Biology subjects

Feldman, T.

Publications and source records attributed to Feldman, T..

5 recordsLinked to original sources

bioRxiv2025

Interrogation of retinal lipofuscin by fluorescence lifetime imaging microscopy

Age-related macular degeneration is a disease that affects the middle part of the vision and involves pathological alterations in the retinal pigment epithelium. Accurate and timely evaluation of the retinal pigment epithelium is a cornerstone of effective treatment planning. In this study, we present the development of a preclinical method for early diagnostics of age-related macular degeneration using time and spectral characteristics of fluorescence of lipofuscin granules from retinal pigment epithelium. Using the unique system based on asuperconducting single-photon detector and time-correlated single-photon counting electronics integrated in the confocal laser scanning microscope we determined the parameters of fluorescence (distribution long and short fluorescence lifetime components and their contribution to the total fluorescence signal as well as fluorescence spectral shift) that have a diagnostic value for differentiation of the normal and pathological states in the degenerative diseases of the retina and retinal pigment epithelium.

biophysics↗

bioRxiv2022

DNA Methylation is stable after MMEJ and NHEJ double strand break repair

DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred. Author SummaryDNA holds the genetic information. Modifications on the DNA molecule such as DNA methylation are crucial for the genetic regulation. DNA damage is harmful to the cell and needs to be repaired. Different repair mechanisms may result in mutations that can be identified according to their sequence signature. In the work presented here we examined changes in DNA methylation after different types of DNA repair, MMEJ and NHEJ. We found that DNA methylation is highly stable after DNA repair regardless to the repair mechanism and genomic context.

genetics↗

bioRxiv2022

The Colon Mucosal Sialylglycome Is Redox-Regulated by the Golgi Enzyme QSOX1

Mucus shields the intestinal epithelium from pathogens and provides a supportive environment for commensal bacteria. Mucus is composed of enormous, heavily glycosylated proteins called mucins, which become disulfide crosslinked in a multi-step biosynthetic pathway culminating in the Golgi apparatus and secretory granules of goblet cells. We observed that knockout mice lacking the Golgi-localized disulfide catalyst QSOX1 produced poorly protective colon mucus, were hypersensitive to induced colitis, and had an altered microbiome. The initial hypothesis arising from these observations was that QSOX1 catalyzes disulfide crosslinking of mucins. Contrary to this hypothesis, the disulfide-mediated polymerization of mucins and related glycoproteins proceeded normally without QSOX1. Instead, we found that QSOX1 forms regulatory disulfides in Golgi glycosyltransferases and thereby promotes effective sialylation of the colon glycome. Our findings reveal that enzymatic control of Golgi redox state impacts glycan elaboration in goblet cells, and that this pathway is crucial for maintaining mucosal function.

cell biology↗

bioRxiv2020

Recurrent pre-leukemic deletions in myeloid malignancies are the result of DNA double-strand breaks followed by microhomology-mediated end joining

The mechanisms underlying myeloid malignancies deletions are not well understood, nor is it clear why specific genomic hotspots are predisposed to particular deletions. In the current study we inspected the genomic regions around recurrent deletions in myeloid malignancies, and identified microhomology-mediated end-joining (MMEJ) signatures in recurrent deletions in CALR, ASXL1 and SRSF2 loci. Since MMEJ deletions are the result of DNA double-strand breaks (DSBs), we introduced CRISPR Cas9 DSBs into exon 12 of ASXL1, successfully generating recurrent ASXL1 deletion in human hematopoietic stem and progenitor cells (HSPCs). A systematic search of COSMIC dataset for MMEJ deletions in all cancers revealed that recurrent deletions enrich myeloid malignancies. Despite this myeloid predominance, we provide evidence that MMEJ deletions occur in multipotent HSCs. An analysis of DNA repair pathway gene expression in single human adult bone marrow HSPCs could not identify a subpopulation of multipotent HSPCs with increased MMEJ expression, however exposed differences between myeloid and lymphoid biased progenitors. Our data indicate an association between MMEJ-repaired DSBs and recurrent MMEJ deletions in human HSCs and in myeloid leukemia. A better understanding of the source of these DSBs and the regulation of the HSC MMEJ repair pathway might aid with preventing recurrent deletions in human pre-leukemia.

genomics↗

bioRxiv2019

Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

Single-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors, to the mechanical response of soft materials within the cell. To expand the single-molecule toolbox, we have developed a surface-free force spectroscopy assay based on a high-speed hydrodynamic trap capable of applying extremely high tensions for long periods of time. High-speed single-molecule trapping is enabled by a rigid and gas-impermeable microfluidic chip, rapidly and inexpensively fabricated out of glass, double-sided tape and UV-curable adhesive. Our approach does not require difficult covalent attachment chemistries, and enables simultaneous force application and single-molecule fluorescence. Using this approach, we have induced a highly extended state with twice the contour length of B-DNA in regions of partially intercalated double-stranded (dsDNA) by applying forces up to 250 pN. This highly extended state resembles the hyperstretched state of dsDNA, which was initially discovered as a structure fully intercalated by dyes under high tension. It has been hypothesized that hyperstretched DNA could also be induced without the aid of intercalators if high-enough forces were applied, which matches our observation. Combining force application with single-molecule fluorescence imaging is critical for distinguishing hyperstretched DNA from single-stranded DNA that can result from peeling. High-speed hydrodynamic trapping is a powerful yet accessible force spectroscopy method that enables the mechanics of biomolecules to be probed in previously difficult to access regimes.

biophysics↗