In situ laser-induced breakdown spectroscopy measurements during laser welding of superalloy.

Laser-induced breakdown spectroscopy (LIBS) has been utilized for in situ diagnostics of the laser welding process. The influence of different weld spot areas (melt pool, solid weld) on LIBS signals and plasma properties has been studied in detail. Liquid metal sampling and high target surface temperature of the melt enhance LIBS plasma intensity and increase plasma temperature. The influence of laser welding process parameters on LIBS measurements has been studied in order to differentiate optimal and defective laser welding. In case of defective laser welding, the melt pool was intensively boiling, so we have observed greater LIBS signals but poor reproducibility. For the first time, the LIBS technique was demonstrated to detect defective laser welding during in situ measurements utilizing atomic and ionic line comparison by paired sample t-test hypotheses testing.

Phospholipase Superfamily: Structure, Functions, and Biotechnological Applications.

Enzymes of the phospholipase superfamily are involved in lipid metabolism, as well as regulation of membrane composition, cell signaling, and inflammation. This review provides an insight into the structure, functional properties, and biotechnological application of phospholipase A2 and phospholipases in general.

Expression, purification and biophysical characterization of recombinant Streptomyces violaceoruber phospholipase PLA2 overproduced in Pichia pastoris.

Aim: The main purpose of this work was to develop new protocols for high yield purification of secretory phospholipase A2 (PLA2) and to investigate its biophysical properties.Materials and methods: We have used a Pichia pastoris expression system for PLA2 expression and two-stage chromatography for its purification. The biophysical properties of PLA2 were investigated by circular dichroism.Results: A scalable method for high yield purification of recombinant Streptomyces violaceruber PLA2 was developed. The PLA2 from S. violaceruber was expressed in the methylotrophic yeast P. pastoris. Functional active phospholipase A2 with specific activity 73 U/mg was purified with a concentration of at least 3 mg/mL. The role of different divalent ions in PLA2 thermostability were evaluated. Ca2+ and Ba2+ ions significantly increased thermostability of the enzyme.

[Characteristic of infectious status in patients with acutely decompencated chronic heart failure and its impact on annual prognosis].

AIM: The assessment of infectious status in patients with acutely decompensated chronic heart faiure (ADCHF) without evident signs of acute inflammatory stress and its impact on the 1 year prognosis. MATERIAL AND METHODS: Totally, 65 patients with ADCHF of ischemic origin investigated, age 67,3±2,3 y.o. All patients were taken markers of phagocytosis and inflammatory stress as well as antibodies to Streptococcus, Cytomegalovirus (CMV), Epstein-Barr virus (VEB), Candida albicans, Toxoplasma gondii, Aspergillus, Mycoplasma hominis and pneumonia and also level of lipopolysaccharids (LPS) of gram-negative bacteriae. RESULTS: More often LPS of gram-negative bacteriae were revealed in patients with ADCHF and further in decreasing order - antibodies to CMV, VEB, Streptococcus, Candida, Aspergillus and LPS. All patients have been infected by at least 2 pathogens, more than 90 % of them had 3 ones or more. Mortality in first 12 months observation correlated with quantity of patient`s pathogenic patterns (r=0,52, p=0,004). Dependency of one-year mortality from degree of viral-bacterial mixt contamination was almost linear. CMV was a monopathogen with strongest correlation with mortality (r=0,39, p=0,001). In patients with more significant infection bigger rate of re-hospitalizations about new ADCHF correlated with number of pathogens was observed (r=0,61, p=0,001). CONCLUSION: Chronic latent infection with a significant number of pathogens is characteristic of patients with low-ejection ADCHF of ischemic genesis with a significant number of pathogens: more than 90 % of patients had three or more. The most common exogenous pathogens in the study sample of patients with chronic obstructive heart failure were CMV, EBV, and hemolytic streptococcus, of the potentially endogenous ones, gram-negative intestinal bacteria. The number of infectious agents in patients with chronic obstructive heart failure has a direct correlation with deaths and re-admission to hospital with total heart failure within 1 year after discharge from the hospital.

[Clinical case of thromboembolism of small branches of the pulmonary artery on the background of the single ventricle id type with the opening of the сava-caval collaterals].

A three-compartment heart - a severe congenital defect, can develop after genetic mutation or teratogenic influence on fetus at early stages of embryogenesis. According to the literary data, the survival of such patients without surgery is 6-7 %. This publication demonstrates clinical case of the patient who has single ventricle of the heart and lived up to 56 without surgery.

Data in support of toxicity studies of structurally modified plant virus to safety assessment.

This data article is related to the research article entitled "Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies" (Nikitin et al., 2018), devoted to the safety study of structurally modified plant virus - spherical particles (SPs). SPs are generated by thermally denatured tobacco mosaic virus (TMV) coat protein and act as effective adjuvant for development of new vaccine candidates. This article reports the additional results on the toxicity studies of TMV SPs. The weight coefficients of laboratory animals internal organs complements the data of the subchronic toxicity studies. Also plaque-forming cell assay, delayed-type hypersensitivity test and peritoneal macrophage assay as a part of immunotoxicity studies of TMV SPs are presented.

Assessment of structurally modified plant virus as a novel adjuvant in toxicity studies.

Spherical particles (SPs) generated by thermally denatured tobacco mosaic virus (TMV) coat protein can act as an adjuvant, as they are able to enhance the magnitude and longevity of immune responses to different antigens. Here, the toxicity of TMV SPs was assessed prior to it being offered as a universal safe adjuvant for the development of vaccine candidates. The evaluation included nonclinical studies of a local tolerance following the single administration of TMV SPs, and of the local and systemic effects following repeated administrations of TMV SPs. These were conducted in mice, rats and rabbits. General health status, haematology and blood chemistry parameters were monitored on a regular basis. Also, reproductive and development toxicity were studied. No significant signs of toxicity were detected following single or repeated administrations of the adjuvant (TMV SPs). The absence of toxicological effects following the injection of TMV SPs is promising for the further development of recombinant vaccine candidates with TMV SPs as an adjuvant.

Optimizing laser crater enhanced Raman scattering spectroscopy.

The laser crater enhanced Raman scattering (LCERS) spectroscopy technique has been systematically studied for chosen sampling strategy and influence of powder material properties on spectra intensity enhancement. The same nanosecond pulsed solid state Nd:YAG laser (532 nm, 10 ns, 0.1-1.5 mJ/pulse) was used for laser crater production and Raman scattering experiments for l-aspartic acid powder. Increased sampling area inside crater cavity is the key factor for Raman signal improvement for the LCERS technique, thus Raman signal enhancement was studied as a function of numerous experimental parameters including lens-to-sample distance, wavelength (532 and 1064 nm) and laser pulse energy utilized for crater production. Combining laser pulses of 1064 and 532 nm wavelengths for crater ablation was shown to be an effective way for additional LCERS signal improvement. Powder material properties (particle size distribution, powder compactness) were demonstrated to affect LCERS measurements with better results achieved for smaller particles and lower compactness.

[High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein].

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.

[Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain].

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.

[In silico analysis of the restriction fragments length distribution in the human genome]. / Analiz in silico raspredelenie restriktnykh fragmentov genoma cheloveka po dlinam.

The Restriction On Computer (ROC) program (freely available at http://www.mcb.harvard.edu/gilbert/ROC) was developed and used to analyze the restriction fragment length distribution in the human genome. In contrast to other programs searching for restriction sites, ROC simultaneously analyzes several long nucleotide sequences, such as the entire genomes, and in essence simulates electrophoretic analysis of DNA restriction fragments. In addition, this program extracts and analyzes DNA repeats that account for peaks in the restriction fragment length distribution. The ROC analysis data are consistent with the experimental data obtained via in vitro restriction enzyme analysis (taxonomic printing). A difference between the in vitro and in silico results is explained by underrepresentation of tandem DNA repeats in genomic databases. The ROC analysis of individual genome fragments elucidated the nature of several DNA markers, which were earlier revealed by taxonomic printing, and showed that L1 and Alu repeats are nonrandomly distributed in various chromosomes. Another advantage is that the ROC procedure makes it possible to analyze the nonrandom character of a genomic distribution of short DNA sequences. The ROC analysis showed that a low poly(G) frequency is characteristic of the entire human genome, rather than of only coding sequences. The method was proposed for a more complex in silico analysis of the genome. For instance, it is possible to simulate DNA restriction together with blot hybridization and then to analyze the nature of markers revealed.

Process of biosynthetic protein folding determines the rapid formation of native structure.

Biosynthetic folding, beginning with the growing nascent chain and leading to the biologically active structure within its proper cellular context, is one function shared by all proteins. We show that the bacterial luciferase beta subunit reaches its final native form in the alphabeta heterodimer much more rapidly during biosynthetic folding than during refolding from urea. The rate of formation of active enzyme is determined by a short-lived folding intermediate, which is able to associate with the alpha subunit very rapidly following release from the ribosome. This intermediate appears to involve a transient interaction of the C-terminal region of the beta subunit, a region distant from the subunit interface, but intimately involved in heterodimerization. Refolding of the beta subunit under similar conditions proceeds much more slowly. We have characterized both pathways and show that the basic difference between biosynthetic folding and refolding from urea is that the newly synthesized beta subunit enters the folding pathway at a point beyond the slow, rate-determining step that limits the rate of the renaturation process and constitutes a kinetic trap. This mechanism embodies a major strategy, the avoidance of slow-folding intermediates and kinetic traps, that may be employed by many proteins to achieve fast and efficient biosynthetic folding.

Variable and invariable DNA repeat characters revealed by taxonprint approach are useful for molecular systematics.

A specially optimized restriction analysis of highly repetitive DNA elements, called DNA taxonprint, was applied for phylogenetic study of primates and lizards. It was shown that electrophoretic bands of DNA repeats revealed by the taxonprint technique have valuable properties for molecular systematics. Approximately half of taxonprint bands (TB) are invariable and do not disappear from the genomes during evolution or change spontaneously. Presumably these invariable bands are restriction fragments of dispersed DNA repeats. Another group represents variable taxonprint bands that differ even between closely related species. These variable bands are probably represented by tandem DNA repeats and could be used as species-specific markers. It was shown that taxonprint bands are independent characters since the appearance of a new taxonprint band does not change the previous band pattern. Phylogenetic reconstruction carried out on taxonprint data demonstrated that this approach could be of general utility for molecular systematics and species identification.

Restriction endonuclease analysis of highly repetitive DNA as a phylogenetic tool.

Multiple band patterns of DNA repeats in the 20-500-nucleotide range can be detected by digesting genomic DNA with short-cutting restriction endonucleases, followed by end labeling of the restriction fragments and fractionation in nondenaturing polyacrylamide gels. We call such band patterns obtained from genomic DNA "taxonprints" (Fedorov et al. 1992). Here we show that taxonprints for the taxonomic groups studied (mammals, reptiles, fish, insects-altogether more than 50 species) have the following properties: (1) All individuals from the same species have identical taxonprints. (2) Taxonprint bands can be subdivided into those specific for a single species and those specific for groups of closely related species, genera, and even families. (3) Each restriction endonuclease produces unique band patterns; thus, five to ten restriction enzymes (about 100 bands) may be sufficient for a statistical treatment of phylogenetic relationships based on polymorphisms of restriction endinuclease sites. We demonstrate that taxonprint analysis allows one to distinguish closely related species and to establish the degree of similarity among species and among genera. These characteristics make taxonprint analysis a valuable tool for taxonomic and phylogenetic studies.

GroE modulates kinetic partitioning of folding intermediates between alternative states to maximize the yield of biologically active protein.

The central issue of chaperone function is the mechanism whereby partitioning of folding polypeptides along the productive pathway may be maximized, while non-productive folding pathways are minimized. We have found that the GroE chaperone is capable of accelerating the rate of the productive pathway of bacterial luciferase alphabeta heterodimer formation. At intermediate temperatures at which the productive pathway and non-productive pathways leading to dimerization-incompetent monomeric forms of the subunits coexist, GroE enhances the yield of native enzyme while minimizing the yield of misfolded protein. These results suggest that GroE releases the subunits in forms capable of achieving the native structure faster than the forms initially bound by the chaperone. At higher temperatures, at which the native enzyme is stable but the dimerization reaction is diminished, GroE is unable to force the productive folding reaction to occur. However, the chaperone decreases the rate of formation of the heterodimerization-incompetent species, thereby enhancing the final yield of active enzyme when the temperature is reduced to the permissive range. Our results suggest a mechanism by which the chaperone functions to maximize the yield of the biologically active form of the protein while maintaining or even accelerating the essential rapid kinetics of folding reactions.

[Ca2+ ion transport in the sarcoplasmic reticulum of the gastrocnemius muscles of white rats under treatment of the sciatic nerve with a colchicine solution]. / Transport ionov Ca2+ v sarkoplazmaticheskom retikulume ikronozhnykh myshts belykh krys pri obrabotke sedalischnogo nerva rastvorom kolkhitsina.

In different time after application of 12.5 microM colchicine-inhibitor of exoplasmic current to the ischiadicus the quantity of amino acid residues of methionine and cysteine in the sarcoplasmic reticulum decreases, its protein content changes. The Ca(2+)-ATPase activity increases on the 3d day after colchicine application and decreases on the 14th day. Analogical changes are observed in the capacity of the sarcoplasmic reticulum to transport calcium ions, the Michaelis constant in this case being stable. Passive outflow of Ca2+ from the sarcoplasmic reticulum vesicules changes in such a way: the Michaelis constant decreases with a simultaneous increase in the maximal velocity after 14 and 28 days of colchicine application.

[Comparison of repeat DNA sequences in the Erinaceidae mammal family by restriction analysis]. / Sravnenie povtoriaiushchikhsia posledovatel'nostei DNK mlekopitaiushchikh semeistva Erinaceidae metodom restriktaznogo analiza.

Hedgehog (Erinaceidae) DNA was digested with teh Sau 96 I, Bsp 143 I, Csp 6 I, Taq I, Hinf I, Msp I, Eco 130I, Bcn I, BsuR I restriction endonucleases. The obtained products were end-labeled and electrophoretically separated in polyacrylamide gel. DNA fragments consisting of highly repetitive genomic sequences were detected as a set of bands corresponding to fragments between 30 and 500 bp in length. Comparison of DNA restriction patterns of the species analyzed revealed the presence of species-specific bands as well as common bands. Phylogenetic trees were constructed by means of the maximum parsimony method and the bootstrap procedure. Our data suggest that hedgehog species from arid areas are clearly distinguished from forest species.