Biology subjects

Kumar, P.

Publications and source records attributed to Kumar, P..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Designing and bioengineering of CDRs with higher affinity against receptor-binding domain (RBD) of SARS-CoV-2 Omicron variant

The emergence of the SARS-CoV-2 Omicron variant highlights the need for innovative strategies to address evolving viral threats. This study bioengineered three nanobodies H11-H4, C5, and H3 originally targeting the Wuhan RBD, to bind more effectively to the Omicron RBD. A structure-based in silico affinity maturation pipeline was developed to enhance their binding affinities. The pipeline consists of three key steps: high-throughput in silico mutagenesis of complementarity-determining regions (CDRs), protein-protein docking for screening, and molecular dynamics (MD) simulations for assessment of the complex stability. A total of 741, 551, and 684 mutations were introduced in H11-H4, C5, and H3 nanobodies, respectively. Protein-protein docking and MD simulations shortlisted high-affinity mutants for H11-H4(6), C5(5), and H3(6). Further, recombinant production of H11-H4 mutants and Omicron RBD enabled experimental validation through Isothermal Titration Calorimetry (ITC). The H11-H4 mutants R27E, S57D, S107K, D108W, and A110I exhibited improved binding affinities with dissociation constant (KD) values ranging from [~]8.8 to [~]27 {micro}M, compared to the H11-H4 nanobody KD of [~]32 {micro}M, representing a three-fold enhancement. This study demonstrates the potential of the developed in silico affinity maturation pipeline as a rapid, cost-effective method for repurposing nanobodies, aiding the development of robust prophylactic strategies against evolving SARS-CoV-2 variants and other pathogens. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/613860v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1ea8ff7org.highwire.dtl.DTLVardef@91d567org.highwire.dtl.DTLVardef@51629corg.highwire.dtl.DTLVardef@1212752_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

An integrated view of the structure and function of the human 4D nucleome

The dynamic three-dimensional (3D) organization of the human genome (the "4D Nucleome") is closely linked to genome function. Here, we integrate a wide variety of genomic data generated by the 4D Nucleome Project to provide a detailed view of human 3D genome organization in widely used embryonic stem cells (H1-hESCs) and immortalized fibroblasts (HFFc6). We provide extensive benchmarking of 3D genome mapping assays and integrate these diverse datasets to annotate spatial genomic features across scales. The data reveal a rich complexity of chromatin domains and their sub-nuclear positions, and over one hundred thousand structural loops and promoter-enhancer interactions. We developed 3D models of population-based and individual cell-to-cell variation in genome structure, establishing connections between chromosome folding, nuclear organization, chromatin looping, gene transcription, and DNA replication. We demonstrate the use of computational methods to predict genome folding from DNA sequence, uncovering potential effects of genetic variants on genome structure and function. Together, this comprehensive analysis contributes insights into human genome organization and enhances our understanding of connections between the regulation of genome function and 3D genome organization in general.

genomics↗

bioRxiv2024

Electron tomography visualization of HIV-1 virions trapped by fusion inhibitors to host cells in infected tissues

HIV-1 delivers its genetic material to infect a cell after fusion of the viral and host cell membranes, which takes place after the viral envelope (Env) binds host receptor and co-receptor proteins. Binding of host receptor CD4 to Env results in conformational changes that allow interaction with a host co-receptor (CCR5 or CXCR4). Further conformational rearrangements result in an elongated pre-hairpin intermediate structure in which Env is anchored to the viral membrane by its transmembrane region and to the host cell membrane by its fusion peptide. Although budding virions can be readily imaged by electron tomography (ET) of HIV-1-infected tissues and cultured cells, virions that are fusing (attached to host cells via pre-hairpin intermediates) are not normally visualized, perhaps because the process of membrane fusion is too fast to capture by EM. To image virions during fusion, we used fusion inhibitors to prevent downstream conformational changes in Env that lead to membrane fusion, thereby trapping HIV-1 virions linked to target cells by prehairpin intermediates. ET of HIV-1 pseudovirions bound to CD4+/CCR5+ TZM-bl cells revealed presumptive pre-hairpin intermediates as 2-4 narrow spokes linking a virion to the cell surface. To extend these results to a more physiological setting, we used ET to image tissues and organs derived from humanized bone marrow, liver, thymus (BLT) mice infected with HIV-1 and then treated with CPT31, a high-affinity D-peptide fusion inhibitor linked to cholesterol. Trapped HIV-1 virions were found in all tissues studied (small intestine, mesenteric lymph nodes, spleen, and bone marrow), and spokes representing pre-hairpin intermediates linking trapped virions to cell surfaces were similar in structure and number to those seen in the previous pseudovirus and cultured cell ET study. IMPORTANCETrapped and untrapped HIV-1 virions, both mature and immature, were distinguished by localizing spokes via 3D tomographic reconstructions of HIV-1 infected and fusion-inhibitor treated tissues of humanized mice. The finding of trapped HIV-1 virions in all tissues examined demonstrates a wide distribution of the CPT31 inhibitor, a desirable property for a potential therapeutic. In addition, the presence of virions trapped by spokes, particularly in vascular endothelial cells, demonstrates that fusion inhibitors can be used as markers for potential HIV-1-target cells within tissues, facilitating the mapping of HIV-1 target cells within the complex cellular milieu of infected tissues.

microbiology↗

bioRxiv2024

Structures of the multi-domain oxygen sensor DosP: remote control of a c-di-GMP phosphodiesterase by a regulatory PAS domain

The heme-based direct oxygen sensor DosP degrades c-di-GMP, a second messenger nearly unique to bacteria. In stationary phase Escherichia coli, DosP is the most abundant c-di-GMP phosphodiesterase. Ligation of O2 to a heme-binding PAS domain (hPAS) of the protein enhances the phosphodiesterase through an allosteric mechanism that has remained elusive. We determined six structures of full-length DosP in its aerobic or anaerobic conformations, with or without c-di-GMP. DosP is an elongated dimer with the regulatory heme and phosphodiesterase separated by nearly 180 [A]. In the absence of substrate, regardless of the heme status, DosP presents an equilibrium of two distinct conformations. Binding of substrate induces DosP to adopt a single, ON-state or OFF-state conformation depending on its heme status. Structural and biochemical studies of this multi-domain sensor and its mutants provide insights into signal regulation of second-messenger levels.

biochemistry↗

bioRxiv2024

CyNET- a network analysis framework for high dimensionality, system level analyses of the functional Immunome

The immune system is a complex "Network of Networks", in which various immune cell subsets interact and influence each others functions, ultimately determining immune competence and the control or onset of disease. The coordinated interactions between these cell subsets determine whether the outcome is a normal physiological state or a pathological condition. Established statistical procedures largely ignore the interactions between subsets and rely on statistically significant changes in the frequency of cell subsets. We developed CyNET (Cytometry Network)-- an analysis platform based on a network science approach to understand the immune system holistically. CyNET enables us to quantify the systems level and subsets level properties. We show that changes in the centrality of the nodes (immune subsets) reflect better biological functions than changes in frequency. We used CyNET to analyze the immune development along the chronological age gradient. Peripheral blood cells from healthy newborns (cord blood), adults (20 to 55 years), and elderly (70 years and above) human subjects were further used for validation using single-cell transcriptomics. We found that network edge density, degree centralization, and assortativity score reflect the maturation and development of the immune system along the age axis, thus enabling the characterisation of the functional architecture of the Immunome and the identification of key functional hubs within the immune networks.

immunology↗

bioRxiv2024

Cortical inhibitory parvalbumin interneurons exhibit metabolic specializations coordinated by PGC-1α that are lost in rodents and humans after traumatic brain injury

Parvalbumin-positive interneurons (PV-INs) regulate neuronal and circuit activity, and their dysfunction is observed across neurological conditions, including traumatic brain injury (TBI), epilepsy, Alzheimers disease, and schizophrenia. PV-INs are particularly vulnerable to cell loss, potentially due to their increased metabolic demands arising from their uniquely high level of electrical activity, which render them susceptible to metabolic pressure. Here, we use single-nucleus RNA-sequencing (snRNAseq) data from a rodent model of TBI, as well as human TBI data, and demonstrate PV-INs have unique metabolic specializations that are lost after injury and can be rescued by in vivo treatment with the glycolytic inhibitor, 2-deoxyglucose. We generated a novel PV-IN transcriptional identity module comprised primarily of genes encoding specialized ion channels, metabolic enzymes, and synaptic machinery, that identifies heterogenous subsets of injury-associated PV-INs with loss of PV-IN transcriptional identity. We show that changes in metabolic specialization are coupled to changes in transcriptional identity in PV-INs and implicate the PV-IN-enriched transcriptional co-activator, Ppargc1a, as a key driver of PV-IN transcriptional metabolic dysfunction. We also identify a family of long non-coding RNAs enriched in this subset of transcriptionally dysfunctional PV-INs that negatively correlates with PV-IN metabolic specialization. Lastly, we utilize these tools to interrogate a published human TBI snRNAseq data set and find nearly identical changes, underscoring the importance of PV-IN metabolic dysfunction in the pathology of TBI.

neuroscience↗

bioRxiv2024

The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

CGGBP1 is a GC-rich DNA-binding protein which is important for genomic integrity, gene expression and epigenome maintenance through regulation of CTCF occupancy and cytosine methylation. It has remained unclear how CGGBP1 integrates multiple diverse functions with its simple architecture of only a DNA-binding domain tethered to a C-terminal tail with low structural rigidity. We have used truncated forms of CGGBP1 with or without the DNA-binding domain (DBD) to assay cytosine methylation and global gene expression. Proximal promoters of CGGBP1-repressed genes, although significantly GC-poor, contain GC-rich transcription factor binding motifs and exhibit base compositions indicative of low C-T transition rates due to prevention of cytosine methylation. Genome-wide analyses of cytosine methylation and binding of CGGBP1 DBD show that CGGBP1 restricts cytosine methylation in a manner that depends on its DBD and its DNA-binding. The CGGBP1-repressed genes show an increase in promoter cytosine methylation alongside a decrease in transcript abundance when the DBD-deficient CGGBP1 is expressed. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss and thereby regulates gene expression. By analysing orthologous promoter sequences we show that restriction of cytosine methylation is a function of CGGBP1 progressively acquired during vertebrate evolution. A superimposition of our results and evolution of CGGBP1 suggests that mitigation of cytosine methylation is majorly achieved by its N-terminal DBD. Our results position CGGBP1 DNA-binding as a major evolutionarily acquired mechanism through which it keeps cytosine methylation under check and regulates TFBS retention and gene activity.

molecular biology↗

bioRxiv2024

Causal network perturbation analysis identifies known and novel type-2 diabetes driver genes

The molecular pathogenesis of type 2 diabetes mellitus (T2DM) involves environmental and genetic factors that remain poorly understood. Increased insulin resistance leads to pancreatic {beta}-islet cell exhaustion and failure, leading to more severe T2DM. As such, targeting {beta}-cell dysfunction is an attractive pathway for T2DM treatment. Single-cell RNA sequencing can help identify differentially expressed genes (DEGs) that may contribute to disease-associated pathophysiology, but cannot capture the full spectrum of disease pathophysiology, as some genes that are not differentially expressed may still play important roles in disease progression or mechanisms. As such, we investigated single-cell gene expression changes in {beta}-cells from healthy (C57BL/6J) and diabetic (NZO/HlLtJ) mice fed with normal or high-fat, high-sugar diet (HFHS) across the spectrum of T2DM development (prediabetic, mild or severely diabetic) using an innovative integration of the causal network perturbation assessment (ssNPA) framework with meta-cell transcriptome analysis to identify driver genes for T2DM. By generating a reference causal network and conducting in silico perturbations, we identified three classes of genes implicated in T2DM pathophysiology: (1) DEGs that were not perturbed (e.g, Igf2bp2 strongly linked to {beta} cell dysfunction and glucose regulation), (2) perturbed non-DEGs (e.g., Glp1r a therapeutic target of modern diabetes drugs) that were perturbed, and (3) DEGs that were perturbed (e.g., Pdx1 a master {beta} cell transcription factor), and validated some of the targets using the KOMP database. Together, these results show that each analytic layer captures complementary mechanisms underlying T2DM severity, and their synthesis provides a more complete view of the disease pathophysiology.

bioinformatics↗

bioRxiv2024

Enhancing affinity of neutralizing SARS-CoV-2 nanobody through facile structure-guided mutations in CDRs

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for development of the next generation therapeutics. This study delves into the refinement and engineering of CDRs through in silico affinity maturation followed by binding validation using ITC and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of RBD of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Subsequent biophysical studies using ITC provided insights into binding affinity and key thermodynamic parameters. Consistent with in silico findings, seven single mutations resulted in enhanced affinity. The mutants were further tested for neutralization activity against SARS-CoV-2 pseudovirus. L106T, L106Q, S107R, and S107Q generated mutants were more effective in virus-neutralizing with IC50 values of [~]0.03 {micro}M, [~]0.13 {micro}M, [~]0.14 {micro}M, and [~]0.14 {micro}M, respectively as compared to the native nanobody (IC50 [~]0.77 {micro}M). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.

bioengineering↗

bioRxiv2024

Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

biochemistry↗

bioRxiv2024

Repeat modules and N-linked glycans define structure and antigenicity of a critical enterotoxigenic E. coli adhesin

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as human volunteers challenged with ETEC, diarrheal severity is significantly increased severity in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of human volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected human volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design. Author summaryEnterotoxigenic E. coli (ETEC), a leading cause of diarrhea disproportionately affecting young children in low-income regions, are a priority for vaccine development. Individuals possessing A blood-type are more susceptible to severe cholera-like disease. EtpA, a secreted, immunogenic, blood group A binding protein, is a current vaccine target antigen. Here, we determined the atomic structure of EtpA in complex with protective as well as non-protective monoclonal antibodies targeting two different domains of the protein, allowing us to pinpoint key regions involved in blood-group A antigen recognition and uncover the mechanism of antibody-based protection. In addition, we show through mass-spectrometry that EtpA is extensively and heterogeneously glycosylated at surface-exposed asparagine residues by a promiscuous and low-fidelity glycosyltransferase, EtpC, and that this unique form of bacterial glycosylation is critical for to development of protective immune responses. Lastly, polyclonal antibodies from vaccinated mice as well as monoclonal antibodies obtained from ETEC-infected human volunteers revealed that the highly antigenic surface of EtpA exhibits both protective and non-protective epitopes. These results greatly expand our understanding of ETEC pathogenesis, and the immune responses elicited by these common infections, providing valuable information to aid in the rational design and testing of subunit vaccines.

microbiology↗

bioRxiv2024

MicroRNA hsa-miR-203a-3p promotes H1N1 and NDV Virus Replication by suppressing the IFNA signaling.

MicroRNAs (miRNAs) are small non-coding molecules that act as essential post-transcriptional regulators in various biological processes. Many studies suggest that miRNAs may modulate the hosts immune response during virus infections. We analyzed publicly available transcriptomics data involving infection with different RNA viruses and selected the most prominent candidate, miR-203a-3p. miR-203a-3p is upregulated during H7N1, HCV, and HIV+HPV infections. Interestingly, pathway analysis of microRNA-targeted genes shows that miR-203a-3p targets the type-I interferon pathway. In this study, we report that the expression of miR-203a-3p is elevated in response to polyinosinic-polycytidylic acid [poly(I:C)] transfection and infection with RNA viruses like Newcastle Disease Virus (NDV) and H1N1 influenza virus. We found that overexpression of miR-203-3p promotes the replication of H1N1 virus by suppressing the hosts type-I interferons and interferon-stimulated genes. In addition, miR-203a-3p overexpression reduced the expression of ISGs, and is attributed to the binding of miR-203a-3p to 3 UTRs of Janus-activated kinase 1 (JAK1), STAT1, SOCS3, and multiple IFNA transcripts, as shown by luciferase and Ago-2 pulldown assays. Altogether, these findings strongly suggest that miR-203a-3p acts as a pro-viral molecule during H1N1 and NDV infection by targeting the hosts IFN signaling pathways.

immunology↗

bioRxiv2024

CD4 downregulation precedes Env expression and protects HIV-1-infected cells from ADCC mediated by non-neutralizing antibodies

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly-neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA-flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively-infected cells that already downmodulated CD4. This suggest that CD4 downmodulation precedes env mRNA expression. Consequently, productively-infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1 infected humanized mice with the ADCC mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively-infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/592003v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1c34d91org.highwire.dtl.DTLVardef@11975c0org.highwire.dtl.DTLVardef@81b5beorg.highwire.dtl.DTLVardef@1c2e0b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Directed differentiation of functional corticospinal-like neurons from endogenous SOX6+/NG2+ cortical progenitors

Corticospinal neurons (CSN) centrally degenerate in amyotrophic lateral sclerosis (ALS), along with spinal motor neurons, and loss of voluntary motor function in spinal cord injury (SCI) results from damage to CSN axons. For functional regeneration of specifically affected neuronal circuitry in vivo, or for optimally informative disease modeling and/or therapeutic screening in vitro, it is important to reproduce the type or subtype of neurons involved. No such appropriate in vitro models exist with which to investigate CSN selective vulnerability and degeneration in ALS, or to investigate routes to regeneration of CSN circuitry for ALS or SCI, critically limiting the relevance of much research. Here, we identify that the HMG-domain transcription factor Sox6 is expressed by a subset of NG2+ endogenous cortical progenitors in postnatal and adult cortex, and that Sox6 suppresses a latent neurogenic program by repressing proneural Neurog2 expression by progenitors. We FACS-purify these progenitors from postnatal mouse cortex and establish a culture system to investigate their potential for directed differentiation into CSN. We then employ a multi-component construct with complementary and differentiation-sharpening transcriptional controls (activating Neurog2, Fezf2, while antagonizing Olig2 with VP16:Olig2). We generate corticospinal-like neurons from SOX6+/NG2+ cortical progenitors, and find that these neurons differentiate with remarkable fidelity compared with corticospinal neurons in vivo. They possess appropriate morphological, molecular, transcriptomic, and electrophysiological characteristics, without characteristics of the alternate intracortical or other neuronal subtypes. We identify that these critical specifics of differentiation are not reproduced by commonly employed Neurog2-driven differentiation. Neurons induced by Neurog2 instead exhibit aberrant multi-axon morphology and express molecular hallmarks of alternate cortical projection subtypes, often in mixed form. Together, this developmentally-based directed differentiation from cortical progenitors sets a precedent and foundation for in vitro mechanistic and therapeutic disease modeling, and toward regenerative neuronal repopulation and circuit repair.

neuroscience↗

bioRxiv2024

Beyond A and B Compartments: how major nuclear locales define nuclear genome organization and function

Models of nuclear genome organization often propose a binary division into active versus inactive compartments yet typically overlook nuclear bodies. Here we integrated analysis of sequencing and image-based data to compare genome organization in four human cell types relative to three different nuclear locales: the nuclear lamina, nuclear speckles, and nucleoli. Whereas gene expression correlates mostly with nuclear speckle proximity, DNA replication timing correlates with proximity to multiple nuclear locales. Speckle attachment regions emerge as DNA replication initiation zones whose replication timing and gene composition vary with their attachment frequency. Most facultative LADs retain a partially repressed state as iLADs, despite their positioning in the nuclear interior. Knock out of two lamina proteins, Lamin A and LBR, causes a shift of H3K9me3-enriched LADs from lamina to nucleolus, and a reciprocal relocation of H3K27me3-enriched partially repressed iLADs from nucleolus to lamina. Thus, these partially repressed iLADs appear to compete with LADs for nuclear lamina attachment with consequences for replication timing. The nuclear organization in adherent cells is polarized with nuclear bodies and genomic regions segregating both radially and relative to the equatorial plane. Together, our results underscore the importance of considering genome organization relative to nuclear locales for a more complete understanding of the spatial and functional organization of the human genome.

genomics↗

bioRxiv2024

Deciphering Oxygen Distribution and Hypoxia Profiles in the Tumor Microenvironment: A Data-Driven Mechanistic Modeling Approach

The distribution of hypoxia within tissues plays a critical role in tumor diagnosis and prognosis. Recognizing the significance of tumor oxygenation and hypoxia gradients, we introduce mathematical frameworks grounded in mechanistic modeling approaches for their quantitative assessment within a tumor microenvironment. Our approach provides a non-invasive method to measure and predict hypoxia using known blood vasculature. Formulating a reaction-diffusion model for oxygen distribution, we apply it to derive the corresponding hypoxia profile. The modeling and simulations successfully replicate the observed inter- and intra-tumor heterogeneity in experimentally obtained hypoxia profiles across various tumor tissues (breast, ovarian, and pancreatic) in our dataset. Employing a data-driven approach, we propose a method to deduce partial differential equation (PDE) models with spatially dependent parameters, enabling us to comprehend the variability of hypoxia profiles within a tissue. The versatility of our framework lies not only in capturing diverse and dynamic behaviors of tumor oxygenation but also in categorizing states of vascularization. These categories are distinguished based on the dynamics of oxygen molecules, as identified by the model parameters.

cancer biology↗

bioRxiv2024

Aligning with the predecessors and counterarguments: A systematic review of the anatomical correlates for the newly discovered meningeal layer in the existing literature

A recent study reported the existence of a subarachnoid lymphatic-like membrane (SLYM) -an intermediate leptomeningeal layer between the arachnoid and pia mater in mice and human brains - dividing the subarachnoid space (SAS) into two functional compartments. Despite being a macroscopic structure, how it missed detection in previous studies is surprising. We systematically reviewed the published reports in animals and humans to explore whether prior descriptions of this meningeal layer have existed. An electronic search was conducted in PubMed/Medline, EMBASE, Google Scholar, Science Direct, and Web of Science databases using combinations of MeSH terms and keywords with Boolean operators from inception until 31st Dec 2023. We found at least eight studies that provided structural evidence of an intermediate leptomeningeal layer in the brain or spinal cord. However, unequivocal descriptions for this layer all along the central nervous system were scarce. Obscured names were used to describe it, i.e., the epipial layer, intermediate meningeal layer, intermediate lamella, and outer pial layer. Its microscopic/ultrastructural details closely resembled the SLYM. Further, we examined the counterarguments in current literature that are skeptical of this layers existence. Considering the significant physiological/clinical implications, exploring further structural and functional details of the new meningeal layer is a need of the hour.

neuroscience↗

bioRxiv2024

Single-cell and bulk transcriptional profiling of mouse ovaries reveals novel genes and pathways associated with DNA damage response in oocytes.

Immature oocytes enclosed in primordial follicles stored in female ovaries are under constant threat of DNA damage induced by endogenous and exogenous factors. Checkpoint kinase 2 (CHEK2) is a key mediator of the DNA damage response in all cells. Genetic studies have shown that CHEK2 and its downstream targets, p53 and TAp63, regulate primordial follicle elimination in response to DNA damage, however the mechanism leading to their demise is still poorly characterized. Single-cell and bulk RNA sequencing were used to determine the DNA damage response in wildtype and Chek2-deficient ovaries. A low but oocyte-lethal dose of ionizing radiation induces a DNA damage response in ovarian cells that is solely dependent on CHEK2. DNA damage activates multiple ovarian response pathways related to apoptosis, p53, interferon signaling, inflammation, cell adhesion, and intercellular communication. These pathways are differentially employed by different ovarian cell types, with oocytes disproportionately affected by radiation. Novel genes and pathways are induced by radiation specifically in oocytes, shedding light on their sensitivity to DNA damage, and implicating a coordinated response between oocytes and pre-granulosa cells within the follicle. These findings provide a foundation for future studies on the specific mechanisms regulating oocyte survival in the context of aging, as well as therapeutic and environmental genotoxic exposures.

cell biology↗