Biology subjects

Ghosh, K.

Publications and source records attributed to Ghosh, K..

39 records · Page 3Linked to original sources

bioRxiv2020

The role of dermis resident macrophages and their interaction with neutrophils in the early establishment of Leishmania major infection transmitted by sand fly bite

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from the inflammatory and immune reactions initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite. SummarySand flies transmit Leishmania major which causes cutaneous leishmaniasis in humans and in non-human hosts. Our analyses of sand fly transmission sites of L. major in the mouse skin revealed that dermis resident macrophages (TRM) were the predominant phagocytes to take up the parasite within the first 24 hr post-bite. The early involvement of neutrophils varied depending on the proximity of deposited parasites to the site of tissue damage around which the neutrophils coalesced. By intra-vital microscopy, some of the dermal TRMs could be visualized acquiring their infections by direct transfer from or phagocytosis of parasitized neutrophils. The involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these cellular interactions and in sustaining the anti-inflammatory functions of dermal TRMs was supported by the reduced parasite burdens but more severe pathology observed in Axl-/-Mertk-/- mice. The heterogeneity of sand fly transmission sites with respect to the dose of parasites and the early cellular interactions described here likely contribute to the wide range of infection outcomes that are associated with natural transmission of L. major observed in mouse models and possibly humans.

immunology↗

bioRxiv2018

Maximum Caliber can build and infer models of oscillation in a three-gene feedback network

Single-cell protein expression time trajectories provide rich temporal data quantifying cellular variability and its role in dictating fitness. However, theoretical models to analyze and fully extract information from these measurements remain limited for three reasons: i) gene expression profiles are noisy, rendering models of averages inapplicable, ii) experiments typically measure only a few protein species while leaving other molecular actors - necessary to build traditional bottom-up models - unnoticed, and iii) measured data is in fluorescence, not particle number. We have recently addressed these challenges in an alternate top-down approach using the principle of Maximum Caliber (MaxCal) to model genetic switches with one and two protein species. In the present work we address scalability and broader applicability of MaxCal by extending to a three-gene (A, B, C) feedback network that exhibits oscillation, commonly known as the repressilator. We test MaxCals inferential power by using synthetic data of noisy protein number time traces - serving as a proxy for experimental data - generated from a known underlying model. We notice that the minimal MaxCal model - accounting for production, degradation, and only one type of symmetric coupling between all three species - reasonably infers several underlying features of the circuit such as the effective production rate, degradation rate, frequency of oscillation, and protein number distribution. Next, we build models of higher complexity including different levels of coupling between A, B, and C and rigorously assess their relative performance. While the minimal model (with four parameters) performs remarkably well, we note that the most complex model (with six parameters) allowing all possible forms of crosstalk between A, B, and C slightly improves prediction of rates, but avoids ad-hoc assumption of all the other models. It is also the model of choice based on Bayesian Information Criteria. We further analyzed time trajectories in arbitrary fluorescence (using synthetic trajectories) to mimic realistic data. We conclude that even with a three-protein system including both fluorescence noise and intrinsic gene expression fluctuations, MaxCal can faithfully infer underlying details of the network, opening up future directions to model other network motifs with many species.

synthetic biology↗

bioRxiv2018

Ancient Thioredoxins Evolved to Modern Day Stability-Function Requirement by Altering Native State Ensemble

Thioredoxins (Thrxs) - small globular proteins that reduce other proteins - are ubiquitous in all forms of life, from archaea to mammals. Although ancestral Thioredoxins share sequential and structural similarity with the modern day (extant) homologs, they exhibit significantly different functional activity and stability. We investigate this puzzle by comparative studies of their (ancient and modern day Thrxs) native state ensemble, as quantified by the Dynamic Flexibility Index (DFI), a metric for the relative resilience of an amino acid to perturbations in the rest of the protein. Clustering proteins using DFI profiles strongly resembles an alternate classification scheme based on their activity and stability. The DFI profiles of the extant proteins are substantially different around the 3, 4 helices and catalytic regions. Likewise, allosteric coupling of the active site with the rest of the protein is different between ancient and extant Thrxs, possibly explaining the decreased catalytic activity at low pH with evolution. At a global level, we note that the population of low flexibility (called hinges) and high flexibility sites increases with evolution. The heterogeneity (quantified by the variance) in DFI distribution increases with the decrease in the melting temperature typically associated with the evolution of ancient proteins to their modern-day counterparts.

biophysics↗