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Lata, S.

Publications and source records attributed to Lata, S..

10 recordsLinked to original sources

IFN and IFN mimetics prevent IFN-I-mediated TB susceptibility by regulating iron metabolism and lipid peroxidation

Type I interferons (IFN-I) and IFN{gamma} exert divergent effects during tuberculosis, but the mechanisms that determine whether macrophage activation promotes host defense or inflammatory pathology remain incompletely understood. Here, we dissect the interplay between IFN-I and IFN{gamma} in macrophage activation using genetically susceptible B6.Sst1S macrophages. We show that, during tumor necrosis factor (TNF) stimulation, susceptible macrophages enter a persistent pathological activation state (pPAS) characterized by sustained lipid peroxidation and super-induction of IFN-I responses. This pathological state is maintained by autocrine IFN-I signaling. In contrast, IFN{gamma} priming prevents pPAS development by enhancing macrophage resilience to oxidative stress, in part through regulation of iron metabolism and induction of ferritin expression. Computational cell state transition assessment and regulation (cSTAR) analysis identified pathways and small molecules predicted to promote the transition of susceptible macrophages toward an IFN{gamma}-induced, Mtb-resistant state. Consistent with these predictions, the CDK4/6 inhibitor trilaciclib reduced lipid peroxidation by regulating iron metabolism, whereas retinoic acid signaling enhanced GPX4 expression and lipid biosynthesis programs. Combined CDK4/6 inhibition and retinoic acid receptor activation efficiently prevented the pathological activation state. Together, these findings delineate a mechanism of IFN-I/IFN{gamma} crosstalk during macrophage activation and identify pharmacologic strategies to prevent IFN-I-dominant, lipid peroxidation-driven macrophage pathology.

immunology↗

ANCHOR : Atlas of Neurochemical Characterization of the Human Brainstem with 3D Reconstruction

The human brainstem is a complex division of the brain comprised of more than 200 nuclei and fiber tracts. The brainstem is essential for the functioning of the entire body. We introduce here the most detailed human brainstem Atlas across the human lifespan: fetus, child, adult. ANCHOR, the Atlas of Neurochemical Characterization of the Human Brainstem, is an online platform that includes more than 800 serial histological sections, stained for Nissl and seven immunochemical (IHC) markers, from the human brainstem of three ages: 25 fetal gestational weeks (GW), 9 years old, and 54 years old. This makes ANCHOR the most comprehensive human brainstem Atlas to date. In these three brainstems, we identified and manually annotated over 200 structures. We further characterized these structures with the seven IHC markers. We specifically describe the catecholaminergic groups in the human brainstem across all three age groups. In addition, we identified the protoplasmic commissural dendrites of the hypoglossal nucleus and we describe the pretectal nuclei in the Nissl-stained fetal 25 GW brainstem. ANCHOR includes an online viewer that integrates multimodal data, from magnetic resonance imaging and block face imaging to Nissl- and IHC-stained serial sections and 3D reconstruction of the entire brainstem. For the 9-year-old specimen, the online viewer allows simultaneous navigation of annotated sections with corresponding IHC, for viewing the specific region-wise cellular features accessible at https://anchor.humanbrain.in/.

neuroscience↗

ECLIPSE: Exploring the dark proteome of ESKAPE pathogens through the sequence similarity network of the Protein Universe Atlas

MotivationThe accelerating crisis of antimicrobial resistance among the critical, so-called ESKAPE bacterial pathogens demands the urgent identification of novel molecular targets. However, a substantial fraction of ESKAPE proteomes remains functionally uncharacterized, with many genes annotated as encoding hypothetical proteins. These protein sequences often lack significant similarity to known protein families when using conventional homology-based annotation methods and thus remain "dark". This limits our ability to explore their role in pathogenicity, and it is thus crucial to bridge this substantial gap in pathogen biology by developing novel strategies to illuminate these "dark" regions of the ESKAPE pan-proteomes. ResultsWe introduce ECLIPSE (ESKAPE Connectome Linkage and Inference for Proteome Sequence Exploration), a network-based computational framework that systematically identifies and prioritises functionally dark protein families in ESKAPE pan-proteomes. ECLIPSE embeds target ESKAPE pathogen proteomes within the global sequence similarity network of the Protein Universe Atlas (Durairaj et al. 2023). It detects connected components composed entirely of unannotated proteins, called the "dark proteome". As a case study, we applied ECLIPSE to a pan-proteome of 3,460,657 protein sequences from 635 strains of Pseudomonas aeruginosa (PA). ECLIPSE identified 120,985 proteins (4%) residing in completely dark connected components. Furthermore, we performed a taxonomic diversity analysis using normalized Shannon indices to characterize each dark component by its enrichment in ESKAPE pathogens. The analysis utilized the evenness (E) value (see Methods 2.1), which distinguishes Pseudomonas-specific (target-specific) from ESKAPE-enriched dark components. We then developed the Dark Proteome Prioritization Score (DPPS), a composite multi-dimensional scoring framework (see Methods 2.5). It ranks these dark components by biological relevance across four orthogonal axes: (i) functional darkness, (ii) P. aeruginosa proportion in the Atlas, (iii) AMR-clade taxonomic restriction, and (iv) conservation across the 635 P. aeruginosa strains. This framework outputs a robust four-tier scoring system; the prioritized Tier I components were validated by weight sensitivity analysis and remained stable across 500 Monte Carlo weight perturbations. Structural characterization of one of the top-ranked ESKAPE-enriched dark component revealed that it belongs to the {square}-barrel fold DUF1302 (PF06980) family for which no experimentally solved three-dimensional structure exists in the PDB. The genomic context analysis indicates that it is co-localized with a LuxR-type transcriptional regulator. Collectively, ECLIPSE identifies evolutionarily conserved, structurally defined, and functionally dark proteins enriched across ESKAPE pathogens; these candidates can further facilitate the experimental characterization of dark proteins as an alternative antimicrobial target. Availability and implementationThe source code and dataset are available for free at: Github: https://github.com/surabhilata/ECLIPSE.git Zenodo: DOI: https://doi.org/10.5281/zenodo.21064323

bioinformatics↗

A conformationally heterogeneous bending pivot enables bent-to-straight transition in the central helix of mycobacterial FtsZ

Conformational changes in the central helix at the inter-domain cleft of bacterial treadmilling motor protein FtsZ are coupled to polymerization. Central helix of mycobacterial FtsZ interconverts between a bent and a straight form, with an unknown mechanism. We probed the mechanism of this conformational switching in the central helix of mycobacterial FtsZ using multi-temperature synchrotron crystallography at 20 {o}C, 30 {o}C, 37 {o}C and -173 {o}C temperatures. A comparison of the resultant crystal structures of FtsZ revealed altered conformations at the bending pivot of the bent central helix inside the inter-domain cleft. Further, ensemble modeling of FtsZ structure shows that this bending pivot is labile at near-physiological temperatures. Conformational fluctuations in this pivot region resulted in breakage of regular alpha helical hydrogen bonds that likely made the central helix easily bendable. These fluctuations are largely arrested in the straightened form of the central helix in comparison to the bent form. To summarize, multi-temperature crystallography combined with ensemble modeling suggest that conformational heterogeneity and associated perturbations of helix-forming interactions in the bending pivot can trigger bent-to-straight conformational transition in the central helix of mycobacterial FtsZ. This work demonstrates the effectiveness of multi-temperature crystallography in delineating the mechanisms of conformational changes in protein machines.

biophysics↗

Mechanistically explainable AI model for predicting synergistic cancer therapy combinations

This study introduces a Large Language Model (LLM)-based framework that combines drug combination data with a knowledge graph to predict synergistic oncology drug combinations with mechanistic insights. Using a retrieval-augmented generation (RAG) approach, over 50,000 in vitro drug pair assay results and 1,631 human clinical trial or preclinical test entries were integrated to enhance predictive accuracy and explainability. Validation achieved an F1 score of 0.80, demonstrating the frameworks potential to streamline drug discovery and improve translational strategies in cancer treatment.

cancer biology↗

Protocol for Developing a Mouse Model of Post Primary Pulmonary Tuberculosis after Hematogenous Spread in Native Lungs and Lung Implants

This protocol describes a mouse model of post-primary pulmonary tuberculosis (PTB) that develops after hematogenous spread from the primary lesion in native lungs and subcutaneous lung implants. It demonstrates that virulent Mycobacterium tuberculosis (Mtb) disseminates to lymphoid tissue in many organs, but selectively damages the lungs. This approach demonstrates a particular vulnerability of the lung tissue to virulent Mtb independent of the route of infection and provides a robust platform for examining lung-specific mechanisms driving TB pathology. HighlightsO_LIMouse model for studying mechanisms driving post-primary pulmonary TB progression in immune hosts C_LIO_LIModels a hematogenous spread of virulent Mycobacterium tuberculosis to the lungs from a primary site of infection C_LIO_LIAllows for the investigation of lung-specific mechanisms of TB susceptibility using lung tissue implants. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/640830v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@1ce5384org.highwire.dtl.DTLVardef@cd3b88org.highwire.dtl.DTLVardef@1ff7ce7org.highwire.dtl.DTLVardef@614598_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

3D Multiplexed Immunohistochemistry Using Opal-TSA Amplification for Enhanced Imaging of Pulmonary TB Lesions

Mycobacterium tuberculosis hijacks the host immune system and persists for several years before the onset of active disease. Spatial characterization of epithelial compartments, immune cell populations and bacteria simultaneously within tissue specimens provide significant information about host pathogen interactions. Here, we present a protocol to detect multiple protein markers using Opal-TSA conjugated fluorescent dyes in free floating 10% neutral buffered formalin fixed thick tissue sections (50-100 m), with minimal additional tissue processing not requiring specialized equipment. Use of thick sections provides more information as compared to classic thin microtomy sections (3-10 m), including the capacity for Z stacking and three-dimensional image rendering. Importantly, reduced tissue processing of samples with this method preserves endogenous fluorescent reporter signal. Use of Opal-TSA conjugated fluorescent dyes enhances the sensitivity of low expressing proteins and supports the use of primary antibodies raised in the same species. Before you beginThis protocol describes the specific use of Mtb infected mouse lungs, but is applicable to any tissue type and species of origin. Institutional permissionsObtain institutional permission to perform animal studies and collect tissues under an approved Institutional Animal Care and Use Committee (IACUC) or Institutional Review Board protocol. Our protocol was approved by Boston Universitys Institutional Animal Care and Use Committee (IACUC protocol number PROTO201800218). MiceB6J.C3-Sst1C3HeB/Fej Krmn and B6. Sst1S, ifnb-YFP mice were developed in our laboratory (available from MMRRC stock # 043908-UNC). HighlightsO_LICompatible with Opal-TSA conjugated fluorescent dyes resulting in enhanced signal sensitivity with low background noise C_LIO_LIReduced tissue processing preserves endogenous fluorescent reporter signals C_LIO_LICompatible with primary antibodies raised in the same species C_LIO_LIDoesnt require specialized automated instruments C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/619885v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@3e3439org.highwire.dtl.DTLVardef@1d6ba85org.highwire.dtl.DTLVardef@6f9595org.highwire.dtl.DTLVardef@1c573ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

Combining in-line chromatography coupled SAXS and AIpredicted structures to dissect the mechanism of ParB1parS1 partition assembly formation

Coupling of solution SAXS and AI-predicted structures can be a powerful strategy for delineating subtle conformational changes and self-association in protein switches. ParB, which is a condensate-forming DNA clamp that aids in bacterial chromosomal origin segregation, undergoes CTP-induced conformational switching to enable DNA sliding. The nature of parS DNA-induced conformational change in full-length ParB, and the structural features that govern self-association of ParB for partition assembly condensate formation, remains sparsely understood. We combined chromatography-coupled SAXS, rigid domains obtained from Alphafold model of ParB1 from Vibrio cholerae, and synthetic SAXS data describing known domain interfaces, to build integrative models of conformational states of full-length ParB1. These integrative models revealed how parS1 DNA loading primed ParB1 for clamping and sliding. The CTPase domains in ParB1 were moved nearer upon DNA loading to facilitate clamping, and a lumen lined with a weak DNA binding site was formed below parS1 binding site for capturing the sliding DNA. Furthermore, we showed that an N-terminal segment of ParB1 undergoes concentration-dependent oligomerization. An intrinsically disordered linker joining this oligomerization-prone N-terminal segment and the C-terminal domain curbs self-association of full-length ParB1, which is likely relevant for ParB1-mediated higher order partition assembly formation. To summarize, SAXS and Alphafold were effectively combined to provide unique insights into context-specific domain rearrangements and self-association in ParB1 for the mechanistic understanding of partition assembly formation.

biophysics↗

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure 1) to properly sequester intracellular iron and 2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled Ifn{beta} superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

immunology↗

Aberrant macrophage activation and failed regeneration of pulmonary epithelium promote tuberculosis progression uniquely in lung tissue

Pulmonary tuberculosis (PTB) represents 85% of the disease burden caused by Mycobacterium tuberculosis (Mtb) and promotes aerosol transmission infecting about a quarter of people globally. Most Mtb infections are effectively limited within primary granulomatous lesions. Containment failures lead to hematogenous spread and the formation of post-primary destructive PTB lesions. Factors that favor Mtb survival and replication in the lungs after hematogenous spread despite systemic immunity represent appealing targets for host-directed TB therapies, but are currently unknown. We developed a novel mouse model that mimics progression of chronic post-primary PTB in humans: wherein PTB lesions form after hematogenous spread from a remote primary lesion in immunocompetent but TB-susceptible B6.Sst1S mice. The B6.Sst1S mice developed PTB lesions featuring granulomatous pneumonia, bronchogenic expansion and broncho-occlusion closely resembling post-primary PTB in humans. Using spatial transcriptomic and fluorescent multiplexed immunochemistry, we demonstrated the expansion of myeloid cell populations with the appearance of alternatively activated macrophages, dissolution of initial lymphoid follicles, and accumulation of de-differentiated lung epithelial cells in the advanced PTB lesions. To determine whether lung parenchymal cells or lung oxygenation were necessary for the pulmonary TB progression, we implanted lung and spleen fragments subcutaneously to serve as potential targets for hematogenous spread. The lung (but not spleen) implants displayed characteristic organized granulomas with necrosis and Mtb replication demonstrating that deleterious interactions of aberrantly activated macrophages with the inflammation-injured lung resident cells, and possibly hypoxia, not oxygenation, are critical determinants of PTB progression in immunocompetent hosts. Necrotic TB lesions also developed in subcutaneous implants of human lung tissue in mice with human immune system after respiratory infection. These animal models may serve to further dissect the lung-specific mechanisms of host susceptibility to virulent Mtb and for testing therapeutic interventions targeting these mechanisms.

immunology↗