Biology subjects

Prakhar, P.

Publications and source records attributed to Prakhar, P..

4 recordsLinked to original sources

bioRxiv2026

Deep learning-guided design of cell type-specific AAV promoters

Precise cell type targeting is critical for both clinical and experimental applications of adeno-associated viral (AAV) vectors, yet engineering vectors with cell type-specific activity remains a challenge. Here, we compared three strategies leveraging single-cell chromatin accessibility data to design cell type-specific AAV promoters, including a deep learning-based method to generate de novo regulatory sequences. When applied to target retinal ganglion cells or horizontal cells in mouse retina, deep learning-guided design consistently outperformed rational approaches, yielding synthetic promoters with stronger and more specific expression in vivo. Synthetic AAV promoters supported diverse transgenes, enabling the recording and ablation of targeted cells. Promoter activity was also maintained in human retinal organoids, suggesting that deep learning-designed sequences may be suitable for translation. Our findings highlight the potential of deep learning to synthesize cell type-specific AAV promoters and establish a versatile platform for cell type targeting with broad implications for gene therapy and basic research.

synthetic biology↗

bioRxiv2024

Induction of a Muller glial-specific protective pathway safeguards the retina from diabetes induced damage

Diabetes can lead to cell-type-specific responses in the retina, including vascular lesions, glial dysfunction and neurodegeneration, all of which contribute to retinopathy. However, the molecular mechanisms underlying these cell type-specific responses, and the cell types that are sensitive to diabetes have not been fully elucidated. Employing single cell transcriptomic analyses, we profiled the transcriptional changes induced by diabetes in different retinal cell types in diabetic rat models as the disease progressed. Rod photoreceptors, a subtype of amacrine interneurons, and Muller glial cells exhibited rapid responses to diabetes at the transcript levels. Genes associated with ion regulation were upregulated in all three cell types, suggesting a common response to diabetes. Furthermore, focused studies revealed that while Muller glial cells initially increased the expression of genes playing protective roles, they cannot sustain this beneficial effect as the disease progressed. We explored one of the candidate protective genes, Zinc finger protein 36 homolog (Zfp36), and observed that depleting Zfp36 in rat Muller glial cells in vivo using AAV-based tools exacerbated early diabetes-induced phenotypes, including gliosis, neurodegeneration, and vascular defects. Notably, the over-expression of Zfp36 slowed the development of phenotypes associated with diabetic retinopathy. In summary, this work unveiled retinal cell types that are sensitive to diabetes and demonstrated that Muller glial cells can mount protective responses through Zfp36. The failure to maintain Zfp36 levels contributes to the development of diabetic retinopathy.

neuroscience↗

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↗