Crosstalk between CST and RPA regulates RAD51 activity during replication stress

Replication stress causes replication fork stalling, resulting in an accumulation of single-stranded DNA (ssDNA). Replication protein A (RPA) and CTC1-STN1-TEN1 (CST) complex bind ssDNA and are found at stalled forks, where they regulate RAD51 recruitment and foci formation in vivo. Here, we investigate crosstalk between RPA, CST, and RAD51. We show that CST and RPA localize in close proximity in cells. Although CST stably binds to ssDNA with a high affinity at low ionic strength, the interaction becomes more dynamic and enables facilitated dissociation at high ionic strength. CST can coexist with RPA on the same ssDNA and target RAD51 to RPA-coated ssDNA. Notably, whereas RPA-coated ssDNA inhibits RAD51 activity, RAD51 can assemble a functional filament and exhibit strand-exchange activity on CST-coated ssDNA at high ionic strength. Our findings provide mechanistic insights into how CST targets and tethers RAD51 to RPA-coated ssDNA in response to replication stress.

Application of Multivariate Approaches to the Hydro-chemical Assessment of the Ghodaghodi Lake, Sudurpaschim Province, Nepal

Wetlands are considered the most diverse and productive ecosystems in the world, providing life-sustaining ecological services. Hydrochemical variables of the Ghodaghodi Lake situated in Far west Nepal were studied using multivariate statistical approaches to investigate the characteristics of water quality of the lake. A total of 10water samples were collected and analyzed for water temperature, pH, electrical conductivity, total dissolved solids, total hardness, salinity, dissolved oxygen, major ions, and dissolved Si in the pre-monsoon season during 2017.The water quality assessment was carried out by applying sodium percentage, sodium adsorption ratio, permeability index, Kelly’s ratio, magnesium adsorption ratio, action ratio of soil structural stability, and water quality index. Multivariate statistical techniques were used to evaluate the geochemical and anthropogenic processes and to identify factors influencing the ionic concentrations. The results revealed that the water was slightly alkaline with low ionic strength and remarkable spatial variations. The hydrochemistry of the lake was mainly controlled by rock weathering as the dominant Ca2+-HCO3- facies. The principal component analysis provided three major components exhibiting the diverse sources of natural and anthropogenic chemicals, including agricultural fertilizers, leakage of sewages near the human settlements, etc. The measured hydrochemical parameters indicate that lake water lies within the safe drinking water and irrigational standards. However, special consideration should be taken to control the concentrations of NO3- due to increasing anthropic activities.

Quercetin and Vitamin E ameliorate cardio-apoptotic risks in diabetic rats

Background: Apoptosis is upregulated in all forms of diabetes, with the mitochondria acting as a target in diabetes pathophysiology. Quercetin and vitamin E have shown usefulness in the delay of progression of diabetes-induced complications. However, their effect on the apoptotic process in diabetes mellitus is unknown. We hypothesize that quercetin treatment in diabetes may decrease the propensity for cardio-myocytic death via regulation of the mitochondria permeability transition (mPT) pore opening. Methods: Hearts from normal and streptozotocin-induced diabetic rats were used for the study. Low-ionic strength heart mitochondria were used for swelling assay, and mitochondrial lipid peroxidation (mLPO) activity was spectrophotometrically assessed. Levels of cytochrome c, caspase 3 and 9 were determined by immunohistochemistry, while lesions assessed by histology.Discussion: Diabetic heart mPT pore showed larger amplitude swelling than control, while mLPO levels was increased in diabetic rats relative to control, this resulted in cytochrome c release. This initiated increased caspase 3 and 9 activity in diabetic rats (p<0.05). Histology showed haemorrphagic lesions in diabetic rat hearts. Quercetin and vitamin E treatment reversed these effects, suggestive of their anti-apoptotic effect. Conclusions: Quercetin and vitamin E protection in diabetes is mediated by mPT pore inhibition and modulation of mitochondrial-mediated apoptosis.

Calibration-Less DNA Concentration Measurements Using EOF Volumetric Flow and Single Molecule Counting

Nanopore sensing is a promising tool well suited to capture and detect DNA and other single molecules. DNA is a negatively charged biomolecule that can be captured and translocated through a constricted nanopore aperture under an applied electric field. Precise assessment of DNA concentration is of crucial importance in many analytical processes and medical diagnostic applications. Recently, we found that hydrodynamic forces can lead to DNA motion against the electrophoretic force (EPF) at low ionic strength. This study utilized glass nanopores to investigate the DNA capture mechanism and detect DNA molecules due to volumetric flow at these low ionic strength conditions. We measured the DNA capture rate at five different pico-molar concentrations. Our findings indicated that the translocation rate is proportional to the concentration of DNA molecules and requires no calibration due to the volumetric flow rate and DNA counting directly correlates with concentration. Using finite element analysis, we calculated the volumetric flow and proposed a simple, straightforward approach for accurate DNA quantification. Furthermore, these experiments explore a unique transport mechanism where one of the most highly charged molecules enters a pore against electric field forces. This quantitative technique has the potential to provide distinct insight into nanopore-based biosensing and further enhance the nanopore’s capability as a biomolecule concentration sensor.

Structural Characterization of EnpA D,L-Endopeptidase from Enterococcus faecalis Prophage Provides Insights into Substrate Specificity of M23 Peptidases

The best-characterized members of the M23 family are glycyl-glycine hydrolases, such as lysostaphin (Lss) from Staphylococcus simulans or LytM from Staphylococcus aureus. Recently, enzymes with broad specificities were reported, such as EnpACD from Enterococcus faecalis, that cleaves D,L peptide bond between the stem peptide and a cross-bridge. Previously, the activity of EnpACD was demonstrated only on isolated peptidoglycan fragments. Herein we report conditions in which EnpACD lyses bacterial cells live with very high efficiency demonstrating great bacteriolytic potential, though limited to a low ionic strength environment. We have solved the structure of the EnpACD H109A inactive variant and analyzed it in the context of related peptidoglycan hydrolases structures to reveal the bases for the specificity determination. All M23 structures share a very conserved β-sheet core which constitutes the rigid bottom of the substrate-binding groove and active site, while variable loops create the walls of the deep and narrow binding cleft. A detailed analysis of the binding groove architecture, specificity of M23 enzymes and D,L peptidases demonstrates that the substrate groove, which is particularly deep and narrow, is accessible preferably for peptides composed of amino acids with short side chains or subsequent L and D-isomers. As a result, the bottom of the groove is involved in interactions with the main chain of the substrate while the side chains are protruding in one plane towards the groove opening. We concluded that the selectivity of the substrates is based on their conformations allowed only for polyglycine chains and alternating chirality of the amino acids.

The Impact of Universal Transport Media and Viral Transport Media Liquid Samples on a SARS-CoV-2 Rapid Antigen Test

The impact of universal transport media (UTM) and viral transport media (VTM) liquid samples on the performance of the Healgen Scientific Rapid COVID-19 Antigen Test was investigated. Twelve different UTM/VTM liquid samples were added at different dilutions to the extraction buffer, and 2 of 12 generated false-positive results. To understand the cause of these false-positive results, the effect of extraction buffer dilution on sample pH, surfactant concentration, and ionic strength were investigated. The most important factor in UTM/VTM liquid sample dilution of the extraction buffer was ionic strength as measured by conductivity. Dilutions with conductivity below ~17 mS/cm can induce a false-positive result. It was also noted that the ionic strength of UTM/VTMs can vary, and those with low ionic strength can be problematic. To rule out the effect of other common components found in UTMs/VTMs, several materials were mixed with extraction buffer and tested at high concentrations. None was shown to produce false-positive results.

Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.