HIV-1 usurps mixed-charge domain-dependent CPSF6 phase separation for higher-order capsid binding, nuclear entry and viral DNA integration.

HIV-1 integration favors nuclear speckle (NS)-proximal chromatin and viral infection induces the formation of capsid-dependent CPSF6 condensates that colocalize with nuclear speckles (NSs). Although CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro, the contributions of its different intrinsically disordered regions, which includes a central prion-like domain (PrLD) with capsid binding FG motif and C-terminal mixed-charge domain (MCD), to LLPS activity and to HIV-1 infection remain unclear. Herein, we determined that the PrLD and MCD both contribute to CPSF6 LLPS activity in vitro. Akin to FG mutant CPSF6, infection of cells expressing MCD-deleted CPSF6 uncharacteristically arrested at the nuclear rim. While heterologous MCDs effectively substituted for CPSF6 MCD function during HIV-1 infection, Arg-Ser domains from related SR proteins were largely ineffective. While MCD-deleted and wildtype CPSF6 proteins displayed similar capsid binding affinities, the MCD imparted LLPS-dependent higher-order binding and co-aggregation with capsids in vitro and in cellulo. NS depletion reduced CPSF6 puncta formation without significantly affecting integration into NS-proximal chromatin, and appending the MCD onto a heterologous capsid binding protein partially restored virus nuclear penetration and integration targeting in CPSF6 knockout cells. We conclude that MCD-dependent CPSF6 condensation with capsids underlies post-nuclear incursion for viral DNA integration and HIV-1 pathogenesis.

CD47 and IFT57 Are Colinear Genes That Are Highly Coexpressed in Most Cancers and Exhibit Parallel Cancer-Specific Correlations with Survival.

An association between high CD47 expression and poor cancer survival has been attributed to its function on malignant cells to inhibit phagocytic clearance. However, CD47 mRNA expression in some cancers lacks correlation or correlates with improved survival. IFT57 encodes an essential primary cilium component and is colinear with CD47 across amniote genomes, suggesting coregulation of these genes. Analysis of The Cancer Genome Atlas datasets identified IFT57 as a top coexpressed gene with CD47 among 1156 human cancer cell lines and in most tumor types. The primary cilium also regulates cancer pathogenesis, and correlations between IFT57 mRNA and survival paralleled those for CD47 in thyroid and lung carcinomas, melanoma, and glioma. CD47 ranked first for coexpression with IFT57 mRNA in papillary thyroid carcinomas, and higher expression of both genes correlated with significantly improved overall survival. CD47 and IFT57 mRNAs were coordinately regulated in thyroid carcinoma cell lines. Transcriptome analysis following knockdown of CD47 or IFT57 in thyroid carcinoma cells identified the cytoskeletal regulator CRACD as a specific target of IFT57. CRACD mRNA expression inversely correlated with IFT57 mRNA and with survival in low-grade gliomas, lung adenocarcinomas, and papillary thyroid carcinomas, suggesting that IFT57 rather than CD47 regulates survival in these cancers.

Sparse Deep Neural Network for Encoding and Decoding the Structural Connectome.

Brain state classification by applying deep learning techniques on neuroimaging data has become a recent topic of research. However, unlike domains where the data is low dimensional or there are large number of available training samples, neuroimaging data is high dimensional and has few training samples. To tackle these issues, we present a sparse feedforward deep neural architecture for encoding and decoding the structural connectome of the human brain. We use a sparsely connected element-wise multiplication as the first hidden layer and a fixed transform layer as the output layer. The number of trainable parameters and the training time is significantly reduced compared to feedforward networks. We demonstrate superior performance of this architecture in encoding the structural connectome implicated in Alzheimer's disease (AD) and Parkinson's disease (PD) from DTI brain scans. For decoding, we propose recursive feature elimination (RFE) algorithm based on DeepLIFT, layer-wise relevance propagation (LRP), and Integrated Gradients (IG) algorithms to remove irrelevant features and thereby identify key biomarkers associated with AD and PD. We show that the proposed architecture reduces 45.1% and 47.1% of the trainable parameters compared to a feedforward DNN with an increase in accuracy by 2.6 % and 3.1% for cognitively normal (CN) vs AD and CN vs PD classification, respectively. We also show that the proposed RFE method leads to a further increase in accuracy by 2.1% and 4% for CN vs AD and CN vs PD classification, while removing approximately 90% to 95% irrelevant features. Furthermore, we argue that the biomarkers (i.e., key brain regions and connections) identified are consistent with previous literature. We show that relevancy score-based methods can yield high discriminative power and are suitable for brain decoding. We also show that the proposed approach led to a reduction in the number of trainable network parameters, an increase in classification accuracy, and a detection of brain connections and regions that were consistent with earlier studies.

Effects of a humanized CD47 antibody and recombinant SIRPα proteins on triple negative breast carcinoma stem cells.

Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.

Esophagogastrectomy for Long-Segment Recurrent Esophageal Stricture Following Hyperemesis Gravidarum.

An esophageal stricture is an abnormal tightening of the esophageal lumen. Benign strictures are often caused by gastroesophageal reflux disease (GERD) and are more common in patients over 40 years. When caused by GERD, these strictures develop when acid from the stomach regurgitates into the esophagus, leading to inflammation, fibrosis, and eventual narrowing of the lumen. This case report aims to highlight the importance of obtaining a detailed history in discovering the underlying cause of these strictures. We present a unique case of a young female presenting with dysphagia several months after experiencing Hyperemesis gravidarum. She was found to have a long esophageal stricture that would eventually recur within a few weeks of therapeutic intervention. The pathological report confirmed benign disease, but she ultimately would require surgical intervention for her condition.

Human CCR6+ Th Cells Show Both an Extended Stable Gradient of Th17 Activity and Imprinted Plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.

Efficacy of the Fruit and Vegetable Peels as Substrates for the Growth and Production of α-Amylases in Marine Actinobacteria.

Enzymes from haloalkaliphilic microorganisms have recently focused attention on their potential and suitability in various applications. In this study, the growth and production of extracellular amylases in the marine actinomycetes, using kitchen waste as the raw starch source, have been investigated. Actinobacteria were isolated from the seawater of the Kachhighadi Coast near Dwarika, Gujarat. Seven Actinobacterial isolates of pre-monsoon, monsoon, and post-monsoon seasons belonging to different strains of Nocardiopsis genera were screened and selected for amylase production. The amylase production was initially assessed on the solid media supplemented with the extracts of different fruits and vegetable peels as a substrate by agar plate assay. The strains Kh-2(13), Kh-2(1), and Kh-3(12) produced maximum amylase with potato peel as a substrate, while no significant differences were found with the media containing other peels. Nevertheless, all strains produced amylases at a significant level with other raw substrates as well. For the optimization of the growth and enzyme production, the selected two isolates Kh-2(13) and Kh-3(12) of the monsoon and winter seasons were cultivated in a liquid medium under the submerged fermentation conditions, with potato peel as a substrate. In both organisms, the optimum amylase production was observed in the stationary phase of growth. For amylase production, the effect of different physical and chemical parameters was evaluated. The optimum growth and amylase production was achieved in 2% inoculum size, at pH 8.0, 28℃, and 5% salt concentration. On the basis of the amylase production index (API) (a ratio of the amylase units and cell growth), both isolates produced significant amylase with the only extract of potato peels, without any other supplements. The trends further indicated that while additional complex sources, such as yeast extract and peptone can enhance the cell growth of the actinobacteria, the amylase production remained unaltered. The study projects the significance of waste raw materials for the production of enzymes in extremophilic microorganisms.

Heterologous Expression and Structural Elucidation of a Highly Thermostable Alkaline Serine Protease from Haloalkaliphilic Actinobacterium, Nocardiopsis sp. Mit-7.

A highly thermostable alkaline serine protease gene (SPSPro, MN429015) obtained from haloalkaliphilic actinobacteria, Nocardiopsis sp. Mit-7 (NCIM-5746), was successfully cloned and overexpressed in Escherichia coli BL21 under the control of the T7 promoter in the pET Blue1 vector leading to a 20-kDa gene product. The molecular weight of the recombinant alkaline protease, as determined by SDS-PAGE and the Mass Spectrometer (MALDI-TOF), was 34 kDa. The structural and functional attributes of the recombinant thermostable alkaline serine protease were analyzed by Bioinformatic tools. 3D Monomeric Model and Molecular Docking established the role of the amino acid residues, aspartate, serine, and tryptophan, in the active site of thealkaline protease.The activity of the recombinant alkaline protease was optimal at 65 °C, 5 °C higher than its native protease. The recombinant protease was also active over a wide range of pH 7.0-13.0, with a maximal activity of 6050.47 U/mg at pH 9. Furthermore, the thermodynamic parameters of the immobilized recombinant alkaline protease suggested its reduced vulnerability against adverse conditions under which the enzyme has to undergo varied applications.

COVID-19-Associated Rhino-orbital mucormycosis: Presentation and outcome.

We are reporting four accounts of rhino-orbital mucormycosis in patients during and after recovery from SARS-CoV-2 infection. The patients were diagnosed and treated for COVID-19 according to the current treatment protocols, following which they presented with sudden proptosis, ophthalmoplegia, and conjunctival injection, confirmed by magnetic resonance imaging and histopathological examination. The patients were treated with intravenous liposomal amphotericin B, and the outcome was observed. Early diagnosis and prompt intervention can substantially reduce the morbidity and mortality rates in these patients.

Solvent tolerant enzymes in extremophiles: Adaptations and applications.

Non-aqueous enzymology has always drawn attention due to the wide range of unique possibilities in biocatalysis. In general, the enzymes do not or insignificantly catalyze substrate in the presence of solvents. This is due to the interfering interactions of the solvents between enzyme and water molecules at the interface. Therefore, information about solvent-stable enzymes is scarce. Yet, solvent-stable enzymes prove quite valuable in the present day biotechnology. The enzymatic hydrolysis of the substrates in solvents synthesizes commercially valuable products, such as peptides, esters, and other transesterification products. Extremophiles, the most valuable yet not extensively explored candidates, can be an excellent source to investigate this avenue. Due to inherent structural attributes, many extremozymes can catalyze and maintain stability in organic solvents. In the present review, we aim to consolidate information about the solvent-stable enzymes from various extremophilic microorganisms. Further, it would be interesting to learn about the mechanism adapted by these microorganisms to sustain solvent stress. Various approaches to protein engineering are used to enhance catalytic flexibility and stability and broaden biocatalysis's prospects under non-aqueous conditions. It also describes strategies to achieve optimal immobilization with minimum inhibition of the catalysis. The proposed review would significantly aid our understanding of non-aqueous enzymology.

Human CCR6 + Th cells show both an extended stable gradient of Th17 activity and imprinted plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related (type 17) cells and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo . By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The CCR6 + cells' phenotypes and epigenomes are stable across cell divisions under homeostatic conditions. Nonetheless, activation in polarizing and non-polarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the continuum to yield the unusual plasticity ascribed to type 17 cells.

Chemokine positioning determines mutually exclusive roles for their receptors in extravasation of pathogenic human T cells.

Pro-inflammatory T cells co-express multiple chemokine receptors, but the distinct functions of individual receptors on these cells are largely unknown. Human Th17 cells uniformly express the chemokine receptor CCR6, and we discovered that the subgroup of CD4+CCR6+ cells that co-express CCR2 possess a pathogenic Th17 signature, can produce inflammatory cytokines independent of TCR activation, and are unusually efficient at transendothelial migration (TEM). The ligand for CCR6, CCL20, was capable of binding to activated endothelial cells (ECs) and inducing firm arrest of CCR6+CCR2+ cells under conditions of flow - but CCR6 could not mediate TEM. By contrast, CCL2 and other ligands for CCR2, despite being secreted from both luminal and basal sides of ECs, failed to bind to the EC surfaces - and CCR2 could not mediate arrest. Nonetheless, CCR2 was required for TEM. To understand if CCR2's inability to mediate arrest was due solely to an absence of EC-bound ligands, we generated a CCL2-CXCL9 chimeric chemokine that could bind to the EC surface. Although display of CCL2 on the ECs did indeed lead to CCR2-mediated arrest of CCR6+CCR2+ cells, activating CCR2 with surface-bound CCL2 blocked TEM. We conclude that mediating arrest and TEM are mutually exclusive activities of chemokine receptors and/or their ligands that depend, respectively, on chemokines that bind to the EC luminal surfaces versus non-binding chemokines that form transendothelial gradients under conditions of flow. Our findings provide fundamental insights into mechanisms of lymphocyte extravasation and may lead to novel strategies to block or enhance their migration into tissue.

CD47-Dependent Regulation of Immune Checkpoint Gene Expression and MYCN mRNA Splicing in Murine CD8 and Jurkat T Cells.

Elevated expression of CD47 in some cancers is associated with poor survival related to its function as an innate immune checkpoint when expressed on tumor cells. In contrast, elevated CD47 expression in cutaneous melanomas is associated with improved survival. Previous studies implicated protective functions of CD47 expressed by immune cells in the melanoma tumor microenvironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell function that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was upregulated in CD47-deficient cells but downregulated in CD47-deficient cells following activation. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms, as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified a significant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Thrombospondin-1 inhibited the induction of TIGIT, CD40LG, and MCL1 mRNAs following T cell activation in vitro. Increased mRNA expression of these T cell transcripts and MYCN in melanomas was associated with improved overall survival.

Microbial community and predictive functionalities associated with the marine sediment of Coastal Gujarat.

Marine sediments are complex ecosystems where structures and functions constantly change due to natural and anthropogenic influences. In this investigation, a comprehensive and comparative analysis of the bacterial communities and their functional potential of the pristine and polluted marine sediments were carried out using MiSeq. The phylum Proteobacteria was dominant in all study sites. Other phyla were Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Chloroflexi, Nitrospirae, Cyanobacteria, Verrucomicrobia, Tenericutes, and Chlorobi. Interestingly, about 50% of genera belong to the unclassified categories. The key genera were identified as Acinetobacter, Bacillus, Pseudomona, Idiomarina, Thalassospira, and Marinobacter, Halomonas, Planctomyces, Psychrobacter, and Vogesella. PICRUSt analysis revealed that major functions are associated with the metabolism category. Additionally, metabolism related to amino acids, carbohydrates, energy generation, xenobiotics degradation, nitrogen, sulfate, and methane were prominent. Similarly, the predicted metabolisms by COG and KEGG were observed in the microbial communities of the marine sediments. To date, a comprehensive description of the microbial life with metabolic potential in these study sites has not been investigated. This study therefore significantly adds to our understanding of the microbiome and its functional attributes of marine sediments.

Epigenetic Silencing of p16INK4a gene in Sporadic Breast Cancer.

Epigenetic alterations of tumor suppressor genes (TSG) involved in the onset and progression of Breast Cancer (BC) may serve as biomarkers for early detection and prediction of disease prognosis. We have herein tried to determine the methylation status of TSG, p16INK4a, in our 50 BC patients and their association with clinicopathological parameters. The methylation status of the p16INK4a gene in fresh tissue samples from 50 patients with BC was assessed by methylation-specific polymerase chain reaction (MS-PCR). The mean age of BC patients was 49.30 ± 9.75 years. Of 50 BC samples tested, 21 (42%) had methylated p16INK4a gene. p16INK4a gene hypermethylation was significantly associated with age ≤ 50 years, premenopausal status and advanced BC stage. Multivariate analysis revealed a strong association between advanced BC stage (Stage III and Stage IV) and p16INK4a hypermethylation (P = 0.008, RR = 5.996, 95% CI = 1.581-22.739). p16INK4a methylation was significantly associated with Triple Negative BC (TNBC) (P = 0.045, OR = 4.181, 95% CI = 1.030-16.981). These findings indicate that p16INK4a hypermethylation frequently occurs in BC. Hypermethylation of p16INK4a in young, premenopausal, TNBC and with advance stage in BC patients suggests its association with aggressive BC.

Microorganisms under extreme environments and their applications.

Extremophiles are group of microorganisms that possess ability to tolerate and live under the extremes of physico-chemical, geological and nutritional conditions. Such microorganisms are evolutionary relics and have evolved adaptation strategies at cellular, biochemical and molecular levels. They produce enzymes that are capable to maintain stability and function under the multitudes of extremities. These organisms also produce variety of other molecules and metabolites, such as extremolytes and surface-active compounds to protect against extremes of salinity, pH, pressure, temperatures and solar radiation. Investigations on these microorganisms can further open new avenues and opportunity for research and biotechnological applications in the areas of waste water treatment, bio-plastics, biofuel, cosmetics, agriculture, food and pharmaceuticals. Further, extremophiles have potential roles to play in bioremediation, astrobiology and biorefinery.

Comparative analysis of the catalysis and stability of the native, recombinant and metagenomic alkaline proteases in organic solvents.

The effect of organic solvents on alkaline proteases was assessed for native, recombinant, and metagenomically derived alkaline proteases. Their stability and the effects of physicochemical parameters were studied in the presence of hexane. The native enzyme was comparatively more resistant against the organic solvents than the recombinant counterparts. On the other hand, the metagenomically derived alkaline protease was minimally resistant against solvents. A similar trend was apparent for the stability of enzyme in organic solvents. The novelty of this study lies in the fact that the majority of the studies on the solvent tolerance have focused on the mesophilic enzymes, while those from the haloalkaliphilic bacteria have received little attention. The comparative tolerance of the native, recombinant, and metagenomic alkaline proteases against the organic solvent has practical importance. The phylogenetic relatedness among the various protease sequences will be described.

Bile Acids Improve Psoriasiform Dermatitis through Inhibition of IL-17A Expression and CCL20-CCR6-Mediated Trafficking of T Cells.

Bile acids (BAs), produced in the liver and further transformed in the gut, are cholesterol-derived molecules involved in essential physiological processes. Recent studies suggest that BAs regulate T helper 17 cell function, but the underlying mechanism of this action and their therapeutic value in disease models remains unclear. Using an IL-23 minicircle DNA-based murine model of psoriasiform dermatitis, we showed that oral administration of secondary BAs, including lithocholic acid (LCA), deoxycholic acid, and 3-oxoLCA, significantly improved psoriasiform dermatitis without inducing apparent hepatotoxicity. Of the BAs tested, LCA possessed the greatest potency in treating psoriasiform dermatitis. Intravenous administration of LCA at a much lower dosage (compared with oral treatment) showed a comparable antipsoriatic effect and markedly suppressed the IL-17A response. Ex vivo experiments revealed that LCA reduced IL-17A production in IL-23-stimulated murine T cells in the absence of BA receptors TGR5 or FXR. Strikingly, BAs inhibited CCL20 expression in keratinocytes, which led to reduced migration of CCR6-expressing Jurkat cells cultured in the conditioned medium of stimulated keratinocytes. Thus, BAs improve psoriasiform dermatitis with minimal toxicity via direct inhibition of IL-17A production and blockade of CCL20-mediated trafficking, supporting the potential use of BAs in psoriasis.

Amylases from thermophilic bacteria: structure and function relationship.

Amylases hydrolyze starch to diverse products including dextrins and progressively smaller polymers of glucose units. Thermally stable amylases account for nearly 25% of the enzyme market. This review highlights the structural attributes of the α-amylases from thermophilic bacteria. Heterologous expression of amylases in suitable hosts is discussed in detail. Further, specific value maximization approaches, such as protein engineering and immobilization of the amylases are discussed in order to improve its suitability for varied applications on a commercial scale. The review also takes into account of the immobilization of the amylases on nanomaterials to increase the stability and reusability of the enzymes. The function-based metagenomics would provide opportunities for searching amylases with novel characteristics. The review is expected to explore novel amylases for future potential applications.