Biology subjects

Bisht, K.

Publications and source records attributed to Bisht, K..

25 records · Page 2Linked to original sources

bioRxiv2022

Transient RNA structures cause aberrant influenza virus replication and innate immune activation

During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVG) and mini viral RNAs (mvRNA). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid-inducible gene I (RIG-I) to viral RNAs. However, not all influenza A virus RNAs are strong RIG-I agonists. Here we show that potent innate immune activation by mvRNAs is determined by transient RNA structures, called template loops (t-loop) that stall the viral RNA polymerase. The effect of t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and their effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings provide a mechanism that links aberrant viral replication to the activation of the innate immune response.

microbiology↗

bioRxiv2021

Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling

Microglia are brain-resident immune cells with a repertoire of functions in the developing, mature and pathological brain. Their wide-ranging roles in physiology include the clearance of cellular debris, elimination of excess synapses, regulation of neuronal activity and contributions to blood vessel development. Despite these known roles for microglia, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, using in vivo acute and longitudinal two-photon imaging in transgenic mice combined with electron microscopy, fixed tissue immunohistochemistry, pharmacological treatments and laser speckle imaging, we document the steady-state interactions between ramified CX3CR1+ myeloid cell somata and capillaries in the brain. We first confirm that these myeloid cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing and contrasting them with parenchymal microglia (PCMs) in their static, dynamic and chronic morphological activities including during microglial depletion and repopulation. Molecularly, we identify microglial-specific purinergic P2RY12 receptors as a receptor regulating CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, to elucidate roles for microglia in vascular structure and function, we eliminated microglia and showed that this triggered capillary dilation, blood flow increase, and impaired vasodilative responses. We find that P2RY12-/- and PANX1-/- mice recapitulate these vascular impairments suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function.

neuroscience↗

bioRxiv2021

Differential impact of a dyskeratosis congenita mutation in TPP1 on mouse hematopoiesis and germline

Telomerase extends chromosome ends in somatic and germline stem cells to ensure continued proliferation. Mutations in genes critical for telomerase function result in telomeropathies such as dyskeratosis congenita (DC), frequently resulting in spontaneous bone marrow failure. While knockout of telomerase in mice has been instrumental in highlighting the importance of telomere length maintenance at an organismal level, it may not be representative of human telomeropathy mutations in vivo. A DC mutation in the shelterin protein TPP1 (K170{Delta}) that compromises telomerase recruitment to telomeres but leaves other functions of TPP1 and the integrity of the telomerase holoenzyme intact is a physiologically relevant tool to evaluate telomerase-dependent telomere length maintenance in mice. We used CRISPR-Cas9 to generate a mutant mouse knocked in for the equivalent of the TPP1 K170{Delta} mutation (TPP1 K82{Delta}) and investigated both its bone marrow and germline compartments in unprecedented detail. TPP1 K82{Delta} caused progressive telomere erosion with increasing generation number but did not induce steady-state hematopoietic defects. Strikingly, K82{Delta} caused mouse infertility, consistent with gross morphological defects in the testis and sperm, the appearance of either empty or severely disorganized seminiferous tubules, and a decrease in both spermatogonia and spermatocytes. It is intriguing that both TPP1 K82{Delta} mice and previously characterized telomerase knockout mice show no spontaneous bone marrow failure but rather succumb to a robust infertility phenotype at steady state. We speculate that telomere length maintenance contributes differently to the evolutionary fitness of humans and mice. Telomere length maintenance in the human bone marrow can ensure progression to reproductive age, while that in the mouse germline can help meet the elevated demand for sperm to produce multiple offspring.

cell biology↗

bioRxiv2020

Heterochromatin Replication: Direct Interaction of DNA replication machinery with heterochromatin code writer Clr4/Suv39 and reader Swi6/HP1 in S. pombe

The establishment of heterochromatin in fission yeast involves methyltransferase Clr4-mediated H3-Lys9 methylation, which is bound specifically by Swi6/HP1. However, the mechanism of propagation of heterochromatin through multiple cell divisions is not known. A role of DNA replication in propagating the heterochromatin is envisaged. Studies in S. pombe have indicated a direct interaction between DNA Pol and Swi6/HP1 and between DNA Pol{varepsilon} and Rik1-Dos2 complex, suggesting a coupling between DNA replication and heterochromatin assembly. Here, we show that like DNA Pol, Pol{delta}, which plays a role in both leading and lagging strand replication, also plays a role in silencing at mating type and centromere. We show that both the polymerases and {delta} interact directly with both Clr4 and Swi6/HP1. Mutations in both the polymerases lead to decrease in H3-Lys9 methylation and Swi6 at the mating type and left outer repeats of centromeres I and II, with a reciprocal increase in their level at the central element, cnt, at all the three centromeres. These mutations also cause defects in chromosome segregation, recruitment of Cohesin and chromosome dynamics during mitosis and meiosis. Thus, our results indicate that a tight coordination between DNA replication machinery and propagation of the heterochromatin-specific epigenetic mark.

genetics↗

bioRxiv2020

A genome engineering resource to uncover principles of cellular organization and tissue architecture by lipid signalling

Phosphoinositides (PI) are key regulators of cellular organization in eukaryotes and genes that tune PI signalling are implicated in human disease mechanisms. Biochemical analyses and studies in cultured cells have identified a large number of proteins that can mediate PI signalling. However, the role of such proteins in regulating cellular processes in vivo and development in metazoans remains to be understood. Here we describe a set of CRISPR based genome engineering tools that allow the manipulation of each of these proteins with spatial and temporal control during metazoan development. We demonstrate the use of these reagents to deplete a set of 103 proteins individually in the Drosophila eye and identify several new molecules that control eye development. Our work demonstrates the power of this resource in uncovering the molecular basis of tissue homeostasis during normal development and in human disease biology.

genetics↗

bioRxiv2019

Essential omega-3 fatty acids tune microglial phagocytosis of synaptic elements in the developing brain

Omega-3 fatty acids (n-3 polyunsaturated fatty acids; n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in epidemiological studies, but the mechanisms by which a n-3 PUFA dietary imbalance affects CNS development are poorly understood. Active microglial engulfment of synaptic elements is an important process for normal brain development and altered synapse refinement is a hallmark of several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development. Our results show that maternal dietary n-3 PUFA deficiency increases microglial phagocytosis of synaptic elements in the developing hippocampus, through the activation of 12/15- lipoxygenase (LOX)/12-HETE signaling, which alters neuronal morphology and affects cognition in the postnatal offspring. While women of child bearing age are at higher risk of dietary n-3 PUFA deficiency, these findings provide new insights into the mechanisms linking maternal nutrition to neurodevelopmental disorders.\n\nOne Sentence SummaryLow maternal omega-3 fatty acids intake impairs microglia-mediated synaptic refinement via 12-HETE pathway in the developing brain.

neuroscience↗

bioRxiv2019

Multiple Myeloma DREAM Challenge Reveals Epigenetic Regulator PHF19 As Marker of Aggressive Disease

While the past decade has seen meaningful improvements in clinical outcomes for multiple myeloma patients, a subset of patients do not benefit from current therapeutics for unclear reasons. Many gene expression-based models of risk have been developed, but each model uses a different combination of genes and often involve assaying many genes making them difficult to implement. We organized the Multiple Myeloma DREAM Challenge, a crowdsourced effort to develop models of rapid progression in newly diagnosed myeloma patients and to benchmark these against previously published models. This effort lead to more robust predictors and found that incorporating specific demographic and clinical features improved gene expression-based models of high risk. Furthermore, post challenge analysis identified a novel expression-based risk marker and histone modifier, PHF19, which featured prominently in several independent models. Lastly, we show that a simple four feature predictor composed of age, International Staging System stage (ISS), and expression of PHF19 and MMSET performs similarly to more complex models with many more gene expression features included.\n\nKey pointsO_LIMost comprehensive and unbiased assessment of prognostic biomarkers in MM resulting in a robust and parsimonious model.\nC_LIO_LIIdentification of PHF19 as the expression based biomarker most strongly associated with rapid progression in MM patients.\nC_LI

cancer biology↗