Biology subjects

Bhatt, B.

Publications and source records attributed to Bhatt, B..

8 recordsLinked to original sources

bioRxiv2026

Sense-and-response to intracellular signaling via nucleocytoplasmic localization control of a synthetic transcription factor platform

Many therapeutically relevant signaling pathways are sensed inside the cell, yet a modular synthetic biology platform for responding to intracellular signaling at the post-translational level is lacking. Signaling pathways control the nuclear localization and activity of native transcription factors via phosphorylation of nuclear localization/export or cytoplasmic anchor sequences. Here, we apply this concept to develop a platform of nucleocytoplasmic localization tags (NCtags) that control the nuclear translocation of a synthetic transcription factor (synTF) for transgene induction in engineered cells. We identify human-based synTF components that are basally cytoplasmic and induce transgenes under the control of NCtags with minimal background. We demonstrate the modular nature of this platform through transcriptional reporter induction in response to a calcium-inducible NFAT-derived NCtag, and development of novel NCtags responsive to hyperosmolarity, cAMP, or logical combinations of cAMP and calcium. Multiple factors in the tumor microenvironment elevate cAMP in T cells, leading to reduced function. We demonstrate that NCtag-synTF fusions expressed in T cells can respond to endogenous sources of cAMP or calcium in breast cancer spheroids to induce transgene expression. Our use of human components and an all-in-one lentiviral vector encoding the NCtag-synTF-transgene system establishes this as a novel platform for sense-and-response inside therapeutic cells.

synthetic biology↗

bioRxiv2023

Ketogenic diet prevents sucrose withdrawal-induced anxiety-like behaviour in male mice

Sugar bingeing has been shown to induce withdrawal effects when sugar is no longer available in the diet. In this study, we investigated the sex differences in sugar-addiction-like behaviour and the effects of the ketogenic diet on these outcomes. We used the two-bottle sucrose choice paradigm as a pre-clinical sucrose overeating and withdrawal model. Female mice consumed more sucrose than males when given free access to water and 10% sucrose for four weeks. One week of sucrose withdrawal after four weeks of consecutive sucrose overeating showed anxiety-like behaviour in male mice. However, the ketogenic diet did not affect sucrose overconsumption in males and females but prevented the sucrose withdrawal-induced anxiety-like behaviour in male mice with a concomitant increase in cFos mRNA in the prefrontal cortex. These findings provide evidence of sex differences in sucrose addiction-like behaviour and also indicate that a ketogenic diet may prevent sucrose withdrawal-induced anxiety-like behaviour in males. However, further research is needed to explore the underlying molecular mechanisms driving sex differences in sugar-addiction-like behaviour and the anxiolytic effects of the ketogenic diet.

neuroscience↗

bioRxiv2021

Combinatorial transcriptional codes specify macrophage polarization destinations

Macrophages are driven to form distinct functional phenotypes in response to different immunological stimuli, in a process widely referred to as macrophage polarization. Transcriptional regulators that guide macrophage polarization in response to a given trigger remain largely unknown. In this study, we interrogate the programmable landscape in macrophages to find regulatory panels that determine the precise polarization state that a macrophage is driven to. Towards this, we configure an integrative network analysis pipeline that utilizes macrophage transcriptomes in response to 28 distinct stimuli and reconstructs contextualized human gene regulatory networks, and identifies epicentres of perturbations in each case. We find that these contextualized regulatory networks form a spectrum of thirteen distinct clusters with M1 and M2 at the two ends. Using our computational pipeline, we identify combinatorial panels of epicentric regulatory factors (RFs) for each polarization state. We demonstrate that a set of three RFs i.e., CEBPB, NFE2L2 and BCL3, is sufficient to change the polarization destination from M1 to M2. siRNA knockdown of the 3-RF set in THP1 derived M0 cells, despite exposure to an M1 stimulant, significantly attenuated the shift to M1 phenotype, and instead increased the expression of M2 markers. Single knockdown of each RF also showed a similar trend. The siRNA-mediated knockdown of the 3-RF set rendered the macrophages hyper-susceptible to Staphylococcus aureus infection, demonstrating the importance of these factors in modulating immune responses. Overall, our results provide insights into the transcriptional mechanisms underlying macrophage polarization and identify key regulatory factors that may be targeted to modulate immune responses.

systems biology↗

bioRxiv2021

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling

RAS is the most commonly mutated oncogene in human cancers and RAS-driven tumors are amongst the most difficult to treat. Historically, discovery of therapeutics targeting this protein has proven challenging due to a lack of deep hydrophobic pockets to which a small molecule might bind. The single such pocket available is normally occupied by GDP or GTP which have millimolar cellular concentrations and picomolar affinities for KRAS and hence is challenging to target. The recent FDA approval of sotorasib, a covalent modifier of the KRASG12C mutant protein, has clinically validated KRAS as an oncology target. However, traditional challenges remain for targeting the more common KRAS mutations such as G12D and G12V. As an alternative approach, we investigated the superior binding capacity of macrocyclic peptides to identify KRAS inhibitory molecules. We focused on the recently reported disulfide-cyclized arginine-rich peptide KRpep-2d, discovered through phage display and previously independently confirmed by us as a bona fide KRAS binder. To mitigate intracellular disulfide reduction and loss of binding, we identified an alternate cyclization motif by inverting the configuration of Cys5 and linking it to Cys15 through a thioacetal bridge. The corresponding peptide bound KRAS through cis isomerization of the peptide bond between D-Cys5 and Pro6 as observed through x-ray crystallography. Prototypic molecules displayed measurable cellular inhibition of RAS signaling without membrane lysis and counter-screen off-target activity. An analogue containing azido-lysine confirmed the cell penetrant nature of this molecule using our recently reported NanoClick assay. To improve cellular activity, we sought to improve proteolytic stability. Structure-activity relationship studies identified key scaffold residues critical for binding and revealed that replacement of N- and C-terminal arginine residues with D-arginines and introduction of an -methyl moiety at Ser10 resulted in a molecule with improved proteolytic stability as indicated by its persistence in whole cell homogenate. The resulting peptide MP-3995 had improved and sustained cell-based efficacy. On-target activity was validated through confirmation of target engagement, lack of signaling in irrelevant pathways, and use of non-binding control peptides. Mechanism of action studies suggested that peptide binding to both GDP- and GTP-states of KRAS may contribute to cellular activity. Although validated as bona fide and on-target inhibitors of cell based KRAS signaling, this series is unlikely to advance to the clinic in its current form due to its arginine-dependent cell entry mechanism. Indeed, we showed a strong correlation between net positive charge and histamine release in an ex vivo assay making this series challenging to study in vivo. Nonetheless, these molecules provide valuable templates for further medicinal chemistry efforts aimed at leveraging this unique inhibitory binding site on KRAS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/459913v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17f4764org.highwire.dtl.DTLVardef@12818daorg.highwire.dtl.DTLVardef@177bdf0org.highwire.dtl.DTLVardef@57046c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Preexisting mycobacterial infection modulates Candida albicans-driven pyroptosis

Active tuberculosis patients are at high risk of co-infection with opportunistic fungal pathogen C. albicans. However, the molecular mechanisms that orchestrate pathogenesis of Mycobacterium tuberculosis (Mtb)-C. albicans co-infection remains elusive. In the current study, we utilise a mouse model to demonstrate that Mtb promotes macrophage environment conducive for C. albicans survival. Mtb-dependent PKC{zeta}-WNT signalling axis induces expression of an E3 ubiquitin ligase, COP1. A secondary infection of C. albicans in such Mtb-infected macrophages causes COP1 to mediate the proteasomal degradation of IRF9, a cardinal factor that we identified to arbitrate an inflammatory programmed cell death, pyroptosis. In vivo experiments mimicking a preexisting Mtb infection demonstrate that inhibition of pyroptosis in mice results in increased C. albicans burden and aberrant lung tissue architecture, leading to increased host mortality. Together, our study reveals the crucial role of pyroptosis regulation for manifesting a successful C. albicans-Mtb co-infection.

microbiology↗

bioRxiv2021

G9a and Sirtuin6 epigenetically modulate host cholesterol accumulation to facilitate mycobacterial survival

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that a functional amalgamation of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and Sirt6 (H3K9 deacetylase) orchestrate cholesterol build-up in in-vitro and in-vivo models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while Sirt6 represses the genes involved in cholesterol efflux. The accumulated cholesterol promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and in vivo by pharmacological inhibition; or utilization of BMDMs derived from Sirt6 KO mice or in vivo infection in Sirt6 heterozygous mice; hampers host cholesterol accumulation and restricts Mtb burden. These findings shed light on the novel roles of G9a and Sirt6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

microbiology↗

bioRxiv2020

A Multi-perspective Analysis of Retractions in Life Sciences

The aim of this study is to explore trends in retracted publications in life sciences and biomedical sciences over axes like time, countries, journals and impact factors, and topics. Nearly seven thousand publications, which comprise the entirety of retractions visible through PubMed as of August 2019, were used. This work involved sophisticated data collection and analysis techniques to use data from PubMed, Wikipedia, and WikiData, and study it with respect to the above mentioned axes. Importantly, I employ state-of-the-art analysis and visualization techniques from natural language processing (NLP) to understand the topics in retracted literature. To highlight a few results, the analyses demonstrate an increasing rate of retraction over time and noticeable differences in the publication quality (as measured by journal impact factor) among top countries. Moreover, while molecular biology and cancer dominate retractions, we also see a number of retractions not related to biology. The methods and results of this study can be applied to continuously understand the nature and evolution of retractions in life sciences, thus contributing to the health of this research ecosystem.

scientific communication and education↗

bioRxiv2019

Partial loss of CFIm25 causes aberrant alternative polyadenylation and learning deficits

We previously showed that NUDT21-spanning copy-number variations (CNVs) are associated with intellectual disability (Gennarino et al., 2015). However, the patients CNVs also included other genes. To determine if reduced NUDT21 function alone can cause disease, we generated Nudt21+/- mice to mimic the human state of decreased expression. We found that although these mice have 50% reduced Nudt21 mRNA, they only have 30% less of its cognate protein, CFIm25. Despite this partial protein-level compensation, the Nudt21+/- mice have learning deficits and cortical hyperexcitability. Further, to determine the molecular mechanism driving neural dysfunction, we partially inhibited NUDT21 in human stem cell-derived neurons to reduce CFIm25 by 30%. This reduction in CFIm25 was sufficient to induce misregulated alternative polyadenylation (APA) and protein levels in hundreds of genes, dozens of which cause intellectual disability when mutated. Altogether, these results indicate that disruption of NUDT21-regulated APA events in the brain can cause intellectual disability.

genomics↗