Biology subjects

Pradhan, P.

Publications and source records attributed to Pradhan, P..

17 recordsLinked to original sources

bioRxiv2026

An R-Based Adaptive Quadtree Spatial Tiling Workflow for Boundary-Exact GBIF Species Occurrence Mining within User-Defined KML Polygons

Global Biodiversity Information Facility (GBIF) occurrence retrievals for an irregularly shaped region are limited by the API spatial query capabilities - rectangular envelopes or size/vertex-limited WKT polygons - neither of which conform to protected areas, sacred groves, wetlands, panchayat or municipal boundaries or any other arbitrary KML polygon of interest queried by users. This paper presents and validates an open, self-contained, adaptive spatial-tiling protocol that (i) ingests any KML polygon of any shape, size and location on earth, breaks it into a set of GBIF API-compatible rectangular tiles, (ii) queries, cleans and clips the individual records to the target polygon, and (iii) summarises the inventory with a generic diversity-completeness-rarefaction module, with minimal manual re-parameterisation between sites. The protocol implements an iterative quadtree refinement algorithm that adapts tile number, size and location to the target polygon geometry, is combined with a fault-tolerant pagination/retry query system, a boundary-exact two-step clipping procedure and a Chao1-based completeness assessment to ensure statistical comparability between sites of different spatial extent and sampling intensity. The algorithm is implemented in open R source (sf, terra, rgbif, tidyverse) with the tiling algorithm controlled by the four parameters only (initial cell size, area floor, tile overlap threshold, recursion limit), with default settings on a new site by simply changing the input file path. This paper describes in detail its five main components - (i) polygon input and validation, (ii) quadtree adaptive tiling, (iii) polygon coverage verification, (iv) tile-wise GBIF query with retry/shrink pagination and partial data retention, (v) boundary-exact deduplication, clipping and diversity estimation. A downstream generic module estimates diversity, Chao1 richness/completeness and Hurlbert rarefaction, for each taxonomic rank and generates rank-ordered diversity tables as output. The generalisability of algorithm to multiple sites has been demonstrated with second polygon (Sonamukhi Sal forest dominated stretch, Bankura district, West Bengal; approx. 610 sq km) that differs from the first (Bishnupur Sal forest dominated stretch; 938 sq km) in both size and complexity (10 vs 34 KML vertices) and report the tiling and diversity metrics comparable results across the two polygons. With no parameter changes, the algorithm generated 135 adaptive query tiles for Sal forest dominated stretch adjoining Bishnupur, and 86 tiles for Sal forest dominated stretch Sonamukhi SDFP, covering completely the area of both polygons. The number of tiles per 100 sq km is comparable between the two runs (14.4 vs 14.1 tiles) despite the 35% difference in polygon size and 3.4x vertex count. The tile-wise querying with retry/shrink pagination retrieved 6,169 GBIF records (excluding errors) with boundary-exact clipping across 404 species for Bishnupur and 1,222 GBIF records (excluding errors) across 271 species for Sonamukhi; the generic diversity module processed the records without further parameter changes and generated comparable metrics for each rank at both sites. The protocol addresses a general bioinformatic challenge in polygon-based GBIF queries, is provided as an open, reusable, documented method which has been validated on two sites. Because the protocol has so far been validated on only two polygons that differ markedly in size, shape and observer regime, it may be regarded as an initial cross-site validation rather than a comprehensive benchmark, and recommend testing on a broader, globally distributed set of polygons before the approach is treated as a general-purpose standard.

ecology↗

bioRxiv2026

Protein import-driven mitochondrial hyperactivation dictates angiogenesis independently of HIF-1α

Angiogenesis is essential for sustained neoplastic progression, yet its initiation has largely been framed through hypoxia-dependent HIF signalling. In OXPHOS driven tumours, how mitochondrial function contributes to angiogenic induction remains poorly understood. Here, we uncover a non-canonical mechanism linking tumour cell mitochondrial hyperactivity to a pro-angiogenic response. Mitochondrial function critically depends on the import of nuclear-encoded proteins, largely mediated by TIM23 translocase. We found Timm44, the central scaffold of this translocase, to be broadly upregulated across angiogenesis-dependent malignancies. Using gain-of-function system that recapitulate tumour-associated Timm44 amplification, we enhanced mitochondrial protein import capacity, thereby remodelling mitochondrial gene expression and driving hyperactivation of the electron transport chain, particularly Complex I. This hyperactive mitochondrial state generated a signalling-competent ROS-enrich environment that induces VEGFA expression independently of HIF-1 stabilization, via a redox-licensed TRX-ASK1-p38MAPK-SP1 axis, thereby promoting endothelial sprouting and proliferation. These findings define protein import-driven mitochondrial hyperfunction as an alternative driver of tumour angiogenesis, extending current paradigms beyond classical hypoxia-dependent mechanisms and highlighting new therapeutic vulnerabilities.

cancer biology↗

bioRxiv2025

Harnessing Lytic Phages for Biofilm Control in Carbapenem-Resistant Klebsiella pneumoniae Causing Urinary Tract Infection

BackgroundKlebsiella pneumoniae is a major opportunistic pathogen with rising multidrug resistance and biofilm-related infections. Molecular and phage characterization is crucial to understand resistance mechanisms and explore alternative therapies such as phage therapy. MethodsWe performed whole-genome sequencing and antibiotic susceptibility testing of hospital-isolated Klebsiella pneumoniae (KP6697). MLST, plasmid replicon analysis, and resistance gene identification were conducted using bioinformatics. Phage isolation, electron microscopy-based morphological and biofilm analysis, and evaluation of lytic activity, stability, and host range were performed. Phage genome sequencing and annotation identified functional genes. ResultsThe host strain Klebsiella pneumoniae (KP6697) was multidrug-resistant, exhibiting resistance to 18 of 22 tested antibiotics, and genome analysis identified ST16 with eight plasmid replicons and 23 resistance genes, including blaCTX-M-15, blaNDM-5, and blaOXA-181. Functional annotations revealed extensive metabolic versatility and a rich repertoire of genes for biofilm formation, quorum sensing, secretion systems, and stress response. A lytic phage, Phage_KP6697_Omshanti, was isolated and classified as a Caudoviricetes member with a 45.3kb genome encoding lysis, replication, and structural genes. It demonstrated short latency, high burst size, thermal and pH stability, and broad host range against CRKP and other MDR strains. Importantly, microscopy confirmed its ability to inhibit and degrade biofilms at multiple stages, highlighting strong therapeutic potential. ConclusionComprehensive analysis of carbapenem-resistant K. pneumoniae (KP6697) revealed multidrug resistance and strong biofilm formation. The lytic phage Phage_KP6697_Omshanti, with depolymerase and endolysin activity, disrupted biofilms, and its stability, high burst size, and genomic traits suggest potential as an anti-CRKP agent, especially with antibiotics IMPORTANCEKlebsiella pneumoniae is increasing multidrug resistance and robust biofilm formation pose severe clinical challenges, limiting treatment options. Understanding the molecular basis of its resistance and exploiting bacteriophages with strong biofilm-disrupting properties provide promising alternative therapeutic strategies. This study highlights the isolation and genomic characterization of a lytic phage with potent anti-biofilm activity against carbapenem-resistant K. pneumoniae, underscoring its potential in combating resistant infections.

microbiology↗

bioRxiv2025

NTCmatch: R package for searching synonyms of plants listed under list of Normally Traded as Commodities

Equitable sharing of benefits from the commercial use of biological resources is a cornerstone of the Convention on Biological Diversity (CBD). This principle is pivotal in advancing the other two CBD objectives: conserving biological diversity and its sustainable use. India, as a CBD signatory, enacted the Biological Diversity Act in 2002 (amended in 2023) and established a three-tiered implementation system involving the National Biodiversity Authority (NBA), State Biodiversity Boards (SBBs), and Union Territory Biodiversity Councils (UTBCs). A critical regulatory framework within this system is the Access and Benefit Sharing (ABS) mechanism, guided by the Nagoya Protocol and NBA guidelines. This study addresses challenges in the identification and processing of biological resources listed as Normally Traded as Commodities (NTC), particularly when discrepancies in scientific nomenclature or taxonomic errors are present. We developed an R package, NTCmatch, designed to facilitate the identification of plant synonyms within the NTC list, enhancing the accuracy and efficiency of the ABS mechanism. The package was created using devtools and roxygen2, with readxl for data management. Results demonstrate the utility of NTCmatch in synonym matching, although the manual verification of plant parts and sources remains crucial. The package aims to streamline workflows for NBA, SBBs, and UTBCs, ensuring compliance with the Biological Diversity Act and facilitating the equitable sharing of benefits.

ecology↗

bioRxiv2025

Decoding Breast Cancer in X-ray Mammograms: A Multi-Parameter Approach Using Fractals, Multifractals, and Structural Disorder Analysis

We explored the fractal and multifractal characteristics of breast mammogram micrographs to identify quantitative biomarkers associated with breast cancer progression. In addition to conventional fractal and multifractal analyses, we employed a recently developed fractal-functional distribution method, which transforms fractal measures into Gaussian distributions for more robust statistical interpretation. Given the sparsity of mammogram intensity data, we also analyzed how variations in intensity thresholds, used for binary transformations of the fractal dimension, follow unique trajectories that may serve as novel indicators of disease progression. Our findings demonstrate that fractal, multifractal, and fractal-functional parameters effectively differentiate between benign and cancerous tissue. Furthermore, the threshold-dependent behavior of intensity-based fractal measures presents distinct patterns in cancer cases. To complement these analyses, we applied the Inverse Participation Ratio (IPR) light localization technique to quantify structural disorder at the microscopic level. This multi-parametric approach, integrating spatial complexity and structural disorder metrics, offers a promising framework for enhancing the sensitivity and specificity of breast cancer detection.

cancer biology↗

bioRxiv2024

N6-methyladenosine in DNA promotes genome stability

DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), and unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, and these results were confirmed using an orthogonal approach, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). METTL3 and 6mA were required for repair of lesions driven by additional base damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA in promoting genome stability in mammalian cells, especially in response to base damage.

molecular biology↗

bioRxiv2022

Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine

Despite recent success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability, remain. Although mRNA, pDNA, and viral-vector based vaccines are being administered, no protein subunit-based SARS-CoV-2 vaccine is approved. Molecular adjuvants targeting pathogen-recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulate various PRRs, including toll-like receptors (TLRs) and retinoic-acid-inducible gene I-like receptors (RIG-I). We hypothesized that targeting the same PRRs using adjuvants on nanoparticles along with a stabilized spike (S) protein antigen could provide broad and efficient immune responses. Formulations targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer-nanoparticles (NPs) were combined with the S1 subunit of S protein and assessed in vitro with isogeneic mixed lymphocyte reactions (iso-MLRs). For in vivo studies, the adjuvanted nanoparticles were combined with stabilized S protein and assessed using intranasal and intramuscular prime-boost vaccination models in mice. Combination NP-adjuvants targeting both TLR and RIG-I (MPLA+PUUC, CpG+PUUC, or R848+PUUC) differentially increased proinflammatory cytokine secretion (IL-1{beta}, IL-12p70, IL-27, IFN-{beta}) by APCs cultured in vitro, and induced differential T cell proliferation. When delivered intranasally, MPLA+PUUC NPs enhanced local CD4+CD44+ activated memory T cell responses while MPLA NPs increased anti-S-protein-specific IgG and IgA in the lung. Following intramuscular delivery, PUUC-carrying NPs induced strong humoral immune responses, characterized by increases in anti-S-protein IgG and neutralizing antibody titers and germinal center B cell populations (GL7+ and BCL6+ B cells). MPLA+PUUC NPs further boosted S-protein-neutralizing antibody titers and T follicular helper cell populations in draining lymph nodes. These results suggest that SARS-CoV-2-mimicking adjuvants and subunit vaccines could lead to robust and unique route-specific adaptive immune responses and may provide additional tools against the pandemic. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/478507v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@7b8174org.highwire.dtl.DTLVardef@701721org.highwire.dtl.DTLVardef@feb860org.highwire.dtl.DTLVardef@fae094_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

How synonymous mutations alter enzyme structure andfunction over long time scales

The specific activity of enzymes can be altered over long time scales in cells by synonymous mutations, which change an mRNA molecules sequence but not the encoded proteins primary structure. How this happens at the molecular level is unknown. Here, we investigate this issue by applying multiscale modeling to three E. coli enzymes - type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B, and dihydrofolate reductase. This modeling involves coarse-grained simulations of protein synthesis and post-translational behavior, all-atom simulations as a test of robustness, and QM/MM calculations to characterize enzymatic function. We first demonstrate that our model predicts experimentally measured changes in specific activity due to synonymous mutations. Then, we show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. These long-lived states exhibit reduced catalytic activity, as demonstrated by their increased activation energies for the reactions they carry out. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. The localized misfolding involves noncovalent lasso entanglements - a topology in which the protein backbone forms a loop closed by noncovalent native contacts which is then threaded by another portion of the protein. Such entanglements are often kinetic traps, as they can require a large proportion of the protein to unfold, which is energetically unfavorable, before they can disentangle and attain the native state. The near-native structures of these misfolded states allow them to bypass the proteostasis machinery and remain soluble, as they exhibit similar hydrophobic surface areas as the native state. These entangled structures persist in all-atom simulations as well, indicating that these conclusions are independent of model resolution. Moreover, the structures of long-lived entangled states are supported by agreement with limited-proteolysis mass spectrometry results. Thus, synonymous mutations cause shifts in the co- and post-translational structural ensemble of proteins, whose altered subpopulations lead to long-term changes in the specific activities of some enzymes. The formation of entangled subpopulations is a plausible mechanism through which changes in translation elongation rate alter ensemble-averaged specific activities, which can ultimately affect the efficiency of biochemical pathways and phenotypic traits.

biophysics↗

bioRxiv2020

Single-Cell Transcriptomic Attributes and Unbiased Computational Modeling for the Prediction of Immunomodulatory Potency of Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are currently being tested in numerous clinical trials as potential cell therapies for the treatment of various diseases and due to their potential immunomodulatory, pro-angiogenic, and regenerative properties. However, variabilities in tissue sources, donors, and manufacturing processes and the lack of defined critical quality attributes (CQAs) and clinically relevant mechanism of action (MoA) pose significant challenges to identify MSC cell therapy products with a predictable therapeutic outcome. This also hinders regulatory considerations and broad clinical translation of MSCs. MSC products are often administered to the patient immediately after thawing from cryopreserved vials (out-of-thaw). However, the qualifying quality-control assays are either performed before cryopreservation, or after culturing the post-thaw cells for 24-48 hours (culture-rescued), none of which represent the out-of-thaw product administered to patients. In this study, we performed a broad functional characterization of out-of-thaw and culture-rescue MSCs from bone marrow (BM-MSCs) and cord tissue (CT-MSCs) using macrophage activation and T cell proliferation-based in vitro potency assays and deep phenotypic characterization using single-cell RNA-sequencing. Using this data, we developed unbiased computational models, specifically symbolic regression (SR) and canonical correlation analysis (CCA) models to predict the immunomodulatory potency of MSCs. Overall, our results suggest that manufacturing conditions (OOT vs. CR) have a strong effect on MSC-function on MSC interactions with macrophages and T cells. Furthermore, single-cell RNA-seq analyses of out-of-thaw BM and CT-MSCs indicate a tissue of origin-dependent variability and heterogeneity in the transcriptome profile. Using symbolic regression modeling we identified specific single-cell transcriptomic attributes of MSCs that predict their immunomodulatory potency. In addition, CCA modeling predicted MSC donors with high or low immunomodulatory potency from their transcriptome profiles. Taken together, our results provide a broad framework for identifying predictive CQAs of MSCs that could ultimately help in better understanding of their MOAs and improved reproducibility and manufacturing control of MSCs.

immunology↗

bioRxiv2020

Single-Cell RNAseq of Out-of-Thaw Mesenchymal Stromal Cells Shows Striking Tissue-of-Origin Differences and Inter-donor Cell-Cycle Variations

Mesenchymal stromal cells (MSCs) from a variety of tissue sources are widely investigated in clinical trials, and the MSCs are often administered immediately after thawing the cryopreserved product. While previous reports have examined the transcriptome of freshly-cultured MSCs from some tissues, little is known about the single-cell transcriptomic profiles of out-of-thaw MSCs from different tissue sources. Such understanding could help determine which tissue origins and delivery methods are best suited for specific indications. Here, we characterized cryopreserved MSCs, immediately post-thaw, from bone marrow (BM) and cord tissue (CT), using single-cell RNA sequencing (scRNA-seq). We show that out-of-thaw BM-vs. CT-MSCs have significant differences in gene expression. Gene-set enrichment analyses implied divergent functional potential. In addition, we show that MSC-batches can vary significantly in cell cycle status, suggesting different proliferative vs. immunomodulatory potentials. Our results provide a comprehensive single-cell transcriptomic landscape of clinically and industrially relevant MSC products. HighlightsO_LISingle cell gene expression comparison between Bone-marrow derived MSCs and Cord-tissue derived MSCs C_LIO_LIDonor effects and cell heterogeneity on tissue-specific MSC gene expression C_LIO_LISingle Cell Pooling Enhances Differential Expression Analysis for Bone marrow and Cord tissue MSC samples C_LIO_LIGene ontology reveals tissue specific unique molecular function and pathways C_LI

cell biology↗

bioRxiv2020

TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation

Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). PAMPs and PAMP-analogs are also used as immune-adjuvants to enhance vaccine efficacy by activating various Pattern Recognition Receptors (PRRs), like Toll-like receptors (TLRs). Various combinations of PAMP adjuvants can trigger synergistic immune responses, but the underlying molecular mechanisms driving that synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with MPLA (TLR4-adjuvant) and CpG (TLR9-adjuvant) as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for the integrated, dual-adjuvant immune response. PLP-based co-presentation of MPLA and CpG to mouse bone marrow derived antigen-presenting cells (BM-APCs) elicited synergistic Type-I Interferon (IFN-{beta}) and IL-12p70 responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that the adapter protein MyD88 and the Interferon-Regulatory-Factor-5 (IRF-5), but not the canonical factors IRF-3 or IRF-7, were necessary for production of both IFN-{beta} and IL12p70. TRIF signaling was required to elicit the synergistic response; the absence of TRIF abolished synergy. Importantly, both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling (TRIF-TRAF-IRF5 pathway kinetics) drove the observed synergy. These results identify not only the key signaling mechanism that cooperates to generate a combinatorial response to MPLA-CpG dual engagement in BM-APCs, but they also underscore the critical role that signaling kinetics and biophysical presentation plays in integrated responses to combination adjuvants.

immunology↗

bioRxiv2020

TLR7 and RIG-I dual-adjuvant loaded nanoparticles drive broadened and synergistic responses in dendritic cells and boost cellular immunity in influenza vaccination

Although the existing flu vaccines elicit strong antigen-specific antibody responses, they fail to provide effective, long term protection - partly due to the absence of robust cellular memory immunity. We hypothesized that co-administration of combination adjuvants, mirroring the flu-virus related innate signaling pathways, could elicit strong cellular immunity. Here, we show that the small molecule adjuvant R848 and the RNA adjuvant PUUC, targeting endosomal TLR7s and cytoplasmic RLRs respectively, when delivered together in polymer nanoparticles (NP), elicits a broadened immune responses in mouse bone marrow-derived dendritic cells (mBMDCs) and a synergistic response in both mouse and human plasmacytoid dendritic cells (pDCs). In mBMDCs, NP-R848-PUUC induced both NF-{kappa}B and interferon signaling. Interferon responses to co-delivered R848 and PUUC were additive in human peripheral blood mononuclear cells (PBMCs) and synergistic in human FLT3-differentiated mBMDCs and CAL-1 pDCs. Vaccination with NPs loaded with H1N1 Flu antigen, R848, and PUUC increased percentage of CD8+ T-cells in the lungs, percentage of antigen-specific CD4+T-cells in the spleen, and enhanced overall cytokine-secreting T cell percentages upon antigen restimulation. Also in the spleen, T lymphopenia, especially after in vitro restimulation, was observed. Our results demonstrate that simultaneous engagement of TLR7 and RIG-I pathways using particulate carriers is a potential approach to improve cellular immunity in flu vaccination. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/207423v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1599111org.highwire.dtl.DTLVardef@1431395org.highwire.dtl.DTLVardef@baa40dorg.highwire.dtl.DTLVardef@16877cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Mutation landscape of SARS-CoV-2 reveals three mutually exclusive clusters of leading and trailing single nucleotide substitutions

The COVID-19 pandemic has spread across the globe at an alarming rate. However, unlike any of the previous global outbreaks the availability of a large number of SARS-CoV-2 sequences provides us with a unique opportunity to understand viral evolution in real time. We analysed 1448 full-length (>29000 nt) sequences available and identified 40 single-nucleotide substitutions occurring in >1% of the genomes. Majority of the substitutions were C to T or G to A. We identify C/Gs with an upstream TTT trinucleotide motif as hotspots for mutations in the SARS-CoV-2 genome. Interestingly, three of the 40 substitutions occur within highly conserved secondary structures in the 5 and 3 regions of the genomic RNA that are critical for the virus life cycle. Furthermore, clustering analysis revealed unique geographical distribution of SARS-CoV-2 variants defined by their mutation profile. Of note, we observed several co-occurring mutations that almost never occur individually. We define five mutually exclusive lineages (A1, B1, C1, D1 and E1) of SARS-CoV-2 which account for about three quarters of the genomes analysed. We identify lineage-defining leading mutations in the SARS-CoV-2 genome which precede the occurrence of sub-lineage defining trailing mutations. The identification of mutually exclusive lineage-defining mutations with geographically restricted patterns of distribution has potential implications for diagnosis, pathogenesis and vaccine design. Our work provides novel insights on the temporal evolution of SARS-CoV-2. ImportanceThe SARS-CoV-2 / COVID-19 pandemic has spread far and wide with high infectivity. However, the severeness of the infection as well as the mortality rates differ greatly across different geographic areas. Here we report high frequency mutations in the SARS-CoV-2 genomes which show the presence of linage-defining, leading and trailing mutations. Moreover, we propose for the first time, five mutually exclusive clusters of SARS-CoV-2 which account for 75% of the genomes analysed. This will have implications in diagnosis, pathogenesis and vaccine design

genomics↗

bioRxiv2020

Uncanny similarity of unique inserts in the 2019-nCoV spike protein to HIV-1 gp120 and Gag

We are currently witnessing a major epidemic caused by the 2019 novel coronavirus (2019-nCoV). The evolution of 2019-nCoV remains elusive. We found 4 insertions in the spike glycoprotein (S) which are unique to the 2019-nCoV and are not present in other coronaviruses. Importantly, amino acid residues in all the 4 inserts have identity or similarity to those in the HIV-1 gp120 or HIV-1 Gag. Interestingly, despite the inserts being discontinuous on the primary amino acid sequence, 3D-modelling of the 2019-nCoV suggests that they converge to constitute the receptor binding site. The finding of 4 unique inserts in the 2019-nCoV, all of which have identity /similarity to amino acid residues in key structural proteins of HIV-1 is unlikely to be fortuitous in nature. This work provides yet unknown insights on 2019-nCoV and sheds light on the evolution and pathogenicity of this virus with important implications for diagnosis of this virus.

evolutionary biology↗

bioRxiv2020

Development of Systemic Immune Dysregulation in a Rat Trauma Model with Biomaterial-Associated Infection

Orthopedic biomaterial-associated infections remain a large clinical challenge, particularly with open fractures and segmental bone loss. Invasion and colonization of bacteria within immune-privileged canalicular networks of the bone can lead to local, indolent infections that can persist for years without symptoms before eventual catastrophic hardware failure. Host immunity is essential for bacterial clearance and an appropriate healing response, and recent evidence has suggested an association between orthopedic trauma and systemic immune dysregulation and immunosuppression. However, the impact of a local infection on this systemic immune response and subsequent effects on the local response is poorly understood and has not been a major focus for addressing orthopedic injuries and infections. Therefore, this study utilized a model of orthopedic biomaterial-associated infection to investigate the effects of infection on the long-term immune response. Here, despite persistence of a local, indolent infection lacking outward symptoms, there was still evidence of long-term immune dysregulation with systemic increases in MDSCs and decreases in T cells compared to non-infected trauma. Further, the trauma only group exhibited a regulated and coordinated systemic cytokine response, which was not present in the infected trauma group. Locally, the infection group had attenuated macrophage infiltration in the local soft tissue compared to the non-infected group. Our results demonstrate widespread impacts of a localized orthopedic infection on the systemic and local immune responses. Characterization of the immune response to orthopedic biomaterial-associated infection may identify key targets for immunotherapies that could optimize both regenerative and antibiotic interventions, ultimately improving outcomes for these patients.

bioengineering↗

bioRxiv2019

Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways

For decades, cationic polymer nanoparticles have been investigated for nucleic acid delivery. Despite promising in vitro transfection results, most formulations have failed to translate into the clinic due to significant in vivo toxicity - especially when delivered intravenously. To address this significant problem, we investigated the detailed mechanisms that govern the complex in vivo systemic toxicity response to common polymeric nanoparticles. We determined that the toxicity response is material dependent. For branched polyethylenimine (bPEI) nanoparticles - toxicity is a function of multiple pathophysiological responses - triggering of innate immune sensors, induction of hepatic toxicity, and significant alteration of hematological properties. In contrast, for chitosan-based nanoparticles - systemic toxicity is primarily driven through innate immune activation. We further identified that modification of primary amines to secondary and tertiary amines using the small molecule imidazole-acetic-acid (IAA) ameliorates in vivo toxicity from both nanocarriers by different, material-specific mechanisms related to Toll-like receptor 4 activation (for bPEI) and complement activation driven neutrophil infiltration (for chitosan), respectively. Our results provide a detailed roadmap for evaluating in vivo toxicity of nanocarriers and identifies potential opportunities to reduce toxicity for eventual clinical translation.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC=\"FIGDIR/small/647305v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (27K):\norg.highwire.dtl.DTLVardef@63c7a7org.highwire.dtl.DTLVardef@c8ac52org.highwire.dtl.DTLVardef@a9589dorg.highwire.dtl.DTLVardef@1f8ec62_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2017

A synthetic stroma-free germinal center niche using material-surface driven polyvalent signaling efficiently induces antigen-specific humoral immunity ex vivo

B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial \"follicular T helper cells (TFH)\" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40; and by mimicking artificial \"follicular dendritic cells (FDC)\" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.

bioengineering↗