Biology subjects

Golubeva, A.

Publications and source records attributed to Golubeva, A..

3 recordsLinked to original sources

bioRxiv2024

Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissues

Current scRNA-seq studies of solid tissues mostly rely on enzymatic dissociation of fresh samples or the fallback on nuclei isolation from frozen or partially fixed samples. However, due to the complex tissue organization or cell fragility, it could be challenging to apply these approaches to the sensitive endocrine tissues. That is, dissociating intact cells from such problematic fresh-frozen samples routinely collected by biobanks remains challenging. In this study, we adapted the acetic-methanol dissociation method - ACME High Salt (ACME HS) to effectively isolate intact single cells from fresh-frozen endocrine tumor samples, including adrenal gland neoplasms, thyroid carcinomas, and pituitary neuroendocrine tumors. We compared the ability of enzymatic, ACME HS, and nuclear isolation methods to preserve the integrity of major cell types and gene expression across 41 tissue samples of different origins. We demonstrated that ACME HS simultaneously dissociates and fixes cells, thus preserving morphology and a high RNA integrity number in problematic cell types. This finding renders the ACME HS dissociation method a valuable alternative in scRNA-seq protocols for challenging tissues where obtaining live cell suspension is difficult or impossible.

genomics↗

bioRxiv2020

Loss of vacuolar acidity results in iron sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wildtype yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.

molecular biology↗

bioRxiv2018

Isolation and characterization of extracellular vesicles from Caenorhabditis elegans for multi-omic analysis.

Cells from bacteria to human release vesicles into their extracellular environment. These extracellular vesicles (EVs) contain multiple classesof molecules, including nucleic acids, proteins, and lipids. The isolation and analysis of EV cargos from mammalian cell culture and liquid biopsysamples has become a powerful approach for uncovering the messages that are packaged into these organelles. However, this approach has not been tenable in invertebrate model systems due to lack of sufficient amounts of pure EVs. Here we report a robust and reproducible procedure to isolateEVs from Caenorhabditis elegans with yields similar to those obtained from human cell culture. Through nanoparticle tracking, transmission electron microscopy, flow cytometry, mass spectrometry, RNAseq, and immunoaffinity analysis we provide the first ever detailed characterization of C. elegans EV composition and demonstrate that C. elegans EVs share fundamentally similar properties with their mammalian counterparts. These include vesicle size, enrichment for lipid rafts, and similar types of RNA and protein cargos. This ability of isolate pure EVs on ascale amenable to multiple types of downstream analyses permits, multi-omics characterization of EV cargos in an invertebrate model system.

cell biology↗