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Confirmatory diagnosis of childhood tuberculosis (TB) remains a challenge mainly due to its dependence on sputum samples and the paucibacillary nature of the disease. Thus, only ~ 30% of suspected cases in children are diagnosed and the need for minimally invasive, non-sputum-based biomarkers remains unmet. Understanding host molecular changes by measuring blood-based transcriptomic markers has shown promise as a diagnostic tool for TB. However, the implication of sex contributing to disease heterogeneity and therefore diagnosis remains to be understood. Using publicly available gene expression data (GSE39939, GSE39940; n = 370), we report a sex-specific RNA biomarker signature that could improve the diagnosis of TB disease in children. We found four gene biomarker signatures for male (SLAMF8, GBP2, WARS, and FCGR1C) and female pediatric patients (GBP6, CELSR3, ALDH1A1, and GBP4) from Kenya, South Africa, and Malawi. Both signatures achieved a sensitivity of 85% and a specificity of 70%, which approaches the WHO-recommended target product profile for a triage test. Our gene signatures outperform most other gene signatures reported previously for childhood TB diagnosis.

Predictions of xenobiotic hepatic clearance in humans using in vitro-to-in vivo extrapolation methods are frequently inaccurate and problematic. Multiple strategies are being pursued to disentangle responsible mechanisms. The objective of this work is to evaluate the feasibility of using insights gained from independent virtual experiments on two model systems to begin unraveling responsible mechanisms. The virtual culture is a software analog of hepatocytes in vitro, and the virtual human maps to hepatocytes within a liver within an idealized model human. Mobile objects (virtual compounds) map to amounts of xenobiotics. Earlier versions of the two systems achieved quantitative validation targets for intrinsic clearance (virtual culture) and hepatic clearance (virtual human). The major difference between the two systems is the spatial organization of the virtual hepatocytes. For each pair of experiments (virtual culture, virtual human), hepatocytes are configured the same. Probabilistic rules govern virtual compound movements and interactions with other objects. We focus on highly permeable virtual compounds and fix their extracellular unbound fraction at one of seven values (0.05–1.0). Hepatocytes contain objects that can bind and remove compounds, analogous to metabolism. We require that, for a subset of compound properties, per-hepatocyte compound exposure and removal rates during culture experiments directly predict corresponding measures made during virtual human experiments. That requirement serves as a cross-system validation target; we identify compound properties that enable achieving it. We then change compound properties, ceteris paribus , and provide model mechanism-based explanations for when and why measures made during culture experiments under- (or over-) predict corresponding measures made during virtual human experiments. The results show that, from the perspective of compound removal, the organization of hepatocytes within virtual livers is more efficient than within cultures, and the greater the efficiency difference, the larger the underprediction. That relationship is noteworthy because most in vitro-to-in vivo extrapolation methods abstract away the structural organization of hepatocytes within a liver. More work is needed on multiple fronts, including the study of an expanded variety of virtual compound properties. Nevertheless, the results support the feasibility of the approach and plan.

BackgroundOnce-daily, orally administered, co-formulated glecaprevir (NS3/4A protease inhibitor) and pibrentasvir (NS5A inhibitor) (G/P) demonstrated pangenotypic activity and high sustained virologic response (SVR) rates in studies outside Japan. Here we report safety and efficacy in a subset of Japanese patients with chronic HCV infection who received G/P 300/120 mg in a phase 3, open-label, multicenter study (CERTAIN-1).MethodsThis analysis focuses on three difficult-to-treat subgroups: HCV GT1/2-infected patients who failed to achieve SVR after treatment with a direct acting antiviral (DAA)-containing regimen; GT1/2-infected patients with severe renal impairment (estimated glomerular filtration rate < 30 mL/min/1.73 m2); and GT3-infected patients. Patients in the renal impairment and GT3 cohorts were treatment-naive or interferon treatment-experienced. Noncirrhotic GT1/2-infected, DAA-naïve patients in the renal impairment cohort received G/P for 8 weeks; all other patients were treated for 12 weeks. Primary outcome was SVR (HCV RNA < 15 IU/mL) 12 weeks post-treatment (SVR12).ResultsThe study enrolled 33 GT1/2-infected patients who failed previous DAA treatment (four with cirrhosis); 12 GT1/2-infected patients with severe renal impairment (two with cirrhosis); and 12 GT3-infected patients (two with cirrhosis). SVR12 was achieved by 31/33 (93.9%), 12/12 (100%), and 10/12 (83.3%) patients, respectively. One serious adverse event (fluid overload, not related to G/P) occurred in a patient on chronic intermittent hemodialysis.ConclusionsG/P achieved high SVR12 rates and was well tolerated in three difficult-to-treat patient subgroups with limited treatment options in Japan (DAA-experienced patients, patients with severe renal impairment, and GT3-infected patients). These results support the potential suitability of this regimen for these special populations in Japan.

BackgroundThe once-daily, all oral, RBV-free, pangenotypic direct-acting anti-viral regimen consisting of co-formulated NS3/4A protease inhibitor glecaprevir and NS5A inhibitor pibrentasvir (G/P), demonstrated high rates of sustained virologic response (SVR) in phase 2 and 3 studies outside Japan.MethodsCERTAIN-1 is a phase 3, open-label, multicenter study assessing the safety and efficacy of G/P (300/120 mg) once daily in Japanese patients with chronic HCV GT1 infection. Patients without cirrhosis received 8 weeks of G/P or 12 weeks of ombitasvir/paritaprevir/ritonavir (OBV/PTV/r, 25/150/100 mg); patients with cirrhosis received G/P for 12 weeks. The primary efficacy endpoint was non-inferiority of G/P compared to OBV/PTV/r by assessing SVR at post-treatment week 12 (SVR12) among non-cirrhotic patients without the NS5A Y93H polymorphism.ResultsSVR12 was achieved by 128/129 (99.2%; one patient lost to follow-up) non-cirrhotic patients in the 8-week G/P Arm (including 23/23 patients with the NS5A Y93H polymorphism) and 52/52 (100%) patients in the 12-week OBV/PTV/r Arm. No patients from the G/P Arm prematurely discontinued the study drug or experienced a treatment-emergent serious adverse event (TESAE). Three patients from the OBV/PTV/r Arm experienced five TESAEs and one of these patients discontinued the study drug due to TESAEs. All 38 (100%) patients with compensated cirrhosis achieved SVR12; in this group, no TESAEs were reported and one patient discontinued treatment due to an AE.ConclusionsCERTAIN-1 study results demonstrate high efficacy and favorable tolerability of G/P in GT1-infected Japanese patients including those with the NS5A Y93H polymorphism, with no virologic failures observed.

Discoveries on nonprotein-coding RNAs have induced a paradigm shift in our overall understanding of gene expression and regulation. We now understand that coding and noncoding RNA machinery work in concert to maintain overall homeostasis. Based on their length, noncoding RNAs are broadly classified into two groups—long (>200 nt) and small noncoding RNAs (<200 nt). These RNAs perform diverse functions—gene regulation, splicing, translation, and posttranscriptional modifications. MicroRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) are two classes of small noncoding RNAs that are now classified as master regulators of gene expression. They have also demonstrated clinical significance as potential biomarkers and therapeutic targets for several diseases, including cancer. Despite these similarities, both these RNAs are generated through contrasting mechanisms, and one of the aims of this review is to cover the distance travelled since their discovery and compare and contrast the various facets of these RNAs. Although these RNAs show tremendous promise as biomarkers, translating the findings from bench to bedside is often met with roadblocks. The second aim of this review therefore is to highlight some of the challenges that hinder application of miRNA and piRNA as in guiding treatment decisions.

One of the most abundant, yet least explored, classes of RNA is the small nucleolar RNAs (snoRNAs), which are well known for their involvement in post-transcriptional modifications of other RNAs. Although snoRNAs were only considered to perform housekeeping functions for a long time, recent studies have highlighted their importance as regulators of gene expression and as diagnostic/prognostic markers. However, the prognostic potential of these RNAs has not been interrogated for breast cancer (BC). The objective of the current study was to identify snoRNAs as prognostic markers for BC. Small RNA sequencing (Illumina Genome Analyzer IIx) was performed for 104 BC cases and 11 normal breast tissues. Partek Genomics Suite was used for analyzing the sequencing files. Two independent and proven approaches were used to identify prognostic markers: case-control (CC) and case-only (CO). For both approaches, snoRNAs significant in the permutation test, following univariate Cox proportional hazards regression model were used for constructing risk scores. Risk scores were subsequently adjusted for potential confounders in a multivariate Cox model. For both approaches, thirteen snoRNAs were associated with overall survival and/or recurrence free survival. Patients belonging to the high-risk group were associated with poor outcomes, and the risk score was significant after adjusting for confounders. Validation of representative snoRNAs (SNORD46 and SNORD89) using qRT-PCR confirmed the observations from sequencing experiments. We also observed 64 snoRNAs harboring piwi-interacting RNAs and/or microRNAs that were predicted to target genes (mRNAs) involved in tumorigenesis. Our results demonstrate the potential of snoRNAs to serve (i) as novel prognostic markers for BC and (ii) as indirect regulators of gene expression.

Aims/hypothesis Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca 2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca 2+ signalling with defective insulin maturation remain incompletely understood. Methods We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca 2+ ATPase-2 (SERCA2) deletion (βS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca 2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. Results βS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca 2+ levels and glucose-stimulated Ca 2+ synchronicity were reduced in βS2KO islets. Islets isolated from βS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of βS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in βS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. Conclusions/interpretation Taken together, these data highlight an important link between ER Ca 2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca 2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. Data availability RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498). Graphical Abstract

Background Renewable energy for sustainable development is a subject of a worldwide debate since continuous utilization of non-renewable energy sources has a drastic impact on the environment and economy; a search for alternative energy resources is indispensable. Microalgae are promising and potential alternate energy resources for biodiesel production. Thus, our efforts were focused on surveying the natural diversity of microalgae for the production of biodiesel. The present study aimed at identification, isolation, and characterization of oleaginous microalgae from shola forests of Nilgiri Biosphere Reserve (NBR), the biodiversity hot spot of India, where the microalgal diversity has not yet been systematically investigated. Results Overall the higher biomass yield, higher lipid accumulation and thermotolerance observed in the isolated microalgal strains have been found to be the desirable traits for the efficient biodiesel production. Species composition and diversity analysis yielded ten potential microalgal isolates belonging to Chlorophyceae and Cyanophyceae classes. The chlorophytes exhibited higher growth rate, maximum biomass yield, and higher lipid accumulation than Cyanophyceae. Among the chlorophytes, the best performing strains were identified and represented by Acutodesmus dissociatus (TGA1), Chlorella sp. (TGA2), Chlamydomonadales sp. (TGA3) and Hindakia tetrachotoma (PGA1). The Chlamydomonadales sp. recorded with the highest growth rate, lipid accumulation and biomass yield of 0.28 ± 0.03 day −1 (μ exp ), 29.7 ± 0.69% and 134.17 ± 16.87 mg L −1  day −1 , respectively. It was also found to grow well at various temperatures, viz. , 25 °C, 35 °C, and 45 °C, indicating its suitability for open pond cultivation. The fatty acid methyl ester (FAME) analysis of stationary phase cultures of selected four algal strains by tandem mass spectrograph showed C16:0, C18:1 and C18:3 as dominant fatty acids suitable for biodiesel production. All the three strains except for Hindakia tetrachotoma (PGA1) recorded higher carbohydrate content and were considered as potential feed stocks for biodiesel production through hydrothermal liquefaction technology (HTL). Conclusions In conclusion, the present investigation is a first systematic study on the microalgal diversity of soil and water samples from selected sites of NBR. The study resulted in isolation and characterization of ten potent oleaginous microalgae and found four cultures as promising feed stocks for biodiesel production. Of the four microalgae, Chlamydomonadales sp. (TGA3) was found to be significantly thermo-tolerant and can be considered as promising feedstock for biodiesel production.

Background Limited understanding of the diversity of variants in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene across ancestries hampers efforts to advance molecular diagnosis of cystic fibrosis (CF). The consequences pose a risk of delayed diagnoses and subsequently worsened health outcomes for patients. Therefore, characterizing the spectrum of CFTR variants across ancestries is critical for revolutionizing molecular diagnoses of CF. Methods We analyzed 454,727 UK Biobank (UKBB) whole-exome sequences to characterize the diversity of CFTR variants across ancestries. Using the PanUKBB classification, the participants were assigned into six major groups: African (AFR), American/American Admixed (AMR), Central South Asia (CSA), East Asian (EAS), European (EUR), and Middle East (MID). We segregated ancestry-specific CFTR variants, including those that are CF-causing or clinically relevant. The ages of certain CF-causing variants were determined and analyzed for selective pressure effects, and curated phenotype analysis was performed for participants with clinically relevant CFTR genotypes. Results We detected over 4000 CFTR variants, including novel ancestry-specific variants, across six ancestries. Europeans had the most unique CFTR variants [ n  = 2212], while the American group had the least unique variants [ n  = 23]. F508del was the most prevalent CF-causing variant found in all ancestries, except in EAS, where V520F was the most prevalent. Common EAS variants such as 3600G > A, V456A, and V520, which appeared approximately 270, 215, and 338 generations ago, respectively, did not show evidence of selective pressure. Sixteen participants had two CF-causing variants, with two being diagnosed with CF. We found 154 participants harboring a CF-causing and varying clinical consequences (VCC) variant. Phenotype analysis performed for participants with multiple clinically relevant variants returned significant associations with CF and its pulmonary phenotypes [Bonferroni-adjusted p  < 0.05]. Conclusions We leveraged the UKBB database to comprehensively characterize the broad spectrum of CFTR variants across ancestries. The detection of over 4000 CFTR variants, including several ancestry-specific and uncharacterized CFTR variants, warrants the need for further characterization of their functional and clinical relevance. Overall, the presentation of classical CF phenotypes seen in non-CF diagnosed participants with more than one CF-causing variant indicates that they may benefit from current CFTR modulator therapies.

Interpretations of elevated blood levels of alanine aminotransferase (ALT) for drug-induced liver injury often assume that the biomarker is released passively from dying cells. However, the mechanisms driving that release have not been explored experimentally. The usefulness of ALT and related biomarkers will improve by developing mechanism-based explanations of elevated levels that can be expanded and elaborated incrementally. We provide the means to challenge the ability of closely related model mechanisms to generate patterns of simulated hepatic injury and ALT release that scale (or not) to be quantitatively similar to the wet-lab validation targets, which are elevated plasma ALT values following acetaminophen (APAP) exposure in mice. We build on a published model mechanism that helps explain the generation of characteristic spatiotemporal features of APAP hepatotoxicity within hepatic lobules. Discrete event and agent-oriented software methods are most prominent. We instantiate and leverage a small constellation of concrete model mechanisms. Their details during execution help bring into focus ways in which particular sources of uncertainty become entangled with cause-effect details within and across several levels. We scale ALT amounts in virtual mice directly to target plasma ALT values in individual mice. A virtual experiment comprises a set of Monte Carlo simulations. We challenge the sufficiency of four potentially explanatory theories for ALT release. The first of the tested model theories failed to achieve the initial validation target, but each of the three others succeeded. Results for one of the three model mechanisms matched all target ALT values quantitatively. It explains how ALT externalization is the combined consequence of lobular-location-dependent drug-induced cellular damage and hepatocyte death. Falsification of one (or more) of the model mechanisms provides new knowledge and incrementally shrinks the constellation of model mechanisms. The modularity and biomimicry of our explanatory models enable seamless transition from mice to humans.