[Specific features of the enteric microbiota composition in patients with alcoholic liver cirrhosis].

AIM: To establish the specific features of the taxonomic and functional composition of the enteric microbiota in patients with alcoholic liver cirrhosis (LC). SUBJECTS AND METHODS: Metagenomic analysis was used to study the taxonomic composition and functional potential of the enteric microbiota in 20 patients with alcoholic LC. Total DNA was isolated from the patients' fecal samples; thereafter full genome sequencing was carried out. The metagenomic analysis yielded the results of the relative taxonomic and functional abundance of microbial species in the test samples. These were comparatively analyzed with the previously published metagenomic datasets of healthy population cohorts in the Russian Federation, as well as in Denmark, China, and the USA. RESULTS: In the majority of patients, the dominant part of the intestinal community represented bacterial species constituting the normal human intestinal flora. At the same time, abnormal gut microbiota composition, which was suggestive of marked dysbacteriosis, was identified in a number of patients. In addition, pooled analysis of the data could identify a number of species with a statistically significantly increase and decrease in the relative abundance as compared to the control groups. Thus, the enteric microbiota of the patients with alcoholic LC showed a high proportion of bacteria characteristic of the oral cavity. Analysis of the pooled metabolic potential of the microbiota in these patients demonstrated the higher abundance of enzyme genes involved in alcohol metabolism. CONCLUSION: In the patients with alcoholic LC, the microbiota composition changes identified in individual bacterial species may be associated with gastrointestinal comorbidities, such as chronic erosive gastritis, chronic pancreatitis, and gastric ulcer. The alterations occurring in alcoholic cirrhosis promote the penetration and generation of oral cavity-specific microorganisms in the human intestine. This may a potential biomarker for the diagnosis of liver diseases. The bacterial enzyme genes involved in alcohol metabolism have an increased abundance in patients with alcoholic LC and healthy volunteers from the Russian Federation.

An accessory chromophore in red vision.

In the absence of a red-sensitive visual pigment, some deep-sea fish use a chlorophyll derivative in their green-sensitive rod cells in order to see deep-red light. Here we show that living rods extracted from a salamander can also accumulate an exogenous chlorophyll derivative, chlorin e6, that renders them as sensitive to red light as they are to green. This vision enhancement by an unbleachable chlorophyll derivative might therefore be a general phenomenon in vertebrate photoreception.

Complete genome and proteome of Acholeplasma laidlawii.

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.

Proteome analysis of chloroplasts from the moss Physcomitrella patens (Hedw.) B.S.G.

Intact chloroplasts were prepared from protoplasts of the moss Physcomitrella patens according to an especially developed method. They were additionally separated into stroma and thylakoid fractions. The proteomes of intact plastids, stroma, and thylakoids were analyzed by 1D-electrophoresis under denaturing conditions followed by protein digestion and nano-LC-ESI-MS/MS of tryptic peptides from gel bands. A total of 624 unique proteins were identified, 434 of which were annotated as chloroplast resident proteins. The majority of proteins belonged to a photosynthetic group (21.3%) and to the group of proteins implicated in protein degradation, posttranslational modification, folding, and import (20.6%). Among proteins assigned to chloroplasts, the following groups are prominent combining proteins implicated in metabolism of: amino acids (6.9%), nucleotides (2.5%), lipids (2.2%), carbohydrates (2.4%), hormones (1.5%), isoprenoids (1.25%), vitamins and cofactors (1%), sulfur (1.25%), and nitrogen (1%); as well as proteins involved in the pentose-phosphate cycle (1.75%), tetrapyrrole synthesis (3.7%), and redox processes (3.6%). The data can be used in physiological and photobiological studies as well as in further studies of P. patens chloroplast proteome including structural and functional specifics of plant protein localization in organelles.

Correlating diameter, mechanical and structural properties of poly(l-lactide) fibres from needleless electrospinning.

The development and application of nanofibres requires a thorough understanding of the mechanical properties on a single fibre level including respective modelling tools for precise fibre analysis. This work presents a mechanical and morphological study of poly-l-lactide nanofibres developed by needleless electrospinning. Atomic force microscopy (AFM) and micromechanical testing (MMT) were used to characterise the mechanical response of the fibres within a diameter range of 200-1400 nm. Young's moduli E determined by means of both methods are in sound agreement and show a strong increase for thinner fibres below a critical diameter of 800 nm. Similar increasing trends for yield stress and hardening modulus were measured by MMT. Finite element analyses show that the common practice of modelling three-point bending tests with either double supported or double clamped beams is prone to significant bias in the determined elastic properties, and that the latter is a good approximation only for small diameters. Therefore, an analytical formula based on intermediate boundary conditions is proposed that is valid for the whole tested range of fibre diameters, providing a consistently low error in axial Young's modulus below 10%. The analysis of fibre morphology by differential scanning calorimetry and 2D wide-angle X-ray scattering revealed increasing polymer chains alignment in the amorphous phase and higher crystallinity of fibres for decreasing diameter. The combination of these observations with the mechanical characterisation suggests a linear relationship between Young's modulus and both crystallinity and molecular orientation in the amorphous phase. STATEMENT OF SIGNIFICANCE: Fibrous membranes have rapidly growing use in various applications, each of which comes with specific property requirements. However, the development and production of nanofibre membranes with dedicated mechanical properties is challenging, in particular with techniques suitable for industrial scales such as needleless electrospinning. It is therefore a key step to understand the mechanical and structural characteristics of single nanofibres developed in this process, and to this end, the present work presents changes of internal fibre structure and mechanical properties with diameter, based on dedicated models. Special attention was given to the commonly used models for analyzing Young's modulus of single nanofibers in three-point bending tests, which are shown to be prone to large errors, and an improved robust approach is proposed.

Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study.

BACKGROUND: The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N-carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods. RESULTS: The first step in catalysis by DTBS is the formation of an enzyme-substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg2+ dependent. The kinetic constants in the presence and absence of Mg2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg2+, substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the alpha- and gamma-phosphates of ATP in Mg2+ binding and in the subsequent steps of the reaction. CONCLUSIONS: Mg2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS-substrate complex, when Mg2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.

Mechanistic implications and family relationships from the structure of dethiobiotin synthetase.

BACKGROUND: Biotin is the vitamin essential for many biological carboxylation reactions, such as the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA in fatty acid biosynthesis. Dethiobiotin synthetase (DTBS) facilitates the penultimate, ureido ring closure in biotin synthesis, which is a non-biotin-dependent carboxylation. DTBS displays no sequence similarity to any other protein in the database. Structural studies provide a molecular insight into the reaction mechanism of DTBS. RESULTS: We present the structure of DTBS refined to 1.80 A resolution with an R-factor of 17.2% for all terms plus unrefined data on the binding of the substrate, 7,8-diaminopelargonic acid and the product, dethiobiotin. These studies confirm that the protein forms a homodimer with each subunit folded as a single globular alpha/beta domain. The presence of sulphate ions in the crystals and comparisons with the related Ha-ras-p21 oncogene product are used to infer the ATP-binding site, corroborated by the difference electron density for the ATP analogue AMP-PNP. CONCLUSIONS: This study establishes that the enzyme active site is situated at the dimer interface, with the substrate binding to one monomer and ATP to the other. The overall fold of DTBS closely resembles that of three other enzymes, adenylosuccinate synthetase (purA), Ha-ras-p21, and nitrogenase iron protein, that are unrelated by sequence or function, indicating that DTBS is a member of a diverse family of enzymes.

Novel fusion transcripts in bladder cancer identified by RNA-seq.

Urothelial carcinoma (UC) is the most common type of bladder cancer and is the second most frequently diagnosed genitourinary tumor. The identification of fusion genes in bladder cancer might provide new perspectives for its classification and significance. In this study, we present a thorough search on three UC samples for novel fusion transcripts in bladder cancer using high-throughput RNA sequencing. We used stringent requirements for 819 fusion candidates and nominated 10 candidate fusion transcripts. Among them four novel fusion genes SEPT9/CYHR, IGF1R/TTC23, SYT8/TNNI2 and CASZ1/DFFA were validated and characterized in 48 formalin-fixed paraffin-embedded (FFPE) specimens of bladder cancer. Chromosomal rearrangements of regions 17q25, 15q26.3 and 1p36.22 resulting in the fusion transcripts SEPT9/CYHR, IGF1R/TTC23 and CASZ1/DFFA, appeared to be rare or unique events because they were not detected in the 48 UC samples. In contrast, the SYT8/TNNI2 fusion transcript resulting from transcription-induced chimerism by read-through mechanisms was a rather common and tumor-specific event occurring in 37.5% (18/48) of the UC specimens. Further investigation of functional and clinical relevance of novel fusion genes remains to be elucidated to reveal their role in bladder carcinogenesis.

The crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-dependent, acyl-CoA-condensing enzyme.

8-Amino-7-oxononanoate synthase (or 8-amino-7-ketopelargonate synthase; EC 2.3.1.47; AONS) catalyses the decarboxylative condensation of l-alanine and pimeloyl-CoA in the first committed step of biotin biosynthesis. We have cloned, over-expressed and purified AONS from Escherichia coli and determined the crystal structures of the apo and PLP-bound forms of the enzyme. The protein is a symmetrical homodimer with a tertiary structure and active site organisation similar to, but distinct from, those of other PLP-dependent enzymes whose three-dimensional structures are known. The critical PLP-binding lysine of AONS is located at the end of a deep cleft that allows access of the pantothenate arm of pimeloyl-CoA. A cluster of positively charged residues at the entrance to this cleft forms a putative diphosphate binding site for CoA. The structure of E. coli AONS enables identification of the key residues of the PLP-binding site and thus provides a framework with which to understand the biochemical mechanism, which is similar to that catalysed by 5-aminolevulinate synthase and two other alpha-oxoamine synthases. Although AONS has a low overall sequence similarity with the catalytic domains of other alpha-oxoamine synthases, the structure reveals the regions of significant identity to be functionally important. This suggests that the organisation of the conserved catalytic residues in the active site is similar for all enzymes of this sub-class of PLP-dependent enzymes and they share a common mechanism. Knowledge of the three-dimensional structure of AONS will enable characterisation of the structural features of this enzyme sub-family that are responsible for this important type of reaction.

The 2.3 A X-ray crystal structure of S. cerevisiae phosphoglycerate mutase.

The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined. This structure shows important differences from the lower resolution structure deposited in 1982. The crystal used to determine the new structure was of a different form, having spacegroup P2(1). The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction. The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts. Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides. The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase. However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal. An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge. The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure. Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation.

Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis.

The enzyme dethiobiotin synthetase (EC 6.3.3.3) has been cloned and over-expressed in Escherichia coli in such a way that milligram quantities are available. The purified enzyme has been subjected to a number of physical and chemical studies, sequenced and most notably it has been crystallized in a form that is suitable for X-ray structure determination. The cell dimensions are a = 72.8 A, b = 49.2 A, c = 61.4 A, beta = 106.2 degrees. The systematic absences are consistent with the monoclinic space group C2 with one polypeptide chain in the asymmetric unit.

[Metagenomic analysis of taxonomic and functional changes in gut microbiota of patients with alcoholic dependence syndrome].

Here we present the first metagenomic study of gut microbiota in patients with alcohol dependence syndrome (ADS) performed in the whole-genome ("shotgun") format. Taxonomic analysis highlighted changes in community "drivers" abundance previously associated with inflammatory processes (including increase in Ruminococcus gnavus and torques, as well as decrease in Faecalibacterium and Akkermansia). Microbiota of alcoholics manifested presence of specific opportunistic pathogens rarely detected in healthy control subjects of the world. Differential analysis of metabolic potential basing on changes in KEGG Orthology groups abundance revealed increase in pathways associated with response to oxidative stress. Analysis of two specific gene groups--alcohol metabolism and virulence factors--also showed increase in comparison with the control groups. We suggest that gut microbiota distinct in alcoholics by both taxonomic and functional composition plays role in modulating the effect of alcohol on host organism.

The structure of poly(dA).poly(dT) as revealed by an X-ray fibre diffraction.

X-ray diffraction in fibres revealed that the calcium salt of poly(dA).poly(dT) is a 10-fold double helix with a pitch of 3.23 nm. The opposite sugar-phosphate chains in the refined model are characterized by a complete conformational equivalence and contain sugars in a conformation close to C2'-endo. As a result a new model of the sodium salt of poly(dA).poly(dT) has been constructed, which is different from the Heteronomous DNA proposed earlier (S. Arnott et al., Nucl. Acids Res. 11, 4141 (1983)). The new model of Na-poly(dA).poly(dT) has conformationally similar opposite chains; it is a structure of the B-type, rather like that of Ca-poly(dA).poly(dT).

Bh-DNA: variations of the poly[d(A)].poly[d(T)] structure within the framework of the fibre diffraction studies.

A refinement of the recent results for poly[d(A)].poly[d(T)] (Alexeev et al., J. Biomol. Struct. Dyn. 4,989 (1987)) involving additional parameters of the base-pair structure and of the sugar-phosphate backbone expands the conformational potential of this polynucleotide of the B type to include the possibility of bifurcated hydrogen bonds of the kind recently discovered in crystalline deoxyoligonucleotide with lone d(A)n.d(T)n stretch (Nelson et al., Nature 330, 221 (1987)). Still, analysis of the available data and energy calculations do not seem to indicate that the bifurcated H-bonds are a crucial factor responsible for the anomalous structure of the d(A)n.d(T)n sequence. The unique structural properties of poly[d(A)].poly[d(T)] can hardly be explained without taking into account its interactions with the double-layer hydration spine in the minor groove. In view of the hydration mechanism stabilizing poly[d(A)].poly[d(T)] and of the polynucleotide's heteronomous prehistory (Arnott et al., Nucleic Acids Res. 11,4141 (1983)) we suggest that this B-type structure be called Bh.

EXAFS studies of the calcium salts of natural DNA and polydA:polydT.

Preliminary EXAFS experiments were carried out on film of the Ca salts of the synthetic polynucleotide polydA:polydT at 95%, 81%, and 76% relative humidity (r.h.) and for the Ca salt of chicken erythrocyte DNA at 81% r.h. (approximately 43% GC pairs). Detailed analysis of EXAFS data shows that the Ca2+ ion is in fairly close proximity (within 4 A) to a number of phosphorous atoms. This is in contradiction with the recently proposed model, which assumes a close coordination between the cations and the purine and pyrimidine bases deep inside the polynucleotide molecule, so that the distance to the nearest phosphorous atoms must not be less than 5 A. Instead, the EXAFS results suggest that the Ca2+ ions are, for the most part, located at the periphery of individual polydA:polydT (or DNA) molecules, possibly serving as intermolecular links.

Patterson methods in fibre diffraction analysis.

The potential of the Patterson methods for X-ray diffraction studies of textures is examined in DNA fibres. Patterson analysis, which is rarely used in these situations, is shown to yield important information on the preliminary interpretation of diffraction patterns and to increase the reliability of the three-dimensional structural pattern obtained for polymeric molecules. We also show how the screw symmetry of helical molecules can be used to calculate their electron density by means of the three-dimensional Patterson function.

SOD2 gene polymorphism and muscle damage markers in elite athletes.

Exercise-induced oxidative stress is a state that primarily occurs in athletes involved in high-intensity sports when pro-oxidants overwhelm the antioxidant defense system to oxidize proteins, lipids, and nucleic acids. During exercise, oxidative stress is linked to muscle metabolism and muscle damage, because exercise increases free radical production. The T allele of the Ala16Val (rs4880 C/T) polymorphism in the mitochondrial superoxide dismutase 2 (SOD2) gene has been reported to reduce SOD2 efficiency against oxidative stress. In the present study we tested the hypothesis that the SOD2 TT genotype would be underrepresented in elite athletes involved in high-intensity sports and associated with increased values of muscle and liver damage biomarkers. The study involved 2664 Caucasian (2262 Russian and 402 Polish) athletes. SOD2 genotype and allele frequencies were compared to 917 controls. Muscle and liver damage markers [creatine kinase (CK), creatinine, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP)] were examined in serum from 1444 Russian athletes. The frequency of the SOD2 TT genotype (18.6%) was significantly lower in power/strength athletes (n = 524) compared to controls (25.0%, p = 0.0076) or athletes involved in low-intensity sports (n = 180; 33.9%, p < 0.0001). Furthermore, the SOD2 T allele was significantly associated with increased activity of CK (females: p = 0.0144) and creatinine level (females: p = 0.0276; males: p = 0.0135) in athletes. Our data show that the SOD2 TT genotype might be unfavorable for high-intensity athletic events.

Purification and characterisation of recombinant Bacteroides fragilis toxin-2.

Fragilysin (BFT) is metalloprotease that is secreted by enterotoxigenic Bacteroides fragilis. Studying the mechanism of BFT interaction with intestinal epithelial cells requires a pure protein sample. In this study, we cloned DNA-fragments coding for the catalytic domain of fragilysin-2 and profragilysin-2 into an E. coli expression vector. Purification methods for the recombinant fragilysin-2 catalytic domain and profragilysin-2 were developed. In addition, we obtained mature active fragilysin-2 from recombinant proprotein by limited tryptic digestion. We tested the biological activity of the recombinant protein samples and revealed that E-cadherin was cleaved when HT-29 cells were treated with mature fragilysin-2 but not with profragilysin-2. Azocoll, azocasein and gelatin were not proteolytically cleaved by mature fragilysin-2. Proteins released in culture medium after HT-29 cells treatment with mature active BFT-2 were identified.

The structural principles of multidomain organization of the giant polypeptide chain of the muscle titin protein: SAXS/WAXS studies during the stretching of oriented titin fibres.

Elasticity of titin is a key parameter that determines the mechanical properties of muscle. These include reversibility, i.e., the muscle's capacity to change its length many-fold and return to its original state, and the transduction of passive tension generated by the stretched muscle. The morphology and elastic properties of oriented fibres of titin molecules were studied using SAXS and WAXS (small- and wide-angle X-ray scattering, respectively) and mechanical techniques. We succeeded in obtaining oriented filaments of purified titin suitable for diffraction measurements. Our X-ray data suggest a model of titin as a nanoscale, morphological, and aperiodical array of rigid Ig- and Fn3-type domains covalently connected by conformationally variable short loops. The line group symmetry of the model can be defined as SM with axial translation tau(infinity). Both tension transduction and high elasticity of titin can be explained in terms of crystalline polymer physics. Titin stretching experiments show that each individual titin macromolecule can adopt a novel two-phase state within the fibre. Conversion between high elasticity and strength can be explained as a phase transition under external tension. In the terms of the concept of orientational melting the origin of the functional heterogeneity along the titin strand becomes interpretable.

Correlation function method in protein crystallography.

A weighted correlation function as a method for computing electron-density maps is proposed to reduce the errors of the Fourier syntheses performed on inaccurate and/or incomplete data. The formulae are revised for the difference Patterson vector search, for multiple isomorphous replacement (MIR) and single isomorphous replacement (SIR) syntheses and for the difference Fourier synthesis. The examples show that the correlation-function approach has the potential to provide more reliable results than those obtained by conventional Fourier syntheses.