Biology subjects

Bhatt, K.

Publications and source records attributed to Bhatt, K..

11 recordsLinked to original sources

bioRxiv2026

Polyketide synthase 12 is an in vivo essential source of novel mycolyl lipids in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a major pathogen worldwide that infects and transmits only among humans, yet nearly all in vivo virulence research occurs in non-human hosts. To overcome this central challenge in tuberculosis research, we leveraged a dataset of more than 50,000 sequenced isolates to identify Mtb genes that that are functionally preserved during natural infection, disease causation, and transmission between humans. This whole-genome ranking identified polyketide synthase 12 (pks12 ) as an exceptionally in vivo essential gene in human tuberculosis, which we validated in zebrafish and mouse models. Though Pks12 produces a mycoketide lipid in only trace amounts, pks12 deletion severely altered the host-facing surface and arabinoglycan architecture of Mtb. This amplified effect was explained through the discovery of mannosyl-{beta}-1-phosphomycoketide monomycolate (MPMMM), which is synthesized from the known Pks12 product at higher abundance by antigen 85 mycolyltransferases. Thus, we used a new host-facing genomic-metabolomic-phenotypic approach to discover the functions of an in vivo essential Mtb gene, which controls the physical structure of the Mtb-host interface.

microbiology↗

bioRxiv2026

Deciphering the Multiscale Morphology of Somatic Oncogenic Alterations in Hepatocellular Carcinoma

Somatic oncogenic mutations are typically defined by their molecular alterations, yet how they reorganize cellular architecture within intact tissues remain largely unknown. Here, we demonstrate that distinct oncogenic drivers produce unique multiscale architectural phenotypes that can be quantitatively resolved in intact liver tissue. Using iterative expansion microscopy, multiscale light-sheet imaging, and three-dimensional morphometric analysis, we systematically mapped structural remodeling from single-cell morphology to mitochondrial architecture in mosaic mouse models of hepatocellular oncogene activation. NRAS and CTNNB1 induced fundamentally different morphological programs. NRAS activation drove extensive remodeling of cell morphology, membrane curvature, and surface irregularity, whereas CTNNB1 activation largely preserved global cell morphology while selectively altering mitochondrial organization and shape. We first established a zonation-aware reference state for interpreting oncogene-associated organelle remodeling by resolving mitochondrial differences between periportal and pericentral hepatocytes. Within this framework, CTNNB1 activation shifted mitochondrial features toward a pericentral-like state, consistent with the role of Wnt/{beta}-catenin signaling in hepatic zonation and metabolic identity. Furthermore, integrating cellular morphology, membrane geometry, and mitochondrial architecture improved discrimination of oncogenic states beyond any individual structural feature, demonstrating that mutation-specific phenotypes arise through coordinated remodeling across multiple biological scales. Together, these findings establish multiscale structural phenotyping as a framework for linking oncogenic genotype to three-dimensional cellular organization and reveal that distinct oncogenic drivers remodel different architectural compartments during liver oncogene activation.

cancer biology↗

bioRxiv2026

Stress adaptation of free-living microbes generates novel benefits to plant hosts

Most microbes that live in or on hosts are not obligate symbionts. Instead, they often cycle between host-associated and free-living phases and experience selection in both environments. The benefits that microbes confer to hosts are often assumed to be a product of host-microbe (co)evolution, but microbial benefits to hosts may also evolve independent of the host-microbe interaction, while the microbe is free-living. We investigated this hypothesis by experimentally evolving a beneficial Allorhizobium bacterium we had previously isolated from duckweed (Lemna japonica). We evolved this Allorhizobium strain in the absence of any host at high and low salinity and at high and low nitrogen, and then tested how the evolved strains performed as symbionts when host-associated. Microbial salinity adaptation drove the emergence of novel benefits to host plants in high-salinity environments. However, these locally adaptive benefits emerged only when microbes evolved under low-nitrogen conditions; microbial adaptation to high nitrogen reduced plant growth. Bacterial phenotyping indicated that the same microbial traits that underlie salinity adaptation mediate host benefits. Whole-genome sequencing of the evolved strains revealed significant genomic shifts between selective treatments, including plasmid variation and point mutations associated with osmotic regulation. The emergence of the microbial benefits to hosts, as a byproduct of microbial adaptation, highlights that these benefits do not require targeted host-microbe co-evolution. Rather, predicting the evolutionary trajectory of these symbioses may require understanding both the abiotic and biotic selective agents acting on key microbial traits mediating the host-microbe interaction.

microbiology↗

bioRxiv2026

Oxygen-generating cryogel vaccines help overcome tumor antigen tolerance and induce durable anti-tumor immunity in prostate cancer

Therapeutic cancer vaccines represent a promising approach to boost patients own immune system to fight cancer. However, many vaccine candidates have shown limited success in clinical trials in large part due to the insufficient antigen delivery to overcome tolerance and hypoxia mediated immunosuppressive mechanisms. Cryogel-based delivery scaffolds have emerged as a promising platform for cancer vaccines due to their biocompatibility and macroporous structure that allows for effective delivery to infiltrating antigen-presenting cells. However, these systems are limited by rapid, diffusion-mediated burst release of encapsulated recombinant proteins and local hypoxia-driven immunosuppression within the scaffold. Herein, we demonstrate that click conjugation of a tumor-associated protein within cryogel-based vaccines, combined with our new O2-generating platform (Click O2-CryogelVAX), helps overcome immune suppression and weak antigenicity and primes effective anti-cancer immune responses. Sustained antigen delivery promotes cellular memory and Th1-mediated anti-cancer responses. By reversing hypoxia-driven immunosuppression, O2 acts as a powerful co-adjuvant to enhance humoral immunity. Together, Click O2-CryogelVAX supports a robust antitumor response that inhibits tumor growth and prolongs survival in a therapeutic prostate cancer model. These findings support the further research and development of Click O2-CryogelVAX as an effective delivery platform for therapeutic cancer vaccines.

bioengineering↗

bioRxiv2026

Modulation of Oncogenic KRAS Signaling by Branched Actin-driven Cell Membrane Protrusions

For over three decades, we have known that oncogenic RAS alters the actin cytoskeleton organization and cell surface morphology1,2. RAS activates the GTPase RAC1, which triggers the growth of branched actin networks to promote cell membrane protrusions3,4. In melanoma, the hyperactive RAC1 mutant, Rac1P29S, was recently shown to drive extended lamellipodia, which then empower cell proliferation through sequestration and localized inhibition of the merlin tumor suppressor5. This discovery illustrates cell morphological programs not only as outputs but also as regulators of human oncogenic signals. Hence, we wondered whether the pronounced branched actin-driven membrane protrusions (BAMPs) downstream of oncogenic RAS are not mere outputs of RAS signaling but rather an active component in mediating the oncogenic penetrance of RAS mutants. We used volumetric light sheet microscopy and biochemical approaches to investigate the role of BAMPs in regulating the molecular signaling of oncogenic KRAS in pancreatic and lung cancer models. We found that elevated BAMP formation regulated the interaction of oncogenic KRAS with downstream effectors, specifically with the RAC1 GEF TIAM1. This implies that BAMPs amplify their own upstream regulators in a positive feedback. This meritorious cycle upregulates cyclin D1 expression by inactivating the merlin tumor suppressor, independently of the mitogen activated protein kinase pathway (MAPK). In the absence of BAMPs, cells carrying oncogenic KRAS mutations are unable to attain their full penetrance in proliferation. Overall, this work unveils the long-overlooked role of branched actin-driven cell morphology in the functionalization of KRAS mutants as potent oncogenes.

cancer biology↗

bioRxiv2024

Caveolin-1 regulates context-dependent signaling and survival in Ewing Sarcoma.

Plasticity is a hallmark function of cancer cells, yet the mechanisms that enable dynamic switching between survival states remain incompletely understood. Here, we identify Caveolin-1, a membrane-domain scaffolding protein, as a context-dependent regulator of survival signaling in Ewing sarcoma (EwS). Single-cell analyses reveal a distinct subpopulation of EwS cells marked by high CD99 and elevated Caveolin-1 expression. These CD99High cells exhibit unique morphology, transcriptional programs, and markedly enhanced survival both under chemotherapeutic challenge and in vivo. Importantly, CD99High and CD99Low states are reversible, providing EwS cells with a flexible route to survival-oriented plasticity. Mechanistically, we show that Caveolin-1 in CD99High cells orchestrates PI3K/AKT survival signaling by modulating the spatial organization of PI3K activity on the plasma membrane. We propose that the CD99High state establishes a Caveolin-1-driven signaling architecture that supports survival through mechanisms distinct from those used by CD99Low cells. These findings uncover a dynamic state transition in EwS cells and position Caveolin-1 as a key driver of context-specific survival signaling.

cancer biology↗

bioRxiv2023

Controlling pericellular oxygen tension in cell culture reveals distinct breast cancer responses to low oxygen tensions

Oxygen (O2) tension plays a key role in tissue function and pathophysiology. O2-controlled cell culture, in which the O2 concentration in an incubators gas phase is controlled, is an indispensable tool to study the role of O2 in vivo. For this technique, it is presumed that the incubator setpoint is equal to the O2 tension that cells experience (i.e., pericellular O2). We discovered that physioxic (5% O2) and hypoxic (1% O2) setpoints regularly induce anoxic (0.0% O2) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O2 consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O2 tensions, we developed a reaction-diffusion model that predicts pericellular O2 tension a priori. This model revealed that the effect of cellular O2 consumption is greatest in smaller volume culture vessels (e.g., 96-well plate). By controlling pericellular O2 tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs. hypoxia. MCF7 also expressed higher levels of HIF2A, CD73, NDUFA4L2, etc. and lower levels of HIF1A, CA9, VEGFA, etc. in response to hypoxia vs. anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs. anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O2, suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O2 tension in cell culture incubators is insufficient to control O2 in cell culture and introduce the concept of pericellular O2-controlled cell culture.

bioengineering↗

bioRxiv2023

Keratin isoform shifts modulate motility signals during wound healing

Keratin intermediate filaments form strong mechanical scaffolds that confer structural stability to epithelial tissues, but the reason this function requires a protein family with 54 isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. How this change modulates cellular function to support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising epidermal stability by activating myosin motors. This pathway depended on isoform-specific interaction between intrinsically disordered keratin head domains and non-filamentous vimentin shuttling myosin-activating kinases. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as isoform-tuned signaling scaffolds that organize signal transduction cascades in space and time to influence epithelial cell state.

cell biology↗

bioRxiv2023

HIF-stabilizing Biomaterials: from Hypoxia-mimicking to Hypoxia-inducing

Recent advances in our understanding of hypoxia and hypoxia-mediated mechanisms shed light on the critical implications of the hypoxic stress on cellular behavior. However, tools emulating hypoxic conditions (i.e., low oxygen tensions) for research are limited and often suffer from major shortcomings, such as lack of reliability and off-target effects, and they usually fail to recapitulate the complexity of the tissue microenvironment. Fortunately, the field of biomaterials is constantly evolving and has a central role to play in the development of new technologies for conducting hypoxia-related research in several aspects of biomedical research, including tissue engineering, cancer modeling, and modern drug screening. In this perspective, we provide an overview of several strategies that have been investigated in the design and implementation of biomaterials for simulating or inducing hypoxic conditions--a prerequisite in the stabilization of hypoxia-inducible factor (HIF), a master regulator of the cellular responses to low oxygen. To this end, we discuss various advanced biomaterials, from those that integrate hypoxia-mimetic agents to artificially induce hypoxia-like responses, to those that deplete oxygen and consequently create either transient (< 1 day) or sustained (> 1 day) hypoxic conditions. We also aim to highlight the advantages and limitations of these emerging biomaterials for biomedical applications, with an emphasis on cancer research.

bioengineering↗

bioRxiv2023

Hypoxia-inducing cryogels uncover key cancer-immune cell interactions in an oxygen-deficient tumor microenvironment

Hypoxia, an important feature of solid tumors, is a major factor shaping the immune landscape, and several cancer models have been developed to emulate hypoxic tumors. However, to date, they still have several limitations, such as the lack of reproducibility, inadequate biophysical cues, limited immune cell infiltration, and poor oxygen (O2) control, leading to non-pathophysiological tumor responses. As a result, it is essential to develop new and improved cancer models that mimic key features of the tumor extracellular matrix and recreate tumor-associated hypoxia while allowing cell infiltration and cancer-immune cell interactions. Herein, hypoxia-inducing cryogels (HICs) have been engineered using hyaluronic acid (HA) as macroporous scaffolds to fabricate three-dimensional microtissues and model a hypoxic tumor microenvironment. Specifically, tumor cell-laden HICs have been designed to deplete O2 locally and induce long-standing hypoxia. This state of low oxygen tension, leading to HIF-1 stabilization in tumor cells, resulted in changes in hypoxia-responsive gene expression and phenotype, a metabolic adaptation to anaerobic glycolysis, and chemotherapy resistance. Additionally, HIC-supported tumor models induced dendritic cell (DC) inhibition, revealing a phenotypic change in plasmacytoid B220+ DC (pDC) subset and an impaired conventional B220- DC (cDC) response in hypoxia. Lastly, our HIC-based melanoma model induced CD8+ T cell inhibition, a condition associated with the downregulation of pro-inflammatory cytokine secretion, increased expression of immunomodulatory factors, and decreased degranulation and cytotoxic capacity of T cells. Overall, these data suggest that HICs can be used as a tool to model solid-like tumor microenvironments and identify a phenotypic transition from cDC to pDC in hypoxia and the key contribution of HA in retaining cDC phenotype and inducing their hypoxia-mediated immunosuppression. This technology has great potential to deepen our understanding of the complex relationships between cancer and immune cells in low O2 conditions and may pave the way for developing more effective therapies.

cancer biology↗

bioRxiv2021

Inference of Granger-causal relations in molecular systems -- a case study of the functional hierarchy among actin regulators in lamellipodia

Many cell regulatory systems implicate nonlinearity and redundancy among components. The regulatory network governing lamellipodial and lamellar actin structures is prototypical of such a system, containing tens of actin-nucleating and -modulating molecules with functional overlap and feedback loops. Due to instantaneous and long-term compensation, phenotyping the system response to perturbation provides limited information on the roles the targeted component plays in the unperturbed system. Accordingly, how individual actin regulators contribute to lamellipodial dynamics remains ambiguous. Here, we present a perturbation-free reconstruction of cause-effect relations among actin regulators by applying Granger-causal inference to constitutive image fluctuations that indicate regulator recruitment as a proxy for activity. Our analysis identifies distinct zones of actin regulator activation and of causal effects on filament assembly and delineates actin-dependent and actin-independent regulator roles in controlling edge motion. We propose that edge motion is driven by assembly of two independently operating actin filament systems. A record of this papers Transparent Peer Review process is included in the Supplemental Information.

cell biology↗