were 142 (40.5%) males and 208 (59.4%) females. In cases, there were 155 (57%) females and 115 (43%) males, whereas in controls, there were 54 (67%) females and 26 (33%) males. In both males and females, the median age was 20 years. Median age of cases was 20 years and median age of controls was 21 years. Ninety-Seven (36%) of patients had mild disease, 108 (40%) had moderate disease and 65 (24%) had severe disease.In the comparison of hormonal levels between cases and controls, there was a statistically significant difference  tested bIn the correlation of serum levels of hormones with severity, there was no significant correlation .In the comparison of hormonal levels between different grades of severity, there was a significant difference in case of Testosterone and DHEAS . AlthougIn this study, the median age was 20 years. In the study by Kiyani et al.4Thirty six percent patients had mild, 40% moderate and 24% had severe disease. The highest proportion comprised of patients with mild disease. This is similar to the study by Kaiyani et al.The levels of androgenic hormones are given in Marynick SP et al.In this study, there was a significant difference in androgen levels between cases and controls . In anotThere was no significant correlation of androgens with severity  with SpeAdityan BIn this study, comparison of androgens between the three grades of acne revealed significant difference in Testosterone and DHEAS levels , but notLucky AW et al.25Although androgenic hormones may be raised in the acne patients as compared to controls, only Testosterone and DHEA-S levels serve as markers of acne severity. Any therapy targeted towards acne and its severity can, therefore, be directed towards the ovarian androgen production, or blocking of androgen receptors in the pilosebaceous unit. Thus hormonal therapy can be an adjuvant to early treatment in acne.UI: Conceived, designed, and performed statistical analysis.NC: Helped in analysis and interpretation of data, final approval of the manuscript."}
+{"text": "Citrobacter freundii strain isolated from the cecum of a house sparrow that was found dead in Berlin-Lichtenberg, Germany, in 2017. This isolate exhibits increased MICs for several antimicrobials and a comprehensive set of acquired resistance determinants potentially involved in horizontal gene transfer.Here, we announce the genome of an extended-spectrum \u03b2-lactamase-producing  Citrobacter freundii strain isolated from the cecum of a house sparrow that was found dead in Berlin-Lichtenberg, Germany, in 2017. This isolate exhibits increased MICs for several antimicrobials and a comprehensive set of acquired resistance determinants potentially involved in horizontal gene transfer.Here, we announce the genome of an extended-spectrum \u03b2-lactamase-producing  Citrobacter freundii is a Gram-negative,opportunistic pathogen of the family Enterobacteriaceae. It is widely distributed in the environment and is also present in the intestine of wildlife, livestock, and humans. In the past, the pathogenic potential of this bacterium was considered low, but C. freundii is now recognized as an important nosocomial pathogen causing both superficial wound infections and life-threatening infections  C. freundii clones, especially in health care units, are major public health issues . Among all isolated C. freundii, strain 236-17-2 attracted attention because of a comprehensive antimicrobial resistance pattern, with high MIC(s) for ampicillin (>64\u2009mg/liter), chloramphenicol (>128\u2009mg/liter), ciprofloxacin (0.5\u2009mg/liter), nalidixic acid (>128\u2009mg/liter), sulfamethoxazole (>1024\u2009mg/liter), tetracycline (64\u2009mg/liter), trimethoprim (>32\u2009mg/liter), cefepime (0.5\u2009mg/liter), ertapenem (0.12\u2009mg/liter), cefotaxime (32\u2009mg/liter), cefoxitin (>64\u2009mg/liter), and ceftazidime (32\u2009mg/liter). Here, we announce the draft genome of this isolate, which was recovered from the intestine of a house sparrow (Passer domesticus) in Berlin-Lichtenberg (Germany) in 2017.Extended-spectrum \u03b2-lactamase (ESBL)-producing fotaxime . Represefotaxime  and asseDe novo genome assembly was performed using the PATRIC database (https://www.patricbrc.org/) and resulted in 36 contigs with an average sequence coverage of 15 per consensus base. The resulting genome consists of 4,973,288\u2009bp with an average G+C content of 51.51%. For initial genome annotation, the automated Prokaryotic Genome Annotation Pipeline of the NCBI database (in silico detection of the acquired resistance genes for aminoglycosides (strA and strB), beta-lactams (blaCMY-67 and blaTEM-1A), phenicols (catA1), sulfonamides (sul1), tetracyclines [tet(A)], and trimethoprim (dfrA1) using ResFinder3.0 (https://cge.cbs.dtu.dk/services/ResFinder/). Further analyses will be necessary to determine the locations of the resistance determinants on potential mobile elements  and their role in the transmission to other bacteria of the Enterobacteriaceae.Genome sequencing was performed using DNA extracted with the PureLink Genomic DNA minikit . Short-read sequencing  was conducted on a MiSeq benchtop sequencer using sequencing libraries prepared with the Nextera XT DNA sample preparation kit as previously described (C. freundii strain 236-17-2 genome was deposited in GenBank under accession number PQFB00000000.The complete genome sequence of the"}
+{"text": "The understanding and institutionalisation of the seamless link between urban critical infrastructure and disaster management has greatly helped the developed world to establish effective disaster management processes. However, this link is conspicuously missing in developing countries, where disaster management has been more reactive than proactive. The consequence of this is typified in poor response time and uncoordinated ways in which disasters and emergency situations are handled. As is the case with many Nigerian cities, the challenges of urban development in the city of Abeokuta have limited the effectiveness of disaster and emergency first responders and managers. Using geospatial techniques, the study attempted to design and deploy a spatial database running a web-based information system to track the characteristics and distribution of critical infrastructure for effective use during disaster and emergencies, with the purpose of proactively improving disaster and emergency management processes in Abeokuta. The world\u2019s contemporary urban settlements are undergoing massive and unprecedented change both in their complexity and function , failed to establish the needed link between critical infrastructure and disaster management. The works of Elias and Omojola , Samuel et al. , Anifowo , medical facilities, security and emergency response outfit , fuel stations, pipe-borne water network, financial institutions  and roads. These major sectors of critical infrastructure, their subsectors and interaction taking place amongst them were modelled in the designed database. The choice of use of these classes of critical infrastructure was based on the availability of some level of skeletal structure of the data. Because of the problems inherent in the acquisition and storage of crucial spatial data, a lot of extensive work was carried out to build up the database used for the study. Hand-held Global Positioning System receivers were used to update the spatial location of medical facilities, police stations, fire stations, road safety outposts and ATMs whilst their attribute data were obtained from interviews conducted with relevant agencies in charge of these facilities. Existing data on the other hand were obtained from the archives  of some of the agencies overseeing these facilities. For instance, water (distribution) network map and electricity network were obtained from the State Water Corporation and the Power Holding Company of Nigeria, respectively.Data acquired were structured into a spatial database that served the dual purpose of inventorying existing critical infrastructure in the study area and of being deployed to disaster and emergency management, thereby improving on the status quo. Creating the geospatial database involved two major sections of work; the first being the design and the second being the actual creation. The design phase of the geospatial database involved four stages vividly described in design of the geospatial section design of the geospatial database and summarised in This phase involves abstracting the geographic features of interest from the actual reality, and it requires a sound understanding of the reality to be modelled. However, because of the complexity of the real world, there is need for a more concise, compact and easy-to-follow representation of this complex reality , schools and markets.x and y coordinates.To vividly capture the abstracted view of reality, basic geometric and thematic components of the data sets were defined. The conceptual stage also involved a concise description of the thematic components of the reality and the semantic relationships that exist amongst the entities. In this application, the entity\u2013relationship (ER) data model, which is a high-level modelling language, was employed to map the relationships and constraints existing between identified entities in the study. In other words, the ER diagram graphically maps important semantic information about objects of interest in a database, the representation of relationship existing between the objects in reality and possible access to the database, given the constraints between the objects . That is, the representation of the logical design in the format of the implementing DBMS. This stage also involved the declaration of storage and access paths in which DBMS provides data access methods or access paths that accelerate data retrieval, query processing and optimising and concurrency or recovery, which guarantees security and consistency of the database     }Fuel_Station: {    }Transformer: {  }Powerline: {  }ATM: {    }Medicals: {    }Drainage: {   }Bridge: {  }Market: {    }Water_Pipeline: {  }Hydrants: {   }Dam: {   }Stream: {  }Security/Safety: {    }Security/Safety: {     }.A critical infrastructure information system was built from the created spatial database to track possible damages to critical infrastructure during disaster and emergency situations. The information system, which was christened Abeokuta Critical Infrastructure Information System (ACIIS), was developed as a web service using client\u2013server architecture. This implies that the system acts like a piece of software running on a client computer and makes requests to a remote server. The thick server, built using web programming techniques, hosts the bulk of the services and processes the data, whilst the thin client is the browser used to access the service. As later explained at the implementation of the geospatial database section, the database that runs the ACIIS was built using PostGIS, which was queried with spatial enabled SQL language, whilst PHP and Java were used to build the web applications. Google map API was used to present the spatial data on the browser, and Environmental System Research Institute (ESRI) web service was used as the service framework.The ACIIS application has two main panels. The first is the data frame where the data, query performed on them as well as the results of the query are displayed. Second is the query panel, where the query to be executed on the data are structured. The geospatial database was designed to keep track of critical infrastructure and other facilities in the city of Abeokuta. The database was implemented in PostGIS, an open-source, spatial database extender for PostgreSQL object-relational DBMS. The choice of PostGIS is based on the fact that aside from being available at no cost, except for Internet connection, it spatially enables the PostgresSQL server by allowing it to be used as a backend spatial database for geographic information systems. This implies that the adaptive capability of PostGIS to model spatial features, using very simple feature specification for SQL, informed its choice of use.To ensure that the data conform to completeness accuracy, the database was viewed through the PgAdmin interface  and thenExamples of spatial analyses carried out on the database included spatial query, which was carried out to have an overview of the total number of facilities under each class of critical infrastructure. z-scale of 0.03 and p value of 0.98. Also, the geographic centre of the concentration of gas stations, shown in The spatial spread of some critical infrastructure was appraised from the geospatial database. This was carried out to evaluate their locations in the event of possible emergency situations. Basic clustering analyses were performed on gas stations, substation transformers, hospitals, ATMs and fire hydrant locations. The spatial distribution of the gas stations (represented in red) is random at a z-score value of -2.58, there is less than 1% likelihood that there exists a clustered relationship amongst the electricity substations. This is explained by the fact that electricity distribution, especially the presence of a transformer, in many Nigerian cities is a pull factor for other facilities and subsequent development of areas they serve  in A fire incident was simulated to test the spatial database use in optimal route finding and closest facility analysis. For the optimal route finding, the database returned an optimal route (in green) from the nearest fire station to the fire incident location (in red) as shown in The critical infrastructure information developed was also tested to see the workability of the spatial database created in tracking critical infrastructure during disaster and emergency situations. As shown in To achieve the purpose of effective use during emergency management, results from the ACIIS could also be refined to show critical infrastructure within proximity of the initiating point of an emergency point. For instance, affected critical infrastructure and other facilities in the area could be selected by rolling the mouse over the fire or by clicking the features as shown in Working from the background of a developing country where much committed resources to disaster and emergency situations yield little results, the study attempted to model a critical infrastructure-driven spatial database that could be deployed for proactive management of disaster and emergencies. As is the case with many Nigerian cities, the challenges of urban development in Abeokuta have limited the effectiveness of disaster and emergency first responders and managers. The design and deployment of both the spatial database and the information system that runs on it is expected to proactively improve disaster and emergency management in the city of Abeokuta, and by extension, it is expected to be prototype for other urban areas in Nigeria. Nigeria is characterised by haphazard urban development, which in most cases has had serious consequences on disaster and emergency management in the country. The study dwelt on technical issues surrounding the development of a repository of geospatial data, to bridge the gaps identified in the disaster management process of urban disaster in the country. By emphasising the dynamic link between critical infrastructure, disaster and its management, the study tries to prepare the platform for proactive management of urban disaster in the country to achieve an effective and efficient time-sensitive response to extreme events and emergency situations. Although the study attempted to highlight the roles played by critical infrastructure in disaster management, the lack of up-to-date crucial spatial data sets was a major challenge. However, despite the poor organisation and coordination in the acquisition, storage and use of essential spatial data on critical infrastructure in the study area, the database designed for this study is expected to form the basis for a more detailed and integrated spatial database that could be used for proactive disaster management in Nigeria. Also, by creating the necessary awareness on the significance of critical infrastructure on disaster management in Nigeria and many other developing countries, it is strongly believed that stakeholders concerned with disaster management in developing countries would adopt the template provided by this study to maintain subsequent spatial data that would be acquired on critical infrastructure and disaster management."}
+{"text": "The European Society of Paediatric Radiology (ESPR) research committee was established to initiate, drive forward and foster excellence in paediatric imaging, paediatric image-guided intervention and radiation protection research, by facilitating more evidence-based standards, protocols and multi-institutional collaborations. The ESPR Strategic Research Agenda outlines our current research approach, highlighting several areas of paediatric imaging where the society can help guide current and future research, and emphasizing those areas where early research (\u201cseed\u201d) funding may need to be allocated by this and other societies as precursors to larger grant applications. The key aims are to evaluate normal variation in order to be able to confidently diagnose disease states, develop robust image-based classification systems to aid diagnosis and treatment monitoring, and help develop evidence-based clinical guidelines using current literature and experience to identify knowledge gaps. For this reason, the development of evidence-based imaging pipelines, broken down step-by-step to include diagnosis, classification and clinical effectiveness, should be the end goal for each disease entity for each affected child. Here, we outline the 2019 ESPR Strategic Research Agenda along three points in the clinical imaging pipeline: clinical referral, disease diagnosis and evolution, and clinical therapeutic evaluation and effectiveness. Through multicentre trials, using existing high-level experience and expertise, and nurturing the next generation of researchers, we will be able to achieve these aims. Paediatric radiology is the skill of accurate and appropriate imaging of childhood disease, a key component being safety , 2. The Whilst several countries have dedicated large children\u2019s hospitals with specialist radiology staff with paediatric skills, much of paediatric imaging is undertaken by nonspecialists. The most recent publication from the Royal College of Radiologists in the United Kingdom estimates that 75% of children\u2019s radiographs and scans are taken in smaller, nonspecialist hospitals , by radiThe importance of this child-centric and child-specific approach has been addressed in several recent publications showing a significant rate of major disagreements between interpretations of paediatric imaging studies by general radiologists and those of specialty radiologists at a tertiary care paediatric hospital , 6. NotaBy strengthening research in paediatric radiology, we will increase the impact of our subspecialty and form a research basis for the foundation for paediatric radiology clinical practice. With this in mind, the ESPR research committee was established to initiate, advance and foster excellence in paediatric imaging, including paediatric image-guided intervention and radiation protection research. It would do this by facilitating the progression away from individual, locally isolated projects toward more evidence-based standards, protocols and multi-institutional collaborations, which could be used on both a national and international basis.. In order to achieve our aim, the ESPR research committee underwrites the policy in which research data are Findable, Accessible, Interoperable, and Reusable (FAIR) [The ESPR research committee has identified several areas of paediatric imaging for essential research, but until now has not had a formal Strategic Research Agenda under which to coordinate these projects. This document acts not only as a strategic agenda for the society to help guide current and future research, ensuring that it aligns closely with wider European Society of Radiology (ESR)\u2019s European Institute for Biomedical Imaging Research and European commission funding calls, but also to highlight those particular areas where early research (\u201cseed\u201d) funding may need to be allocated by this and other societies as precursors to larger grant applications. Through multicentre trials, existing high-level experience and expertise, and nurturing the next generation of researchers, we will be able to achieve these aims. This research agenda is mission orientated and impact focused, and necessitates the free movement of data among researcherse (FAIR) .Research that can clearly measure or emphasize the advantages of specialized imaging is the future of our specialty. Investment in well-designed research trials to address today\u2019s challenges and questions will improve the health care of tomorrow\u2019s children. Clearly, all resources are finite, so time and funding should be prioritized to the most urgent and far-ranging problems. Areas of current study that warrant further work include non-oncological bowel imaging in children, management of radiation exposure in paediatric imaging and the application of advanced new imaging methods to complex paediatric disease.Improve imaging-based paediatric research through multicentre trials and collaborative working, including data sharing;Document normal variation in imaging findings to be able to confidently differentiate disease from normal;Develop robust image-based classification systems to aid diagnosis and treatment monitoring, andHelp develop evidence-based clinical guidelines using current literature and experience to identify knowledge gaps.The key aims of the ESPR research committee are to:Evidence-based clinical imaging pathways are the ultimate end point of successful imaging research as they provide an efficient system of the maximum and minimum imaging requirements to make a diagnosis in a particular clinical situation, using the best available literature at the time. If implemented correctly, they also allow for a reduction in practice variation leading to overall improvement in clinical care. For this reason, the development of evidence-based imaging pipelines, broken down step-by-step to include diagnosis, classification and interval monitoring, should be the end goal for each disease entity for each child.Clinical referral and diagnostic criteria: risk factors and clinical presentation.Disease diagnosis and evolution: image acquisition and analysis.Clinical impact: therapeutic evaluation and clinical effectiveness.The three steps in any imaging pipeline, loosely based on Fryback and Thornbury framework , are Fi:Fig. 1EuThe appropriate imaging for the correct clinical scenario is still led by experience rather than evidence. In many cases, imaging algorithms have developed through patient and clinician experience and expertise without being validated through comprehensive studies. Clinical imaging referral guidelines play a fundamental role in enhancing appropriateness and thus the implementation of the principle of justification. Modern systematic reviews and meta-analyses provide a critical review of the existing literature and can provide much needed insight, but expert opinion is often the resource used when evidence is lacking. Literature studies or questionnaires also help to highlight current knowledge and practice gaps, and can initiate new research projects.Pediatric Radiology. Several of these have become implemented as paediatric imaging referral guidelines in the recent ESR iGuide clinical decision support system for referrers and radiologists [The ESPR recognizes the value of expert opinion by supporting several imaging task forces that have been active in evaluating the literature within their areas of expertise and generating consensus documentation. Here, the ESPR abdominal  task force can serve as an excellent example. Over the past 10\u00a0years, in close collaboration with other international societies such as the European Society of Uroradiology, the task force has provided imaging recommendations that can be implemented in daily practice , 16. Theologists .One of the fundamental aspects of paediatric imaging research is the ability to be able to share data sets among centres, particularly in cases of rare disease. However, the inability to standardize image acquisition (operating protocols and specific sequence acquisitions) hampers the ability to analyse comparable data across institutions, occasionally resulting in additional imaging tests being performed. It is for this reason that the standardization of technical parameters is paramount going forwards, but also perhaps the largest hurdle to overcome. This challenge occurs on many levels, includes the standardization of imaging approaches , imaginPaediatric oncology and musculoskeletal imaging are good examples of how this has been achieved through international guidelines, but this is lacking in several other areas. For example, the combined efforts of the OMERACT (Outcome Measures in Rheumatology) and Health e-Child have developed working guidelines for imaging of the wrist and knee in juvenile idiopathic arthritis \u201323. WithDecisions regarding which imaging modality is best to answer which clinical question often involve trade-offs between ease of access to imaging types, radiation dose and the level of detail required. This is all the more difficult when studies are not only multicentre but also multinational, as there is unequal access to more advanced imaging techniques  between centres and countries. Meanwhile, reducing the radiation dose of ionizing radiation modalities  remains a priority, while maintaining or improving image quality . Improvihttp://www.eurosafeimaging.org/pidrl). This multi-partner European Consortium-lead project headed by the ESR was intended to provide European Diagnostic Reference Levels (DRLs) for paediatric examinations. By doing so, their use would be promoted to advance the optimization of radiation protection of paediatric patients, with a focus on CT, interventional procedures using fluoroscopy, and digital radiographic imaging. Their first steps were to agree on a methodology for establishing and using dose reference levels for paediatric imaging, and to update and extend the European DRLs to cover more procedures and a wider patient age/weight range. The final document including European guidelines on dose reference levels for paediatric imaging has been endorsed and published by the European Commission and is available in the Radiation Protection Series [Good examples of the significant work done across Europe in this regard include the ESPR\u2019s pivotal role in the PiDRL project  advises that clinical indication-specific, rather than examination-specific, dose reference levels are desirable. For example, CT examinations of the same anatomical regions can be performed with different techniques, and consequently different dose exposures, depending on the clinical indication. However, there is very limited information about clinical-indication specific dose reference levels for medical imaging in children. The next project to establish new European clinical dose reference levels in children is long awaited.Once image acquisition standardisation is achieved, simultaneously there must be image analysis standardisation, i.e. of classifications, measurements and scoring systems. Key priorities for image analysis (or assessment of diagnostic performance) are to develop robust methods of diagnosing a specific disease state, developing normal standards that stand up to rigorous testing, validation and repeatability assessment, and classification or scoring criteria with validation.Normal reference appearances and values are essential to correctly interpret diagnostic images, particularly in children as normal appearances change during growth. The ability to distinguish normal variations from abnormal disease in its earliest form is the cornerstone of paediatric imaging, but this is largely experiential rather than evidential in its current practice, leading to wide variations in interpretation without evidence to the contrary. All trainee radiologists will be familiar with Keats\u2019 standard textbook of normal variants which mimic disease, but the \u201cevidence\u201d behind this book is experiential . Normal https://www.generationr.nl/researchers/data-collection/).Areas for future study would include wide variation of ventricular size in healthy newborns and young children, which is crucial knowledge to identify hydrocephalus , or the Many of the aspects of image acquisition and analysis apply equally, whether it is the first or last scan in a series of complex patient interactions. But there are more questions than answers to be explored here: When to image? What is the appropriate time interval to image in certain diseases? What are the risks in doing so (missing early disease states or relapse) versus those of imaging too early (latency bias)? Imaging is frequently used to evaluate disease evolution despite a lack of evidence for its clinical utility. Accurate imaging markers of effective therapy are essential for disease reassessment to be worthwhile. Imaging is frequently used in an attempt to evaluate a patient\u2019s response to therapy, whether pharmacological or surgical. Whilst it is relatively straightforward to image and observe change associated with treatment, proving that a change has occurred outside of normal variation and that it is causally linked to the treatment is not always clear. Clinical improvement may not correspond to imaging changes, and vice versa, or may be temporally displaced. Worse still, imaging may identify incidental lesions with the risk of overtreatment. How accurately we can predict disease monitoring is challenging, and imaging frequency may need to be tailored to individual disease states and risk factors.Imaging is heavily used in paediatric oncology assessment and reassessment, and has already demonstrated problem areas. For example, imaging following tumour resection can be challenging: Marginal amounts of residual tumours may not be visible using current imaging techniques, postsurgical contrast enhancement around resection margins can be physiological or reactive and they do not necessarily infer residual tumour. Reduction of tumour volume of >50% following treatment may be termed \u201cpartial response,\u201d but the exact nature of the residual tissue is currently not determinable.Within the European paediatric soft-tissue sarcoma group, several recent retrospective studies have evaluated the impact of imaging findings on patient care, using rhabdomyosarcoma patients as the example group. One study showed that early radiologic response to chemotherapy (volumetric tumour reduction) did not infer a survival advantage in patients with rhabdomyosarcoma . AnotherOne example of a current and ongoing specific disease pipeline is the current pan-European research initiative into juvenile idiopathic arthritis (JIA). The Euro-Im-JIA, launched in 2013\u201314, addresses the current lack of imaging markers for JIA through developing precise, validated child-specific imaging biomarkers and scoring systems to allow for evidence-based clinical practice as well as for robust drug trials. The entire project is founded on multidisciplinary collaboration across several well-established research groups across Europe, including paediatric rheumatologists, radiologists, dentists and oral surgeons as well as medical physicists and mathematicians. This project proposes a standardisation of the assessment of active and permanent change in JIA, allowing better assessment of disease evolution and therapeutics , 23. If Cancer imaging is another such pipeline likely to improve in several domains for children\u2019s services in the near future. Hybrid imaging is improving sensitivity across different imaging modalities, and may simultaneously improve specificity, particularly for metastatic disease , 40. Newhttps://www.bbmri.nl/health-ri/) may help coordinate these studies in the future. The idea of a single imaging repository with anonymised access to vast amounts of verified clinical data is a long way from being possible with current technology and border controls, including industry, consent and ethical issues.Some of the data needed to be able to address these vital questions are already acquired and currently sit on disparate hospital networks across different countries and institutions. There are several issues with data access and coordinated studies, but multiuser sharing platforms such as those provided by the European Initiative in Biomedical Imaging Research, the QUARTET network, and the Dutch Health Research Infrastructure  are shared with other paediatric imaging representatives, such as the Paediatric Imaging Research Committee of the American College of Radiology (White paper ) and we Fostering clinical research collaboration at an international level is the ESPR research committee\u2019s goal over the next 5\u00a0years to facilitate improvements in paediatric health care. Disease prevention and other aspects of health care have not been neglected nor will support for other initiatives be abandoned. These are simply the areas that we collectively believe as a society require the most intellectual and financial input in the next 5\u00a0years in order to develop imaging markers of disease that can be relied upon for diagnosis, monitoring and therapeutic evaluation. Fundamentally, we are investing in these priorities as the ESPR\u2019s strategic agenda for 2019\u20132022. We look forward to the progress that will be made on these and other areas and to updating this research strategy in 3\u20135 years."}
+{"text": "We systematically reviewed current guidelines for managing rheumatoid arthritis (RA) to evaluate their range and nature, assess variations in their recommendations and highlight divergence in their perspectives.We searched Medline and Embase databases using the terms \u2018clinical practice guidelines\u2019 and \u2018rheumatoid arthritis\u2019 from January 2000 to January 2017 together with publications of national and international bodies. We included guidelines providing recommendations on general RA management spanning a range of treatments and published in English. We undertook narrative assessments due to the heterogeneity of the guidelines.We identified 529 articles; 22 met our inclusion criteria. They were primarily developed by rheumatologists with variable involvement of patient and other experts. Three dealt with early RA, one established RA and 18 all patients. Most guidelines recommend regular assessments based on the Outcome Measures in Rheumatology core dataset; 18 recommended the disease activity score for 28 joints. Twenty recommended targeting remission; 16 suggested low disease activity as alternative. All guidelines recommend treating active RA; 13 made recommendations for moderate disease. The 21 guidelines considering early RA all recommended starting disease modifying drugs (DMARDs) as soon as possible; methotrexate was recommended for most patients. Nineteen recommended combination DMARDs when patients failed to respond fully to monotherapy and biologics were not necessarily indicated. Twenty made recommendations about biologics invariably suggesting their use after failing conventional DMARDs, particularly methotrexate. Most did not make specific recommendations about using one class of biologics preferentially. Eight recommended tapering biologics when patients achieved sustained good responses.Five general principles transcend most guidelines: DMARDs should be started as soon as possible after the diagnosis; methotrexate is the best initial treatment; disease activity should be regularly monitored; give biologics to patients with persistently active disease who have already received methotrexate; remission or low disease activity are the preferred treatment target. Guidelines for the management of rheumatoid arthritis (RA) produced by expert groups based on assessments of the research evidence have been produced for over 25\u2009years \u20134. They The existence of multiple guidelines raises several questions. First, as they have all had access to the same research data, albeit at different time-points, are there recommendations similar or are there substantial differences between them? Second, why are there different guidelines dealing with the same issue \u2013 how best to treat RA? Thirdly, what is the impact of these guidelines on clinical practice? Finally, what guidelines will be needed in future years?We have systematically reviewed current RA guidelines. Our overall aims were to evaluate the range and nature of guidelines currently available, to assess the variations in their recommendations about RA management, and highlight any divergence in their perspectives. The specific questions we considered were: (a) to examine their recommendations about composite assessments of disease activity; (b) to identify their management targets with drug therapy; (c) to define the categories of drug treatments considered. As a consequence of these assessments we sought to provide insights into the value and relevance of different guidelines.We searched Medline and Embase databases using the terms \u2018clinical practice guidelines\u2019 and \u2018rheumatoid arthritis\u2019. We also searched national bodies including the Scottish Intercollegiate Guidelines Network (SIGN) and the National Institute For Health and Care Excellence and national and international specialist societies including the British Society for Rheumatology, the American College of Rheumatology and the European League Against Rheumatism. Finally we searched lists of references from identified guidelines.Our inclusion criteria comprised: (a) publications that identified themselves as guidelines; (b) guidelines that provided recommendations on the general management of RA; (c) guidelines that included a range of different drug treatments; (d) guidelines published from January 2000 to January 2017; (e) guidelines published in English. Our exclusion criteria comprised: (a) guidelines and appraisals that dealt with specific areas of management, such as safety monitoring of drugs; (b) guidelines or appraisals of single drugs or technologies. When there were several versions of guidelines from the same organisation, only the latest guideline was included.Two researchers  independently assessed studies for eligibility and extracted data onto a predefined template. The data included: (a) year of publication; (b) format (who was involved); (c) quality method followed; (d) systematic review of evidence; (e) patient groups considered; (f) area of management included; (g) composite activity assessments; (h) prognostic assessments; (i) treatment targets; (j) and range of treatments considered. When there were differences between assessors, they reviewed the reports together and came to a joint conclusion.We sought evidence that individual guidelines had followed nationally or internationally accepted quality methods in their development; we did not assess their quality as part of this report. Firstly, we recorded who had been involved in developing the guideline, including the involvement of specialists, other experts and patients. Secondly we evaluated whether they had used recognised quality methods such as Agree and Agree II , Adapte We followed the general PRISMA recommendations  and otheThe guidelines were very heterogeneous in terms of the areas covered, the approaches taken in their development and the presentation of their recommendations. Consequently we undertook narrative assessments of their recommendations. Initially we assessed the areas covered by the guidelines, whether they included statements of principles and needs, their intended audiences and their overall structure, including whether they dealt with specific questions or recommendations. We then focussed on three predefined areas related to our specific aims. These comprised; (a) recommendations about composite assessments of disease activity and other assessments; (b) management targets with drug therapy including the impact of prognostic assessments; (c) and the categories of drug treatments considered. We considered this approach would enable us to assess the variations in their recommendations about RA management and identify divergences in their perspectives. We did not set out to produce any single optimal set of recommendations for RA management from our analyses of these guidelines. We considered management from the perspective of conventional disease modifying anti-rheumatic drugs (DMARDs) like methotrexate, biologic DMARDs like tumour necrosis factor inhibitors, Janus Kinase (JAK) inhibitors and glucocorticoids (steroids).We identified 529 potential guidelines articles: 80 were assessed in detail; 22 guidelines \u201335 selecThe 59 excluded guidelines articles included 5 superseded guidelines and one separately published summary article, 32 guidelines that dealt with single drugs or drug classes, 18 that dealt with non-drug treatments and 3 patient-related articles.These are summarised in Table\u00a0The guidelines varied substantially in the ways they were constructed. Three guidelines , 22, 28 The approaches to assessing clinical research evidence supporting the guidelines also varied. The two EULAR guidelines , 34 commTwo guidelines dealt with early RA under 2 years duration , 20 and All the guidelines dealt with drug treatment, though they did not all cover the same aspects of drug therapy. Eleven guidelines also covered diagnosis , 29\u201332 aGuidelines often included a range of statements of general principles, the specific need for the guideline and the audience the guideline was intended to inform. These statements were so diverse that it is not possible to provide a succinct summary of them.The EULAR guidelines  providedTwenty guidelines outlined, to a greater or lesser extent, their intended audience , 33\u201335. 13/22 guidelines dealt with specific questions or recommendations , 34, 35;18/22 guidelines , 21\u201335 rThe importance of frequent assessment is stressed in most guidance. Some guidelines gave relatively specific suggestions. For example EULAR guidelines recommend assessing patients every 1 to 3 months, at least in the early stages of their RA. Many guidelines indicated patients should be assessed by rheumatologists at least annually. The English  guideline gives a very specific recommendation for annual review. The ACR guideline recommended annual assessments of function.Twenty guidelines recommended remission as a treatment target and 16 guidelines recommended using low disease activity as an alternative target Table\u00a0. Two guiRemission was defined in various ways, in keeping with current international criteria . DAS28-dAll guidelines recommend treating active RA. There was less unanimity about treating moderately active disease. Thirteen guidelines made specific recommendations about treating moderate disease. Four guidelines gave implied guidance about treating moderate disease in that they indicated what treatment policies were needed until patients achieved remission. Five guidelines made no recommendations about treating moderate disease.Sixteen guidelines specifically included assessments of prognostic factors to help guide management decisions about treatments \u201333, 35. Twenty one guidelines dealt with the management of early RA; all of these recommended starting conventional DMARDs as soon as possible after diagnosis. Methotrexate, which is often described as the \u201canchor\u201d drug for RA, was recommended for most patients in 19/22 guidelines , 31\u201335 . In 13/2When there are contraindications to methotrexate or if there are clinically significant adverse events to methotrexate all 19 guidelines that suggested methotrexate as initial treatment recommend considering alternative conventional DMARDs. Sulfasalzine, leflunomide and hydroxychloroquine were all considered potentially appropriate; there was no consistent pattern in these recommendations. Other rarely used conventional DMARDs, such as azathioprine, though not excluded were not specifically recommended.Three guidelines considered DMARDs generically without giving recommendations about which drugs to use; these were the British Guidelines for established  and earlThe way individual guidelines outlined the initial treatment for RA varied considerably. EULAR guidelines recommend that methotrexate should be part of the first treatment strategy. ACR guidelines recommend that DMARD monotherapy is generally more acceptable and better tolerated than combination DMARD therapy and that methotrexate should be the preferred initial DMARD for most early RA patients. Canadian guidelines recommend that initial combination therapy with traditional DMARD should be considered, particularly in patients with poor prognostic features, moderate-high disease activity and in patients with recent-onset disease. English  guidelines recommended that in people whose RA is active, patients should be offered a combination of DMARDs  as first-line treatment.Twenty guidelines considered the use of combinations of conventional DMARDs; 19 of these guidelines recommended using them in some patients \u201333, 35. The one exception was the EULAR guidelines which do not specifically recommend using them. However, EULAR did not exclude their use, and mention them briefly. The EULAR guidelines also provide an extensive commentary on the divergence of expert opinion on this issue, highlighting potential toxicities and difficulties dissociating the impact of methotrexate, short-term glucocorticoids (steroids) and other conventional DMARDs in combinations.Only 4 guidelines consider the use of Janus Kinase inhibitors; this mainly reflects whether they were developed after these drugs became available. Those guidelines that consider them recommend their use as an alternative to biologics in some patients with established RA. They are usually recommended to be used in combination with methotrexate.Twenty guidelines recommended using glucocorticoids in some RA patients; these were usually patients with early RA who were starting DMARD treatment. In the main only short-term courses of low dose glucocorticoids (steroids) were recommended. The EULAR treat to target guideline implied glucocorticoids (steroids) should be used within the treatment strategy in some patients but did give any recommendations about specific therapies. The British guidelines for established RA did not consider glucocorticoids (steroids). In addition some guidelines gave advice about the role of glucocorticoids (steroids) in specific clinical settings, particularly in the management of some comorbidities.Twenty guidelines made recommendations about using biologics. Three guidelines made generic recommendations about biologics and the other 17 that dealt with them considered individual biologics and classes of biologics. The 2 guidelines that did not were for primary care clinicians who should not usually prescribe these treatments. All the guidelines that dealt with biologics recommended their use in patients who had failed to respond to conventional DMARDs, particularly methotrexate. They also recommended using them in combination with methotrexate whenever possible. Most guidelines did not make specific recommendations about using one class of biologics preferentially. However, some guidelines such as the Canadian ones, recommend using tumour necrosis factor inhibitors as an initial biological treatment. In patients who have continuing disease activity despite biologic treatment or adverse events to biologics starting an alternative biologic was recommended. In most instances no particular sequences of biologics were recommended in the different guidelines.Eight guidelines recommended considering tapering biologic treatment in patients who had achieved sustained good responses and remissions. A further two guidelines implied this was appropriate without giving detailed recommendations.Thirteen guidelines made recommendations about the use of NSAIDs and 12 about using analgesics to control symptoms. Those guidelines which consider the use of NSAIDs invariably focus on minimising exposure to these treatments. For example the Scottish Guidelines suggest using the lowest NSAID dose compatible with symptom relief, and indicate that treatment should be reduced and if possible withdrawn as soon as possible and that gastro-protection should be included when using them. When analgesics such as paracetamol were mentioned for symptom relief though the evidence supporting their use is noted to be minimal by current standards.Our overview of 22 different RA management guidelines shows that several general principles transcend the majority of them. Firstly DMARDs should be started as soon as possible after the diagnosis has been established. Secondly disease activity should be regularly monitored using composite indices such as DAS28, which relates to our initial aim which was our initial specific question. Thirdly methotrexate is the best initial treatment, and that this can be usefully supplemented with short-term glucocorticoid (steroid) therapy. Fourthly biologic DMARDs should be given to patients with persistently active disease who have already received methotrexate and, in some instances another conventional DMARD. These principles relate to another of our specific questions. Fifthly remission or low disease activity is a suitable target and that treatment can be tapered in patients who have achieved sustained remissions. This principle relates to our final specific question. We consider that applying these general principles to RA management in all clinical settings is likely to achieve good overall clinical outcomes.There is considerable uncertainty about the value and place for using combinations of conventional DMARDs. The most recent EULAR guidance is particularly uncertain about its value. Other guidance including the ACR guidance is more definite it is perspective. The reasons for this difference are unclear. In part it may be presentational; EULAR guidance does not exclude using such combinations and ACR guidance does not explicitly recommend them; consequently much of the apparent difference may represent the way in which the information is presented. There has been correspondence about this particular aspect of the EULAR guidelines , 47. HowThere is also relatively little overall consensus about treating moderately active RA. The ACR guidance makes the strongest recommendation on this point. Other guidance has either not considered it or may have been published prior to much evidence becoming available. Despite the limitations of explicit recommendations, those guidelines which consider moderate disease recommend treating it intensively.The guidelines differ in the formality of their approach and in the extent of systematic reviews commissioned specifically for them. The EULAR, ACR and Royal College of Physicians guidelines were the most detailed and involved the greatest amount of preparatory work including a number of detailed systematic reviews. Specialist rheumatologists were involved in almost all guidelines; varying numbers of other experts and patients were involved. The impact that these non-rheumatologists would be able to make to the guidelines was uncertain.The limitations of clinical guidelines have been described in detail \u201352. We dOur own assessment of RA guidelines has its own limitations. Firstly, some of the guidelines were developed over 10\u2009years or longer and the older ones cannot have included the more recent clinical evidence. Therefore comparisons need to take this into account. Secondly, there are different types of guidelines. We have included general ones. Many others focus on single drugs or treatment modalities including surgery. It is difficult to draw a clear line between which ones to include and which to omit. Not all experts would necessarily agree with our approach to inclusion. Thirdly, we have only provided a narrative assessment of them. They are too diverse in their approaches to allow any synthesis of their various conclusions and recommendations. Fourthly we have focussed on issues in the guidelines we consider to be of most importance. Other experts may have considered different aspects of the guidelines in more detail and overlooked some of the matters we have dealt with. Finally, systematic reviews of guidelines are not one of the current PRISMA extensions  though wOur analysis shows several things. Firstly, the recommendations in the guidelines are broadly similar, though they differ in some points of detail; for example the use of combinations of conventional DMARDs. Such minor variations most likely reflect the challenges in balancing evidence of benefits against evidence of risks. Secondly, although guidelines deal with the same issue, they bring together different groups of experts and it is likely the production of guidelines enhances clinical practice. Consequently multiple guidelines appear to be needed. Thirdly, although it is difficult to judge accurately the impact of guidelines on clinical practice, there is evidence that RA outcome have improved significantly during the last 10\u201320\u2009years and in part this is likely to reflect the impact of guidelines in improving the quality of clinical practice. Finally, as new treatments are introduced, particularly new JAK inhibitors, guidelines will need to be continually updated and, potentially produced by different groups.We anticipate that many of the existing guidelines will be updated in future years. We believe it important to do so to maintain their relevance to clinical practice. The frequency of review will reflect the timing of new clinical information. Looking back at the earliest guidelines from the 1990s  shows juOne important role of guidelines is to suggest potential future research questions. Our own research in the TITRATE research programme, of which this systematic review in a single component, was based on the absence of evidence on the benefits of intensive management in moderately active RA . InteresAlthough a number of differences exist between guidelines, there are some general principles. These include starting DMARDs soon after diagnosis; methotrexate should be used first line; disease activity should be monitored regularly; biologics therapies should be used where there is persistently active disease; and remission or low disease activity is the preferred target."}
+{"text": "It has a profound role in determining disease changes and facilitating drug research and development, thus creating new medical modalities to monitor human health. At present, a variety of different molecular imaging techniques have their advantages, disadvantages, and limitations. In order to overcome these shortcomings, researchers combine two or more detection techniques to create a new imaging mode, such as multimodal molecular imaging, to obtain a better result and more information regarding monitoring, diagnosis, and treatment. In this review, we first describe the classic molecular imaging technology and its key advantages, and then, we offer some of the latest multimodal molecular imaging modes. Finally, we summarize the great challenges, the future development, and the great potential in this field.Molecular imaging has emerged at the end of the last century as an interdisciplinary method involving  Molecular imaging has emerged at the end of the last century and consists of a combination of in vivo imaging and molecular biology aiming at identifying or describing living biological process at a cellular and molecular level using noninvasive procedures. It is especially addressed to reveal abnormalities in cells and molecules which cause the disease, rather than the final anatomical and structural abnormality caused by cellular or molecular changes . VariousIn clinical research, CT, MRI, PET, SPECT, US, and optical imaging are usually among the choices of the imaging modalities. Each imaging modality has its own unique strength and intrinsic limitations, such as spatial/depth resolution and sensitivity, making the achievement of precise and reliable information at the disease site difficult. In order to compensate these weak aspects, multimodal molecular imaging has been considered in recent years . MultimoAt present, molecular imaging models mainly include CT, MRI, radionuclide, ultrasound, and optical imaging.CT is a technique producing images reflecting human anatomy, thanks to differential levels of X-ray attenuation by tissues within the body. CT is widely available, and it has some advantages such as high spatial resolution, strong penetration depth, fast acquisition time, low cost, clinical utility, and relative simplicity. However, CT also has some disadvantages: one is the high radiation dose, which often limits the scan time, and another is the low-quality soft tissue contrast, compared with MRI . In addiThe application of CT molecular imaging requires high quality imaging agents, after injection, in order to achieve the target site for a change in X-ray attenuation. Currently, most CT molecular contrast agents are designed to combine a maximum number of X-ray-absorbing atoms with a nanoparticle, which includes emulsion, liposomes, lipoproteins, and polymeric nanoparticles \u201317.2 S3) nanoparticle as an injectable CT imaging agent, which is used for enhanced in vivo imaging of the vasculature, the liver, and lymph nodes in normal mice. Next, Hyafil et al. -2-fluoro-2-deoxy-glucose ([18F]-FDG) \u2013104. ForIn addition to its clinical practicality, PET has extensive applications in the basic and preclinical researches. PET can be used to study basic physiological and molecular mechanisms of human diseases by using the appropriate radiolabeled-imaging agents . Bretin 99mTc [123I [99mTc-TRODAT-1 imaging in the evaluation of patients with early-stage Parkinson's disease [111In-DTPA-octreotide in tumors of various sizes [99mTc-labeled C2A domain of synaptotagmin I in a mouse model. Moscaroli et al. [111In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single-photon emission tomography (SPECT). Tang et al. [SPECT imaging agents use energy between 85 and 500\u2009Kev; radiographic tomography is a technique for projection reconstruction of faulty images, which are similar to X-ray and CT imaging. SPECT uses nuclides such as 99mTc \u2013114, andTc [123I , 116 thrTc [123I . Some ex disease , the theus sizes , and theus sizes . Besidesus sizes , 122, ani et al.  presenteg et al.  develope18F radiotracer and examined its properties both in vitro and in vivo, for PET imaging of folate receptor-positive tumors. Xing et al. [64Cu. Thus, the nuclear medical imaging is associated with near-infrared imaging. Finally, the authors show that the PET signal is highly coincided with quantum dot near-infrared image.Since biochemical changes always occur before anatomical changes in disease, both PET and SPECT have clear diagnostic strength over anatomical techniques such as classical CT and MRI. However, PET and SPECT have a key weakness, that is, the lack of an anatomical reference frame. This weakness may be eliminated through the combination of these instruments with either CT or MRI, producing a single scanner capable of accurately identifying molecular events with precise correlation to anatomical findings . This meg et al.  built a So far, nuclear medicine molecular imaging technology is one of the widely used technologies in clinical molecular imaging technology and plays an important role in the study of personalized medical care due to its unique technology \u2013133. PETUltrasound imaging, like MRI and CT, has been used as a morphological imaging modality. Medical ultrasound imaging is a unique imaging modality that exploits the properties and behavior of high-frequency sound waves as they travel through biological tissue, and it can be used both for diagnostic imaging and as a therapeutic tool. Compared with traditional imaging techniques such as radionuclide imaging and optical imaging, ultrasound imaging has some advantages such as economy, convenience, and real-time imaging , 137. Fuin vivo simulation of immunohistochemistry or in situ hybridization techniques targets biomolecules to highlight the pathological changes of diseased tissue. Thus, it can visualize the real pathogenesis and significantly improve the sensitivity and accuracy of imaging diagnosis. These aspects are actually the current clinical research central issues.Traditional ultrasound contrast agents, that are a few micrometers in diameter and in the terms of gas-filled microbubbles, are often coated with lipids or biopolymers, and they are available for enhancing the reflection signal-to-noise ratio for blood . These cAt present, targeted microbubbles are being used in preclinical investigations of both inflammation and angiogenesis. For example, microbubble shells have been attached to endothelial cell adhesion molecules for visualization of P-selectin, supplying foresight on molecular aspects of inflammation . DeshpanWith the emergence of ultrasound molecular imaging, the early diagnosis and specific treatment of malignant tumors gained some research achievements. Kim et al.  used inkIn the near future, ultrasound molecular imaging is expected to pass from a preclinical modality to a fully clinically useful technique through the use of different clinically translatable instrumentation, such as endoscopes and novel US-compatible imaging agents that are able to exudate , 157. AmOptical imaging is a method of obtaining biological information by using optical detection means combined with optical detection molecules to imaging cells or tissues or even organisms. If the biological optical imaging is limited to the visible and near-infrared range, different biological optical imaging methods can be divided into fluorescence imaging, bioluminescence imaging, photoacoustic imaging, and optical tomography. Nowadays, molecular imaging becomes more popular and is combined with classical optical imaging techniques.Fluorescence imaging technology is marked with a fluorescent report group including inorganic materials, such as upconversion, quantum dots, and other organic materials, such as green fluorescent protein, red fluorescent protein, or fluorescent dye. It uses excitation light to make the report group reach a higher level of molecular level and then emit a longer wavelength visible light to form biological light source in vivo and detect it. At present, common fluorescent groups include various small molecule fluorescent dyes, green fluorescent protein, and red fluorescent protein. In recent years, fluorescence technology has been extensively used in the study of molecular biology and the metabolism of small molecules in small molecules. There is a rapidly expanding list of fluorescent agents which includes near-IR Cy 5.5, turnip green, quantum dots (QDots), the Alexa dye series, and all kinds of fluorescent proteins. Besides, lanthanide-based imaging agents were added to this list . There aBioluminescence imaging technology uses luciferase gene to label cells or DNA and exploits sensitive optical detection instrument to directly monitor cell activity and gene behavior in living subjects. This technique has these following advantages: (1) noninvasive, (2) continuous repeated detection, (3) fast real-time scanning imaging, and (4) high sensitivity. Bioluminescence imaging has been used to study numerous enigmatic protein-protein interactions. One such study uses a firefly luciferase-based protein fragment complementation assay to visualize luciferase-expressing bone marrow cells in brain inflammation in living mice . In 2006Photoacoustic imaging (PA) by using optical absorption and transformation between the tissues of the light and sound energy is a nondestructive imaging method developed in recent years. It combines the high penetration characteristics of pure optical imaging and high contrast characteristics by light into the ultrasound, and it can provide tissue imaging with high resolution and high contrast. Based on the technology of photoacoustic effect of time-domain photoacoustic spectrum, it partially overcomes the effect of strong scattering in the optical transmission in the organization when optical and acoustic are organically combined. Therefore, photoacoustic technology has well biological penetration, the characteristics of high resolution, and no side effects. Its main application direction can effectively carry out biological tissue structure and function imaging, providing an important means for studying the morphological structure, physiological characteristics, pathological characteristics, and metabolic function of biological tissue. For example, Ding et al.  developeAt present, there are still some defects in the living organism imaging system. Many in vivo optical imaging are also just stay in the phantom and small animal experimental stage, has not yet entered into clinical application, and need a further improvement in many aspects. It is an important task for the future to find new high quantum efficiency fluorophores, improve the reconstruction algorithm and image resolution, and expand the new optical imaging technology. In vivo bioluminescent imaging technology has become an indispensable tool in the research of nuclear small-animal models. It studies the pathological process, drug development, and drug efficacy from a unique perspective. In fact, the biological optical imaging technology has had a significant impact on the basic and applied medical research.Multimodal molecular imaging combines two kinds or more detection technologies to form a new way of imaging, which is convenient for obtaining some further information in diagnosis, treatment, and monitoring. At present, multimodal molecular imaging has been widely used to optimize medical research and clinical practice. In practice, multimodal molecular imaging has been helpful for cardiovascular diseases , 166, neA few specific examples are as follows: (1) cardiovascular diseases: Yoo  presenteIn summary, multimodal molecular imaging has a bright future. The development of this field will bring a major breakthrough in medical imaging and molecular biology. Although molecular imaging remains at its initial stage, a broader space for further developments is still possible."}
+{"text": "The recognition of selected anions including carbonate, hydrogen phosphate, perchlorate, oxalate, picrate, and EDTA was conducted using electrochemical impedance spectroscopy with ferricyanide redox probe. For the potentiometric sensor assembling, the ionic liquids were stabilized by multiwalled carbon nanotubes and carbon black deposited on the glassy carbon electrode. The influence of support, steric factors and modification conditions on the sensor performance has been investigated. As was shown, potentiometric sensors developed make it possible to selectively determine hydrogen phosphate anion within the concentration range from 1 \u00d7 10\u22122 to 1 \u00d7 10\u22126 M and limit of detection of 2 \u00d7 10\u22127\u22121 \u00d7 10\u22126 M with unbiased selectivity coefficients varied from 1.2 \u00d7 10\u22121 to 1.0 \u00d7 10\u22128 .New solid-contact potentiometric sensors have been developed for hydrogen phosphate recognition on the basis of ionic liquids containing tetrasubstituted derivatives of thiacalix[4]arene in  To the best of our knowledge, this is a first example of application of ILs with calixarene core for potentiometric determination of hydrogen phosphate.tert-butyl-25,26,27,28-tetrakis [ammoniumpropylcarbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene tetra [bis(trifluoromethylsulfonyl)imide] in cone (1) and 1,3-alternate (2) conformation, and 5,11,17,23- tetra-tert-butyl\u221225,26,27,28-tetrakis[ ammoniumethyl)carbamoylmethoxy]-2,8,14,20-tetrathiacalix[4]arene tetra[bis(trifluoromethylsulfonyl)imide] in cone (3) and 1,3-alternate (4) conformation, were synthesized at the Organic Chemistry Department of Kazan Federal University as described elsewhere 6] and K4[Fe(CN)6], MWCNTs were purchased from Sigma-Aldrich, Germany, CB from IMERYS, Belgium, succinic acid from VitaChim, Russia, glutaric and glycolic acids from TauRus, Russia, malonic acid from AquaChim, Russia. All the reagents were of analytical grade. All the solutions were prepared using deionized Milli-Q\u00ae water.Oxalic acid, potassium ferricyanides KHome-made glassy carbon electrode consisted of 2 cm long rod 1.7 mm in diameter inserted in the polytetrafluoroethylene tube was used for solid-contact ion-selective electrode assembling. Threaded connection made of stainless steel was fixed from the opposite end of the electrode. All the potentials are given against double-junction Ag/AgCl/3 M KCl reference electrode (Metrohm Autolab).3 and 0.5 mL of the mixture of 0.1 M K3[Fe(CN)6] and 0.1 M K4[Fe(CN)6] at 0.265 V using FRA2 module of the potentiostat-galvanostat PGSTAT 302N . The glassy carbon electrode was first mechanically polished and cleaned by acetone, sulfuric acid, NaOH and twice with deionized Milli-Q\u00ae water. Then, 2 \u03bcL of 1.0 mM solution of the IL in acetone were placed on the working surface and allowed to dry at ambient temperature. The amplitude of the applied sine potential was 5 mV and the frequency varied from 100 kHz to 0.04 Hz with a sampling rate of 12 points per decade. The capacitance and charge transfer resistance were calculated by fitting experimental data using Randles equivalent circuit (1) with the NOVA software (Metrohm Autolab).EIS measurements were performed in working cell containing 4.5 mL of 0.1 M KNORs is solution resistance, Ret charge transfer resistance, ZW Warburg impedance and C is constant phase element which is here equal to interface capacitance.Here Prior to measurement, freshly prepared electrode covered with the IL film was repeatedly heated to 80\u00b0C in solid-state thermostat and cooled to ambient temperature to equalize the surface layer. After equalization, an aliquot of the analyte solution was placed on the working surface for 10 min. Then the electrode was washed and the impedance was measured in the presence of the ferricyanide redox probe. The EIS parameters were calculated and averaged for three replications.The glassy carbon electrode was first mechanically polished and cleaned as described above in section Impedimetric Measurements. In layer-by-layer deposition, 1 \u03bcL of 1 mg/mL CB suspension in dimethylformamide (DMF) was placed on its surface and dried at 60\u00b0C for 30 min. Then, 2 \u03bcL of 1.0 mM IL solution in acetone were placed on the electrode surface and dried again. Alternatively, a mixture of the CB suspension and IL solution in 10:1 or 20:1 v/v ratio was prepared and the same aliquot (2 \u03bcL) were spread on the working surface as described above.2SO4 and HNO3 in 3:1 vol. ratio and sonicated at 40\u00b0C for 4 h . Then they were centrifuged, washed with distilled water and dried at 80\u00b0C for 2 h. The MWCNTs dispersion was prepared by 15 min unltrasonication of oxidized MWCNTs in DMF.MWCNTs were deposited on the surface of electrode in a similar manner. They were first mixed with concentrated HSEM images of the CB and MWCNTs coatings were obtained with the high-resolution field emission scanning electron microscope Merlin\u2122 (Carl Zeiss). Highly ordered pyrolytic graphite plate 12 \u00d7 12 \u00d7 2 mm  was used as substrate for deposition of carbonaceous materials.If not used, the potentiometric sensors were stored in dry conditions in alumina foil cover at ambient temperature. Prior to use, dry electrodes were conditioned in deionized water and appropriate salt containing the anion to be determined. were determined by separate solution method (SSM). For this purpose, standard potentials of primary  and interfering  ions were calculated from the dependence of the electrode potential on their activities, Si is the slope of calibration curve. The  values were calculated from Equation (2) adapted for anion determination . For sensor calibration, a series of standard solutions with concentration varied from 0.1 \u03bcM to 10.0 mM was prepared in deionized water. The pH was adjusted to 8.0 with NaOH. The pH of solutions was measured using glass pH-electrode (Econix-Expert). Calibration curves were obtained by step addition of appropriate salt solutions to the working cell under continuous magnetic stirring. The unbiased potentiometric selectivity coefficients  Bakker,  and aver values are calculated in accordance with Equation (3), where  is the activity of the primary ion in reference solution, ai is its activity after addition of an aliquot, aj is the activity of interfering ion resulted in the same shift of the potential.Besides, matched potential method (MPM) was used for selectivity coefficient determination to take into account non-ideal behavior of primary and interfering ions  with CD conductivity detector.Ret first monotonously increased by 10\u201320 k\u03a9. In deionized water, the Ret was stabilized at about 200 k\u03a9. In the salts of inorganic and organic acids, stationary value was remarkably higher and depended on the analyte nature. Increase in the concentration of sodium phosphate, oxalate and carbonate in the range from 1.0 \u00d7 10\u22126 to 1.0 \u00d7 10\u22122 M decreased the Ret. Between measurements, the potentiometric sensor was shortly heated above IL melting point (60\u201380\u00b0C) and sharply cooled to ambient temperature to accelerate the stabilization of the EIS parameters. Typical Nyquist diagram and changes in the charge transfer resistance are shown in Figure 1 as example. The slope of appropriate graphs in the plots of Ret vs. logc increased from 21 to 24 k\u03a9/pC  to 65\u201370 k\u03a9/pC (carbonate and EDTA). Other anions tested influenced the Ret value only at their high concentrations. Thus, perchlorate suppressed the Ret value in concentrations exceeding 1.0 \u00d7 10\u22123 M, thiocyanate, chloride, bromide anions in those higher than 1.0 \u00d7 10\u22122 M.Although, the ILs assume different mechanisms of potentiometric response toward target analytes, ion exchange remains most frequently discussed. To estimate the anion exchange capabilities of the ILs studied, EIS technique was used. In these experiments, the IL was deposited alone on the surface of glassy carbon electrode and the EIS spectra were recorded in the presence of ferricyanide redox probe. Reproducible results were obtained after equalization of the freshly prepared sensor in water. Commonly, in consecutive measurements the charge transfer resistance Ret values can be attributed to the ion exchange on the surface of the IL film. Partial substitution of bis(trifluoromethylsulfonyl)imide (Tf2N\u2212) anion with a smaller ferricyanide ion bearing higher charge results in electrostatic repulsion of redox probe and hence increase of the Ret value. In the presence of other anions, the implementation of ferricyanide ions becomes lower and thus the resistance changes in the opposite direction. The lower the size of the anion and the higher its charge are the more the Ret value changes with its addition. The highest sensitivity corresponds to the three basic EDTA anion and carbonate. The latter one can additionally alter the accessibility of ferricyanide ion due to formation of molecular form CO2\u00b7H2O in the measurement conditions. All the anions mentioned above affect the Ret value in the whole range of concentrations tested, i.e., from 1.0 \u00d7 10\u22126 to 1.0 \u00d7 10\u22122 M.Changes in the Direct deposition of the ILs on the glassy carbon electrode allowed impedimetric detection of some anions able to ion exchange on the electrode interface. However, the lifetime of the sensor was limited by 15\u201320 measurements due to partial deterioration of the surface layer especially on the stage of heating and following solidification. This calls for searching possible supports for mechanical hardening of the surface layer and improving its robustness in routine measurements.Carbonaceous materials, e.g., CB  or in one step by preliminary mixing of the suspension of CB (MWCNTs) in DMF and IL solution in acetone. The amounts of CB and MWCNTs were chosen to reach full coverage of the electrode surface. Higher quantities of modifiers made worsen electron exchange conditions on the electrode interface and were less mechanically stable. In the series of consecutive drying-wetting steps, excessive amounts of particles left the electrode. This resulted in dramatic shift of the stationary potential of the sensor. As could be seen from Figure The IL film improved the mechanical strength of the layer and about fully excluded deterioration of carbonaceous particles within the sensor lifetime. In some cases, crack appeared after washing the electrode, which could be removed by short heating them over 60\u00b0C and cooling to ambient temperature.2HPO4. Prior to use, freshly prepared sensor was conditioned overnight in 0.01 M solution of the analyte. The same results were obtained in series of heating\u2014cooling steps as described above for impedimetric measurements. After addition of the hydrogen phosphate, the sensor potential shifts within 10\u201312 s in accordance with anionic sensitivity of the response. The dynamic response time insignificantly alters with the amounts of CB (MWCNTs) and IL present in the surface layer and tends to increase to 15\u201320 s with the analyte concentrations decreasing to 1.0 \u00d7 10\u22126 M. Reverse return after washing is slower but can be accelerated by melting IL in a short heating of the sensor to 80\u00b0C. The reversibility of the signal was demonstrated in measurements of alternating concentrations (10.0 mM and 10.0 \u03bcM) with the same sensor . In both cases, difference in the slopes of the curves can be attributed to different steric hindrance of the anion access to the cationic center of the IL. Contrary to that, CB and MWCNTs did not show significant difference in the hydrogen phosphate determination. Higher deviation of the signals observed for CB can be related to the lower reproducibility of the surface morphology and specific surface influencing the ion exchange. The LOD values were determined in accordance with the IUPAC recommendation .Using this potentiometric sensor, unbiased selectivity coefficients were determined by SSM and MPM methods (see section Potentiometric Sensor Preparation and Application). The results obtained are presented in Table 1 was used in all the measurements, the results were averaged for three replications. As could be seen, the potentiometric sensor developed can be used for the assessment of the phosphate content in fresh waters. Minor underestimation against chromatography results can be attributed to the different sample treatment protocol including some other phosphate sources  undetectable with potentiometric sensors. In potentiometric experiment, water samples were filtered to exclude organic phosphate sources and their pH was adjusted to 8.0 with NaOH solution.The potentiometric sensors developed were tested in the determination of hydrogen phosphate anions in fresh waters samples taken from natural lakes in the suburb of Kazan City. The content of anions established by ionic chromatography and the results of potentiometric determination are presented in Table Application of ILs in the assembly of solid-state potentiometric sensors offers some attractive advantages, e.g., small size, flexibility of the design, no need in internal filling and long conditions prior to use. Besides, the ILs do not need in additional implementation of lipophilic salt and plasticizers and are applicable with modern technologies of sensor manufacture (screen-printing technique). Meanwhile, the number of successful examples of IL based potentiometric sensors is rather limited due to difficulties in their synthesis and some difficulties of introduction in the sensor assembly including moderate solubility in water and in some cases insufficient mechanical durability of the surface layer.2N\u2212 anion instead of bromide or nitrate decreased melting point to meet the IL definition  and those added to the solution for the binding sites of the IL layer. The comparison of the effect achieved for different salts made it possible to preliminarily conclude on the selective recognition of hydrogen phosphate anions. The following experiments with carbonaceous materials as supports for ILs confirmed the possibility for reliable and sensitive determination of HPOPP and IS synthesized ILs based on thiacalixarene platform, characterized their purity and conformation and participated in the manuscript preparation. AP and GE performed electrochemical measurements (EIS and potentiometry).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": "Nos3  was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non\u2010resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGF\u03b2\u2010induced pro\u2010fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGF\u03b2\u2010induced lung fibroblast activation in 2D and 3D co\u2010cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non\u2010resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro\u2010fibrotic and reduced vascular homeostasis gene expression. Among the latter,  Bleomycin\u2010induced lung injury promotes transient fibrosis accompanied by increased capillary density in young mice. In contrast, persistent fibrosis, capillary rarefaction, loss of endothelial cell identity, and reduction of Nos3 are observed in aged mice. eNOS/NO signal is an important driver of fibroblast quiescence and fibrosis resolution, that is lost with aging. Lung vascular bed plays a critical role during lung repair and fibrosis resolution. Analysis of body weight and survival curves showed no significant differences for these parameters between the two groups Figure . As showBecause aging\u2010induced functional and structural alterations of the microcirculation contribute to the pathogenesis of a range of age\u2010related diseases  and aged mice (N\u00a0=\u00a04) at 30\u00a0days following bleomycin challenge. Next, we measured expression of endothelial genes by using a commercially available qPCR endothelial biology array  , eNOS is predominantly expressed in ECs and belongs to a family of enzymes catalyzing the production of nitric oxide (NO), a gaseous molecule that binds and activates the receptor soluble guanylate cyclase (sGC) in multiple cell types including fibroblasts  and endothelial (TEK and VWF) transcripts showed no differences in NOS3\u2010silenced HLMECs compared to control cells  that have been previously treated with a siRNA targeting eNOS. As shown in Figure s Figure . Becauses Figure , its abis Figure . FACS\u2010so3IPF is characterized by the excessive accumulation of extracellular matrix that leads to distortion of lung architecture and loss of organ function. Myofibroblasts are the main source of ECM in IPF lungs and their sustained pathological activation is primarily responsible for the progressive worsening of the disease  relaxation has been investigated in great depth  mice were provided by Dr. Zvonimir S. Katusic.Female and male Col1\u03b11\u2010GFP transgenic mice (FVB strain) were provided by Dr. Derek Radisky. Female and male wild\u2010type (C57BL6) and eNOS4.2Normal primary human lung fibroblasts, HLFs  were used between passages 3 and 7. Normal human lung microvascular endothelial cells, HLMECs (Lonza) were used within passage 4. In experiments involving siRNA, serum was reduced to 0.1%.4.3All animal experiments were carried out conforming to the ARRIVE guidelines. Mice were anesthetized with ketamine/xylazine solution and bleomycin (1\u00a0U/kg) or PBS was intratracheally delivered on day 0 as described in Appendix 4.4Mice were anesthetized with ketamine/xylazine solution and perfused via left ventricle with cold PBS. The lungs were immediately harvested and the single cell suspension was obtained as detailed in Appendix 4.5Hydroxyproline content was measured using a hydroxyproline assay kit (Biovision), comparing the samples to a hydroxyproline standard curve as described in Appendix 4.6Lung tissue from patients with IPF and from non\u2010fibrotic healthy controls was obtained from Dr. Steven Huang at the University of Michigan. Diagnoses of patients with IPF were established by clinic\u2010pathologic criteria and confirmed by multidisciplinary consensus conference. All IPF tissues were derived from explanted lungs obtained at the time of transplantation. Normal control lungs were obtained from deceased donors  whose lungs were deemed unsuitable for transplant. All patient samples were obtained with informed consent and were approved by the University of Michigan IRB (IRB #: HUM00105694). Mouse and human lung tissues were stained with Collagen 1\u03b11  or PECAM\u20101  antibodies as described in Appendix 4.7NOS3 siRNA (M\u2010006490\u201000\u20100005) by using Lipofectamine RNAiMAX reagent  as described in Appendix RNA interference was performed with siGENOME Non\u2010Targeting Control siRNA Pool #1 (D\u2010001206\u201013\u201005) or siGENOME Human 4.8NOS3 siRNA. Six hours after transfection, the cells were lifted and plated into the outer minor wells of the \u03bc\u2010Slide. After 72\u00a0hr, HLFs were processed and stained as described in Appendix Co\u2010cultivation of HLFs and HLMECs and \u03b1SMA staining were performed by using \u03bc\u2010Slide 2 well Co\u2010culture . HLFs were primed with TGF\u03b2 (2\u00a0ng/ml) for 24\u00a0hr and then transferred into the inner minor well of the \u03bc\u2010Slide. HLMECs were transfected with Non\u2010Targeting or 4.9A mixture of FACS\u2010sorted Col1\u03b11\u2010GFP mice fibroblasts and HLMECs was plated in a 1:1 solution of Matrigel Matrix  and endothelial cell growth basal medium  as detailed in Appendix 4.10Mouse lung tissue sections (7\u00a0\u03bcm) were permeabilized, blocked, and stained with PECAM\u20101 antibody , and nuclei were counterstained with DAPI  as described in Appendix 4.11Each well of a 48\u2010well culture plate was coated with 250\u00a0\u03bcl of rat tail collagen I . Fresh lung explants were embedded into the collagen I layers, cultured for 7\u00a0days in presence of VEGFA (20\u00a0ng/ml) and analyzed as described in Appendix 4.12Western blotting analysis of whole lung tissue or cell lysates was performed using eNOS  and GAPDH  antibodies, as described in Appendix 4.13t test, one\u2010way analysis of variance (followed by Tukey's post hoc test), or non\u2010parametric Mann\u2013Whitney test as detailed in Appendix p\u00a0<\u00a00.05.Individual data points are shown in all plots and represent data from independent mice or biological replicates from cell culture experiments. Statistical analysis was performed using Student's None declared.N.C., D.J.T., and G.L. designed the study. N.C., J.A.M., A.A., D.L.J., S.A.A., T. X. P., A.J.H., K.M.C., Q.T., and A.H. performed experiments. N.C. and G.L. analyzed data. The manuscript was drafted by N.C., D.J.T., and G.L. and revised by N.C., S.K.H., Z.S.K., D.J.T., and G.L. All authors participated in manuscript preparation and provided final approval of the submitted work.Supplementary MaterialClick here for additional data file."}
+{"text": "The NPTG and HPTG performed 50 min of Pilates training using a tubing band for 12 weeks (3 days/week) in their respective environmental conditions  = 20.9%; HPTG: moderate hypoxic condition, FiO2 = 14.5%). The CON maintained their daily lifestyle without intervention. All subjects underwent body composition, blood pressure, arterial stiffness, vascular endothelial function, cardiometabolic biomarker, hemorheological function, and aerobic performance measurements before and after the intervention. The HPTG showed a significant improvement in diastolic blood pressure, total cholesterol and triglyceride concentrations, flow-mediated dilation, and erythrocyte deformability and aggregation  compared with the CON and NPTG. However, compared with the CON and NPTG, the HPTG did not show improvement in other parameters. Hypoxic Pilates intervention is a novel and successful method for promoting endothelial and hemorheological functions in women with obesity.This study examined the effect of Pilates training under hypoxia, a novel treatment method, for obesity. Thirty-two Korean women with obesity (age: 34\u201360 (47.5 \u00b1 7.5) years) were randomly assigned to control (CON;  Obesity, a phenomenon caused by excessive body fat accumulation, is recognized as a global health problem and is reported to increase the prevalence and risk of death because it is a major cause of various metabolic and cardiovascular diseases . AccordiThe prevalence rate of obesity in South Korea is increasing rapidly with age   \u00d7 100%) were used to evaluate the diameter and blood flow rate were used.All cardiometabolic biomarkers  were analyzed by the Seegene medical foundation (an organization certified by the Korea government). The concentrations of TC, HDL-C, LDL-C, TG, FFA, glucose, and insulin were quantified. An 8-mL sample of venous blood was collected into a serum separating tube (SST) for serum. Clot formation was ensured in the SST by centrifuging the sample at 3500 rpm for 10 min. The TC concentration was determined by an enzyme kinetic assay using the Cobas C702 . The HDL-C and LDL-C concentrations were detected by homogeneous enzymatic colorimetric assay using the Cobas C702 . The glucose concentration was determined by an enzyme kinetic assay using the Cobas8000 C702  and the insulin concentration was detected by an electrochemiluminescence immunoassay using the Cobas8000 e602 . HOMA-IR and HOMA-\u03b2 were calculated using the following formulas: HOMA-IR = (glucose [mg/dL] \u00d7 insulin [\u00b5U/mL])/405 and HOMA-\u03b2 = (360 \u00d7 insulin [\u00b5U/mL])/. A heart rate (HR) sensor was attached to the earlobe, and each subject\u2019s information  was entered into a bicycle ergometer. Thereafter, following a break until HR stabilization, subjects performed the exercise. All subjects exercised on the bicycle ergometer at a rate of 50 rpm until the HR reached 75% maximal heart rate (HRmax) by the Lamb protocol . VO2max was calculated using the regression equation (VO2 = 9.386 watt + 289.6) [Estimated VO+ 289.6) .t-test was performed to compare the post-intervention versus pre-intervention values of dependent parameters in each group separately. We used Cohen\u2019s d (effect size), which reflects the value of a statistic calculated from a sample of data and standardized mean differences. Statistical difference in the means  was determined with significance level (p < 0.05) and 95% confidence interval (CI). Cohen\u2032s d effect size  were used to assess the significant effects. All analyses were performed using SPSS Statistics 21.0 .Means and standard deviation were calculated for each primary dependent parameter. The normality of distribution of all outcome parameters was verified using the Shapiro\u2013Wilk W-test prior to the parametric tests. A two-way analysis of covariance (\u201cgroup\u201d \u00d7 \u201ctime\u201d) with repeated measures for time-dependent pretest value was performed to analyze the effects of the 12-week intervention on each dependent parameter. If a significant interaction or main effect within time was found, a Bonferroni post-hoc test was performed to identify within-group change over time. Additionally, the paired p = 0.032, \u03b72 = 0.154) and BMI   . Howeverp = 0.006, \u03b72 = 0.244), DBP , and MAP . Post-hoc analysis revealed a significant decrease in DBP  in the HPTG. However, post-hoc analysis revealed no significant difference in SBP and MAP due to the 12-week intervention.p = 0.005, \u03b72 = 0.315). Additionally, significant main effects within time were found for TC , FFA , and glucose . Post-hoc analyses revealed a significant decrease in the TC  and TG  concentrations in the HPTG and a significant increase in the glucose concentration in the NPTG .The pre- and post-intervention concentrations of cardiometabolic biomarkers in all groups are presented in p < 0.001, \u03b72 = 0.841), which corresponds to vascular endothelial cell function (p < 0.05) and significant enhancement of FMD in the NPTG  and HPTG . However, a greater improvement in FMD was observed in the HPTG than in the NPTG (NPTG vs. HPTG: 15.6% vs. 51.2%).A significant interaction was found for FMD . Additionally, a significant main effect within time was found for the AI_3 Pa . Post-hoc analyses revealed a significant improvement in the erythrocyte EI_3 Pa  and erythrocyte AI_3 Pa  in the HPTG.2max. Compared with the control and normoxic Pilates intervention, the moderate hypoxic Pilates intervention for 12 weeks had no effect on aerobic performance (estimated VO2max).Aerobic performance of all groups is presented in iO2: 14.5%) enhanced the endothelial and hemorheological functions in women with obesity. Additionally, hypoxic Pilates training improved DBP and the TC and TG concentrations. However, no changes were found in body composition and aerobic performance.In this study, compared with the 12-week Pilates intervention under normoxia, the 12-week Pilates intervention under moderate hypoxia  on body composition [Hypoxic training has been designed to improve the performance of individual- and team-sports athletes ,24,25,26position ,32. Howeposition . Hence, iO2 and 21% FiO2, with the heart rate measured at 3 mmol/L lactate during pre-exercise testing. Consequently, the hypoxic training group showed greater improvements in the TG and insulin concentrations, HOMA-index, and area under the curve for insulin during the oral glucose tolerance test, although the absolute workload under hypoxia (1.4 watts/kg) was lower than that under normoxia (1.7 watts/kg). Wisner et al. [2max under moderate hypoxia (FiO2: 15%) was effective in decreasing body fat mass, insulin level, and HOMA-index, although the absolute exercise load under hypoxia was lower than that under normoxia. In the present study, we confirmed that hypoxic Pilates training was more effective in reducing the TC and TG concentrations than normoxic Pilates training. Our findings on cardiometabolic risk factors are consistent with those of previous studies. However, no outstanding change in other cardiometabolic biomarkers  was noted. This is thought to be because of our Pilates program, which did not provide sufficient stimulation to cardiometabolic function, and therefore, had a lower exercise intensity than other aforementioned aerobic exercises.Exercise under hypoxia results in a higher cardiometabolic response than that under normoxia for similar workloads ,33. The r et al.  reportediO2: 14%) during exercise on BP, NO metabolites, and hypoxia inducible factor-1 alpha (HIF-1\u03b1) over a 6-week period. They confirmed that exercise under hypoxia reduces SBP and increases the NO metabolites and HIF-1\u03b1 concentrations. These results suggest that exercise under moderate hypoxia can be used as an alternative therapeutic strategy for preventing and treating obesity by improving endothelial and hemorheological functions. Based on this mechanism, our present research team previously investigated the effect of an acute Pilates program under moderate hypoxia on vascular endothelial function in Pilates participants [An exercise training program induces augmented blood flow and subsequently increases vasodilation and upregulates endothelial NO synthase. The release of various vasodilators such as adenosine, prostaglandin, and NO during exercise contributes to the mechanism responsible for vasodilation. Particularly, hypoxia induces an increase in NO bioavailability resulting in vasodilation and a reduction in total systemic vascular resistance and thereby BP ,38. Moreicipants . Althougicipants , hypoxicicipants . In the 2max) was noted. This is because the Pilates intervention performed in the present study had a low positive impact on cardiopulmonary, vascular, and hemorheological functions owing to lower workload than those (moderate-intensity aerobic exercise programs) in previous studies. Therefore, we believe that providing a higher-workload Pilates program for the Pilates intervention under moderate hypoxia will have a more pronounced positive effect on body composition, cardiovascular risk factors, and aerobic performance.However, although vascular endothelial and hemorheological function improved, no remarkable positive effect of the Pilates intervention under moderate hypoxia on aerobic performance (estimated VOSome limitations of the present study should be considered when interpreting results. The normality of distribution of all outcome parameters was verified using the Shapiro\u2013Wilk W-test prior to the parametric tests, however the small sample size was a limitation to elicit the effectiveness of hypoxic Pilates intervention on improving endothelial and hemorheological function in obese women. In addition, the trial period in the present study was relatively short of 12-weeks, and the dietary intake and daily activity of participants during the intervention period were not investigated .The 12-week Pilates intervention under moderate hypoxia elicited a decrease in BP and the TC and TG concentrations and an increase in FMD and erythrocyte deformability and erythrocyte aggregation in women with obesity compared with the Pilates intervention under normoxia. However, it did not affect the body composition, arterial stiffness, and aerobic performance owing to a lower workload than that in the moderate intensity aerobic exercise program. Thus, our findings suggest that the 12-week Pilates intervention under moderate hypoxia improved cardiometabolic parameters, vascular endothelial function, and hemorheological function in the present study. The hypoxic Pilates intervention may be an efficient exercise strategy for preventing and treating obesity by improving endothelial and hemorheological functions and minimizing the possibility of injury to joints and muscles."}
+{"text": "The bilirubin nanoparticles improved liver function and activated the hepatic \u03b2-oxidation pathway by increasing PPAR\u03b1 and acyl-coenzyme A oxidase 1. The bilirubin nanoparticles also significantly elevated plasma levels of the ketone \u03b2-hydroxybutyrate and lowered liver fat accumulation. This study demonstrates that bilirubin nanoparticles induce hepatic fat utilization, raise plasma ketones, and reduce hepatic steatosis, opening new therapeutic avenues for NAFLD.The inverse relationship of plasma bilirubin levels with liver fat accumulation has prompted the possibility of bilirubin as a therapeutic for non-alcoholic fatty liver disease. Here, we used diet-induced obese mice with non-alcoholic fatty liver disease treated with pegylated bilirubin (bilirubin nanoparticles) or vehicle control to determine the impact on hepatic lipid accumulation. The bilirubin nanoparticles significantly reduced hepatic fat, triglyceride accumulation,  Therape burning .\u03b2-oxidation and fat utilization, which reduces hepatic fat and NAFLD (\u03b2-hydroxybutyrate (BOHB)  . The BOHe (BOHB) . Fat acced NAFLD . While ied NAFLD . While ped NAFLD . These sin vivo models  and promote the formation of highly water-soluble bilirubin nanoparticles . PEG-BR o models . However\u03b2-oxidation, fat utilization, and BOHB production using bilirubin nanoparticles. We found that hepatic PPAR\u03b1 induced and liver fat content was lower, which correlated with higher plasma BOHB levels and lower serum triglycerides. Our results demonstrate a possible role for bilirubin nanoparticles in the protection against obesity-induced fatty liver disease.Here, we wanted to determine bilirubin\u2019s functionality on obesity-induced hepatic steatosis and NAFLD and determine whether it activates Animals. The experimental procedures and protocols of this study conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. C57BL/6J mice were purchased from Jackson Labs  and placed on a 60% high-fat diet  for 24\u00a0weeks with full access to tap water. After this time, mice were randomly assigned to either a treatment group consisting of pegylated bilirubin nanoparticles  or vehicle  for 4\u00a0weeks while continuing on the high-fat diet.Pegylated bilirubin synthesis. The synthesis of PEG-BR was done at the Research Institute of Pharmaceutical Sciences at the University of Mississippi . The PEG-BR was prepared from bilirubin-IX-alpha  and mPEG2000-NH2  as previously described . The sizLiver composition. Liver composition was measured at the end of the study using magnetic resonance imaging . MRI measurements were performed on whole livers placed in a thin-walled plastic cylinder. Liver fat and lean mass were obtained and expressed as a percent of total liver weight.g) for 2\u00a0min. Tissue triglyceride levels were measured using a colorimetric assay kit according to manufactures\u2019 guidelines . Tissue triglyceride are expressed as mM. Samples from individual mice were run in duplicate and averaged, and the averages used to obtain group averages.Liver triglyceride measurement. Triglycrides were measured from 100\u00a0mg of liver tissue homogenized in 1\u00a0ml of 5% NP-40 in water. Homogenized tissues were then heated to 95\u00b0C for 5\u00a0min and then centrifuged  staining were performed as previously described . The OilAST/ALT measurements. Plasma alanine transaminase (ALT) and aspartate transaminase (AST) were measured using a Vet Axcel serum chemistry analyzer  from 30\u00a0\u03bcL of plasma. Samples were measured in duplicate with standards supplied by the manufacturer. Data are presented as Units (U)/L.Quantitative Real-Time PCR Analysis. Total RNA was harvested from the animals by lysing livers using a Qiagen Tissue Lyser LT  and then extraction by 5-Prime PerfectPure RNA Tissue Kit . Total RNA was read on a NanoDrop 2,000 spectrophotometer  and cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit . PCR amplification of the cDNA was performed by quantitative real-time PCR using TrueAmp SYBR Green qPCR SuperMix  for gene-specific primers as previously described . The theg at 4\u00b0C. Protein samples were resolved by SDS polyacrylamide gel electrophoresis and electrophoretically transferred to Immobilon-FL membranes. Membranes were blocked at room temperature for 2\u00a0h in TBS (10\u00a0mM Tris-HCl (pH 7.4) and 150\u00a0mM NaCl) containing 3% BSA. Subsequently, the membranes were incubated overnight at 4\u00b0C with the following antibodies: ACOX1 , fatty acid synthase (FAS) , SCD1, or heat shock protein 90 (HSP90) . After three washes in TBS + 0.1% Tween 20, the membrane was incubated with an infrared anti-rabbit  or anti-mouse  secondary antibody labeled with IRDye infrared dye (LI-COR Biosciences)  for 2\u00a0h at 4\u00b0C. Immunoreactivity was visualized and quantified by infrared scanning in the Odyssey system (LI-COR Biosciences).Gel Electrophoresis and Western Blotting\u2014Mouse tissues were flash frozen in liquid nitrogen during harvesting and stored at \u221280\u00b0C. For gel electrophoresis, 50\u2013100\u00a0mg of cut tissue was then resuspended in three volumes of CelLytic Buffer  plus 10% protease inhibitor cocktail  and Halt phosphatase inhibitor cocktail , and then incubated on ice for 30\u00a0min. The livers were lyzed using a Qiagen Tissue Lyser LT  and then centrifuged at 100,000 \u00d7 In-Vitro Diagnostics research (IVDr) protocol. Lipoprotein subclass analysis was performed using regression analysis of the NMR data as previously described  spectroscopy as part of the Bruker IVDr platform , as previously described . Plasma escribed .p < 0.05 was considered to be significant. All analyses were performed with GraphPad Prism eight software .Statistics. All bar graph data are presented as mean \u00b1 S.E.M. Box and whisker plots display whiskers from the minimum or maximum, with a vertical line in the box to indicate the median. Differences between treatment groups were determined using student t-test or one-way analysis of variance with a post hoc test (Dunnett\u2019s). A p = 0.2823). After the 4-weeks treatment, the plasma bilirubin levels were increased in the PEG-BR treated (0.45 \u00b1 0.08\u00a0mg/dl) compared to the vehicle (0.13 \u00b1 0.05\u00a0mg/dl) (p < 0.0001). After the 4-weeks PEG-BR and vehicle treatments, the percent body weight change was greater in the vehicle but not the PEG-BR (103.4% \u00b1 2.5 for vehicle and 96.3% \u00b1 2.7 for PEG-BR) (p = 0.0058). The PEG-BR group had a 7% reduction in body weight gain compared to the vehicle treated animals. There was no difference between the groups for the liver weight  reduced expression compared to VEH (p < 0.001) reduced with PEG-BR compared to VEH groups. The ALT level was also reduced with PEG-BR was not reduced but showed a trend toward reduction that did not reach statistical significance. Overall, these data demonstrate that PEG-BR improved hepatic function and reduced inflammation.To better understand how the bilirubin nanoparticles impact hepatic function, the livers from both groups of mice were analyzed by hematoxylin and eosin (H&E) staining for observable differences in treated animals and hepatic dysfunction biomarkers. The H&E staining revealed that the VEH treated animals possibly had inflammation, which were found to be lower in the PEG-BR treated group . This wad to VEH . We measd to VEH . However\u03b2-hydroxybutyrate (3-hydroxybutyric acid) were measured in the two groups. Analysis showed that only elevated plasma BOHB levels had a significant (p < 0.05) difference for PEG-BR treated compared to the vehicle groups  mRNA expression was increased in the PEG-BR treated animals compared to the vehicle group  higher in the PEG-BR compared to vehicle-treated animals. Also, the PPAR\u03b1-target genes for long-chain fatty acid transporters FATP1 (gene Slc27a1) and FATP2 (gene Slc27a2)  reduced with PEG-BR (p = 0.0137) higher in the PEG-BR treated animals compared to the vehicle (de novo lipogenesis proteins fatty acid synthase (FAS) and stearoyl-Coenzyme A desaturase 1 (SCD1) were significantly  reduced with PEG-BR compared to vehicle treatments. These indicate that PEG-BR enhances fatty acid uptake and lipid utilization for hepatic \u03b2-oxidation increasing plasma BOHB and inhibits de novo lipogenesis, improving fatty liver.Next, we determined how PEG-BR treatments affect plasma metabolites of amino acids by NMR spectroscopy using the Bruker IVDr platform. We found no significant differences in amino acids measured in the plasma of the PEG-BR or vehicle-treated groups . Exercise groups . There wle group . Huang en, Acox1 , and p45pathways . Here, w Cyp4a12  were sigSlc27a2) , were siSlc27a2) . The liph PEG-BR . Immunob vehicle . The de PparaHepKO) had worsened hepatic steatosis on a HFD that also caused significantly higher plasma triglycerides and ApoB100 levels  expression  lower in the PEG-BR compared to vehicle treated animals  enzyme that forms fatty acids and lipid synthesis  improved NAFLD, which is potentially due to fat-burning mechanisms, as demonstrated by reduced hepatic triglyceride content, changes in gene expression in line with increased fat utilization and to PPAR\u03b1 , which ixidation  that supxidation . Patientxidation , which iynthesis . A deficynthesis . Activatde novo lipogenesis and activation of \u03b2-oxidation  on a HFD had no changes in serum cholesterol, HDL, LDL, or ApoA proteins  or HFD showed no differences (Mttp) assists ApoB100 for excretion of the VLDL molecule from the liver  and 1R01DK126884-01 (DS), the National Heart, Lung and Blood Institute K01HL-125445 (TH) and P01 HL05197-11 (DS), and the National Institute of General Medical Sciences P20GM104357-02 (DS). This project was partially supported by Grant Number P30GM122733 (Chemistry and DMPK Core Faculty)-funded by the National Institute of General Medical Sciences (NIGMS) a component of the National Institutes of Health (NIH) as one of its Centers of Biomedical Research Excellence (COBRE). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.TH and DS have submitted patents on bilirubin and obesity related disorders.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."}
+{"text": "Cognitive disorders are increasing in prevalence. Nutritional or metabolic stressors during early life, and female sex, are predisposing conditions towards the development of cognitive diseases, including Alzheimer\u2019s disease. Though there is evidence that breastfeeding may play a beneficial role in children\u2019s neurocognitive development, the literature remains controversial. In this study we aimed at assessing the association between exclusive breastfeeding and children\u2019s cognitive development from six months to five years of age, addressing sex differences. In 80 mother-child pairs from the Pisa birth cohort (PISAC), we measured cognitive development in groups of children of 6, 12, 18, 24, 36, and 60 months by Griffiths Mental Development Scales, parents\u2019 intelligence quotient (IQ) by Raven\u2019s progressive matrices, and maternal and infants\u2019 anthropometric parameters. We found that exclusive breastfeeding was associated with higher hearing-language development in five years old girls, independent of maternal IQ, age and BMI (body mass index). Exclusive breastfeeding in the first three months of life seemed sufficient to establish this positive relationship. In conclusion, our data indicate that exclusive breastfeeding is a positive predictor of cognitive development in preschool-age girls, paving the way for the implementation of sex-specific cognitive disease risk detection and prevention strategies from early life. Further studies are warranted to explore causality and longer term effects. Exposure to metabolic, stress and nutritional factors during early life can shape neuronal development and brain function in later life. Among these factors, early feeding, in particular lactation, has raised research interest because the developing brain is greatly susceptible to postnatal nutritional deficits ,2.Breastfeeding was shown to be protective against later development of obesity and metabolic diseases in offspring when compared to formula feeding ,4,5. ConSeveral methodological issues can contribute to explain discrepancies among studies. One source of heterogeneity is represented by the different definitions of breastfeeding. Breastfeeding can be measured by duration, exclusivity or volume of breast milk consumed. The duration criterion carries an intrinsic confounder due to the frequent addition of formula in variable proportions (and different brands), whereas the objective measurement of breast milk consumed is technically very challenging.Other methodological issues include the diverse children\u2019s ages and the methods used for assessing children\u2019s cognition, as well as the confounders that need to be considered . PreviouIn the present study, we aimed at assessing the relationship between breastfeeding and cognitive development from the age of six months to five years in a subset of the Pisa birth cohort (PISAC), including families that were recruited during pregnancy and followed-up to the children\u2019s age of five years.To overcome the above biases, we stratified our population in two groups, separating children who had received exclusively breast milk from those who had received any combination of breast and formula milk before weaning. Moreover, we measured cognitive scores of both parents and children objectively, by standard tests, and we recorded mothers\u2019 and offsprings\u2019 body sizes as potential confounders. We focused on preschool age to avoid e.g., exposure to the school environment and other social stimuli, which make it more difficult to dissect early determinants of cognitive development. Finally, we accounted for the epidemiological observation that women have a higher risk to develop cognitive disease, including Alzheimer\u2019s disease . Sex infThe study was conducted in the Pisa birth cohort (PISAC). The cohort includes 90 families, i.e., mothers, fathers, and infants born 2011\u20132014, 42/48 females/males, followed up from birth. Families were recruited at the beginning of pregnancy visits or at delivery (41/49) through hospital pregnancy visits in the area of Massa, Tuscany, Italy, to investigate the effects of maternal obesity during pregnancy on offspring cardiometabolic and cognitive health. The cohort is intended to represent the general population, and therefore inclusion criteria are broad, namely (1) mothers within the first trimester of pregnancy at the first visit or at delivery; (2) any parents\u2019 age; (3) any BMI; (4) willingness of mothers and fathers to participate and to actively collect questionnaires and samples; (5) capacity of mothers and fathers to understand the study and its implications; (6) signature of the informed consent by mothers and fathers and (7) absence of major diseases (mothers and fathers) and perinatal complications. Exclusion criteria are (1) history of major diseases in the mother and in the father ; (2) major health complications during the perinatal period and (3) failure to understand study implications, comply with the study schedule or sign the consent form. Follow-up visits were carried out from birth to the children\u2019s age of five years and included measurement of anthropometric parameters, blood pressure, complete echocardiographic evaluation, cognitive assessment and collection of nutritional questionnaires and biological samples . Families were given the option to participate in all or only part of the assessments and measurements included at each age-point. In addition, we prioritized different health aspects at different age points, namely cardiac health in the first two years and cognitive health in preschool five-year old children. Therefore, the sample size varied across age point and measurements. At age five years, all families were called for a new follow-up round, only six decided to drop-out, while the others participated in all or some measurements, or asked to be considered for future follow-up rounds. The cognitive follow-up at five years included 56 mother-offspring dyads that were homogenous with the enrolled population in terms of mothers\u2019 age (34.0 \u00b1 0.5 vs. 33.3 \u00b1 0.5 years) and fathers\u2019 age (36.6 \u00b1 0.6 vs. 36.2 \u00b1 0.5 years). Distribution of mothers\u2019 or fathers\u2019 job categories was not related to the type of feeding categories , nor to adherence to any of the cognitive follow-up visits. Body sizes were collected at the beginning and during pregnancy in mothers, and in mothers and infants at multiple time points after delivery. In women, and in 36 and 60 months-old children, body weight (in kg to the nearest 0.1 kg) and length (in cm to the nearest 0.5 cm) were measured by weight scale and stadiometer, with participants wearing light clothes and standing straight without shoes and with heels close together. In infants up to age two years, weight was measured by using a hospital-grade pediatric weighting scale (in kg to the nearest 0.01 kg) removing clothes, shoes and diaper from the infants, and recumbent length (in cm to the nearest 0.1 cm) was recorded. Additional body size measurements were collected from pediatric records. Information on feeding was collected at three and six months, children\u2019s and parents\u2019 cognitive scores were measured starting from the infants\u2019 age of six months, as reported in the following paragraph.The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Massa and Carrara, and latest amendments by the Ethical Committee of the Area Vasta Nord-Ovest (CEAVNO), Pisa, Italy . Parents gave their written informed consent before inclusion.Information on early feeding were collected via parents\u2019 interview during follow-up visits or by phone at three and six months of age, and they were used to stratify children in two groups, i.e., one receiving exclusive and one receiving nonexclusive breastfeeding before weaning. Infants receiving only maternal milk  before the introduction of any complementary food were defined as exclusively breastfed. Conversely, the nonexclusive breastfeeding category included infants who received only formula milk or a combination of breast and formula milk in any proportion. Eighty of the 90 families provided information on early feeding practices and were included in this work. These 80 families were homogenous with the enrolled population in terms mothers\u2019 age (33.4 \u00b1 0.5 vs. 33.3 \u00b1 0.5 years) and fathers\u2019 age (36.4 \u00b1 0.5 vs. 36.2 \u00b1 0.5 years).N = 24, 26, 26, 23, 27). At the age of five years, cognitive development was measured again in a larger subset (N = 56). The GDMS measures the rate of development of infants and young children in six domains, i.e., locomotor, personal-social, hearing and language, eye and hand coordination, performance and practical reasoning domains (the latter from age 36 months). Mothers\u2019 and fathers\u2019 intelligence quotients (IQ) were measured by the Raven\u2019s progressive matrices .Children\u2019s cognitive development was assessed by a trained childhood psychologist by using the Griffiths Mental Development Scales (GMDS) in subgroups of children at the age of 6, 12, 18, 24 and 36 months  was used to incorporate covariates . To assess the sex-specific relationship between breastfeeding and cognitive development, we repeated each analysis in girls and boys separately. The Chi-Square test was applied to examine relationships between categorical variables. These tests were selected to capture group differences in cognitive outcomes at each separate age point, maximizing power. Considering that not all children had cognitive measures at all-time points, changes over time by repeated measure testing was not performed to avoid sample size reduction. Results are presented as mean \u00b1 sem, and p-values \u2264 0.05 were regarded as statistically significant.SPSS for Mac  was used for statistical analyses. Variables distribution was tested by the Shapiro-Wilk test. A two-tailed n = 26) received both maternal and formula milk between birth and weaning (mean age 5.4 \u00b1 0.1 months in the whole population). Exclusive formula milk feeding from birth to weaning occurred in 35% (n = 14), while 27% (n = 11) of the infants were started on exclusive formula feeding at the age of three months. The prevalence of maternal gestational diabetes, as well as children\u2019s sex distribution and delivery mode were not different between the two groups of children.In our population, 50% of children were exclusively breastfed. General characteristics of the stratified population are reported in p = 0.046) and at five years in the eye and hand coordination domain (102.4 \u00b1 2.7 vs. 97.3 \u00b1 1.7 p = 0.040) and in the performance (114.0 \u00b1 1.3 vs. 110.5 \u00b1 1.3 p = 0.025) domain.Cognitive scores measured in six different domains were in the normal range , both inp = 0.049) and locomotor development at 36 months (p = 0.036). In separate sex group analyses , with no significant difference in other scales and other ages.In the whole population, accounting for sex as a covariate, exclusive breastfeeding was associated with higher scores in hearing and language at six months  and at 36 months , with practical reasoning at 36  and at 60 months , and with personal-social scores at 36 months , while being negatively correlated with the infants\u2019 personal-social  and performance  scores at six months. Fathers\u2019 IQ was directly associated with offspring performance at 36 months  only. However, the IQ was measured in a smaller subset of fathers, and was not used in covariate analysis to avoid sample size and power reduction.In our population, the maternal IQ was directly associated with the hearing and language scores at 18 months  and negatively with the locomotor  and performance  scores at six months. Fathers\u2019 age was negatively correlated with the locomotor score at 36 months .Parental age was also related to children\u2019s cognitive development. In particular, maternal age was directly associated with the performance score at 60 months , in which exclusive breastfeeding was able to explain 20% of the difference between children\u2019s groups. Likewise, in sex-specific analyses, the adjustment for maternal IQ and parents\u2019 age did not abolish the difference observed in hearing and language scores at five years in girls , in which exclusive breastfeeding was able to explain 22% of the difference between groups.In the whole children\u2019s population, the adjustment for maternal IQ and/or parents\u2019 age abolished the association between exclusive breastfeeding and locomotor scores at 36 months, but not the association between exclusive breastfeeding and hearing and language scores at six months , suggesting that the first trimester of life may be sufficient to predict the development of hearing and language skills in girls.p-values between 0.012\u20130.040 in sex-specific analyses). However, the maternal BMI did not correlate with cognitive outcomes in the offspring at any time point. Instead, a lower weight gain in infants between three and six months of age was associated with exclusive breastfeeding , and negatively correlated with hearing and language scores at five years in the whole population , showing a trend in females .Exclusive breastfeeding was significantly less common in women with a high than low BMI in our maternal population , indepenChildren\u2019s body weight, length (not reported), BMI and cranial circumferences at birth and at 3, 6, 12, 18, 24, 36 and 60 months of age were similar between the two groups and not related to cognitive outcomes.p = 0.033) and of children\u2019s weight gain in the first six months of life (p = 0.04), accounting for 20% of the group difference  shows a strong and positive association with hearing and language scores in girls reaching the age of five years. Moreover, exclusive breastfeeding in the first three months of life seems sufficient to observe this effect.We also showed that exclusive breastfeeding is associated with lower maternal BMI and lower infants\u2019 weight gain between three and six months of age. A higher weight gain in this time window negatively predicts hearing and language scores at five years of age. Moreover, parents\u2019 IQ and age predict offspring cognitive development. However, none of these factors affected the main finding.Several authors have explored the role of early feeding practices on children\u2019s cognitive development, producing sparse and controversial results. Some studies found positive associations between breastfeeding duration and exclusivity and cognitive and school achievements during childhood ,7,8,9 anAnother source of discordant results is given by the heterogeneous definitions of breastfeeding and the lack of adjustment for important confounders. For example, in an old study, no difference in phonologic development at the age of three to four years was observed between children who had been breastfed or bottle fed . A largeSeveral authors ,17 suggeThe duration of breastfeeding, besides its exclusivity, could also be relevant to predict children\u2019s cognitive development. Stratification of our population based on exclusive breastfeeding at three rather than six months, resulted in similar associations between breastfeeding and cognitive outcomes, which suggests that the first three months could be sufficient to positively predict hearing and language development. In agreement with our results, exclusive breastfeeding even for only three months was related to higher intelligence quotient in more than 450 Polish children of prescholar age . In the Our results extend previous observations in preschool children since we explored multiple ages within the first five years of life. One additional novel observation is the sex difference emerging from our study. In fact, we showed a positive independent correlation between exclusive breastfeeding and cognitive outcomes in girls but not in boys. The analysis of specific differences between males and females is mostly neglected in the literature addressing early nutrition. It is now established that neurodevelopment during fetal and subsequent life is influenced by hormones ,33, and Obesity, either in the mother during pregnancy or in the children, is considered a negative predictor of cognitive outcomes ,20,38,39In our study, exclusive breastfeeding was significantly less common in women with a high than low BMI, in line with the established inverse association between maternal obesity and low rates of initiation, duration and exclusivity of breastfeeding, as published by Li et al.  in a popOf note, in our study the positive correlation between exclusive breastfeeding and hearing and language scores in five-year old girls were independent of the above negative predictors, i.e., maternal pregravid and gravid BMI, and children\u2019s weight gain.n = 56 children of five years. In addition, multiple correlations bear the risk of chance findings, though our main observation was robust and remained fully significant after adjusting for confounders. The second limitation, which is a frequent criticism in breastfeeding targeted research, is the intrinsic bias of feeding groups, since infants are not randomly allocated to groups, and parents\u2019 and family characteristics may be responsible for both feeding choices and cognitive outcomes. To mitigate this risk, we controlled for maternal features associated with cognitive scores and/or feeding practices, i.e., BMI and IQ. The strengths of our study are that both parents\u2019 and children\u2019s cognitive scores were objectively measured and not estimated from proxy variables such as school or job attainment or questionnaires, and that cognition was examined at different ages in preschool children.We acknowledge that our study has limitations. The small sample size, and the fact that most children could not undergo all repeated measures, might have prevented our analyses from finding more significant differences among cognitive domains before the age of five years. In fact, relationships were stronger in the most numerous group of In conclusion, we found that exclusive breastfeeding predicts higher hearing and language outcomes in five-year old girls, independent of parents\u2019 age, maternal weight and IQ, or children\u2019s weight and weight gain. Knowing that cognitive decline is frequent in women, our finding of a sex-selective association offers an opportunity to identify individuals at risk in a very early stage of life, requiring nutritional attention. Considering that formula feeding is not a choice of families in many cases, but depends on an insufficient production of breast-milk, or the return to work after the first postnatal months, it was important to show that three months of breast-feeding already predicted the beneficial outcome. Caution should be used in the interpretation of data, which are mainly correlative and do not imply causality or long term benefits, representing the challenges of future studies."}
+{"text": "Three-dimensional culture systems and suitable substrates topographies demonstrated to drive stem cell fate in vitro by mechanical conditioning. For example, the Nichoid 3D scaffold remodels stem cells and shapes nuclei, thus promoting stem cell expansion and stemness maintenance. However, the mechanisms involved in force transmission and in biochemical signaling at the basis of fate determination are not yet clear. Among the available investigation systems, confocal fluorescence microscopy using fluorescent dyes enables the observation of cell function and shape at the subcellular scale in vital and fixed conditions. Contrarily, nonlinear optical microscopy techniques, which exploit multi-photon processes, allow to study cell behavior in vital and unlabeled conditions. We apply confocal fluorescence microscopy, coherent anti-Stokes Raman scattering (CARS), and second harmonic generation (SHG) microscopy to characterize the phenotypic expression of mesenchymal stem cells (MSCs) towards adipogenic and chondrogenic differentiation inside Nichoid scaffolds, in terms of nuclear morphology and specific phenotypic products, by comparing these techniques. We demonstrate that the Nichoid maintains a rounded nuclei during expansion and differentiation, promoting MSCs adipogenic differentiation while inhibiting chondrogenesis. We show that CARS and SHG techniques are suitable for specific estimation of the lipid and collagenous content, thus overcoming the limitations of using unspecific fluorescent probes. Mesenchymal stem cells (MSCs) are largely used in regenerative medicine thanks to their location in several tissues , to their multilineage potential and immunomodulatory effect ,2. MSCs Several biochemical techniques are conventionally used to identify markers of cell differentiation, such as real-time polymerase chain reaction (RT-PCR), DNA sequencing, transcriptome analyses, and protein content (Western blotting) and cyto/histopathological assays. Despite their high information content, these techniques require invasive and, often, destructive treatments like fixation, permeabilization, and extraction of lysates. Optical techniques, on the other hand, are non-destructive and enable three-dimensional observations of viable cells and the study of their functional and morphological properties. Confocal fluorescence is the gold standard in optical microscopy, widely employed in biological laboratories, relying on a wide variety of dyes for live measurements and labelled antibodies for immunofluorescence studies. However, the addition of fluorescent labels can in some cases affect the biochemical phenomenon of interest. Label-free functional biological assessments are useful to overcome these limitations. Nonlinear optical (NLO) microscopy comprises several modalities such as: two-photon excited fluorescence (TPEF) , second-In this work, we study MSCs grown in an innovative 3D scaffold, named Nichoid, designed by our group ,29,30 prTo investigate the effect of the Nichoid scaffold on MSCs morphology during adipogenic and chondrogenic differentiation, fluorescence analyses were performed since the scaffold allows optical inspections. a and b, and with a vertical semi-axis, c. The effect of the Nichoid scaffold on MSCs nuclei resulted in a more compact nuclear shape with a tendency to induce a spherical morphology since the ratio between the minor (b) and the vertical (c) semi-axes and the major (a) and the vertical (c) semi-axes is higher than 0.5 , a long-chain dialkylcarbocyanine commonly used to stain lipids in live cells, for fluorescence confocal microscopy  was employed  was provided in addition to nuclear (Hoechst 33342) and F-actin  staining A,B. HighF-actin organization and nuclear shape. In this study an investigation of the effect of Nichoids on MSCs during long-term differentiation was performed by optical techniques comparing fluorescence microscopy with label-free and vital nonlinear optical microscopy. Adipogenesis and chondrogenesis of MSCs were considered since tissue characteristics are slightly different in stiffness and genesis  and seve\u22121 enabled a time-evolution study of adipogenic differentiation, replicating the trend of oil red-O analysis in viable and unstained 3D samples. The analysis of CARS images allowed to quantify and measure the size of the lipid vesicles, thus suggesting that these underwent a fusion from the first to the second week of differentiation, becoming larger . Nichoids were fabricated in serial blocks constituted by squared matrixes of 25 elements, 450 \u03bcm \u00d7 450 \u03bcm \u00d7 30 \u03bcm each, to cover a whole circular area of 8 mm in diameter. Glass substrates were previously covered with 30 \u03bcL of SZ2080 photoresist by drop-casting and then exposed to localized infrared radiation to polymerize single structures. Nichoid microfabrication through two-photon polymerization was performed as detailed in . To remo2 . MSCs were differentiated towards adipogenic and chondrogenic phenotypes by adding a specific cocktail of growth factors to a basal medium composed by Dulbecco\u2019s modified essential medium (DMEM) phenol red-free  supplemented with 10% of fetal bovine serum, 1% L-glutamine (2mM), 1% penicillin (10 units/mL), and streptomycin (10 \u00b5g/mL)).Rat bone marrow-derived MSCs at low passages (maximum passage 2) were initially expanded with standard alpha-MEM phenol red-free culture medium  supplemented with 20% of fetal bovine serum, 1% L-glutamine (2mM), penicillin (10 units/mL), and streptomycin (10 \u00b5g/mL) at 37 \u00b0C and in 5% CO2 on Nichoids and 10,000 cells/cm2 on glass flat substrates. Cells were maintained in culture with a medium supplemented with 5 \u03bcg/mL human insulin , 1 \u03bcM dexamethasone (Sigma-Aldrich), 0.5 \u03bcM 3-isobutyl-1-methylxanthine (Sigma-Aldrich) and 50 \u03bcM indomethacin (Sigma-Aldrich) for 72 h and, after, with insulin for 24 h. This procedure was cyclically repeated for the 2 weeks of the experiment [MSCs adipogenic differentiation was performed by seeding 20,000 cells/cmperiment .2 increased in a second experiment to 50,000 cells/cm2 on Nichoids and 5000 cells/cm2 on glass flat substrates. MSCs were fed for 3 weeks with a chondrogenic induction medium made by mixing 100 nM dexamethasone, 10 ng/mL transforming growth factor-\u03b21 , insulin-transferrin-selenium-G 1X (Thermo Fisher), 50 \u03bcg/mL ascorbic acid\u20142-phosphate to the basal medium [MSCs chondrogenic differentiation was obtained by seeding initially 20,000 cells/cml medium .To quantify the level of differentiation of MSCs after 0, 7, and 14 days from the beginning of the adipogenic induction, oil red-O staining was performed on 3 Nichoids and 3 glass flat samples exposed to adipogenic medium and on the same number of samples exposed to basal medium as a control. At each time point, 2 Nichoids exposed respectively to adipogenic and basal medium and 2 flat glass substrates, in the same conditions, were fixed and colored with oil red-O assay (Sigma Aldrich) 0.1% in isopropanol 60% for 10 min. Before oil red-O extraction, samples were imaged through brightfield microscopy . Then, samples were exposed to isopropanol 100% and 100 \u03bcL of the extraction production were collected for triplicate reads in a 96-well plate. The absorbance measurement of the lysate was taken at 490 nm with plate reader .To observe and quantify the lipid content of MSCs differentiated towards adipogenic phenotype, 2 Nichoids and 2 glass flat substrates were analyzed, respectively at day 7 and 14 from the beginning of the differentiation through confocal fluorescence microscopy. Cells were stained with 1 \u03bcg/mL of Hoechst 33342 for nuclei identification and 5 \u03bcg/mL of lipophilic tracers -DiO  for lipids. Image size was set at 212.13 \u03bcm \u00d7 212.13 \u03bcm (512 \u00d7 512 pixels) and multiple scans along the Z direction with 1 \u03bcm step were captured to ensure a complete volumetric observation.\u22121. Image size was set at 140 \u03bcm \u00d7 140 \u03bcm (200 \u00d7 200 pixels) and multi-stacks along Z direction were captured with 2 \u03bcm of step for 3D Nichoids. The lipid content was measured by analyzing 3 images per sample type at each time point, by manually drawing a circular region of interest (ROI) through Fiji-ImageJ software . The area of each ROI was extrapolated through the Measure tool of Fiji-ImageJ. Then, the number of lipid droplets and the total area covered by lipids was obtained by summing the areas of single droplets for each image.For vital and label-free 3D nonlinear imaging of MSCs exposed to adipogenic differentiation medium, CARS microscopy was performed on a couple of Nichoids and flat controls. Lipid droplets were imaged at day 7 and 14 both on Nichoids and on flat glass substrates through label-free CARS microscopy at the vibrational Raman frequency of 2845 cmWe performed immunofluorescence staining to characterize actin organization and nuclear shape of MSCs during adipogenic and chondrogenic differentiation with respect to cells exposed to basal medium, and to quantify the production of collagen-I during chondrogenesis. Experiments were performed on 6 standard Nichoids, 2 Nichoids with a revised geometry, and 6 flat glass substrates. After 3 weeks of cell culture, samples were washed in phosphate buffered saline solution , Italy) three times, fixed in paraformaldehyde 4% for 10 min, rinsed with PBS, permeabilized with 0.25% Triton X-100 (Sigma Aldrich)-PBS for 15 min at room temperature, and finally processed for indirect immunofluorescence analysis. Cells were first incubated for 3 h with a solution of PBS, 2% bovine serum albumin  and tween 0.1% (Sigma-Aldrich), then incubated with mouse monoclonal anti-collagen-I antibody 2 \u03bcg/mL (Thermo Fisher Scientific) at 4 \u00b0C, overnight. After washing 3 times the samples were incubated for 1h at room temperature with 1 \u03bcg/mL secondary antibody . After three other washes in PBS, 1 \u03bcg/mL phalloidin-FITC (Sigma Aldrich) was added and incubated for 30 min to stain F-actin. After three washes, cell nuclei were stained with a 10-min incubation using 1 \u03bcg/mL of Hoechst 33342  in PBS. Samples were then mounted with Mowiol (Dabco) on standard microscope slides and sealed to undergo fluorescence imaging by the use of confocal microscope . Images were collected with a pinhole aperture of 1.5 Airy unit, over a 212.13 \u03bcm \u00d7 212.13 \u03bcm (512 \u00d7 512 pixels) field of view and multiple scans along Z direction with 1 \u03bcm step to ensure a full volume observation (40 \u03bcm for Nichoids and 20 \u03bcm for controls).Fluorescence images were manipulated with the open-source software Fiji-ImageJ and the measured data collected and analyzed through the software Excel .c), the major (a) and minor (b) semi-axes, the Feret diameter and the nuclear aspect ratio were measured through the Measure tool of Fiji-ImageJ. To calculate the maximum projected area of nuclei per sample type, the mean value among each population was calculated with the relative standard deviation. The nuclear volume was calculated for each nucleus considering the formula of the ellipsoid volume (1).Characterization of the nuclear shapes. Three images per sample type i.e., adipogenic and chondrogenic differentiation and control cells grown with basal medium respectively in Nichoid and in flat control, were analyzed to extract data on nuclear morphology. The count of the nuclei was obtained by manually drawing a polygonal ROI for each of the nuclei of each image and, to avoid errors, a single stack analysis was perform to separate overlapped nuclei and to better exclude the autofluorescent signal from the scaffold. Considering ten nuclei per image in randomized positions the mean area, the vertical . To evaluate the size of collagen deposits, the percentage of area occupied by the collagen with respect to the area of the image was measured by the Measure tool Area in the Fiji-ImageJ measurement settings. Furthermore, a ratio between the number of nuclei and the number of collagen deposits was calculated and compared in all the cases. Statistical differences among culture configurations and media conditioning were established after normality test and Student t test.To establish whether Nichoids inhibited collagen type-I synthesis during MSCs chondrogenic differentiation, high-density Nichoids were studied through SHG microscopy in label-free and vital conditions. Here, a standard Nichoid and a Nichoid with a revised geometry were imaged after 3 weeks of differentiation. Secondly, differentiated samples with the new Nichoid configuration were studied at day 14 and 21 of differentiation. SHG images were captured approximately at 10 \u03bcm of vertical distance from the glass  sized about 200 \u03bcm \u00d7 200 \u03bcm (200 \u00d7 200 pixels) with a pixel dwell time of 10 ms. Since SHG microscopy depends on the polarization of the light with respect to the orientation of the molecular dipoles, all the samples analyzed were positioned in the same direction. Five SHG images per sample were then analyzed through Fiji-ImageJ to quantify the amount of collagen produced and to establish the effect of Nichoids with respect to flat areas: the SHG signal intensity was measured and compared with the signal from the internal pores of the scaffolds. Statistical analyses were performed after normality test and student Our nonlinear multimodal microscope capable to perform in parallel CARS/TPEF/SHG and SRS imaging by the use of two near-infrared ultrashort pulsed laser sources was previously described in . This cuIn the field of tissue engineering and regenerative medicine, the use of MSCs is growing thanks to their adhesive, immunoregulatory and stemness properties. Their multilineage potential, combined with a larger availability in adult mammalians with respect to other cell types, makes MSCs a valid candidate for scaffold-based constructs in bioengineering applications . The dev"}
+{"text": "COL1A1gene in both affected individuals, although heterozygous variants in theCOL1A1are known to cause EDS. Recently, only one report showed homozygous variant as an underlying cause of the EDS in two Saudi families. This is the second report of a homozygous variant in theCOL1A1gene in a family of Saudi origin. Heterozygous carriers ofCOL1A1variant are asymptomatic. Interestingly, the homozygous variant identified previously and the one identified in this study are same (c.2050G\u2009>\u2009A). The identification of a unique homozygous mutation (c.2050G\u2009>\u2009A) in three Saudi families argues in favor of a founder effect.Ehlers\u2013Danlos syndrome (EDS) is a group of clinically and genetically heterogeneous disorder of soft connective tissues. The hallmark clinical features of the EDS are hyperextensible skin, hypermobile joints, and fragile vessels. It exhibits associated symptoms including contractures of muscles, kyphoscoliosis, spondylodysplasia, dermatosparaxis, periodontitis, and arthrochalasia. The aim of this study is to determine the exact subtype of EDS by molecular genetic testing in a family segregating EDS in an autosomal recessive manner. Herein, we describe a family with two individuals afflicted with EDS. Whole exome sequencing identified a homozygous missense mutation  in the Ehlers\u2013Danlos syndrome (EDS) is an inherited disorder of connective tissues and comprises a wide range of clinical conditions involving skin, joints, and vessels. EDS manifests as joint hypermobility, skin hyperextensibility, and vascular fragility.COL1A1,COL1A2,COL3A1,COL5A1,COL5A2,COL12A1,C1R, andC1S.B4GALT7, B3GALT6, SLC39A13, ADAMTS2, TNXB, PLOD1, FKBP14 ZNF469, PRDM5 CHST14, DSE,andAEBP1genes have been shown to cause autosomal recessive form of the EDS.COL1A1gene can also lead to an autosomal recessive form of EDS.Inheritance pattern in EDS is both an autosomal recessive as well as an autosomal dominant. The disorder is genetically heterogeneous and at least six autosomal dominant and seven autosomal recessive forms of the EDS have been identified and, except in one case, the corresponding genes have been discovered. The autosomal dominant form of the EDS have been shown to result from mutations in the genesCOL1A1as an underlying cause of EDS in the family.Here, we presented a five generations consanguineous Saudi family with suspected symptoms of EDS. Considering the huge genetic heterogeneity, whole exome sequencing strategy was undertaken. Genetic data analysis identified a homozygous mutation in theEthical approval to commence the work was obtained from the ethical review committee of Taibah University Almadinah Almunawarah, Kingdom of Saudi Arabia. A 5-generation Saudi consanguineous family with two affected individuals was referred to the Center for Genetics and Inherited Diseases (CGID) from Madinah Maternity and Children Hospital (MMCH) by a pediatric consultant for detailed molecular investigation. Informed written consents were taken from all the members of the family; parents gave consents in case of children. Family history of the disease was enquired from the parents, and a pedigree was drawn based upon the information taken from elders of the family .A complete clinical checkup of the affected members was conducted at MMCH by a pediatric consultant. Skin and musculoskeletal system was evaluated. A Beighton score was used to assess the joint hypermobility. Full blood count and echocardiography were performed.Approximately 5\u2009mL peripheral blood sample was collected from affected as well as unaffected members of the family in EDTA blood vacutainers. Genomic DNA from these blood samples were extracted using QIAquick DNA extraction kit. Nanodrop spectrophotometer and gel electrophoresis were used to evaluate the quality and integrity of the extracted DNA, respectively.Whole genome SNP genotyping array was performed using Illumina iScan platform and HumanOmni 2.5M bead chip, genotyping 2.5 million SNPs. Approximately 200\u2009ng genomic DNA of two affected and two unaffected members were taken for genotyping as per protocol described elsewhere.In an attempt to identify the underlying genetic variant causing EDS, entire coding region (whole exome) was sequenced using Nextera rapid capture exome kit and Illumina NextSeq500 machine. A total of 70\u2009ng of the genomic DNA from both the affected members were used as a starting material, while library preparation and exome enrichment were done using Nextera rapid capture exome kit. Detailed protocol used for WES was the same as described elsewhere.Genetic analyzer ABI3500 was used to validate the genetic variant discovered by WES. Primer 3The family was clinically evaluated by a consultant at MMCH. Affected members of the family manifested typical symptoms of the EDS type 1, including skin loosening (hyperextensivity of the skin), tissue fragility, and bruised skin with variable degree of expression amongst the two members. Unaffected members of the family including normal siblings and parents did not show any of these disease symptoms. A Beighton score of 5 was obtained, indicating a generalized joint hypermobility. Full blood count was normal and clotting disorders were excluded.A common region of homozygosity shared by both the affected members of the family was identified by whole genome SNP genotyping array with the help of 2.5M Illumina iScan platform. Healthy members of the family did not share this homozygosity region. The shard homozygous region is of 4\u2009Mb on chromosome 17q21.33.COL1A1gene was identified. The geneCOL1A1is present in the shared homozygous region. Besides targeting this shared homozygous region, different filter options like pathogenicity, allele frequency, genomic position, nonsynonymous variants, protein effect, etc., were used to further filter the entire 90,000 genetic variants. However, we failed to detect any other notable, relevant genetic variants responsible for the EDS phenotype. Therefore, a missense variant  in exon 31 of theCOL1A1gene was considered as the genetic defect underlying EDS phenotype in the family.An average coverage (covering 214405 exons and splice sites) of 98.3% was obtained from exome sequencing. Different suitable filter options were employed to analyze WES data generated by Illumina NextSeq500 platform. The shared homozygous region identified by genome-wide SNP genotyping array was primarily targeted due to its disease relevance, in order to identify underlying gene of interest. A missense homozygous variant (c.2050G\u2009>\u2009A) in the exon 31 of theCOL1A1gene was validated by Sanger sequencing approach. Data analysis validated the presence of the variant (c.2050G\u2009>\u2009A) and its complete segregation within the family. Variant (c.2050G\u2009>\u2009A) was found in homozygous state in the DNA of affected individuals, while unaffected members of the family were either heterozygous or wild type  inCOL1A1have not been observed in our cases. This report provides further evidence that COL1A1 homozygous variants may cause EDS like phenotype, and the variant identified in this study is a founder in Saudi population.cEDS with typical disease symptoms included exceptional high skin extensibility and fragility, hypermobility of joints, atrophic scared and bruised skin. Molecular investigations led to the identification of a homozygous missense variant (c.2050G\u2009>\u2009A) in the"}
+{"text": "In animal models of pain, HSK21542 significantly inhibited acetic acid-, hindpaw incision- or chronic constriction injury-induced pain-related behaviors, and the efficacy was comparable to CR845 at 15\u00a0min post-dosing. HSK21542 had a long-lasting analgesic potency with a median effective dose of 1.48\u00a0mg/kg at 24\u00a0h post-drug in writhing test. Meanwhile, the antinociceptive activity of HSK21542 was effectively reversed by a KOR antagonist nor-binaltorphimine. In addition, HSK21542 had powerful antipruritic activities in compound 48/80-induced itch model. On the other hand, HSK21542 had a weak ability to produce central antinociceptive effects in a hot-plate test and fewer effects on the locomotor activity of mice. HSK21542 didn\u2019t affect the respiratory rate of mice. Therefore, HSK21542 might be a safe and effective KOR agonist and promising candidate for treating pain and pruritus.Kappa opioid receptor (KOR) agonists have been promising therapeutic candidates, owing to their potential for relieving pain and treating intractable pruritus. Although lacking morphine-like central nervous system (CNS) effects, KOR agonists do elicit sedation, dysphoria and diuresis which seriously impede their development. Peripherally-restricted KOR agonists have a poor ability to penetrate into the CNS system, so that CNS-related adverse effects can be ameliorated or even abolished. However, the only approved peripherally-restricted KOR agonist CR845 remains some frequent CNS adverse events. In the present study, we aim to address pharmacological profiles of HSK21542, with an expectation to provide a safe and effective alternative for patients who are suffering from pain and pruritus. The  Kappa opioid receptor (KOR), one of the classical opioid receptors, is an inhibitory G-protein coupled receptor (GPCR) that is distributed in both the central nervous system (CNS) and the peripheral tissues . Due to To avoid these adverse side effects, other approaches have been attempted for developing the KOR agonists, and the biased KOR agonists and peripherally-restricted KOR agonists among these have gained much attention. The development of biased KOR agonists was based on the concept that G-protein coupled receptors (GPCRs) can selectively signal in different contexts . It is eThe actions of peripherally-restricted KOR agonists are restricted to peripheral sites due to their low penetration into the brain, and the CNS-associated side effects associated with this can be significantly ameliorated or even completely abolished. The peripherally-restricted KOR agonists had analgesic, anti-inflammatory and antipruritic effects . Till noin vitro assays, including [3H]diprenorphine binding assay, cAMP accumulation assay, and a SafetyScreen panel. Its ability to penetrate into CNS tissues was detected with a brain/plasma distribution study. Four different animal models of pain and compound 48/80-induced scratching mouse model of pruritus were used to evaluate the in vivo pharmacological activities of HSK21542, and the CNS side effects associated with it were also detected. Further, pharmacological profiles of HSK21542 and CR845 were compared.With the aim to develop more effective and safer peripherally-restricted KOR agonist, HSK21542 , was synthesized. Several studies were conducted to comprehensively address the pharmacological profiles of HSK21542. The biological activity and selectivity of HSK21542 were examined using ICR mice weighing 18\u201322\u00a0g and Sprague Dawley (SD) rats weighing 160\u2013180\u00a0g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). C57BL/6J mice weighing 18\u201322\u00a0g were obtained from Chengdu DOSSY Laboratory Animal Technology Co., Ltd. (China). All animals were aged between 8 and 10\u00a0weeks at the start of the experiments. Animals were maintained on a standard 12\u00a0h light/12\u00a0h dark cycle in a temperature-and humidity-controlled facility with free access to food and water. The investigators were blinded to the treatment conditions. All animal care and experimental procedures were performed in accordance with the guidelines of National Institutes of Health for the handling and use of laboratory animals and approved by the Guideline of the Institutional Animal Care and Use Committee of Haisco Pharmaceutical Group Co., Ltd. [HSK-(HEISCO-I-17)-2-1-2001-01].3H]diprenorphine , U69593 , morphine sulfate , nor-binaltorphimine , nalfurafine hydrochloride  and compound 48/80 . For in vivo experiments, all the test compounds were solubilized in normal saline, and intravenously administered with a volume of 10\u00a0\u03bcL/g, except for morphine and nor-binaltorphimine (subcutaneously) when the animals were not under anesthesia and were awake. All other reagents used were of analytical grade unless otherwise stated.HSK21542 and CR845 were synthesized in Sichuan Haisco Pharmaceutical Co., Ltd. (China). The synthesis and physicochemical characteristics of HSK21542 have been described in a patent (WO2019015644). The chemical structures of HSK21542 and CR845 are shown in 2. HEK-293 cells that stably express human \u03ba opioid receptor were established in our laboratory and used in this assay. The cell membranes were prepared in 50\u00a0mM Tris-HCl buffer (pH 7.4). An equivalent of 30\u00a0\u03bcg of membranes was incubated with compounds and 0.6\u00a0nM [3H]diprenorphine (an opioid antagonist) at 25\u00b0C for 60\u00a0min (inhibitory effect) or the multiple time points (binding kinetics). Nonspecific binding was estimated in the presence of 10\u00a0\u03bcM naloxone. The bound and free fractions were separated by vacuum filtration through a GF/B filter that was pretreated with 0.3% polyetherimide. The filters were washed with ice-cold buffer and then were counted to specifically determine the bound radioligand  = / \u00d7 100, where CPMtotal = total [3H]diprenorphine bound (membrane +0.6\u00a0nM [3H]diprenorphine) and CPMnon-specific = non-specific [3H]diprenorphine bound . For the unlabeled compounds, the association/dissociation constants were calculated by fitting the data using equations as described by Motulsky and Mahan (HEK-293 cells (ATCC) were maintained in Eagle\u2019s Minimum Essential Medium with 10% FBS, and incubated at 37\u00b0C in humidified air containing 5% COioligand . The pernd Mahan .\u00ae U2OS OPRK1 \u03b2-Arrestin cell line (DiscoverX) was maintained in McCoy\u2019s 5A with 10% FBS, 250\u00a0\u03bcg/ml Hygromycin B and 500\u00a0\u03bcg/ml G418. When the cells reached to 80\u201390% confluence, they were collected and resuspended in HBSS (1X) containing 50\u00a0mM HEPES, 5\u00a0mM IBMX and 1% BSA stabilizer  by adjusting the cell density to 3 \u00d7 105 cells/mL. The cells were divided into 384-well white plate (Corning\u00ae3572) at a volume of 5\u00a0\u03bcL, and were treated with compounds and 2\u00a0\u03bcM forskolin (an inducer of intracellular cAMP formation) for 30\u00a0min at room temperature. Subsequently, 5\u00a0\u03bcL of Eu-cAMP Tracer Working Solution and 5\u00a0\u03bcL of Ulight-anti-cAMP Working Solution per well were added to reach a final volume of 20\u00a0\u03bcL. The plate was incubated for 1\u00a0h at room temperature in the dark. The cAMP levels were then determined by using a microplate reader with TR-FRET assay  \u00d7 100.PathHunterET assay . The resIn vitro off-target pharmacological activities of HSK21542 were evaluated on 86 targets using a SafetyScreen panel  and the corresponding methods could be found at https://www.eurofinsdiscoveryservices.com/.SD rats  were intravenously given a single dose of 0.3\u00a0mg/kg HSK21542. The samples were collected at 0.083, 0.5, 1.5 and 4\u00a0h after dosing. The rats were anesthetized with isoflurane and then sacrificed by taking blood from the abdominal aorta. The whole brains were rapidly removed from the crania. The plasma (\u223c100\u00a0\u03bcL) was separated from the blood by centrifugating at 2000 \u00d7g for 10\u00a0min at 4\u00b0C. The brains were rinsed with ice-cold normal saline, blotted dry, weighted and placed into a plastic tube. For 1.0\u00a0g of brain sample, 4\u00a0ml of acetonitrile-ultrapure water solution  was added to the tube. The brain samples were then homogenized for 120\u00a0s at 50\u00a0Hz and ultrasound was performed for 5\u00a0min. The plasma and the brain samples were analyzed using a LC-MS/MS assay as detailed in supplementary materials.The plasma or brain homogenate was ice thawed. After 30\u00a0\u03bcL of plasma or brain homogenate was transferred into a centrifuge tube, 50\u00a0\u03bcL of internal standard  and 120\u00a0\u03bcL of acetonitrile were added. The mixture was vortexed for 10\u00a0min and centrifuged at 2000 \u00d7g for 10\u00a0min at 4\u00b0C. The collected supernatant (150\u00a0\u03bcL) was placed in a 96-well plate and dried under nitrogen. The residue was reconstituted with 150\u00a0\u03bcL of ultrapure water and vortexed for 10\u00a0min. The resulting solution was then analyzed to determine the concentrations of HSK21542 on a LC-MS/MS system, which consisted of a DGU-20A5R degasser, a LC-30AD pump, a SIL-30AC autosampler, a CTO-20A column oven  and an AB Sciex Triple Quad 5500 mass spectrometer . The LC system was coupled to mass spectrometer by using an electro-spray ionization (ESI) source . ChromatICR mice  were used in this assay. Fifteen or 30\u00a0min (only for morphine) after test compounds were given, each mouse was intraperitoneally injected with 0.6% acetic acid at a volume of 0.4\u00a0ml. Subsequently, each animal was individually maintained in a Plexiglas chamber and the pain-induced writhing behaviors were observed for 15\u00a0min . A writhThe surgery was conducted as reported previously . Male SDThe CCI surgery was performed as described previously with slight modifications . Male SDvs. time) was calculated using a trapezoid rule.The rats were placed individually in Plexiglas chambers on a metallic mesh floor and allowed to acclimatize for 30\u201360\u00a0min . MechaniAfter acclimatization for 30\u201360\u00a0min in Plexiglas chambers, the male ICR mice were given vehicle or test compounds. Compound 48/80  was subcutaneously injected into the back of the neck at 15\u00a0min after drug administration . The micThe hot-plate test was performed according to previous reports . Female 3) for 2\u00a0days. 15 or 30\u00a0min (only for morphine) after either vehicle or test compounds were administered, each rat was returned to the cage, and then allowed to explore the field for 1\u00a0h. The data were collected using an ANY-maze video tracking system and the total distance traveled was analyzed . Experiments were performed after animals were acclimatized to a rectangular experimental cage  was used to measure respiration in freely moving ICR mice  as described previously  with somin vitro experiments, the IC50 or EC50 value was determined by non-linear, least squares regression analysis. The binding kinetic curves were fitted by a competitive binding model. In this model, the K1 (the association rate of [3H]diprenorphine) and K2 (the dissociation rate of [3H]diprenorphine) were constrained to 1.44 \u00d7 108\u00a0M\u22121 min\u22121 and 0.0257\u00a0min\u22121, respectively. The association kinetic curves of [3H]diprenorphine are shown in . For most of the in vivo experiments, a parametric analysis  was performed if the Bartlett\u2019s test for variance homogeneity showed no significance at 1% level, and the treated groups were compared to the vehicle group using Dunnett\u2019s test when F achieves the necessary level of statistical significance (the null hypothesis: there was no difference among the treated groups and the vehicle group). Otherwise, a non-parametric analysis  was performed, and the treated groups were compared to the vehicle group using Dunn\u2019s test when necessary. Planned comparison was done between the two groups using student\u2019s t-test (with same variance) or Mann-Whitney test. The original data from chronic constriction injury model and the measurement of respiration in mice were analyzed by two-way analysis of variance (ANOVA) using the treatment conditions and time as factors. Then, post-hoc Dunnett\u2019s test was performed at different time points if there was an interaction effect. The criterion for statistical significance was set at p < 0.05.All study endpoints were expressed as means \u00b1 SD and no data have been excluded. Statistical comparisons were made using GraphPad Prism 8.3.0 software . No statistical methods were used to predetermine the sample sizes, but the choice of sample sizes was based on our previous studies and the sample sizes are similar to those that are typically used in the field. For 3H]diprenorphine binding assay was performed to investigate the inhibitory effects of HSK21542 on [3H]diprenorphine competition binding and determine the binding kinetics of unlabeled HSK21542. U69593, a positive control, obviously prevented [3H]diprenorphine binding to KOR with an IC50 value of 14.72\u00a0nM (95% CI: 9.08\u201322.38\u00a0nM). As anticipated, HSK21542 significantly inhibited [3H]diprenorphine binding to KOR with an IC50 value of 0.54\u00a0nM (95% CI: 0.38\u20130.75\u00a0nM), while CR845 had an IC50 value of 1.16\u00a0nM  and 0.23\u00a0nM (95% CI: 0.17\u20130.26\u00a0nM), respectively , which was found to be longer than that of CR845 . On the other hand, HSK21542 significantly inhibited forskolin-induced cAMP accumulation in HEK-293 cells that stably expressed human \u03ba opioid receptor with an EC50 value of 2.41 pM (95% CI: 1.43\u20134.67 pM), which was 12.4-fold and 747-fold lower than those of CR845 and U69593, respectively . However, the efficacy of 10\u00a0mg/kg morphine had vanished at 24\u00a0h post-dosing (t(18) = 1.25, p = 0.30), which was corresponding to the pharmacological profile of morphine. Fifteen minutes after drug administration, HSK21542 inhibited acetic acid-induced writhing response in a dose-dependent manner  = 41.18, p < 0.001), and there was no obvious sex difference  = 3.40, p = 0.075). HSK21542 at a dose of 0.03\u00a0mg/kg induced an inhibitory rate of 27.46% on writhing behaviors, and there was a statistically significant difference in writhing responses between 0.03\u00a0mg/kg HSK21542-treated group and vehicle group (p = 0.023). Therefore, the dose of 0.03\u00a0mg/kg was defined as the minimum effective dose (MED), which was 3.33-fold lower than that of CR845 (0.1\u00a0mg/kg). Moreover, the ED50 values of HSK21542 and CR845 were both 0.09\u00a0mg/kg (95% CI: 0.06\u20130.12\u00a0mg/kg), and the inhibitory activity of HSK21542 on writhing response was comparable to that produced by CR845 at the same doses. Finally, nor-binaltorphimine , a kappa opioid receptor antagonist which was given at 24\u00a0h before drug administation, reversed the antinociceptive effects produced by 0.3 or 1\u00a0mg/kg HSK21542 , a positive control, completed suppressed acetic acid-induced pain behaviors at 30\u00a0min post-dosing (HSK21542 .p = 0.02). At the doses of 1, 3, 10 and 30\u00a0mg/kg, HSK21542 induced inhibitory rates of 49.67, 55.60, 68.12 and 75.16%, respectively. However, as for CR845, a dose of 30\u00a0mg/kg was needed to maintain the antinociceptive effects at 24\u00a0h post-drug (p < 0.001). The ED50 values of HSK21542 and CR845 were 1.48\u00a0mg/kg (95% CI: 0.62\u20132.45\u00a0mg/kg) and 24.62\u00a0mg/kg (95% CI: 13.90\u201342.55\u00a0mg/kg), respectively.To explore the duration of action of a single dose of HSK21542, the antinociceptive effects of HSK21542 were detected at 24\u00a0h post-drug. Surprisingly, 0.3\u00a0mg/kg of HSK21542 still significantly inhibited the writhing responses with an inhibitory rate of 32.75% , while morphine had no any effect on pain behaviors at 24\u00a0h post-dosing .The hindpaw incision model and chronic constriction injury (CCI) model in rats, which were widely used for evaluating the analgesic property, were employed to determine the antiallodynic effects of HSK21542. Morphine, a potent analgesic, was given as a positive control. Obviously, morphine (10\u00a0mg/kg) completely inhibited hindpaw incision- or CCI-induced mechanical allodynia at 15\u00a0min after dosing . At a dose of 1\u00a0mg/kg, HSK21542 induced a 10.5-fold increase of 50% PWT  at 15\u00a0min post-dosing. The MED value of HSK21542 was 1\u00a0mg/kg, which was comparable to that achieved by CR845 at 15\u00a0min after dosing, and 10\u00a0mg/kg HSK21542 induced the maximum antiallodynic activity (p = 0.01) and 10\u00a0mg/kg (p < 0.001), which were similar to those of CR845  exerted a dose-dependent inhibitory effect on incision-induced mechanical allodynia  = 245.0, p < 0.001). At a dose of 0.3\u00a0mg/kg, HSK21542 induced a 5.15-fold increase of 50% PWT (6.62\u00a0g vs. 1.29\u00a0g in the vehicle group) at 15\u00a0min post-dosing. In 1 or 3\u00a0mg/kg HSK21542-treated group, the 50% PWT value reached a peak at 2\u00a0h post-dosing, and then gradually faded (p < 0.001). Therefore, the dose of 0.3\u00a0mg/kg was defined as the MED value of HSK21542. On the other hand, there was no obvious difference in mechanical pain thresholds between 0.1\u00a0mg/kg HSK21542-treated group and vehicle-treated group at any time points post-dosing. Moreover, although the antiallodynic effects of CR845 could still persist until 24\u00a0h post-administration at a dose of 3\u00a0mg/kg (p < 0.001), the effects of CR845 had completely vanished at a dose of 1\u00a0mg/kg (p = 0.13).In the chronic constriction injury model, intravenous injection of HSK21542 (0.1\u20133\u00a0mg/kg) suppressed CCI-induced mechanical allodynia in rats in a dose-dependent manner . HSK21542 (0.01\u20133\u00a0mg/kg) inhibited the scratching responses to a similar extent as did CR845 in a dose-dependent manner at 15\u00a0min post-drug  = 27.82, p < 0.001). At a dose of 0.03\u00a0mg/kg, HSK21542 induced an inhibitory rate of 34.89%, and the number of scratching bouts was statistically less than that in the vehicle-treated group (p = 0.015). Therefore, the MED value of HSK21542 was designated as 0.03\u00a0mg/kg. At a dose of 1\u00a0mg/kg, the antipruritic activity of HSK21542 reached a peak with an inhibitory rate of 99.78%. In the 0.1 and 0.3\u00a0mg/kg HSK21542-treated groups, the inhibitory rates of 53.02 and 73.75% were observed, respectively. Furthermore, the analysis of dose-response curve showed that HSK21542 had an ED50 value of 0.09\u00a0mg/kg (95% CI: 0.04\u20130.16\u00a0mg/kg), and this was comparable to that of CR845 .KOR agonist has been validated as an effective therapy for pathological itch . The antin vivo pharmacological effects of HSK21542 are mediated by a peripheral mechanism, a hot-plate test was employed in mice to evaluate the central antinociceptive effects of HSK21542 at 15\u00a0min post-drug. As one of central analgesics, 10\u00a0mg/kg morphine (a positive control) showed an almost complete efficacy. HSK21542 at 3.75\u00a0mg/kg did not evoke significant antinociceptive effects (p = 0.12), although 7.5\u00a0mg/kg HSK21542 induced a percent maximum possible effect (% MPE) of 29.60%, which was statistically higher than that in the vehicle-treated group (p = 0.007). However, CR845 displayed significant antinociceptive effects at a dose of 3.75\u00a0mg/kg (p = 0.008). In addition, the effects of HSK21542 at 7.5\u00a0mg/kg were comparable to that of CR845 at 3.75\u00a0mg/kg  = 0.42, p = 0.68). The ED50 values of HSK21542 and CR845 were 10.49\u00a0mg/kg (95% CI: 7.58\u201315.37\u00a0mg/kg) and 6.76\u00a0mg/kg (95% CI: 4.70\u20138.69\u00a0mg/kg), respectively .As a supraspinal model for acute pain, the hot-plate test is considered as one of the experimental methods for differentiating the central and peripheral antinociceptive effects . To valip < 0.001) and there was no sex difference  = 4.19, p = 0.052). Although HSK21542 at 2\u00a0mg/kg induced an obvious sedative effect (p = 0.005), the lower dose of HSK21542 (0.4\u00a0mg/kg) did not significantly affect the locomotor activity of mice (p = 0.16). On the other hand, 0.4\u00a0mg/kg CR845 induced an obvious decrease in the total distance traveled by mice (p < 0.001) with comparable efficacy to the higher dose of HSK21542 or CR845 . As shown in p < 0.001). There were no obvious effects on respiration when HSK21542 was given at a dose of as high as 2\u00a0mg/kg (p > 0.05). In contrast, CR845 caused significant decrease in the respiratory rate at a dose of 2\u00a0mg/kg at 25\u00a0min post-administration and the effects reached a peak at 45\u00a0min post-drug (p < 0.01).To directly explore the CNS effects of HSK21542, its effects on sedation and respiration rate were measured. The sedative effects of HSK21542 and CR845 at 15\u00a0min post-dosing were evaluated using a locomotor activity test. The results showed that 10\u00a0mg/kg morphine reduced remarkedly the distance traveled by mice  of HSK21542 was higher than the dose (1\u00a0mg/kg) needed to produce maximum antinociceptive effects in writhing test. The EDof 116.6 . In contof 116.6 . Therefohttp://www.chinadrugtrials.org.cn/clinicaltrials.searchlist.dhtml).However, it is noteworthy that there is a huge challenge in preclinical-to-clinical translation for analgesic and antipruritic candidates. One of the main causes is the discordance in endpoints between animal and human studies. In animals, the pain- or itch-stimulated behaviors are recorded to label analgesic or antipruritic candidates, making it unavoidable that the false-positive effects might exist, resulting from non-selective drug effects such as sedation and paralysis . Therefoin vitro findings revealed that HSK21542 is a selective KOR agonist with a higher potency than CR845. The brain/plasma distribution study showed that HSK21542 has an extremely poor ability to penetrate into the CNS system. The in vivo pharmacological activities supported the translational potential of HSK21542 as a safe and effective analgesic and antipruritic candidate. Generally, HSK21542 has the ability to avoid adverse CNS effects that are associated with centrally penetrating KOR agonists and MOR agonist, and might provide an effective alternative for treating patients with pain or pruritus.In conclusion, the"}
+{"text": "Traditional methods for creating food composition databases struggle to cope with the large number of products and the rapid pace of turnover in the food supply. This paper introduces Food Label Information Program (FLIP), a big data approach to the evaluation of the Canadian food supply and presents the latest methods used in the development of this database.The Food Label Information Program (FLIP) is a database of Canadian food and beverage package labels by brand name. The latest iteration of the FLIP, FLIP 2020, was developed using website \u201cscraping\u201d to collect food labeling information  on all foods and beverages available on seven major Canadian e-grocery retailer websites between May 2020 and February 2021.The University of Toronto's Food Label Information Program (FLIP) 2020 was developed in three phases: Phase 1, database development and enhancements; Phase 2, data capture and management of food products and nutrition information; Phase 3, data processing and food categorizing. A total of 74,445 products available on websites of seven retailers and 2 location-specific duplicate retailers were collected for FLIP 2020. Of 57,006 food and beverage products available on seven retailers, nutritional composition data were available for about 60% of the products and ingredients were available for about 45%. Data for energy, protein, carbohydrate, fat, sugar, sodium and saturated fat were present for 54\u201365% of the products, while fiber information was available for 37%. Food products were classified under multiple categorization systems, including Health Canada's Table of Reference Amounts, Health Canada's sodium categories for guiding benchmark sodium levels, sugar-focused categories and categories specific to various global nutrient profiling models.FLIP is a powerful tool for evaluating and monitoring the Canadian food supply environment. The comprehensive sampling and granularity of collection provides power for revealing analyses of the relationship between nutritional quality and marketing of branded foods, timely observation of product reformulation and other changes to the Canadian food supply. The study of nutritional epidemiology relies on understanding the association between nutrient consumption and health outcomes and usually involves monitoring the nutritional quality of food consumed by a population . Thus, sNational food composition databases are expensive to develop, construct and maintain , 8. The The packaged food and beverage industry is also characterized by fast-moving continuous turnover as new products are introduced and/or reformulated, some to replace less-favored or discontinued products , 15\u201318. To address these research gaps, we aimed to develop a product- and brand-specific comprehensive database containing nutrition information for a diverse array of packaged foods and beverages in the Canadian food supply. Such a database allows for identifying important levers for promoting healthy diets, prioritizing nutritional interventions for public health policy, evaluating the impact of population-level policies such as Sodium Reformulation  or TransPreviously, we have developed three versions of FLIP datasets in 2010, 2013, and 2017, described in detail elsewhere \u201328. HoweThe University of Toronto's Food Label Information Program (FLIP) 2020 was developed in three phases: Phase 1, database development and enhancements; Phase 2, data capture and management of food products and nutrition information ; Phase 3, data processing and food categorization .n = 10,487)   (The FLIP is a database of Canadian pre-packaged food and beverage package labels by brand name that was updated every 3\u20134 years at the University of Toronto (UofT), Toronto, ON, Canada. The purpose of the FLIP is to provide comprehensive food product nutrition information to allow for assessment and monitoring over time. To date, three previous versions of the FLIP datasets have been completed in 2010, 2013, and 2017 and are described in detail elsewhere \u201328. Brie 10,487)  was coll 17,671)  data wer 17,671) , 75%   of groce 17,671) .The FLIP website enables users to generate data outputs and reports in Microsoft Excel for further analyses. The FLIP website contains a user tutorial, user guides, and a dashboard with the FLIP version number and details on the latest updates. The information captured on each product is described in The FLIP is hosted on a cloud-based infrastructure located in Virginia, USA and Quebec, Canada. Raw data for each product page with date and time of data collection is stored separately for audit and data verification purposes, and to provide a mechanism for re-extracting data in the event that data was previously extracted incorrectly, or additional data points are required. At present, the FLIP website is available to the L'Abb\u00e9 Lab nutritional sciences researchers at the University of Toronto, as well as national and international university and government researchers with whom the University of Toronto has set up data sharing agreements.The latest phase, FLIP 2020, is described in this manuscript. The FLIP 2020 contains nutrition information for 74,445 product listings, representing 48,829 unique universal product codes (UPC). Food information from the leading grocery retailers in Canada with online information were acquired from their respective websites and digitalized to enhance ease and efficiency of collection and analysis. Food composition database software  (web and mobile) was developed for FLIP 2020, resulting in a shorter and more efficient food collection and data processing approach .\u00ae, Costco Wholesale Canada Ltd., Nepean, ON, Canada; Grocery Gateway by Longo's, Longo's Brothers Fruit Markets Inc., Empire Company Ltd., Stellarton, NS, Canada; Loblaws\u00ae, Loblaws Companies Ltd., Brampton, ON, Canada; Metro, Metro Inc., Montreal, QB, Canada; No Frills\u00ae, Loblaws Companies Ltd., Brampton, ON, Canada; Voil\u00e0 by Sobeys, Empire Company Ltd., Stellarton, NS, Canada; and Walmart, Walmart Canada Corp., Mississauga, ON, Canada), representing over 80% of the grocery retail market share  as available, price , dietary or allergen information  (if available on packaging as part of the ingredient list), and date and location/store information of sampling . Each prn = 7,400 products) were manually validated by FLIP staff and students, for this version to determine accuracy of the AI-OCR technology.After web-scraping the product information, foods were automatically assigned a product ID  and photos and web data were uploaded onto the FLIP website for data management and processing. Each product's ID is used for identifying and tracking unique products over time. Artificial Intelligence (AI)-enhanced/powered Optical Character Recognition (OCR) (AI-enhanced OCR) technology was used to automatically extract data available in photo format only . In the AI-enhanced OCR process, each image of the product was scanned for text and a text parsing algorithm determined which image had text that resembled the NFt or ingredient list, followed by extraction of that particular text only. All NFt data extracted from OCR technology . The no change in barcodes and product codes were used as indicators of no significant product change, therefore, the matching process allowed for any empty data fields in FLIP 2020 to be populated by FLIP 2017 . However, food products from Costco, Walmart, Grocery Gateway, No Frills and Voil\u00e1 could not be linked to FLIP 2017 as the previous versions of FLIP did not contain any Costco, Walmart, Grocery Gateway and No Frills products and Voil\u00e1 did not contain any barcodes on the website. All product matches were manually validated by two Research Assistants and the following information was transferred over for the matched products: Table of Reference Amount (TRA) categories, sodium and sugar categories, Company/Parent Company, As Prepared NFts , container size, serving size g/mL conversion factors  and free sugars.Barcodes of food products from Metro, Walmart, and Grocery Gateway and store-specific product numbers from Loblaws and No Frills in FLIP 2020 were matched to those in FLIP 2017 barcodes and store-specific product codes, respectively . If one of these products was missing NFt or ingredients information, its data fields would be populated using a linked product with the most complete data. The FLIP log tracks when data is transferred from one product to another and the source.In phase 3, food products were classified using Health Canada's Table of Reference Amounts (TRA) , 33, andAdditional automation algorithms were developed for classifying foods into sodium-focused  and suga\u00a9 smartphone application, as described in detail elsewhere (\u00a9 app categories consist of categorizing the FLIP database into product specific major categories (n = 19), sub-categories (n = 101) and minor categories (n = 397) in order to allow consumers to easily locate products in consumer-friendly categories. Categorization of foods for FoodFlip\u00a9 is based on merging Health Canada's TRA categories  and outliers to check for erroneous values.Additional data extraction or processing, dependent on research objectives and analyses, are ongoing or will be conducted  .n = 3,724) were collected between August 2017 and May 2018 from three leading groceries stores located in the province of Buenos Aires and Buenos Aires city  were collected from two grocery stores located in San Jose during and January-August 2018  and Peruvian data  were collected during the Summer 2017 and December 2019. The Canadian FLIP or the FLIP-LAC have been used for research, food supply monitoring, policy evaluation and modeling , Costco (n = 735), Walmart , Grocery Gateway , Loblaws , Loblaw's Maple Leaf Gardens , No Frills , No Frills Joe's , and Voila . However, food products from Loblaw's Maple Leaf Gardens  and No Frills Joe's  were omitted from the current analysis as they are duplicate outlets of the same data discussed in this manuscript. There were 1,261 (from seven retailers) and 1,409 (from nine retailers) non-food products , which were removed from further analysis. In total, 25,980 of the FLIP 2020 products  were matched to 8,646 of the FLIP 2017 products. FLIP 2020 products may have been matched to multiple 2017 products, and vice versa. Therefore, the total number of matches was 26,395.A total of 74,445 products were collected from Metro, Costco, Walmart, Grocery Gateway (Longo's), Loblaws, Loblaw's Maple Leaf Gardens (a specific location in a metropolitan Toronto area), No Frills, No Frills Joe's (a specific location in a metropolitan Toronto area) and Voila. The number of products for each store was as follows: Metro .Using automated techniques  to collect data from e-grocery retailers can result in food composition databases with far greater coverage and temporality than have been achieved in the past , allowinThe FLIP 2020 data collection via web-scraping showed that from about 73,000 foods, about 60% of products had NFt information, suggesting that automatically and repeatedly scraping data from online e-retailers websites can produce food composition databases with sufficient information on nutrients and ingredients with reliability to allow for monitoring/evaluating a highly dynamic food and beverage supply. In comparison, a study from UK on foodDB, with over 97,368 products, found data on specific nutrients were present for over 90% of nutrient declaration tables, with data on ingredients available for >80% of the foods and drinks . ConsideThe need for branded food databases as well as the challenges of creating such tools are recognized by researchers and policymakers , 6. ThesSome limitations to our approach are related to the continued evolution and changes to the e-retailers product availability and websites, in order to ensure the data on these products are up-to-date. Although, the use web-scraping with OCR and AI/ML for data collected in FLIP 2020 were key innovations of our database that provided up-to-date product-specific nutrient information in a systematic and comprehensive manner, it was also a key limitation. E-grocery retailers may detect web-scraping, enabling OCR blockers and other techniques to make it difficult to scrape the data. Furthermore, there are no e-grocery food labeling regulations to mandate and standardize the availability and presentation of product information resulting in poor availability and wide inconsistencies in food labeling information, including missing information, number and quality of images, NFt and ingredients in the e-grocery retail environment in Canada .The current FLIP2020 does not capture local regional and geographical variability of food and drink availability within individual online grocery retailers nor does it capture regional and local ethnic supermarkets that once catered to immigrant communities but are serving non-immigrant consumers seeking new products. Furthermore, convenience stores and large drugstore chains are introducing new product ranges that often include foods, which are not currently captured by the FLIP database.Some tasks needed for research or monitoring remain time- and labor-intensive. For example, creating scores for some nutrient profiling models, automatic mapping of categories and subcategories and parsing of ingredients in any database remain, although work is underway to apply AI/ML to such tasks.The automation of FLIP 2020 is a first step in providing real time nutritional data on foods. Web-scraping coupled with AI-powered OCR technology are important tools in automating the collection of real-time foods and nutrition information. The automated data collect process, using AI-enhanced OCR, provides FLIP with distinct analytical advantages compared to previous versions of FLIP and the generic food composition database in Canada (CNF) and takes the burden off manual processing by staff and students. A systematic methodology was established, based on previous versions of FLIP, to validate and categorize information, thereby enhancing the collection, storage, processing and management of nutrition information for each product. The use of web-scraping and automation further lowers the cost for future collections and allows for regularity in data capture on products. These features can also be implemented for future collections of FLIP databases, such as the FLIP-LAC and can be useful for other nutrition databases. Automating the systematic and consistent data capture will ensure sustainability and feasibility of maintaining large-scale branded food composition databases as new products and other changes to product formulation are introduced and others discontinued.FLIP 2020 is an automated methodological step forward for food composition databases, which are the bedrock of nutritional epidemiology. Web-scraping coupled with OCR technology (AI/ML) are important tools in automating the collection of real-time food and nutrition information. The FLIP 2020 data collection demonstrated that automatically scraping data from online supermarkets can produce a food composition database with sufficient accuracy, transparency, granularity and flexibility to regularly monitor a highly dynamic food and drink marketplace. Such information are important in understanding the relationships between the nutritional quality of food products and measurements of policy impacts and health over time.mary.labbe@utoronto.ca through data sharing agreements. Requests to access the datasets should be directed to Mary L'Abbe, mary.labbe@utoronto.ca.The datasets presented in this article are not readily available because complete database for non-commercial use can be obtained from the corresponding author at MA, AS, and ML'A conceived and designed the study. AS, JL, and MW collected the data. MA, AS, JL, MW, and BF-A processed the different phases of the FLIP database. All authors contributed to interpreting the data as well as writing and editing the manuscript and have read and agreed to the published version of the manuscript.This research was supported by funds from the Canadian Institute of Health Research Project Grant - Policy Impact (2016PJT-378415)(ML'A). MA was supported by a Postdoctoral Fellowship from the Joannah and Brian Lawson Center for Child Nutrition at University of Toronto, Toronto, ON, Canada. JL was supported by CIHR Doctoral Fellowship and the Banting and Best Diabetes Center at University of Toronto, Toronto, ON, Canada. BF-A was supported by a Postdoctoral Fellowship at Ontario Tech University. MW was supported by a Banting and Best Graduate Studentship Award.ML'A and MA report receiving a competitive research grant from IAFNS formerly ILSI NA to analyze NHANES data to determine the intakes and sources of sodium in the diets of Americans 2021\u20132022. None of these companies/organizations had any involvement in the present research. 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.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": "As we all know, Coronavirus Disease 2019 spread all over the world and had became a public international event of global concern. Among Coronavirus Disease 2019 patients in China, two doctors, Yi Fan and Weifeng Hu, were noticed with their skin pigmentary disorder due to polymyxin B. However, we found that polymyxin E has almost no reports of skin hyperpigmentation, but polymyxin B was reported about skin hyperpigmentation, although the number of relevant reports was small, what causes the difference between the polymyxin B and polymyxin E?Polymyxin is a general term for a group of basic peptide antibiotics, including A, B, C, D and E mainly. At present, the two commonly used clinically are polymyxin B and polymyxin E.2 receptor on the surface of melanocytes inducing the stimulation of melanin synthesis1. Polymyxin B activates mast cells to release histamine, which binds to the HThe mechanism of polymyxin B causing skin hyperpigmentation may include the following aspects:2 receptor on the surface of melanocytes in the basal layer to induce the production of cyclic adenosine monophosphate and the activation of protein kinase a in melanocytes, which leads to phosphorylation of members of the cyclic adenylate response element binding protein transcription factor family. Cyclic AMP response element binding protein activates a variety of genes and induces the transcription of a variety of enzymes and proteins related to melanin synthesis. Eventually leads to increased melanin synthesis in the cytoplasm  as the prodrug is predominantly cleared by renal excretion, with only a relatively small fraction of the dosage converted to the active antibacterial in renally healthy individuals (2. The skin inflammation process is related to the activation of melanocytesPolymyxin E can also release histamine equivalent to polymyxin B , the reaividuals , it is eividuals . Is the Histological and immunohistochemical results of pigmented skin in patients with polymyxin Btreatment showed an abundant melanocyte-pigmented dendritic network. Langerhans cells\u2019 hyperplasia and dermal IL-6 overexpression were also found, presumably for an inflammatory process due to polymyxin B use ; At the Matzneller et al. reported that polymyxin E could decrease inflammatory cytokines, including IL-6, in the blood of L lipopolysaccharide-challenged healthy volunteers in a model of human endotoxiemia . And acc3. Oxidative stress is also considered to be one of the mechanisms of pigmentation According to these findings, the effects of polymyxin B and polymyxin E on IL6 are different. We speculate that it may explain the difference in pigmentation between the two drugs.4. Phenylalanine increases melanin synthesis Compared with polymyxin E, polymyxin B has a different structure at position 6. Polymyxin B is phenylalanine at position 6, and polymyxin E is leucine. According to Martindale records, vitiligo can be treated with phenylalanine, and more than 60% of patients have skin pigmentation during the treatment process . This coThe nephrotoxicity and neurotoxicity induced by polymyxin B have been proved to be dose-dependent , but theIt should be pointed out that there have been no reports of inhalation of polymyxin B-induced skin hyperpigmentation so far, the administration for all patients suffered from skin hyerpigmentation induced by polymyxin B were intravenous , which iAccording to the findings above, we speculate boldly that polymyxin B needs to reach a certain concentration in blood to cause pigmentation and that reducing the dosage may be an effective way to prevent skin hyperpigmentation, however, reducing the dosage of polymyxin B may be likely to reduce the efficacy and even lead to bacterial resistance. It should be point out that there is no definite evidence that the occurrence of pigmentation is related to the increased concentration of polymyxin B in blood, and there is no report of dosage adjustment after the occurrence of skin pigmentation.Regarding the difference between polymyxin B and polymyxin E in causing skin pigmentation, further research is needed in the future, and further research is needed on how to prevent and treat polymyxin B-induced skin pigmentation. However, the current related reports can remind us that we should pay attention to monitoring related adverse reactions when applying polymyxin B. If skin hyerpigmentation occurs, provide corresponding psychological counseling to the patient, or take corresponding treatment measures such as laser cosmetic therapy and topical whitening agents, or adjust the dosing regimen if necessary."}
+{"text": "Esophageal cancer (EC) is a common and lethal carcinoma; however, the effectiveness and feasibility of the chemo- and radio-therapy (CRT) for the elderly patients (\u2265\u200970\u00a0years) with surgery have not been fully discussed. The purpose of this study was to investigate the potential effect of CRT on the prognosis.A total of 1085 patients (534 CRT patients vs. 551 non-CRT patients) from 1998 to 2016 were collected from the Surveillance, Epidemiology, and End Results database according to the inclusion and exclusion criteria. Using the competing risk regression and survival analysis, an overall estimation of the effectiveness of CRT was performed on a well-balanced cohort via performing propensity score matching. Then, the specific impact of CRT on high- (n\u2009=\u2009557) and low-risk (n\u2009=\u2009528) cohorts derived from the nomogram\u2019s risk quantification for every patient were further evaluated respectively. Additionally, the advantages of the nomogram model and the conventional tumor, node, metastasis  staging system were compared.P\u2009<\u20090.001), especially for those with tumors characterized by poor differentiation, large tumor size, advanced T staging, lymphatic metastasis, and distant metastasis , while no benefit was observed among the low-risk patients. Furthermore, the newly established nomogram model might be better than the TNM (6th revision) staging system but more data needed.A better survival outcome was observed among patients receiving both surgery and CRT than those who underwent surgery alone (HR: 0.55, 95% CI 0.45\u20130.68, Aggressive treatments, such as surgery, chemotherapy, and radiotherapy, were considered effective for selected elderly patients with EC according to the newly established nomogram model.The online version contains supplementary material available at 10.1186/s12876-021-02016-9. Esophageal cancer (EC) is a common upper gastrointestinal tract carcinoma with high morbidity and mortality worldwide, with approximately 5.5% of new digestive tumor cases and 2.7% of tumor mortality in 2020 . The incRecently, to improve the quality of life and prolong the survival time, progress on treatments has been made, including minimally invasive surgery for early stage EC , neoadjuTo facilitate clinical decision-making for elderly patients with EC, we analyzed the impact of chemo- and radio-therapy  on the elderly patients with EC undergoing surgery based on a cohort from the Surveillance Epidemiology and End Results (SEER) database. First, we estimate the general impact of CRT based on two well-comparable cohorts processed by propensity score matching (PSM). To further identify the specific patient subgroup that might benefit from CRT, a nomogram model was established to measure the risk for every patient based on individual characteristics, which could be further labeled as high risk (high score) or low risk (low score) according to the median score of the whole cohort. Finally, predictive models were created to measure the effects of CRT on a specific population.The study was approved by the Institutional Ethical Committee of Shengzhou People's Hospital .This study collected data from the patients registered in the United States SEER cancer registries from 1998 to 2016. The inclusion criteria were as follows: (a) patients who underwent esophagectomy; (b) histologically diagnosed esophageal squamous cell carcinoma or esophageal adenocarcinoma; and (c) age at diagnosis \u2265\u200970\u00a0years. The exclusion criteria were as follows: (a) patients with missing demographic or clinical information ; (b) received chemotherapy or radiotherapy alone; and (c) survival time shorter than 1\u00a0month. Following the inclusion and exclusion criteria, 1085 eligible patients (534 CRT patients vs. 551 non-CRT patients) were selected for the further analysis  [Cases histologically confirmed with EC according to the International Classification of Diseases in Oncology (ICD\u2010O\u20103) from 1998 to 2016 were identified from the SEER database, an open database available at n 8.3.8) . The \u201cSETo facilitate statistical analysis, some demographics and clinical covariates were modified based on our clinical and research experiences: race was defined as \u201cwhite\u201d or \u201cother races ,\u201d histological type was defined as \u201csquamous cell carcinoma\u201d or \u201cadenocarcinoma,\u201d and histological grade was defined as \u201cI\u2009+\u2009II (well or moderate differentiation grade)\u201d or \u201cIII\u2009+\u2009IV (poorly or undifferentiation grade) grade.\u201d Then, a matrix was built containing the information for race, age at diagnosis, marital status at diagnosis, primary tumor location, histological type and grade, TNM staging system (6th), tumor size, treatment record, survival status, survival time, and cause of death for every patient.2 test, and P\u2009<\u20090.05 was considered statistically significant.R-software packages of \u201cMatchIt\u201d and \u201ccobalt\u201d were used to conduct the PSM; packages of \u201csurvival,\u201d \u201ccmprsk,\u201d and \u201csurvminer\u201d were used to establish competing risk models; packages of \u201cmstate,\u201d \u201criskRegression,\u201d and \u201crmda\u201d were used to build nomogram, calibration curves, and decision curve analysis. Categorical variables described as counts and relative percentages were compared using the \u03c72 tests were performed to assess the effectiveness of PSM. Next, the whole-population cohort was randomized into training and validation sets at a ratio of 7:3. Based on the training set, univariate and multivariate competing risk models were established to select the key risk factors for CSD. Then, high- and low-risk groups were derived according to the risk score from the nomogram model built on the above risk factors. The impact of CRT on the two specific groups was then estimated. Furthermore, in both the training and validation sets, concordance indexes (c-index) were calculated to show the discrimination, and calibration curves were drawn to measure the consistency between the actual and expected values. Finally, a decision curve analysis was conducted to measure the superiority of the nomogram model over the conventional TNM staging system.Next, the whole-population cohort was divided into CRT and non-CRT groups through 1:1 nearest neighbor PSM with a caliper of 0.08. The sub-distribution hazard ratio (sHR) and hazard ratio (HR) were calculated to estimate the probability of cancer-specific death (CSD) and overall survival (OS), and the results were presented using the cumulative incidence function curves and Kaplan\u2013Meier curves , 19, resP\u2009=\u20090.2), race (P\u2009=\u20090.372), primary tumor location (P\u2009=\u20090.225), tumor histological type (P\u2009=\u20090.154), and distant metastasis (P\u2009=\u20090.173) between the CRT and non-CRT cohorts were similar. However, all variables\u2019 distributions became balanced after matching  and better survival outcomes  than those who did not receive CRT.The competing regression and Cox regression models were constructed to identify the patients who received CRT that had lower probabilities of CSD  patients, the 1085 patients were randomly divided into training (n\u2009=\u2009761) and validation n\u2009=\u2009324) sets. In the training set, the univariate and multivariate analysis of competing risk models were performed to estimate probabilities of CSD; as a result, poorly or un-differentiated grade, tumor size \u2265\u200933\u00a0mm, T stage >\u2009T1, positive lymph nodes, and metastasis diseases had been associated with increased risk of CSD (Table 4 sets. IP\u2009<\u20090.001) and better overall survival outcome . However, no marked benefits were observed among the low-risk patients who received additional treatment . In addition, the calibration curves showed that the 1-, 3-, and 5-year predictive probabilities of CSD matched well with the actual ones both in the training  [In the present study, based on the post-PSM cohort, we found that CRT could have a positive impact on CSD and OS in elderly patients. Univariate and multivariate competing regression models were performed to identify poor histological differentiation, and advanced T stage and positive lymph node status were significantly correlated with higher probability of CSD. Furthermore, tumor size and distant metastasis were also considered key factors in predicting the prognosis of EC. Kamel et al. indicated that the size of malignancy (HR\u2009=\u20091.005) was a significant independent predictor of CSD in T1N0M0 patients based on the SEER database . Malnutr=\u20090.016) . Further=\u20090.016)  Regardin=\u20090.016) . Finally=\u20090.016) .P\u2009=\u20090.006), alopecia (P\u2009<\u20090.001), neutropenia (P\u2009<\u20090.001), nausea (P\u2009=\u20090.029), and leukopenia (P\u2009<\u20090.001) [P\u2009=\u20090.003). Contrary to the above findings, a multicenter randomized phase III trial of FFCD 9901 involving 195 patients with a median age of approximately 60\u00a0years, investigated the effect of neoadjuvant chemoradiotherapy on patients with early stage EC and found no significant positive impact on the rate of R0 resection or survival prognosis; although the mortality after surgery was increased [Based on the Fig.\u00a0<\u20090.001) . Regardi<\u20090.001) . The medncreased .Generally, it is widely accepted that both the chemotherapy and radiotherapy could exert a positive effect on the survival of elderly patients with EC. For instance, after reviewing 21,593 EC patients aged\u2009\u2265\u200970\u00a0years from the National Cancer Database, Gregory et al. pointed out that any cancer-related treatment could play a positive role in prolonging the survival of elderly patients with EC . SimilarThe present study has several limitations. First, it was a retrospective study based on the SEER database, covering only 30% of the population of the United States  and withBased on the post-PSM cohorts, we found that CRT could decrease the probability of CSD and improve OS in elderly (\u2265\u200970\u00a0years) EC patients in general. Further analysis of the sub-cohort marked as the high- and low-risk groups derived from a nomogram indicated that high-risk patients with tumors characterized as middle- and upper-third of the tumor location, poor and undifferentiated histological grade, \u2265\u200933\u00a0mm tumor size, advanced T stage, positive lymph node, and distant metastasis could be considered beneficial for aggressive anti-tumor treatment. However, for low-risk patients whose tumors were characterized as lower-third of the tumor location, well and moderately differentiated histological grade, <\u200933\u00a0mm of tumor size, early T stage, negative lymph node, and non-metastasis, CRT could not bring therapeutic benefits on the survival outcomes. Despite this, well-designed trials are still needed to validate our conclusions based on real-world clinical practice.Additional file 1: Figure S1. Standardized mean differences of variables between the pre- and post-matching cohorts based on the CRT or not. aCRT: chemo- and radio-therapy.Additional file 2: Figure S2. A, C, E: The calibration curves of nomogram for predicting 1-, 3-, and 5-year probabilities of cancer-specific death (CSD) in the training set. B, D, F: The calibration curves of nomogram for predicting 1-, 3-, and 5-year probabilities of CSD in the validation set. Nomogram-predicted CSD is plotted on the x-axis; actual CSD is plotted on the y-axis. The imaginary line indicates a perfect calibration model in which the predicted probabilities are identical to the actual incidence.Additional file 3: Figure S3. Decision curve analysis (DCA) of the nomogram and 6th edition of the American Joint Committee on Cancer\u2019s (AJCC) tumor, node, metastasis (TNM) staging system for 1-year (A), 3-year (B), and 5-year (C) overall survival. The x-axis measures the threshold probabilities, and the y-axis represents the net benefit. The horizontal line along the x-axis assumes that overall death occurred in no patients, while the solid gray line assumes that all patients will have overall death at a specific threshold probability. The blue solid line represents the 6th edition of the AJCC TNM staging system. The red solid line represents the nomogram."}
+{"text": "Chronic quadriceps tendon rupture is technically challenging for surgeons due to scarring and tendon retraction. The presence of concurrent ipsilateral knee osteoarthritis compounds the issue even further. Although a combined presentation is rare, treatment options to manage each coexisting pathology simultaneously are scarcely reported. We describe the case of a 67-year-old Caucasian male who had such a presentation, and was subsequently treated with a one-stage extensor mechanism autograft reconstruction and total knee replacement with computer navigation.2. Otherwise, there were no risk factors for tendon rerupture. Clinical examination later revealed a large palpable gap in the right suprapatellar region and weakness of active knee extension. No traumatic cause for this new presentation was identified. Suspicion of a chronic quadriceps tendon rupture was confirmed on radiological imaging, but the investigations also noted the presence of severe tricompartmental osteoarthritis of the ipsilateral, native knee joint. The combined procedure took place in one surgical sitting. The total knee replacement with patella resurfacing was performed first and assisted by computer navigation. The quadriceps tendon reconstruction was then conducted sequentially using the patient\u2019s hamstring tendons (semitendinosus and gracilis). The tensile strength was reinforced with use of a Ligament Augmentation and Reconstruction System\u00a0(LARS) ligament. Initial outcomes were excellent, and these results were maintained at 6\u00a0months postoperatively, with the patient reporting no pain and having full range of movement.The patient was a 67-year-old male Caucasian, who had previously sustained an acute rupture of his right quadriceps tendon that was adequately repaired 6\u00a0months prior. Despite an initial satisfactory result, he reported deterioration in his mobility in the few months thereafter, with worsening knee pain. His comorbidities consisted of hypertension, asthma, and a body mass index of 40.4\u00a0kg/mOur techniques used have not previously been reported, but are successful options in treating coexisting chronic quadriceps tendon rupture and ipsilateral knee osteoarthritis. The advantage of using computer navigation with an extramedullary femoral jig can lead to improved accuracy of bone cuts, which is important in the presence of anatomical disruption. Chronic failures of the extensor mechanism require different approaches depending on the inherent and underlying pathology. We feel that the multidisciplinary team approach to the management and use of two surgeons with differing expertise added to the successful outcome of this complex case. The incidence of isolated quadriceps tendon rupture is low and is associated with certain risk factors such as smoking, steroid use, diabetes mellitus, inflammatory conditions, connective tissue disorders, and sporting overuse . Such inChronic or neglected quadriceps tendon rupture is often a clinical conundrum for surgeons. The technical challenges posed by the nature of the lesion, scarring, and tendon retraction necessitate complex surgical procedures and augmentation techniques for reconstruction . The difet\u00a0al. [n\u2009=\u200924) for quadriceps tendon rupture postoperatively in a cohort of 23,800 primary TKR patients. They concluded that such injury is an uncommon sequel [However, although a combined presentation is uncommon, the management of chronic quadriceps tendon rupture and concurrent knee OA is poorly documented in the orthopedic literature. Most relevant studies only comment on tendon rupture as a complication after TKR surgery for OA, rather than as a coexisting pathology preoperatively. For example, Dobbs et\u00a0al.  reportedet\u00a0al. differed greatly to ours. Our patient underwent a combined tendon reconstruction (with hamstring autograft) and TKR (assisted with computer navigation) in one sitting. This case report will discuss the procedural aspects and challenges of having to perform a dual procedure in this way. The techniques used have not previously been reported. Given the paucity of evidence describing similar cases, this report adds to current orthopedic literature and presents a unique way for how surgeons may choose to proceed with this complex and challenging presentation.We present a rare case of a middle-aged Caucasian gentleman with chronic rupture of his right quadriceps tendon that was complicated by concurrent severe right knee OA. At the time of submission, a literature search revealed only one other case  describi2 .The patient was a 67-year-old Caucasian gentleman, who was assigned an American Society of Anesthesiologists (ASA) grade of 3 . He was classified as severely obese in accordance with his body mass index (BMI) of 40.4\u00a0kg/mAside from these comorbidities, there were otherwise no risk factors for tendon rupture or clinical evidence of inflammatory conditions that could have increased his overall risk. He initially had an acute, nontraumatic rupture of his right quadriceps tendon without any precipitating cause, and the tendon was repaired in the private sector in November 2019 after a delayed presentation of more than 6\u00a0weeks.The patellofemoral joint (PFJ) showed evidence of grade IV osteoarthritis.The rupture was seen to have extended into the vastus lateralis and lateral retinaculum.The torn ends of the tendon were degenerate and of poor quality.At the time of the initial surgery, there were several observations of note:Subsequently, the injured ends of the quadriceps tendon were freshened, debrided, and repaired with a modified Kessler\u2019s technique (using an Arthrex fibertape) and reinforced with a continuous vicryl end-to-end suture. The medial parapatellar retinaculum was also repaired.The intraoperative result was satisfactory, and flexion of the knee joint up to 90\u00b0 was possible. He was discharged with a hinged knee brace, which was locked in extension for the first 4\u00a0weeks postoperatively, with a plan to gradually increase his range of motion (ROM) thereafter. Following an intense course of physiotherapy and rehabilitation, a good outcome was achieved at first. According to the patient, he returned to full unaided mobilization and was able to flex and extend his knee without any difficulty .In December 2020 (over 6\u00a0months following discharge), he was referred to our care by his general practitioner after presenting to them with signs and symptoms that were consistent with a chronic rupture of his quadriceps tendon. No traumatic cause for this presentation was identified. Symptomatically, he now reported moderate discomfort in his right knee as well, but was pain free on the contralateral side and in his hip joints. Despite this, his mobility was impaired to the extent that he relied on a walking stick to assist with his activities of daily living (ADLs). He was clinically well otherwise, with no history of fever or infection either locally  or systemically.Clinical examination demonstrated an unsteady gait. On closer inspection, there was a mature, well-healed surgical scar over his right knee, with no evidence of infection or inflammation. There was a large palpable gap in the suprapatellar area but minimal swelling and effusion in the knee joint itself. Wasting of the ipsilateral quadriceps muscle was also noted. He was found to have weakness of active knee extension and was unable to straight leg raise (SLR). However, he had a reasonable flexion range from 0\u00b0 to 100\u00b0, which was mostly asymptomatic. Further examination was unremarkable.Considering these findings, the patient proceeded to have a magnetic resonance imaging (MRI) scan of his right knee, which confirmed chronic rupture of his quadriceps tendon. It showed that the ruptured tendon had retracted proximally, leaving a big residual space in the suprapatellar region with patella baja. Significant scarring and fluid signal were also seen within the rupture gap, which were radiographic signs consistent with the diagnosis Fig. . Of signFollowing the results of these investigations, the patient was reviewed again in May 2021. An open discussion was held to ascertain their preferences regarding the surgical management of their dual problem of chronic quadriceps tendon rupture and severe knee OA. At this point, it was noted that his knee pain had deteriorated further as well.In the meantime, a multidisciplinary team (MDT) discussion had been conducted in our unit to determine how best to proceed. We considered whether it was best to reconstruct the tendon first and perform the TKR at a later stage, or to perform both procedures in one sitting. Due to the severity of the OA and chronicity of the tendon rupture with subsequent retraction and scarring, the expert consensus was in favor of performing the two procedures in one sitting. This would thereby improve the patient\u2019s chances of a good outcome. Furthermore, if only an isolated tendon repair was to be performed, a TKR would not be feasible for a prolonged period thereafter to allow for the full extensor mechanism to recover sufficiently. Considering the patient\u2019s worsening symptoms, the dual procedure was deemed to be in their best interests.The patient agreed with the proposed plan and was counseled accordingly. The expectations of surgery (including the possibility of permanent stiffness with limited knee flexion), intraoperative plan, and rehabilitation goals were discussed. The patient was also made aware that if the operation were to be unsuccessful, the only remaining options would be conservative management or a knee arthrodesis. Willing to accept the risks presented to him, the patient gave his informed consent to proceed with a right quadriceps tendon reconstruction and primary TKR in one sitting.After preoperative assessment and counseling, the planned procedure took place. A spinal block with light sedation was the anesthetic of choice. The patient was positioned supine on the operating table with the appropriate foot and lateral supports for optimal surgical access Fig. . A high The TKR was performed initially by one of the senior authors (AA), through a standard medial parapatellar approach over the healed surgical scar and using the Knee 3 Brainlab computer navigation, with a pin-less femoral extramedullary jig for assistance. The DePuy PFC Sigma CR Knee was the favored prosthesis, and the patella was also resurfaced. The wound was irrigated meticulously, and fresh drapes were reapplied to minimize the risk of wound contamination Figs.  and 5.FiThe quadriceps tendon reconstruction was then conducted sequentially by the second senior author (DK). The torn ends of the tendon were initially mobilized and released. Scar tissue was debrided to help improve the position and tracking of the patella after the reconstruction. The hamstring tendons (semitendinosus and gracilis) were harvested through the same surgical incision used for the TKR Fig. . The twoThe knee ROM intraoperatively was 0\u2013100\u00b0, with minimal tension over the reconstructed tendon, although there was still some residual patella baja observed. The retinaculum and rest of the wound were closed in layers after liberal washout with normal saline. The patient also received two doses of antibiotics as per local guidelines.The knee was then placed in a ROM brace, which was to be locked in extension to allow for full weight-bearing for 4\u00a0weeks but able to flex up to 50\u00b0 passively while sitting or engaging with physiotherapy during that timeframe. He was also advised to avoid any straight leg raise activity during the same period to protect the extensor mechanism. After an uncomplicated postoperative stay of a few days, the patient was discharged home.The clinical assessments at various follow-up intervals are summarized as follows:Skin clips were removed, and the condition of the wound was satisfactory.He had some residual postoperative swelling and pain with a visual analogue scale (VAS) of 2/10.Initial postoperative x-rays showed good position and implantation of the prostheses Fig. . There wThe patient was advised to continue physiotherapy and weight reduction measures.Day 10 postoperativelyThe wound was completely healed with no signs of infection.He was completely pain free with VAS of 0/10.He achieved a flexion of 60\u00b0 and an SLR of 0\u201330\u00b0. .The brace was further unlocked to allow for full flexion, and he was advised to continue physiotherapy.Six\u00a0weeks postoperativelyThe wound was completely healed.The patient was able to walk unaided without any symptoms or difficulty.There was active knee flexion of up to 90\u00b0 (without the ROM brace), an SLR of 45\u00b0, and an extension lag of less than 10\u00b0 Figs.  and 14.The patient was very pleased with his clinical results, and he was allowed to resume activities as per his premorbid status.Three\u00a0months postoperativelyAbsence of pain (VAS 0/10) was maintained.He had continued with full unaided mobilization.He demonstrated flexion of at least 100\u00b0.The extension lag had marginally increased to 10\u201315\u00b0. Some weakness of his quadriceps muscle was noted, and he was given exercises to strengthen his extensor compartment accordingly.The patient remained pleased with his surgery and did not feel limited with his ADLs.Six\u00a0months postoperativelyHe will continue to be followed up for a minimum of 2\u00a0years, and his recovery is expected to remain uncomplicated.We present a case of chronic quadriceps tendon rupture in a middle-aged Caucasian patient with ipsilateral knee osteoarthritis (OA), which were both managed at the same time with quadriceps tendon reconstruction and computer-navigated total knee replacement (TKR). Although a combined presentation is rare, information regarding the treatment of coexistent quadriceps tendon rupture and ipsilateral knee OA is very scarce in the orthopedic literature, with most publications focusing on extensor mechanism disruption following TKR . Althouget\u00a0al. [Only Piatek et\u00a0al.  have repet\u00a0al. . Althouget\u00a0al. [A relevant but unidentical case was presented by Zhang et\u00a0al. , who deset\u00a0al. [On the other hand, there is a growing body of literature about extensor mechanism disruption following TKR surgery, as highlighted by Vyas et\u00a0al.  in a recet\u00a0al. .,  with or\u2019 tendon . Many st [et\u00a0al. , on exteLike Piatek\u2019s case, we also adopted a combined surgery as it was advantageous to perform both procedures simultaneously. Only a single anesthetic and hospital admission were required, which reduced unnecessary exposure for the patient and the risk of additional hospital-acquired complications or morbidity during the recovery period. This strategy is particularly important given the ever-changing climate amidst the current global COVID-19 pandemic. A single admission is more convenient for patients as well, by allowing them to fulfill any working commitments or social activities without the need to take additional time off from work and family.Unlike Piatek\u2019s case , howeverWith regards to the tendon reconstruction itself, we chose to perform this with autograft instead of allograft by harvesting the ipsilateral hamstring tendons (semitendinosus and gracilis). This was then strengthened with the synthetic\u00a0Ligament Augmentation and Reconstruction System (LARS) ligament, as we felt that this would help to promote earlier mobilization postoperatively. The technique was found to be very secure and allowed knee flexion of up to 100\u00b0 during the procedure. The autograft was taken through the same knee incision and so there was minimal donor site morbidity. Although there is clinical evidence to support the use of drill holes for tunneling and securing the autograft through the patella , we wereTo the best of our knowledge, the intraoperative techniques described in this case are novel ways for managing coexisting chronic quadriceps tendon rupture and ipsilateral knee OA. With the ever-increasing technological advances in orthopedic surgery, we hope that our report is of educational value to the readers and something that may be considered in similar case presentations.The techniques that we adopted had several advantages. The use of computer navigation with an extramedullary femoral jig led to improved accuracy of bone cuts, which was important in the presence of anatomical disruption. The hamstring tendon autograft was also easily accessible through the same surgical incision as the TKR, which allowed for the combined surgery to take place simultaneously.Such techniques should be easy to teach and reproduce. Moreover, we were able to demonstrate efficacy with a good functional outcome and patient satisfaction at 6-months follow-up. On the other hand, more information will be needed on this topic to help guide best practice moving forwards, when faced with these two challenging and complex problems at the same time."}
+{"text": "During an inflammatory process, shift in the cellular metabolism associated with an increase in extracellular acidification are well-known features. This pH drop in the inflamed tissue is largely attributed to the presence of lactate by an increase in glycolysis. In recent years, evidence has accumulated describing the role of lactate in inflammatory processes; however, there are differences as to whether lactate can currently be considered a pro- or anti-inflammatory mediator. Herein, we review these recent advances on the pleiotropic effects of lactate on the inflammatory process. Taken together, the evidence suggests that lactate could exert differential effects depending on the metabolic status, cell type in which the effects of lactate are studied, and the pathological process analyzed. Additionally, various targets, including post-translational modifications, G-protein coupled receptor and transcription factor activation such as NF-\u03baB and HIF-1, allow lactate to modulate signaling pathways that control the expression of cytokines, chemokines, adhesion molecules, and several enzymes associated with immune response and metabolism. Altogether, this would explain its varied effects on inflammatory processes beyond its well-known role as a waste product of metabolism. Lactate is a hydroxycarboxylic acid that is present as two stereoisomers in mammals, the left-handed (L-lactate) and the right-handed (D-lactate) forms, with L-lactate being the predominant form produced during anaerobic glycolysis , 2. SeveStreptococcus bovis, which metabolizes carbohydrates to L (+) and D (-) lactate. Bovines with ARA develop several lesions, including ruminitis, polioencephalomalacia , liver abscess, and lameness  response \u201325. (LDH is a nicotinamide adenine dinucleotide (NAD+) dependent enzyme that mediates the bidirectional conversion of pyruvate and lactate concomitantly with the oxidation and reduction of the cofactor NAD+. LDH is a tetramer made up of two subunits, LDH-A and LDH-B, LDH-A having higher affinity and Vmax for pyruvate than LDH-B. Thus, LDH-A, and particularly the LDH-5 tetramer (consisting of 4 subunits of LDH-A), catabolizes pyruvate to lactate. In contrast, LDH-B transform lactate into pyruvate, allowing cells to use lactate as a source of nutrients for oxidative metabolism . Both L- . In cont , 25 givi .An increase of LDH-A expression is key in the metabolism of cancer cells and is considered a negative prognostic biomarker\u00a0,\u00a033. LDH. MG is a by-product of glycolysis, produced by the fragmentation of dihydroxyacetone phosphate (DHAP), and glyceraldehyde 3-phosphate (G3P)    . G3P is te (G3P) . Furtherte (G3P) .The production of MG from the oxidation of fatty acids occurs by conversion of acetone to MG in two steps catalyzed by acetone and acetyl monooxygenase (AMO). MG can also be produced by semicarbazide-sensitive amine oxidase (SSAO)-catalyzed aminoacetone deamination from the catabolism of L-threonine and glycine . GlyceroStreptococcus, Lactobacillus, and Lactococcus has been observed in gastric cancer patients. This could involve the supply of exogenous lactate, which is an energy source for cancer cells that favor inflammation, angiogenesis, metastasis, epithelial-mesenchymal transition, and immune evasion  exert health-promoting functions such as immunomodulatory improvement of intestinal integrity, resistance to pathogens, prevention of lactose intolerance, anticancer effects, reduction of depression and anxiety symptoms, anti-obesity and anti-diabetic activities, and decrease serum cholesterol levels . L-lacta evasion . LABs ar. For monocarboxylate transporters, members of the 16A solute carrier family proton-bound monocarboxylic acid symporters, i.e., MCT1 (SLC16A1), MCT2 (SLC16A7), MCT3 (SLC16A8), and MCT4 (SLC16A3) and two sodium-coupled lactate cotransporters  have been described (Lactate can cross the cell membrane by three known pathways: 1)\u00a0free diffusion of undissociated acid, 2) exchange for another anion, and 3) transport escribed , 63.In humans, MCTs have been reported in retina, muscle, kidney, brain capillary endothelial cells, cardiac myocytes, enterocytes, hepatocytes, erythrocytes, thymocytes, placenta, and nervous tissue , 65. In MCT1 plays an essential role in neuroinflammation since lipopolysaccharide (LPS) was shown to increase the expression of MCT1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 in microglia obtained from the brain of C57BL/6 mouse . The knoThe transport kinetics for both stereoisomers have been measured in frog oocytes expressing MCT1 and MCT4. The Km values for MCT1 determined for L-lactate and D-lactate were 4.4 and >60 mM and for MCT4 were 28 and 519 mM, respectively . DespiteTo carry out the inflammatory response, immune cells must activate metabolic pathways as part of host defense responses . ConversIt has recently been demonstrated in neutrophils that lactate can be used as non-glucose substrates to generate glycogen stores . In addiEndothelial cells (ECs) control the extravasation of circulating immune cells into tissues through the production of cytokines, chemokines, and adhesion molecules. It has been proposed that lactate can activate signaling pathways in endothelial cells and modulate the inflammatory response.via MCT1 induces the activation of NF-\u03baB  in the regulation of the lactate-induced inflammatory response in ECs  and neutrophil extracellular traps (NETs) . However .Inhibition of lactate formation by sodium oxamate, an LDH inhibitor, reduces tachyzoite-induced NETs . FurtherD-lactate has been shown to have a pro-inflammatory effect on bovine neutrophils since it induces the release of MMP-9, increases the expression of CD11b and decreases the expression of L-selectin, which favors endothelial adhesion , 105. CoLPS induces differentiation to M1-type macrophages, a proinflammatory phenotype that generates ATP and lactate release through aerobic glycolysis . Furthervia HIF1\u03b1 stabilization  increase the expression and secretion of IL-8 and IL-6 in an NF-\u03baB-dependent manner in bFLS  as potential lactate and proton sensors, could additionally explain the varied responses seen with lactate in inflammation , 142.via protein kinase A (PKA) or G\u03b2\u03b3, can inhibit NF-\u03baB and reduces cytokine expression (via G\u03b1i/o reduces cAMP favoring apoptosis through the pro-apoptotic protein BAD (.Hydroxycarboxylic acid receptor 2 (HCA2) is a GPCR also known as PUMA-G (upregulated protein in macrophages by IFN-\u03b3) , HM74A, pression , whereastein BAD . In sepstein BAD   localized in the cytoplasmic membrane. GPR81 is coupled to G\u03b1i-type G proteins . After l) . In macria ARRB2  (Table . In macepatitis . In GPR8epatitis . GPR81 hepatitis . It has eability .in vitro (LPS activates bone marrow (BM) neutrophils and induces lactate release through increased glycolysis. Lactate released acts on GPR81 expressed by endothelial cells to increase vascular permeability inducing the mobilization of BM neutrophils . Lactatein vitro . Additioin vitro . AccordiThe activation of GPR81 inhibits the secretion of the vascular cellular adhesion molecule (VCAM)-1 and endothelial selectin (E-selectin), which suppress monocyte attachment to the endothelium . ConversAcidosis is a hallmark of the microenvironment of inflammatory pathologies , 167, whGPR132 is described in neutrophils  and macr  is expressed in T cells, B cells, neutrophils, and eosinophils and is coupled to the Gs/adenyl cyclase/cAMP pathway , 183. GP (GPR68 (OGR1) is a proton-sensing receptor that can detect decreases in extracellular pH during inflammation. It is expressed in macrophages , dendrit , 192. GP . GPR68 e , 193. Fu . Extrace , 196.Lactate, more than a product of metabolism, exerts modulating effects on the immune response and, depending on the cell type, could interfere or promote the inflammatory response. Apparently, the diversity of effects would depend on the pathway by which lactate is generated or metabolized. In addition, during the development of inflammatory processes, various changes occur in cell metabolism, in this scenario the presence of lactate can contribute to enhance or interfere with the immune response, depending on the pathological context analyzed. Furthermore, the evidence suggests that lactate may play as pleiotropic physiological signaling agent, modulating several signal transduction pathways and transcription factors. Lactate can mediate its effects directly through lactate-sensitive G-protein coupled receptors or indirectly through its effects on extracellular acidification, which would stimulate different proton-sensitive receptors. The activation of these receptors can contribute or interfere with the inflammatory process.Taken together, all the currently available evidence suggest that lactate possesses a myriad of biological effects, which could explain dissimilar responses observed in inflammatory processes.All listed authors have made a direct and substantial contribution to this work and approved the final manuscript.This work was supported by the Agencia Nacional de Investigaci\u00f3n y Desarrollo (ANID) Scholarship 21171843, and Fondo Nacional de Desarrollo Cient\u00edfico y Tecnol\u00f3gico (FONDECYT) 1210754.The authors declare that the research was conducted in the absence of any commercial or financial relationship 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": "Many indigenous communities reside in biodiverse environments replete with natural food sources but show \u200bpoor access and utilization.n\u2009=\u2009211) from 17 villages in the Godda district of Jharkhand, India. Survey methods included household surveys, dietary intake assessment (24 HDR) and micronutrient and inflammatory biomarkers' estimation.To understand the links between indigenous food access, dietary intakes, and biomarkers, we conducted a cross-sectional study among women of the Santhal Community (baari)\u00a0was positively associated with retinol-binding protein levels and negatively with inflammatory biomarkers, while access to ponds was positively associated with ferritin levels.The diversity in access to foods from different natural sources expressed as Food access diversity index was low. This led to poor consumption and thus a low Minimum Dietary Diversity. The mean nutrient intake was less than the estimated average requirement for all nutrients. Women with higher dietary diversity scores had higher nutrient intakes. Thiamine and calcium intakes were significantly higher in women consuming indigenous foods than non-consumers. One-fourth of the women had elevated levels of inflammatory biomarkers. The prevalence of iron deficiency was approximately 70%. Vitamin A insufficiency (measured as retinol-binding protein) was observed in around 33.6% women, while 28.4% were deficient. Household access to natural food sources was associated with specific biomarkers. The access to\u00a0kitchen garden (The findings highlight the role of access to diverse natural foods resources, including indigenous foods, for improving nutrition security in indigenous communities. Nutrition and health programs promoting indigenous food sources should include the assessment of biomarkers for effective monitoring and surveillance.The online version contains supplementary material available at 10.1186/s40795-023-00669-1. Micronutrient deficiency or hidden hunger is a crucial global health issue affecting more than 2 billion people worldwide . It has India is home to 104 million indigenous people spread across 705 indigenous communities, constituting 8.6% of the country's population. These are designated as \"Scheduled Tribes\" by article 342 of the Indian Constitution . CompareMany programs for the development and protection of indigenous people exist globally. Likewise, in India, development of indigenous people has been at the center stage of every 5-year plan by the government. However, a wide gap exists in achieving desired nutrition and health indicators  owing toNational surveys play a crucial role in informing, designing, and implementing sound public health programs and monitoring the progress of ongoing programs, however these data sources rely primarily on self-reported dietary intake data . More reBiomarkers of nutritional intake objectively and precisely quantify nutritional status and are not subject to measurement errors from recall bias and misreporting. They can be measured in multiple tissue types and help to validate traditional dietary assessment methods , 22. HenJharkhand, a central-eastern state of India, is one of the most biodiverse regions having a large indigenous population constituting 26.3 percent of the state's total population. In Jharkhand, Santhal is the most populous indigenous community representing 34 percent of the total ST population, and is spread over vast areas of Giridih, Dumka, Purbi Singhbhum, Pakaur, and Sahibganj districts. The majority of the Santhals in Jharkhand are cultivators (48.6%) and agricultural laborers (38.4%) . Many stThe work described in this paper is a part of a larger project that examined the contribution of IF consumption towards dietary diversity and food security among WRA and children of indigenous communities of Jharkhand. This paper reports the iron and vitamin A levels along with the status of inflammation among the Santhal WRA group using selected biomarkers. Further, it explores the associations of biomarker levels with dietary intake, with a special emphasis on indigenous food intake and dietary diversity; nutritional intake; sociodemographic and economic factors; and access to different food sources.The present study is part of a larger project, with the comprehensive description and methodological approaches reported elsewhere . In the This exploratory cross-sectional study was conducted in July\u2013August 2018 in seventeen selected villages of Godda district of Jharkhand. The population of Santhal community in Godda district was 224,068 as per the census conducted in 2011 .A two-stage cluster sampling was used wherein blocks (an administrative subdivision of a district) were purposively chosen , followed by random selection of a total of 17 villages  using probability proportional to size (PPS) sampling from the selected blocks. This was followed by the second stage, in which a house listing of Santhal households (HH) was carried out, and a sampling frame of all eligible HHs was constructed. Based on the larger objective of the project, all HHs with at least one non-pregnant WRA group (15\u201349\u00a0years) and one child (6 to 54\u00a0months) were selected for the study. This paper reports the observations on all eligible women who fulfilled the inclusion criteria . If more than one eligible woman was present in a HH, one woman was randomly selected using a Kish table .n\u2009=\u2009327). The HH survey could be completed in 275 HHs, a dietary survey in 266 women, and blood sampling could be completed in 238 women. After data collation, cleaning, and removal of outliers, a common pool of 211 women were included for analysis.The sample size for the present study is based on the\u00a0objective of the larger study, which aimed at assessing the differences in micronutrient intake between IF consumers and IF non-consumers. The present study, however, did not analyze the data on the basis of IF consumers and non-consumers. Iron intake data from our previous study on same indigenous community  was usedWe conducted three different research activities, i.e., the Household survey, dietary survey, and collection of capillary blood samples to analyze biomarkers. An overview of the sampling framework and study procedures is given in Fig.\u00a0baari), access to forests for gathering food, access to water bodies (pond/river), etc. was also collected using HH survey tool and agriculture diversity tool (agri-tool) (on a sub-sample).A pretested structured questionnaire developed on an electronic data capture (EDC) platform using the software CS Pro (Version 7.1) and Samsung tablets (Model SM\u2010T385) was administered\u00a0at HH level to gather information on HH characteristics. At the HH level, any adult member of the family, preferably a woman in the HH, who had information on household food access and other HH characteristics, was administered the HH survey. This survey elicited information on socioeconomic and demographic characteristics such as age, educational and occupational status of HH members, type of house, number of rooms, presence of separate kitchen, access to water and sanitation facilities, and some other features. Besides this, information on availability and access to different food sources such as HH ownership of agricultural land and kitchen garden  was conducted on the eligible woman from each HH to estimate nutrient intake. A food recall kit consisting of standard utensils and a food picture flipbook for portion size estimation was used and the participant was asked to recall the food items consumed in each meal over the past 24\u00a0h with their corresponding approximate amounts, the ingredients used, and the method of preparation followed for each recipe consumed. In order to minimize the error in self-reporting, a final probing was done to make the woman recall if any additional food was consumed but not reported. This information on the self-reported amounts of raw foods was used to calculate the daily intake of individual food groups. Nutritionists and nutrition interns administered a paper form after due training for dietary recalls.A clinical biochemist provided training on sampling and processing procedures to the field worker and a laboratory technician. During the daily surveys, the capillary blood collection procedure included cleaning the ring finger of the non-dominant hand with an alcohol swab, pricking the finger using a lancet, collecting the blood in labeled microtainers, and storing them in an icepack lined icebox. Simultaneously, spotting of drawn blood (20 \u00b5l) on Whatman paper was also done to estimate hemoglobin from dried blood spots (DBS). At the end of each day's collection, serum of collected samples was separated by centrifugation; multiple aliquots were made and stored in cryoboxes in the freezer compartment of the refrigerator at the field site laboratory. These samples were then transported in dry ice to Kolkata via road and subsequently to the Bangalore laboratory (Certificate of incorporation no (CIN): U74900KA2015PTC079960; Start-up registration number: KBITS/SK-REGN/2016/165) and AIIMS, New Delhi via air shipment.The collected blood samples were analyzed for micronutrients and inflammatory markers- namely ferritin, soluble transferrin receptor (sTfR), Retinol Binding Protein (RBP), alpha-1-acid glycoprotein (AGP), C-Reactive Protein (CRP), and hemoglobin. An in-house assay was developed and validated for measuring these biomarkers in partnership with the laboratory at Bangalore. The method was developed using Luminex technology on a Luminex xMAP \u00ae platform along with the drop array technology. More details have been included elsewhere For Hemoglobin estimation, a colorimetric method based on the formation of cyanmethemoglobin (Benesphera\u2122) was used as per manufacturers' protocol, and all DBS samples were processed in a single batch. The DBS was cut out, transferred to test tubes containing 5\u00a0ml of Drabkin's solution, and kept at room temperature (20\u201325\u00b0) for 6\u00a0h. After that, one mL of the Drabkin's solution was measured on a spectrophotometer at 540\u00a0nm (OD540). A standard curve was constructed using hemoglobin standards provided with the kit and used for calculating the hemoglobin concentrations in the DBS samples.A HH wealth index score was obtained using principal component analysis (PCA). Variables such as type of housing, ownership of selected assets, number of rooms, presence of separate kitchen, source of electricity, access to water and sanitation facilities, and monthly expense on food were used to generate HH Wealth Index. Depending upon the quantiles of the HH wealth index score, the HHs were classified into five groups.A modified and adapted version of the crop diversity index (CDI)  namely tWhere n is the total number of foods grown, gathered, or accessed and animals raised in a particular HH and N is the maximum possible number of foods grown, gathered, accessed, or raised in a specific village. Detailed information on FADI has been reported elsewhere .The Minimum Dietary Diversity for Women (MDD-W) was calculated as per FAO and USAID guidelines. It was A validated software, DietCal , converted food intake data into nutrient intakes. As the data on nutrient intake was skewed, the box cox method was deployed to transform the distribution\u00a0into a\u00a0normal distribution. The transformed data was then used in the linear mixed effect regression (LMER) model, and the values thus obtained were back-transformed to calculate the 'usual nutrient intake' . The estTable In order to obtain the ferritin values in ng/ml, antilog of log(ferritin)adj values were determined. Similarly, log (sTfR) was predicted deploying a linear regression model using log (AGP) and the sTfR values were obtained using the estimated regression coefficients in the following equation:p-value\u2009<\u20090.05 were transferred to the Linear Mixed Effects Regression (LMER) model for multivariate analysis.Data entry and collation were done using Microsoft Excel and Stata SE version 15.1 . The catEthical approval for the study was obtained from Institutional Ethics Committee at the Indian Institute of Public Health\u2010Delhi, Public Foundation of India, and The All India Institute of Medical Sciences, New Delhi. Administrative approvals were also taken from the authorities at the district level. Cluster level consent from the village leader was obtained before any data collection. Informed consent  was taken before the administration of the interview, and collection of blood sample and for publication of the data. Participation in the study was voluntary and small incentives, procured from the local markets were given to the participants.n\u2009=\u2009211). The mean age of the women was 27.2\u2009\u00b1\u20097\u00a0years, with a majority having a primary or lower level of education (78.2%). The sample population was divided into five quintiles based on the HH wealth index, the mean wealth score of the lowermost, and the uppermost quintiles, were -2.8\u2009\u00b1\u20090.7 and 3.2\u2009\u00b1\u20091.4 respectively.Table baari) (71.6%), water bodies (ponds and rivers) (55.5%), and forests (54.5%). The food items accessed from different food sources are listed in Table p\u2009<\u20090.001) with higher mean value of FADI in joint family  as compared to nuclear (mean\u2009=\u20090.24\u2009\u00b1\u20090.23), suggesting higher diversity in access to different foods in HHs with joint family.The households reported accessing various food sources such as agricultural land (92.9%), kitchen garden (forests 5.5%. The The main source of drinking water for a majority of households was tube well/hand-pump (63.5%), and a majority of them reported defecating in open field/jungle (91.5%) Table .Table Based on the 2\u00a0day-24 HDR, Table The mean dietary diversity score obtained was 2.3\u2009\u00b1\u20090.6 with a median of 2  which is lower than the recommended minimum dietary diversity (MDD-W) of more than or equal to five food groups for the WRA group. The mean IF score obtained was 0.9\u2009\u00b1\u20090.7 with a median of 1.0 .There was a trend of increasing nutrient intake  with increasing MDD-W score Table . The IF Table The following section reports the association between various factors or determinants that contribute to the micronutrient status and the level of biomarkers that reflect the micronutrient status. We analyzed the associations aligned to the conceptual framework Fig.\u00a0 adapted p\u2009>\u20090.05). However, a statistically significant difference between group means of RBP was observed between the two levels of education: primary and below (group 1) and above primary (group 2), wherein the mean RBP was higher in group 1  as compared to group 2 . There were no significant differences in mean values of biomarkers among people with different drinking water sources and place of defecation (p\u2009>\u20090.05).When a one-way ANOVA test or t-test was applied to find the association between sociodemographic variables and the biomarkers, no statistically significant difference was found in group means of the biomarker levels across the wealth quintiles (p\u2009=\u20090.01), and access to baari was significantly associated with the ferritin levels (p\u2009=\u20090.02), RBP levels (p\u2009=\u20090.02) and level of inflammatory biomarkers, i.e., CRP (p\u2009=\u20090.02) and AGP-1a (p\u2009=\u20090.01). Access to ponds and forests was significantly associated with the ferritin levels (p\u2009=\u20090.004 and p\u2009=\u20090.02 respectively for pond and forest).There was no statistically significant correlation between agroforestry diversity index-FADI and the biomarkers as determined by the Karl Pearson correlation coefficient test. Concurrently, when access to different food sources was compared with the biomarkers using t-test, it was found that access to agricultural land was significantly associated with hemoglobin levels  and negatively correlated with the sTfR levels , but the correlation was not significant. The correlation between folate intake and ferritin  and folate intake and sTfR  was statistically significant. Intake of iron, folate, and vitamin C was also positively correlated with the hemoglobin level, with the correlation between folate intake and hemoglobin level being statistically significant  , with the correlation being marginally significant.No statistically significant correlation was found on exploring the correlation between MDD-W scores, indigenous food consumption, and biomarkers Table . However1) Table . The enep\u2009<\u20090.05) were transferred to the multivariate linear regression model, it was found that women having above primary level of education had 15.3% lower RBP level than women having a level of education of primary or below . HH access to baari was positively associated with the RBP level and negatively associated with inflammatory biomarkers, while access to ponds was positively associated with the ferritin levels. The women who had HH access to baari had 18.53% higher level of RBP than those who did not access baari . Some of the Vitamin A-rich foods accessed from the baari included mustard leaves, and pumpkin, accessed by 66.9%, and 22.5% of the HHs respectively as determined by the HH survey  and 13.06% lower level of AGP-1a than those who did not have access to the same . Women who had HH access to the pond had 27.12% higher ferritin levels than women who did not have access to the same . Some iron-rich food items accessed from ponds included indigenous flesh foods like fish, snail, turtle, etc.  and hemoglobin changed by 0.28% . The folate intake was negatively associated with the sTfR levels and per unit increase in folate intake led to a 0.59% decrease in sTfR levels .When the variables that showed significant association in the bivariate analysis . Higher levels of iron deficiency ranging from 36 to 76% \u201351 are aWe did not find any significant correlations between the enabling determinants like sociodemographic and economic profiles and micronutrient status. Data from extensive global studies show a variable contribution of sociodemographic factors like education, household assets, healthcare access, sanitation and income quintiles on micronutrient and inflammatory biomarkers \u201358.baari,\u00a0e.g., mustard leaves and pumpkin, by a substantial number of HHs, have significant amounts of beta-carotene  and food supplementation programs such as supplementary feeding initiatives under ICDS can be done. These activities can be strongly backed by behaviour change communication strategies including nutrition education programs, and activities to promote indigenous food consumption in the habitual diets of Santhal women.Due to the cross-sectional nature of the study design, our findings represent only the associations, we cannot draw causal inferences from the same. There is also a possibility of unmeasured confounding factors in this study. Seasonal fluctuations, which are often known to affect nutrient consumption, were not included in the 24- hour DR of the respondent women. There may be some inaccuracy in reported portion size estimates, although standard food recall kits and a portion size estimation flipbook were employed during the survey. The nutrient calculation database used by us, calculates nutrients based on the nutritive value of raw foods using the Indian food composition table. However, it does not consider the nutrient retention factors ascribed to the effect of cooking on the nutrient content. The information on access to different natural food sources was gathered at the HH level, and it may not reflect the actual access/utilization of the women, although we made specific efforts to interview an adult woman during the HH survey. The markers of inflammation chosen by us could be affected by malaria and other infections. While we did ensure that only healthy, afebrile women were chosen as participants; we did not have biomarker-based information on recent malaria infection.Additional file 1.\u00a0Rawdataset."}
+{"text": "Medullary thyroid carcinoma (MTC) is a well-known neuroendocrine carcinoma, derived from C cells of the thyroid gland. Additionally, MTC is an uncommon aggressive carcinoma that metastasizes to lymph nodes, bones, lungs and liver. For MTC, the 10-year general survival ratio of patients with localized disease is about 95%, whereas that of patients with local phase disorder is around 75%. Only 20% of patients with distant metastasis to lung at diagnosis survive 10 years, which is notably lower than survival for well-differentiated thyroid carcinoma (WDTC). The management of MTC with distant metastasis to lung could be re-surgery or chemotherapy. In this research, we planned to assess the in vitro and in vivo combinational anticancer effect of a novel combination of low-dose cisplatin and sorafenib in patient-derived MTC. The patient-derived MTC cell lines YUMC-M1, M2, and M3 were isolated and treated with a combination of cisplatin and sorafenib or either agent alone. Cisplatin and sorafenib acted in combination to forward tumor restraint compared with each agent administered alone at a low dose. Therefore, a combination of cisplatin and sorafenib could be a new therapeutic approach for MTC. Thyroid carcinoma (TC), which accounts for more than 90% of total endocrine carcinomas, is the most common endocrine malignancy, and its incidence has increased over the last four decades . TC is cc-mediated apoptosis in response to the combinational anticancer treatment of cisplatin and sorafenib at a low dose compared to each agent administered alone. These findings could be clinically noticeable for the growth of novel combinatorial strategies effectively targeting highly malignant cells such as metastatic MTC. In particular, based on these findings, we are investigating the fundamental mechanism of anticancer drug resistance to forward the expansion of new pharmaceutical composition to address this problem.In this study, we aimed to investigate whether aggressive MTC can be suppressed via the induction of cytochrome This study was a retrospective, single-center analysis of patients diagnosed with MTC (between January 2003 and December 2019), as detailed in our previous study [Patients who were diagnosed with MTC were pathologically confirmed either through surgery or open/core needle biopsy. Patients were followed up for at least 1\u20135 years or until death.Fresh tumor tissues were collected from patients with histologically and biochemical proven MTC who were treated at the Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. Fresh tumors were collected throughout surgical excision of MTC metastatic and primary sites.YUMC-M1 was a 24-year-old woman with sporadic MTC (stage II). The patient underwent bilateral total thyroidectomy with central compartment neck dissection and bilateral modified radical neck dissection and has been followed up for 71 months without recurrence or metastasis.YUMC-M2 was a 19-year-old female MEN2B MTC (stage IVa) patient with RET-gene mutation. The patient underwent bilateral total thyroidectomy with bilateral modified radical neck dissection and later confirmed her pheochromocytoma and underwent left adrenalectomy. The patient has no metastasis to other organs, but she has been followed for 76 months without her serum calcitonin normalization.YUMC-M3 was a 57-year-old man with sporadic MTC with lung, mediastinal and axillary lymph nodes metastases (stage IVc). The patient underwent bilateral total thyroidectomy with bilateral modified radical neck dissection, mediastinal dissection via partial sternotomy and axillary lymph node dissection. Since then, metastasis to the axillary lymph nodes, retropharyngeal space, and lung has progressed, and he was treated with a tyrosine kinase inhibitor, vandetanib.The patient-derived cancer cells were obtained from fresh tumors of patients. YUMC-M1, M2 and -M3 and -M1 were obtained from medullary thyroid cancer patients treated at the Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. These patients were diagnosed with medullary thyroid cancer through fine-needle biopsy before surgery and histologically confirmed after surgery. Specimens were maintained and transferred to the laboratory. Fat and non-tumor parts were eliminated and washed using 1\u00d7 Hank\u2019s Balanced Salt Solution. Cell viability was evaluated with the trypan blue dye. Isolated tumor cells were authenticated with short tandem repeat profiling, karyotyping, and isoenzyme analysis. Mycoplasmal contamination was checked for with the Lookout Mycoplasma PCR detection kit . Further protocol details are as described in our previous article .We pre-processed the raw values from the sequencer to discard low attribute and adapter sequences before analysis and revised the procedure reads to Homo sapiens (GRCh37) using HISAT v2.1.0. The detailed protocol can be found in our previous article .The comparative abundances of genes were calculated as read counts through StringTie. We carried out the statistical analysis to discover differentially expressed genes with evaluations of expression for each gene in the samples. The detailed protocol can be found in our previous article .t-test raw p < 0.05. The whole data analysis was carried out with R 3.5.1  .Hierarchical clustering was carried out with complete connect and Euclidean distance as a determination of resemblance to show the expression patterns of separately shown transcripts, which are gratified with |fold change| \u2265 2 and separate https://string-db.org/, accessed on 20 October 2021) and visualized with Cytoscape 3.8.2 . PPI was classified by a combination score  and connecting whole genes counts (>3 counts).Protein\u2013protein interaction (PPI) was performed through the String database . The detailed protocol can be found in our previous article .3 cells per well. These cells were cultured overnight to accomplish 70\u201380% confluency. The indicated drugs were added to achieve final concentrations of 0\u201340 \u03bcM. The detailed protocol can be found in our previous article [Cell viability was estimated by the MTT -2,5-Diphenyl tetrazolium Bromide) assay. Tumor cells were cultured in 96-well plates at 7 \u00d7 10 article .Cells were treated with a combination of cisplatin and sorafenib or either agent alone in RPMI-1640 medium containing 15% FBS for 40 h. The cells were then harvested by trypsinization and fixed in 70% ethanol. Further protocol details can be found in our previous article .c was evaluated by the immunofluorescence staining assay. The primary antibody for the assay was anti-cytochrome c  incubated in 3% bovine serum albumin in PBS. Nuclei were stained with Hoechst 33342 . Images for immunofluorescence were acquired using a confocal microscope  and were analyzed with Zeiss LSM Image Browser, version 4.2.0121.The level of cytochrome Cellular fractionation was performed with the NEPER Nuclear and Cytoplasmic Extraction kit  in conformity with the manufacturer\u2019s instructions.c, and histone H2B purchased from Abcam ; and caspase 3 and \u03b2-actin purchased from Santa Cruz  were maintained overnight at 4 \u00b0C. Blots were developed with ECL reagents (Pierce) and exposed using Kodak X-OMAT AR Film  for 3\u20135 min.Primary antibodies against CHOP  purchased from Cell Signaling ; Bcl-2, cytochrome 6 cells/mouse) were cultured in vitro and then injected subcutaneously into the upper left flank region of female NOD/Shi-scid, IL-2R\u03b3 KOJic (NOG) mice. After 15 days, tumor-bearing mice were grouped randomly (n = 10 per group) and treated with 5 mg/kg cisplatin (p.o.) alone, 80 mg/kg sorafenib (p.o.) alone, and 2.5 mg/kg cisplatin and 40 mg/kg sorafenib in combination. Tumor size was measured every three days using calipers. The detailed protocol can be found in our previous article [All experiments were approved by the Animal Experiment Committee of Yonsei University. YUMC-M1, M2, and M3 patient-derived MTC cells  were used to report consecutive results. Survival curves were produced through the Kaplan\u2013Meier method established on the log-rank test. The detailed protocol can be found in our previous article .RET mutation was confirmed in 25% patients. Median preoperative serum calcitonin and carcinoembryonic antigen levels were 151.5 pg/dL (range: 1.5\u20138030.0 pg/dL) and 10.0 ng/mL (range: 0.8\u2013642.7 ng/mL), respectively. Serum calcitonin normalization  was found in 67.1% patients with MTC. Persistent and recurrent lesions were identified in 49 patients; of these patients, 32 underwent surgery, 3 received external radiation therapy, and 7 received chemotherapy. Of the patients who received chemotherapy, four received vandetanib, one received sorafenib and adriamycin, one received cabozantinib, and one received epirubicin and carboplatin. The median overall survival for 143 patients with MTC was 72.4 months , SNAIL (Zinc finger protein SNAI1), and TWIST (twist family bHLH transcription factor)) were compared with YUMC-F1 (first isolated patient-derived FTC), as shown in FGF (Fibroblast growth factor), FGFR, ZEB, SNAIL, and TWIST (twist family bHLH transcription factor)  B. Kyoto  factor) C.Taken together, the findings regarding MTC can be of outstanding value to therapeutic trials and the administration of patients with aggressive TC.50) than that with cisplatin or sorafenib treatment alone in YUMC-M1, M2, and M3 cells  stress markers (C/EBP homologous protein (CHOP)) and apoptotic signaling pathway markers (B-cell lymphoma 2 (Bcl-2) and cleaved caspase 3) in YUMC-M1, M2, and M3 cells using flow cytometry, immunoblotting (whole-cell lysate or cellular fractionation), and immunofluorescence analyses. The combination of cisplatin and sorafenib at low doses significantly induced the sub-G pathway D. Immunont alone E. To sumTo assessment the combinational in vivo anticancer effectiveness of the co-treatment with cisplatin and sorafenib, we developed a mouse xenograft model with YUMC-M1, M2, and M3 patient-derived MTC cell lines . We discTaken together, the combination of cisplatin and sorafenib indicated potent anticancer effects in an aggressive patient-derived MTC cell xenograft model.c release from mitochondria is a critical event for apoptotic death [c release was significantly induced in the caspase-dependent apoptosis signaling pathway [c from mitochondria most significantly increased apoptosis, and progressively amassed in the nucleus as proven by both immunofluorescence and cellular fractionation after medication of cisplatin and sorafenib at a low dose. Clinically aggressive MTCs lead to poor prognosis [The survival ratio of thyroid carcinoma is well-known as being the best among general endocrine-mediated malignancies , and itsic death ,44. Dist pathway ,46. We irognosis , considerognosis . AccordiIn this research, we showed a prospective new therapy scheme for advanced MTC . Of courAccording to the guidelines of the National Cancer Institute, cisplatin and sorafenib are already well-known anticancer drugs. Although, in conventional cancer therapy, there is no definite solution for treatment of recurrent or metastasis by refractory MTC. In these research findings, the combinational anticancer consequences of the cisplatin and sorafenib were more capable at low doses of each agent alone against patient-derived metastatic MTC. These results could be practical in the design of prospective therapeutic trials and aid the expansion of valuable therapies for patients with refractory MTC. However, research on patient-derived refractory cancer cells is challenging and limited. However, these challenges could be cause to a breakthrough in, and clinical solution to, refractory cancer therapy."}
+{"text": "Ash2l both H3K4 mono- and tri-methylation  were deregulated. In particular, loss of H3K4me3 at promoters correlated with gene repression, especially at CpG island promoters. Ash2l loss resulted in increased loading of histone H3 and reduced chromatin accessibility at promoters, accompanied by an increase of repressing and a decrease of activating histone marks. Moreover, we observed altered binding of CTCF upon Ash2l loss. Lost and gained binding was noticed at promoter-associated and intergenic sites, respectively. Thus, Ash2l loss and reduction of H3K4me3 correlate with altered chromatin accessibility and transcription factor binding. These findings contribute to a more detailed understanding of mechanistic consequences of H3K4me3 loss and associated repression of gene transcription and thus of the observed cellular consequences.Changes in gene expression programs are intimately linked to cell fate decisions. Post-translational modifications of core histones contribute to control gene expression. Methylation of lysine 4 of histone H3 (H3K4) correlates with active promoters and gene transcription. This modification is catalyzed by KMT2 methyltransferases, which require interaction with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, for catalytic activity. Ash2l is necessary for organismal development and for tissue homeostasis. In mouse embryo fibroblasts (MEFs), Ash2l loss results in gene repression, provoking a senescence phenotype. We now find that upon knockout of  These shape chromatin by controlling the density and composition of nucleosomes, modulating the functions of core histones by post-translational modifications (PTMs), and influencing higher-order chromatin organization. Together, these activities allow a multitude of different functional states, which are only incompletely understood. The methylation of histone H3 at lysine 4 (H3K4) has been extensively studied. Trimethylation (H3K4me3) is typically found near transcriptional start sites (TSS) and correlates with accessible promoters7. Together with H3K27 acetylation (H3K27ac), H3K4me3 marks active promoters, while in combination with H3K27me3, referred to as bivalent chromatin, promoters are poised9. H3K4me1 is located at enhancers but is also found in promoter regions, where it appears to have a repressive function12.Cells need to respond to endogenous and exogenous signals to modify and adapt their activities to support tissue and organismal functions. The integration of such signals involves complex changes in gene expression programs that can be short-term or long-lasting as for example in response to stress or during differentiation processes, respectively. Central to executing these regulatory programs are sequence-specific transcription factors (sTFs), which exert their effect on chromatin and ultimately on polymerase activity by recruiting transcriptional cofactors6. Six KMT2 enzymes in mammals (MLL1-4 and SET1A and B) associate with the WRAD core complex composed of WDR5, RBBP5, ASH2L and DPY30, as well as other subunits that are complex specific. KMT2 complexes are functionally important, for example, all subunits that have been evaluated in knock-out studies are essential13. In particular, Ash2l is necessary for organismal development, tissue homeostasis, and cell proliferation and differentiation18. Moreover, increasing evidence points to critical roles of KMT2 complexes in various diseases19. For instance, the core subunit Ash2l, which interacts with the oncoprotein c-MYC20, has been linked to cancer24.Methylation of H3K4 is catalyzed by complexes that contain group 2 lysine methyltransferases (KMT2). These enzymes function in protein complexes referred to as COMPASS (complex of proteins associated with Set1)25. For example, binding to GC-rich sequences, including CpG islands (CGIs), has been observed6. CFP1, an essential subunit of SET1A/B complexes, binds to non-methylated CpG-rich DNA28. Some KMT2 complexes, containing SET1A/B, associate with the C-terminal domain of RNA-polymerase II, thereby stimulating the recruitment of these complexes to transcribed genes30. The function of H3K4me3 at promoters is debated. It has been reported that H3K4me3 is read by the plant homeodomain (PHD) finger of TAF3, a subunit of the general transcription factor complex TFIID. This led to the suggestion that H3K4me3 helps recruit the RNA-polymerase II complex33. This is supported by the finding that promoting H3K4me3 using a dCas9 system was sufficient to induce gene expression34. Moreover, broad and strong H3K4me3 levels at promoters are linked to high transcription36. Importantly, promoter-associated H3K4me3 can also serve as memory for gene expression37. Others find that H3K4me3 might serve as a mark at promoters of transcribed genes, only acquired once transcription has started38. However, in Xenopus blocking transcription using \u03b1-amanitin has little effect on H3K4me339. In yeast, reduction in H3K4me3 has rather small effects on gene transcription. It was noted that gene silencing was most affected and not gene activation as expected45. Yeast Set1 interacts with active RNA Pol II-dependent on Ser5 phosphorylation of the C-terminal domain (CTD) and thus modifies the 5\u2019-end of transcribed regions46. In Drosophila, replacing all histone H3 versions with H3K4R mutants, which cannot be methylated by KMT2 complexes, reduced cell proliferation but did not affect development47. Together, these findings suggest roles for H3K4me3 in both activating and maintaining gene transcription.The recruitment of KMT2 complexes to chromatin is only partially understood. Several mechanisms have been suggested to be relevant, including the interaction of KMT2 complexes with DNA sequence motifs and structural DNA elements, and the binding to transcription factors and histone marksAsh2l and of Dpy30 in murine tissues reduces overall methylation of H3K4, which correlates with altered gene expression48. Using mouse embryo fibroblasts (MEFs), the loss of Ash2l results in broad repression of gene transcription18. Phenotypically, the cells stop proliferating and induce a senescence program characterized by a conserved set of downregulated genes. We have now expanded on this work and analyzed H3K4 methylation using chromatin immunoprecipitation combined with next generation sequencing (ChIP-seq). Ash2l loss reduced H3K4me3 at promoters, which was particularly obvious at CGI promoters. Moreover, a general reduced accessibility of chromatin was observed, particularly at promoters, correlating with loss of H3K4me3. The in silico studies of altered transcription factor binding sites of our ATAC-seq data indicated that the transcriptional regulator and chromatin organizer CCCTC-binding factor (CTCF51) was one of the factors possessing increased DNA binding. Indeed, ChIP-seq experiments supported this notion as CTCF binding increased in intra- and intergenic regions but decreased at promoters. Together, these findings suggest that the loss of Ash2l affects chromatin compaction and that H3K4me3 is involved in maintaining an open chromatin state at promoters.The knockout of 18. Immortalized MEF cells with the Ash2lfl/fl/Cre-ER genotype (KO cells) were treated with 5\u00a0nM 4-hydroxytamoxifen (+\u2009HOT)  or with vehicle  to induce recombination of exon 4. This results in loss of protein expression18.Primary mouse fibroblasts were obtained from d13 embryos, immortalized, cultured and manipulated as described before18. These data were used for the bioinformatic studies described here.RNA was obtained from KO cells treated with and without HOT (for 5\u00a0days) and sequenced as reported previously. The results were deposited in the Gene Expression Omnibus under the accession number GSE165458The antibodies that were used are listed in Table 18. In brief, immunoprecipitations (IPs) were performed with 2\u00a0\u03bcg of specific antibodies recognizing histone H3 or distinct histone marks. For CTCF 5\u00a0\u00b5g of a specific antibody was used. The IgG controls were carried out with respective amounts. For IPs 10\u2013100\u00a0\u00b5g sheared chromatin with a mean size of 500\u00a0bp was used. For ChIP-qPCR undiluted 2\u00a0\u03bcl IP samples and diluted 2\u00a0\u03bcl input samples were applied in duplicates. A SYBR Green reaction mix  was employed for the quantitative PCR (qPCR) analyses in a RotorGene 6000 cycler (Corbett/Qiagen). Results were calculated by determining percent input of IPs considering dilution factors. For histone marks a further normalization was performed to percent input obtained with the H3 antibody. The PCR reactions were carried out with an initial step at 95\u00a0\u00b0C for 2\u00a0min, followed by 40 cycles at 95\u00a0\u00b0C for 10\u00a0s, 60\u00a0\u00b0C for 10\u00a0s, and 72\u00a0\u00b0C for 5\u00a0s and a melting curve analysis. One exception was the primer pair CTCF Chr 11 . DNA concentrations in IP and input samples were measured using a Qubit fluorometer (Thermo Fisher Scientific) according to the manufacturer\u2019s instructions. The libraries were prepared using the Next Ultra II DNA library prep kit for Illumina  and the sequencing was performed as single reads for 75 cycles with a NextSeq 500/550 High Output kit v2.5  according to the manufacturer\u2019s recommendations. Multiplex single end sequencing was performed at the EMBL Genomic Core Facility in Heidelberg and at the Genomics Facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen of the Faculty of Medicine at RWTH Aachen University.For ChIP-seq experiments, 100\u00a0\u03bcg chromatin was used per IP, with 10\u00a0\u03bcg chromatin retained as input control. After immunoprecipitation, as described in the manual of the used Diagenode kit, the complexes were washed in 1\u2009\u00d7\u2009ChIP buffer, taken up in 200\u00a0\u03bcl TE  and incubated with 1\u00a0\u03bcl RNase A (10\u00a0mg/ml) at 37\u00a0\u00b0C for 30\u00a0min. After pelleting, the beads were resuspended in 150\u00a0\u03bcl\u00a0EB buffer  and incubated with 1\u00a0\u03bcl proteinase K (50\u00a0\u00b5g/ml) for 2\u00a0h at 68\u00a0\u00b0C. The beads were centrifuged, and the supernatant was transferred to a DNA low binding reaction tube. The beads were resuspended again in 100\u00a0\u03bcl\u00a0EB and incubated for additional 5\u00a0min at 68\u00a0\u00b0C. The beads were pelleted, and the two supernatants were pooled. Input DNA was purified by precipitation with five volumes 100% ethanol, incubated for 10\u00a0min on ice and centrifuged for 10\u00a0min at 10,000\u00d752, with adaptions53. In brief, crude nuclear extracts were prepared by adding 50\u00a0\u00b5l of ice-cold ATAC-RSB  supplemented with 0.1% NP40, 0.1% Tween-20, 0.01% Digitonin to 5\u2009\u00d7\u2009104 cells. Samples were prepared in two technical replicates. Cells were incubated for 3\u00a0min on ice. Cooled 1\u00a0ml Wash buffer (ATAC-RSB\u2009+\u20090.1% Tween-20) was added, tubes inverted and centrifuged at 500\u2009\u00d7\u2009g for 10\u00a0min at 4\u00a0\u00b0C. With the pellets of the crude nuclear extracts, a transposition procedure was performed. 50\u00a0\u00b5l Transposition mix  was added to the nuclei and incubated for 1\u00a0h at 37\u00a0\u00b0C with shaking. Purification of transposed DNA was carried out with the Qiagen MinElute PCR Purification Kit (#28004).The basic protocol was performed as describedTransposed DNA was barcoded using i5/i7 primers of the Nextera Rapid Capture Custom Enrichment Kit (FC-140-1007 by Illumina) and NEBNext Ultra II Q5 Master Mix (#M0544) from New England Labs. Amplification with barcoding primers was done for initial 5 cycles. After this, an aliquot of PCR product was taken and supplemented with SYBR Green in DMSO  together with the matching set of primers per sample and fresh Q5 polymerase. Another 20 cycles were performed with this aliquot and the amplification was monitored in a real-time application  to assess the progress of the library preparation after the initial 5 cycles in the thermocycler. For both\u2009+\u2009HOT treated and one -HOT treated sample 4 additional and for the second -HOT replicate 3 additional amplification cycles were needed to perform to extend the initial 5 cycles of preparation. After library preparation was done, samples were purified with the Qiagen MinElute PCR Purification Kit (#28004).The libraries were sequenced as paired-end reads for 75 cycles with a NextSeq 500/550 High Output kit v2.5  according to the manufacturer\u2019s recommendations. Sequencing and de-multiplexing were done by the Genomics Facility of the Faculty of Medicine at RWTH Aachen University.Error bars represent standard deviation (SD) of the mean, unless otherwise indicated. Statistical significance was evaluated by multiple t-test using GraphPadPrism software, unless otherwise indicated.https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). They were then aligned against the reference genome (mm9) using BWA54. We used the view, sort and index functions of SAMtools to convert the Sam to Bam files and sort and index the mapped reads55. The complexity of the genomic sequencing library was checked using preseq (https://github.com/smithlabcode/preseq). The duplicates were marked using Picard MarkDuplicates (https://github.com/broadinstitute/picard/blob/master/src/main/java/picard/sam/markduplicates/MarkDuplicates.java). We filtered the un-mapped reads, PCR-duplicates, blacklist region as defined by ENCODE and, in case of paired-end sequencing (ATAC-seq), the un-paired reads using the view function of SAMtools55. We checked the enrichment and quality of ChIP-seq  using the plotFingerprint function of DeepTools56 and Rscript run_spp.R in the phantumpeakqualtools package .For both ChIP-seq and ATAC-seq, sequences were trimmed using Trim_Galore . MultiQC was used to merge all reports from the same experiment57. Narrow peaks (ChIP-seq (H3K4me3), ATAC-seq and ChIP-seq (CTCF)) and Broad peaks (ChIP-seq (H3K4me1)) were called using Macs258. In ChIP-seq (CTCF) experiments, motif-analysis of CTCF consensus sites at topologically associating domain (TAD) boundaries was performed using the FIMO  program from the MEME suite59. TAD boundaries were obtained from published data60 (bed-files at http://chromosome.sdsc.edu/mouse/hi-c/index.html). The overlap was examined by considering a resolution of\u2009\u00b1\u200920\u00a0kb regarding the published Hi-C data. Intersecting between different experiments was done using BEDTools61. The computeMatrix and the plotheatmap functions of DeepTools were used to calculates the scores per genome region in each sample and then ploted the heatmaps56. These were normalized using CPM (count per Million) in ChIP-seq (H3K4me1 and me3) and ATAC-seq . For ChIP-seq (CTCF), the BigWig tracks were normalized using the scale factors obtained by Deseq2.The quality of the ATAC-seq was evaluated by checking the insert size distribution using the CollectMultipleMetrics function of Picard (62. For ChIP-seq (H3K4me1 and me3), the counts were normalized to the lowest coverage and the logFC was calculated manually for each biological replicate (KO1 and KO2). Individual logFC threshold to call gained and lost peaks in\u2009+\u2009HOT compared to -HOT for each of the above-mentioned sequencing experiments was determined after visualization in IGV . The called gained and lost peaks were annotated using Homer (http://homer.ucsd.edu/homer/ngs/annotation.html). The information about the distance to the nearest promoter provided by Homer after the annotation was used to annotate the peaks as promoters (\u00b1\u20093000\u00a0bp of the TSS). We also grouped the counts of the H3K4me3 binding sites at promoters by their A value (log(counts in -HOT and\u2009+\u2009HOT)) in KO1 and KO2, which is estimated from the MA plots  based on promoter sequences 1000\u00a0bp upstream and 100\u00a0bp downstream of the TSS. Motifs were obtained from Jaspar version 202063. Promoter sequences 1000\u00a0bp upstream to 100\u00a0bp downstream the TSS were enriched for TATA box and GC-rich motifs provided by the Eukaryotic promoter database64. The IGV genome browser was used to produce screenshots of selected genomic locations. The enhancers\u2019 genomic locations were obtained from the EnhancerAtlas 2.0 (http://www.enhanceratlas.org/indexv2.php)65. Coordinates of\u00a0CpG\u00a0islands\u00a0were\u00a0obtained from UCSC (https://hgdownload.soe.ucsc.edu/goldenPath/mm9/database/cpgIslandExt.txt.gz).For both ChIP-seq (CTCF) and ATAC-seq, we used DEseq2 to normalize the raw counts in the two technical replicates of each condition and to perform differential analysis between -HOT and\u2009+\u2009HOThttps://github.com/broadinstitute/picard/blob/master/src/main/java/picard/sam/MergeSamFiles.java). TF-footprinting and thereafter the TF-differential analysis were performed using RGT-HINT53. Part of the codes used in this manuscript were modified from nf-core (https://nf-co.re/chipseq/ and https://nf-co.re/atacseq).In ATAC-seq, the two technical replicates were merged before the transcription factor (TF)-footprinting analysis using Picard MergeSamFiles . 2. Accession number GSE205230 for ATAC-seq. 3. Accession number GSE205231 for ChIP-seq (CTCF).All sequencing data are available in NCBI\u2019s Gene Expression Omnibus (GEO)18. In MEF cells this correlates with the induction of senescence. To further evaluate H3K4 methylation, we performed ChIP-seq of 2 pairs of Ash2l KO and WT immortalized MEF cells  at day 5 after 4-hydroxytamoxifen (HOT) treatment, resulting in deletion of exon 4 of Ash2l and loss of Ash2l protein. Genome-wide, 22,344 and 122,781 H3K4me3 and me1 modified regions, respectively, were identified. A large number of H3K4me3 marked chromatin sites  showed loss of signal upon HOT treatment of KO cells   Fig.\u00a0a, consis12. Of the large number of H3K4me1 modified regions, fewer than 600 lost signals  and thus are enriched for GC-rich binding sites. These include the GC box, originally defined as SP1 binding site72, and more general sites for SP as well as Kr\u00fcppel-like factors (KLF)76. We compared the presence of TATA and GC boxes in promoters of up- and downregulated genes. Downregulated genes were increased for promoters with GC boxes while TATA boxes were reduced , which lost H3K4me3 upon Ash2l KO  binding sites. We noticed that a few sites were strongly linked to altered accessibility Fig.\u00a0e. For fuset Fig.\u00a0. Overall94. Moreover, in murine cells two- to threefold more CTCF sites were noticed when compared to human cells95. Of those sites that showed altered binding upon knockout of Ash2l , a loss was observed at 719 and a gain at 1682 binding sites 60, as no defined positions of annotated TAD boundaries for MEFs were available. Therefore, this comparison has to be interpreted with caution. We found that both gained and lost CTCF peaks were associated with potential TADs in MEF cells  boundariesTo further compare the different data sets, we used the 1682 CTCF binding sites that gained binding in response to Ash2l loss in the ChIP-seq experiments and asked how this increased binding affected the neighboring chromatin. We observed increased accessibility around the CTCF binding sites Fig.\u00a0a. This w102. It also results in a redistribution of CTCF binding from promoters to intergenic sites, which suggests that higher-order chromatin organization may be affected by Ash2l loss. Although these findings correlate with altered H3K4 methylation, it is not understood whether the loss of H3K4me3 at promoters is necessary for the local reduced chromatin accessibility. Multiple H3K4me3 readers have been identified, which include protein complexes with histone acetyltransferase and chromatin remodeling activity105. Thus, the loss of H3K4me3 may cause direct effects on the accessibility of chromatin at promoters. However, it is important to note that Ash2l is an abundant protein. The analysis in HeLa cells suggests that Ash2l is considerably more abundant than all KMT2 subunits together106. It is possible that Ash2l possesses additional functions that do not rely on KMT2 complex activities and thus may be independent of H3K4 methylation. Future work will need to address whether so far unknown functions can be attributed to Ash2l. This will be important to clarify the contribution of H3K4 methylation to the complex phenotypes associated with Ash2l loss. Such studies will also be useful in further defining the functions of H3K4me3, in particular regarding the discussion whether this histone mark is a determinant of initiation of gene transcription or a consequence of gene transcription, for example by facilitating polymerase reinitiating and/or effects on RNA processing.Our findings suggest that Ash2l loss and concomitant reduction in H3K4 methylation results in chromatin compaction. This is exemplified by the increased histone H3 ChIP-qPCR signals at selective promoters and the overall decreased accessibility of promoters in the ATAC-seq experiments. This is consistent with the observation that active TSSs are preferentially found in open chromatinAsh2l in mice in hematopoietic cells results in the accumulation of so-called LSK (lin\u2212Sca1+Kit+) cells in the bone marrow. LSK cells are highly enriched in hematopoietic stem and multi-potent progenitor cells. Importantly, these cells are unable to differentiate, both in vivo and in tissue culture, and as a consequence essential mature hematopoietic cells are lacking in the animals15. These LSK cells accumulate over several days with strongly reduced overall H3K4me3. Thus, we suppose that the decrease in H3K4me3, most likely at promoters, results in the inability of the cells to adapt their gene expression programs for efficient differentiation. This is consistent with the above discussed functions of this histone mark as a modification that allows gene activation. While the LSK cells are arrested in G2/M, the MEF cells, both KO1 and KO2, do not respond by accumulating at a defined cell cycle stage18. Nevertheless, these cells stop proliferating. Phenotypically, the cells appear senescent. This is somewhat unexpected as senescence requires typically the activation of a specific gene expression program, which includes SASP (senescence-associated secretory phenotype)108. Indeed, SASP gene activation could not been observed, consistent with the broad loss of H3K4me3 at promoters. Instead, a set of downregulated genes is associated with senescence18. Thus, we suspect that the upregulation of SASP genes is similarly impaired as differentiation-associated genes in LSK cells. Together, these findings support the notion that H3K4me3 is important for de novo gene activation.The biological responses to loss of Ash2l and H3K4 methylation are consistent with the broad effects on promoters and gene transcription. The knockout of Supplementary Information."}
+{"text": "Clostridium genus showpromise as industrial solvent producers and cancer therapeutic deliveryvehicles. Previous development of shuttle plasmids and genome editingtools has aided the study of these species and enabled their exploitationin industrial and medical applications. Nevertheless, the precisecontrol of gene expression is still hindered by the limited rangeof characterized promoters. To address this, libraries of promoters(native and synthetic), 5\u2032 UTRs, and alternative start codonswere constructed. These constructs were tested in Escherichiacoli K-12, Clostridium sporogenes NCIMB 10696, and Clostridium butyricum DSM 10702, using \u03b2-glucuronidase (gusA) asa gene reporter. Promoter activity was corroborated using a secondgene reporter, nitroreductase (nmeNTR) from Neisseria meningitides. A strong correlation wasobserved between the two reporters. In C. sporogenes and C. butyricum, respectively, changesin GusA activity between the weakest and strongest expressing levelswere 129-fold and 78-fold. Similar results were obtained with the nmeNTR. Using the GusA reporter, translation initiationfrom six alternative (non-AUG) start codons was measured in E. coli, C. sporogenes, and C. butyricum. Clearly, species-specificdifferences between clostridia and E. coli in translation initiation were observed, and the performance ofthe start codons was influenced by the upstream 5\u2032 UTR sequence.These results highlight a new opportunity for gene control in recombinantclostridia. To demonstrate the value of these results, expressionof the sacB gene from Bacillus subtilis was optimized for use as a novel negative selection marker in C. butyricum. In summary, these results indicateimprovements in the understanding of heterologous gene regulationin Clostridium species and E. coli cloning strains. This new knowledge can be utilized for rationallydesigned gene regulation in Clostridium-mediatedindustrial and medical applications, as well as fundamental researchinto the biology of Clostridium species.Several species from the  Clostridium is a highlydiverse genus of Gram-positivebacteria, unified by their ability to form endospores and their inabilityto grow in the presence of oxygen. Although several hundred speciesare taxonomically classified as clostridia, more recent phylogeneticanalysis suggests that a much smaller group of bacteria are evolutionarilyrelated, the so-called \u201cClostridium sensu stricto\u201d.1 Historically, species of thisgenus were synonymous with infection, caused by species such as Clostridium botulinum, Clostridiumtetani, Clostridium perfringens, and Clostridioides difficile. However,most clostridia are completely benign.2 Beyond the domain of human health, solventogenic clostridia havebeen utilized in the industrial production of petrochemicals due totheir ability to produce acetone\u2013butanol\u2013ethanol (ABE)by microbial fermentation ,3 while cellulolytic, biofuel-producing strains,such as Clostridium cellulolyticum and Clostridium cellulovorans, are now attracting attentionto satisfy the increasing demand for cleaner fuels.5 Oncolyticstrains, such as Clostridium sporogenes and Clostridium novyi, selectivelygerminate in the hypoxic/necrotic regions of tumors and their endogenousoncolytic activity can result in partial destruction of the tumor.7 Subsequent studies have highlighted distinct advantages to usingcertain Clostridium species as a therapeutic vectorin bacterial-mediated cancer therapy.10 The addition, throughgenetic engineering, of clinically approved heterologous products,such as antibodies,11 proinflammatory cytokines,13 and checkpoint inhibitors, could further increase their therapeuticvalue. An alternative approach is clostridia-directed enzyme prodrugtherapy (CDEPT), which has seen significant advances in recent years.18 Thus, demand for synthetic strains of these species has highlightedthe need for genetic tools that will enable the precise control ofsynthetic products of industrial or therapeutic value. This need hasbeen met to a degree in the form of an Escherichiacoli\u2013Clostridium shuttle plasmids,2 the bacterial group II intron technology (ClosTron),19 and CRISPR-based editing systems.2122 Intratumoraldelivery of therapeutics will have similar demands. However, promotersreported in the literature are typically of Clostridium origin and from a very limited number of species, such as the promotersof the thl (thiolase), ptb (phosphotransbutyrylase),and araE (arabinose sugar\u2013proton symporter)genes of C. acetobutylicum ATCC 82425 and of the fdx (ferredoxin) gene of C. sporogenes NCIMB 10696.26 Due to the limited range of strengths and the multitude of regulatoryproteins that can interfere with native promoters, the focus of researchershas shifted to the generation of synthetic promoter libraries.27 In a recent study, the widely used constitutivepromoter of C. acetobutylicum, Pthl, was employed to generate synthetic promoters by randomizationof the regions surrounding the consensus \u221235 and \u221210elements. The apparent stringent requirements for promoters in Clostridium reduce the number of functional promoters insynthetic libraries, highlighting the challenge of controlling geneexpression in this genus.29 In addition to controllingtranscript production by the promoter, the 5\u2032 untranslatedregion (5\u2032 UTR) of the resultant mRNA, essential for translation,is another element that can be exploited for controlling gene expression.30 Moreover, the start codon is recognized by theribosome to initiate translation31 andis a key modulator of translation.32Accurate quantification of available gene promoters enablessyntheticbiologists to control recombinant bacteria precisely. In the industrialcontext, this enables microorganisms to be modified for maximum productyield and minimum cellular biomass.Clostridium in an established GusA assay, whichshowed good stability and sensitivity. Second, consensus promotersequences, based on whole genome promoter alignment, were predicted.In combination with different 5\u2032 UTR sequences, this promoter-5\u2032UTR library was evaluated for gene expression in two Clostridiumsensu stricto species: C. sporogenes-NT12 and C. butyricum DSM 10702.33 This library was cross-validatedusing a previously reported nitroreductase of Neisseriameningitidis, NmeNTR14 in C. sporogenes-NT. In addition, we measured translation initiation from alternative(non-AUG) start codons in E. coli, C. sporogenes-NT, and C. butyricum by the GusA assay. Finally, to demonstrate the value of these results,we tuned the expression of the sacB gene, encodinglevansucrase, from Bacillus subtilis and reported its use as a novel negative selection maker in C. butyricum.Here, we describe the generation of promoters, 5\u2032 UTRs,and alternative start codon libraries that enable genes to be expressedat different levels in clostridia. The promoter-5\u2032 UTR librarybuilds on previously published work, while the alternative start codonlibrary is novel and adds to the repertoire of elements for gene controlin clostridia. First, we optimized the construction of a glucuronidase(GusA) reporter system for the rapid generation expression variants.Using this system, we directly compared frequently cited native promotersof 34 FAST,35 HaloTag, and SNAP-tag proteins.36 Fluorescent reporters are nonenzymatic and nonamplifying,which could reduce their sensitivity for quantifying gene expressioncompared to enzyme-based reporter assays.37 Similarly, chloramphenicol acetyl transferase (CAT) of the antibioticselection marker is a popular reporter in clostridia40 that has been used previously to generate a synthetic promoter library.29 Due to the lack of sensitivity, relatively weakpromoters may fall below the detection limit of CAT, limiting itsuse as a reporter.41 Enzymatic reportershave been widely used in clostridia, including \u03b2-galactosidase(LacZ)44 and glucuronidase (GusA).Since the GUS reporter system of plants was reviewed in 1989, mostresearchers in clostridia have employed the GusA reporter to evaluatepromoter strength.48 Based on these considerations,we here chose GusA as a sensitive and reliable reporter that allowsthe comparison of strong and weak promoters covering a broad dynamicrange of gene expression.A range of gene products has been utilized as gene expression reporters.To serve this purpose, the protein should be stable, sensitive enoughto detect the signal of weak promoters above background noise, inexpensive,and simple to assay. Fluorescent proteins, such as GFP or RFP, areappealing due to the simplicity of signal detection, but the oxygenrequirement of these reporters limits their use in anoxic conditions.Recently, oxygen-independent fluorescent reporters have been developedin clostridia, including iLOV,BsaI restrictionsites in the multiple cloning site was generated. The first versionof this vector (pGG2151) was based on the high-copy E. coli\u2013Clostridium shuttlevector, pMTL82151. Initial attempts to use this vector in Golden Gateassembly reactions yielded low-efficiency assembly with a high rateof single nucleotide polymorphisms (SNPs). Sanger sequencing showedthat the SNPs frequently occurred in the ribosome-binding site (RBS)and the open reading frame (ORF) of gusA gene. Wespeculated that due to the high-copy number of the Gram-negative repliconColE1 in E. coli,49 the resulting high expression level of the gusA gene was harmful to the host and resulted in cloning difficulties.Thus, the replicon was swapped to low-copy p15a replicon  were assembled utilizing the Golden Gate reactionand transformed into E. coli K-12 strainJW1609 (gusA\u2013)50 or E. coli S17-1for conjugation into Clostridium spp. Clostridium transconjugants were confirmed by Sanger sequencing before beingtested in the GusA assay. This workflow enabled the library to begenerated faster and more efficiently, as compared to conventionalcloning techniques.600 = 1) across all constructsfor both E. coli and Clostridium strains harboring GusA expression plasmids. As controls, strainscarrying an empty vector pGG2121 or a promoter-less gusA vector (pGG2121-gusA) were assayed. Both constructsdemonstrated very low GusA activity in E. coligusA\u2013, C. sporogenes-NT, and C. butyricum .To enable comparison of expression levels, all samples were obtainedat the same growth point  in our study,we added the native 5\u2032 UTR of Pthl (tU) tothe 3\u2032 end of the promoters, resulting in the promoter-5\u2032UTR fusions miniP4_tU, Psyn_tU andPj23119_tU. As shown in C. sporogenes-NT. miniP4_tU showed a similar level of expression to Pthl79%-26_tU, which was the strongest promoter in a previous Pthl mutant library.28 The resultssuggest that promoter prediction by bioinformatics tools could bean efficient method for finding functional promoters of differentstrengths in the Clostridium genus.We initially tested four native fdx exhibited the highest GusA activityin our current library (specific activity of 3000). Both processesof transcription and translation affect gene expression level. The5\u2032 UTR plays a critical role in a fine balance between transcription,transcript stability, and translation.30 To investigate the influence of the 5\u2032 UTR on gene expression,we directly exchanged tU with the 5\u2032 UTR of Pfdx (fU), which is considerably shorter than tU  and Pfdx (Pfdx_fU), obtainingmodified promoter-5\u2032 UTR fusions Pthl_fU andPfdx_tU. In C. sporogenes-NT, the GusA activity of Pthl_fU was higher thanthat of the Pthl_tU native promoter, while Pfdx_tU showed a significant decrease in GusA activity comparedto native Pfdx_fU. The reverse was observed in E. coli .58 The resulting promoter miniP4 produceda very high level of gene expression in our reporter assay, suggestingthat this method can generate strong promoter candidates that performwell between related species. Thus, to create novel predicted consensuspromoters using the same method, we used the genomes of C. sporogenes NCIMB 10696 (NZ_CP009225.1) and C. butyricum DSM 10702 (NZ_CP040626.1) as input datafor the PePPER algorithm. For each species, 363 and 142 promoters,respectively, were identified (Table S3). These were aligned to create two 29-nucleotide-long DNA logosusing WebLogo,66 named miniPc.sp and miniPc.b, respectively , over 1.8-fold higher than that ofnative promoter Pfdx. The 16 promoters-5\u2032UTR of the library in C. sporogenes showed a broad range of GusA expression, with a 129-fold changebetween the weakest (Psyn_fU) and the strongest (miniPc.sp_fU) promoters-5\u2032 UTR .A native \u201cconsensus\u201d promoter can be determined bythe alignment of predicted native promoters using the webserver PePPER.ectively 4a. The lClostridium genus, all 16 promoters-5\u2032UTR expressingGusA were transferred into the distantly related Clostridium species C. butyricum DSM 10702. Thisspecies is a butyric acid-producing strain of industrial relevance67 and is widely used as a probiotic.69 All 16 promoters-5\u2032 UTR were active and exhibited a widerange of strengths , functional intwo divergent Clostridium species, was generatedusing native promoters, bioinformatics promoter prediction, and native5\u2032 UTR exchange.To demonstrate the portability and utility of thislibrary in the utyricum 4c, suggeClostridium-mediatedmedical applications, eight promoters  were selected fromthe library to express the previously characterized nitroreductasegene from Neisseria meningitidis, nmeNTR, as a therapeutic gene. NmeNTR was studied as a prodrugconverting enzyme (PCE) in Clostridium-directed enzymeprodrug therapy (CDEPT) and in combination with prodrug CB1954 orPR-104 showed significant antitumor efficacy.15 The promoter-5\u2032 UTR sequences and nmeNTR coding sequence were cloned into pGG2121, as described previouslyand confirmed by PCR screening and Sanger sequencing. The resultingseries of pGG2121-Promoter-nmeNTR plasmids were transformed into E. coli S17-1 and then conjugated into C. sporogenes-NT. After confirmation of Clostridium transconjugantsby Sanger sequencing, 7 h subcultures were harvested and the menadionenitroreductase assay  and strongest (miniP4_tU) promoters-5\u2032UTR . In addition, all of thepromoter-5\u2032 UTR sequences in our study resulted in a high geneexpression level in E. coli. In thecontext of gene products that are toxic to E. coli, this may prevent cloning of the gene of interest. To further tunethe gene expression between clostridia and E. coli, we set out to investigate the effect of the start codon sequenceon heterologous gene expression in E. coli, C. sporogenes, and C. butyricum. Previously, non-AUG start codons areannotated as initiation codons in 69 bacterial genomes including GUG,UUG, CUG, AUC, AUU, and AUA, from which translation initiated at 0.1\u2013100%of AUG in E. coli.32 Inspired by this finding, we changed the AUG start codonof gusA gene with six alternative start codons  and used the promoter-5\u2032 UTR sequencesof native Pfdx_fU and the Pfdx_tUto drive the expression, obtaining a series of vectors pGG2121-Pfdx_fU-non-AUGgusA and pGG2121-Pfdx_tU-non-AUGgusA.Anideal promoter for fdx_fU, GusA expression initiated from allsix alternative start codons showed only a 0.04\u20133% activityof AUG in E. coli , GusAexpression showed only a 0.1\u20130.3% activity of AUG in E. coli  could create out-of-framecanonical start codons.72 The regulationof translation initiation from the 5\u2032 UTR is complicated, andit is likely to vary between species. However, the aforementionedresults indicate that translation initiation from these alternativestart codons \u2014at least in the two contextsthat were tested\u2014is more efficient in clostridia than in E. coli. In bacteria, translation initiation requiresinitiation factors  and the initiator tRNA (fMet-tRNAfMet), in which IF3 increases the accuracy of initiator tRNAselection, monitoring codon\u2013anticodon interactions.74 We reasoned that compared to E. coli, the initiation factors in clostridia might be less stringent inselecting the initiator tRNA. Another explanation is that the initiatortRNA may be more abundant in clostridia than in E.coli. Therefore, we encourage reconsidering gene annotationsin Clostridium genomes and further exploration oftranslation initiation in clostridia. Overall, combined with our promoter-5\u2032UTR library before, the library of alternative start codons couldfurther tune the gene expression between clostridia and E. coli.Driven by thenative P E. coli 6a, whileectively 6b,c. The E. coli 6d, whileutyricum 6e and a orogenes 6f. These E. coli 6d, which E. coli 6a. We resacB genefor expression in C. butyricum. The sacB gene originates from B. subtilis and encodes the secreted levansucrase that converts sucrose intolevans, which have toxic effects on bacterial cells.76 The sacB gene has been used as a negative selectionmarker to screen for double-crossover events in E.coli since the early 1990s and in numerous Gram-negativebacteria subsequently.77 Although certainspecies, such as Corynebacterium glutamicum and species of genus Mycobacterium, show sucrosesensitivity under sacB expression,78 to date there is no report of using sacB as a negative selection marker in clostridia. Thus, sacB with its native promoter PsacB  would notbe functional in C. butyricum. Thus,we selected the Pthl_fU, shown to have a medium\u2013highstrength in our promoter-5\u2032 UTR library of C.butyricum . This cloning difficulty is similarto previous reports in ilvB (encoding acetohydroxyacidsynthase) clones of E. coli.79 The Pthl_fU can express a significantGusA level in E. coli , and cloning the native sacB expression cassette can result in fragility of the E. coli envelopes, as previously demonstrated.80 Thus, we reasoned that the high level of sacB expression by the Pthl_fU increasedthe toxic effect in E. coli and resultedin cloning difficulties of the expression cassette Pthl_fU-AUGsacB. To selectively downregulate sacB expression in E. coli, we used the UUG start codon for translation initiation, previouslyshown to reduce GusA expression to a 3% activity of AUG in E. coli, driven by the native Pfdx_fU  in this study were used for the cloning, GusA assay, and conjugation,respectively. In addition, E. coli strainscontaining plasmids (Table S1) were grownat 37 \u00b0C in LB broth supplemented with 12.5 \u03bcg/mL of chloramphenicolor on LB agar plates supplemented with 25 \u03bcg/mL of chloramphenicol.No-toxic strain of C. sporogenes NCIMB10696 was created previously by deleting the putative StreptolysinS (SLS) operon.12C. butyricum-type strain DSM 10702 was purchased from DSMZ-German Collection.These Clostridium strains in this study were grownin peptone yeast thioglycolate media12 withthe addition of 10 g/L of d-glucose (PYTG), supplementedwith d-cycloserine (250 \u03bcg/mL) and thiamphenicol (15\u03bcg/mL) when necessary. Then, the culture was incubated at 37\u00b0C in an anaerobic cabinet .Details ofthe used strain are all listed in Table S1). All of the plasmidsconstructed in this study were confirmed by PCR screening of M13-F/Rand Sanger sequencing. The shuttle vectors pMTL82151 and pMTL82121were obtained from Prof. Minton .The vector pMTL82151/pMTL82121 was digested by MreI/NheI to remove the terminator CD0164 and multiplecloning site (MCS), and the \u223c4 kb fragment was purified asthe backbone. The fragment of terminator tyrS wasamplified by primers with Golden Gate BsmBI sites.In addition, new MCS was amplified by primers with Golden Gate BsaI sites, universal primer pairs M13-F/R sequences, andGolden Gate BsmBI sites. According to the GoldenGate assembly protocol (BsmBI-v2) , these three fragments were ligated and transformedinto E. coli 10-\u03b2, obtainingthe Golden Gate assembly plasmid pGG2121. Two primers with GoldenGate BsaI sites were designed to amplify gusA gene using pRPF185 as a template. To enable the rapidand precise cloning of the GusA reporter system, the ORF of gusA gene was blunt-cloned into vector pMiniT 2.0 by PCRCloning Kit . The resulting plasmid pMiniT 2.0-gusA  , gusA fragment in pMiniT 2.0-gusA and promoters were ligated into plasmid pGG2121. Similarly, nmeNTR fragment14 and sacB fragment were amplified and ligated with selected promotersinto plasmid pGG2121 by Golden Gate assembly. Plasmids were transformedby heat shock into E. coli strainsand transferred to Clostridium strains by conjugation,as described previously.2Details ofthe used plasmids are all listed in 2.0-gusA 1 was seqE. coligusA\u2013 and Clostridium spp. was evaluated as described by Pawe\u0142 and John.28 The overnight culture of strains containingGusA expression plasmids was inoculated into a fresh PYTG media (1:100).As OD600 of the cultures grow to about 1.0, samples of1.5 mL were harvested by centrifugation and the pellets were frozenat \u221280 \u00b0C. Similarly, the pellet testing was administratedas described and the specific activity of GusA was calculated in theunit of \u0394Abs405/OD600/min by dividingthe absorbance at 405 nm by sample OD600 nm and incubationtime.The glucuronidase (GusA)activity in 81 the nitroreductase (NTR) activityin C. sporogenes was determined usingmenadione  as an enzymatic substrate and bovinecytochrome c  as an electronacceptor and also a colorimetric chemical. Briefly, the overnightculture of strains containing NmeNTR expression plasmids was inoculatedinto a fresh PYTG media (1:100). After 7 h, samples of 2 mL were harvestedby centrifugation and the pellets were frozen at \u221280 \u00b0C.As for stock solutions, 1 mM of menadione was made up in dimethylsulfoxide (DMSO) and stored at room temperature. The other solutionsof 10 mM of NADH  and 700 \u03bcM of bovinecytochrome c were dissolved in 10 mM of tris\u2013HClbuffer, pH 7.5 and stored at \u221220 \u00b0C. Before testing, thepellets were lysed and the soluble proteins inside were extractedin 300 \u03bcL of BugBuster Master Mix/protease solution , which then were diluted in 10 mM of tris\u2013HClbuffer. Moreover, using a flat-bottom 96-well plate, 150 \u03bcL/wellsof reaction master mix  was preparedand preheated at 37 \u00b0C for about 10 min in a multimode microplatereader . Then, 10 \u03bcL of diluted lysates with40 \u03bcL of 10 mM tris\u2013HCl buffer was added into wells at37 \u00b0C. During the incubation, the increase of absorbance at 550nm for 1 min was recorded and the rate divided by the volume (in mL)of lysate used was 14.79 (the extinction coefficient of cytochrome c in cm\u20131 \u03bcM\u20131), obtaining the menadione nitroreductase activity expressed in unitsper mL (U/mL). Finally, the menadione nitroreductase activity testedabove was normalized to units per g (U/mg) by dividing the total proteinconcentration (mg/mL) inside the used lysate, which was determinedas the BCA Protein Assay Kit  described.According to Knox etal.,"}
+{"text": "Amlitelimab, an anti-OX40L antibody, was studied in a 12-week treatment phase 2a clinical trial, with a significant efficacy response observed within 2 weeks. At week 16, amlitelimab groups reached the EASI mean percentage change from the baseline of \u221269.9% and \u221280.1% versus the placebo . Among the responders, 68% of amlitelimab patients were sustained 24 weeks following the last dose. Both treatments were shown to be safe and well tolerated. Current evidence points to OX40-OX40L inhibitors as future options for atopic dermatitis treatment with potential disease-modifying effects.Despite the recent emergence of targeted therapeutic options, there are still unmet needs concerning moderate-to-severe atopic dermatitis treatment. This review aims to discuss the OX40-OX40L pathway as a therapeutic target for the treatment of atopic dermatitis. OX40 and OX40L are two checkpoint molecules that bind to potentiate pro-inflammatory T-cell responses that are pivotal to atopic dermatitis pathogenesis. Two OX40-OX40L inhibitors, rocatinlimab and amlitelimab, are being developed for the treatment of atopic dermatitis. Rocatinlimab, an anti-OX40 antibody, was evaluated in phase 2b, a randomized, placebo-controlled clinical trial. At week 16, rocatinlimab groups achieved a greater reduction in the EASI percentage change from the baseline (\u221248.3% to \u221261.1%) against the placebo (\u221215.0%;  Atopic dermatitis (AD) is a chronically relapsing, inflammatory skin disease with a great impact on the patient\u2019s quality of life, especially due to pruritus. With a prevalence of 2\u20135% in young adults and up to 20% in children, AD is one of the most common skin diseases ,2.The currently available treatment armamentarium includes topical therapy , phototherapy, and systemic therapy . Conventional systemic immunosuppressive therapies may have limited efficacy and harbor long-term toxicity, which makes them not appropriate for continuous use ,4. FortuNonetheless, some patients are still a therapeutic challenge, whether because they do not respond/lose clinical response or are unable to receive treatment due to tolerability and safety issues. These unmet needs require that new mechanisms of action and new targeted drugs continue to be developed.This review aims to discuss the OX40-OX40L pathway as a potential therapeutic target for the treatment of AD, focusing on two OX40-OX40L inhibitors: rocatinlimab and amlitelimab.A complex interplay between skin barrier dysfunction, skin inflammation, and dysbiosis contributes to AD development and chronicity . The acuOX40\u2013OX40L interaction (two co-stimulatory immune checkpoint molecules) plays a central role in the pathogenesis of AD. Immune checkpoint molecules have co-stimulatory and co-inhibitory roles in adaptative immune responses. They are primarily classified into two groups: (1) the immunoglobulin superfamily and the (2) the tumor necrosis factor superfamily (TNFSF) and its receptors (TNFRSFs). OX40  and its ligand OX40L  are two of the TNFSF/TNFRSF co-stimulatory immune checkpoint molecules .The co-stimulatory T-cell receptor OX40 is expressed predominantly on effector and regulatory T-cells. Its ligand, OX40L, is expressed on activated antigen-presenting cells, including dendritic cells (DCs), endothelial cells, macrophages, and activated B-cells. OX40\u2013OX40L engagement is key to potentiating the expansion of effector T-cells and the prolongation of their survival by suppressing apoptosis, enhancing T-cell effector functions, such as cytokine production, and generating T helper memory cells. Na\u00efve T-cells are activated by antigen-presenting cells through co-stimulatory molecule interaction, such as CD80/CD86 and CD28. The activated effector Th1 and Th2 T-cell expansion are sustained by OX40-OX40L ligation. Even though resting memory T-cells do not express OX40, upon reactivation, they become effector memory T-cells, and they start expressing it. OX40-OX40L ligation promotes the expansion of these cells ,18.Preclinical studies of skin inflammation and asthma models have supported that OX40-OX40L signaling interactions are pivotal to the efficiency of the responses that are regulated by memory Th2 cells ,20. TSLPIn patients with AD, the surface expression of OX40 and its ligand on the peripheral blood of mononuclear cells is higher in comparison with healthy adults. Strong correlations were observed between the disease activity scores and Th2-associated markers, TSLPR, and OX40L ,23.p < 0.05) and 71 (p < 0.001). A significant reduction in mRNA cytokines such as IL-31, CCL11, CCL17, and S100 was also demonstrated. GBR 830 was well tolerated, with an equal treatment-emergent adverse events (TEAE) distribution . One serious event was reported in the GBR 830 group, but it was deemed unrelated to the study of the drug. The most reported TEAE was a headache, with no clinically meaningful differences between the groups. Myalgias were only reported in the GBR 830 group (6.5% [3/46]) ) ,25.No further studies have been developed with this drug.Rocatinlimab, formerly known as AMG 451/KHK4083, is a fully human, non-fucosylated, immunoglobulin G1 (IgG1) anti-OX40 monoclonal antibody currently under investigation for the treatment of moderate-to-severe AD. It has been shown to selectively deplete OX40+ activated T-cells and suppress clonal T-cells and is expected to control Th2-driven conditions .A phase 1, single-center, open-label trial evaluated 22 patients with moderate-to-severe AD through a 6-week treatment period with repeated intravenous infusions of 10 mg/kg KHK4083 every 2 weeks  and a 16-week follow-up period (NCT03096223) . KHK4083p < 0.001). Higher proportions of patients in the rocatinlimab groups achieved EASI75 . Among the rocatinlimab patients, 36.5% to 55.8% achieved a 4-point improvement or greater from the baseline in the pruritus NRS score (placebo: 19.3%). On each endpoint, greater improvements were observed at week 16 for rocatinlimab: 300 mg Q2W in comparison to other doses [A phase 2b multicenter, randomized, double-blind, parallel-group, placebo-controlled clinical trial (NCT03703102) was posteriorly developed to further evaluate the efficacy and safety of rocatinlimab in subjects with moderate-to-severe atopic dermatitis with an inadequate response to topical treatments . The 274er doses . Efficacer doses . Througher doses .A clinical biomarker sub-study aimed at analyzing transcriptomic and proteomic profiles in skin biopsy specimens and serum samples of the participating AD patients. Skin biopsy specimens were collected from 20/150 Japanese patients at the baseline, week 8, week 16, week 36, and week 52, and the genomic profile showed significant and robust changes from the baseline throughout treatment, approaching that of the non-lesional skin. Quantitative polymerase chain reaction analysis revealed a reduced OX40 mRNA expression and the downregulation of Th2, Th1/Th17, and Th22-related genes after rocatinlimab treatment. The effects of rocatinlimab on gene expression persisted after the discontinuation of treatment at week 36, through week 52. A reduction in Th2/Th22 and pruritus-related molecules were also observed in proteomic analysis using serum samples at week 16 .A post hoc analysis evaluated rocatinlimab\u2019s efficacy concerning head and neck disease. The head and neck EASI score was calculated with the adjustment of 0.1, equating to 10% of the weighting of the head and neck region in the total EASI score. Reported results were consistent with the main analysis as all rocatinlimab doses resulted in greater head and neck EASI score improvements versus the placebo until week 56, 20 weeks after treatment had ceased .A phase 3, 52-week, multicenter, randomized, double-blind, placebo-controlled trial is currently in progress  to further evaluate two different doses in four different treatment schemes (NCT05398445-ROCKET-IGNITE) .Amlitelimab, also known as KY1005/SAR445229, is another OX40-OX40L pathway inhibitor but with a different mechanism of action. It is a non-depleting IgG4 human anti-OX40L monoclonal antibody that binds OX40L and blocks interactions with OX40. By targeting OX40L, amlitelimab aims to restore immune homeostasis between pro-inflammatory and anti-inflammatory T-cells. In addition to blocking antigen-presenting T-cell activation, amlitelimab also blocks T-cell independent antigens that present the cells\u2019 pro-inflammatory activity via the inhibition of OX40L back signaling, thus blocking both type 2 and Th1/17/22 inflammation ,32.n = 190) or moderate severity (n = 16) and were self-resolving without sequalae, a headache being the most reported one. There were no clinically significant changes in any safety laboratory parameters [A phase 1 trial NCT03161288) was first conducted with 64 healthy subjects to evaluate its safety, tolerability, and immunogenicity profile. Subjects were enrolled into eight cohorts, and in each of them, they were randomized to the intravenous administration of KY1005 (different doses) or a placebo. Two subjects per cohort started as a sentinel group, and if no safety issues arose within 48 h after dosing, the remaining six subjects received treatment. At 4 and 8 weeks after the initial administration, the subjects received two maintenance doses (50% of the loading dose). One subject in the 12 mg/kg cohort did not receive the second and third KY1005 doses due to a possible mild and self-limited hypersensitivity reaction. All treatment-emergent adverse events were of mild (61288 wasn = 29), high dose , or a placebo (n = 29) until week 12. At the end of week 16, there were 59 evaluable patients, and at the end of week 36, there were 50. The main reason for the study discontinuation was a withdrawal of consent (15/29) [A phase 2a (NCT03754309) double-blind, randomized, controlled trial was conducted for 16 weeks with 89 moderate-to-severe AD patients who were intolerant or had an inadequate response to topical treatments. They were randomized 1:1:1 to an intravenous amlitelimab low dose  ,33.p = 0.009 and p = 0.072, respectively). EASI\u221275 was reached by 59.3% of patients in the amlitelimab LD group, 51.9% in the amlitelimab HD group, and 25.0% in the placebo group. The onset of response was as early as week 2 for both amlitelimab groups. Additionally, 44% of patients treated with amlitelimab-LD and 37% of patients treated with amlitelimab-HD achieved a score of 0 (clear) or 1  on the validated Investigator\u2019s Global Assessment (vIGA) scale compared with 8% with placebo (p < 0.001 both LD and HD). At week 36, 68% of patients who achieved a vIGA score of 0 or 1 at week 16 maintained their response 24 weeks after their last dose. Pruritus NRS \u2265 4-point improvement at week 16 was reached by 57.9% in amlitelimab LD, 62.5% in amlitelimab HD, and 38.1% in the placebo group [According to non-peer-reviewed data, at week 16, the mean percentage change from the baseline in EASI was significantly greater in patients receiving amlitelimab LD (\u221280.1%) and HD (\u221269.9%) vs. the placebo , and no significant differences were observed between the groups. At week 16, a significant decrease in IL-22 serum levels was observed in patients treated with amlitelimab but not in the placebo (p = 0.381). An amlitelimab-induced decrease in IL-22 levels was maintained until week 36 in those defined as vIGA 0/1 responders at week 16, 24 weeks after their last dose. On the other hand, there were no significant disparities in the baseline IL-22 between responders and non-responders [Considering the importance of IL-22 in AD pathogenesis and its hypothesized association with the OX40-OX40L pathway, a specific analysis was performed to evaluate its behavior during treatment. The IL-22 baseline serum levels correlated significantly with the severity of the disease  to further evaluate the impact of amlitelimab, when given subcutaneously, in patients with moderate-to-severe atopic dermatitis. It is an interventional, randomized, parallel-group, phase 2b, double-blind, 5-arm trial, which aims to assess the effect of amlitelimab in adult patients with moderate-to-severe atopic dermatitis. Additionally, a single group, phase 2, long-term extension study (NCT05492578) will characterize the safety and efficacy of amlitelimab in treated adult participants with moderate-to-severe AD who have previously been enrolled in the study (NCT05131477) .Amlitelimab is also under research for the treatment of moderate-to-severe asthma .Atopic dermatitis poses a therapeutic challenge for clinicians, particularly in moderate-to-severe forms of the disease. Systemic corticosteroids and classic immunosuppressants, such as cyclosporine, methotrexate, mycophenolate mofetil, and azathioprine, have their prolonged use limited by safety concerns, variable efficacy response, and the need for frequent laboratory monitoring. Increased knowledge of atopic dermatitis complex pathophysiology has allowed the consideration of more immunological pathways as potential therapeutic targets. The currently available targeted options include biologic therapy, dupilumab, tralokinumab, and oral JAK inhibitors. Dupilumab, an IL-4 and IL-13 pathway inhibitor, was the first available biologic agent for the treatment of atopic dermatitis, and it is currently approved by FDA for adults and children aged 6 months and older with moderate to severe diseases that are not adequately controlled with topical therapies. Tralokinumab is an anti-IL13 antibody recently approved for adult patients. JAK inhibitors, such as baricitinib, upadacitinib and abrocitinib, are efficient new oral small molecules but with a less specific mechanism of action, raising more safety concerns, especially in high-risk patients ,35,36. OGBR 830 was the first-in-class monoclonal antibody against OX40 to present good efficacy and safety results, but its investigation has not been pursued. Rocatinlimab, an anti-OX40 monoclonal antibody, recently completed a phase 2b trial in which four different doses were evaluated, all of them with good efficacy results. The patients kept improving until week 36 of treatment. A post hoc head and neck analysis revealed that the therapeutic effect seemed to persist for 5 months after the treatment had ceased, which was supported by skin transcriptomic analysis. This durability of the response with rocatinlimab potentially reflects how this mechanism of action may induce disease modification [Amlitelimab is another OX40-OX40L pathway inhibitor that binds OX40L and blocks the interaction with OX40. It presented a rapid and marked clinical improvement in patients with moderate-to-severe AD, with a good safety profile. These efficacy results were maintained for 6 months after the treatment ceased, which also could suggest a disease-modifying effect. This could indicate a long and sustained response following the last dose, opening up the opportunity for extended dosing. The sustained reduction in IL-22 serum levels in amlitelimab-treated patients strongly indicates that amlitelimab effectively targets immune dysregulation in AD. It supports the hypothesis that targeting OX40L on antigen-presenting cells modulates not only type 2 response but also other T-cell pathways, including Th22 .Both rocatinlimab and amlitelimab presented no major safety issues. Pyrexia and chills, after the first administration of rocatinlimab, were frequent, but they were not reported in subsequent administrations. No hypersensitivity or tolerability events were reported with amlitelimab. However, long-term studies will be essential to determine the potential risks. Animal OX40 deficient models seem to have impaired interferon-\u03b3 production and Th1 differentiation. On the other hand, in the viral infection model, virus-specific antibody production and the virus-specific cytotoxic T-cell response were not affected. Ongoing long-term extension and phase 3 trials will clarify whether the increased risk of infections will be a concern .The evidence thus far points to OX40-OX40L inhibitors as a future efficient and safe option for the treatment of AD. Their potential disease-modifying effect could be ground-breaking and life-changing for our patients. Peer-reviewed information and further investigation with large phase 3 trials will be fundamental to understanding this class of drugs\u2019 positioning in the atopic dermatitis therapeutic armamentarium."}
+{"text": "Evidence suggests that rates of occupational injuries in the US are decreasing. As several different occupational injury surveillance systems are used in the US, more detailed investigation of this trend is merited. Furthermore, studies of this decrease remain descriptive and do not use inferential statistics. The aim of this study was to provide both descriptive and inferential statistics of temporal trends of occupational injuries treated in US emergency departments (EDs) for 2012 to 2019.Monthly non-fatal occupational injury rates from 2012 to 2019 were estimated using the national electronic injury surveillance system\u2014occupational supplement (NEISS-Work) dataset, a nationally representative sample of ED-treated occupational injuries. Rates were generated for all injuries and by injury event type using monthly full-time worker equivalent (FTE) data from the US Current Population Survey as a denominator. Seasonality indices were used to detect seasonal variation in monthly injury rates. Trend analysis using linear regression adjusted for seasonality was conducted to quantify changes in injury rates from 2012 to 2019.Occupational injuries occurred at an average rate of 176.2 (95% CI\u2009=\u2009\u2009\u00b1\u200930.9) per 10,000 FTE during the study period. Rates were highest in 2012 and declined to their lowest level in 2019. All injury event types occurred at their highest rate in summer months (July or August) apart from falls, slips, and trips, which occurred at their highest rate in January. Trend analyses indicated that total injury rates decreased significantly throughout the study period . Significant decreases were also detected for injuries associated with contact with foreign object and equipment , transportation incidents , and falls, slips, and trips .This study supports evidence that occupational injuries treated in US EDs have decreased since 2012. Potential contributors to this decrease include increased workplace mechanization and automation, as well as changing patterns in US employment and health insurance access.The online version contains supplementary material available at 10.1186/s40621-023-00423-y. Non-fatal occupational injuries represent a significant source of morbidity for workers in the\u00a0United States (US), with an estimated 1,108,300 non-fatal occupational injuries requiring time away from work in 2019  obtains the data for NEISS-Work through an inter-agency agreement with the Consumer Product Safety Commission (CPSC), the agency responsible for collecting the NEISS-Work data. For the purposes of NEISS-Work, an occupational injury is defined as an injury for which an ED chart or other hospital record indicates that the injury involved a non-institutionalized civilian who was injured while working for pay or compensation of any kind, working on a farm, or volunteering for an organization  codes or workers compensation billing status  Division of Safety Research Data for 2012\u20132019 were chosen as this was the longest period for which data for injury event were all comparably coded to the same version (v 2.01) of the BLS Occupational Injury and Illness Classification System (OIICS). BLS OIICS codes are used to assign injury event and diagnosis codes in NEISS-Work using a narrative comment field developed by coders through review of ED chart and hospital admission data. Data for years prior to 2012 were coded based on the BLS OIICS v 1.01  Division of Safety Research All data were stored on a secure drive accessible only to the study team. Statistical analyses were performed in Rstudio version 4.0.1  (Hyndman and Athanasopoulos p\u2009<\u20090.05) and was necessary to control for serial correlation. Finally, significance of each model\u2019s Ljung-Box Q statistic was observed to ensure proper model fit, with a non-significant value considered a properly fit model  modeling. This analysis was conducted using both monthly total injury rate estimates and monthly estimates stratified by injury event type. In data violating the linear regression assumption of no autocorrelation, ARIMA models are used to control for serial correlation  by including lagged dependent variable values and errors, including in studies of injury data  and lowest in February (0.87) Table . With thTable Using the NEISS-Work dataset, one of the primary workplace injury surveillance programs\u00a0in the US, we analyzed rates of occupational injuries treated in US EDs from 2012 to 2019. We found that injury rates during the study period were greatest in 2012  and lowest in 2019 . ED-treated injuries displayed a marked seasonal pattern, with seasonality indices at their greatest in summer months (July or August) and lowest during winter months . Seasonality indices for rates stratified by injury event type followed a similar pattern, apart from falls, slips, and trips, which had a peak seasonality index in January. Additionally, we observed a decrease in estimated rates of occupational injuries treated in US EDs of 18.5% (95%\u2009=\u2009\u2009\u00b1\u200914.5%) throughout the study period.The BLS SOII, another major US occupational injury surveillance program, also reported a decrease in occupational injury rates throughout our study period. However, SOII recorded annual injury rates of 3.7 and 3.0 per 100 FTE for 2012 and 2019, respectively, nearly double the rates estimated in our study for those years  and lowest in a winter month  which has been attributed in other studies to increased heat and humidity, as well as an influx of temporary workers and increased construction during summer months  are from the National Emergency Injury Surveillance System\u2014Occupational Supplement (NEISS-Work) dataset and were produced using the R packages \u201csurvey\u201d and \u201csrvyr.\u201d Denominator data (FTE) were obtained from the Current Population Survey (CPS) via the NIOSH Employed Labor Force querying system. Variances from both numerator and denominator data were used to calculate for injury rate 95% CI using a Taylor series expansion.Additional file 2: Fig. S2. Numerator data (monthly ED-treated transportation injury count estimates) are from the National Emergency Injury Surveillance System\u2014Occupational Supplement (NEISS-Work) dataset and were produced using the R packages \u201csurvey\u201d and \u201csrvyr.\u201d Denominator data (FTE) were obtained from the Current Population Survey (CPS) via the NIOSH Employed Labor Force querying system. Variances from both numerator and denominator data were used to calculate for injury rate 95% CI using a Taylor series expansion.Additional file 3: Fig. S3. Numerator data  are from the National Emergency Injury Surveillance System\u2014Occupational Supplement (NEISS-Work) dataset and were produced using the R packages \u201csurvey\u201d and \u201csrvyr.\u201d Denominator data (FTE) were obtained from the Current Population Survey (CPS) via the NIOSH Employed Labor Force querying system. Variances from both numerator and denominator data were used to calculate for injury rate 95% CI using a Taylor series expansion.Additional file 4: Fig. S4. Numerator data (monthly ED-treated injury count estimates associated with exposure to harmful substances or environments) are from the National Emergency Injury Surveillance System\u2014Occupational Supplement (NEISS-Work) dataset and were produced using the R packages \u201csurvey\u201d and \u201csrvyr.\u201d Denominator data (FTE) were obtained from the Current Population Survey (CPS) via the NIOSH Employed Labor Force querying system. Variances from both numerator and denominator data were used to calculate for injury rate 95% CI using a Taylor series expansion.Additional file 5: Fig. S5. Numerator data (monthly ED-treated injury count estimates associated with contact with objects and equipment) are from the National Emergency Injury Surveillance System\u2014Occupational Supplement (NEISS-Work) dataset and were produced using the R packages \u201csurvey\u201d and \u201csrvyr.\u201d Denominator data (FTE) were obtained from the Current Population Survey (CPS) via the NIOSH Employed Labor Force querying system. Variances from both numerator and denominator data were used to calculate for injury rate 95% CI using a Taylor series expansion.Additional file 6: Fig. S6. Numerator data (monthly ED-treated injury count estimates associated with overexertion and other bodily reaction) are from the National Emergency Injury Surveillance System\u2014Occupational Supplement (NEISS-Work) dataset and were produced using the R packages \u201csurvey\u201d and \u201csrvyr.\u201d Denominator data (FTE) were obtained from the Current Population Survey (CPS) via the NIOSH Employed Labor Force querying system. Variances from both numerator and denominator data were used to calculate for injury rate 95% CI using a Taylor series expansion."}
+{"text": "Optimizing interfacial defects and absorber layers to a high standard is essential in improving the efficiency of Sb23 solar cells. In particular, the electron transport layer (ETL) greatly affects the final device performance of the superstrate structure. In this study, a simple and effective hydrazine hydrate (N2H4) solution post\u2010treatment is proposed to modify CdS ETL in order to enhance Sb23 solar cell efficiency. By this process, oxides and residual chlorides, caused by CdCl2 treated CdS under a high temperature over 400\u00a0\u00b0C in air, are appropriately removed, rendering smoother and flatter CdS ETL as well as high\u2010quality Sb23 thin films. Furthermore, the interfacial energy band alignment and recombination loss are both improved, resulting in an as\u2010fabricated FTO/CdS\u2010N2H4/Sb23/spiro\u2010OMeTAD/Au solar cell with a high PCE of 10.30%, placing it in the top tier of Sb\u2010based solar devices. This study provides a fresh perspective on interfacial optimization and promotes the future development of antimony chalcogenide\u2010based planar solar cells.Antimony selenosulfide (Sb 2H4) assisted solution treatment (HHST) is first introduced to improve CdS electron transport layers (ETLs) quality, which enables over 10.30% planar Sb23 solar cells, achieves a smoother CdS and Sb23 surface, tailors the elemental composition, optimizes the defect properties and improves interfacial quality.In this paper, a new method termed hydrazine hydrate (N It is well\u2010known that the scarcity and toxicity of Ga and Cd elements impede their long\u2010term or future development. Kesterite Cu2ZnSn4 (CZTSSe) materials have also been considered as promising candidates for high PCE devices. However, due to its complex elemental constituents and defects, the maximum obtained PCE has remained at 12.6% for several years. Recently, antimony selenosulfide 3), an alloyed binary compound with a high absorption coefficient (>105 cm\u22121), earth\u2010abundant non\u2010toxic constituents, and a tunable bandgap (through varying the atom ratio of Se and S elements), has been proposed as a potential next\u2010generation solar cell material. Meanwhile, it is encouraging that in only 6 years, the PCE based on this material at laboratory level has reached \u224810%. Impressively, CdS is used as the principal electron transport layer (ETL) in all Sb\u2010based solar cells, achieving a high efficiency of over 9%, as well as performing well in CIGS and CdTe solar cells. According to a theoretical study, the CdS/Sb23 interface plays an influential role in Sb\u2010based solar device performance, where interface recombination is the main loss mechanism, and an increase in defect density will probably result in a significant reduction in performance. Consequently, optimizing the CdS/Sb23 interface contact while reducing interfacial defect density are the most important initiatives to improve device efficiency in the future.Metal chalcogenides, including CuInGaSeover 22%.1, 22 post\u2010deposition treatment (PDT) process. However, for the new Sb\u2010based devices with superstrate structure, treating the CdS ETL with CdCl2 has a proven positive effect on the superstructure CdS/Sb2Se3 solar cell. Moreover, the CdCl2 post\u2010treatment following chemical bath deposition (CBD)\u2010prepared CdS is used in most high\u2010efficiency Sb\u2010based solar devices. However, the CdCl2\u2010PDT process is executed by exposing CdCl2 treated CdS film to temperatures of over 400\u00a0\u00b0C in air. Cadmium chloride oxides form easily on the CdS thin film surface and have been proved to increase resistance while reducing corresponding device performance. Unfortunately, as Sb\u2010based solar cells are new, attempts to address the above problem are limited. Furthermore, exactly how cadmium chloride oxides affect CdS ETL as well as corresponding device performance is still not clear.In Sb\u2010based solar cells with a substrate structure or mature developed CIGS devices, the CdS ETL does not need to consider a CdCl process.12 How2H4) assisted solution treatment (HHST) is applied to CdS ETLs to improve the performance of Sb23 thin\u2010film solar cells. Specifically, CdS thin film treated with CdCl2 solution is designated as the control sample. The control CdS is then further modified by immersing it in hydrazine hydrate (N2H4) solution for a specified period (labeled W\u2010HHST), followed by a hydrothermal deposition of Sb23 thin film. Finally, a typical superstrate solar device with an FTO/ETL/Sb23/spiro\u2010OMeTAD/Au structure is built. The fine reducibility and high corrosive properties of N2H4 solution improve the CdS surface quality and the subsequently deposited Sb23 absorber makes it smoother and flatter. Moreover, it is discovered through element composition analysis, that the remaining Cd oxychlorides on the CdS thin film surface are removed during the HHST process. Consequently, the interfacial energy band alignment and recombination loss are both improved. This results in an as\u2010fabricated FTO/CdS\u2010N2H4/Sb23/spiro\u2010OMeTAD/Au solar cell with a high PCE of 10.30%, which could be placed in the top tier of Sb\u2010based solar devices. This research provides a feasible alternative CdS layer post\u2010treatment and a new interfacial optimization perspective, demonstrating the potential of high\u2010efficiency antimony chalcogenides solar cells with CdS ETLs.In this study, an effective hydrazine hydrate 3 is also regarded as the main interface recombination for the carriers. The CdS ETL plays an important role in the final device efficiency, particularly in planar structure Sb23 solar cells. In order to eliminate any side effect from the above elements on the interfacial properties, treating the CdS ETLs with a HHST is proposed to improve the CdS/Sb23 thin\u2010film solar cell performance. The HHST procedure for the CdS ETLs, and Sb23 solar cells with a typical structure of glass/FTO/CdS/Sb23/spiro\u2010OMeTAD/Au, fabrication process is schematically illustrated in Figure23 solar cells. CdCl2\u2010PDT is then performed on the pristine CdS thin films, in air at over 400\u00a0\u2103. During the HHST process, the CdCl2\u2010treated CdS samples were immersed in hydrazine hydrate solution for various times at room temperature. The Sb23 absorber layers are then synthesized by a typical hydrothermal deposition method. Finally, the planar Sb23 thin\u2010film solar cells are assembled by spin\u2010coated spiro\u2010OMeTAD and deposition of a thermal evaporation Au layer.As previously reported, the CdS ETLs are fabricated using a CBD method, which is treated with CdClar cells.13 In 23 film. Thus, to investigate the evolution of the CdS film morphology with HHST, surface characterization of the CdS film was conducted via scanning electron microscopy (SEM). As shown in Figure2 solution treatment. It is well\u2010known that a smooth and uniform surface is vital in thin\u2010film solar cells, used in high\u2010performance devices. Therefore, this rough CdS ETL surface may not be suitable for depositing Sb23 film, and could reduce the efficiency of the final solar device. To enhance the results while considering the solubility of cadmium chlorides, Figure 2H4 post\u2010treatment. Increased water affinity may prove favorable in subsequent Sb23 thin\u2010film depositions in water.As previously predicted, the HHST process influences the surface morphology and element ratios of the CdS film, changing the subsequently deposited Sb23 layer. The reason for higher transmittance is probably the corrosive ability of N2H4 solution to clear any particles formed on the surface, leading to a smoother CdS film surface. Grazing incidence angle X\u2010ray diffraction (GIXRD) tests are performed to investigate the HHST phase evolution, as shown in Figure 23 thin\u2010film crystallization  is conducted to further identify the effect of HHST on element composition of the CdS surface.To better understand the CdS film changes and evolution of these particles, other CdS thin\u2010film characteristics, including optical properties, crystalline structure and elemental composition, are further investigated via UV\u2013vis, XRD and energy dispersive spectroscopy (EDS), respectively. As shown in Figure\u00a0Figure However, no Cl 2p peaks are detected in the HHST\u2010treated CdS film spectrum. This indicates that the residual surface Cd oxychloride caused by CdCl2\u2010PDT, is decreased by the HHST process, which is consistent with the EDS results. In terms of the S 2p XPS spectra shown in Figure\u00a0 While for the Cd 3d XPS spectra in Figure\u00a0 and the extra peaks at 406.8 and 413.2\u00a0eV originate from the Cd\u2014O in CdS. This demonstrates a significant decrease in the Cd\u2014O peak following HHST. It was predicted that the N2H4 solution etched the control CdS film, causing the intensity difference on Cd 3d XPS spectra. This phenomenon is more obvious when comparing the XPS spectra of O 1s. In Figure\u00a0 and the peak at 533.5\u00a0eV is geared to C\u2014O. Cd\u2014O from the cadmium oxide is expected during the high\u2010temperature annealing for CdS film, and the C\u2014O mainly contributes to the CdS film surface pollution, causing the O 1s peak in both samples. On comparing the peaks at 531.8\u00a0eV, we find that the Cd\u2014O peak intensity dropped slightly and has a larger full width at half maximum (FWHM) for the treated CdS sample, reflecting the reduction of Cd oxychloride content by the HHST. Moreover, to enlarge the etching effect, we also measure the evolution of the O 1s peak of the CdS\u2010HHST film over a long time (\u224845\u00a0min). It clearly demonstrates that the Cd\u2014O peak intensity becomes weaker with increased HHST time for the CdS films, as presented in Figure\u00a0The XPS full survey spectra and fine spectra data of Cl 2p, S 2p, Cd 3p, and O 1s for the control and HHST processed CdS films are shown in 23 absorbed layers by hydrothermal deposition method on CdS films with or without HHST have also been compared in Figure23 film also becomes much flatter than the control sample, due to fewer dust particles and higher water affinity in CdS with HHST, which is more favorable for the adsorption and growth of Sb23. According to the literature surveys, during a typical hydrothermal deposition process, the Sb23 homogeneous and heterogeneous nucleation rates decrease exponentially with homogeneous Gibbs free energy (\u0394GHom) and heterogeneous Gibbs free energy (\u0394GHet), respectively. The homogeneous and the heterogeneous Gibbs free energy can be expressed by the following equations:rk is the critical radius of the nuclei, n is the number of critical nuclei per unit volume, \u0394Gv is the change in free energy between the nuclei and solution per unit volume, \u03b3SL is the particle surface energy, and \u03b8 is the contact angle. Theoretically, according to the Equation\u00a03 particles are formed on the surface of the Sb23 layer for untreated CdS film, which could adversely affect the spin\u2010coating process for the spiro\u2010OMeTAD layer and the final Sb23 solar cell efficiency.The surface SEM images of the subsequently deposited Sb23 absorber layers are obtained through the HHST approach, its effects on the photovoltaic properties of Sb23 solar cells is unclear. Two types of CdS ETL\u2010based Sb23 solar cells with an FTO/CdS/Sb23/spiro\u2010OMeTAD/Au planar structure. Figure\u00a023 and spiro\u2010OMeTAD layers are \u224850, \u2248280, and \u224880\u00a0nm, respectively. The HHST process parameters are carefully and systematically studied, including the N2H4 solution concentration and HHST duration time on the CdS thin films. The current density\u2013voltage (J\u2013V) characteristics of the Sb23 solar cells are investigated under one sun illumination (AM 1.5G). In Figure\u00a02H4 solution at room temperature for only 4\u00a0min delivers the best device performance, which is attributed to the significantly improved current density (Jsc) and fill factor (FF) value due to the increased transmission and reduced impurities of the CdS film. In this case, we also optimize the deposition temperature 3 film based on the CdS layer with the best HHST process parameters. Finally, Sb23 solar cells based on the control and HHST\u2010CdS ETLs exhibit PCEs of 8.97% and 10.30%, respectively, corresponding J\u2013V curves are presented in Figure\u00a0JSC values, from 22.33 to 23.63\u00a0mA cm\u22122, and fill factor (FF) values, from 63.74% to 66.07%. The detailed photovoltaic parameters of each device are displayed in Table23 devices, external quantum efficiency (EQE) measurements are taken and the results are displayed in Figure\u00a023 device spectrum increases in short wavelengths below 500\u00a0nm, which can be explained by the increase in HHST\u2010treated CdS film optical transmittance. More importantly, the spectrum of Sb23 device with HHST also shows a slight increase between 600 and 750\u00a0nm. This may be due to the improvement of interfacial property between HHST optimized CdS and Sb23 film. Furthermore, as illustrated in Figure 2H4 etching. Therefore, the breaking of the CdS ETL surface is proposed as one explanation for the decreased Sb23 solar cell performance with increasing N2H4 concentrations.Although high\u2010quality CdS ETLs and Sbdensity Jsc and fiI), VOC, and JSC is investigated /q. The VOC versus log\u2010scaled intensity is shown in FigureVOC increases linearly with the logarithm of light intensity. By fitting, each device shows \u025b values of 1.42 (Control) and 1.35 (W\u2010HHST), respectively. A lower \u025b value for the W\u2010HHST device indicates that there is less trap\u2010assisted Shockley\u2013Read\u2013Hall recombination in Sb23 solar cells. Besides, the dependence of JSC on I can be defined as JSC\u221d I\u03b1 . As usual, the \u03b1 value is less than 1, and higher \u03b1 values suggest a better charge collection in the device. Here, the relationship of JSC with I is shown in Figure\u00a0\u03b1 values of 0.862 and 0.861, which are consistent with previously reported values of high\u2010efficiency Sb23 solar cells.To gain more insight into the carrier transport and recombination information, the relationship between light intensity 3 solar cells, J\u2013V characteristics under dark for each device are also measured and analyzed. Figure\u00a0J\u2013V plots for two devices based on the control and HHST\u2010CdS ETLs. By plots fitting, the shun conduction (G), diode ideality factor (A), series resistance (R), and reverse saturation current density (J0) can be calculated according to the following Equation\u00a0, the A value can be calculated by the slope of AkT/q, and R can be obtained from the intercept. As illustrated in Figure\u00a0A and R are determined as 2.38 and 5.63 \u03a9 cm2 for the control device, and 1.85 and 5.24 \u03a9 cm2 for the device by employing HHST treated CdS, respectively. Moreover, A and J0 values can be extracted from fitting the curves of ln(J + JSC \u2212 GV) versus (V \u2212 RJ), as presented in Figure\u00a0A values for each device are similar to those obtained from Figure\u00a0J0 decreases from 6.4 \u00d7 10\u22125 to 1.0 \u00d7 10\u22125\u00a0mA cm\u22122 due to the effective action of HHST on the CdS ETL. In summary, the HHST action on the CdS ETL results in smaller J0 and A, and larger shun resistance, which induces a lower recombination probability and less carrier loss. Therefore, HHST\u2010optimized CdS ETLs with a smooth surface and less oxychloride probably lead to an improved CdS/Sb23 heterojunction, thus yielding an enhancement in JSC and FF.To clearly understand the reduced recombination loss in W\u2010HHST based SbJsc enhancement in W\u2010HHST based Sb23 solar cells, ultraviolet photoelectron spectroscopy (UPS) measurement was used to investigate the interfacial charge transfer. As a result, the Fermi level of untreated CdS film is calculated to be \u22123.95\u00a0eV, and the valence band is \u22482.39\u00a0eV under the Fermi level 3 are determined as \u22123.74 and \u22125.26\u00a0eV, respectively. Thus, the upshift of Fermi level for CdS sample with HHST is owing to the reduction of CdO in the CdS film, resulting from the reducibility and corrosivity of hydrazine hydrate solution. Finally, the band alignment of CdS ETLs with the Sb23 absorber is illustrated in Figure\u00a023 solar cells based on CdS ETLs treated just with water, indicating that cadmium chlorides solubleness in water is irrelevant. Therefore, the values of FF and Jsc are significantly improved by etching the impurities on CdS surface with HHST, making interface contact between the CdS and Sb23 layer to be more matching in the present solar cell.In order to explain the underlying mechanism behind the FF and el Figure6a. Cons23 films. Additionally, we selected CdS/Sb23 and HHST\u2010CdS/Sb23 films as samples for the comparison, which directly reflect that the electron extraction efficiency of the CdS ETL. According to the TAS mappings in Figure\u00a023 films, according to the previously reported TA evolution of the CdS/Sb23 sample. We then extracted the decay kinetics of two films at the wavelength of 632\u00a0nm 3 and HHST\u2010CdS/Sb23 films are 8.50 and 6.89\u00a0ns 3 exactly indicates that the electrons transferred from the Sb23 absorber to the HHST\u2010CdS ETL is faster than in the control sample, resulting in less recombination with holes. Hence, the TAS study further explains the lower recombination probability and improved device performance of the HHST\u2010CdS/Sb23 device, consistent with previous electrical property characterizations.To further explore the effect of the HHST process on the carrier transport dynamics, we performed transient absorption spectroscopy (TAS) characterizations. Here, the time window of time\u2010resolved transient spectra is 0\u20138000\u00a0ps. A 400\u00a0nm pulse laser was used to illuminate the Sbm Figure\u00a0. The kin323 thin\u2010film solar cells. It conclusively points out that using the HHST technique improves the device performance in three aspects: i) the obliteration of residual Cd oxychloride by HHST decreases the number of dust particles on the CdS surface, creating a smoother surface, higher optical transparency of CdS film and an obvious increment in the EQE of the final solar device; ii) CdS with HHST facilitates the deposition of Sb23 thin films with high quality, which benefits carrier collection; and iii) HHST improves the energy band alignment and increase the high electron transportation efficiency between CdS and Sb23 interface, indicating a positive effect on the reduction in the carrier recombination loss. Finally, by carefully optimizing the relevant fabrication parameters, a PCE of 10.30% was achieved for HHST\u2010based Sb23 solar cell. This study offers an effective approach for the surface passivation of CdS ETLs, which benefits the future application of high\u2010performance chalcogenide thin\u2010film solar cells.In summary, we propose an effective HHST for CdS films and reveal its positive effects on the properties of CdS ETLs as well as the performance improvement of Sb42 (FTO) substrate using a traditional CBD method. The FTO substrates were cleaned sequentially with deionized water, acetone and ethanol, respectively. During the CdS film CBD process, a precursor solution was prepared by mixing 20\u00a0mL Cd(NO3)2 (1.5\u00a0mm), 13\u00a0mL thiourea (1.5 m), 26\u00a0mL ammonia (25\u201328%), and 140\u00a0mL deionized water. The deposition process was set at 63\u00a0\u00b0C for 16\u00a0min with the substrate placed face down in the growth solution. After growth, the CdS film CdCl2\u2010PDT was executed by spin\u2010coating a CdCl2\u03872.5H2O methanol solution with a 20\u00a0mg mL\u22121 concentration at 3000\u00a0rpm for 30 s, followed by annealing at 430\u00a0\u00b0C for 15\u00a0min, in air.The CdS film was deposited on an F\u2010doped SnO2\u2010treated CdS film had cooled to room temperature naturally, the samples were immersed in hydrazine hydrate solution for different durations at room temperature. The relevant experimental parameters were then optimized, including the N2H4 solution concentration and treatment time. It should be noted that a 50% mass fraction of hydrazine hydrate solution diluted at a 5\u00a0% volume ratio is simply referred to as 5%. After being removed from the N2H4 solution, the samples were rinsed with deionized water. Finally, the as\u2010fabricated FTO/CdS\u2010N2H4 samples were blow\u2010dried using nitrogen gas.When the CdCl23 films were synthesized utilizing a typical hydrothermal method with precursor materials including KSbC4H4O7\u00b70.5H2O, Na2S2O3\u00b75H2O, CH4N2Se, and deionized water. In brief, 4\u00a0mmol KSbC4H4O7\u00b70.5H2O and 16\u00a0mmol Na2S2O3\u00b75H2O were mixed and stirred in 200\u00a0mL deionized water. 0.8\u00a0mmol CH4N2Se was then added and the mixture was stirred until the solution clarified. It was then transferred into autoclave Teflon tanks containing samples with two different CdS thin film types. The autoclaves were placed in an air oven for 110\u00a0min at 120\u00a0\u00b0C to deposit the Sb23 film. Subsequently, the as\u2010grown Sb23 films were annealed at 350\u00a0\u00b0C for 10\u00a0min in a nitrogen atmosphere. For the device fabrication, 2,2\u2032,7,7\u2032\u2010tetrakis\u20109,9\u2032\u2010spirobifluorene (spiro\u2010OMeTAD), fabricated according to previous reports, was selected as the hole transport layer. Typical superstrate Sb23 planar solar cells with the structure of glass/FTO/ETLs/Sb23/spiro\u2010OMeTAD/Au, were constructed after thermal evaporation using gold electrodes. Finally, a mask with an aperture area of 0.0735 cm was adopted to define the active device area.Sb reports,36 was23 films were characterized using a SEM , equipped with EDS. The element composition and chemical states of the CdS films were analyzed via X\u2010ray photoelectron spectroscopy (XPS) . The crystallinity and phase of the CdS samples were detected using grazing incidence angle X\u2010ray diffraction (GIXRD). The J\u2013V curves of the Sb23 devices were measured under AM 1.5G, 100\u00a0mW cm\u22122 conditions, with illumination provided by a solar simulator , and were recorded using a source meter . The EQE was investigated using a quantum efficiency (QE) measurement system . The energy band positions of two types of CdS thin film were determined via ultraviolet photoelectron spectroscopy (UPS)  with a bias of \u22125\u00a0V. The optical transmittance of the samples was detected by ultraviolet\u2010visible near\u2010infrared absorption spectrum . The ultrafast transient absorption (TA) of the CdS/Sb23 films was performed on a pump\u2010probe system  with a maximum time delay of \u22488\u00a0ns using a motorized optical delay line under ambient conditions. The pump pulses at 400\u00a0nm (\u2248200 \u00b5W average power at the sample) were delivered by an ultrafast optical parametric amplifier (OPera Solo) excited by a regenerative amplifier , seeded with a mode\u2010locked Ti:sapphire oscillator , and pumped with an LBO laser . A small number of 800\u00a0nm femtosecond pulses from the regenerative amplifier were used to pump a sapphire crystal, which created a 420\u2013780\u00a0nm white light continuum as the probe pulses. The data was fitted by a multi\u2010exponential function:t is the probe time delay, and Ai and \u03c4i are the amplitude and decay lifetime. The number of components N to satisfactorily fit the experimental data is two. The average lifetime \u03c4ave was estimated from the fitting parameters according to the following equation \u03c4ave = \u03a3Ai\u03c4i2/\u03a3Ai\u03c4i.The surface and sectional morphologies of CdS and SbThe authors declare no conflict of interest.Supporting informationClick here for additional data file."}
+{"text": "The cephalocaudal progression (CCP) of neonatal jaundice is a well-known phenomenon, but quantitative information on CCP in preterm infants is absent. In this study, CCP was quantified in preterm infants as a function of postnatal age and body location.N\u2009=\u20091.113).5.693 transcutaneous bilirubin (TcB) measurements were performed in 101 preterm infants from birth until postnatal day seven at five body locations . Multi-level linear regression analysis was performed to evaluate the CCP as a function of body location and postnatal age. TcB measurements at all body locations and postnatal days were compared to total serum bilirubin (TSB) levels  of jaundice in preterm infants, assessed by transcutaneous bilirubin (TcB) measurements, is substantial and rather stable over postnatal day 0 to 7.To the best of our knowledge, this study is the first to investigate CCP of jaundice in preterm infants as a function of postnatal age in preterm infants.Our results demonstrate that TcB measurements at the tibia and ankle differ from the TSB beyond the clinically used TcB safety margins. We advise to perform TcB measurements only at locations cephalic from the tibia; i.e., hipbone, forehead, and sternum. It affects up to 80% of preterm and 60% of the term newborn infants.12 More insight into this phenomenon can improve our in-depth understanding of the progression and diagnosis of hyperbilirubinemia. From a more practical point of view, it is important to understand the influence of CCP on the correlation between the TcB and TSB. Current transcutaneous bilirubin (TcB) meters are designed for forehead and/or sternum measurements. However, measurements on other body locations are gaining popularity, e.g., on the interscapular site to avoid the influence of ambient light13 and at the hipbone underneath the diaper to reduce the influence of photototherapy.14 CCP potentially influences the reliability of the TcB at these non-standard body locations.The yellow skin discoloration associated with neonatal jaundice is often first observed at the face and only later in the extremities. This phenomenon is known as the cephalocaudal progression (CCP) of jaundice. The physiological causes and spatiotemporal dependency of CCP are still not fully understood.15 Knudson demonstrated a significantly higher TcB at the forehead compared to the sole (ratio unknown) for newborn infants with a median GA of 40 weeks;16 Purcell reported a significantly higher TcB value on the head compared to the sole (ratio unknown) for newborn infants with an average GA of 38.9 weeks;17 and Kamphuis found a foot/ forehead ratio of 0.44 (SD 0.15) for newborn infants with a median GA of 37.1 weeks.18Previous studies assessed CCP based on TcB measurements in jaundiced newborn infants. Hegyi found a ratio of TcB measurements of the sole compared to the forehead of 0.58 (SD 0.07) for clinically healthy newborn infants );Besides term infants, preterm infants are also often subjected to TcB measurements. To the best of our knowledge, the CCP of jaundice has not yet been investigated in preterm infants. As CCP may also influence the reliability of TcB measurements in this patient group, our aim was to evaluate the presence of CCP in preterm infants and to quantify it as a function of time  and body location.19Preterm infants (\u226528 weeks GA), born between December 2017\u2013September 2019 admitted to the NICU at Isala Women and Children\u2019s hospital  were included after informed consent was obtained from the parents. Hypothermia treatment was an exclusion criterium, since it may influence bilirubin metabolism and thereby the transcutaneous measurement outcome.This study was approved by the Medical Ethical Committee of Isala Hospital in Zwolle, the Netherlands (number 170317).20 Care was taken to use the same TcB meter per measurement series, in order to avoid the potential influence of low inter-device reproducibility.21Transcutaneous measurements were performed using the transcutaneous meter type JM-105 , which is widely used in pediatric and neonatal departments across the Netherlands. The accuracy of the TcB measurements specified by the manufacturer is 25.5\u2009\u03bcmol/L (>35 weeks GA) and 27.4\u2009\u03bcmol/L (>24 weeks GA).22 Measurements were performed on uncovered skin. Hipbone (spina iliaca anterior superior), tibia  and ankle  measurements were performed at the best reachable side of the body (left or right), depending on patient orientation. Each TcB measurement was the average of three repeated measurements per location, as suggested in the manufacturer\u2019s instructions.20 This means that 15 measurements (5 locations\u2009x\u20093 repeated measurements) were performed per patient, per TcB measurement series. Our experienced staff could finish these 15 measurements within a few minutes.From birth  until postnatal day seven, TcB measurement series at different body locations were ideally performed at least three times a day for each patient. Owing to practical reasons, it was not always possible to perform three measurements every day. The TcB measurement series were performed by the nurse or attending physician during planned care , preventing any avoidable disturbance. All nurses and physicians on the NICU were trained in the proper use of the TcB meter. To evaluate the presence of CCP of jaundice, every measurement series included five different body locations , representing each dermal zone described by Kramer.x, t)) was quantified as the ratio of the TcBx,t and the corresponding TcBtforehead, minus the reference ratio of the forehead :Cephalocaudal progression  CCP implies that TcBx,t is lower than TcBtforehead, and vice versa for a positive (+) progression. The CCP was determined for all postnatal ages combined and per postnatal day.The subscript p-value of <0.05 was considered statistically significant.Since multiple measurement series were performed over time, a multi-level linear regression analysis  was applied to assess CCP with its 95% confidence interval (CI). For this analysis, measurements were clustered per patient. To check for effect modification of CCP, interaction terms of the following variables were added to the analyses; sex, GA, birthweight, phototherapy and TSB. TSB was analyzed both as a continuous and dichotomous measure, for which the threshold <170\u2009\u03bcmol/L was used as cut-off value. A Rrep) using mrcorrShiny .24 For all repeated measures analysis the measurements were clustered per patient.Three repeated measurements per location were performed. Repeated measurement correlation was applied to determine how well the individual TcB measurements correlated with each other . TcB measurements were paired in time as much as possible with routine invasive blood sampling. Measurements of TSB and TcB within a time frame of 1\u2009h were labeled as \u201cpaired\u201d Table\u00a0. Since I\u2013TcBx,t) and depicted in Bland-Altman plots, adjusted for repeated measures for all body locations, using MedCalc .25 Repeated measurement correlation was applied to determine how well the TcB measurements correlated with the paired TSB measurements  using mrcorrShiny .24 For all repeated measures analysis the measurements were clustered per patient.The agreement between TSB and TcB levels was calculated  over all postnatal ages combined was not significantly different from 0  for the sternum  and the hipbone   at the sternum  and the hipbone  was not significantly different from 0 on all individual postnatal days, except on postnatal days 1 and 7 for the sternum and day 1 for the hipbone  was significantly lower than 0 for the tibia  and the ankle  on all postnatal days.The CCP, whereas the repeated measurement correlation between the individual TcB measurements at the ankle was substantially weaker (Rrep\u2009=\u20090.67)  Table\u00a0.Table 3PRTcB,TSB\u2009=\u20090.84, 0.74, 0.80 and 0.81, respectively), whereas the correlation between the TSB and TcB at the ankle was substantially lower   Table\u00a0.Fig. 3Blx,t) over all postnatal ages combined was 0.04 for the sternum, 0.05 for the hipbone, and significantly lower for the tibia (\u22120.33) and the ankle (\u22120.62). Sex, gestational age, birthweight, phototherapy and TSB were no effect modificators of the CCP ratio changes between the locations. Furthermore, TcB measurements at the forehead, sternum and hipbone were in agreement with the TSB within the reported accuracy by the manufacturer (27.4\u2009\u03bcmol/L).20 On body locations caudal from the hipbone, the TcB underestimated the TSB.The main purpose of this study was to evaluate the cephalocaudal progression of jaundice in preterm infants as a function of time  and body location. Hereto, we evaluated TcB measurements on five cephalocaudal body locations . We showed that CCP was present and stable over time. The CCP(+3 weeks) are in line with previous studies on term newborn infants.18 CCP ratios of TcB measurements on caudal sites were similar to the reference ratio at the forehead and decreased towards the caudal side of the body.18 For instance, our CCP ratio of the ankle compared to the forehead of 0.38 (SD 0.40) is comparable to Kamphuis 0.44 (SD 0.15).18 In accordance with previous studies, our study demonstrated a good correlation between TSB and TcB measurements on forehead, sternum and hipbone.16 In addition, we found a good correlation on the tibia.Our results on the CCP of jaundice on preterm infants , bone depth and potentially skin temperature can influence the measured TcB significantly.32 For this study, skin maturity at the same postnatal day can be considered to be a constant factor in a patient over different body locations. Other studies confirm that this factor does not differ significantly between the evaluated body locations.33 Therefore, we assume that only bone depth and skin temperature can have a potential influence on our cephalocaudal TcB measurements. Since TcB measurements are underestimated beyond the accuracy specified by the manufacturer 27.4\u2009\u03bcmol/L20 for bone depths <1.1\u2009mm,32 we expect that this effect would be most prominent for the most premature infants in our study population. However, we did not observe any influence of gestational age on our results. To fully take into account the influence of local skin anatomy and skin temperature on cephalocaudal TcB measurements, follow up research may benefit from the use of spectroscopic high-resolution skin imaging and local skin temperature measurements. High-resolution skin imaging can be achieved with either high-resolution ultrasound34 optical coherence tomography.35 The latter combines high-resolution skin images with spatially confined bilirubin measurements, which may provide even further in-depth understanding into the relation between microcirculatory perfusion and the extravasation of bilirubin into the skin and local temperature measurements.35In our recent work, we demonstrated that local skin anatomy can influence TcB readouts,6 Therefore, healthcare providers should be aware that the decision to do an additional TSB determination can be influenced by the TcB measurement location. Based on our observations, we advise to only use measurement locations cephalic from the tibia; i.e., hipbone, sternum and forehead.This study provides insight into the CCP of jaundice in preterm infants as a function of postnatal age and body location. With a CCP of the ankle up to \u22120.70 and a mean underestimation of the TSB at the ankle of 80\u2009\u03bcmol/L, our results demonstrate that body location affects the measured TcB. Furthermore, our results demonstrate that the clinically accepted safety margin (50\u2009\u03bcmol/L below the phototherapy threshold) can be exceeded on more caudal body locations.In this study, we demonstrated the presence of cephalocaudal progression of jaundice in preterm infants during the first week of life. The TcB at the tibia and ankle differed significantly from the TcB at the forehead throughout the evaluated period. The measured CCP ratios remained relatively stable over time. No effect modification by sex, gestational age, birthweight, phototherapy and TSB was found. The deviation of the TcB from the TSB exceeded the clinically accepted safety margins for the ankle and tibia with an average underestimation of 45 and 80\u2009\u03bcmol/L, respectively. Based on the results of this study, we advise to use only TcB measurement locations cephalic from the tibia; i.e., hipbone, sternum, and forehead."}
+{"text": "BMI exerted a significant effect on the frequency of PPH (p\u2009=\u20090.004). Compared to patients with BMI 18.5\u201324.9\u00a0kg/m2, patients with BMI between 25 and 59.9\u00a0kg/m2 had an increased odds of PPH. The odds of PPH in patients with BMI\u2009>\u200960\u00a0kg/m2 was not increased compared to patients with BMI 18.5\u201324.9\u00a0kg/m2. Obesity was associated with a decreased odds of blood transfusion . In conclusion, higher BMI was associated with PPH yet a lower odds of transfusion after CD.We aimed to evaluate the association between obesity and postpartum hemorrhage (PPH) after cesarean delivery (CD). This was a retrospective cohort study using a multicenter database of 20 hospitals in the United States. We analyzed 27,708 patients undergoing CD from 2015 to 2019. The exposure of interest was BMI, and the primary outcome was PPH (estimated blood loss [EBL]\u2009\u2265\u20091000\u00a0mL). Simple logistic regression was used to evaluate the relationship between obesity and intrapartum complications. Multivariable logistic regression was used to adjust for any confounding demographic variables. Hosmer and Lemeshow\u2019s purposeful selection algorithm was adapted to develop a multivariable logistic regression model of PPH. Analyses were conducted using STATA 16.1  with  In addition, the prevalence of obesity and severe obesity (BMI of 40\u00a0kg/m2 or greater) is increasing in the United States2. Obesity has been associated with multiple adverse pregnancy outcomes, including pregnancy loss, gestational diabetes, venous thromboembolism, and increased risk for cesarean delivery (CD)6.Obesity  is the leading cause of maternal mortality worldwide, accounting for more than 80 thousand maternal deaths in 2015The primary objective of our study was to determine if obesity is associated with an increased odds of PPH or hemorrhage-related complications (including blood transfusion) during delivery. To eliminate the potential confounding effect of mode of delivery and its associated variables on PPH, only women undergoing CD were eligible for inclusion in this study. To our knowledge, this is the first multicenter retrospective cohort study specifically examining the effect of obesity on PPH after CD.Data were obtained from a multicenter database of women admitted to labor and delivery units from 2015 to 2019 in the Universal Health Services (UHS) system in the United States. All patients who underwent CD during their admission were eligible for inclusion in this study . A total of 8252 patients were excluded from analysis due to missing data for BMI at time of delivery as this was the exposure of interest. Of those excluded for missing BMI at delivery, approximately 65% were missing data for\u2009\u2265\u200960% of analysis-relevant variables. An additional 2374 patients in the dataset with vacuum- or forceps-assisted delivery were also excluded to ensure that patients who had a vaginal delivery were not included in the analysis. A total of 27,708 patients were eligible for inclusion in the analysis. CD were reported by 20 hospitals across the Eastern , Western , and Central  United States. The requirement for informed consent was waived by The George Washington University Committee on Human Research Institutional Review Board due to the retrospective nature of the study, and all data were de-identified prior to analysis. The study was determined to be research that is exempt from IRB review by The George Washington University Committee on Human Research Institutional Review Board under Department of Health and Human Services (DHHS) regulatory category 4 (IRB number NCR203136). All methods were carried out in accordance with relevant guidelines and regulations. No experimental protocols were performed as this was a retrospective study.15, of which 5 were employed for descriptive and demographic analysis (Table 2). The primary outcome was PPH, defined as EBL\u2009\u2265\u20091000\u00a0mL for the purpose of analysis. Additional outcomes such as estimated gestational age (EGA) less than 37\u00a0weeks, intrapartum complications including pre-eclampsia and chorioamnionitis, abnormal hematologic parameters including thrombocytopenia, and pharmacologic interventions including oxytocin use were evaluated as secondary outcomes. Descriptive statistics included counts, proportions, means, and standard deviations calculated as necessary using standard definitions. To evaluate the association between BMI classification and other patient-level variables, we used chi-square or Fischer\u2019s exact test (when N\u2009\u2264\u20095) for categorical variables and analysis of variance for continuous variables16.The main exposure of interest was BMI at time of delivery. BMI at delivery was sorted into six categories  according to guidelines from the National Institutes of Health and World Health Organization18. Candidate variables were screened with simple logistic regression, using the Wald test and a p-value cutoff of\u2009\u2264\u20090.25 to exclude variables from consideration. During the iterative process of variable selection, covariates were sequentially removed from the model if they were not significant at the 0.1 alpha level and did not exert a confounding influence. We tested for multicollinearity between covariates by determining the variance inflation factor. A literature review was also used to support the choice of covariates as potential confounders influencing the odds of PPH23.We employed simple logistic regression to evaluate the relationship between obesity and intrapartum complications, including PPH. We used multivariable logistic regression to adjust for any confounding demographic characteristics that were associated with obesity at the 0.1 alpha level. To determine the demographic and intrapartum variables most associated with PPH, we adapted Hosmer and Lemeshow\u2019s purposeful selection algorithm to develop a multivariable logistic regression model of PPH24. Missing data were also observed for race/ethnicity, marital status, insurance type, and parity. Missing data from these variables were included in the multivariable models as dummy variables. All analyses were conducted using STATA 16.1  with p\u2009\u2264\u20090.05 considered significant.EBL was not reported for 1918 deliveries (6.92%), maternal age was not reported for 227 deliveries (0.82%), and EGA was not reported for 451 deliveries (1.63%), with approximately 9% of deliveries missing any combination of these variables. Because these data could not be assumed to be missing completely at random, we treated them as missing at random and employed multiple imputation by chained equations with 10 total imputations to account for statistical uncertainty while addressing missing datap\u2009<\u20090.001 for all)  system across the United States. The majority of patients included in our study were White (55.0%) and between ages 20 and 34\u00a0years (75.5%); 49.1 percent were married. A large proportion of the patients in our cohort had private insurance (42.4%) and delivered at hospitals in the West Coast (48.0%). The majority of patients (79.9%) had no prior CD. We compared demographic and medical characteristics across 5 BMI categories, with several differences noted between groups. The prevalence of asthma, pre-gestational diabetes, and chronic hypertension increased as BMI increased between groups (2) did not significantly influence the odds of EBL\u2009\u2265\u20091000\u00a0mL. Increasing BMI category was, however, associated with EBL\u2009\u2265\u20091000\u00a0mL (p\u2009<\u20090.001) . The greatest odds of EBL\u2009\u2265\u20091000\u00a0mL was observed among patients with BMI 50\u201359.9\u00a0kg/m2 (aOR 1.69 [1.28\u20132.21]), relative to patients with BMI 18.5\u201324.9\u00a0kg/m2 , and any antibiotic use (p\u2009=\u20090.001) (Table p\u2009<\u20090.001) and NICU admission (p\u2009<\u20090.001) , gestational diabetes (1) Table . Conside1) Table . Categor1) Table .Table 4S2 BMI to define obese versus non-obese is not itself predictive of PPH. We found an association between obesity and other intrapartum complications, including pre-eclampsia and gestational diabetes. Despite an increased odds of EBL\u2009\u2265\u20091000\u00a0mL, we found a decreased odds of blood transfusion in patients with obesity after CD.In our cohort of 27,708 CD across the United States, we observed an association between increasing BMI and EBL\u2009\u2265\u20091000\u00a0mL after adjusting for maternal demographic characteristics known to be associated with PPH. The observation that further discretized BMI was associated with increased odds of EBL\u2009\u2265\u20091000\u00a0mL while categorical obesity was not suggests that the BMI cutoff of\u2009\u2265\u200930\u00a0kg/m9. In addition, the absolute event rate for PPH was 2.8%, which was lower than the frequency observed in our cohort (11.9%)9. Paglia et al. found a reduced risk of severe PPH among women with obesity after controlling for mode of delivery; of note, this was a case\u2013control study of women with PPH who received blood components, indicating severe hemorrhage11. Our study focused on EBL\u2009\u2265\u20091000\u00a0mL, which may or may not have clinical effects.Our findings contrast with prior work investigating the relationship between obesity and PPH. Butwick et al. conducted a large population-based cohort study that found an increased odds of hemorrhage after vaginal delivery and decreased odds of hemorrhage after CD for women with obesity; however, the effect was small and without strong evidence of a dose\u2013response relationship between BMI and PPH25. This finding also lends support to the notion that estimated blood loss is an unreliable indicator of clinically significant hemorrhage, and variation exists among hospitals in the use of quantitative over estimated blood loss. One formula devised for estimating allowable blood loss prior to transfusion includes allowable blood loss as directly proportional to the patient\u2019s estimated blood volume, implying that patients with obesity with a higher estimated blood volume are able to tolerate a higher estimated blood loss prior to requiring blood transfusion26. However, there is little consensus in the literature regarding the impact of obesity on maternal blood volume, with one study reporting similar circulating blood volume between gravidas with and without obesity27.Interestingly, despite an increased odds of PPH, we observed a decreased odds of blood transfusion among women with obesity. This finding implies that the increased blood loss we observed in patients with obesity may lack clinical relevance, and is consistent with prior work demonstrating greater blood volume and hemoglobin mass in individuals with obesityWe found that women with obesity in our cohort more frequently used oxytocin, dinoprostone, misoprostol, and magnesium sulfate, lending support to the theory that the mechanism by which obesity could increase PPH risk is related to uterine atony. Future work may examine variation in the etiology of PPH among women with and without obesity, as well as variations in PPH management based on BMI. The impact of obesity on maternal blood volume and the threshold for estimated blood loss at which clinically significant changes in hematologic parameters or transfusion risk occurs in women with obesity should also be investigated further due to our finding that despite a higher estimated blood loss, transfusion was not more frequent among women with obesity.28.Limitations of our study include limitations common to studies using large databases, including potential misclassification errors and missing data. To address this limitation, we treated missing data as missing at random and employed multiple imputation by chained equations to account for statistical uncertainty while addressing missing data. Our dataset was unable to distinguish quantitative blood loss from estimated blood loss, which limits the accuracy of the blood loss reported and may contribute to variation in reporting blood loss among the hospitals in our dataset. Pre-pregnancy BMI and gestational weight gain were not reliably captured in our dataset, yet may affect pregnancy outcome more significantly than BMI at the time of delivery. Our study is also limited by the lack of ability to capture postpartum complications that occur after hospital discharge, such as delayed PPH, which is a potential explanation for the lack of association we observed between obesity and venous thromboembolism in our study despite the known increased risk of venous thromboembolism among gravidas with obesity29. Our results demonstrate several potential risk factors for PPH after CD that can be used for population-based risk assessment, which can aid clinicians in identifying groups of patients that are at highest risk for PPH as part of an ongoing effort to lower hemorrhage-related morbidity and mortality.Despite these limitations, our study has a number of strengths. We included a large number of patients across multiple sites in different geographic regions across the United States, increasing the generalizability of our results. We excluded patients with missing BMI data since this variable was the focus of our study. We focused on including only CD to eliminate the potential confounding effect of mode of delivery in the relationship between BMI and blood loss. Our findings have significant clinical implications due to the rising prevalence of obesity in the United States as well as the high number of CD performed annually, currently accounting for more than 30 percent of all deliveries in the United StatesIn conclusion, we found an association between BMI and estimated blood loss after CD, with elevated BMI associated with an increased frequency of PPH. Despite a higher estimated blood loss, we did not find a higher frequency of blood transfusion among women with obesity after CD, implying that there may be no difference in clinically significant blood loss between women with and without obesity . Future Supplementary Information."}
+{"text": "Since the successful application of messenger RNA (mRNA) vaccines in preventing COVID-19, researchers have been striving to develop mRNA vaccines for clinical use, including those exploited for anti-tumor therapy. mRNA cancer vaccines have emerged as a promising novel approach to cancer immunotherapy, offering high specificity, better efficacy, and fewer side effects compared to traditional treatments. Multiple therapeutic mRNA cancer vaccines are being evaluated in preclinical and clinical trials, with promising early-phase results. However, the development of these vaccines faces various challenges, such as tumor heterogeneity, an immunosuppressive tumor microenvironment, and practical obstacles like vaccine administration methods and evaluation systems for clinical application. To address these challenges, we highlight recent advances from preclinical studies and clinical trials that provide insight into identifying obstacles associated with mRNA cancer vaccines and discuss potential strategies to overcome them. In the future, it is crucial to approach the development of mRNA cancer vaccines with caution and diligence while promoting innovation to overcome existing barriers. A delicate balance between opportunities and challenges will help guide the progress of this promising field towards its full potential. Based on the most recent statistical report, about 5370 new occurrences, and 1670 deaths each day are expected during 2023 in the United States , 5. ICIsRecently, the successful applications of COVID-19 global pandemic offered a great opportunity for messenger RNA (mRNA)vaccines in antitumor therapy \u201310. TherIn this review, we evaluate the benefits of therapeutic mRNA cancer vaccines while providing a succinct overview of their classification and mechanism. Particularly, we highlight recent advancements from preclinical studies and clinical trials that identify the obstacles associated with the development of mRNA cancer vaccines and discuss potential strategies to overcome them.2in vivo, leading to the expression of the encoded related protein  technology and is based on messenger RNA . This inA phase 1/2 clinical trial (NCT04382898) is currently underway to evaluate the effectiveness of the cancer vaccine BNT112. This vaccine encodes five different tumour-associated antigens and is being administered alone or in combination with cemiplimab to patients with metastatic castration-resistant prostate cancer. Another trial is a randomised phase 2 study (NCT04534205) evaluating the anti-human papillomavirus (HPV)-16-derived oncoprotein-encoding mRNA BNT113 vaccine in HPV16-positive, PD-L1-positive head and neck squamous cell carcinoma. BNT113 is also being tested in a two-arm phase 1/2 vaccine dose-escalation study (NCT03418480) for patients with previously treated or advanced HPV16-positive head and neck squamous cell carcinoma. Another phase 1 study (NCT04163094) is being conducted to evaluate the BNT115 that encodes ovarian-specific tumour-associated antigens. This vaccine is being administered both before and in combination with adjuvant and neoadjuvant chemotherapies to patients with ovarian cancer. Finally, a fifth clinical trial (NCT05142189) is evaluating the FixVac vaccine BNT116 in combination with cemiplimab or docetaxel in a phase 1 study for patients with advanced or metastatic non-small cell lung cancer.4.3The profound potential of mRNA cancer vaccines stems from their ability to encode a wide range of proteins, including immunostimulants that can modify the tumor immune microenvironment (TME) and enhance the efficacy of immune checkpoint inhibitors . This pr5Numerous clinical trials are currently under development or in progress to assess the safety and efficacy of mRNA cancer vaccines . Despite5.1The complexity of treatment decisions is enhanced by tumour heterogeneity, which can be divided into spatial and temporal heterogeneity . TemporaTo overcome spatial heterogeneity in tumours, one strategy is to use tumour tissue multipoint sampling to identify differences between tumour regions and inform the design of personalised mRNA cancer vaccines. Another approach involves using mRNA cancer vaccines that target multiple antigens expressed across various tumour regions, thereby compensating for spatial heterogeneity. Meanwhile, monitoring disease progression and adjusting treatment plans accordingly may help to address temporal heterogeneity. However, these strategies not only increase the complexity of vaccine design and administration but also raise costs and increase treatment time for patients. Another promising method is the use of artificial intelligence algorithms such as MHC-binding prediction, quantification of mutated transcript expression, and clonality of the mutation to predict neoantigens based on tumour genomic data, then used to prioritize these mutations as vaccine candidates based on their likelihood to elicit a T cell response, which could improve the efficiency and accuracy of vaccine design and overcome the heterogeneity of tumour \u201364. Howe5.2Tumour initiation, progression and maintenance depend highly on interactions between the tumour and the associated microenvironment . Tumour Combining mRNA cancer vaccines with other anti-cancer treatments, which is being constantly tried in cancer patients, can be an effective strategy for overcoming the immunosuppressive microenvironment. One commonly used combination therapy is to combine mRNA vaccines with immune checkpoint inhibitors, which can \u201crelease the brakes\u201d on the immune system, allowing it to attack cancer cells more effectively , 70. Ado5.3in vivo  technology has garnered significant attention as a novel tool in assessing the immunotherapy response of cancer patients . The scRRecently, the circulating tumour DNA (ctDNA) test has the advantages of sensitivity, flexibility, repeatability, and safe gain much attention to help the evaluation of treatment responses, and the application of ctDNA detection in monitoring mRNA cancer vaccine therapeutic response is a worthwhile attempt . Accumul6Altogether, mRNA cancer vaccines present a promising new approach to anticancer therapies with both opportunities and challenges. The highly personalized and specific nature of this technology offers tremendous potential for precision medicine in the fight against cancer. However, further clinical trials are necessary to fully establish the safety and efficacy of mRNA cancer vaccines and additional preclinical studies are warranted to explore the combined use of mRNA cancer vaccine and other anticancer therapies. Additionally, addressing issues such as tumoral heterogeneity, routes of administration and development of methods to assess the efficacy processes will be critical for advancing this technology toward meaningful clinical outcomes. With continued research and investment, mRNA cancer vaccines hold great promise as a transformative therapy for cancer patients.BW and JL collected relevant literature and wrote the manuscript. BW and JY conceptualized the main structure of this review, and revised and validated the final version. JP, SX and BW contributed to the literature analysis and manuscript editing. All authors contributed to the article and approved the submitted version."}
+{"text": "Currently, cancer immunotherapy is rapidly developing, including the use of monoclonal antibodies, adoptive T-cells, and mRNA vaccines. Due to their extensive use during the COVID-19 pandemic, mRNA vaccines are proving to be a promising therapeutic strategy for various medical fields, such as oncology. A number of clinical trials aim to evaluate the safety and efficacy of these vaccines in the treatment of solid tumors. Several studies are in the process of investigating mRNA vaccines when combined with other immunotherapeutic drugs and methods. Research has yet to provide practice-changing results considering NSCLC. However, based on the existing outcomes of the ongoing studies, an increase in clinician awareness of this therapeutic approach is significantly important. Therefore, the goal of this review is to highlight the progress and obstacles concerning the development of mRNA vaccines for the treatment of NSCLC and present ways in which they could be used in clinical practice.The introduction of immune checkpoint inhibitors in the therapeutics of non-small cell lung cancer (NSCLC) has been a game-changer in the management of patients with lung cancer; however, challenges do exist since a non-negligible subset of patients does not respond to therapy. Various immunotherapeutic anticancer strategies have been increasingly developed in recent years, including monoclonal antibodies, adoptive T-cell therapy, and vaccines. Fueled by their rapid drug development and successful implementation during the COVID-19 pandemic, messenger RNA (mRNA) vaccines represent an emerging therapeutic approach in other fields of medicine, including oncology. Several clinical trials are currently being conducted to assess the safety and efficacy of mRNA vaccines regarding a variety of solid tumors. Combining mRNA vaccines with other immunotherapeutic approaches has also been suggested and is currently under investigation. Although, in the case of NSCLC, the investigation is still in its early stages, the initial results raise the need for clinician awareness of these promising therapies. To this end, in the present review, we aim to summarize current advances in the development of mRNA vaccines in NSCLC therapeutics and discuss pragmatic challenges regarding their drug development and the different opportunities for implementation. The introduction of immune checkpoint inhibitors (ICIs) in the therapeutics of non-small cell lung cancer (NSCLC) during the last ten years has been a game-changer in the management of patients with lung cancer, leading to durable responses and significant survival benefits ,2,3,4. FIt should be noted that apart from ICIs, other modalities of immunotherapy have been developed in anticancer research with the aim of activating the host\u2019s antitumor immunity, modifying the suppressive tumor microenvironment (TME), and ultimately improving the anticancer response, including T-cell therapy and vaccines ,12,13. IAnticancer vaccines with prophylactic intent have mainly been developed in the case of a known causative agent, the highlight being the human papillomavirus (HPV) vaccines that provide high levels of protection against cervical cancer and other HPV-related neoplasms . Such vaThe emerging and widespread use of mRNA technology vaccines has boosted many clinical trials regarding their use in cancer immunotherapy ,19. AlthFirstly, a comprehensive search in the clinicaltrials.gov database was performed (accessed on 25 August 2023). The clinical trials were retrieved from the clinicaltrials.gov database using the following query: \u201clung cancer AND mRNA vaccines\u201d. All clinical trials, either completed or ongoing, related to the therapeutic treatment of NSCLC based on mRNA vaccines were included. Additionally, a literature review was performed in the PubMed database with combinations of the following keywords \u201ccancer immunotherapy\u201d, \u201cmRNA vaccine\u201d, \u201clung cancer\u201d, and \u201cNSCLC\u201d. Non-English literature was excluded. Among the retrieved abstracts, we included recent (last three years) preclinical studies regarding the investigation of mRNA vaccines in the context of NSCLC. This search was followed by a manual search of the reference list of the included articles to identify additional studies. Finally, we included representative comprehensive reviews on the topic in order to highlight the background research in this field.The main types of anticancer vaccines are cell-based, protein/peptide-based, viral vectors, and gene-based vaccines (either DNA or RNA). Using mRNA in vaccines, despite its theoretical advantages, such as easy and cost-effective laboratory production and direct translation to protein in the cytoplasm without entering the nucleus, was long considered a challenging approach due to the molecule\u2019s instability ,22. ImprAn additional issue of mRNA vaccine technology is that the elongated single-stranded chain of mRNA makes it difficult to achieve high cellular encapsulation efficiency and drug loading; thus, applicable formulation strategies are usually required for effective delivery . In receRegarding the delivery systems, the main platforms for mRNA vaccines in older studies were the dendritic cells (DCs) . DCs areFurthermore, the primary goal of mRNA anticancer vaccines is to stimulate the immune response against cancer by harnessing one or multiple tumor antigens. Importantly, tumor antigens can be categorized into two main groups: tumor-associated antigens (TAAs), which are self-antigens that are abnormally expressed in cancer cells but also expressed in normal cells, and tumor neoantigens, which are the repertoire of peptides that are expressed in the surface of tumor cells but are not expressed in normal tissues and are recognized by antigen-specific T cell receptors (TCRs) through the cooperation of major histocompatibility complex (MHC) molecules . Most deThe review of the trials revealed a number of studies with mRNA anticancer vaccines that were addressed either to patients with a variety of solid tumors, including NSCLC, or were addressed exclusively to patients with NSCLC. All the studies are phase I or II clinical trials. Details of the retrieved clinical trials are summarized in The trial known as NCT00004604 was conducted in the early 2000s and was designed to assess the safety and dose-limiting toxicity of a vaccine of DCs loaded with mRNA encoding for CEA. The study enrolled patients with CEA-expressing metastatic malignancies, including lung cancer . More spAnother phase I/II trial (NCT02688686) initiated in China was planned to assess the safety and efficacy of a DC vaccine combined with cytokine-induced killer (CIK) cells in patients with advanced NSCLC and bone metastases. The cells were transfected with adenovirus type 5 (Ad5) vectors containing mRNA molecules encoding for three fixed tumor antigens: suppressor of cytokine signaling (SOCS) 1, MUC1, and survivin. However, there are no published results from this trial. Another interesting concept involves trials investigating mRNA vaccines that target Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, which are frequent genetic alterations in several neoplasms. The study NCT03948763 is a phase I trial using an LNP-formulated mRNA vaccine (mRNA-5671/V941), either alone or in combination with pembrolizumab, in patients with KRAS-mutant NSCLC, colorectal cancer, or pancreatic adenocarcinoma. The vaccine is tetravalent and includes mRNA molecules corresponding to G12D, G12V, G13D, or G12C driver mutations in the KRAS gene, with the aim of enhancing antigen-specific T-cell responses following vaccination. The purpose of the study is to determine the recommended phase 2 dose. The study has been completed with an enrollment of 70 patients, but the results are not published yet. Similarly, the NCT05202561 trial is currently recruiting patients with advanced malignancies and KRAS mutations , including lung cancer. This is a phase I single-arm, open-label study to evaluate the mRNA tumor vaccine\u2019s safety, tolerability, antitumor activity, immunoreactivity, and pharmacokinetics. The vaccine\u2019s safety is investigated either alone or in combination with a PD-1 inhibitor. Furthermore, CV9201 and CV9202 are two mRNA-based cancer vaccines developed for NSCLC, containing mRNAs encoding for a set of different tumor antigens to stimulate an adaptive cellular and humoral immune response. The NCT00923312 trial used CV9201, an mRNA-based vaccine with free and protamine-complexed full-length mRNAs that encodes for five NSCLC-related antigens: New York esophageal squamous cell carcinoma-1 (NY-ESO-1), melanoma antigen family C1 and C2, survivin, and trophoblast glycoprotein (5T4). A phase I/IIa dose-escalation trial was conducted, which enrolled 46 patients with locally advanced or metastatic NSCLC and who experienced at least stable disease after first-line treatment . The patIn the trial NCT01915524, the researchers investigated whether a similar lung cancer vaccine (CV9202 or BI1361849) could be safely administered, in combination with local radiation therapy (RT), for the consolidation and maintenance of treatment of stage IV NSCLC after first-line chemotherapy or therapy with an EGFR tyrosine kinase inhibitor . The vacThe same vaccine (CV9202 or BI1361849) has been evaluated in an open-label, multicenter, two-armed study (NCT03164772) to assess the safety and preliminary efficacy of adding the vaccine to one or two ICIs for NSCLC, namely, the anti-PD-L1 antibody durvalumab and the anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody tremelimumab . The patCurrently, the NCT03908671 trial is evaluating a personalized neoantigen mRNA vaccine. The vaccine is used as a monotherapy in patients with advanced esophageal cancer and NSCLC after the failure of standard treatment. The main objective of the trial is to evaluate the safety and tolerability of an mRNA personalized tumor vaccine and the secondary objective is the preliminary assessment of the efficacy of the vaccine in this patient population. The trial is currently enrolling patients. One of the main challenges in cancer vaccines consists of identifying the relevant tumor antigens that will maximize the potential of the patient\u2019s anticancer immune response and eliminate immune escape possibilities; therefore, several recent translational studies have focused on finding the optimal tumor antigens that could be used for mRNA vaccine development in NSCLC . Those aA set of lung adenocarcinoma-related genes, namely, GPRIN1, MYRF, PLXNB2, SLC9A4, TRIM29, UBA6, and XDH, were reported as candidate genes for mRNA vaccines in a recent study . Xu et aSun et al. selected a set of somatic genetic alterations after tissue-profiling a mouse lung cancer model and subsequently generated a neoantigen\u2013RNA vaccine . The vacThe present literature review revealed that research on mRNA vaccines for NSCLC is in the early stages but has evolved in recent years. In the past decades, trials  mostly used mRNA vaccines encoding for a single, pre-fixed tumor antigen . GradualAll trials are in early phases (phase I and II), primarily evaluating the toxicity, establishing the recommended doses for further investigation, and assessing the immune responses of patients, as well as making a preliminary assessment of efficacy. Regarding toxicity, the adverse events associated with mRNA vaccines are mainly mild and include flu-like symptoms, fever, fatigue, diarrhea, and injection-site reactions such as erythema ,33. ImmuThe development of mRNA anticancer vaccines will likely expand because of their profound advantages . It shouWith regard to NSCLC therapeutics in particular, a major issue is the identification of the most suitable phenotypes and the appropriate clinical scenario(s) so that mRNA vaccines might find their place in the armamentarium of the disease. Preclinical research has been focused on the prediction of the candidate targets of mRNA vaccines. These studies aim to guide the production of mRNAs that encode the corresponding proteins of the studied targets. Hence, mRNA molecules, coupled with efficient delivery platforms, could induce tailored T-cell responses. However, the candidate targets of mRNA vaccines are inconsistent among studies, indicating the need to validate the results of single studies ,36,37,38Finally, the optimal clinical scenario(s) to employ the mRNA vaccination of NSCLC has not yet been defined\u2014and it might not be only one disease setting (as illustrated in In conclusion, it seems that the development of mRNA vaccines for the therapeutic treatment of NSCLC is still in its infancy; however, clinical investigation and pre-clinical research have recently been enriched with new ideas on the topic. mRNA vaccination in NSCLC is being explored as a monotherapy or in combination with other therapeutic modalities. Establishing the optimal formulation of mRNA vaccines for NSCLC and solid tumors, in general, is a work in progress. Close collaborations between basic scientists and physicians, as well as between academia and industry, are warranted in order to maximize the potential new mRNA applications in thoracic oncology."}
+{"text": "The continuous progress in vaccine development witnessed in the last decades, culminated with the development of vaccines against cancers, is set to change how various cancers are treated. Cancer vaccines can be divided into two main categories: prophylactic and therapeutic cancer vaccines.Prophylactic vaccines are given to individuals at risk of developing certain types of tumors to reduce global disease morbidity and mortality . Such vaThe first-ever therapeutic cancer vaccine dates back to 1980 when Hoover Jr et al. developed an autologous vaccine against colorectal cancer . SubsequIn addition to their solo use in cancer treatment, therapeutic cancer vaccines can be combined with other immunotherapies to achieve better results. For example, the combination of therapeutic vaccines with immune checkpoint blockade (ICB) has yielded promising results due to the prevention of T cell exhaustion by immune checkpoint molecules, thus potentiating the anti-tumor response . An examFour cell-based cancer vaccine platforms currently exist: peptide-based, nucleic acid-based, and virus-based .Cell-based vaccines include tumor cell and immune cell vaccines. The former contains whole TAAs and is further classified into autologous or allogenic . The expVirus-based vaccines include inactivated, live attenuated, and subunit vaccines and stimulate an anti-tumor response by triggering both innate and adaptive immune responses . The mosOn the contrary, peptide-based vaccines are weaker in their immune response; therefore, adjuvants are mostly needed to enhance their immunogenicity . These aLastly, nucleic acid vaccines are composed of a group of pathogen antigens (carriers) and the encoding gene. They can be in the form of ribonucleic acid (RNA), deoxyribonucleic acid (DNA) , or, mosThe success of mRNA vaccines in the fight against the COVID-19 pandemic has sparked hope in the potential anti-tumor effects of these vaccines. mRNA vaccines have proven to be an effective alternative to DNA, DC, and protein-based vaccines. This is because they have the advantage of being devoid of insertional mutagenesis, being able to encode for multiple antigens (and thus potentiating the anti-tumor response), and having better tolerability, fewer adverse effects, and the possibility of rapid, low-cost, and large-scale manufacturing. Moreover, they possess a higher protein expression rate than DNA vaccines, making them the nucleic acid vaccines with the highest future potential . NeverthAlthough therapeutic cancer vaccines represent an exciting frontier in the race to solve the age-old cancer treatment puzzle, identifying vaccine platforms that can achieve high immunogenicity is crucial. Moreover, addressing individual variations in tumor antigens is needed for better anti-tumor response.In conclusion, future research should focus on improving immunogenicity by optimizing combination therapy and refining vaccine platforms for better clinical outcomes."}
+{"text": "This commentary discusses a ground-breaking study on the use of personalized mRNA cancer vaccines for treating pancreatic ductal adenocarcinoma (PDAC), a highly malignant form of cancer. The study, which capitalizes on lipid nanoparticles for mRNA vaccine delivery, aims to induce an immune response against patient-specific neoantigens and offers a potential ray of hope for improving patient prognosis. Initial results from a Phase 1 clinical trial indicated a significant T cell response in half of the subjects, opening new avenues for PDAC treatment. However, despite the promising nature of these findings, the commentary emphasizes the challenges that remain. These include the complexity of identifying suitable antigens, the possibility of tumor immune escape, and the requirement for extensive large-scale trials to confirm long-term safety and efficacy. This commentary underscores the transformative potential of mRNA technology in oncology while highlighting the hurdles that need to be overcome for its widespread adoption. Pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC), which accounts for over 95% of all pancreatic cancer cases, is notoriously challenging to diagnose and treat due to its high malignancy. Its incidence has been notably rising in recent years, with the majority of cases only diagnosed at advanced stages, earning it the chilling moniker of \"the king of all cancers\" due to its low survival rates and poor prognosis .As it stands, surgery is the only effective method of treating PDAC. Still, approximately 90% of patients experience relapse within 7\u20139\u00a0months post-operation (median time), with a 5-year overall survival rate standing at a mere 8\u201310%. Adjuvant chemotherapy after surgery can delay PDAC recurrence, but almost 80% of patients still relapse around 14\u00a0months post-operation, and the 5-year overall survival rate is less than 30%. Radiation, biological agents, and targeted therapies have shown no significant efficacy, and PDAC has proven almost entirely insensitive to immune checkpoint inhibitors with a response rate less than 5%. This insensitivity can be partially attributed to the low mutation rate of PDAC and the consequent scarcity of neoantigens, resulting in weak antigenicity of PDAC.However, recent studies have indicated that most PDACs actually harbor more neoantigens than previously predicted. Moreover, research on long-term PDAC survivors suggests that neoantigens can potentially stimulate T cells within PDAC. Therefore, strategies to deliver these neoantigens might induce neoantigen-specific T cells, thereby improving patient prognosis .mRNA cancer vaccines represent an exciting frontier in oncology, harnessing the power of the immune system to recognize and destroy cancer cells. The approach builds on the success of mRNA technology used in COVID-19 vaccines, such as those developed by Pfizer-BioNTech  and ModeThe fundamental idea behind mRNA cancer vaccines is the ability to direct the immune system towards specific tumor-associated antigens (TAAs) or neoantigens . TAAs arOne major advantage of mRNA vaccines is their flexibility. The mRNA sequence can be quickly and easily altered in response to the unique antigenic profile of a patient's tumor, allowing for a highly personalized treatment approach. This is particularly relevant in the context of neoantigens, which can vary widely between individuals and even within different regions of the same tumor.Lipid nanoparticles (LNPs)\u00a0have emerged as a critical component in the development and success of mRNA vaccines, most notably demonstrated in the COVID-19 vaccines developed by Pfizer-BioNTech and Moderna. They play a central role in the effective delivery of the mRNA payload to cells, promoting the production of the protein that will ultimately trigger an immune response.mRNA is inherently unstable and prone to degradation. Furthermore, it's negatively charged, which prevents it from easily crossing the lipid bilayer of cells. LNPs help to overcome these challenges by encapsulating the mRNA, protecting it from premature degradation, and facilitating its delivery into the cytoplasm of cells .A key component for mRNA LNP is an ionizable cationic lipid, which carries a positive charge at low pH (as in the conditions during LNP synthesis) but is neutrally charged at physiological pH. This property allows the LNPs to form complexes with the negatively charged mRNA during the formulation process and to release the mRNA once inside the cell .Other components of LNPs include phospholipids, cholesterol, and PEGylated lipids. The phospholipids and cholesterol contribute to the stability and fluidity of the nanoparticles, while the PEGylated lipids extend their circulation time in the body by reducing clearance by the immune system.Moreover, the small size of LNPs  allows them to be taken up by cells through endocytosis and captured in the endosome. The acidic environment of the endosome causes the ionizable lipid to take on a positive charge, which leads to localized disruption of the endosomal membrane and release of the mRNA into the cytoplasm, where it can be translated into protein .The development and success of LNP technology has been a game changer for mRNA vaccines, enabling the successful delivery of mRNA into cells and contributing to the unprecedented speed of COVID-19 vaccine development. Future applications of this technology are expected to extend beyond vaccines to other areas of medicine, such as gene therapy and cancer immunotherapy .Personalized RNA neoantigen vaccines: On May 10, 2023, a research team comprised of the Memorial Sloan Kettering Cancer Center, BioNTech, and Genentech (a subsidiary of Roche) published a paper in the prestigious academic journal Nature titled \"Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer\" [As aforementioned, PDAC is generally recognized as a weakly antigenic tumor with few infiltrating T cells . However+ and CD8+ T cells, resulting in anti-tumor immune responses [Therapeutic mRNA vaccine technology has paved the way for the development of personalized neoantigen vaccines. Neoantigens can be identified by individual tumor exosome sequencing, and mRNA vaccines targeting multiple patient-specific neoantigens can be rapidly produced for personalized vaccination , 9. Uponesponses . Importaesponses .In this Phase 1 clinical trial, the research team injected a personalized mRNA vaccine\u2014autogene cevumeran\u2014expressing up to 20 neoantigens, delivered using LNPs via intravenous injection, in 16 PDAC patients after surgical resection, in combination with chemotherapy (mFOLFIRINOX regimen) and immune checkpoint therapy . A significant T cell response was observed in 50% of the patients, indicating that this personalized mRNA vaccine could trigger enhanced immune responses.These results highlight the potential of personalized mRNA vaccines in treating PDAC, providing evidence of their general effectiveness as a therapeutic tool. Even with the low mutation rate of PDAC, mRNA vaccines can still induce T cell activity against new antigens produced by PDAC. The research team states that despite the limited sample size, these preliminary results suggest the need for broader clinical studies on this PDAC vaccine.In summary, this study provides preliminary clinical trial evidence that this personalized mRNA neoantigen vaccine, Autogene cevumeran (BNT122), when used in conjunction with Atezolizumab  and mFOLFIRINOX (the standard adjuvant chemotherapy regimen after PDAC surgery), induces significant T cell activity in PDAC patients who have undergone surgical resection and are at risk of delayed recurrence.This personalized mRNA neoantigen vaccine, Autogene cevumeran (BNT122) , jointlyEarly clinical trials of mRNA cancer vaccines have shown promise . HoweverFinally, as with any new therapeutic approach, the long-term safety and efficacy of mRNA cancer vaccines remain to be established through large-scale clinical trials. Despite these challenges, the potential of mRNA technology to revolutionize cancer treatment is clear, and this is an area of intense research and development."}
+{"text": "A 60-year-old man who underwent uncomplicated staged bilateral total hip arthroplasty for femoral head osteonecrosis presented with mechanical catching of his left total hip arthroplasty 3\u00a0years after index surgery. Radiographs revealed eccentricity of the left femoral head, concerning the failure of a modern moderately cross-linked polyethylene liner. Catastrophic polyethylene liner failure with significant wear, fragmentation, and femoral head abrasion was noted during revision surgery. The original liner and head were replaced, and the patient has exhibited no complications, pain, or difficulty ambulating 6 months postoperatively. This report highlights one potential novel mechanism for the failure of the Exactech Connexion GXL liner , an implant recently reported to have a higher-than-expected failure rate, potentially due to insufficient packaging and increased oxidative processes. Total hip arthroplasty (THA) is an incredibly effective treatment option for hip osteoarthritis and is considered one of the most cost-effective and successful surgeries in all of orthopaedics . In factCross-linked polyethylene acetabular liners are one example of implant technology that have dramatically improved the longevity\u00a0and survivorship of THA. One meta-analysis of 8 studies confirmed that cross-linked polyethylene has substantially reduced radiological wear . MeanwhiRecently, there have been reports of early polyethylene failure and wear resulting in significant osteolysis with the recently recalled Exactech Connexion GXL liner  . In ordeThe reporting of this study conforms to the Case Report guidelines . The pat2 and a medical history of hypertension, hyperlipidemia, and prediabetes with no smoking history. He presented in November 2018 for chronic worsening left hip pain that was no longer responsive to conservative measures. Radiographic imaging  at 2\u00a0months postoperatively that improved to no pain at 8\u00a0months in addition to full strength on hip abduction, flexion, and extension. Radiographs at 2 and 8\u00a0months postoperatively continued to demonstrate well-fixed components without hardware complications.In January 2020, the patient presented with chronically worsening right hip pain that was no longer responsive to conservative measures. Radiographic imaging and an magnetic resonance imaging revealed severe right hip osteoarthritis with edema and osteonecrosis of the right femoral head and neck and a well-positioned left THA 10 months postoperatively. In October 2020, the patient underwent right THA through the posterior approach for right hip osteoarthritis and osteonecrosis. The implants used were a 56\u00a0\u00d7 40 mm Exactech Alteon XLE vitamin E liner, Exactech Alteon tapered femoral stem, 56 mm Exactech Alteon acetabular shell, and a ceramic 40 mm Exactech Biolox Delta Novation femoral head. The Exactech Alteon XLE, a highly cross-linked vitamin E liner, had recently been acquired by the home institution and was used instead of the Connexion GXL liner to provide low wear while maintaining mechanical strength and reducing free radicals and oxidative degeneration. After obtaining postoperative radiographic imaging demonstrating well-positioned bilateral THAs without evidence of hardware failure , the patIn April 2022 (3\u00a0years after primary left THA), the patient presented after acutely experiencing mechanical catching and grinding in the left hip in addition to mild left hip pain over the past few days. He had difficulty with ambulation but did not require an assistive device. The patient denied experiencing any inciting event, trauma, falls, excessive physical activity, instability, or dislocation, in addition to any recent redness, swelling, or inflammation near the incision site. On physical exam, there was no swelling or erythema near the incision; mild pain and mechanical catching were reproduced with full ROM and full strength of hip abduction, flexion, and extension. The patient showed no signs of systemic or surgical site infection on physical exam or in laboratory workup. Radiographs  and a coThe patient underwent revision THA through the posterior approach for acute hardware failure due to polyethylene liner wear with mechanical problems of the left THA, 3\u00a0years after implantation. Upon exposure, there were fragmented polyethylene liner pieces in the joint. In addition, there was minimal head and cup articulation at the fractured site of the liner, resulting in metallosis and dark-colored fluids. Also, there was a linear scratch on the surface of the femoral head and significant wear and failure of the polyethylene liner . The oriIn May and October 2022, the patient presented for his 1- and 6-month follow-up for left revision THA. Radiographs revealed well-aligned bilateral THA with no signs of hardware failure . He was The patient in this report underwent early revision THA due to early catastrophic failure and fragmentation of an Exactech Connexion GXL moderately cross-linked polyethylene liner. While this patient did exhibit factors that could be associated with revision of primary THA such as male sex, osteonecrosis, and obesity ,15, we dThomas et\u00a0al. reviewed their institutional database from 2009 to 2019 for patients presenting with osteolysis in the setting of a THA utilizing the Exactech Connexion GXL polyethylene liner. They found 9 of the 12 patients in their investigation underwent revision surgery for early polyethylene wear and secondary osteolysis at an average postoperative time of 55.9 months (range: 12-120 months). In addition, they reviewed the Manufacturer and User Facility Device Experience database from 2009 to 2019 and found 22 reported cases of wear-related failure. They suggested the locking mechanism or manufacturing characteristics as the potential cause for failure . YakkantThere have been reports of reasonable outcomes using this liner. In a letter to the editor, Godoy-Monzon and Cid-Casteulani reported that the Exactech Connexion GXL liner had demonstrated success in 2 of their midterm studies . In a prExactech Inc. recently issued a recall for several implants including the Connexion GXL liner for insufficient packaging. They have disclosed that many inserts were packaged into vacuum bags lacking a secondary oxygen barrier layer, which normally augments oxygen resistance and prevents oxidation . ConsequThis report of a catastrophic early failure and fragmentation of a modern moderately cross-linked polyethylene liner appears to be a novel mechanism of failure of the Exactech Connexion GXL liner. This failure may be related to the recent Exactech recall, which indicated certain liners were insufficiently packaged, predisposing them to high levels of oxygen exposure and consequent oxidation. Surgeons should be aware of the concerns with this implant as well as potential failure mechanisms.J.A. Browne serves as a board or committee member of the American Association of Hip and Knee Surgeons, American Joint Replacement Registry (American Academy of Orthopaedic Surgeons), Southern Orthopedic Association, Hip Society, and Knee Society; received royalties and serves as a paid consultant to Enovis; serves on editorial or governing board and received financial support from the Journal of Arthroplasty; serves as a paid consultant to OsteoRemedies and Kinamed; has stock or stock options held in RadLink; and received financial support from Elsevier and the Journal of Bone and Joint Surgery. Q. Cui received research support from Depuy and Exactech; received financial support from Elsevier; serves on editorial or governing boards of the Journal of Arthroplasty and the Journal of Orthopedic Research; and serves as a board or committee member for the Association Research Circulation Osseous. None of the following authors or any immediate family members has received anything of value from or has stock or stock options held in a commercial company or institution related directly or indirectly to the subject of this article: N. Barakat.https://doi.org/10.1016/j.artd.2023.101161.For full disclosure statements refer to The author(s) confirm that written informed consent has been obtained from the involved patient(s) or if appropriate from the parent, guardian, power of attorney of the involved patient(s); and, they have given approval for this information to be published in this case report (series)."
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