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Background: Challenges exist in the diagnosis and management of autoimmune rheumatic diseases in low and middle income countries due to factors, such as poverty and under-resourced healthcare infrastructure. Furthermore, other contributory factors such as societal, cultural and religious practices influence health seeking behaviour which has a bearing on access and delivery of healthcare., Objectives: To examine the health seeking behaviour and referral patterns of Ghanaian patients with autoimmune rheumatic diseases and assess the associated factors that influence these., Method: A cross-sectional study using an explanatory sequential mixed method design was carried out in a Rheumatology clinic at a national referral centre. 110 participants were purposively recruited for the quantitative phase. The qualitative phase comprised 10 participants for in-depth interviews and 10 participants for a focus group discussion. Analysis using descriptive statistics, t-tests and logistic regression models were performed. Transcripts generated from the interviews and focused group discussion were analysed using thematic analysis., Results: Median duration from onset of symptoms until seeking help was 1 week (IQR = 12); from seeking help until obtaining a final diagnosis was 12 months (IQR = 33). Multiple factors determined the choice of first facility visited, X2 (12, N = 107) = 32.29, p = .001. Only twenty-one participants (19.6%) had knowledge of their disease prior to diagnosis. Education predicted prior knowledge [OR = 2.6 (95% CI = .66-10.12), p < .021]. Unemployed participants had increased odds of seeking help after a month compared to those who were employed [Odds ratio = 2.60 (95% CI = 1.14-5.90), p = .02]. Knowledge of autoimmune rheumatic diseases was low with multiple causative factors such as biomedical, environmental and spiritual causes determining where patients accessed care. Forty (36.4%) participants utilised complementary and alternative treatment options., Conclusion: We observed that knowledge about autoimmune rheumatic diseases among Ghanaian patients was low. Patients sought help from numerous medical facilities, traditional healers and prayer camps often contributing to a delay in diagnosis for most patients. This was influenced by individual perceptions, cultural beliefs and socioeconomic status. Active awareness and educational programmes for the public and healthcare workers are required, as well as strategic planning to integrate the biomedical and traditional care services to enable earlier presentation, accurate diagnosis and better clinical outcomes for the patients., Competing Interests: The authors have declared that no competing interests exist.

Malnutrition continues to be a primary concern for researchers and policymakers in India. There is limited scientific research on the effect of agriculture on child nutrition in the country using a large representative sample. To the best of our knowledge, no study has examined the spatial clustering of child malnutrition and its linkage with agricultural production at the district-level in the country. The present study aims to examine agricultural production's role in improving the nutritional status of Indian children through child feeding practices. The nutritional indicators of children from the National Family Health Survey-4 (2015-16) and the agricultural production data for all the 640 districts of India obtained from the District-Wise Crop Production Statistics (2015-16), published by the Ministry of Agriculture, Government of India were used for the analysis. The statistical analysis was undertaken in STATA (version 14.1). ArcMap (version 10.3), and GeoDa (version 1.8) were used for the spatial analysis. The study found a higher prevalence of malnutrition among children who had not received Minimum Meal Frequency (MMF), Minimum Dietary Diversity (MDD), and Minimum Acceptable Diet (MAD). Further, child feeding practices- MMF, MDD, and MAD- were positively associated with high yield rates of spices and cereals. The yield rate of cash crops, on the contrary, harmed child feeding practices. Production of pulses had a significant positive effect on MDD and MAD. Districts with high cereal yield rates ensured that children receive MMF and MAD. There is a significant spatial association between child feeding practices and malnutrition across Indian districts. The study suggests that adopting nutrient-sensitive agriculture may be the best approach to improving children's nutritional status., Competing Interests: The authors have declared that no competing interests exist.

The tumor immune microenvironment is emerging as a critical player in predicting cancer prognosis and response to therapies. However, the prognostic value of tumor-infiltrating immune cells in Gingivo-Buccal Oral Squamous Cell Carcinoma (GBOSCC) and their association with tumor size or lymph node metastases status require further elucidation. To study the relationship of tumor-infiltrating immune cells with tumor size (T stage) and lymph node metastases (N stages), we analyzed the density of tumor-infiltrating immune cells in archived, whole tumor resections from 94 patients. We characterized these sections by immune-histochemistry using 12 markers and enumerated tumor-infiltrating immune cells at the invasive margins (IM) and centers of tumors (CT). We observed that a higher density of CD3+ cells in the IM and CT was associated with smaller tumor size (T1-T2 stage). Fewer CD3+ cells was associated with larger tumor size (T3-T4 stage). High infiltration of CD3+and CD8+ cells in IM and CT as well as high CD4+ cell infiltrates in the IM was significantly associated with the absence of lymph node metastases. High infiltrates of CD3+ and CD8+ cells in CT was associated with significantly improved survival. Our results illustrate that the densities and spatial distribution of CD3+ and CD8+ cell infiltrates in primary GBOSCC tumors is predictive of disease progression and survival. Based on our findings, we recommend incorporating immune cell quantification in the TNM classification and routine histopathology reporting of GBOSCC. Immune cell quantification in CT and IM may help predict the efficacy of future therapies., Competing Interests: NO authors have competing interests.

The effects of climate on wildland fire confronts society across a range of different ecosystems. Water and temperature affect the combustion dynamics, irrespective of whether those are associated with carbon fueled motors or ecosystems, but through different chemical, physical, and biological processes. We use an ecosystem combustion equation developed with the physical chemistry of atmospheric variables to estimate and simulate fire probability and mean fire interval (MFI). The calibration of ecosystem fire probability with basic combustion chemistry and physics offers a quantitative method to address wildland fire in addition to the well-studied forcing factors such as topography, ignition, and vegetation. We develop a graphic analysis tool for estimating climate forced fire probability with temperature and precipitation based on an empirical assessment of combustion theory and fire prediction in ecosystems. Climate-affected fire probability for any period, past or future, is estimated with given temperature and precipitation. A graphic analyses of wildland fire dynamics driven by climate supports a dialectic in hydrologic processes that affect ecosystem combustion: 1) the water needed by plants to produce carbon bonds (fuel) and 2) the inhibition of successful reactant collisions by water molecules (humidity and fuel moisture). These two postulates enable a classification scheme for ecosystems into three or more climate categories using their position relative to change points defined by precipitation in combustion dynamics equations. Three classifications of combustion dynamics in ecosystems fire probability include: 1) precipitation insensitive, 2) precipitation unstable, and 3) precipitation sensitive. All three classifications interact in different ways with variable levels of temperature.

Motivation: Proper management of fecal sludge has significant positive health and environmental externalities. Most research on managing onsite sanitation so far either simulates the costs of, or the welfare effects from, managing sludge in situ in pit latrines. Thus, designing management strategies for onsite rural sanitation is challenging, because the actual costs of transporting sludge for treatment, and sources for financing these transport costs, are not well understood., Methods: In this paper we calculate the actual cost of sludge management from onsite latrines, and identify the contributions that latrine owners are willing to make to finance the costs. A spreadsheet-based model is used to identify a cost-effective transport option, and to calculate the cost per household. Then a double-bound contingent valuation method is used to elicit from pit-latrine owners their willingness-to-pay to have sludge transported away. This methodology is employed for the case of a rural subdistrict in Bangladesh called Bhaluka, a unit of administration at which sludge management services are being piloted by the Government of Bangladesh., Results: The typical sludge accumulation rate in Bhaluka is calculated at 0.11 liters/person/day and a typical latrine will need to be emptied approximately once every 3 to 4 years. The costs of emptying and transport are high; approximately USD 13 per emptying event (circa 14% of average monthly income); household contributions could cover around 47% of this cost. However, if costs were spread over time, the service would cost USD 4 per year per household, or USD 0.31 per month per household-comparable to current expenditures of rural households on telecommunications., Conclusion: This is one of few research papers that brings the costs of waste management together with financing of that cost, to provide evidence for an implementable solution. This framework can be used to identify cost effective sludge management options and private contributions towards that cost in other (context-specific) administrative areas where onsite sanitation is widespread.

Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order) and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP) holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon), within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons are expressed independent of rRNA synthesis under specific conditions where further synthesis of ribosomes is not needed., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) suffer from significantly more cardiovascular comorbidity and mortality than would be anticipated from conventional risk factors. The aim of this study was to determine whether COPD patients have a higher coronary artery calcium score (CACS) and epicardial fat burden, compared to control subjects, and their association with cardiovascular events., Methods: From a registry of 1906 patients 81 patients with clinically diagnosed COPD were one-to-one matched to 81 non-COPD control subjects with a smoking history, according to their age, sex, and the number of classic cardiovascular risk factors (arterial hypertension, diabetes mellitus, dyslipidemia, family history of premature coronary artery disease). CACS, epicardial fat, and subsequent major adverse cardiovascular events (MACE) during follow-up were compared between groups., Results: Patients with COPD (Global Initiative for Chronic Obstructive Lung Disease-classification I: 5%, II: 23%, III: 16% and IV: 56%) showed no difference in CACS (median difference 68 Agatston Units [95% confidence interval -176.5 to 192.5], p=0.899) or epicardial fat volume (mean difference -0.5 cm3 [95% confidence interval -20.9 to 21.9], p=0.961) compared with controls. After a median follow-up of 42.6 months a higher incidence of MACE was observed in COPD patients (RR=2.80, p=0.016) compared with controls. Cox proportional hazard regression identified cardiac ischemias and CACS as independent predictors for MACE., Conclusion: COPD patients experienced a higher MACE incidence compared to controls despite no baseline differences in coronary calcification and epicardial fat burden. Other mechanisms such as undersupply of medication seem to account for an excess cardiovascular comorbidity in COPD patients.

Background: The clinical uses of 2-chloro-2'-deoxyadenosine (2-CDA) or cladribine which was initially prescribed to patients with hematological and lymphoid cancers is now extended to treat patients with multiple sclerosis (MS). Previous data has shown that 2-CDA has high affinity to the brain and readily passes through the blood brain barrier reaching CSF concentrations 25% of that found in plasma. However, whether long-term administration of 2-CDA can lead to any adverse effects in patients or animal models is not yet clearly known., Methodology: Here we show that exposure of 2-CDA to CHO cells stably expressing wild-type APP751 increased generation and secretion of amyloid β peptide (Aβ) in to the conditioned medium. Interestingly, increased Aβ levels were noticed even at non-toxic concentrations of 2-CDA. Remarkably, chronic treatment of APdE9 mice, a model of Alzheimer's disease with 2-CDA for 60 days increased amyloid plaque burden by more than 1-fold. Increased Aβ generation appears to result from increased turnover of APP as revealed by cycloheximide-chase experiments. Additionally, surface labeling of APP with biotin and immunoprecipitation of surface labeled proteins with anti-biotin antibody also indicated increased APP at the cell surface in 2-CDA treated cells compared to controls. Increased turnover of APP by 2-CDA in turn might be a consequence of decreased protein levels of PIN 1, which is known to regulate cis-trans isomerization and phosphorylation of APP. Most importantly, like many other oncology drugs, 2-CDA administration led to significant delay in acquiring a reward-based learning task in a T maze paradigm., Conclusions: Taken together, these data provide compelling evidence for the first time that chronic 2-CDA administration can increase amyloidogenic processing of APP leading to robustly increased plaque burden which may be responsible for the observed deficits in learning skills. Thus chronic treatment of mice with 2-CDA can have deleterious effects in vivo.

Background: The Inositol 1,4,5-trisphosphate receptor (InsP(3)R) is an InsP(3) gated intracellular Ca(2+)-release channel. Characterization of Drosophila mutants for the InsP(3)R has demonstrated that InsP(3)-mediated Ca(2+) release is required in Drosophila larvae for growth and viability., Methodology/principal Findings: To understand the molecular basis of these growth defects a genome wide microarray analysis has been carried out with larval RNA obtained from a strong InsP(3)R mutant combination in which 1504 independent genes were differentially regulated with a log(2) of fold change of 1 or more and P<0.05. This was followed by similar transcript analyses from InsP(3)R mutants where growth defects were either suppressed by introduction of a dominant suppressor or rescued by ectopic expression of an InsP(3)R transgene in the Drosophila insulin like peptide-2 (Dilp2) producing cells., Conclusions/significance: These studies show that expression of transcripts related to carbohydrate and amine metabolism is altered in InsP(3) receptor mutant larvae. Moreover, from a comparative analysis of genes that are regulated in the suppressed and rescued conditions with the mutant condition, it appears that the organism could use different combinations of pathways to restore a 'normal' growth state.

Background: Methane (CH(4)) is a potent greenhouse gas (GHG), having a global warming potential 21 times that of carbon dioxide (CO(2)). Methane emissions from agriculture represent around 40% of the emissions produced by human-related activities, the single largest source being enteric fermentation, mainly in ruminant livestock. Technologies to reduce these emissions are lacking. Ruminant methane is formed by the action of methanogenic archaea typified by Methanobrevibacter ruminantium, which is present in ruminants fed a wide variety of diets worldwide. To gain more insight into the lifestyle of a rumen methanogen, and to identify genes and proteins that can be targeted to reduce methane production, we have sequenced the 2.93 Mb genome of M. ruminantium M1, the first rumen methanogen genome to be completed., Methodology/principal Findings: The M1 genome was sequenced, annotated and subjected to comparative genomic and metabolic pathway analyses. Conserved and methanogen-specific gene sets suitable as targets for vaccine development or chemogenomic-based inhibition of rumen methanogens were identified. The feasibility of using a synthetic peptide-directed vaccinology approach to target epitopes of methanogen surface proteins was demonstrated. A prophage genome was described and its lytic enzyme, endoisopeptidase PeiR, was shown to lyse M1 cells in pure culture. A predicted stimulation of M1 growth by alcohols was demonstrated and microarray analyses indicated up-regulation of methanogenesis genes during co-culture with a hydrogen (H(2)) producing rumen bacterium. We also report the discovery of non-ribosomal peptide synthetases in M. ruminantium M1, the first reported in archaeal species., Conclusions/significance: The M1 genome sequence provides new insights into the lifestyle and cellular processes of this important rumen methanogen. It also defines vaccine and chemogenomic targets for broad inhibition of rumen methanogens and represents a significant contribution to worldwide efforts to mitigate ruminant methane emissions and reduce production of anthropogenic greenhouse gases.

Background: Cancer stem cells exhibit close resemblance to normal stem cells in phenotype as well as function. Hence, studying normal stem cell behavior is important in understanding cancer pathogenesis. It has recently been shown that human breast stem cells can be enriched in suspension cultures as mammospheres. However, little is known about the behavior of these cells in long-term cultures. Since extensive self-renewal potential is the hallmark of stem cells, we undertook a detailed phenotypic and functional characterization of human mammospheres over long-term passages., Methodology: Single cell suspensions derived from human breast 'organoids' were seeded in ultra low attachment plates in serum free media. Resulting primary mammospheres after a week (termed T1 mammospheres) were subjected to passaging every 7th day leading to the generation of T2, T3, and T4 mammospheres., Principal Findings: We show that primary mammospheres contain a distinct side-population (SP) that displays a CD24(low)/CD44(low) phenotype, but fails to generate mammospheres. Instead, the mammosphere-initiating potential rests within the CD44(high)/CD24(low) cells, in keeping with the phenotype of breast cancer-initiating cells. In serial sphere formation assays we find that even though primary (T1) mammospheres show telomerase activity and fourth passage T4 spheres contain label-retaining cells, they fail to initiate new mammospheres beyond T5. With increasing passages, mammospheres showed an increase in smaller sized spheres, reduction in proliferation potential and sphere forming efficiency, and increased differentiation towards the myoepithelial lineage. Significantly, staining for senescence-associated beta-galactosidase activity revealed a dramatic increase in the number of senescent cells with passage, which might in part explain the inability to continuously generate mammospheres in culture., Conclusions: Thus, the self-renewal potential of human breast stem cells is exhausted within five in vitro passages of mammospheres, suggesting the need for further improvisation in culture conditions for their long-term maintenance.