Biology subjects

Goswami, D.

Publications and source records attributed to Goswami, D..

9 recordsLinked to original sources

bioRxiv2025

Phosphorylation by the PknB serine/threonine kinase stimulates dimer formation by WalR

WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike other response regulators, WalR is thought to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work we demonstrate similarities between pknB null and the WalRT101M mutants that support a regulatory role for PknB-mediated phosphorylation of WalR. Using a combination of in vitro and in vivo approaches, we confirm the specificity of this posttranslational modification and confirm that phosphorylation at both D53 and T101 is important for dimer formation. Importantly, using an in silico approach, we have discovered that phosphorylation at T101 creates an intermolecular hydrogen bond between the phosphate group and E108, that strengthens contacts along the WalR dimerization interface. This is the first time that a clear molecular role has been assigned to this posttranslational modification and it is the strongest molecular evidence to date for a direct functional relationship between PknB and WalR. We propose that, together with primary activation by WalK, PknB serves as a second potentiating input into the WalKR system, stimulating WalR dimer formation, and target DNA binding and tethering this transcriptional event to important extracellular stimuli.

microbiology↗

bioRxiv2025

Nanoporous Microelectrodes for Neural Electrophysiology Recordings in Organotypic Culture

Organotypic cultures, specifically brain slices, have been used in neuroscience studies for many years to prolong the lifetime of the biological tissue outside of the host organism. However, the cultures must be kept in a sterile environment, maintaining supply of gas/nutrients for tissue survival and physiological relevance. Electrophysiological recordings from cultured tissue are challenging as the conventional approaches implicate a compromise on biological stability or environmental integrity. In this article, a novel approach has been used to design and print nanoporous microelectrodes on culture wells enabling in situ recording of electrophysiological neural activities. Optimized ink formulations are developed for conductive nanocarbon microelectrodes, and furthermore, fluoropolymer (polytetrafluoroethylene-based AF2400) ink has been inkjet printed for the first time acting as an insulator layer for microelectrodes. To keep the biocompatible nanoporous structure of culture wells, the microelectrodes have been printed on the bottom of the culture cells and only small connector pads have been produced on top of the culture membrane. Neural activity has been recorded by such a microelectrode structure for rodent brain slices cultured for three weeks. Furthermore, aerosol jet printing has been used for printing of nanocarbon microelectrodes allowing to produce much smaller size features compared to the inkjet printing.

neuroscience↗

bioRxiv2022

A conserved Plasmodium protein that localizes to liver stage nuclei is critical for late liver stage development.

Malaria, the disease caused by Plasmodium parasites, causes significant mortality and morbidity. Whole parasite vaccination with pre-erythrocytic parasite stages, attenuated through sporozoite irradiation or chemo-attenuation, confers sterilizing immunity against subsequent parasite infection. This provides a rationale for the creation of whole parasite vaccines that are attenuated using gene editing. Here, we report on the creation of a novel genetically attenuated parasite (GAP) by the deletion of Plasmodium LINUP, encoding a liver stage nuclear protein. Epitope-tagging of LINUP in the rodent malaria parasite Plasmodium yoelii showed LINUP expression exclusively in liver stage nuclei after the onset of exo-erythrocytic schizogony. P. yoelii parasites with a gene deletion of LINUP (linup --) suffered an exclusive liver stage phenotype with developmental arrested late in exo-erythrocytic schizogony. Liver stages showed incomplete segregation of nuclei and, mitochondria and apicoplast. These cellular perturbations caused a defect in exo-erythrocytic merozoite formation and a concomitant severe attenuation of liver stage-to-blood stage transition. LINUP gene deletion in Plasmodium falciparum also caused a severe defect in late liver stage differentiation. Importantly, P. falciparum linup -- liver stages showed a severe defect in parasite transitioning from liver stage to viable blood stage infection. These results suggest that P. falciparum LINUPis a useful target for late liver stage attenuation and an additional gene deletion that can be incorporated into a late liver stage-arresting replication competent whole parasite vaccine.

cell biology↗

bioRxiv2022

Lifetime of actin-dependent protein nanoclusters

Protein nanoclusters (PNCs) are dynamic collections of a few proteins that spatially organize in nanometer length clusters. PNCs are one of the principal forms of spatial organization of membrane proteins and they have been shown or hypothesized to be important in various cellular processes, including cell signaling. PNCs show remarkable diversity in size, shape, and lifetime. In particular, the lifetime of PNCs can vary over a wide range of timescales. The diversity in size and shape can be explained by the interaction of the clustering proteins with the actin cytoskeleton or the lipid membrane, but very little is known about the processes that determine the lifetime of the nanoclusters. In this paper, using mathematical modelling of the cluster dynamics, we model the biophysical processes that determine the lifetime of actin-dependent PNCs. In particular, we investigated the role of actin aster fragmentation, which had been suggested to be a key determinant of the PNC lifetime, and found that it is important only for a small class of PNCs. A simple extension of our model allowed us to investigate the kinetics of protein-ligand interaction near PNCs. We found an anomalous increase in the lifetime of ligands near PNCs, which agrees remarkably well with experimental data on RAS-RAF kinetics. In particular, analysis of the RAS-RAF data through our model provides falsifiable predictions and novel hypotheses that will not only shed light on the role of RAS-RAF kinetics in various cancers, but also will be useful in studying membrane protein clustering in general. SignificanceSpatial organization of biomolecules shapes the behavior of a cell. It is particularly important during cell-signaling, where transient, dynamic organization of the biomolecules helps cells process signals and respond to them. Nanoclusters of peripheral membrane proteins, such as KRAS, a specific form of dynamic organization of biomolecules, play a critical part in the modulation of cell signals that control various cellular behaviors including cell growth, proliferation, and differentiation. Although we have made significant progress in understanding the structure, size, and origin of the nanoclusters, very little is known about the biophysical processes that control their lifetime. In this paper, we present a mathematical framework that provides quantitative insights into these processes and explains how oncogenic mutations in KRAS may lead to cancers.

biophysics↗

bioRxiv2020

Potential malaria vector Anopheles minimus (species A) still persisting in North East India

BackgroundVector borne infectious diseases affect two third of the worlds human population and cause mortality in millions each year. Malaria remains one of the major killers in the Indian sub-continent and transmitted uninterruptedly by many efficient vectors and their sibling species. In North East India (NE), Anopheles minimus has been recognized as an important vector which shares majority of malaria cases. This study primarily focuses on to recognize the presence and distribution of sibling species of An. minimus in certain endemic area of NE India. MethodsAnopheles species were collected and identified using available morphological keys. The genomic DNA was extracted from the mosquito specimen and used to perform species specific PCR (ss PCR) for molecular identification of major malaria vector An. minimus sibling species ResultMorphological identification suggested the presence of An. minimus sl in low density in the study area. The specimen of An. minimus subjected to ss PCR confirmed the prevalence of only one sibling species namely, An. minimus A in Sialmari and Chandubi. ConclusionThough in low density, but malaria vector An. minimus is still present in certain endemic areas of NE India. The ss PCR assay employed presently suggested that An. minimus sibling species A is prevailing in the region. Presently used ss PCR assay was simpler, faster, cheaper and more readily interpreted than earlier assays. This information could be useful in understanding of current prevalence and distribution of An. minimus sibling species complex in NE region of India.

ecology↗

bioRxiv2020

CCM signaling complex (CSC) is a master regulator governing homeostasis of progestins and their mediated signaling cascades

We demonstrate that a novel signaling network among the CSC and mPRS is dynamically modulated and fine-tuned with intricate feedback regulations in PR negative cells, especially endothelial cells (ECs). Depletion of any of three CCMs (1, 2, 3) genes results in the disruption of non-classic mPRs-mediated signaling in-vitro as well as defective homeostasis of PRG in-vivo. Therefore, we propose the CSC is a master regulator of homeostasis of PRG and its associated classic and non-classic signaling cascades. Assisted with omic approaches, we identified signaling pathways involved and specific biomarkers associated with hemorrhagic events during CCM pathogenesis in-vitro and in-vivo. To our knowledge, this is the first report detailing etiology to predict the occurrence of early hemorrhagic events with a set of serum biomarkers.

neuroscience↗

bioRxiv2020

Platelet Derived Growth Factor Receptor β (PDGFRβ) is a Host Receptor for the human malaria parasite adhesin TRAP

Following their inoculation by the bite of an infected Anopheles mosquito, the malaria parasite sporozoite forms travel from the bite site in the skin into the bloodstream, which transports them to the liver. The thrombospondin-related anonymous protein (TRAP) is a type 1 transmembrane protein that is released from secretory organelles and relocalized on the sporozoite plasma membrane. TRAP is required for sporozoite motility and host infection, and its extracellular portion contains adhesive domains that are predicted to engage host receptors. Here, we identified the human platelet-derived growth factor receptor {beta} (hPDGFR{beta}) as one such protein receptor. Deletion mutants showed that the von Willebrand factor type A and thrombospondin repeat domains of TRAP are both required for optimal binding to hPDGFR{beta}-expressing cells. We also demonstrate that this interaction is conserved in the human-infective parasite Plasmodium vivax, but not the rodent-infective parasite Plasmodium yoelii. We observed expression of hPDGFR{beta} mainly in cells associated with the vasculature suggesting that TRAP:hPDGFR{beta} interaction may play a role in the recognition of blood vessels by invading sporozoites.

microbiology↗

bioRxiv2019

Extensive Mammalian Germline Genome Engineering

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO{middle dot}3KO{middle dot}9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO{middle dot}3KO{middle dot}9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism. One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

synthetic biology↗

bioRxiv2018

Interactome Analysis Reveals Regulator of G Protein Signaling 14 (RGS14) is a Novel Calmodulin (CaM) Effector in Mouse Brain

Regulator of G Protein Signaling 14 (RGS14) is a complex scaffolding protein with an unusual domain structure that allows it to integrate G protein and mitogen-activated protein kinase (MAPK) signaling pathways. RGS14 mRNA and protein are enriched in brain tissue of rodents and primates. In the adult mouse brain, RGS14 is predominantly expressed in postsynaptic dendrites and spines of hippocampal CA2 pyramidal neurons where it naturally inhibits synaptic plasticity and hippocampus-dependent learning and memory. However, the signaling proteins that RGS14 natively interacts with in neurons to regulate plasticity are unknown. Here, we show that RGS14 exists as a component of a high molecular weight protein complex in brain. To identify RGS14 neuronal interacting partners, endogenous RGS14 immunoprecipitated from mouse brain was subjected to mass spectrometry and proteomic analysis. We find that RGS14 interacts with key postsynaptic proteins that regulate neuronal plasticity. Gene ontology analysis reveals that the most enriched RGS14 interacting proteins have functional roles in actin-binding, calmodulin(CaM)-binding, and CaM-dependent protein kinase (CaMK) activity. We validate these proteomics findings using biochemical assays that identify interactions between RGS14 and two previously unknown binding partners: CaM and CaMKII. We report that RGS14 directly interacts with CaM in a calcium-dependent manner and is phosphorylated by CaMKII in vitro. Lastly, we detect that RGS14 associates with CaMKII and with CaM in hippocampal CA2 neurons by proximity ligation assays in mouse brain sections. Taken together, these findings demonstrate that RGS14 is a novel CaM effector and CaMKII phosphorylation substrate thereby providing new insight into cellular mechanisms by which RGS14 controls plasticity in CA2 neurons.

biochemistry↗