Probing the MvaI Methyltransferase Region that Interacts with DNA: Affinity Labeling with the Dialdehyde-Containing DNA Duplexes

Abstract

Affinity labeling of methyltransferase MvaI by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine or 1-(β-D-galactopyranosyl)thymine residues was performed. Partial chemical hydrolysis of the covalently bound methylase in the conjugates with the dialdehyde-containing DNA allowed us to determine the amino acid region in the C terminus of methylase MvaI that interacts with DNA.     

Automated Assessment of Upper Extremity Movement Impairment due to Stroke

Current diagnosis and treatment of movement impairment post-stroke is based on the subjective assessment of select movements by a trained clinical specialist. However, modern low-cost motion capture technology allows for the development of automated quantitative assessment of motor impairment. Such outcome measures are crucial for advancing post-stroke treatment methods. We sought to develop an automated method of measuring the quality of movement in clinically-relevant terms from low-cost motion capture. Unconstrained movements of upper extremity were performed by people with chronic hemiparesis and recorded by standard and low-cost motion capture systems. Quantitative scores derived from motion capture were compared to qualitative clinical scores produced by trained human raters. A strong linear relationship was found between qualitative scores and quantitative scores derived from both standard and low-cost motion capture. Performance of the automated scoring algorithm was matched by averaged qualitative scores of three human raters. We conclude that low-cost motion capture combined with an automated scoring algorithm is a feasible method to assess objectively upper-arm impairment post stroke. The application of this technology may not only reduce the cost of assessment of post-stroke movement impairment, but also promote the acceptance of objective impairment measures into routine medical practice.     

Association of the insulin-like growth factor II (IGF2) gene with human cognitive functions

Active search for candidate genes whose polymorphisms are associated with human cognitive functions has been in progress in the past years. The study focused on the role that the insulin-like growth factor II (IGF2) gene may play in the variation of cognitive processes related to executive functions. The ApaI polymorphism of the IGF2 gene was tested for association with selective attention during visual search, working memory/mental control, and semantic verbal fluency in a group of 182 healthy individuals. The ApaI polymorphism was associated with the general cognitive index and selective attention measure. Carriers of genotype AA displayed higher values of the two parameters than carriers of genotype GG. It was assumed that the ApaI polymorphism of the IGF2 gene influences the human cognitive functions, acting possibly via modulation of the IGF-II level in the central nervous system.     

Soluble fibrin and D-dimer at normal pregnancy and pregnancy with risk of miscarriage

ELISA for soluble fibrin (SF) quantification has been elaborated on the basis of our fibrin-specific monoclonal antibodies (mAb). Epitope for these mAb is localized in fibrin fragment Bbeta118-134. The method was used on the blood plasma of healthy pregnant women (control group) and pregnant women with the risk of fetal loss (RFL). The increased mean values of SF concentrations were observed at pregnancy with RFL as compared to the normal pregnancy at the terms from 4 to 24 weeks (17.87 +/- 3.15 mkg/ml and 9.03 +/- 1.58 mkg/ml accordingly, p < 0.05). A weak negative correlation between SF concentration and pregnancy term was found at RFL (r = -0.201, n=35), while there was no correlation between these variables in control group (r = 0.004, n=28). The mean values of SF concentration estimated by semiquantitative test (by phosphates salting out of SF) were also higher at the pregnancy with RFL as compared to the normal pregnancy. However, the absolute values of SF concentrations determined by salting out method were essentially higher than in the case of ELISA. Immunoblot analysis with mAb 2d-2a (epitope for which in fibrin molecule encompasses peptide bond Bbeta14-15), showed that the main molecular component of SF at normal pregnancy and RFL was oligomeric fibrin desAA with possible incorporation of fibrinogen and/or fibrin desA which was not stabilized by factor XIIIa. D-dimer concentrations determined in blood plasma samples of pregnant women by ELISA varied in the range of 1-224 ng/ml at the pregnancy period from 4 to 37 weeks. There was positive correlation between D-dimer concentration and pregnancy term both at normal pregnancy and pregnancy with RFL (r = 0.765, n=33 and r = 0.712, n=44 correspondingly). The mean values of D-dimer concentration at various terms of normal pregnancy and pregnancy with RFL did not vary considerably. Thus SF but not D-dimer quantification may give useful diagnostic information at the pregnancy with RFL.     

Temporal Proteome and Lipidome Profiles Reveal Hepatitis C Virus-Associated Reprogramming of Hepatocellular Metabolism and Bioenergetics

Proteomic and lipidomic profiling was performed over a time course of acute hepatitis C virus (HCV) infection in cultured Huh-7.5 cells to gain new insights into the intracellular processes influenced by this virus. Our proteomic data suggest that HCV induces early perturbations in glycolysis, the pentose phosphate pathway, and the citric acid cycle, which favor host biosynthetic activities supporting viral replication and propagation. This is followed by a compensatory shift in metabolism aimed at maintaining energy homeostasis and cell viability during elevated viral replication and increasing cellular stress. Complementary lipidomic analyses identified numerous temporal perturbations in select lipid species (e.g. phospholipids and sphingomyelins) predicted to play important roles in viral replication and downstream assembly and secretion events. The elevation of lipotoxic ceramide species suggests a potential link between HCV-associated biochemical alterations and the direct cytopathic effect observed in this in vitro system. Using innovative computational modeling approaches, we further identified mitochondrial fatty acid oxidation enzymes, which are comparably regulated during in vitro infection and in patients with histological evidence of fibrosis, as possible targets through which HCV regulates temporal alterations in cellular metabolic homeostasis.     

Blood peptidome-degradome profile of breast cancer

Background

Cancer invasion and metastasis are closely associated with activities within the degradome; however, little is known about whether these activities can be detected in the blood of cancer patients.

Methodology and Principal Findings

The peptidome-degradome profiles of pooled blood plasma sampled from 15 breast cancer patients (BCP) and age, race, and menopausal status matched control healthy persons (HP) were globally characterized using advanced comprehensive separations combined with tandem Fourier transform mass spectrometry and new data analysis approaches that facilitated top-down peptidomic analysis. The BCP pool displayed 71 degradome protein substrates that encompassed 839 distinct peptidome peptides. In contrast, the HP 50 degradome substrates found encompassed 425 peptides. We find that the ratios of the peptidome peptide relative abundances can vary as much as >4000 fold between BCP and HP. The experimental results also show differential degradation of substrates in the BCP sample in their functional domains, including the proteolytic and inhibitory sites of the plasmin-antiplasmin and thrombin-antithrombin systems, the main chains of the extracellular matrix protection proteins, the excessive degradation of innate immune system key convertases and membrane attack complex components, as well as several other cancer suppressor proteins.

Conclusions

Degradomics-peptidomics profiling of blood plasma is highly sensitive to changes not evidenced by conventional bottom-up proteomics and potentially provides unique signatures of possible diagnostic utility.     

The seroresistance of enterobacteria isolated from different sources

395 representatives of the family Enterobacteriaceae, isolated from household and drinking water, from healthy and sick persons, were used as an example to demonstrate that their seroresistance was greatly connected with the type of the source from which these bacteria had been isolated. Experiments showed the phenotypic conversion of Escherichia coli towards an increase in their seroresistance on contact with human blood serum. The study revealed the pleiotropic effect produced by the action of serum on a number of other properties, responsible for survival in a macroorganism (anticomplement and antilysozyme activities, adhesive capacity and hydrophobic properties of the outer surface).     

Comparative analysis of the sensitivity of enterobacteria to bile

The analysis of the sensitivity of 195 enterobacterial cultures to bile revealed that their level of resistance decreased in the following row: Shigella > Salmonella > Klebsiella > Escherichia > Providencia. As shown on a sample of 136 E. coli isolates the level of resistance of these bacteria to bile depended on their isolation source: in E. coli isolated from bile in cholecystitis, from urine in pyelonephritis and from feces in intestinal dysbacteriosis resistance was 1.1-1.3 times higher than in E. coli isolated from the water of open reservoirs, from the feces of healthy persons and from extraintestinal foci of purulent inflammation. The level of sensitivity to bile is regarded as a property making it possible for enterobacteria to colonize biliary tracts and the proximal sections of the digestive tract.