Biology subjects

Patel, A. P.

Publications and source records attributed to Patel, A. P..

5 recordsLinked to original sources

bioRxiv2021

Binocular Benefit Following Monocular Subretinal AAV Injection in a Mouse Model of Autosomal Dominant Retinitis Pigmentosa (adRP)

Autosomal dominant retinitis pigmentosa (adRP) is frequently caused by mutations in RHO, the gene for rhodopsin. In previous experiments in dogs with the T4R mutation in RHO, an AAV2/5 vector expressing both an shRNA directed to human and dog RHO mRNA and an shRNA-resistant human RHO cDNA (AAV-RHO820-shRNA820) prevented retinal degeneration for more than 8 months following injection. To confirm that this same vector could protect the retinas of a different species and bearing a different RHO mutation, we injected mice transgenic for human P23H RHO at postnatal day 30 in one eye. For nine months, we monitored their retinal structure using spectral- domain optical coherence tomography (SD-OCT) and retinal function using electroretinography (ERG). We compared these to P23H RHO transgenic mice injected with AAV-GFP. Though retinas continued to thin over time, compared to control injected eyes, AAV-RHO820-shRNA820 slowed the loss of photoreceptor cells and decreased ERG amplitudes in AAV-RHO820-shRNA820 eyes during the nine-month study period. Unexpectedly, we also observed preservation of retinal structure and function in the untreated contralateral eyes of AAV-RHO820-shRNA820 treated mice. PCR analysis and western blots provided evidence that a low amount of vector from injected eyes was present in uninjected eyes.

neuroscience↗

bioRxiv2021

The Aspartyl Protease Ddi1 Is Essential for Erythrocyte Invasion by the Malaria Parasite

Malaria pathology is caused by the exponential replication of Plasmodium parasites in the blood stream. The bottleneck of the parasite life cycle is the invasion of erythrocytes immediately after parasites egress from infected red blood cells. DNA damage-inducible protein 1 (Ddi1) is a conserved eukaryotic proteasome shuttle protein containing an internal retroviral-like protease domain. Using conditional genetics, we now show that the proteolytic activity of the P. falciparum homologue, PfDdi1, is critically required for invasion of red blood cells. Furthermore, PfDdi1 disruption results in the accumulation of highly polyubiquitinated proteins that can be processed by purified PfDdi1 or distant eukaryotic homologues. We also show that PfDdi1 interacts with multiple components of the ubiquitin-proteasome system and that parasites lacking PfDdi1 are more sensitive to proteasome inhibition. Overall, this study establishes PfDdi1 as a key component of the eukaryotic ubiquitin-proteasome system and as a promising antimalarial target.

cell biology↗

bioRxiv2021

High Content and High Throughout Phenotypic Assay for the Hourly Resolution of the Malaria Parasite Erythrocytic Cycle

Over the last 20 years increased funding for malaria research has resulted in very significant technical advances to study the biology of Plasmodium species. High throughput phenotypic assays have been developed to screen millions of compounds and identify small molecules with antiparasitic activity. At the same time, advances in malaria genetic have greatly facilitated the generation of genetically modified parasites, and whole genome genetic screens are now feasible in Plasmodium species. Finally, there has been an increased interest to study malaria parasites at the population level, in particular in the area of drug resistance. Drug resistant field isolates have been collected around the world, and drug resistant strains are routinely generated in the lab to study the mechanisms of drug resistance. As a result, one of the current bottlenecks in malaria research is our ability to quickly characterize the phenotype associated with compound treatment or genetic modification, or to quickly compare differences in intracellular development between strains. Here, we present a high content/high throughput phenotypic assay that combines highly selective RNA, DNA, and RBC membrane dyes to provide hourly resolution of the full erythrocytic cycle for both P. falciparum and P. knowlesi. A flow cytometry assay allows the analysis of samples in a 384-well format and a quick way to determine the parasite developmental stage. On the other hand, the fluorescence microscopy format allows for a detailed visualization of parasite morphology. Finally, using open source software we have developed protocols for the automated cluster analysis of microscopy images. This assay can be applied to any Plasmodium species, requires very little amount of sample, is performed with fixed cells, and is easily scalable. Overall, we believe this assay will be a great tool for the malaria community to study Plasmodium species.

microbiology↗

bioRxiv2020

Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB

Parkinsons disease (PD) is a neurodegenerative disorder characterized by death of midbrain dopamine neurons. The pathogenesis of PD is poorly understood, though misfolded and/or aggregated forms of the protein -synuclein have been implicated in several neurodegenerative disease processes, including neuroinflammation and astrocyte activation. Astrocytes in the midbrain play complex roles during PD, initiating both harmful and protective processes that vary over the course of disease. However, despite their significant regulatory roles during neurodegeneration, the cellular and molecular mechanisms that promote pathogenic astrocyte activity remain mysterious. Here, we show that -synuclein preformed fibrils (PFFs) induce pathogenic activation of human midbrain astrocytes, marked by inflammatory transcriptional responses, downregulation of phagocytic function, and conferral of neurotoxic activity. These effects required the necroptotic kinases RIPK1 and RIPK3, but were independent of MLKL and necroptosis. Instead, both transcriptional and functional markers of astrocyte activation occurred via RIPK-dependent activation of NF-{kappa}B signaling. Our study identifies a previously unknown function for -synuclein in promoting neurotoxic astrocyte activation, as well as new cell death-independent roles for RIP kinase signaling in the regulation of glial cell biology and neuroinflammation. Together, these findings highlight previously unappreciated molecular mechanisms of pathologic astrocyte activation and neuronal cell death with implications for Parkinsonian neurodegeneration.

neuroscience↗

bioRxiv2020

Single-cell analysis of chromatin silencing programs in developmental and tumor progression

Single-cell analysis has become a powerful approach for the molecular characterization of complex tissues. Methods for quantifying gene expression1 and chromatin accessibility2 of single cells are now well-established, but analysis of chromatin regions with specific histone modifications has been technically challenging. Here, we adapt the recently published CUT&Tag method3 to scalable single-cell platforms to profile chromatin landscapes in single cells (scCUT&Tag) from complex tissues. We focus on profiling Polycomb Group (PcG) silenced regions marked by H3K27 trimethylation (H3K27me3) in single cells as an orthogonal approach to chromatin accessibility for identifying cell states. We show that scCUT&Tag profiling of H3K27me3 distinguishes cell types in human blood and allows the generation of cell-type-specific PcG landscapes from heterogeneous tissues. Furthermore, we use scCUT&Tag to profile H3K27me3 in a brain tumor patient before and after treatment, identifying cell types in the tumor microenvironment and heterogeneity in PcG activity in the primary sample and after treatment.

genomics↗