Biology subjects

Kumar, S. S.

Publications and source records attributed to Kumar, S. S..

14 recordsLinked to original sources

bioRxiv2026

Live Droplet Single-Cell RNA Sequencing

Single-cell RNA sequencing has emerged as one of the most influential methods of the past decade, enabling the dissection of cellular heterogeneity and the discovery of rare cell types. However, most workflows require cell lysis and provide only terminal snapshots, limiting their ability to capture dynamic cellular trajectories and temporal variations within the same cell. Herein, we present Live Droplet single-cell RNA sequencing (Live D-seq), a technology that enables scalable transcriptome-wide profiling of individual living cells over time. Live D-seq combines longitudinal intracellular sampling of live, barcoded cells with particle-templated emulsification and barcoded hydrogel beads, enabling repeated samples from the same cell lineage to be linked across time points. We showcase Live D-seq's capabilities by (i) demonstrating minimal cross-contamination in cross-species experiments, (ii) precisely mapping cellular barcodes to hydrogel bead barcodes, and (iii) tracking epithelial-to-mesenchymal transition dynamics in A549 cells over time. Altogether, Live D-seq enables repeated, non-destructive measurements of gene expression in the same single cells, offering a framework to study longitudinal single-cell biology in health and disease.

bioengineering↗

bioRxiv2026

Live Spatial Transcriptomics Enables Longitudinal Tissue Mapping

Spatial transcriptomics has become an important technology in biomedical research by preserving the spatial context of gene expression in tissues and enabling the study of cellular states and interactions in situ. However, most existing methods provide only static snapshots of individual tissue specimens, requiring inference of the molecular dynamics underlying development, disease progression, and therapeutic response from different specimens rather than direct tracking in the same living tissue. Here, we introduce Live Spatial Transcriptomics (Live-ST), a technology that enables longitudinal, transcriptome-wide profiling of living tissue. Live-ST combines electroporation-based intracellular sampling and spatial barcoding to enable repeated, spatially resolved mRNA measurements in intact living tissue through next-generation sequencing. We demonstrate its capabilities by (i) benchmarking spatial mRNA capture in patterned two-dimensional cell systems, (ii) spatially profiling transcriptomes in intact, patient-derived living melanoma tissues while preserving tissue viability, and (iii) longitudinally tracking drug-response dynamics within the tumor microenvironment. Together, Live-ST enables repeated, non-destructive measurements of spatial gene expression, providing a new framework for studying dynamic tissue biology in native physiological and pathological contexts.

bioengineering↗

bioRxiv2025

Tre-DST: A new drug susceptibility test for Mycobacterium tuberculosis using solvatochromic trehalose probes

Tuberculosis (TB) is the most lethal cause of death from a single infectious agent. In 2024, an estimated 10 million people developed TB, nearly half a million of which were infected with drug-resistant tuberculosis (DR-TB). Early detection of infection and drug resistance is critical to controlling DR-TB as this enables rapid engagement into effective care. Currently, bacterial culture and nucleic acid testing remain the primary methods for diagnosing infection, with smear microscopy being phased out. However, these methods present significant limitations for diagnosing drug resistance such as lengthy time-to-result for phenotypic tests, as well as the need for prior knowledge of resistance mutations and prohibitive cost for molecular tests. To address this, we developed a rapid phenotypic TB drug susceptibility test, termed Tre-DST, based on novel trehalose probes, which upon metabolic conversion emit enhanced fluorescence signal, giving them their unique ability to specifically detect live mycobacteria. We used the nonpathogenic Mycobacterium smegmatis and the virulence-attenuated Mycobacterium tuberculosis (Mtb) H37Ra or auxotrophic Mtb to demonstrate a strong correlation between cost-effective plate reader results and flow cytometry data, suggesting the fluorescence plate reader is a suitable fluorescence detector for Tre-DST. We determined that adding a one-week incubation step for Mtb allowed samples originally seeded at 104 CFU/mL to become detectable, over two weeks earlier than colony forming unit analysis. Importantly, we found that Tre-DST reports on drug susceptibility in a drug-agnostic manner, demonstrating loss of fluorescence with frontline TB drugs rifampicin (RIF), isoniazid (INH), and ethambutol, as well as the newer drug bedaquiline. Finally, Tre-DST distinguished RIF-and INH-resistant auxotrophs from susceptible controls and accurately reported resistance activity. Ultimately, because Tre-DST is agnostic to mechanisms of drug resistance, this assay is likely compatible with all WHO-recommended DR-TB drugs as well as any future TB drugs as a diagnostic in reference laboratories.

microbiology↗

bioRxiv2025

Efficacy of DNA Methyltransferase Inhibitor Immune Priming Therapy in Combination with PD-1 Inhibitors to Treat High-Risk Pediatric Brain Tumors

BackgroundDespite intensive therapies, outcomes for high-risk pediatric brain tumors (PBTs) remain dismal, prompting the search for novel treatments. DNA methyltransferase inhibitors (DNMTi) have been shown to prime tumors to improve response to checkpoint inhibition. The aim of this study was to investigate the potential of decitabine (DAC), in combination with a PD-1 inhibitor, to improve survival in pediatric high-risk brain tumor models. MethodsAnalysis of human PBT datasets was performed to determine gene expression levels of immune cell associated markers. Tumor response to DAC, with or without a PD-1 inhibitor, was tested in murine models representing H3-wildtype diffuse intrinsic pontine glioma (DIPG), H3K27-mutant diffuse midline glioma (DMG), atypical teratoid rhabdoid tumor (ATRT), and medulloblastoma (MB). CyTOF analysis of allograft tumors was performed to characterize changes within the tumor microenvironment. ResultsAnalysis of PBT subtypes revealed heterogeneous expression of immune cell markers, checkpoint receptors, and MHC molecules. DAC treatment decreased DNA methylation and increased neoantigen expression in human and mouse tumor cells. DAC alone or in combination with a PD-1 inhibitor resulted in prolonged survival in syngeneic mouse models of DIPG and ATRT but not DMG and MB models. CyTOF analysis of mouse tumors revealed changes in local immune cell infiltration upon combination treatment. ConclusionsDAC in combination with a PD-1 inhibitor can alter the immune microenvironment in mouse tumor models. Changes were observed in H3-wildtype DIPG and ATRT models, suggesting that certain tumor subtypes may respond to checkpoint blockade after immune augmentation with DNMTi. Key PointsO_LIPBTs show heterogenous expression of immune cell infiltrates C_LIO_LIDAC or DAC plus a PD-1 inhibitor shows extension of survival in H3-wildtype DIPG and ATRT mouse models C_LIO_LIMyeloid-derived suppressor cell abundance could be a major contributing factor to treatment response C_LI Importance of the StudyChildren with high-risk PBTs face dismal outcomes. Immune checkpoint inhibitor (ICI) successes have been demonstrated in a variety of adult malignancies; however, such beneficial outcomes have not been realized in PBTs. Here we investigate single and combination treatment of DNMTi and PD-1 checkpoint inhibition in syngeneic mouse models of high-risk PBTs. Our results suggest that some H3-wildtype DIPG and ATRT tumor types may be responsive to checkpoint therapy post immunomodulation and warrant further investigation.

immunology↗

bioRxiv2025

Streptococcus anginosus Activates the NLRP3 Inflammasome to Promote Inflammatory Responses from Macrophages

Chronic inflammation and oral dysbiosis are common features of oral squamous cell carcinoma (OSCC). The commensal streptococci, S. anginosus, is increased in oral diseases including OSCC. Our previous work revealed that S. anginosus promotes inflammatory responses from macrophage cell lines, however the molecular mechanism by which S. anginosus interacts with macrophages to instigate this response remains to be investigated. Here, we expand on our previous findings by investigating the effects of S. anginosus infection of primary bone marrow derived macrophages (BMMs) and during in vivo infection. We found S. anginosus activated primary BMMs, which presented an enlarged cellular area, increased NF-{kappa}B activation and downstream inflammatory cytokines TNF, IL-6 and IL-1{beta} at 24 hours post infection. S. anginosus viability was dispensable for NF-{kappa}B activation, but essential for the induction of downstream inflammatory proteins and cytokines. S. anginosus persisted intracellularly within BMMs and induced the expression of inflammasome sensors AIM2, NLRC4 and NLRP3. Further, BMMs lacking the inflammasome adapter protein ASC (Asc-/-) had significantly diminished IL-1{beta} production compared to wild type BMMs, indicating that S. anginosus activated the inflammasome. S. anginosus primarily triggered the inflammasome through NLRP3 as S. anginosus-infected Nlrp3-/- BMMs and NLRP3 inhibitor (MCC950)-treated wild type BMMs displayed diminished IL-1{beta} production compared to wild type controls. Lastly, S. anginosus-infected Asc-/- and Nlrp3-/-mice displayed reduced weight loss compared to C57BL/6 mice. These overall findings indicate that S. anginosus replicates within macrophages and promotes a proinflammatory response in part through activation of the NLRP3 inflammasome. brief summary sentence: S. anginosus replicates intracellularly within macrophages and is sensed by the NLRP3 inflammasome to promote proinflammatory response.

immunology↗

bioRxiv2024

Single-cell transcriptomics reveal synergistic and antagonistic effects of T21 and GATA1s on hematopoiesis

Trisomy 21 (T21), or Down syndrome (DS), is associated with baseline macrocytic erythrocytosis, thrombocytopenia, and neutrophilia, as well as transient abnormal myelopoiesis (TAM) and myeloid leukemia of DS (ML-DS). TAM and ML-DS blasts both arise from an aberrant megakaryocyte-erythroid progenitor and exclusively express GATA1s, the truncated isoform of GATA1, while germline GATA1s mutations in a non-T21 context lead to congenital cytopenia(s) without a leukemic predisposition. This suggests that T21 and GATA1s both perturb hematopoiesis in multipotent progenitors, but studying their individual effects is challenging due to limited access to relevant human progenitor populations. To dissect individual developmental impacts, we used single-cell RNA-sequencing to interrogate hematopoietic progenitor cells (HPCs) from isogenic human induced pluripotent stem cells differing only by chromosome 21 and/or GATA1 status. The transcriptomes of these HPCs revealed significant heterogeneity and lineage skew dictated by T21 and/or GATA1s. T21 and GATA1s each disrupted temporal regulation of lineage-specific transcriptional programs and specifically perturbed cell cycle genes. Trajectory inference revealed that GATA1s nearly eliminated erythropoiesis, slowed MK maturation, and promoted myelopoiesis in the euploid context, while in T21 cells, GATA1s competed with the enhanced erythropoiesis and impaired megakaryopoiesis driven by T21 to promote production of immature erythrocytes, MKs, and myeloid cells. The use of isogenic cells revealed distinct transcriptional programs that can be attributed specifically to T21 and GATA1s, and how they independently and synergistically result in HPC proliferation at the expense of maturation, consistent with a pro-leukemic phenotype.

molecular biology↗

bioRxiv2023

Multiple TonB Homologs are Important for Carbohydrate Utilization by Bacteroides thetaiotaomicron

The human gut microbiota is able to degrade otherwise undigestible polysaccharides, largely through the activity of the Bacteroides. Uptake of polysaccharides into Bacteroides is controlled by TonB-dependent transporters (TBDT) whose transport is energized by an inner membrane complex composed of the proteins TonB, ExbB, and ExbD. Bacteroides thetaiotaomicron (B. theta) encodes 11 TonB homologs which are predicted to be able to contact TBDTs to facilitate transport. However, it is not clear which TonBs are important for polysaccharide uptake. Using strains in which each of the 11 predicted tonB genes are deleted, we show that TonB4 (BT2059) is important but not essential for proper growth on starch. In the absence of TonB4, we observed an increase in abundance of TonB6 (BT2762) in the membrane of B. theta, suggesting functional redundancy of these TonB proteins. Growth of the single deletion strains on pectin galactan, chondroitin sulfate, arabinan, and levan suggests a similar functional redundancy of the TonB proteins. A search for highly homologous proteins across other Bacteroides species and recent work in B. fragilis suggests that TonB4 is widely conserved and may play a common role in polysaccharide uptake. However, proteins similar to TonB6 are found only in B. theta and closely related species suggesting that the functional redundancy of TonB4 and TonB6 may be limited across the Bacteroides. This study extends our understanding of the protein network required for polysaccharide utilization in B. theta and highlights differences in TonB complexes across Bacteroides species. ImportanceThe human gut microbiota, including the Bacteroides, is required for the degradation of otherwise undigestible polysaccharides. The gut microbiota uses polysaccharides as an energy source and the fermentation products such as short chain fatty acids are beneficial to the human host. This use of polysaccharides is dependent on the proper pairing of a TonB protein with polysaccharide-specific TonB-dependent transporters; however, formation of these protein complexes is poorly understood. In this study, we examine the role of 11 predicted TonB homologs in polysaccharide uptake. We show that two proteins, TonB4 and TonB6, may be functionally redundant. This may allow for development of drugs targeting Bacteroides species containing only a TonB4 homolog with limited impact on species encoding the redundant TonB6.

microbiology↗

bioRxiv2023

Scalable covariance-based connectivity inference for synchronous neuronal networks

We present a novel method for inferring connectivity from large-scale neuronal networks with synchronous activity. Our approach leverages Dynamic Differential Covariance to address the associated computational challenges. First, we analyze spike trains generated from Leaky Integrate-and-Fire network simulations and evaluate the performance of several off-the-shelf multivariate connectivity inference methods. Next, we introduce a new approach, Fractional Dynamic Differential Covariance (FDDC), and demonstrate that it consistently outperforms the other methods. Finally, we apply FDDC to experimental data to assess the topological organization of inferred graphs of in vitro neural network recordings obtained using high-density microelectrode arrays (HD-MEAs). Our results indicate that FDDC-derived graphs exhibit a significant negative correlation between small-worldness and measures of network synchrony. In contrast, graphs inferred through the well-established pairwise correlation method do not show such a correlation. This finding implies that the graphs obtained through FDDC provide stronger evidence in support of the theoretical notion that networks with clustered connections tend to exhibit higher levels of synchronizability. We hypothesize that our findings have implications for the development of scalable connectivity inference methods for large-scale neural network data.

neuroscience↗

bioRxiv2023

Brain network dynamics in transitions of consciousness reorganize according to task engagement

Substantial changes in behavior, physiology, and brain function occur when alertness decreases 1- 5. These changes in brain function involve increased synchronization between cortical areas 6,7 as well as alterations in sensory processing pathways and networks connecting the thalamus and cortex 5,8-11. Cognitive tasks engage overlapping functional networks with sensory pathways facilitating information processing 12,13, and thalamocortical and corticocortical networks supporting task performance 14,15. Frontoparietal circuits play a crucial role in cognitive tasks 16 and states of decreased consciousness 17. To develop an integrated framework of consciousness and cognition, it is important to understand how fluctuations in alertness and cognitive processing interact in these shared circuits 18. Our hypothesis is that during periods of low alertness, individuals who actively maintain task engagement would recruit additional frontoparietal and sensory processing networks, while thalamocortical dynamics that typically change during sleep onset would remain unaffected. Our findings demonstrated that as alertness decreased, passively listening to auditory tones led to increased synchronization in the parietal lobe, whereas actively performing an auditory task resulted in increased long-range frontoparietal synchronization. During decreasing alertness, passive listening (but not active task engagement) was associated with widespread increased synchronization between the thalamus and cortex. In contrast, active task engagement (but not passive listening) led to increased synchronization between the auditory cortex and the rest of the brain. These results reveal the functional mechanisms of the brains flexible reorganization during transitions of consciousness when individuals are actively engaged in cognitive processes.

neuroscience↗

bioRxiv2023

A detailed input-output characterization of single neurons reveals the synaptic basis of spontaneous spiking in recurrent networks

Self-sustained recurrent activity in cortical networks is thought to be important for multiple crucial processes, including circuit development and homeostasis. Yet, the precise relationship between synaptic input patterns and the spiking output of individual neurons remains largely unresolved. Here, we developed, validated and applied a novel in vitro experimental platform and analytical procedures that provide - for individual neurons - simultaneous excitatory and inhibitory synaptic activity estimates during recurrent network activity. Our approach combines whole-network high-density microelectrode array (HD-MEA) recordings from rat neuronal cultures with patch clamping and enables a comprehensive mapping and characterization of active incoming connections to single postsynaptic neurons. We found that, during network states with excitation(E)-inhibition(I) balance, postsynaptic spiking often coincided with the maxima of fast fluctuations in the input E/I ratio. These spike-associated E/I ratio escalations were largely due to a rapid bidirectional change in synaptic inhibition that was modulated by the network-activity level. Our approach also uncovered the underlying circuit architecture and we show that individual neurons received a few key inhibitory connections - often from special hub neurons - that were instrumental in controlling postsynaptic spiking. Balanced network theory predicts dynamical regimes governed by small and rapid input fluctuation and featuring a fast neuronal responsiveness. Our findings - obtained in self-organized neuronal cultures - suggest that the emergence of these favorable regimes and associated network architectures is an inherent property of cortical networks in general.

neuroscience↗

bioRxiv2021

Anti-prothrombin autoantibodies enriched after infection with SARS-CoV-2 and influenced by strength of antibody response against SARS-CoV-2 proteins

Antiphospholipid antibodies (aPL), assumed to cause antiphospholipid syndrome (APS), are notorious for their heterogeneity and detect phospholipids and phospholipid-binding proteins. The persistent presence of Lupus anticoagulant and/or aPL against cardiolipin and/or {beta}2 glycoprotein I have been shown to be independent risk factors for vascular thrombosis and pregnancy morbidity in APS. aPL production is thought to be triggered by - among other factors - viral infections, though infection-associated aPL have mostly been considered non-pathogenic. Recently, the potential pathogenicity of infection-associated aPL has gained momentum since an increasing number of patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been described with coagulation abnormalities and hyperinflammation, together with the presence of aPL. Here, we present data from a multicentric, mixed-severity study including three cohorts of individuals who contracted SARS-CoV-2 as well as non-infected blood donors. We simultaneously measured 10 different criteria and non-criteria aPL (IgM and IgG) by using a line immunoassay. Further, IgG antibody response against three SARS-CoV-2 proteins was investigated using tripartite automated blood immunoassay technology. Our analyses revealed that selected non-criteria aPL were enriched concomitant to or after an infection with SARS-CoV-2. Linear mixed-effects models suggest an association of aPL to prothrombin (PT) with the strength of the antibody response against SARS-CoV-2 and that it is further influenced by SARS-CoV-2 disease severity and sex of the individuals. In conclusion, our study is the first to report an association between disease severity, anti-SARS-CoV-2 immunoreactivity and aPL against PT in patients with SARS-CoV-2.

immunology↗

bioRxiv2020

Multi-Modal Profiling of Human Fetal Liver-Derived Hematopoietic Stem Cells Reveals the Molecular Signature of Engraftment Potential

The human hematopoietic stem cell (HSC) harbors remarkable regenerative potential that can be harnessed therapeutically. During early development, HSCs in the fetal liver (FL) undergo active expansion while simultaneously retaining robust engraftment capacity, yet the underlying molecular program responsible for their efficient engraftment remains unclear. We profiled 26,407 FL cells at both transcriptional and protein levels including over 7,000 highly enriched and functional FL HSCs to establish a detailed molecular signature of engraftment potential. Integration of transcript and linked cell surface marker expression revealed a generalizable signature defining functional FL HSCs and allowed for the stratification of enrichment strategies with high translational potential. This comprehensive, multi-modal profiling of engraftment capacity connects a critical biological function at a key developmental timepoint with its underlying molecular drivers, serving as a useful resource for the field.

cell biology↗

bioRxiv2019

Ictal recruitment of anterior nucleus of thalamus in human focal epilepsy

The thalamic nuclei play diverse roles in the initiation, propagation, and termination of temporal lobe seizures. The role of the anterior nucleus of the thalamus (ANT) - a node that is integral to the limbic network is unclear. The objective of this study was to characterize temporal and - spectral patterns of ANT ictal recruitment in drug-resistant temporal lobe epilepsy (TLE). We hypothesized that seizures localized to the temporolimbic network are likely to recruit ANT, and the odds of recruitment were higher in seizures that had altered consciousness. Ten patients undergoing stereo-electroencephalography (SEEG) were recruited prospectively to record field potentials from the ANT. Using epileptogenicity index and line length, we computed the number of seizures that recruited the ANT (recruitment ratio), the recruitment latencies between the ANT and the epileptogenic zone (EZ), and latency of ANT recruitment to clinical manifestation for seventy-nine seizures. We observed that seizures localized to mesial temporal subregions (hippocampus, amygdala, anterior cingulate) have a higher predilection for ANT recruitment, and the recruitment was faster (ranged 5-12 secs) and preceded clinical onset for seizures that impaired consciousness. Seizures that recruited ANT lasted significantly longer (t=1.795, p=0.005). Recruitment latency was inversely correlated to seizure duration (r=-0.78, p=0.004). Electrical stimulation of the EZ induced seizures, in which early recruitment of ANT was confirmed. Stimulation of ANT did not induce a seizure. Finally, we tested the hypothesis that spectral and entropy-based features extracted from thalamic field potentials can distinguish its state of ictal recruitment from other interictal states (including awake, sleep). For this, we employed classification machine learning that discriminated thalamic ictal state from other interictal states with high accuracy (92.8%) and precision (93.1%). Among the features, the emergence of the theta rhythm (4-8 Hz) maximally discriminated the endogenous ictal state from other interictal states of vigilance. These results prompt a mechanistic role for the ANT in the early organization and sustaining of seizures, and the possibility to serve as a target for therapeutic closed-loop stimulation in TLE.

neuroscience↗

bioRxiv2018

Macronutrients modulate resistance to infection and immunity in Drosophila

Immunity and nutrition are two essential modulators of individual fitness However, while the implications of immune function and nutrition on an individual s lifespan and reproduction are known, the interplay between feeding behaviour, infection, and immune function, remains poorly understood. In this study, we used the fruit fly, Drosophila melanogaster, to investigate how infection through septic injury modulates nutritional intake, and how macronutrient balance affects survival to infection by the pathogenic Gram-positive bacterium Micrococcus luteus. Our results show that infected flies maintain carbohydrate intake, but reduce protein intake, thereby shifting from a protein-to-carbohydrate (P:C) ratio of ~1:4 to ~1:10 relative to non-infected and sham-infected flies. Strikingly, we found that the proportion of flies dying after M. luteus infection was significantly lower when flies were fed a low-P high-C diet, revealing that flies shift their macronutrient intake as means of nutritional self-medication against bacterial infection. This is likely due to the effects of macronutrient balance on the regulation of the constitutive expression of innate immune genes, as a low-P high-C diet was linked to an up-regulation in the expression of key antimicrobial peptides. Together, our results reveal the intricate relationship between macronutrient intake and resistance to infection, and integrate the molecular cross-talk between metabolic and immune pathways into the framework of nutritional immunology.

physiology↗