Biology subjects

Bleher, R.

Publications and source records attributed to Bleher, R..

4 recordsLinked to original sources

bioRxiv2021

Chromatin forms nanoscale three-dimensional packing domains with fractal-like scaling behavior in vitro

Chromatin organization over a wide range of length scales plays a critical role in the regulation of transcription and deciphering the interplay of these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein, we introduce ChromSTEM, a method that utilizes high angle annular dark-field imaging and tomography in scanning transmission electron microscopy combined with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and the statistical packing behavior of the chromatin polymer. Using chromatin mass and density analysis, we observed that chromatin forms spatially well-defined higher-order domains which are around 100 nm in radius, with a radially decreasing mass-density from the center to the periphery. Although the morphological properties of the domains vary within the same cell line, they seem to exhibit greater heterogeneity across cell lines, underlying a potential role of statistical chromatin packing in regulating cell-type-specific gene expression.

genomics↗

bioRxiv2020

In Vitro Analysis of the Anti-viral Potential of nasal spray constituents against SARS-CoV-2

Viral pandemics have taken a significant toll on humanity and the world now is contending with the SARS-CoV-2 epidemic. Readily available economical preventive measures should be immediately explored. Xylitol has been reported to reduce the severity of viral infections as well as the severity of pneumonia, and increase the survivability of animal subjects. Since pneumonia and acute respiratory distress syndrome are potentially fatal complications of COVID-19, the present study tested the in vitro effectiveness of xylitol against SARS-CoV-2. Virus titers and LRV of SARS-CoV-2, were incubated with a single concentration of nasal spray. Toxicity was observed in the top dilution (1/10). Virus was seen below that dilution so it did not affect calculations of virus titer or LRV. After a 25-minute contact time, the nasal spray (11% Pure Xylitol, 0.85%NaCL (Saline), and 0.20% grapefruit seed extract) reduced virus from 4.2 to 1.7 log10 CCID50 per 0.1 mL, a statistically significant reduction (P<0.001) of 2.5 log10 CCID50. STEM Images obtained at the BIoCryo Laboratory revealed virus contained on the cell wall but none intra-cellular, possibly due to D-xylose (xylitol) production of glycoaminoglycans decoy targets. Xylitol and grapefruit seed extract are not exotic nor expensive rare high technology answers to viral epidemics. The potential in saving lives and the economies of the world by using X-GSE combination therapy should inspire large clinical trials, especially in those nations whereas the healthcare system would be dangerously compromised by the adoption of less effective and significantly more financially demanding therapies. Because there are no risk factors in using the X/GSE combination therapy, and the nasal spray is over the counter available without a prescription, and the spray allows for comfortable long term mask-wearing, adoption of this preventive anti-viral therapy should be encouraged.

molecular biology↗

bioRxiv2020

Nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform bridges 4-D chromatin organization with molecular function

In eukaryotic cells, chromatin structure is linked to transcription processes through the regulation of genome organization. Extending across multiple length-scales - from the nucleosome to higher-order three-dimensional structures - chromatin is a dynamic system which evolves throughout the lifetime of a cell. However, no individual technique can fully elucidate the behavior of chromatin organization and its relation to molecular function at all length- and timescales at both a single-cell and a cell population level. Herein, we present a multi-technique nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform that bridges electron tomography and optical superresolution imaging of chromatin conformation and transcriptional processes, with resolution down to the level of individual nucleosomes, with high-throughput, label-free analysis of chromatin packing and its dynamics in live cells. Utilizing nano-ChIA, we observed that chromatin is localized into spatially separable packing domains, with an average diameter of around 200 nm, sub-Mb genomic size, and an internal fractal structure. The chromatin packing behavior of these domains is directly influenced by active gene transcription. Furthermore, we demonstrated that the chromatin packing domain structure is correlated among progenitor cells and all their progeny, indicating that the organization of chromatin into fractal packing domains is heritable across cell division. Further studies employing the nano-ChIA platform have the potential to provide a more coherent picture of chromatin structure and its relation to molecular function.

genomics↗

bioRxiv2019

Quantifying Three-dimensional Chromatin Organization Utilizing Scanning Transmission Electron Microscopy: ChromSTEM

Chromatin organization over a wide range of length scales plays a critical role in the regulation of gene expression and deciphering these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein we introduce ChromSTEM, a method which utilizes high angle annular dark field imaging and tomography in scanning transmission electron microscopy in combination with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and tissue biopsies through local DNA distribution and the scaling behavior of chromatin polymer. We observed that chromatin is densely packed with an average volume concentration of over 30% with heterochromatin having a two-fold higher density compared to euchromatin. Chromatin was arranged into spatially well-defined nanoscale packing domains with fractal internal structure and genomic size between 100 and 400 kb, comparable to that of topologically associated domains. The packing domains varied in DNA concentration and fractal dimension and had one of the distinct states of chromatin packing with differential ratio of DNA content to the chromatin volume concentration. Finally, we observed a significant intercellular heterogeneity of chromatin organization even within a genetically uniform cell population, which demonstrates the imperative for high-throughput characterization of chromatin structure at the single cell level.

genomics↗