Biology subjects

Banerjee, K.

Publications and source records attributed to Banerjee, K..

17 recordsLinked to original sources

bioRxiv2026

Association between breeding-habitat bacterial communities and oviposition behavior of Aedes aegypti: Insights from arboviral outbreak-affected areas of Pune, Western India

Aedes aegypti, an important vector of several arboviral diseases, is highly adapted to human-associated environments and commonly exploits anthropogenic water-holding containers for breeding. Understanding the ecological factors governing oviposition-site selection is essential for developing sustainable vector control strategies. Previous studies have established that microbial communities in breeding waters influence mosquito oviposition-site selection through the production of volatile organic compounds. Importantly, rapid urbanization in India has increased the availability of artificial breeding habitats for Ae. aegypti, potentially facilitating the transmission and persistence of arboviral diseases. However, the composition of bacterial communities in these larval habitats and their potential role in shaping the oviposition behavior of Ae. aegypti remain poorly characterized in the Indian context. Here, we investigated the bacterial diversity and community composition of Aedes larval habitats during the 2024 Zika and chikungunya outbreaks in Pune, India. Water samples, collected from artificial urban containers and natural freshwater habitats, were subjected to 16S rRNA amplicon sequencing. Artificial breeding habitats exhibited significantly higher bacterial diversity and were enriched with Enterobacter spp. compared with natural habitats. Oviposition bioassays further demonstrated a strong preference of gravid Ae. aegypti females for Enterobacter-conditioned water. GCMS analysis of bacterial suspension identified indole as a prominent volatile metabolite associated with Enterobacter, suggesting its possible role in mediating oviposition site selection. Together, these findings provide insights into the microbial and chemical ecology of Ae. aegypti breeding habitats and establish a basis for investigating microbial volatile cues as potential targets for vector surveillance and environmentally sustainable attract-and-kill strategies.

genomics↗

bioRxiv2026

SPICE: A Robust Computational Framework for Identifying Copy Number Variations in Spatial Transcriptomics

Copy number variation (CNV), which alters the number of genomic segments, is a major driver of intratumor heterogeneity, characterized by spatially organized and genetically distinct cell populations. Recent advances in spatially resolved transcriptomic (SRT) technologies, which profile gene expression across thousands of spatially indexed tissue locations, offer a powerful opportunity to reconstruct the CNV architecture and dissect the spatial organization of cancer subclones. Here, we introduce SPICE (spatial inference of CNV events), a probabilistic method for identifying somatic CNVs and allele-specific copy number (ASCN) profiles from SRT data. A key feature of SPICE is its ability to integrate multiple complementary information available in SRT data, including gene expression, spatial coordinates, and heterozygous SNPs inferred from transcriptomic reads, to substantially enhance the accuracy and power of CNV detection. Using datasets generated across different SRT platforms, we first assess the reliability of SNPs derived from SRT data to ensure robust downstream inference. We then demonstrate that SPICE effectively integrates these modalities to deliver accurate and spatially coherent reconstruction of CNV landscapes and subclonal architecture, while maintaining excellent control of false discoveries. Together, SPICE provides a robust and effective solution for dissecting genomic heterogeneity in SRT studies of cancer.

genomics↗

bioRxiv2026

Engineering a pacemaker-driven human mini-heart guided by spatial multi-omics of sinoatrial node development

The human sinoatrial node (SAN) functions as the primary pacemaker of the heart and coordinates the hierarchical electrical activity that drives cardiac contraction. However, experimental systems capable of reconstructing pacemaker-driven cardiac organization in human tissues remain limited. Here we integrate spatial multi-omics of the human fetal SAN with stem-cell engineering to generate pacemaker organoids ("Sinoids") and assemble them into a pacemaker-driven human mini-heart composed of sinoatrial, atrial and ventricular cardiac modules. High-resolution spatial transcriptomics and single-nucleus multi-omic analyses of human fetal SAN tissues identify regulatory pathways guiding pacemaker lineage specification, which we leverage to engineer human pluripotent stem cell-derived SAN organoids with robust pacemaker identity and electrophysiological activity. When integrated with atrial and ventricular cardioids, Sinoids initiate and coordinate electrical activation across assembled cardiac tissues, establishing directional propagation of electrophysiological signals within structured mini-heart organoids. Combining AI-guided perturbation modeling with functional validation further identifies conserved regulatory pathways controlling pacemaker specification and regionalization, including YAP-TEAD and NRG-ERBB signaling. Together, these results establish a multi-omic-guided strategy for engineering pacemaker tissues and reconstructing cardiac conduction hierarchy in vitro. The pacemaker-driven mini-heart platform provides a modular human cardiac system for studying pacemaker biology, modeling arrhythmia mechanisms and enabling electrophysiological drug discovery.

bioinformatics↗

bioRxiv2026

Targeting NF-κB epigenetic activation and DNA repair deficiency in G34-mutant pediatric diffuse hemispheric glioma with nanoparticles combining PARP inhibition and immune stimulation mediated by CpG dinucleotides

Diffuse hemispheric gliomas (DHGs) are highly aggressive and infiltrative CNS tumors that are refringent to treatment, and with a 5-year overall survival of around 20%. A fraction of DHGs is driven by mutations in the histones H3.1 and H3.3. In this study, we demonstrate that the expression of histone H3.3 glycine 34 to arginine mutations (H3.3-G34R) result in the epigenetic and transcriptional activation of the NF-{kappa}B signaling pathway in DHG. To target this vulnerability, we designed high density lipoprotein (HDL) nanoparticles loaded with unmethylated CpG dinucleotides, which mimic the immune stimulatory activity of bacterial DNA. CpG are recognized by Toll-like receptor 9 (TLR9), activating the NF-{kappa}B signaling. The CpG-mediated NF-{kappa}B activation results in the release of immuno-stimulating cytokines that promote an antitumoral response. As we previously established that G34-mutant DHGs are characterized by DNA repair impairment, we combined CpG dinucleotides with a PARP (poly (ADP-ribose) polymerase) inhibitor, olaparib, in the HDL nanoparticles.

cancer biology↗

bioRxiv2025

Reprogramming the Immune Suppressive Tumor Microenvironment in Glioma Enhances the Efficacy of Immune-Mediated Gene Therapy

Gliomas account for ~80% of primary malignant brain tumors. Many CNS WHO grade 2-3 and some grade 4 gliomas harbor mutant isocitrate dehydrogenase 1 (mIDH1), which causes a gain of function mutation (IDH1 R132H) leading to the production of 2-hydroxyglutarate (2HG). Mutant IDH1-induced 2HG, through epigenetic reprogramming elicits an immune-permissive tumor microenvironment (TME). An immunosuppressive mechanism in the glioma TME involves adenosine production via the ectoenzyme CD73. This study investigates mIDH1s influence on CD73 expression and adenosine levels. We demonstrate that mIDH1 glioma cells exhibit reduced CD73 expression, driven by DNA hypermethylation, leading to reduced adenosine levels. Since wtIDH1 gliomas have high CD73 expression, we evaluated CD73 blockade as an immunotherapy target. We show that CD73 inhibition used as monotherapy, did not improve survival in wtIDH1 glioma-bearing mice. However, when combined with immune-stimulatory Ad-TK (adenoviral vectors encoding herpes simplex virus thymidine kinase) and Ad-Flt3L (adenoviral vectors encoding FMS-like tyrosine kinase 3 ligand) gene therapy, CD73 blockade significantly enhanced therapeutic efficacy and increased anti-glioma effector T cell activity. These findings reveal that CD73 inhibition used in combination with immune stimulatory Ad-TK/Ad-Flt3L gene therapy may be an effective treatment for wtIDH1 gliomas, which could be readily translated to the clinical arena.

cancer biology↗

bioRxiv2025

Amoeboid-Mesenchymal Transition and the Proteolytic Control of Cancer Invasion Plasticity

Invasion plasticity allows malignant cells to toggle between collective, mesenchymal and amoeboid phenotypes while traversing extracellular matrix (ECM) barriers. Current dogma holds that collective and mesenchymal invasion programs trigger the mobilization of proteinases that digest structural barriers dominated by type I collagen, while amoeboid activity allows cancer cells to marshal mechanical forces to traverse tissues independently of ECM proteolysis. Here, we use cancer spheroid-3-dimensional matrix models, single-cell RNA sequencing, and human tissue explants to identify the mechanisms controlling mesenchymal versus amoeboid invasion. Unexpectedly, collective/mesenchymal- and amoeboid-type invasion programs - though distinct - are each characterized by active tunneling through ECM barriers, with expression of matrix-degradative metalloproteinases. CRISPR/Cas9-mediated targeting of a single membrane-anchored collagenase, MMP14/MT1-MMP, ablates tissue-invasive activity while co-regulating cancer cell transcriptional programs. Though changes in matrix architecture, nuclear rigidity, and metabolic stress as well as the presence of cancer-associated fibroblasts are proposed to support amoeboid activity, none of these changes restore invasive activity of MMP14-targeted cancer cells. While a requirement for MMP14 is bypassed in low-density collagen hydrogels, invasion by the proteinase-deleted cells is associated with nuclear envelope and DNA damage, highlighting a proteolytic requirement for maintaining nuclear integrity. Nevertheless, when cancer cells confront explants of live human breast tissue, MMP14 is again required to support invasive activity. Corroborating these results, spatial transcriptomic and immunohistological analyses of invasive human breast cancers identified clear expression of MMP14 in invasive cells that were further associated with degraded collagen, underlining the pathophysiologic importance of this proteinase in directing invasive activity in vivo.

cancer biology↗

bioRxiv2025

HDL Nanodiscs Loaded with Liver X Receptor Agonist Decreases Tumor Burden and Mediates Long-term Survival in Mouse Glioma Model

Glioblastoma multiforme (GBM) is highly aggressive primary brain tumor with a 5-year survival rate of 7%. Previous studies have shown that GBM tumors have a reduced capacity to produce cholesterol and instead depend on the uptake of cholesterol produced by astrocytes. To target cholesterol metabolism to induce cancer cell death, synthetic high-density lipoprotein (sHDL) nanodiscs delivering Liver-X-Receptor (LXR) agonists and CpG oligonucleotides for targeting GBM were investigated. LXR agonists synergize with sHDL nanodiscs by increasing the expression of the ABCA1 cholesterol CpG oligonucleotides are established adjuvants used in cancer immunotherapy that work through the toll-like receptor 9 pathway. In the present study, treatment with GW-CpG-sHDL nanodiscs increased the expression of cholesterol efflux transporters on murine GL261 cells leading to enhanced cholesterol removal. Experiments in GL261-tumor-bearing mice revealed combining GW-CpG-sHDL nanodiscs with radiation (IR) therapy significantly increases median survival compared to GW-CpG-sHDL or IR alone. Furthermore, 66% of long-term survivors from the GW-CpG-sHDL +IR treatment group showed no tumor tissue when rechallenged.

cancer biology↗

bioRxiv2024

Epigenetic Reprogramming of Autophagy Drives Mutant IDH1 Glioma Progression and Response to Radiation

Mutant isocitrate dehydrogenase 1 (mIDH1) exhibits a gain of function mutation enabling 2-hydroxyglutarate (2HG) production and epigenetic reprogramming. This leads to enhanced DNA-damage response and radioresistance in mIDH1 gliomas. RNA-seq and ChIP-seq data revealed that human and mouse mIDH1 glioma neurospheres have downregulated gene ontologies (GOs) related to mitochondrial metabolism and upregulated GOs related to autophagy. Decreased mitochondrial metabolism was accompanied by decreased glycolysis, rendering autophagy a source of energy in mIDH1 gliomas. Human and mouse mutant IDH1 glioma cells exhibited increased expression of pULK1-S555 and enhanced LC3 I/II conversion, indicating augmented autophagy. Additionally, scRNA-seq data from human mIDH1 astrocytoma patients samples showed decreased mitochondrial metabolism and increased autophagy. We further demonstrate that inhibiting autophagy in vivo by systemic administration of synthetic protein nanoparticles encapsulating siRNA targeting Atg7 sensitized mIDH1 glioma cells to radiation-induced cell death, resulting in tumor regression, long-term survival, and immunological memory. In summary, our work uncovered that autophagy is a critical pathway for survival in mIDH1 gliomas and by blocking this pathway we can elicit radiosensitivity in vitro in human and mouse mIDH1 glioma cells, and in vivo in genetically engineered mouse models. Our data also highlights that blocking autophagy has significant potential for clinical translation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/584091v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@111f4a9org.highwire.dtl.DTLVardef@1d5bb30org.highwire.dtl.DTLVardef@f467ecorg.highwire.dtl.DTLVardef@1d2e9b5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Our genetically engineered mIDH1 mouse glioma model harbors IDH1R132H in the context of ATRX and TP53 knockdown. The production of 2-HG elicited an epigenetic reprogramming associated with a disruption in mitochondrial activity and an enhancement of autophagy in mIDH1 glioma cells. Autophagy is a mechanism involved in cell homeostasis related with cell survival under energetic stress and DNA damage protection. Autophagy has been associated with radio resistance. The inhibition of autophagy thus radio sensitizes mIDH1 glioma cells and enhances survival of mIDH1 glioma-bearing mice, representing a novel therapeutic target for this glioma subtype with potential applicability in combined clinical strategies. C_FIG

cancer biology↗

bioRxiv2023

H3.3-G34R Mutation-Mediated Epigenetic Reprogramming Leads to Enhanced Efficacy of Immune Stimulatory Gene Therapy in Pediatric High-Grade Gliomas

Diffuse hemispheric glioma (DHG), H3 G34-mutant, representing 9-15% of cases, are aggressive Central Nervous System (CNS) tumors with poor prognosis. This study examines the role of epigenetic reprogramming of the immune microenvironment and the response to immune-mediated therapies in G34-mutant DHG. To this end, we utilized human G34-mutant DHG biopsies, primary G34-mutant DHG cultures, and genetically engineered G34-mutant mouse models (GEMMs). Our findings show that the G34 mutation alters histone marks deposition at promoter and enhancer regions, leading to the activation of the JAK/STAT pathway, which in turn results in an immune-permissive tumor microenvironment. The implementation of Ad-TK/Ad-Flt3L immunostimulatory gene therapy significantly improved median survival, and lead to over 50% long term survivors. Upon tumor rechallenge in the contralateral hemisphere without any additional treatment, the long-term survivors exhibited robust anti-tumor immunity and immunological memory. These results indicate that immune-mediated therapies hold significant potential for clinical translation in treating patients harboring H3.3-G34 mutant DHGs, offering a promising strategy for improving outcomes in this challenging cancer subtype affecting adolescents and young adults (AYA). STATEMENT OF SIGNIFICANCEThis study uncovers the role of the H3.3-G34 mutation in reprogramming the tumor immune microenvironment in diffuse hemispheric gliomas. Our findings support the implementation of precision medicine informed immunotherapies, aiming at improving enhanced therapeutic outcomes in adolescents and young adults harboring H3.3-G34 mutant DHGs.

cancer biology↗

bioRxiv2022

Shear stress induced KLF4 is a key determinant in AVM pathogenesis

BackgroundVascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. For example, FSS synergizes with Bone Morphogenic Protein 9 (BMP9) and BMP10 to promote and maintain vascular stability. Mutation of the BMP receptors ALK1, Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (EC) integrate FSS and BMP signals in normal vascular development and homeostasis, and how mutations give rise to malformations is not well understood. ResultsHere we show that loss of Smad4 in murine ECs increases cells sensitivity to flow and the resulting AVMs are characterized by excessive elongation and polarity against the flow. Smad4 deletion also blocks the anti-proliferative response to high FSS, leading to loss of arterial identity. Our data show that loss of cell cycle arrest leads to loss of arterial identity, which is essential in AVM formation upon Smad4 depletion in ECs. Excessive flow-induced activation of KLF4-PI3K/AKT due to Cyclin dependent Kinase (CDK) activation mediates the aberrant morphological responses to flow triggering AVM formation. ConclusionsOur results show that loss of polarization against the flow is not required for AVM formation upon EC Smad4 depletion. Instead, increased EC proliferation-mediated loss of arterial identity due to flow-induced PI3K/Akt/Cdks hyperactivation and Klf4 over-expression are the main events associated with AVM formation.

molecular biology↗

bioRxiv2022

Zinc Finger MYND-Type Containing 8 (ZMYND8) is epigenetically regulated in mutant Isocitrate Dehydrogenase 1 (IDH1) glioma to promote radioresistance

Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. Establishment of glioma-specific methylation patterns by mIDH1 reprogramming drives oncogenic features of cancer metabolism, stemness and therapeutic resistance. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1. Treatment of glioma cells with mIDH1 specific inhibitor AGI-5198, upregulated gene networks involved in replication stress. Specifically, we found that the expression of ZMYND8, a regulator of DNA damage response was decreased in three patient-derived glioma cell cultures (GCC) after treatment with AGI-5198. ZMYND8 functions as a chromatin reader to modulate enhancer activity and recruit DNA repair machinery. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 knockout (KO) exhibit significant phosphorylation of ATM and sustained {gamma}H2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. The accumulation of DNA damage in ZMYND8 KO mIDH1 GCCs promoted the phosphorylation of cell cycle checkpoint proteins Chk1 and Chk2. In addition, the recruitment of ZMYND8 to sites of DNA damage has been shown to be PARP- dependent. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. Translational RelevanceOur understanding of radioresistance mechanisms in patient-derived glioma cell cultures (GCC) that endogenously express mIDH1-R132H are limited. We have uncovered a novel gene target Zinc Finger MYND-Type Containing 8 (ZMYND8) that is downregulated following treatment of human mIDH1 GCCs with mIDH1 specific inhibitors. We demonstrate that suppression of ZMYND8 expression by shRNA knockdown or genetic knockout reduces the cellular viability of mIDH1 GCCs to ionizing radiation (IR). Our findings reveal an epigenetic vulnerability of mIDH1 GCCs to ZMYND8 knockout (KO) which results in impaired resolution of IR-induced DNA damage and induction of cell cycle arrest. Additionally, ZMYND8 KO mIDH1 GCCs display increased radiosensitivity to inhibition of epigenetic regulators BRD4, HDAC, and PARP which could be mediated by enhanced replicative stress.

cancer biology↗

bioRxiv2021

Systemic delivery of a CXCR4-CXCL12 signaling inhibitor encapsulated in synthetic protein nanoparticles for glioma immunotherapy

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor, with poor prognosis. Major obstacles hampering effective therapeutic response in GBM are tumor heterogeneity, high infiltration of immunosuppressive myeloid cells, and the presence of the blood-brain barrier. The C-X-C Motif Chemokine Ligand 12/ C-X-C Motif Chemokine Receptor 4 (CXCL12/ CXCR4) signaling pathway is implicated in GBM invasion and cell cycle progression. While the CXCR4 antagonists (AMD3100) has a potential anti-GBM effects, its poor pharmacokinetic and systemic toxicity had precluded its clinical application. Moreover, the role of CXCL12/ CXCR4 signaling pathway in anti-GBM immunity, particularly in GBM-mediated immunosuppression has not been elucidated. Here, we developed a synthetic protein nanoparticle (SPNPs) coated with the cell-penetrating peptide iRGD (AMD3100 SPNPs) to target the CXCR4/CXCL12 signaling axis in GBM. We showed that AMD3100 SPNPs effectively blocked CXCR4 signaling in mouse and human GBM cells in vitro as well as in GBM model in vivo. This results in inhibition of GBM proliferation and induction of immunogenic tumor cell death (ICD) leading to inhibition of GBM progression. Our data also demonstrate that blocking CXCR4 sensitizes GBM cells to radiation, eliciting enhanced release of ICD ligands. Combining AMD3100 SPNPs with radiotherapy inhibited GBM progression and led to long-term survival; with 60% of mice remaining tumor-free. This was accompanied by an anti-GBM immune response and sustained immunological memory that prevented tumor recurrence without further treatment. Finally, we showed that systemic delivery of AMD3100 SPNPs decreased the infiltration of CXCR4+ monocytic myeloid-derived suppressor cells to the tumor microenvironment. With the potent ICD induction and reprogrammed immune microenvironment, this strategy has significant potential for future clinical translation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/457953v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@17c9377org.highwire.dtl.DTLVardef@1b5a4dcorg.highwire.dtl.DTLVardef@5aa4eaorg.highwire.dtl.DTLVardef@1011a86_HPS_FORMAT_FIGEXP M_FIG Immunological mechanism targeting Glioblastoma (GBM) upon blocking CXCR4 signaling pathway with AMD3100-conjugated nanoparticles (SPNPs). (1) Radiotherapy induces glioma cell death, followed by Damage-associated molecular patterns (DAMPs) release. Dendritic cells (DC) are activated by DAMPs and migrate to the regional lymph node where they prime cytotoxic T lymphocyte immune response. Tumor-specific cytotoxic T cells infiltrate the tumor and attack glioma cells. (2) Glioma cells express CXCR4, as well its ligand CXCL12. CXCL12 induces glioma cell proliferation and, (3) as well as mobilization in the bone marrow of CXCR4 expressing myeloid MDSC, which will infiltrate the tumor, and inhibit tumor-specific cytotoxic T cells activity. GEMM of glioma when treated systemically with SPNPs AMD3100 SPNPs plus radiation, nanoparticles block the interaction between CXCR4 and CXCL12, thus (4) inhibiting glioma cell proliferation and (5) reducing mobilization in the bone marrow of CXCR4 expressing myeloid MDSC, (6) generating a reduced MDSC tumor infiltration, as well as releasing MDSC inhibition over tumor specific cytotoxic T cell response. C_FIG

cell biology↗

bioRxiv2021

D155Y Substitution of SARS-CoV-2 ORF3a Weakens Binding with Caveolin-1: An in silico Study

The clinical manifestation of the recent pandemic COVID-19, caused by the novel SARS-CoV-2 virus, varies from mild to severe respiratory illness. Although environmental, demographic and co-morbidity factors have an impact on the severity of the disease, contribution of the mutations in each of the viral genes towards the degree of severity needs a deeper understanding for designing a better therapeutic approach against COVID-19. Open Reading Frame-3a (ORF3a) protein has been found to be mutated at several positions. In this work, we have studied the effect of one of the most frequently occurring mutants, D155Y of ORF3a protein, found in Indian COVID-19 patients. Using computational simulations we demonstrated that the substitution at 155th changed the amino acids involved in salt bridge formation, hydrogen-bond occupancy, interactome clusters, and the stability of the protein compared with the other substitutions found in Indian patients. Protein-protein docking using HADDOCK analysis revealed that substitution D155Y weakened the binding affinity of ORF3a with caveolin-1 compared with the other substitutions, suggesting its importance in the overall stability of ORF3a-caveolin-1 complex, which may modulate the virulence property of SARS-CoV-2.

bioinformatics↗

bioRxiv2020

SERUM ANTIBODY RESPONSES AGAINST CARBAPENEM-RESISTANT KLEBSIELLA PNEUMONIAE IN INFECTED PATIENTS

BackgroundCapsular polysaccharide (CPS) heterogeneity within carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strain ST258 must be considered when developing CPS-based vaccines. We sought to characterize CPS-specific antibody responses elicited by CR-Kp infected patients. MethodPlasma and bacterial isolates were collected from 33 hospital patients with positive CR-Kp culture. Isolates capsule were typed by wzi sequencing. Reactivity and measures of efficacy of patient antibodies were studied against 3 prevalent CR-Kp CPS types (wzi29, 154 and 50). ResultsHigh IgG titers against wzi154 and wzi50 CPS were documented in 79% of infected patients. Patient-derived (PD) IgGs agglutinated CR-Kp and limited growth better than naive IgG, and promoted phagocytosis of strains across the serotype isolated from their donors. Additionally, poly-IgG from wzi50 and wzi154 patients promoted phagocytosis of non-concordant CR-Kp serotypes. Such effects were lost when poly-IgG was depleted of CPS-specific IgG. Additionally, mice infected with wzi50, wzi154, and wzi29 CR-Kp strains pre-opsonized with wzi50 patient-derived IgG exhibited lower lung CFU than controls. Depletion of wzi50 Abs reversed this effect in wzi50 and wzi154 infections, whereas wzi154 Ab depletion reduced Poly-IgG efficacy against wzi29 CR-Kp. ConclusionsWe are first to report cross-reactive properties of CPS-specific Abs from CR-Kp patients through both in-vitro and in-vivo models. ImportanceCarbapenem-resistant Klebsiella pneumoniae is a rapidly emerging public health threat that can cause fatal infections in ~40-50% of immunocompromised patients. Due to its resistance to nearly all antimicrobials, development of alternate therapies like antibodies and vaccines are urgently needed. Capsular polysaccharides are emerging as important immunotargets as they are a crucial for Klebsiella pneumoniae pathogenesis. Studying how hosts immune system protect against specific bacteria such as Klebsiella pneumoiniae, with respect to their specific capsule-type is crucial to develop effective anti-CPS immunotherapies. In this manuscript, we are the first to characterize humoral responses developed in infected patients against carbapenem-resistant Klebsiella pneumoniae expressing different wzi-capsule types. This study is the first to report efficacy of cross-reactive properties of CPS-specific Abs in both in-vitro and in-vivo models.

immunology↗

bioRxiv2020

The Role of IgG Subclass in Antibody-Mediated Protection against Carbapenem-Resistant Klebsiella pneumoniae

Monoclonal antibodies (Abs) have the potential to assist in the battle against multidrug-resistant bacteria such as Carbapenem-Resistant Klebsiella pneumoniae (CR-Kp). However, the characteristics by which these Abs function, such as the role of antibody subclass, must be determined before such modalities can be carried from the bench to the bedside. We performed a subclass switch on anti-capsular monoclonal murine IgG3 (mIgG3) hybridomas and identified and purified a murine IgG1 (mIgG1) hybridoma line through sib selection. We then compared the ability of the mIgG1 and mIgG3 antibodies to control CR-Kp ST258 infection both in vitro and in vivo. We found by ELISA and flow cytometry that mIgG3 has superior binding to CR-Kp CPS and superior agglutinating ability compared to mIgG1. The mIgG3 also predictably had better complement-mediated serum bactericidal activity than the mIgG1 and also promoted neutrophil-mediated killing at concentrations lower than the mIgG1. In contrast, the mIgG1 had marginally better activity in improving macrophage-mediated phagocytosis. Comparing their activities in a pulmonary infection model with wild type as well as neutropenic mice, both antibodies reduced organ burden in a non-lethal challenge, regardless of neutrophil status, with mIgG1 having the highest overall burden reduction in both scenarios. However, at a lethal inoculum, both antibodies showed reduced efficacy in neutropenic mice, with mIgG3 retaining the most activity. These findings suggest the viability of monoclonal Ab adjunctive therapy in neutropenic patients that cannot mount their own immune response, while also providing some insight into the relative contributions of immune mediators in CR-Kp protection. ImportanceCarbapenem-resistant Klebsiella pneumoniae is an urgent public health threat that causes life-threatening infections in immunocompromised hosts. Its resistance to nearly all antibiotics necessitates novel strategies to treat it, including the use of monoclonal antibodies. Monoclonal antibodies are emerging as important adjuncts to traditional pharmaceuticals, and studying how they protect against specific bacteria such as Klebsiella pneumoniae is crucial to their development as effective therapies. Antibody subclass is often overlooked but is a major factor in how an antibody interacts with other mediators of immunity. This paper is the first to examine how the subclass of anti-capsular monoclonal antibodies can affect efficacy against CR-Kp. Additionally, this work sheds light on the viability of monoclonal antibody therapy in neutropenic patients, who are most vulnerable to CR-Kp infection.

immunology↗

bioRxiv2019

Loss of microbial diversity and body site heterogeneity in individuals with Hidradenitis Suppurativa

Hidradenitis Suppurativa (HS) is a chronic, scarring, inflammatory skin disease affecting hair follicles in axillae, inguinal, and anogenital regions. Dysbiosis in HS patients compared to healthy subjects is documented. However, whether dysbiosis is specific to particular body sites or skin niches is unknown. We investigated the follicular and skin surface microbiome of the axilla and groin of HS patients (n=11) and healthy individuals (n=10) using 16S rRNA gene sequencing (V3-V4). We sampled non-lesional (HSN) and lesional skin (HSL) of HS patients. {beta}-diversity was significantly decreased (p<0.05) in HSN and HSL skin compared to normal skin with loss of body site and skin niche heterogeneity in HS samples. The relative bacterial abundance of specific microbes was also significantly different between normal and HSN (15 genera) or HSL (21 genera) skin. Smoking and alcohol use influenced the {beta}-diversity (p<0.08) in HS skin. We investigated metabolic profiles of bacterial communities in HS and normal skin using a computational approach. Metabolism, Genetic Information Processing, and Environmental Information Processing were significantly different between normal and HS samples. Altered metabolic pathways associated with dysbiosis of HS skin suggest mechanisms underlying the disease pathology and information about treatment with drugs targeting those pathways.

microbiology↗

bioRxiv2017

Elucidating interplay of speed and accuracy in biological error correction

One of the most fascinating features of biological systems is the ability to sustain high accuracy of all major cellular processes despite the stochastic nature of underlying chemical processes. It is widely believed that such low errors are the result of the error correcting mechanism known as kinetic proofreading. However, it is usually argued that enhancing the accuracy should result in slowing down the process leading to so-called speed-accuracy trade-off. We developed a discrete-state stochastic framework that allowed us to investigate the mechanisms of the proofreading using the method of first-passage processes. With this framework, we simultaneously analyzed speed and accuracy of the two fundamental biological processes, DNA replication and tRNA selection during the translation. The results indicate that speed-accuracy trade-off is not always observed. However, when the trade-off is present, the biological systems tend to optimize the speed rather than the accuracy of the processes, as long as the error level is tolerable. Additional constraints due to the energetic cost of proofreading also play a role in the error correcting process. Our theoretical findings provide a new microscopic picture of how complex biological processes are able to function so fast with a high accuracy.

biophysics↗