The legacy of bio-molecules as a bio-fertilizer: Context of single cell fertilizer Isolation and partial characterization of an amylolytic bacterium

Repeated cultivation of crop plants is the reason for the depletion of nutrients in an agricultural land. Therefore, modern procedures of agriculture cascaded with the addition of organic and inorganic fertilizers, the use of insecticides and pesticides, the addition of proper water, etc. Various inorganic molecules are used as fertilizers. However, the use of organic manures is also in practice. They have many roles such as improving soil porosity, air holding capacity, water holding capacity, structure, texture, etc. Agricultural scientists suggest using organic molecules for many reasons. Bio-fertilizers of many kinds are used by farmers of all nations. However, these fertilizers are unable to cause tremendous effects on the growth and development of crop plants, even though these fertilizers have cumulative effects. The present work focuses on the use of bio-molecules as bio-fertilizer. To make these molecules, an amylolytic bacterium was isolated and partially identified based on microscopic observations and biochemical tests. The optimum pH, temperature, substrate concentration, etc. were studied. The optimum pH and temperature for the growth of the isolate were pH 7.0 and 37.0°C, respectively. However, the organism grows even in 60.0°C. The organism uses four commonly available natural substrates as carbon source. Among these, potato starch is the most conveniently utilized by the organism. The amy gene of the strain was cloned using a vector. It expressed a high amount of amylase (data is not shown). The recombinant organism was used to make bio-molecules. It was grown in the presence of various natural substrates and enzymatic activities, and other associated studies were also carried out. The experimental results obtained in this study showed that the recombinant organism can be utilized to make a huge amount of bio-molecules. It will be a unique fertilizer for future generations

Identification of cellular factors involved in Neisseria gonorrhoea induced enhanced HIV-1 transmission in a cervical tissue based organ culture model

The presence of sexually transmitted infections (STI) such as Neisseria gonorrhoeae (NG) can enhance the transmission of HIV-1. Our goal in this study is to elucidate mechanism by which NG induces enhanced HIV-1 transmission. A cervical tissue based organ culture system was developed to study the interaction between NG and HIV-1 which provided a unique opportunity to elucidate mechanism of NG induced enhanced HIV-1 transmission across cervical mucosa. We demonstrated that the NG exposure on the cervical tissue in the organ culture model showed physical characteristics of NG infection and induced high levels of inflammatory cytokines IL1-β and TNF-α that have been observed during in-vivo NG infection in the cervix. In elucidating the mechanism of NG induced enhancement of HIV-1 transmission, we demonstrated that NG not by itself but the culture fluids from NG exposed tissues (reminiscent of cervical milieu) increased HIV-1 transcription from the HIV- LTR in TZM-bl cells, induced full length HIV-1 from latently infected U1 and ACH2 cells and increased transmission of HIV-1 across cervical mucosa. The whole genome transcriptome analysis using second-generation sequencing of the micro-dissected epithelial layer of the tissues exposed to NG and HIV-1 identified with high statistical significance differentially expressed genes in NG exposed and HIV-1 exposed tissues, and identified common cellular factors as well as pathways involved in cell activation, migration and stimulation expressed in the epithelia. Out of these only two differentially expressed genes that were common between tissues exposed to both NG and HIV-1 are CXCL10 and IL8. Addition of inhibitors of CXCL10 and IL8 suppressed HIV-1 transmission, while addition of CXCL10 and IL8 increased HIV-1 transmission indicating that CXCL10 and IL8 could be involved in HIV-1 transmission across cervical epithelia. IL-1β also increased CXCL10 and IL8 expression in cervical tissues and enhanced HIV-1 transmission Altogether these data are consistent with a model (Figure 42) for NG induced enhanced HIV-1 transmission: NG infection secretes IL-1β which induces increased production of epithelial CXCL10 and IL-8 causing the migration of HIV-1 target cells CD3+T cells and macrophages towards intraepithelial region that fuels HIV-1 replication in submucosa and consequently enhances HIV-1 transmission. Taken together, our results confirm that the risk of acquisition of HIV-1 infection in the ecto-cervical region increases with prior NG infection. From the public health perspective, identification of IL-1β and its target cellular proteins in NG induced enhanced HIV-1 transmission could be useful in development of drugs that impair HIV transmission. Further work in nonhuman primates or humanized mouse models could provide confirmation of the role of CXCL10 and IL-8 in HIV transmission and its modulation by NG secreted proteins like IL-1β in vivo

Synergistic block of SARS-CoV-2 infection by combined drug inhibition of the host entry factors PIKfyve kinase and TMPRSS2 protease

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike (S) protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5- to 10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2. The substantial ∼5-fold or higher decrease of the half-maximal effective concentrations (EC50s) suggests a plausible treatment strategy based on the combined use of these inhibitors.Peer reviewe

SARS-CoV-2 requires acidic pH to infect cells

Publisher Copyright: Copyright © 2022 the Author(s). Published by PNAS.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.Peer reviewe

Testing the HYPE: FIC-mediated Adenylylation/Ampylation in Eukaryotic Signaling

Post-translational modifications (PTMs) are integral regulators of protein function in the cell. Decades of research on PTMs such as phosphorylation, glycosylation, methylation, acetylation and ubiquitination, to name a few, have provided great insight into cellular signal transduction. In comparison, adenylylation, more recently called AMPylation- which entails the covalent addition of an AMP (adenosine monophosphate) to target protein- remains less understood. Recently, a family of enzymes, defined by the presence of a Fic domain, was found to function as a new class of adenylyltransferases. These enzymes are characterized by the presence of a conserved HxFx(D/E)GN(G/K)RxxR sequence motif, which forms the Fic active core. While bacterial Fic proteins are known to regulate pathogenesis by inhibiting host-signaling cascades, studies on eukaryotic Fic proteins conducted by us and others, suggest a role in cellular stress responses. This thesis entails a cellular and enzymatic characterization of the sole human Fic protein, HYPE/FicD and the identification of three novel substrates of HYPE-mediated adenylylation: an Hsp70 molecular chaperone, BiP; α-Synuclein, the protein whose aggregation leads to Parkinson’s disease; and Vimentin, a building block of intermediate filaments. We show that HYPE is upregulated upon endoplasmic reticulum (ER) stress and is critical for maintaining ER homeostasis by regulating a key pathway called the unfolded protein response (UPR). Specifically, we elucidate that HYPE localizes to the ER lumen where it adenylylates BiP, a key sentinel for UPR regulation and protein folding. In vitro, adenylylation enhances BiP’s ATPase activity. Further, we determine the kinetic parameters governing HYPE-mediated adenylylation of BiP, and offer insights for the HYPE-BiP interaction. Finally, our in vitro biochemical assays, tissue culture based cellular assays and in vivo studies reveal that HYPE targets a diverse range of protein substrates, suggesting that adenylylation represents a highly versatile mode of regulating cellular signaling in humans. The goal of this scientific endeavor is to enrich the understanding of this novel PTM in eukaryotes, which has already given us an appreciation of the multifarious nature of HYPE’s physiological substrates and keen insights for future studies directed at targeting adenylylation for disease therapy

Single-Cell Transcriptome Analysis Identifies Subclusters with Inflammatory Fibroblast Responses in Localized Scleroderma

Localized scleroderma (LS) is an autoimmune disease with both inflammatory and fibrotic components causing an abnormal deposition of collagen in the skin and underlying tissue, often leading to disfigurement and disability. Much of its pathophysiology is extrapolated from systemic sclerosis (SSc) since the histopathology findings in the skin are nearly identical. However, LS is critically understudied. Single-cell RNA sequencing (scRNA seq) technology provides a novel way to obtain detailed information at the individual cellular level, overcoming this barrier. Here, we analyzed the affected skin of 14 patients with LS (pediatric and adult) and 14 healthy controls. Fibroblast populations were the focus, since they are the main drivers of fibrosis in SSc. We identified 12 fibroblast subclusters in LS, which overall had an inflammatory gene expression (IFN and HLA-associated genes). A myofibroblast-like cluster (SFRP4/PRSS23) was more prevalent in LS subjects and shared many upregulated genes expressed in SSc-associated myofibroblasts, though it also had strong expression of CXCL9/10/11, known CXCR3 ligands. A CXCL2/IRF1 cluster identified was unique to LS, with a robust inflammatory gene signature, including IL-6, and according to cell communication analysis are influenced by macrophages. In summary, potential disease-propagating fibroblasts and associated gene signatures were identified in LS skin via scRNA seq

Deep neural network automated segmentation of cellular structures in volume electron microscopy

Volume electron microscopy is an important imaging modality in contemporary cell biology. Identification of intracellular structures is a laborious process limiting the effective use of this potentially powerful tool. We resolved this bottleneck with automated segmentation of intracellular substructures in electron microscopy (ASEM), a new pipeline to train a convolutional neural network to detect structures of a wide range in size and complexity. We obtained dedicated models for each structure based on a small number of sparsely annotated ground truth images from only one or two cells. Model generalization was improved with a rapid, computationally effective strategy to refine a trained model by including a few additional annotations. We identified mitochondria, Golgi apparatus, endoplasmic reticulum, nuclear pore complexes, caveolae, clathrin-coated pits, and vesicles imaged by focused ion beam scanning electron microscopy. We uncovered a wide range of membrane–nuclear pore diameters within a single cell and derived morphological metrics from clathrin-coated pits and vesicles, consistent with the classical constant-growth assembly model