Biology subjects

Sharma, S.

Publications and source records attributed to Sharma, S..

At least 217 records · Page 12Linked to original sources

bioRxiv2024

MicroRNA Tae-miR1130p targets wheat ferroportin1 (TaFPN1) in the absence of iron-responsive element/iron-regulatory protein1 module

Ferroportin (FPN) belongs to the Major Facilitator Superfamily of transporters and is a known iron (Fe) exporter in humans, with orthologues also present in plant species. Human FPN is subjected to multi-level regulation at transcriptional, post-transcriptional, and post-translational levels. How plant FPNs are regulated remains to be explored. In the current study, we have characterized wheat FPN1, a plasma-membrane localized protein, for its role in Fe homeostasis. A spatial-temporal expression analysis of wheat FPN1 suggested that its tissue-specific expression is differentially upregulated during Fe deficiency conditions. Unlike human FPN, plant FPN lacks the necessary sites for hepcidin binding, thereby emphasizing the need to explore the transcriptional/post-transcriptional mode of regulation. The lack of Iron Responsive Elements (IRE) in TaFPN1 promoter suggests no direct regulation through the Iron Regulatory Protein (IRP) mechanism like in humans. Further, to explore the miRNA-mediated regulation, we identified Fe-regulated tae-miR1130b-3p capable of targeting TaFPN1 under in-vivo conditions. Transcript expression of tae-miR1130b-3p negatively correlates with the TaFPN1. This alternative regulation pathway suggests a complex network of interactions governing the expression of genes involved in iron homeostasis, highlighting the intricacies of cellular regulatory mechanisms. Altogether, the work will unravel the cellular and physiological role of wheat FPN and contribute to a comprehensive understanding of plant iron homeostasis.

plant biology↗

bioRxiv2024

Elucidating Molecular Mechanism and Chemical Space of Chalcones through Knowledge Graph and Machine Learning: A Combined Bio-Cheminformatic Pipeline

We developed a bio-cheminformatics method, exploring disease inhibition mechanisms using machine learning-enhanced quantitative structure-activity relationship (ML-QSAR) models and knowledge-driven neural networks. ML-QSAR models were developed using molecular fingerprint descriptors and the Random Forest algorithm to explore the chemical spaces of Chalcones inhibitors against diverse disease properties, including antifungal, anti-inflammatory, anticancer, antimicrobial, and antiviral effects. We generated and validated robust machine learning-based bioactivity prediction models ((https://ashspred.streamlit.app/) for the top genes. These models underwent ROC and applicability domain analysis, followed by molecular docking studies to elucidate the molecular mechanisms of the molecules. Through comprehensive neural network analysis, crucial genes such as AKT1, HSP90A1, SRC, and STAT3 were identified. The PubChem fingerprint-based model revealed key descriptors: PubchemFP521 for AKT1, PubchemFP180 for SRC, PubchemFP633 for HSP90, and PubchemFP145 and PubchemFP338 for STAT3, consistently contributing to bioactivity across targets. Notably, chalcone derivatives demonstrated significant bioactivity against target genes, with compound RA1 displaying a predictive pIC50 value of 5.76 against HSP90A and strong binding affinities across other targets. Compounds RA5 to RA7 also exhibited high binding affinity scores comparable to or exceeding existing drugs. These findings emphasize the importance of knowledge-based neural network-based research for developing effective drugs against diverse disease properties. These interactions warrant further in vitro and in vivo investigations to elucidate their potential in rational drug design. The presented models provide valuable insights for inhibitor design and hold promise for drug development. Future research will prioritize investigating these molecules for mycobacterium tuberculosis, enhancing the comprehension of effectiveness in addressing infectious diseases.

bioinformatics↗

bioRxiv2024

Molecular Insights into Zea mays Active Phytochemicals for Diabetes and Inflammation Treatment: A Web App-Based Machine Learning and Network Pharmacology Approach

Withdrawal StatementThe authors have withdrawn their manuscript owing to the inability to complete the revisions or follow up on the manuscript at this time, due to personal circumstances. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

bioinformatics↗

bioRxiv2024

Multimolecular feature-based machine learning: system biology enhanced RF-QSAR modeling for the efficient prediction of the inhibitory potential of diverse SARS CoV-2 3CL Protease inhibitors

bioinformatics↗

bioRxiv2024

Discovering Fragile Clades And Causal Sequences In Phylogenomics By Evolutionary Sparse Learning

Phylogenomic analyses of long sequences, consisting of many genes and genomic segments, infer organismal relationships with high statistical confidence. But, these relationships can be sensitive to excluding just a few sequences. Currently, there is no direct way to identify fragile relationships and the associated individual gene sequences in species. Here, we introduce novel metrics for gene-species sequence concordance and clade probability derived from evolutionary sparse learning models. We validated these metrics using fungi, plant, and animal phylogenomic datasets, highlighting the ability of the new metrics to pinpoint fragile clades and the sequences responsible. The new approach does not necessitate the investigation of alternative phylogenetic hypotheses, substitution models, or repeated data subset analyses. Our methodology offers a streamlined approach to evaluating major inferred clades and identifying sequences that may distort reconstructed phylogenies using large datasets.

evolutionary biology↗

bioRxiv2024

Cryo- EM structure of ribosome from pathogenic protozoa Entamoeba histolytica, reveals unique features of its architecture

Entamoeba histolytica, an anaerobic parasite protozoan, is the causative agent of amoebiasis, the bloody diarrhea, and liver abscesses in humans. Amoebiasis is more predominant in tropical areas with poor sanitation conditions, and it remains the fourth leading cause of death due to a protozoan infection. E. histolytica life cycle spans between an infective cyst stage and an active disease-causing trophozoite stage. We have determined cryo-EM structures of E. histolytica ribosomes, large subunit (LSU), 53S ribosome at 2.8 [A] resolution and associated, 75S ribosome at 3.3 [A] resolution, isolated from its trophozoite stage. The overall core of the ribosome is conserved. However, the periphery has evolved with Entamoeba specific unique features. The most notable features are, the presence of the rRNA triple helix near the peptide exit tunnel on LSU and the co-evolution of rRNA expansion segments and extensions in r-proteins. To the best of our knowledge, this structure reports the presence of RNA triple helix in the ribosome for the first time.

biochemistry↗

bioRxiv2024

STING promotes homeostatic maintenance of tissues and confers longevity with aging

Local immune processes within aging tissues are a significant driver of aging associated dysfunction, but tissue-autonomous pathways and cell types that modulate these responses remain poorly characterized. The cytosolic DNA sensing pathway, acting through cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING), is broadly expressed in tissues, and is poised to regulate local type I interferon (IFN-I)-dependent and independent inflammatory processes within tissues. Recent studies suggest that the cGAS/STING pathway may drive pathology in various in vitro and in vivo models of accelerated aging. To date, however, the role of the cGAS/STING pathway in physiological aging processes, in the absence of genetic drivers, has remained unexplored. This remains a relevant gap, as STING is ubiquitously expressed, implicated in multitudinous disorders, and loss of function polymorphisms of STING are highly prevalent in the human population (>50%). Here we reveal that, during physiological aging, STING-deficiency leads to a significant shortening of murine lifespan, increased pro-inflammatory serum cytokines and tissue infiltrates, as well as salient changes in histological composition and organization. We note that aging hearts, livers, and kidneys express distinct subsets of inflammatory, interferon-stimulated gene (ISG), and senescence genes, collectively comprising an immune fingerprint for each tissue. These distinctive patterns are largely imprinted by tissue-specific stromal and myeloid cells. Using cellular interaction network analyses, immunofluorescence, and histopathology data, we show that these immune fingerprints shape the tissue architecture and the landscape of cell-cell interactions in aging tissues. These age-associated immune fingerprints are grossly dysregulated with STING-deficiency, with key genes that define aging STING- sufficient tissues greatly diminished in the absence of STING. Changes in immune signatures are concomitant with a restructuring of the stromal and myeloid fractions, whereby cell:cell interactions are grossly altered and resulting in disorganization of tissue architecture in STING-deficient organs. This altered homeostasis in aging STING-deficient tissues is associated with a cross-tissue loss of homeostatic tissue-resident macrophage (TRM) populations in these tissues. Ex vivo analyses reveal that basal STING- signaling limits the susceptibility of TRMs to death-inducing stimuli and determines their in situ localization in tissue niches, thereby promoting tissue homeostasis. Collectively, these data upend the paradigm that cGAS/STING signaling is primarily pathological in aging and instead indicate that basal STING signaling sustains tissue function and supports organismal longevity. Critically, our study urges caution in the indiscriminate targeting of these pathways, which may result in unpredictable and pathological consequences for health during aging. HIGHLIGHTSO_LIAging tissues are associated with tissue-autonomous immune fingerprints, primarily driven by interactions of tissue stromal and myeloid populations. C_LIO_LISTING shapes these immune fingerprints of aging tissues in unexpected ways. C_LIO_LILoss of STING alters the location, numbers, and viability of tissue resident macrophages. C_LIO_LISTING signaling is critical for longer lifespans and maintenance of tissue architecture. C_LI

immunology↗

bioRxiv2024

A pathogenic role for IL-10 signalling in capillary stalling and cognitive impairment in type 1 diabetes

Vascular pathology is associated with cognitive impairment in diseases such as type 1 diabetes, but precisely how capillary flow is affected and the underlying mechanisms remain elusive. Here we show that capillaries in the diabetic mouse brain are prone to stalling, with blocks composed primarily of erythrocyte plugs in branches off penetrating venules. Increased capillary obstructions were evident in both sexes and only partially reversed by insulin. Screening for circulating inflammatory cytokines revealed persistently high levels of interleukin-10 (IL-10) in diabetic mice. Contrary to expectation, stimulating IL-10 signalling increased capillary obstructions, whereas inhibiting IL-10 receptors with neutralizing antibodies or endothelial specific knockdown in diabetic mice, reversed these impairments. Chronic IL-10R blocking antibody treatment in diabetic mice also improved stimulus evoked cerebral blood flow, increased capillary widths in lower-order branches and reversed cognitive deficits. These data suggest IL-10 signalling plays an unexpected pathogenic role in cerebral microcirculatory defects and cognitive impairment.

neuroscience↗

bioRxiv2024

Flame retardant tetrabromobisphenol A (TBBPA) disrupts histone acetylation during zebrafish maternal-to-zygotic transition

3,3,5.5-Tetrabromobisphenol A (TBBPA) is a widely used brominated flame-retardant utilized in the production of electronic devices and plastic paints. The objective of this study is to use zebrafish as a model and determine the effects of TBBPA exposure on early embryogenesis. We initiated TBBPA exposures (0, 10, 20 and 40M) at 0.75 h post fertilization (hpf) and monitored early developmental events such as cleavage, blastula and epiboly that encompass maternal-to-zygotic transition (MZT) and zygotic genome activation (ZGA). Our data revealed that TBBPA exposures induced onset of developmental delays by 3 hpf (blastula). By 5.5 hpf (epiboly), TBBPA-exposed (10-20 M) embryos showed concentration-dependent developmental lag by up to 3 stages or 100% mortality at 40 M. Embryos exposed to sublethal TBBPA concentrations from 0.75-6 hpf and raised in clean water to 120 hpf showed altered larval photomotor response (LPR), suggesting a compromised developmental health. To examine the genetic basis of TBBPA-induced delays, we conducted mRNA-sequencing on embryos exposed to 0 or 40 M TBBPA from 0.75 hpf to 2, 3.5 or 4.5 hpf. Read count data showed that while TBBPA exposures had no overall impacts on maternal or maternal-zygotic genes, collective read counts for zygotically activated genes were lower in TBBPA treatment at 4.5 hpf compared to time-matched controls, suggesting that TBBPA delays ZGA. Gene ontology assessments for both time- and stage-matched differentially expressed genes revealed TBBPA-induced inhibition of chromatin assembly- a process regulated by histone modifications. Since acetylation is the primary histone modification system operant during early ZGA, we immunostained embryos with an H3K27Ac antibody and demonstrated reduced acetylation in TBBPA-exposed embryos. Leveraging in silico molecular docking studies and in vitro assays, we also showed that TBBPA potentially binds to P300- a protein that catalyzes acetylation- and inhibits P300 activity. Finally, we co-exposed embryos to 20 M TBBPA and 50 M n-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) -a histone acetyltransferase activator that promotes histone acetylation- and showed that TBBPA-CTPB co or pre-exposures significantly reversed TBBPA-only developmental delays, suggesting that TBBPA-induced phenotypes are indeed driven by repression of histone acetylation. Collectively, our work demonstrates that TBBPA disrupts ZGA and early developmental morphology, potentially by inhibiting histone acetylation. Future studies will focus on mechanisms of TBBPA-induced chromatin modifications.

pharmacology and toxicology↗

bioRxiv2024

Inflammasome-Inhibiting Nanoligomers are Neuroprotective Against Space-Induced Pathology in Healthy and Diseased 3D Human Motor and Pre-Frontal Cortex Brain Organoids

Microgravity and space environment has been linked to deficits in neuromuscular and cognitive capabilities, hypothesized to occur due to accelerated aging and neurodegeneration in space. While the specific mechanisms are still being investigated, spaceflight-associated neuropathology is an important health risk to space astronauts and tourists, and is being actively investigated for the development of appropriate countermeasures. However, such space-induced neuropathology offers an opportunity for accelerated screening of therapeutic targets and lead molecules for treating neurodegenerative diseases. Here we show, a proof-of-concept high-throughput target screening (on Earth), target validation, and mitigation of microgravity-induced neuropathology using our Nanoligomer platform, onboard the 43-day SpaceX CRS-29 mission to the International Space Station (ISS). First, comparing 3D healthy and diseased pre-frontal cortex (PFC, for cognition) and motor neuron (MN, for neuromuscular function) organoids, we assessed space-induced pathology using biomarkers relevant to Alzheimers Disease (AD), Frontotemporal Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Both healthy and diseased PFC and MN organoids showed significantly enhanced neurodegeneration in space, as measured through relevant disease biomarkers, when compared to their respective Earth controls. Second, we tested the top two lead molecules, NI112 which targeted NF-{kappa}B, and NI113 that targeted IL-6. We observed that these Nanoligomers significantly mitigate the AD, FTD, and ALS relevant biomarkers like amyloid beta-42 (A{beta}42), phosphorylated Tau (pTau), Kallikrein (KLK-6), Tar DNA-binding protein 43 (TDP-43), and others. Moreover, the 43-day Nanoligomer treatment of these brain organoids did not appear to cause any observable toxicity or safety issues in the target organoid tissue, suggesting good tolerability for these molecules in the brain at physiologically relevant doses. Together, these results show significant potential for both the development and translation of NI112 and NI113 molecules as potential neuroprotective countermeasures for safer space travel, and demonstrate the usefulness of the space environment for rapid, high-throughput screening of targets and lead molecules for clinical translation. We assert that the use of microgravity in drug development and screening may ultimately benefit millions of patients suffering from debilitating neurodegenerative diseases on Earth.

neuroscience↗

bioRxiv2024

Brain-Penetrant NF-κB and NLRP3 Targeting Nanoligomers are Therapeutic in Amyotrophic Lateral Sclerosis (ALS) and Alzheimers Disease (AD) Human Organoid and Mouse Models

Millions of people suffer worldwide from neurodegenerative diseases ranging from rapidly progressing and fatal motor neuron diseases like Amyotrophic Lateral Sclerosis (ALS) to more chronic illnesses such as frontotemporal dementia (FTD) and Alzheimers disease (AD). A growing number of studies have implicated neuroinflammation as a key and causative phenomenon and an important target for novel therapeutics for these diseases. Neuroinflammation is characterized by reactive glial cells that produce pro-inflammatory neurotoxic cytokines. Our previous studies have shown a brain-penetrant Nanoligomer cocktail (NI112) inhibiting the neuroinflammation mediators nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) is a safe, targeted, and effective neurotherapeutic drug. Here, we show that a four-week NI112 treatment is therapeutic using: 1) an ALS-FTD 3D human motor neuron organoid model of tar DNA binding protein 43 (TDP-43, a key contributor to ALS pathology) overexpression (knock-in); 2) an AD model of APOE4/APOE4 (AD risk allele) double mutation in human neurons comprising a 3D human prefrontal cortex (PFC) organoid; and 3) multiple in vivo (mouse models) of the same/related conditions. In 3D organoids made from healthy motor neurons (HMN negative control) and TDP-43 overexpressing (or ALS organoids), we monitored the mean firing rate using calcium signaling as a functional output, while measuring TDP-43 and other key neurodegeneration biomarkers. After 4 weeks, we observed a massive improvement in the mean firing rate of NI112-treated ALS organoids compared to untreated ALS organoids, which was more comparable to healthy HMN organoids. Similarly, we found a significant decrease in neurodegeneration markers like amyloid beta 42 (A{beta}42) in NI112-treated AD organoids compared to untreated AD organoids (A{beta}42 comparable to healthy PFC organoids). In the mouse ALS (SOD1-G93A) model, we observed behavioral improvements and restoration of motor function (e.g., grip strength) in NI112-treated mice, and in mouse AD model mice (radiation-induced accelerated neuropathology in APP/PS1, and rTg4510 phospho-tau), we observed improved cognition. In both models, we also found an accompanying reduction in neuroinflammation and reduced neuropathology. These results show the promise for further testing and development of neuroinflammation-targeting Nanoligomers to benefit patients suffering from debilitating neurodegenerative diseases like ALS, FTD, and AD.

neuroscience↗

bioRxiv2024

Insight into the regulatory mechanism of the MFS transporter, SCO4121 by the MarR regulator, SCO4122

MarR group of transcriptional regulators are ubiquitous in bacteria and found to be involved in regulation of efflux pumps that confer multidrug resistance phenotype. While most characterized MarR regulators act as transcriptional repressors, we earlier identified a MarR regulator SCO4122 in Streptomyces coelicolor, playing an essential role in transcriptional activation of the MFS transporter SCO4121 in response to multiple substrates of the latter, including streptomycin, ciprofloxacin and chloramphenicol. In this study, using Surface Plasmon Resonance, we demonstrate that SCO4122 interacts directly with the diverse substrates of SCO4121 with the highest affinity for streptomycin with a KD of 0.73 M. Further, in-vitro and in-vivo studies reveal that SCO4122 also binds to the intergenic region between sco4121 and sco4122, where the interaction is dependent on the cooperative binding of SCO4122 to three motifs in this region. A conserved Methionine, M93, in SCO4122 is identified to be an integral amino acid residue that is involved in activation of SCO4121 in response to ciprofloxacin, streptomycin and EtBr but not chloramphenicol. Furthermore, our studies also indicate that upon binding to different substrates, the affinity of SCO4122 to the sco4121 promoter increases 50-1000 fold, thereby leading to enhanced expression of the transporter, SCO4121. This study thus highlights that SCO4122 is a novel MarR regulator that functions as a strong transcriptional activator of an efflux pump, SCO4121, through intricate molecular mechanisms in presence of structurally dissimilar substrates. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/577416v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3394e2org.highwire.dtl.DTLVardef@73afb1org.highwire.dtl.DTLVardef@113c724org.highwire.dtl.DTLVardef@1772ac9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Resolvin D2/GPR18 signaling enhances monocytic myeloid-derived suppressor cell function to mitigate abdominal aortic aneurysm formation

Abdominal aortic aneurysm (AAA) formation is a chronic vascular pathology characterized by inflammation, leukocyte infiltration and vascular remodeling. The aim of this study was to delineate the protective role of Resolvin D2 (RvD2), a bioactive isoform of specialized proresolving lipid mediators, via G-protein coupled receptor 18 (GPR18) receptor signaling in attenuating AAAs. Importantly, RvD2 and GPR18 levels were significantly decreased in aortic tissue of AAA patients compared with controls. Furthermore, using an established murine model of AAA in C57BL/6 (WT) mice, we observed that treatment with RvD2 significantly attenuated aortic diameter, pro-inflammatory cytokine production, immune cell infiltration (neutrophils and macrophages), elastic fiber disruption and increased smooth muscle cell -actin expression as well as increased TGF-{beta}2 and IL-10 expressions compared to untreated mice. Moreover, the RvD2-mediated protection from vascular remodeling and AAA formation was blocked when mice were previously treated with siRNA for GPR18 signifying the importance of RvD2/GPR18 signaling in vascular inflammation. Mechanistically, RvD2-mediated protection significantly enhanced infiltration and activation of monocytic myeloid-derived suppressor cells (M-MDSCs) by increasing TGF-{beta}2 and IL-10 secretions that mitigated smooth muscle cell activation in a GPR18-dependent manner to attenuate aortic inflammation and vascular remodeling via this intercellular crosstalk. Collectively, this study demonstrates RvD2 treatment induces an expansion of myeloid-lineage committed progenitors, such as M-MDSCs, and activates GPR18-dependent signaling to enhance TGF-{beta}2 and IL-10 secretion that contributes to resolution of aortic inflammation and remodeling during AAA formation.

immunology↗

bioRxiv2024

CanLncG4: A database curated for the assessment of G4s in the lncRNAs dysregulated in various human cancers

Long non-coding RNAs (lncRNAs) comprise a substantive part of the human genome and have emerged as crucial participants of cellular processes and disease pathogenesis. Dysregulated expression of lncRNAs in cancer contributes to various hallmarks of the disease, presenting novel opportunities for diagnosis and therapy. G-quadruplexes (G4s) within lncRNAs have gained attention, though their systematic evaluation in cancer biology is yet to be performed. In this work, we have formulated CanLncG4, a comprehensive database integrating experimentally validated associations between lncRNAs and cancer, and detailed predictions of their G4-forming potential. CanLncG4 categorizes predicted G4 motifs into anticipated G4 types and offers insights into the subcellular localization of the corresponding lncRNAs. It provides information on lncRNA-RNA and lncRNA-protein interactions, together with the RNA G4-binding capabilities of these proteins. To ensure the accuracy and validity of the data sourced from various databases, a meticulous examination of the output data was conducted to identify any discrepancies, including incorrect, missing, or duplicate entries. Additionally, scientific literature mining was performed to cross-validate the gathered information. Data from G4-prediction tools was generated using multiple parameter combinations to determine the parameters that yield more relevant and accurate predictions of the G4-forming potential. We validate our in silico G4-prediction pipeline through in vitro experiments, affirming the presence of G4s within specific cancer-dysregulated lncRNAs, thereby illustrating the predictive capability of CanLncG4. CanLncG4 represents a valuable resource for investigating G4-mediated lncRNA functions in diverse human cancers. It is expected to provide distinctive leads about G4-mediated lncRNA-protein interactions. CanLncG4 comprehensively documents 17,666 entries, establishing correlations between 6,408 human lncRNAs encompassing their transcript variants, and 15 distinct types of human cancers. The database is freely available at https://canlncg4.com/, offering researchers a valuable tool for exploring lncRNA and G4 biology towards cancer diagnosis and therapeutics.

bioinformatics↗

bioRxiv2024

Microbiome- and Host Inflammasome-Targeting Inhibitor Nanoliogmers are Therapeutic in Murine Colitis Model

Autoimmune and autoinflammatory diseases account for more than 80 chronic conditions affecting more than 24 million people in the US. Amongst these autoinflammatory diseases, non-infectious chronic inflammation of the gastrointestinal (GI) tract causes inflammatory bowel diseases (IBD), primarily Crohns and Ulcerative Colitis (UC). IBD is a complex disease, and one hypothesis is that these are either caused or worsened by compounds produced by bacteria in the gut. While traditional approaches have focused on pan immunosuppressive techniques (e.g., steroids), low remission rates, prolonged illnesses, and increased frequency of surgical procedures have prompted the search for more targeted and precision therapeutic approaches. IBD is a complex disease resulting from both genetic and environmental factors, but several recent studies have highlighted the potential pivotal contribution of gut microbiota dysbiosis. Gut microbiota are known to modulate the immune status of the gut by producing metabolites that are encoded in biosynthetic gene clusters (BGCs) of the bacterial genome. Here we show, a targeted and high-throughput screening of more than 90 biosynthetic genes in 41 gut anaerobes, through down selection using available bioinformatics tools, targeted gene manipulation in these genetically intractable organisms using Nanoligomer platform, and identification and synthesis of top microbiome-targets as a Nanoligomer BGC cocktail (SB_BGC_CK1, abbreviated as CK1) as a feasible precision therapeutics approach. Further, we used a host-directed immune-target screening to identify NF-{kappa}B and NLRP3 cocktail SB_NI_112 (or NI112 for short) as a targeted inflammasome inhibitor. We used these top two microbe- and host-targeted Nanoligomer cocktails in acute and chronic dextran sulfate sodium (DSS) mouse colitis and in TNF{Delta}ARE/+ transgenic mice that develop spontaneous Crohns like ilitis. The mouse microbiome was humanized to replicate that in human IBD through antibiotic treatment followed by mixed fecal gavage from 10 human donors and spiked with IBD-inducing microbial species. Following colonization, colitis was induced in mice using one week of 3% DSS (acute), or six weeks of 3 rounds of 2.5% DSS induction for a week followed by one week of no DSS (chronic colitis model). Both Nanoligomer cocktails (CK1 and NI112) showed a strong reduction in disease severity, significant improvement in disease histopathology, and profound downregulation of disease biomarkers in colon tissue as assessed by multiplexed ELISA. Further, we used two different formulations of intraperitoneal injections (IP) and Nanoligomer pills in the chronic DSS colitis model. Although both formulations were highly effective, the oral pill formulation demonstrated a greater reduction in biochemical markers compared to IP. A similar therapeutic effect was observed in the TNF{Delta}ARE/+ model. Overall, these results point to the potential for further development and testing of these inflammasome-targeting host-directed therapy (NI112), and more personalized microbiome-cocktails (CK1) for patients with recalcitrant IBD. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/581256v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@834b11org.highwire.dtl.DTLVardef@1b5539corg.highwire.dtl.DTLVardef@1844356org.highwire.dtl.DTLVardef@60f51e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC GRAPHICC_FLOATNO C_FIG

molecular biology↗

bioRxiv2024

Proteomic profiling of intracranial atherosclerotic plaque in the human brain

BackgroundIntracranial atherosclerotic disease (ICAD) is one of the major causes of ischemic stroke and associated with high risk of stroke recurrence. There are no reliable and specific fluid biomarkers for ICAD, and little is known about the proteomic profiling of ICAD. In this study we aimed to explore the feasibility of applying proteomics technology to profile intracranial atherosclerotic plaques extracted from postmortem human brain arteries. MethodsEighteen segments (5-10mm in length) of major arteries from 10 postmortem brains were collected from the Mount Sinai Neuropathology Brain Bank. Among these segments, 5 had no evidence of atherosclerotic disease, and 13 had wall thickening or visible plaques with various degree of stenosis. Proteins were extracted from the vessel segments, quantified, and digested into peptides. Subsequently, the peptides underwent tandem mass tag (TMT) labeling, pooling, and analysis using two-dimensional liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS). Protein identification and quantification were performed using the JUMP software. Differentially expressed proteins (DEPs) were defined as proteins with p.adj < 0.05 and absolute log2 (fold change) > log2 (1.2). ResultsA total of 7,492 unique proteins were detected, and 6,726 quantifiable proteins were retained for further analysis. Among these, 265 DEPs, spanning on 252 unique gene, were found to be associated with ICAD by comparing the arterial segments with vs those without atherosclerotic disease. The top 4 most significant DEPs include LONP1, RPS19, MRPL12 and SNU13. Among the top 50 DEPs, FADD, AIFM1 and PGK1 were associated with atherosclerotic disease or cardiovascular events in previous studies. Moreover, the previously reported proteins associated with atherosclerosis such as APCS, MMP12, CTSD were elevated in arterial segments with atherosclerotic changes. Furthermore, the up-regulation of APOE and LPL, the ICAD GWAS risk genes, was shown to be associated with the plaque severity. Finally, gene set enrichment analysis revealed the DEP signature is enriched for biological pathways such as chromatin structure, plasma lipoprotein, nucleosome, and protein-DNA complex, peroxide catabolic and metabolic processes, critical in ICAD pathology. ConclusionsDirect proteomic profiling of fresh-frozen intracranial artery samples by MS-based proteomic technology is a feasible approach to identify ICAD-associated proteins, which can be potential biomarker candidates for ICAD. Further plaque proteomic study in a larger sample size is warranted to uncover mechanistic insights into ICAD and discover novel biomarkers that may help to improve diagnosis and risk stratification in ICAD.

systems biology↗

bioRxiv2024

DNA methylation-dependent and -independent binding of CDX2 directs activation of distinct developmental and homeostatic genes

Precise spatiotemporal and cell type-specific gene expression is essential for proper tissue development and function. Transcription factors (TFs) guide this process by binding to developmental stage-specific targets and establishing an appropriate enhancer landscape. In turn, DNA and chromatin modifications direct the genomic binding of TFs. However, how TFs navigate various chromatin features and selectively bind a small portion of the millions of possible genomic target loci is still not well understood. Here we show that Cdx2 - a pioneer TF that binds distinct targets in developing versus adult intestinal epithelial cells - has a preferential affinity for a non-canonical CpG-containing motif in vivo. A higher frequency of this motif at embryonic and fetal Cdx2 target loci and the specifically methylated state of the CpG during development allows selective Cdx2 binding and activation of developmental enhancers and linked genes. Conversely, demethylation at these enhancers prohibits ectopic Cdx2 binding in adult cells, where Cdx2 binds its canonical motif without a CpG. This differential Cdx2 binding allows for corecruitment of Ctcf and Hnf4, facilitating the establishment of intestinal superenhancers during development and enhancers mediating adult homeostatic functions, respectively. Induced gain of DNA methylation in the adult mouse epithelium or cultured cells causes ectopic recruitment of Cdx2 to the developmental target loci and facilitates cobinding of the partner TFs. Together, our results demonstrate that the differential CpG motif requirements for Cdx2 binding to developmental versus adult target sites allow it to navigate different DNA methylation profiles and activate cell type-specific genes at appropriate times. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/579850v3_ufig1.gif" ALT="Figure 1"> View larger version (99K): org.highwire.dtl.DTLVardef@12d2253org.highwire.dtl.DTLVardef@289371org.highwire.dtl.DTLVardef@1e81b4borg.highwire.dtl.DTLVardef@157e322_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Large- and Small-Animal Studies of Safety, Pharmacokinetics (PK), and Biodistribution of Inflammasome-Targeting Nanoligomer in the Brain and Other Target Organs

Immune malfunction or misrecognition of healthy cells and tissue, termed autoimmune disease, is implicated in more than 80 disease conditions and multiple other secondary pathologies. While pan-immunosuppressive therapies like steroids offer some relief for systemic inflammation for some organs, many patients never achieve remission and such drugs do not cross the blood-brain barrier making them ineffective for tackling neuroinflammation. Especially in the brain, unintended activation of microglia and astrocytes is hypothesized to be directly or indirectly responsible for Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Parkinsons Disease (PD), and Alzheimers Disease (AD). Recent studies have also shown that targeting inflammasome and specific immune targets can be beneficial for these diseases. Further, our previous studies have shown targeting NF-{kappa}B and NLRP3 through brain penetrant Nanoligomer cocktail SB_NI_112 (abbreviated to NI112) can be therapeutic for several neurodegenerative diseases. Here we show safety-toxicity studies, followed by pharmacokinetics (PK) and biodistribution in small- (mice) and large-animal (dog) studies of this inflammasome-targeting Nanoligomer cocktail NI 112. We conducted studies using four different routes of administration: intravenous (IV), subcutaneous (SQ), intraperitoneal (IP), and intranasal (IN), and identified the drug concentration over time using inductively coupled plasma mass spectrometry (ICP-MS) in the blood serum, the brain (including different brain regions), and other target organs like liver, kidney, and colon. Our results indicate the Nanoligomer cocktail has a strong safety profile, and shows high biodistribution (F [~]0.98) and delivery across multiple routes of administration. Further analysis showed high brain bioavailability with a ratio of NI112 in brain tissue to blood serum [~]30%. Our model accurately shows dose scaling, translation between different routes of administration, and interspecies scaling. These results provide an excellent platform for human clinical translation and predicting therapeutic dosage between different routes of administration.

pharmacology and toxicology↗