Biology subjects

Tyagi, S.

Publications and source records attributed to Tyagi, S..

45 records · Page 3Linked to original sources

bioRxiv2022

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNF, IL-6 and IL-1{beta}) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response. Author SummaryOver-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169+ macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.

immunology↗

bioRxiv2022

GSK2556286 is a novel antitubercular drug candidate effective in vivo with the potential to shorten tuberculosis treatment

As a result of a high-throughput compound screening campaign of Mycobacterium tuberculosis infected macrophages, a new preclinical drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (IC50 = 0.07 {micro}M), is active against extracellular bacteria in cholesterol-containing culture media and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug or extensively-resistant (M/XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. One Sentence SummaryGSK2556286 is a novel preclinical drug candidate for the treatment of tuberculosis with a new mode of action potentially able to contribute to the shortening of TB chemotherapy.

pharmacology and toxicology↗

bioRxiv2022

Glutamine is required for M1-like polarization in response to Mycobacterium tuberculosis infection

In response to Mycobacterium tuberculosis infection, macrophages mount early proinflammatory and antimicrobial responses similar to those observed in M1 macrophages classically activated by LPS and IFN-{gamma}. A metabolic reprogramming to HIF-1-mediated uptake of glucose and its metabolism by glycolysis is required for M1-like polarization, but little is known about other metabolic programs driving M1-like polarization during M. tuberculosis infection. Identification and quantification of labeling patterns of U13C glutamine and U13C glucose-derived metabolites demonstrated that glutamine, rather than glucose, is catabolized in both the oxidative and reductive TCA cycle of M1-like macrophages, thereby generating signaling molecules that include succinate, biosynthetic precursors such as aspartate, and the antimicrobial metabolite itaconate. This conclusion is corroborated by diminished M1 polarization via chemical inhibition of glutaminase (GLS), the key enzyme of the glutaminolysis pathway, and by genetic deletion of GLS in infected macrophages. Furthermore, characterization of the labeling distribution pattern of U15N glutamine in M1-like macrophages indicates that glutamine serves as a nitrogen source for the synthesis of intermediates of purine and pyrimidine metabolism plus amino acids including aspartate. Thus, the catabolism of glutamine, as an integral component of metabolic reprogramming in activating macrophages, fulfills the cellular demand for bioenergetic and biosynthetic precursors of M1-like macrophages. Knowledge of these new immunometabolic features of M1-like macrophages is expected to advance the development of host-directed therapies that will enhance bacterial clearance and prevent immunopathology during tuberculosis. SummaryRecent advances in immunometabolism have stimulated increasing interest in understanding the specific cellular metabolic states of immune cells associated with the various disease states of tuberculosis. As the primary target of Mycobacterium tuberculosis (Mtb) infection, macrophages play essential roles in dictating the progression and final outcome of infection. Previous studies, including our own, show that the upregulation of hypoxia-inducible-factor 1 alpha (HIF-1) and a metabolic reprogramming to the Warburg effect-like state are general features of the host immune cell response to Mtb infection. They are also critical for macrophage polarization to the M1-like phenotype characterized by high-level expression of proinflammatory and antimicrobial molecules against Mtb infection. However, our knowledge about the immunometabolic features of M1-like macrophages is poor. Using widely targeted small metabolite (WTSM) screening (600+ small polar metabolites) and stable isotope tracing of U13 glutamine, U13 glucose, and N15 glutamine, as well as therapeutic and genetic approaches, we report that, in addition to elevated glucose catabolism by glycolysis, glutamine serves as an important carbon and nitrogen source for the generation of building blocks, signaling molecules, and antimicrobial metabolite during macrophage polarization to the M1-like phenotype. The study adds novel insights into the immunometabolic properties of Mtb-infected macrophages.

immunology↗

bioRxiv2021

The Biosynthetic Costs of Amino Acids at the Base of the Food Chain Determine Their Use in Higher-order Consumer Genomes

Dietary nutrient composition is essential for shaping important fitness traits and behaviours. Many organisms are protein limited and for Drosophila melanogaster, this limitation manifests at the level of the single most limiting essential Amino Acid (AA) in the diet. The identity of this AA and its effects on female fecundity is readily predictable by a procedure called exome matching in which the sum of AAs encoded by a consumers exome is used to predict the relative proportion of AAs required in its diet. However, the exome matching calculation does not weight AA contributions to the overall profile by protein size or expression. Here we update the exome matching calculation to include these weightings. Surprisingly, although nearly half of the transcriptome is differentially expressed when comparing male and female flies, we found that creating transcriptome-weighted exome matched diets for each sex did not enhance their fecundity over that supported by exome matching alone. These data indicate that while organisms may require different amounts of dietary protein across conditions, the relative proportion of the constituent AAs remains constant. Interestingly, we also found remarkable conservation of exome matched AA profiles across taxa and that the composition of these profiles could be explained by the metabolic costs of microbial AA synthesis. Thus, it appears that bioenergetic constraints amongst autotrophs shape the relative proportion of AAs that are available across trophic levels and that that this constrains biomass composition.

genomics↗

bioRxiv2021

The heterogeneity of the DNA damage response landscape determines patient outcomes in ovarian cancer

Defective DNA damage response (DDR) pathways allow cancer cells to accrue genomic aberrations and evade normal cellular growth checkpoints. Defective DDR also determines response to chemotherapy. However, the interaction and overlap between the two double strand repair pathways and the three single strand repair pathways is complex, and has remained poorly understood. Here we show that, in ovarian cancer, a disease hallmarked by chromosomal instability, explant cultures show a range of DDR abrogation patterns. Defective homologous recombination (HR) and non-homologous end joining (NHEJ) are near mutually exclusive with HR deficient (HRD) cells showing increased abrogation of the single strand repair pathways compared to NHEJ defective cells. When combined with global markers of DNA damage, including mitochondrial membrane functionality and reactive oxygen species burden, the pattern of DDR abrogation allows the construction of DDR signatures which are predictive of both ex vivo cytotoxicity, and more importantly, patient outcome. SignificanceHolistic assessment of the DDR is possible, shows improved ability to predict response to chemotherapy over single pathway assessment, and is applicable to a variety of ovarian cancer types. Such an assessment has clinical utility in settings of therapeutic dilemma such as retreatment for relapse.

cell biology↗

bioRxiv2021

Hypoxia induces early neurogenesis in human fetal neural stem cells by activating the WNT pathway.

Fetal neural stem cells (FNSCs) are present in the brain of human fetuses that differentiate into cells of neuronal and glial lineages. Difference in oxygen concentration between maternal and fetal circulation indicates that the developing fetus may be exposed to lower oxygen concentrations compared to adults. This physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the sub-ventricular zone of aborted fetal brains (n=5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression, using immunocytochemistry and flowcytometry respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 hours, and hypoxia exposure (n=5) was evaluated by a panel of markers (CA9, PGK1 and VEGF). Whole human genome transcriptomic analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4x44K human array slides), highlighted that genes associated with neurogenesis were getting enriched. The pathway analysis of these enriched genes (using Metacore) showed that WNT signaling played a role in determining the cell fate of FNSCs exposed to hypoxic environment. Microarray analyses was validated using neuronal and glial lineage commitment markers such as Ngn1, Ngn2, ASCL1, DCX, GFAP, Olig2 and Nkx2.2 using qPCR (n=9). This demonstrated upregulation of the neuronal commitment markers on hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n=9), indicated its involvement in mediating neuronal differentiation on exposure to hypoxia.

neuroscience↗

bioRxiv2021

linc2function: A deep learning model to identify and assign function to long noncoding RNA (lncRNA)

MotivationLncRNAs are much more versatile and are involved in many regulatory roles inside the cell than previously believed. Existing databases lack consistencies in lncRNA annotations, and the functionality of over 95% of the known lncRNAs are yet to be established. LncRNA transcript identification involves discriminating them from their coding counterparts, which can be done with traditional experimental approaches, or via in silico methods. The later approach employs various computational algorithms, including machine learning classifiers to predict the lncRNA forming potential of a given transcript. Such approaches provide an economical and faster alternative to the experimental methods. Current in silico methods mainly use primary-sequence based features to build predictive models limiting their accuracy and robustness. Moreover, many of these tools make use of reference genome based features, in consequence making them unsuitable for non-model species. Hence, there is a need to comprehensively evaluate the efficacy of different predictive features to build computational models. Additionally, effective models will have to provide maximum prediction performance using the least number of features in a species-agnostic manner. It is popularly known in the protein world that "structure is function". This also applies to lncRNAs as their functional mechanisms are similar to those of proteins. Generally, lncRNA function by structurally binding to its target proteins or nucleic acid forming complexes. The secondary structures of the lncRNAs are modular providing interaction sites for their interactome made of DNA, RNA, and proteins. Through these interactions, they epigenetically regulate cellular biology, thereby forming a layer of genomic programming on top of the coding genes. We demonstrate that in addition to using transcript sequence, we can provide comprehensive functional annotation by collating their interactome and secondary structure information. ResultsHere, we evaluated an exhaustive list of sequence-based, secondary-structure, interactome, and physicochemical features for their ability to predict the lncRNA potential of a transcript. Based on our analysis, we built different machine learning models using optimum feature-set. We found our model to be on par or exceeding the execution of the state-of-the-art methods with AUC values of over 0.9 for a diverse collection of species tested. Finally, we built a pipeline called linc2function that provides the information necessary to functionally annotate a lncRNA conveniently in a single window. AvailabilityThe source code is accessible use under MIT license in standalone mode, and as a webserver (https://bioinformaticslab.erc.monash.edu/linc2function).

bioinformatics↗

bioRxiv2019

Aging of human endocrine pancreatic cell types is heterogeneous and sex-specific

The human endocrine pancreas must regulate glucose homeostasis throughout the human lifespan, which is generally decades. We performed meta-analysis of single-cell, RNA-sequencing datasets derived from 36 individuals, as well as functional analyses, to characterize age-associated changes to the major endocrine pancreatic cell types. Increasing age was associated with shifts in pancreatic alpha and beta cell identity and loss of nuclear integrity in non-diabetic humans. In non-diabetic individuals [≥] 50 years old, 80% of their beta cells exhibited a transcriptional signature similar to cells from type-2 diabetic (T2D) donors. Surprisingly, [~]5% of beta cells from T2D donors retained a youthful, N.D. transcriptional profile. Furthermore, beta cell function was reduced by 50% during aging in men but not women, which may explain sex-associated differences in diabetes etiology. These analyses reveal that aging of the human endocrine pancreas is sex- and cell-type specific.

physiology↗

bioRxiv2017

Best Practice Data Life Cycle Approaches for the Life Sciences

Throughout history, the life sciences have been revolutionised by technological advances; in our era this is manifested by advances in instrumentation for data generation, and consequently researchers now routinely handle large amounts of heterogeneous data in digital formats. The simultaneous transitions towards biology as a data science and towards a life cycle view of research data pose new challenges. Researchers face a bewildering landscape of data management requirements, recommendations and regulations, without necessarily being able to access data management training or possessing a clear understanding of practical approaches that can assist in data management in their particular research domain.\n\nHere we provide an overview of best practice data life cycle approaches for researchers in the life sciences/bioinformatics space with a particular focus on omics datasets and computer-based data processing and analysis. We discuss the different stages of the data life cycle and provide practical suggestions for useful tools and resources to improve data management practices.

bioinformatics↗