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[This corrects the article DOI: 10.1371/journal.pone.0166400.].

[This corrects the article DOI: 10.1371/journal.pone.0125358.].

BackgroundThe increased use of telemedicine to provide virtual outpatient visits during the pandemic has led to concerns about potential increased emergency department (ED) admissions and outpatient service use prior to such admissions. We examined the frequency of virtual visits use prior to ED admissions and characterized the patients with prior virtual visit use and the physicians who provided these outpatient visits.MethodsWe conducted a retrospective, population-based, cross-sectional analysis using linked health administrative data in Ontario, Canada to identify patients who had an ED admission between July 1 and September 30, 2021 and patients with an ED admissions during the same period in 2019. We grouped patients based on their use of outpatient services in the 7 days prior to admission and reported their sociodemographic characteristics and healthcare utilization.ResultsThere were 1,080,334 ED admissions in 2021 vs. 1,113,230 in 2019. In 2021, 74% of these admissions had no prior outpatient visits (virtual or in-person) within 7 days of admission, compared to 75% in 2019. Only 3% of ED admissions had both virtual and in-person visits in the 7 days prior to ED admission. Patients with prior virtual care use were more likely to be hospitalized than those without any outpatient care (13% vs 7.7.%).InterpretationThe net amount of ED admissions and outpatient care prior to admission remained the same over a period of the COVID-19 pandemic when cases were relatively stable. Virtual care seemed to be able to appropriately triage patients to the ED and virtual visits replaced in-person visits ahead of ED admissions, as opposed to being additive.

Stock market prediction is the process of determining the value of a company's shares and other financial assets in the future. This paper proposes a new model where Altruistic Dragonfly Algorithm (ADA) is combined with Least Squares Support Vector Machine (LS-SVM) for stock market prediction. ADA is a meta-heuristic algorithm which optimizes the parameters of LS-SVM to avoid local minima and overfitting, resulting in better prediction performance. Experiments have been performed on 12 datasets and the obtained results are compared with other popular meta-heuristic algorithms. The results show that the proposed model provides a better predictive ability and demonstrate the effectiveness of ADA in optimizing the parameters of LS-SVM.

Effective dialogue generation for task completion is challenging to build. The task requires the response generation system to generate the responses consistent with intent and slot values, have diversity in response and be able to handle multiple domains. The response also needs to be context relevant with respect to the previous utterances in the conversation. In this paper, we build six different models containing Bi-directional Long Short Term Memory (Bi-LSTM) and Bidirectional Encoder Representations from Transformers (BERT) based encoders. To effectively generate the correct slot values, we implement a copy mechanism at the decoder side. To capture the conversation context and the current state of the conversation we introduce a simple heuristic to build a conversational knowledge graph. Using this novel algorithm we are able to capture important aspects in a conversation. This conversational knowledge-graph is then used by our response generation model to generate more relevant and consistent responses. Using this knowledge-graph we do not need the entire utterance history, rather only the last utterance to capture the conversational context. We conduct experiments showing the effectiveness of the knowledge-graph in capturing the context and generating good response. We compare these results against hierarchical-encoder-decoder models and show that the use of triples from the conversational knowledge-graph is an effective method to capture context and the user requirement. Using this knowledge-graph we show an average performance gain of 0.75 BLEU score across different models. Similar results also hold true across different manual evaluation metrics.

Live-cell imaging has become state of the art to accurately identify the nature of mitotic and cell cycle defects. Low- and high-throughput microscopy setups have yield huge data amounts of cells recorded in different experimental and pathological conditions. Tailored semi-automated and automated image analysis approaches allow the analysis of high-content screening data sets, saving time and avoiding bias. However, they were mostly designed for very specific experimental setups, which restricts their flexibility and usability. The general need for dedicated experiment-specific user-annotated training sets and experiment-specific user-defined segmentation parameters remains a major bottleneck for fully automating the analysis process. In this work we present LiveCellMiner, a highly flexible open-source software tool to automatically extract, analyze and visualize both aggregated and time-resolved image features with potential biological relevance. The software tool allows analysis across high-content data sets obtained in different platforms, in a quantitative and unbiased manner. As proof of principle application, we analyze here the dynamic chromatin and tubulin cytoskeleton features in human cells passing through mitosis highlighting the versatile and flexible potential of this tool set.

Treatment options for men with moderate-to-severe lower urinary tract symptoms (LUTS) due to benign prostatic hyperplasia (BPH) have variable efficacy, safety, and retreatment profiles, contributing to variations in patient quality of life and healthcare costs. This study examined the long-term cost-effectiveness of generic combination therapy (CT), prostatic urethral lift (PUL), water vapor thermal therapy (WVTT), photoselective vaporization of the prostate (PVP), and transurethral resection of the prostate (TURP) for the treatment of BPH. A systematic literature review was performed to identify clinical trials of CT, PUL, WVTT, PVP, and TURP that reported change in International Prostate Symptom Score (IPSS) for men with BPH and a prostate volume ≤80 cm3. A random-effects network meta-analysis was used to account for the differences in patient baseline clinical characteristics between trials. An Excel-based Markov model was developed with a cohort of males with a mean age of 63 and an average IPSS of 22 to assess the cost-effectiveness of these treatment options at 1 and 5 years from a US Medicare perspective. Procedural and adverse event (AE)-related costs were based on 2021 Medicare reimbursement rates. Total Medicare costs at 5 years were highest for PUL ($9,580), followed by generic CT ($8,223), TURP ($6,328), PVP ($6,152), and WVTT ($2,655). The total cost of PUL was driven by procedural ($7,258) and retreatment ($1,168) costs. At 5 years, CT and PUL were associated with fewer quality-adjusted life years (QALYs) than WVTT, PVP, and TURP. Compared to WVTT, the incremental cost-effectiveness ratios (ICERs) for both TURP and PVP were above a willingness-to-pay threshold of $50,000/QALY (TURP: $64,409/QALY; PVP: $87,483/QALY). This study provides long-term cost-effectiveness evidence for several common treatment options for men with BPH. WVTT is an effective and economically viable treatment in resource-constrained environments.

PurposeIt is currently unclear how the shift towards virtual care during the 2019 novel coronavirus (COVID-19) pandemic may have impacted chronic disease management at a population level. The goals of our study were to provide a description of the levels of use of virtual care services relative to in-person care in patients with chronic disease across Ontario, Canada and to describe levels of healthcare utilization in low versus high virtual care users.MethodsWe used linked health administrative data to conduct a population-based, repeated cross-sectional study of all ambulatory patient visits in Ontario, Canada (January 1, 2018 to January 16, 2021). Further stratifications were also completed to examine patients with COPD, heart failure, asthma, hypertension, diabetes, mental illness, and angina. Patients were classified as low (max 1 virtual care visit) vs. high virtual care users. A time-series analysis was done using interventional autoregressive integrated moving average (ARIMA) modelling on weekly hospitalizations, outpatient visits, and diagnostic tests.ResultsThe use of virtual care increased across all chronic disease patient populations. Virtual care constituted at least half of the total care in all conditions. Both low and high virtual care user groups experienced a statistically significant reduction in hospitalizations and laboratory testing at the start of the pandemic. Hospitalization volumes increased again only among the high users, while testing increased in both groups. Outpatient visits among high users remained unaffected by the pandemic but dropped in low users.ConclusionThe decrease of in-person care during the pandemic was accompanied by an increase in virtual care, which ultimately allowed patients with chronic disease to return to the same visit rate as they had before the onset of the pandemic. Virtual care was adopted across various chronic conditions, but the relative adoption of virtual care varied by condition with highest rates seen in mental health.

Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a "donor" ubiquitin and a primary amino group of an "acceptor" ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface.

Information exchange (or signaling) between plants following herbivore damage has recently been shown to affect plant responses to herbivory in relatively simple natural systems. In a large, manipulative field study using three annual plant species (Achyrachaena mollis, Lupinus nanus, and Sinapis arvensis), we tested whether experimental damage to a neighboring conspecific affected a plant's lifetime fitness and interactions with herbivores. By manipulating relatedness between plants, we assessed whether genetic relatedness of neighboring individuals influenced the outcome of having a damaged neighbor. Additionally, in laboratory feeding assays, we assessed whether damage to a neighboring plant specifically affected palatability to a generalist herbivore and, for S. arvensis, a specialist herbivore. Our study suggested a high level of contingency in the outcomes of plant signaling. For example, in the field, damaging a neighbor resulted in greater herbivory to A. mollis, but only when the damaged neighbor was a close relative. Similarly, in laboratory trials, the palatability of S. arvensis to a generalist herbivore increased after the plant was exposed to a damaged neighbor, while palatability to a specialist herbivore decreased. Across all species, damage to a neighbor resulted in decreased lifetime fitness, but only if neighbors were closely related. These results suggest that the outcomes of plant signaling within multi-species neighborhoods may be far more context-specific than has been previously shown. In particular, our study shows that herbivore interactions and signaling between plants are contingent on the genetic relationship between neighboring plants. Many factors affect the outcomes of plant signaling, and studies that clarify these factors will be necessary in order to assess the role of plant information exchange about herbivory in natural systems.

BackgroundThis study reports on physicians' experiences with chronic pain management. For over a decade prescription opioids have been a primary treatment for chronic pain in North America. However, the current opioid epidemic has complicated long-standing practices for chronic pain management which historically involved prescribing pain medication. Caring for patients with chronic pain occurs within a context in which a growing proportion of patients suffer from chronic rather than acute conditions alongside rising social inequities.MethodsOur team undertook an ethnographic approach known as institutional ethnography in the province of Ontario, Canada in order to explore the social organization of chronic pain management from the standpoint of primary care physicians. This paper reports on a subset of this study data, specifically interviews with 19 primary care clinicians and 8 nurses supplemented by 40 hours of observations. The clinicians in our sample were largely primary care physicians and nurses working in urban, rural and Northern settings.FindingsIn their reflections on providing care for patients with chronic pain, many providers describe being most challenged by the work involved in helping patients who also struggled with poverty, mental health and addiction. These frustrations were often complicated by concerns that they could lose their license for inappropriate prescribing, thus shifting their work from providing treatment and care to policing their patients for malingering and opioid abuse.InterpretationOur findings show that care providers find the treatment of patients with chronic pain-especially those patients also experiencing poverty-to be challenging at best, and at worst frustrating and overwhelming. In many instances, their narratives suggested experiences of depersonalization, loss of job satisfaction and emotional exhaustion in relation to providing care for these patients, key dimensions of burnout. In essence, the work that they performed in relation to their patients' social rather than medical needs seems to contribute to these experiences. Their experiences were further exacerbated by the fact that restricting and reducing opioid dosing in patients with chronic pain has become a major focus of care provision.

IntroductionIdentifying and ensuring the Essential Public Health Functions (EPHFs) is one of the core agendas of the World Health Organization to strengthen the health system of a country. The definition of EPHFs varies widely, considering country needs. In Bangladesh, the physician cadres are in the leadership position to oversee the EPHFs at the district and sub-district levels. However, there is a dearth of contextual information regarding the purview of essential EPHFs in the country. The purpose of this study was to document the perception of key stakeholders on what constitute the EPHFs at district and sub-district level and identify the challenges they face in providing the services.MethodsWe carried out a qualitative exploratory study consisting of document review and Key Informant Interviews (KIIs). The desk review included the organograms of the government district and sub-district level health facilities and the job description of Civil Surgeons (CSs) and Upazila Health and Family Planning Officers (UHFPOs). In addition, 15 KIIs with relevant professionals and stakeholders from the Directorate of General of Health Services (DGHS), Ministry of Health and Family Welfare (MoHFW) were conducted. Collected data were analyzed thematically.ResultsThree major categories of EPHFs were identified: i) population-oriented preventive functions, ii) clinical preventive functions, and iii) administrative/management functions. The CSs and UHFPOs need to ensure these wide range of EPHFs at the district level and below. However, at peripheral level, the leadership positions' clinical and public health roles often get amalgamated. Therefore, ensuring public health functions are hampered. Besides, these positions need training and adequate support staff to perform the EPHFs effectively.ConclusionRecognizing the EPHFs in the Bangladesh context is crucial. Revisiting the job descriptions and strengthening appropriate public health services at different tiers in the country health system should be prioritized to achieve health-related Sustainable Development Goals.

Serologic assays developed for SARS-CoV-2 detect different antibody subtypes and are based on different target antigens. Comparison of the performance of a SARS-CoV-2 Spike-Protein ELISA and the nucleocapsid-based Abbott ArchitectTM SARS-CoV-2 IgG assay indicated that the assays had high concordance, with rare paired discordant tests results.

PurposeExisting virtual agents (VAs) present in dialogue systems are either information retrieval based or static goal-driven. However, in real-world situations, end-users might not have a known and fixed goal beforehand for the task, i.e., they may upgrade/downgrade/update their goal components in real-time to maximize their utility values. Existing VAs are unable to handle such dynamic goal-oriented situations.MethodologyDue to the absence of any related dialogue dataset where such choice deviations are present, we have created a conversational dataset called Deviation adapted Virtual Agent(DevVA), with the manual annotation of its corresponding intents, slots, and sentiment labels. A Dynamic Goal Driven Dialogue Agent (DGDVA) has been developed by incorporating a Dynamic Goal Driven Module (GDM) on top of a deep reinforcement learning based dialogue manager. In the course of a conversation, the user sentiment provides grounded feedback about agent behavior, including goal serving action. User sentiment appears to be an appropriate indicator for goal discrepancy that guides the agent to complete the user's desired task with gratification. The negative sentiment expressed by the user about an aspect of the provided choice is treated as a discrepancy that is being resolved by the GDM depending upon the observed discrepancy and current dialogue state. The goal update capability and the VA's interactiveness trait enable end-users to accomplish their desired task satisfactorily.FindingsThe obtained experimental results illustrate that DGDVA can handle dynamic goals with maximum user satisfaction and a significantly higher success rate. The interaction drives the user to decide its final goal through the latent specification of possible choices and information retrieved and provided by the dialogue agent. Through the experimental results (qualitative and quantitative), we firmly conclude that the proposed sentiment-aware VA adapts users' dynamic behavior for its goal setting with substantial efficacy in terms of primary objective i.e., task success rate (0.88).Practical implicationsIn real world, it can be argued that many people do not have a predefined and fixed goal for tasks such as online shopping, movie booking & restaurant booking, etc. They tend to explore the available options first which are aligned with their minimum requirements and then decide one amongst them. The DGDVA provides maximum user satisfaction as it enables them to accomplish a dynamic goal that leads to additional utilities along with the essential ones.OriginalityTo the best of our knowledge, this is the first effort towards the development of A Dynamic Goal Adapted Task-Oriented Dialogue Agent that can serve user goals dynamically until the user is satisfied.

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart, small intestine and dorsal skin were collected for tissue analysis. Female APCHi mice showed the most severe radiation-induced deposition of cardiac collagens but were protected against a radiation-induced loss of microvascular density. Both male and female APCHi mice were protected against a radiation induced upregulation of toll-like receptor 4 in the heart, but this did not translate into a clear protection against immune cell infiltration. In the small intestine, the APCHi genotype had no effect on an increase in the number of myeloperoxidase positive cells (seen mostly in females) or an increase in the expression of T-cell marker CD2 (males). Lastly, both male and female APCHi mice were protected against radiation-induced epidermal thickening and increase in 3-nitrotyrosine positive keratinocytes. In conclusion, prolonged high levels of APC in a transgenic mouse model had little effects on indicators of DEARE in the heart, small intestine and skin, with some differential effects in male compared to female mice.

[This corrects the article DOI: 10.1371/journal.pone.0227828.].

Cotton leaf curl disease (CLCuD), caused by whitefly (Bemisiatabaci) transmitted single-stranded DNA viruses belonging to the Genus, Begomovirus (family, Geminiviridae) in association with satellite molecules; is responsible for major economic losses in cotton in three northwest (NW) Indian states Haryana, Punjab, and Rajasthan. Annual CLCuD incidences during 2012 to 2014 were estimated to be 37.5%, 63.6%, and 38.8% respectively. Cotton leaves were collected from symptomatic plants annually for three years and subjected to DNA isolation, followed by rolling circle amplification (RCA), cloning, and DNA sequencing of apparently full-length begomoviral genomes and associated betasatellites and alphasatellites. Among the thirteen CLCuD-begomoviral genomes recovered, eight were identified as Cotton leaf curl Multan virus-Rajasthan (CLCuMuV-Ra), one as -Pakistan (PK) and another as -Faisalabad (Fai), whereas, three were as Cotton leaf curl Kokhran virus-Burewala (CLCuKoV-Bu), indicating that CLCuMuV-Ra was the most prevalent begomovirus species. Five of the eight CLCuMuV-Ra sequences were found to be recombinants. The CLCuMuV-Ra- associated satellites consisted of Cotton leaf curl Multan betasatellite (CLCuMB), and Gossypium darwinii symptomless alphasatellite (GDarSLA), and Croton yellow vein mosaic alphasatellite (CrYVMoA). The second most abundant helper virus species, CLCuKoV-Bu, was associated with CLCuMB and GDarSLA.

PURPOSE:Developing a Dialogue/Virtual Agent (VA) that can handle complex tasks (need) of the user pertaining to multiple intents of a domain is challenging as it requires the agent to simultaneously deal with multiple subtasks. However, majority of these end-to-end dialogue systems incorporate only user semantics as inputs in the learning process and ignore other useful user behavior and information. Sentiment of the user at the time of conversation plays an important role in securing maximum user gratification. So, incorporating sentiment of the user during the policy learning becomes even more crucial, more so when serving composite tasks of the user. METHODOLOGY:As a first step towards enabling the development of sentiment aided VA for multi-intent conversations, this paper proposes a new dataset, annotated with its corresponding intents, slot and sentiment (considering the entire dialogue history) labels, named SentiVA, collected from open-sourced dialogue datasets. In order to integrate these multiple aspects, a Hierarchical Reinforcement Learning (HRL) specifically options based VA is proposed to learn strategies for managing multi-intent conversations. Along with task success based immediate rewards, sentiment based immediate rewards are also incorporated in the hierarchical value functions to make the VA user adaptive. FINDINGS:Empirically, the paper shows that task based and sentiment based immediate rewards cumulatively are required to ensure successful task completion and attain maximum user satisfaction in a multi-intent scenario instead of any of these rewards alone. PRACTICAL IMPLICATIONS:The eventual evaluators and consumers of dialogue systems are users. Thus, to ensure a fulfilling conversational experience involving maximum user satisfaction requires VA to consider user sentiment at every time-step in its decision making policy. ORIGINALITY:This work is the first attempt in incorporating sentiment based rewards in the HRL framework.

Diagnosis of visceral leishmaniasis (VL) relies on invasive and risky aspirate procedures, and confirmation of cure after treatment is unreliable. Detection of Leishmania donovani antigens in urine has the potential to provide both a non-invasive diagnostic and a test of cure. We searched for L. donovani antigens in urine of VL patients from India and Sudan to contribute to the development of urine antigen capture immunoassays. VL urine samples were incubated with immobilised anti-L. donovani polyclonal antibodies and captured material was eluted. Sudanese eluted material and concentrated VL urine were analysed by western blot. Immunocaptured and immunoreactive material from Indian and Sudanese urine was submitted to mass spectrometry for protein identification. We identified six L. donovani proteins from VL urine. Named proteins were 40S ribosomal protein S9, kinases, and others were hypothetical. Thirty-three epitope regions were predicted with high specificity in the 6 proteins. Of these, 20 were highly specific to Leishmania spp. and are highly suitable for raising antibodies for the subsequent development of an antigen capture assay. We present all the identified proteins and analysed epitope regions in full so that they may contribute to the development of non-invasive immunoassays for this deadly disease.

Trypanosoma cruzi, the protozoan agent of Chagas disease in the Americas, is comprised of six genetic lineages (TcI-TcVI) and a possible seventh (TcBat, related to TcI). Identification of T. cruzi lineages infecting reservoir mammalian species is fundamental to resolving transmission cycles. However, this is hindered by the limited sensitivity and technical complexity of parasite isolation and genotyping. An alternative approach is serology using T. cruzi lineage-specific epitopes, such as those of the trypomastigote small surface antigen (TSSA). For surveillance of T. cruzi lineage infections in mammal species from diverse Brazilian regions, we apply a novel rapid diagnostic test (RDT, Chagas Sero K-SeT), which incorporates the TSSA peptide epitope specific to TcII/V/VI (TSSApep-II/V/VI) and Protein G detection of antibodies. Chagas Sero K-SeT RDT results with sera from experimentally infected mice, from tamarin primates (Leontopithecus spp.) and from canines (Canis familiaris) were concordant with corresponding TSSApep-II/V/VI ELISAs. The Chagas Sero K-Set detected TcII/V/VI infections in Leontopithecus spp. from the Atlantic forest (n = 46), in C. familiaris (n = 16) and Thrichomys laurentius (n = 2) from Caatinga biome and Chiroptera (n = 1) from Acre, Amazonia. The Chagas Sero K-SeT RDT is directly applicable to TcII/V/VI-specific serological surveillance of T. cruzi infection in several different mammalian Orders. It can replace ELISAs and provides efficient, point-of-sampling, low-cost detection of TcII/V/VI infections, with at least equivalent sensitivity, although some mammals may be difficult to trap, and, not unexpectedly, Chagas Sero K-SeT could not recognise feline IgG. Knowledge of sylvatic hosts of T. cruzi can be expanded, new reservoir species discovered, and the ecology of transmission cycles clarified, particularly with adaptation to further mammalian Orders.

Cebpa encodes a transcription factor (TF) that plays an instructive role in the development of multiple myeloid lineages. The expression of Cebpa itself is finely modulated, as Cebpa is expressed at high and intermediate levels in neutrophils and macrophages respectively and downregulated in non-myeloid lineages. The cis-regulatory logic underlying the lineage-specific modulation of Cebpa's expression level is yet to be fully characterized. Previously, we had identified 6 new cis-regulatory modules (CRMs) in a 78kb region surrounding Cebpa. We had also inferred the TFs that regulate each CRM by fitting a sequence-based thermodynamic model to a comprehensive reporter activity dataset. Here, we report the cis-regulatory logic of Cebpa CRMs at the resolution of individual binding sites. We tested the binding sites and functional roles of inferred TFs by designing and constructing mutated CRMs and comparing theoretical predictions of their activity against empirical measurements in a myeloid cell line. The enhancers were confirmed to be activated by combinations of PU.1, C/EBP family TFs, Egr1, and Gfi1 as predicted by the model. We show that silencers repress the activity of the proximal promoter in a dominant manner in G1ME cells, which are derived from the red-blood cell lineage. Dominant repression in G1ME cells can be traced to binding sites for GATA and Myb, a motif shared by all of the silencers. Finally, we demonstrate that GATA and Myb act redundantly to silence the proximal promoter. These results indicate that dominant repression is a novel mechanism for resolving hematopoietic lineages. Furthermore, Cebpa has a fail-safe cis-regulatory architecture, featuring several functionally similar CRMs, each of which contains redundant binding sites for multiple TFs. Lastly, by experimentally demonstrating the predictive ability of our sequence-based thermodynamic model, this work highlights the utility of this computational approach for understanding mammalian gene regulation.

Recent studies have highlighted the extraordinary cell type diversity that exists within mammalian organs, yet the molecular drivers of such heterogeneity remain elusive. To address this issue, much attention has been focused on profiling the transcriptome and epigenome of individual cell types. However, standard cell type isolation methods based on surface or fluorescent markers remain problematic for cells residing within organs with significant connective tissue. Since the nucleus contains both genomic and transcriptomic information, the isolation of nuclei tagged in specific cell types (INTACT) method provides an attractive solution. Although INTACT has been successfully applied to plants, flies, zebrafish, frogs, and mouse brain and adipose tissue, broad use across mammalian organs remains challenging. Here we describe the PAN-INTACT method, which can be used to isolate cell type specific nuclei from fibrous mouse organs, which are particularly problematic. As a proof-of-concept, we demonstrate successful isolation of cell type-specific nuclei from the mouse heart, which contains substantial connective tissue and harbors multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, and epicardial cells. Compared to established techniques, PAN-INTACT allows more rapid isolation of cardiac nuclei to facilitate downstream applications. We show cell type-specific isolation of nuclei from the hearts of Nkx2-5Cre/+; R26Sun1-2xsf-GFP-6xmyc/+ mice, which we confirm by expression of lineage markers. Furthermore, we perform Assay for Transposase Accessible Chromatin (ATAC)-Seq to provide high-fidelity chromatin accessibility maps of Nkx2-5+ nuclei. To extend the applicability of PAN-INTACT, we also demonstrate successful isolation of Wt1+ podocytes from adult kidney. Taken together, our data suggest that PAN-INTACT is broadly applicable for profiling the transcriptional and epigenetic landscape of specific cell types. Thus, we envision that our method can be used to systematically probe mechanistic details of cell type-specific functions within individual organs of intact mice.

Fusarium virguliforme is a soil borne root pathogen that causes sudden death syndrome (SDS) in soybean [Glycine max (L.) Merrill]. Once the fungus invades the root xylem tissues, the pathogen secretes toxins that cause chlorosis and necrosis in foliar tissues leading to defoliation, flower and pod drop and eventually death of plants. Resistance to F. virguliforme in soybean is partial and governed by over 80 quantitative trait loci (QTL). We have conducted genome-wide association study (GWAS) for a group of 254 plant introductions lines using a panel of approximately 30,000 SNPs and identified 19 single nucleotide polymorphic loci (SNPL) that are associated with 14 genomic regions encoding foliar SDS and eight SNPL associated with seven genomic regions for root rot resistance. Of the identified 27 SNPL, six SNPL for foliar SDS resistance and two SNPL for root rot resistance co-mapped to previously identified QTL for SDS resistance. This study identified 13 SNPL associated with eight novel genomic regions containing foliar SDS resistance genes and six SNPL with five novel regions for root-rot resistance. This study identified five genes carrying nonsynonymous mutations: (i) three of which mapped to previously identified QTL for foliar SDS resistance and (ii) two mapped to two novel regions containing root rot resistance genes. Of the three genes mapped to QTL for foliar SDS resistance genes, two encode LRR-receptors and third one encodes a novel protein with unknown function. Of the two genes governing root rot resistance, Glyma.01g222900.1 encodes a soybean-specific LEA protein and Glyma.10g058700.1 encodes a heparan-alpha-glucosaminide N-acetyltransferase. In the LEA protein, a conserved serine residue was substituted with asparagine; and in the heparan-alpha-glucosaminide N-acetyltransferase, a conserved histidine residue was substituted with an arginine residue. Such changes are expected to alter functions of these two proteins regulated through phosphorylation. The five genes with nonsynonymous mutations could be considered candidate SDS resistance genes and should be suitable molecular markers for breeding SDS resistance in soybean. The study also reports desirable plant introduction lines and novel genomic regions for enhancing SDS resistance in soybean.

Time Perspective (TP) is an important area of research within the 'psychological time' paradigm. TP, or the manner in which individuals conduct themselves as a reflection of their cogitation of the past, the present, and the future, is considered as a basic facet of human functioning. These perceptions of time have an influence on our actions, perceptions, and emotions. Assessment of TP based on human language on Twitter opens up a new avenue for research on subjective view of time at a large scale. In order to assess TP of users' from their tweets, the foremost task is to resolve grammatical tense into the underlying temporal orientation of tweets as for many tweets the tense information, and their temporal orientations are not the same. In this article, we first resolve grammatical tense of users' tweets to identify their underlying temporal orientation: past, present, or future. We develop a minimally supervised classification framework for temporal orientation task that enables incorporating linguistic knowledge into a deep neural network. The temporal orientation model achieves an accuracy of 78.7% when tested on a manually annotated test set. This method performs better when compared to the state-of-the-art technique. Secondly, we apply the classification model to classify the users' tweets in either of the past, present or future categories. Tweets classified this way are then grouped for each user which gives rise to unidimensional TP. The valence (positive, negative, and neutral) is added to the temporal orientation dimension to produce the bidimensional TP. We finally investigate the association between the Twitter users' unidimensional and bidimensional TP and their age, education and six basic emotions in a large-scale empirical manner. Our analysis shows that people tend to think more about the past as well as more positive about the future when they age. We also observe that future-negative people are less joyful, more sad, more disgusted, and more angry while past-negative people have more fear.

With the increase in the amount of text information in different real-life applications, automatic text-summarization systems become more predominant in extracting relevant information. In the current study, we formulated the problem of extractive text-summarization as a binary optimization problem, and multi-objective binary differential evolution (DE) based optimization strategy is employed to solve this. The solutions of DE encode a possible subset of sentences to be present in the summary which is then evaluated based on some statistical features (objective functions) namely, the position of the sentence in the document, the similarity of a sentence with the title, length of the sentence, cohesion, readability, and coverage. These objective functions, measuring different aspects of summary, are optimized simultaneously using the search capability of DE. Some newly designed self-organizing map (SOM) based genetic operators are incorporated in the optimization process to improve the convergence. SOM generates a mating pool containing solutions and their neighborhoods. This mating pool takes part in the genetic operation (crossover and mutation) to create new solutions. To measure the similarity or dissimilarity between sentences, different existing measures like normalized Google distance, word mover distance, and cosine similarity are explored. For the purpose of evaluation, two standard summarization datasets namely, DUC2001, and DUC2002 are utilized, and the obtained results are compared with various supervised, unsupervised and optimization strategy based existing summarization techniques using ROUGE measures. Results illustrate the superiority of our approach in terms of convergence rate and ROUGE scores as compared to state-of-the-art methods. We have obtained 45% and 5% improvements over two recent state-of-the-art methods considering ROUGE-2 and ROUGE-1 scores, respectively, for the DUC2001 dataset. While for the DUC2002 dataset, improvements obtained by our approach are 20% and 5%, considering ROUGE-2 and ROUGE-1 scores, respectively. In addition to these standard datasets, CNN news dataset is also utilized to evaluate the efficacy of our proposed approach. It was also shown that the best performance not only depends on the objective functions used but also on the correct choice of similarity/dissimilarity measure between sentences.

BackgroundThe management of complex, multi-morbid patients is challenging for solo primary care providers (PCPs) with limited access to resources. The primary objective of the intervention was to reduce the overall rate of Emergency Department (ED) visits among patients in participating practices.Methods and findingsAn interrupted time series design and qualitative interviews were used to evaluate a multifaceted intervention, SCOPE (Seamless Care Optimizing the Patient Experience), offered to solo PCPs whose patients were frequent users of the ED. The intervention featured a navigation hub (nurse, homecare coordinator) to link PCPs with hospital and community resources, a general internist on-call to provide phone advice or urgent assessments, and access to patient results on-line. Continuous quality improvement (QI) strategies were employed to optimize each component of the intervention. The primary outcome was the relative pre-post intervention change in ED visit rate for patients of participating practices compared with that for a propensity-matched control group of physicians over the contemporaneous period. Themes were identified from semi-structured interviews on PCP's experiences and influential factors in their engagement. Twenty-nine physicians agreed to participate and were provided access to the intervention over an 18-month time period. There were a total of 1,525 intervention contacts over the 18-months (average: 50.6±60.8 per PCP). Both intervention and control groups experienced a trend towards lower rates of ED use by their patients over the study time period. The pre-post difference in trend for the intervention group compared to the controls was not significant at 1.4% per year (RR = 1.014; p = 0.59). Several themes were identified from qualitative interviews including: PCPs felt better supported in the care of their patients; they experienced a greater sense of community, and; they were better able to provide shared primary-specialty care.ConclusionsThis multifaceted intervention to support solo PCPs in the management of their complex patients did not result in a reduced rate of ED visits compared to controls, likely related to variable uptake among PCPs. It did however result in more comprehensive and coordinated care for their patients. Future directions will focus on increasing uptake by improving ease of use, increasing the range of services offered and expanding to a larger number of PCPs.

Knowledge of current genetic diversity and mating systems of crop wild relatives (CWR) in the Fertile Crescent is important in crop genetic improvement, because western agriculture began in the area after the cold-dry period known as Younger Dryas about 12,000 years ago and these species are also wild genepools of the world's most important food crops. Wild pea (Pisum sativum subsp. elatius) is an important source of genetic diversity for further pea crop improvement harbouring traits useful in climate change context. The genetic structure was assessed on 187 individuals of Pisum sativum subsp. elatius from fourteen populations collected in the northern part of the Fertile Crescent using 18,397 genome wide single nucleotide polymorphism DARTseq markers. AMOVA showed that 63% of the allelic variation was distributed between populations and 19% between individuals within populations. Four populations were found to contain admixed individuals. The observed heterozygosity ranged between 0.99 to 6.26% with estimated self-pollination rate between 47 to 90%. Genetic distances of wild pea populations were correlated with geographic but not environmental (climatic) distances and support a mixed mating system with predominant self-pollination. Niche modelling with future climatic projections showed a local decline in habitats suitable for wild pea, making a strong case for further collection and ex situ conservation.

[This corrects the article DOI: 10.1371/journal.pone.0194056.].

BACKGROUND:Improving quality of maternal healthcare services is key to reducing maternal mortality across developing nations, including India. Expanding access to institutionalized care alone has failed to address critical quality barriers to safe, effective, patient-centred, timely and equitable care. Multi-dimensional quality improvement focusing on Person Centred Care(PCC) has an important role in expanding utilization of maternal health services and reducing maternal mortality. METHODS:Nine public health facilities were selected in two rural districts of Uttar Pradesh(UP), India, to understand women's experiences of childbirth and identify quality gaps in the process of maternity care. 23 direct, non-participant observations of uncomplicated vaginal deliveries were conducted using checklists with special reference to PCC, capturing quality of care provision at five stages-admission; pre-delivery; delivery; post-delivery and discharge. Data was thematically analysed using the framework approach. Case studies, good practices and gaps were noted at each stage of delivery care. RESULTS:Admission to maternity wards was generally prompt. All deliveries were conducted by skilled providers and at least one staff was available at all times. Study findings were discussed under two broad themes of care 'structure' and 'process'. While infrastructure, supplies and human resource were available across most facilities, gaps were observed in the process of care, particularly during delivery and post-delivery stages. Key areas of concern included compromised patient safety like poor hand hygiene, usage of unsterilized instruments; inadequate clinical care like lack of routine monitoring of labour progression, inadequate postpartum care; partially compromised privacy in the labour room and postnatal ward; and few incidents of abuse and demand for informal payments. CONCLUSIONS:The study findings reflect gaps in the quality of maternity care across public health facilities in the study area and support the argument for strengthening PCC as an important effort towards quality improvement across the continuum of delivery care.

The cardiac conduction system (CCS) is composed of specialized cardiomyocytes that initiate and maintain cardiac rhythm. Any perturbation to the normal sequence of electrical events within the heart can result in cardiac arrhythmias. To understand how cardiac rhythm is established at the molecular level, several genetically modified mouse lines expressing Cre recombinase within specific CCS compartments have been created. In general, Cre driver lines have been generated either by homologous recombination of Cre into an endogenous locus or Cre expression driven by a randomly inserted transgene. However, haploinsufficiency of the endogenous gene compromises the former approach, while position effects negatively impact the latter. To address these limitations, we generated a Cre driver line for the ventricular conduction system (VCS) that preserves endogenous gene expression by targeting the Contactin2 (Cntn2) 3' untranslated region (3'UTR). Here we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice recombine floxed alleles within the VCS and that Cre expression faithfully recapitulates the spatial distribution of Cntn2 within the heart. We further demonstrate that Cre expression initiates after birth with preservation of native Cntn2 protein. Finally, we show that Cntn23'UTR-IRES-Cre-EGFP/+ mice maintain normal cardiac mechanical and electrical function. Taken together, our results establish a novel VCS-specific Cre driver line without the adverse consequences of haploinsufficiency or position effects. We expect that our new mouse line will add to the accumulating toolkit of CCS-specific mouse reagents and aid characterization of the cell-autonomous molecular circuitry that drives VCS maintenance and function.

In soybean, variegated flowers can be caused by somatic excision of the CACTA-type transposable element Tgm9 from Intron 2 of the DFR2 gene encoding dihydroflavonol-4-reductase of the anthocyanin pigment biosynthetic pathway. DFR2 was mapped to the W4 locus, where the allele containing Tgm9 was termed w4-m. In this study we have demonstrated that previously identified morphological mutants (three chlorophyll deficient mutants, one male sterile-female fertile mutant, and three partial female sterile mutants) were caused by insertion of Tgm9 following its excision from DFR2. Analyses of Tgm9 insertion sites among 105 independent mutants demonstrated that Tgm9 hops to all 20 soybean chromosomes from its original location on Chromosome 17. Some genomic regions are prone to increased Tgm9-insertions. Tgm9 transposed over 25% of the time into exon or intron sequences. Tgm9 is therefore suitable for generating an indexed insertional mutant collection for functional analyses of most soybean genes. Furthermore, desirable Tgm9-induced stable knockout mutants can be utilized in generating improved traits for commercial soybean cultivars.

Phytophthora sojae Kaufmann and Gerdemann, which causes Phytophthora root rot, is a widespread pathogen that limits soybean production worldwide. Development of Phytophthora resistant cultivars carrying Phytophthora resistance Rps genes is a cost-effective approach in controlling this disease. For this mapping study of a novel Rps gene, 290 recombinant inbred lines (RILs) (F7 families) were developed by crossing the P. sojae resistant cultivar PI399036 with the P. sojae susceptible AR2 line, and were phenotyped for responses to a mixture of three P. sojae isolates that overcome most of the known Rps genes. Of these 290 RILs, 130 were homozygous resistant, 12 heterzygous and segregating for Phytophthora resistance, and 148 were recessive homozygous and susceptible. From this population, 59 RILs homozygous for Phytophthora sojae resistance and 61 susceptible to a mixture of P. sojae isolates R17 and Val12-11 or P7074 that overcome resistance encoded by known Rps genes mapped to Chromosome 18 were selected for mapping novel Rps gene. A single gene accounted for the 1:1 segregation of resistance and susceptibility among the RILs. The gene encoding the Phytophthora resistance mapped to a 5.8 cM interval between the SSR markers BARCSOYSSR_18_1840 and Sat_064 located in the lower arm of Chromosome 18. The gene is mapped 2.2 cM proximal to the NBSRps4/6-like sequence that was reported to co-segregate with the Phytophthora resistance genes Rps4 and Rps6. The gene is mapped to a highly recombinogenic, gene-rich genomic region carrying several nucleotide binding site-leucine rich repeat (NBS-LRR)-like genes. We named this novel gene as Rps12, which is expected to be an invaluable resource in breeding soybeans for Phytophthora resistance.

Sudden death syndrome (SDS) is caused by the fungal pathogen, Fusarium virguliforme, and is a major threat to soybean production in North America. There are two major components of this disease: (i) root necrosis and (ii) foliar SDS. Root symptoms consist of root necrosis with vascular discoloration. Foliar SDS is characterized by interveinal chlorosis and leaf necrosis, and in severe cases by flower and pod abscission. A major toxin involved in initiating foliar SDS has been identified. Nothing is known about how root necrosis develops. In order to unravel the mechanisms used by the pathogen to cause root necrosis, the transcriptome of the pathogen in infected soybean root tissues of a susceptible cultivar, 'Essex', was investigated. The transcriptomes of the germinating conidia and mycelia were also examined. Of the 14,845 predicted F. virguliforme genes, we observed that 12,017 (81%) were expressed in germinating conidia and 12,208 (82%) in mycelia and 10,626 (72%) in infected soybean roots. Of the 10,626 genes induced in infected roots, 224 were transcribed only following infection. Expression of several infection-induced genes encoding enzymes with oxidation-reduction properties suggests that degradation of antimicrobial compounds such as the phytoalexin, glyceollin, could be important in early stages of the root tissue infection. Enzymes with hydrolytic and catalytic activities could play an important role in establishing the necrotrophic phase. The expression of a large number of genes encoding enzymes with catalytic and hydrolytic activities during the late infection stages suggests that cell wall degradation could be involved in root necrosis and the establishment of the necrotrophic phase in this pathogen.

A eukaryotic cell contains multiple membrane-bound compartments. Transport vesicles move cargo between these compartments, just as trucks move cargo between warehouses. These processes are regulated by specific molecular interactions, as summarized in the Rothman-Schekman-Sudhof model of vesicle traffic. The whole structure can be represented as a transport graph: each organelle is a node, and each vesicle route is a directed edge. What constraints must such a graph satisfy, if it is to represent a biologically realizable vesicle traffic network? Graph connectedness is an informative feature: 2-connectedness is necessary and sufficient for mass balance, but stronger conditions are required to ensure correct molecular specificity. Here we use Boolean satisfiability (SAT) and model checking as a framework to discover and verify graph constraints. The poor scalability of SAT model checkers often prevents their broad application. By exploiting the special structure of the problem, we scale our model checker to vesicle traffic systems with reasonably large numbers of molecules and compartments. This allows us to test a range of hypotheses about graph connectivity, which can later be proved in full generality by other methods.

The eukaryotic Meiotic Recombination protein 11 (Mre11) plays pivotal roles in the DNA damage response (DDR). Specifically, Mre11 senses and signals DNA double strand breaks (DSB) and facilitates their repair through effector proteins belonging to either homologous recombination (HR) or non-homologous end joining (NHEJ) repair mechanisms. In the human malaria parasite Plasmodium falciparum, HR and alternative-NHEJ have been identified; however, little is known about the upstream factors involved in the DDR of this organism. In this report, we identify a putative ortholog of Mre11 in P. falciparum (PfalMre11) that shares 22% sequence similarity to human Mre11. Homology modeling reveals striking structural resemblance of the predicted PfalMre11 nuclease domain to the nuclease domain of Saccharomyces cerevisiae Mre11 (ScMre11). Complementation analyses reveal functional conservation of PfalMre11 nuclease activity as demonstrated by the ability of the PfalMre11 nuclease domain, in conjunction with the C-terminal domain of ScMre11, to functionally complement an mre11 deficient yeast strain. Functional complementation was virtually abrogated by an amino acid substitution in the PfalMre11 nuclease domain (D398N). PfalMre11 is abundant in the mitotically active trophozoite and schizont stages of P. falciparum and is up-regulated in response to DNA damage, suggesting a role in the DDR. PfalMre11 exhibits physical interaction with PfalRad50. In addition, yeast 2-hybrid studies show that PfalMre11 interacts with ScRad50 and ScXrs2, two important components of the well characterized Mre11-Rad50-Xrs2 complex which is involved in DDR signaling and repair in S. cerevisiae, further supporting a role for PfalMre11 in the DDR. Taken together, these findings provide evidence that PfalMre11 is an evolutionarily conserved component of the DDR in Plasmodium.

Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events.

Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a "donor" ubiquitin and a primary amino group of an "acceptor" ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface.

Numerical results are presented for the effect of first order chemical reaction and thermal radiation on mixed convection flow of Casson fluid in the presence of magnetic field. The flow is generated due to unsteady nonlinearly stretching sheet placed inside a porous medium. Convective conditions on wall temperature and wall concentration are also employed in the investigation. The governing partial differential equations are converted to ordinary differential equations using suitable transformations and then solved numerically via Keller-box method. It is noticed that fluid velocity rises with increase in radiation parameter in the case of assisting flow and is opposite in the case of opposing fluid while radiation parameter has no effect on fluid velocity in the forced convection. It is also seen that fluid velocity and concentration enhances in the case of generative chemical reaction whereas both profiles reduces in the case of destructive chemical reaction. Further, increase in local unsteadiness parameter reduces fluid velocity, temperature and concentration. Over all the effects of physical parameters on fluid velocity, temperature and concentration distribution as well as on the wall shear stress, heat and mass transfer rates are discussed in detail.

A major rate-limiting step for Aβ generation and deposition in Alzheimer's disease brains is BACE1-mediated cleavage (β-cleavage) of the amyloid precursor protein (APP). We previously reported that APP undergoes palmitoylation at two cysteine residues (Cys186 and Cys187) in the E1-ectodomain. 8-10% of total APP is palmitoylated in vitro and in vivo. Palmitoylated APP (palAPP) shows greater preference for β-cleavage than total APP in detergent resistant lipid rafts. Protein palmitoylation is known to promote protein dimerization. Since dimerization of APP at its E1-ectodomain results in elevated BACE1-mediated cleavage of APP, we have now investigated whether palmitoylation of APP affects its dimerization and whether this leads to elevated β-cleavage of the protein. Here we report that over 90% of palAPP is dimerized while only ~20% of total APP forms dimers. PalAPP-dimers are predominantly cis-oriented while total APP dimerizes in both cis- and trans-orientation. PalAPP forms dimers 4.5-times more efficiently than total APP. Overexpression of the palmitoylating enzymes DHHC7 and DHHC21 that increase palAPP levels and Aβ release, also increased APP dimerization in cells. Conversely, inhibition of APP palmitoylation by pharmacological inhibitors reduced APP-dimerization in coimmunoprecipitation and FLIM/FRET assays. Finally, in vitro BACE1-activity assays demonstrate that palmitoylation-dependent dimerization of APP promotes β-cleavage of APP in lipid-rich detergent resistant cell membranes (DRMs), when compared to total APP. Most importantly, generation of sAPPβ-sAPPβ dimers is dependent on APP-palmitoylation while total sAPPβ generation is not. Since BACE1 shows preference for palAPP dimers over total APP, palAPP dimers may serve as novel targets for effective β-cleavage inhibitors of APP as opposed to BACE1 inhibitors.

CGG repeats in the 5'UTR of Fragile X Mental Retardation 1 (FMR1) RNA mediate RNA localization and translation in granules. Large expansions of CGG repeats (> 200 repeats) in FMR1, referred to as full mutations, are associated with fragile X syndrome (FXS). Smaller expansions (55-200 repeats), referred to as premutations, are associated with fragile X tremor ataxia syndrome (FXTAS) and fragile X premature ovarian insufficiency (FXPOI). TMPyP4 is a porphyrin ring compound that destabilizes CGG repeat RNA secondary structure. Here we show that exogenous CGG repeat RNA by itself, lacking the FMRP ORF, microinjected into hippocampal neurons is localized in RNA granules and inhibits translation of ARC RNA, which is localized in the same granules. TMPyP4 rescues translation of ARC RNA in granules. We also show that in human premutation fibroblasts with endogenous CGG repeat expansions in the FMR1 gene, translation of ARC RNA is inhibited and calcium homeostasis is disrupted and both phenotypes are rescued by TMPyP4. Inhibition of granule translation by expanded CGG repeats and rescue of granule translation by TMPy4, represent potential pathogenic mechanism and therapeutic strategy, respectively, for FXTAS and FXPOI.

The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies.

Supernumerary chromosome segments are known to harbor different transposons from their essential counterparts. The aim of this study was to investigate the role of transposons in the origin and evolution of supernumerary segments in the asexual fungal pathogen Fusarium virguliforme. We compared the genomes of 11 isolates comprising six Fusarium species that cause soybean sudden death syndrome (SDS) or bean root rot (BRR), and identified significant levels of genetic variation in A+T-rich repeat blocks of the essential chromosomes and in A+T-neutral regions of the supernumerary segments. The A+T-rich repeat blocks in the essential chromosomes were highly variable between F. virguliforme and non-F. virguliforme isolates, but were scarcely variable between F. virguliforme isolates. The A+T-neutral regions in the supernumerary segments, however, were highly variable between F. virguliforme isolates, with a statistically significant number (21 standard deviations above the mean) of single nucleotide polymorphisms (SNPs). And supernumerary sequence types and rearrangement patterns of some F. virguliforme isolates were present in an isolate of F. cuneirostrum but not in the other F. virguliforme isolates. The most variable and highly expressed region in the supernumerary segments contained an active DNA transposon that was a most conserved match between F. virguliforme and the unrelated fungus Tolypocladium inflatum. This transposon was absent from two of the F. virguliforme isolates. Furthermore, transposons in the supernumerary segments of some F. virguliforme isolates were present in non-F. virguliforme isolates, but were absent from the other F. virguliforme isolates. Two supernumerary P450 enzymes were 43% and 57% identical to their essential counterparts. This study has raised the possibility that transposons generate genetic variation in supernumerary chromosome segments by frequent horizontal transfer within and between closely related species.

Diabetes is a chronic disease commonly managed by family physicians, with the most prevalent complication being cardiovascular disease (CVD). Clinical practice guidelines have been developed to support clinicians in the care of diabetic patients. We conducted a pragmatic cluster randomized controlled trial (RCT) of a printed educational toolkit aimed at improving CVD management in diabetes in primary care, and found no effect, and indeed, the possibility of some harm. We conducted a qualitative evaluation to study the strategy for guideline implementation employed in this trial, and to understand its effects. This paper focuses solely on the qualitative findings, as the RCT's quantitative results have already been reported elsewhere.All family practices in the province of Ontario had been randomized to receive the educational toolkit by mail, in either the summer of 2009 (intervention arm) or the spring of 2010 (control arm).A subset of 80 family physicians (representing approximately 10% of the practices randomized and approached, with records on 1,592 randomly selected patients with diabetes at high risk for CVD) then took part in a chart audit and reflective feedback exercise related to their own practice in comparison to the guideline recommendations. They were asked to complete two forms (one pre- and one post-audit) in order to understand their awareness of the guidelines pre-trial, their expectations regarding their individual performance pre-audit, and their reflections on their audit results. In addition, individual interviews with thirteen other family physicians were conducted. Textual data from interview transcripts and written commentary from the pre- and post-audit forms underwent qualitative descriptive analysis to identify common themes and patterns. Analysis revealed four main themes: impressions of the toolkit, awareness was not the issue, 'it's not me it's my patients', and chart audit as a more effective intervention than the toolkit. Participants saw neither the toolkit content nor its dissemination strategy to be effective, indicating they perceived themselves to be aware of the guidelines pre-trial. However, their accounts also indicated that they may be struggling to prioritize CVD management in the midst of competing demands for their attention. Upon receiving their chart audit results, many participants expressed surprise that they had not performed better. They reported that the audit results would be an important motivator for behaviour change.The qualitative findings outlined in this paper offer important insights into why the intervention was not effective. They also demonstrate that physicians have unperceived needs relative to CVD management and that the chart audit served to identify shortcomings in their practice of which they had been hitherto unaware. The findings also indicate that new methods of intervention development and implementation should be explored. This is important given the high prevalence of diabetes worldwide; appropriate CVD management is critical to addressing the morbidity and mortality associated with the disease.

Fusarium virguliforme causes the serious disease sudden death syndrome (SDS) in soybean. Host resistance to this pathogen is partial and is encoded by a large number of quantitative trait loci, each conditioning small effects. Breeding SDS resistance is therefore challenging and identification of single-gene encoded novel resistance mechanisms is becoming a priority to fight this devastating this fungal pathogen. In this transcriptomic study we identified a few putative soybean defense genes, expression of which is suppressed during F. virguliforme infection. The F. virguliforme infection-suppressed genes were broadly classified into four major classes. The steady state transcript levels of many of these genes were suppressed to undetectable levels immediately following F. virguliforme infection. One of these classes contains two novel genes encoding ankyrin repeat-containing proteins. Expression of one of these genes, GmARP1, during F. virguliforme infection enhances SDS resistance among the transgenic soybean plants. Our data suggest that GmARP1 is a novel defense gene and the pathogen presumably suppress its expression to establish compatible interaction.

Mesenchymal stem cells (MSC) have been proposed as suitable candidates for cell therapy for neurological disorderssince they exhibit good neuronal differentiation capacity. However, for better therapeutic outcomes, it is necessary to isolate MSC from a suitable tissue sourcethat posses high neuronal differentiation. In this context, we isolated MSC from extra ocular muscle (EOM) tissue and tested the in vitro neuronal differentiation potential. In the current study, EOM tissue derived MSC were characterized and compared with bone marrow derived MSC. We found that EOM derived MSC proliferated as a monolayer and showed similarities in morphology, growth properties and cell surface marker expression with bone marrow derived MSC and expressed high levels of NES, OCT4, NANOG and SOX2 in its undifferentiated state. They also expressed embryonic cell surface marker SSEA4 and their intracellular mitochondrial distribution pattern was similar to that of multipotent stem cells. Although EOM derived MSC differentiated readily into adipocytes, osteocytes and chondrocytes, they differentiated more efficiently into neuroectodermal cells. The differentiation into neuroectodermal cellswas confirmed by the expression of neuronal markers NGFR and MAP2B. Thus, EOM derived MSC might be good candidates for stem cell based therapies for treating neurodegenerative diseases.

Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p

Currently, considerable interest exists with regard to the dissociation of close packed aminoacids within proteins, in the course of unfolding, which could result in either wet or dry moltenglobules. The progressive disjuncture of residues constituting the hydrophobic core ofcyclophilin from L. donovani (LdCyp) has been studied during the thermal unfolding of the molecule, by molecular dynamics simulations. LdCyp has been represented as a surface contactnetwork (SCN) based on the surface complementarity (Sm) of interacting residues within themolecular interior. The application of Sm to side chain packing within proteins make it a very sensitive indicator of subtle perturbations in packing, in the thermal unfolding of the protein. Network based metrics have been defined to track the sequential changes in the disintegration ofthe SCN spanning the hydrophobic core of LdCyp and these metrics prove to be highly sensitive compared to traditional metrics in indicating the increased conformational (and dynamical) flexibility in the network. These metrics have been applied to suggest criteria distinguishing DMG, WMG and transition state ensembles and to identify key residues involved in crucial conformational/topological events during the unfolding process.

Visceral leishmaniasis (VL), which is endemic as a major infectious disease in the tropical and subtropical countries, is caused by a protozoan parasite Leishmania donovani. At present, restricted treatment options and lack of vaccines intensify the problem of controlling VL. Therefore, finding a novel immunoprophylactic or therapeutic principle is a pressing need. Here, we report that arabinosylated lipoarabinomannan (Ara-LAM), a TLR2-ligand isolated from Mycobacterium smegmatis, exhibits a strong immunomodulatory property that conferred protection against L. donovani infection. Although, Ara-LAM modulates TLR2 and MAPK signaling, it is not known whether Ara-LAM involves IFN-γ signaling for effective parasite clearance. Because, it is reported that IFN-γ signaling, a principle mediator of NO generation and macrophage and Tcell activation, is hampered during leishmanial pathogenesis. Ara-LAM increases IFN-γ receptor expression and potentiates IFN-γ receptor signaling through JAK-STAT pathway. Moreover, Ara-LAM reciprocally modulates IRF4 and IRF8 expression and reinstates anti-leishmanial Th1 response that eventuates in significantly reduced parasite load in spleen and liver of L. donovani-infected BALB/c mice. IFN-γRα silencing resulted in the suppression of these host-protective mechanisms affected by Ara-LAM. Thus, Ara-LAM-mediated restoration of IFN-γ responsiveness is a novel immuno-modulatory principle for protection against L. donovani susceptible host.