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Biology subjects

Natarajan, S.

Publications and source records attributed to Natarajan, S..

22 records · Page 2Linked to original sources

Drug and siRNA screens identify ROCK2 as a therapeutic target for ciliopathies.

Primary cilia are microtubule-based organelles that act as cellular antennae to mediate vertebrate development and growth factor signalling. Defects in primary cilia result in a group of inherited developmental conditions known as ciliopathies. Ciliopathies often present with cystic kidney disease, a major cause of early renal failure that requires renal replacement therapies. Currently, only one drug, Tolvaptan, is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions for these conditions remain a pressing clinical need. We screened clinical development compounds for positive effects on cilia formation and function and identified fasudil hydrochloride as the top hit. Fasudil is a generic, off-patent drug that is a potent but broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In a parallel whole genome siRNA-based reverse genetics phenotypic screen of positive modulators of cilia formation, we identified ROCK2 as the target molecule. We demonstrate that ROCK2 is a key mediator of cilium formation and function through effects on actin cytoskeleton remodelling. Our results indicate that specific ROCK2 inhibitors such as belumosudil (KD-025) could be repurposed for pharmacological intervention in cystic kidney disease. We propose that ROCK2 inhibition represents a novel, disease-modifying therapeutic approach for heterogeneous ciliopathies.

cell biology↗

Accurate Genomic Variant Detection in Single Cells with Primary Template-Directed Amplification

Improvements in whole genome amplification (WGA) would enable new types of basic and applied biomedical research, including studies of intratissue genetic diversity that require more accurate single-cell genotyping. Here we present primary template-directed amplification (PTA), a new isothermal WGA method that reproducibly captures >95% of the genomes of single cells in a more uniform and accurate manner than existing approaches, resulting in significantly improved variant calling sensitivity and precision. To illustrate the new types of studies that are enabled by PTA, we developed direct measurement of environmental mutagenicity (DMEM), a new tool for mapping genome-wide interactions of mutagens with single living human cells at base pair resolution. In addition, we utilized PTA for genome-wide off-target indel and structural variant detection in cells that had undergone CRISPR-mediated genome editing, establishing the feasibility for performing single-cell evaluations of biopsies from edited tissues. The improved precision and accuracy of variant detection with PTA overcomes the current limitations of accurate whole genome amplification, which is the major obstacle to studying genetic diversity and evolution at cellular resolution.

genomics↗

Single-cell analysis identifies TCF4 and ID3 as a molecular switch of mammary epithelial stem cell differentiation.

It is well known that the expansion of the mammary epithelium during the ovarian cycles in female mammals is supported by the transient increase in mammary epithelial stem cells (MaSCs). However, dissecting the molecular mechanisms that govern MaSC function and differentiation is poorly understood due to the lack of standardized methods for their identification and isolation. The development of robust single-cell mRNA sequencing () technologies and the computational methods to analyze them provides us with novel tools to approach the challenge of studying MaSCs in a completely unbiased way without. Here, we have performed the largest scRNA-seq analysis of individual mammary epithelial cells (~70,000 cells). Our study identified a distinct cell population presenting molecular features of MaSCs. Importantly, further purification and additional in-depth single-cell analysis of these cells revealed that they are not a fully homogenous entity. Instead, we identified three subpopulations representing early stages of lineage commitment. By tracking their molecular evolution through single-cell network analysis we found that one of these subpopulations represents bipotent MaSCs from which luminal and basal lineages diverge. Importantly, we also confirmed the presence of these cells in human mammary glands. Finally, through expression and network analysis studies, we have uncovered transcription factors that are activated early during lineage commitment. These data identified E2-2 (Tcf4) and ID3 as a potential molecular switch of mammary epithelial stem cell differentiation.

developmental biology↗

FLASH irradiation enhances the therapeutic index of abdominal radiotherapy in mice

Peritoneal metastases are the leading cause of morbidity and mortality in ovarian cancer. Despite current surgery, chemotherapy and targeted therapies, the majority of patients diagnosed with advanced epithelial ovarian cancer develop recurrent disease and overall survival rates remain poor. It is known that ovarian cancer is a radiosensitive tumor. Historically, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of chemotherapy sensitive and resistant ovarian cancer. However, TAI fell out of favor due to high toxicity, particularly of the gastrointestinal tract. We have developed a preclinical irradiation platform that allows for total abdominal ultrahigh dose rate FLASH irradiation. We demonstrate that TAI-FLASH reduces radiation-induced intestinal injury in both healthy and tumor-bearing mice compared to conventional dose rate (CONV) irradiation. Single high dose TAI-FLASH reduced mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and decreased cell death in crypt base columnar cells. Importantly, FLASH and CONV irradiation had similar efficacy in the reduction of ovarian cancer peritoneal metastases. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of radiotherapy for the treatment of metastatic ovarian cancer in women.

cancer biology↗