Strategies to enhance stress tolerance in lactic acid bacteria across diverse stress conditions.

Lactic acid bacteria (LAB) hold significant importance in diverse fields, including food technology, industrial biotechnology, and medicine. As basic components of starter cultures, probiotics, immunomodulators, and live vaccines, LAB cells resist a variety of stressors, including temperature fluctuations, osmotic and pH shocks, exposure to oxidants and ultraviolet radiation, substrate deprivation, mechanical damage, and more. To stay alive in these adversities, LAB employ a wide range of stress response strategies supported by various mechanisms, for example rearrangement of metabolism, expression of specialized biomolecules (e.g., chaperones and antioxidants), exopolysaccharide synthesis, and complex repair and regulatory systems. LAB can coordinate responses to various stressors using global regulators. In this review, we summarize current knowledge about stress response strategies used by LAB and consider mechanisms of response to specific stressful factors, supported by illustrative examples. In addition, we discuss technical approaches to increase the stress resistance of LAB, including pre-adaptation, genetic modification of strains, and adjustment of cultivation conditions. A critical analysis of the recent findings in this field augments comprehension of stress tolerance mechanisms in LAB, paving the way for prospective research directions with implications in fundamental and practical areas.

[Multicenter study of gastroesophageal reflux disease symptoms prevalence in outpatients in Russia].

BACKGROUND: Recently, there has been an increase in the prevalence of gastroesophageal reflux disease (GERD) in Northern Europe, North America and East Asia. However data on GERD prevalence in Russian population are very limited. AIM: To determine the prevalence of GERD among the population of Russia, the clinical spectrum of GERD symptoms, the main drugs used for GERD treatment, and the rate of their administration. MATERIALS AND METHODS: The study was conducted from November 2015 to January 2017 in 8 cities of Russia. A survey of patients over the age of 18 years old visiting outpatient medical institutions for any reason, including patients without gastrointestinal complaints was carried out using a short version of the Mayo Clinic questionnaire. RESULTS: In total, 6132 questionnaires of patients aged 1890 years were analyzed [2456 men (40.1%) and 3676 women (59.9%), mean age 46.615.4 years]. The GERD prevalence among the interviewed patients was 34.2%. The incidence of GERD increased depending on body mass index and the age of the patients. Medications used by the patients for heartburn relief included proton pump inhibitors 59.96%, antacids 67.92%, H2-histamine receptor blockers 11.42%, alginates 18.41% of patients. CONCLUSION: The results of this study indicate a high prevalence of GERD among residents of Russian cities applying for primary health care (34.2%). In comparison with previous studies, an increase in the proportion of GERD patients taking proton pump inhibitors was noted; in most cases the regimen of their intake was in accordance with the recommendations.

[CHARACTERISTICS OF FUNGAL STRAINS PRODUCING THERMOSTABLE XYLOGLUCANASES FROM THE RUSSIAN NATIONAL COLLECTION OF INDUSTRIAL MICROORGANISMS.]

Fungal strains degrading plant biomass available from the Russian National Collection of Industrial Microorganisms (VKPM) have been screened for the xyloglucanase activity. Under conditions of submerged cultivation, the thermophilic strains Sporotrichum thermophile VKPM F-972, Myceliophthora thermophila VKPM F-244, and Sporotrichum pruinosum VKPM F-235 produced extracellular xyloglucanases with optimal activity at 60°C, pH 5.0. 88-100% of the initial enzyme activity was retained after l-h incubation at 50°C; 79-84% of the activity was retained after l-h incubation at 60°C. S. thermophile VKPM F-972, M. thermophila VKPM F-244, and S. pruinosum VKPM F-235 strains may be used as the gene sources for construction of highly active producers of the thermostable xyloglucanases.

[Clinical and pathogenetic features of esophageal spasm].

AIM: To comparatively analyze clinical manifestations in patients with primary esophageal spasm (ES) and its concurrence with gastroesophageal reflux disease (GERD) and the results of their instrumental examinations and psychodiagnostic tests. SUBJECTS AND METHODS: A total of 104 patients with the clinical and manometric signs of ES were examined and divided into two groups: 1) 42 patients with primary ES; 2) 62 patients with ES concurrent with GERD. The examination encompassed esophageal manometry, esophagogastroduodenoscopy, 24-hour pH metry, and an interview using a questionnaire to identify autonomic disorders, and the Mini-Mult test. RESULTS: The patients with primary ES compared to those with ES concurrent with GERD significantly more frequently showed severe pain syndrome (p = 0.009) and a paradoxical dysphagia pattern (p = 0.03); manometry revealed an incoordination in the motility of the entire esophagus (p = 0.001). Comparison of the statistical series of values for contraction amplitude and duration in the distal esophagus found no significant difference in the patients of both groups. Autonomic disturbances were detected in 76.0% of the patients with ES; but the intergroup differences were insignificant. Mental maladaptation was observed in 81.7% of the patients in the absence of intergroup differences. CONCLUSION: The etiopathogenetic factor of ES is a psychoautonomic response to chronic stress in both primary ES and its concurrence with GERD. The reflux of gastric contents into the esophagus does not appear to be one of the leading causes of ES. In primary ES, esophageal motor function is generally impaired to a much greater extent than that in ES concurrent with GERD. The degree of motor disorders is embodied in the specific clinical features of the disease.

[Complicated gastroesophageal reflux disease in the presence of congenital esophageal shortening].

The paper presents a clinical case of complicated gastroesophageal reflux disease (GERD) and describes problems in the differential diagnosis of malignant esophageal lesion in patients with GERD and a treatment modality used in this clinical case.

Role of polymorphic loci in HLA-region rs2647012 and rs805288 in the development of non-Hodgkin's malignant lymphomas in Western Siberia.

The data of genome-wide association analysis suggest that human 6p21.3 chromosomal region (localization of HLA genes) contains polymorphic loci influencing the risk of developing non-Hodgkin's lymphomas. We analyzed association of rs2647012 and rs805288 loci with the risk for non-Hodgkin's malignant lymphomas in the population of Western Siberia. Allele and genotype frequencies were determined in the group of 298 patients and in the control group including 551 individuals. Subgroups of diffuse large B-cell lymphoma (86 patients) and follicular lymphoma (25 patients) were analyzed separately. An association of rs2647012 А/А genotype with increased risk of the disease (OR = 2.78, p = 0.002) was detected in the subgroup of diffuse large B-cell lymphoma.

Analysis of the association of polymorphic loci rs917997 in IL18RAP gene and rs187238 in IL18 gene with the risk for non-Hodgkin's malignant lymphomas in Novosibirsk population.

We analyzed the association of polymorphic variants of rs917997 (G/A) locus in IL18RAP gene and rs187238 (G/C) locus in IL18 gene with the risk of malignant non-Hodgkin's lymphomas in Novosibirsk population. Allele and genotype frequencies of the above loci were determined in patients (243 persons) and control group (371 persons) and compared using χ(2) test. None of the analyzed loci showed statistically significant association with the risk of malignant non-Hodgkin's lymphomas.

[The specific features of pancreatic incretory dysfunction in chronic pancreatitis].

AIM: To reveal the specific features of pancreatogenic diabetes mellitus (DM) and to discuss the principles of its medical therapy. SUBJECTS AND METHODS: Sixty-six patients (55 men and 11 women) aged 30 to 65 years with chronic pancreatitis (CP) were examined. The disease was accompanied with pancreatic calcification and cyst formation in 22 and 13 patients, respectively; 5 patients were found to have a pseudotumorous form of CP and 10 had clinically and laboratorily verified DM. 14 resections and 11 drainages for complicated CP were performed. Its diagnosis was established on the basis of clinical, instrumental, and laboratory findings. Pancreatic exocrine function was evaluated from the results of the 13C-trioctanain breath test (BT) that is designed for its in vivo diagnosis. The level of C-peptide was studied by an enzyme immunoassay. RESULTS: The findings suggest that pancreatic exocrine function is diminished in CP patients both with and without complications as compared with the normal value in 44% (24.3 +/- 1.7 and 26.6 +/- 1.3%, respectively), as shown by BT. According to the results of BT, a substantial decrease in the total proportion of a released label was noted in patients with CP and pancreatic calcification, diabetes mellitus, after resection operations for complications of CP and there were also significant differences, as compared to a group of CP patients without complications. In these patient groups, the level of C-peptide fell to a larger extent than that in CP patients without complications and in patients with CP and DM it was decreased to 0.11 +/- 0.02 ng/ml, the normal level being 0.7-1.9 ng/ml. There was a direct correlation between C-peptide levels and BT results in the patients with CP after resection operations. Insulin antibodies were absent in all the examined patients with CP, which proves the specific type of DM in CP. These are detectable only in type 1 DM. Seven patients with CP and DM were found to have calcification, 5 underwent resection operations, 3 had calcification and underwent pancreatic resection operations. CONCLUSION: The development of DM may be predicted in CP patients with formation of pancreatic calcification and resections. In these patients, pancreatic exocrine dysfunction achieves a severe degree.

[Nitric oxide and indicators of oxidative stress in patients with exacerbation of chronic pancreatitis].

The aim of the study was to compare the level of nitric oxide to clinical and laboratory criteria for acute CP, and indicators of oxidative stress in CP. A total of 129 patients with CP (96 males and 33 females), mean age 46,9 +/- 9,2 years, were distributed to the groups with uncomplicated and complicated course. A study of nitric oxide in the blood as an additional criterion for acute CP. Found it significantly increased in patients with CP compared with control values. The content of nitric oxide in the blood during uncomplicated CP was 149,07 +/- 15,4 umol/l, with complicated course increased to 211,5 +/- 17,7 umol/l, which is significantly higher than that in uncomplicated CP (p = 0,042). A significant increase of NO level in the amplification of pain intensity (10-point analogue scale), and also obtained a direct correlation between these criteria (r = 0,69, p = 0,01). Received a significant increase in levels of nitric oxide with an increase in pancreas head size, revealed a direct correlation between these parameters (r = 0,59, p = 0,04). The obtained results allowed using nitric oxide as a criterion of acute HP. For diagnostic levels of nitric oxide made its rise above 120 mmol/liter. Sensitivity and specificity improvement of nitric oxide above 120 umol/L were 97% and 57% respectively when compared with the pain syndrome and 42% and 62% respectively when compared to pancreas head size. Were studied AAO and MDA indices. A significant increase in MDA (t = 2,58, p = 0,012), indicating that activation of LPO. There was a significant increase of MDA in the amplification of the intensity of pain, and also obtained a direct correlation between these criteria (r = 0,30, p = 0,03). Identified a direct correlation between levels of MDA and nitric oxide (r = 0,63, p = 0,01). Study of the level of nitric oxide can be used as an additional criterion of exacerbation of CP. In patients with CP enhanced LPO processes, as evidenced by the increase of MDA in patients with high levels of nitric oxide in the blood. Growth of LP may be an additional pathophysiological factor amplifying damaged pancreas.

[Peculiarities of diabetes mellitus course in chronic pancreatitis].

In order to identify features of the course pancreatic diabetes and discussion of the principles of conservative therapy were examined 66 patients with CP in age of 30 to 65 years (55 men, 11 women). Among them in 22 cases disease was followed with formation of calcification of pancreas, 13 - pancreatic cysts, and 5 revealed pseudo tumor form of CP, 10 patients had clinical and laboratory evidence of diabetes. Concerning CP complicated course were performed 14 resection and 11 draining operations on the pancreas. Based on clinical, instrumental and laboratory data was made the diagnosis of CP. Exocrine pancreatic function was assessed on the results of the breath test, using 13C-trioktanaine, which is applied for exocrine pancreatic function in vivo test. The content of C-peptide was investigated by enzyme-linked immunosorbent assay (ELISA). The data indicate pancreatic exocrine function decrease in patients with CP with complications and without complications in compare with the norm of 44% (24,3 +/- 1,7, 26,6 +/- 1,3%, respectively) according to the breath test. Significant decrease of the cumulative output tags based on the test data of patients with CP and pancreatic calcification, diabetes mellitus, after resection surgery with CP complications, and there were significant differences in compare with a group of patients with CP without complications (p = 0.5). The level of C-peptide in these groups of patients decreased significantly in compare with a group of patients with CP without complications, and patients with CP and Diabetes was reduced to 0,11 +/- 0,02 ng/ml, at a rate range of 0.7-1.9 ng/ml, ie below the minimum values of norm. Obtained a direct correlation between the level of C-peptide and indicators breath test in patients after resection HP (r = 0,84, p = 0,03). Antibodies to insulin in the whole group of studied patients CPs were negative, which proves the specific type of Diabetes at HP. Antibodies to insulin can be detected only at diabetes type 1. In 7 patients with CP and CD detected calcification, 5 patients performed resection surgery, 3 patients had calcification and conducted the pancreas resection. Thus, we can conclude that in patients with CP and formation of pancreas calcification, pancreas resections may predict the development of diabetes.

[Effect of enzyme supportive therapy of the Ermital' on a quality of life of patients with chronic pancreatitis].

The purpose of the study was to evaluate the quality of life in patients with chronic pancreatitis with the presence of complications treated conservatively and surgical treatment. With the help of a questionnaire MOS SF-36 were asked 80 patients with CP, of whom 15 patients were after the operation, the PDR, 10 patients underwent draining operations, 15 patients had a history of pancreatic necrosis, in 20 patients with CP were characterized by complications (cyst calcification, kalkulez, pseudotumoral form of HP, diabetes) and surgical interventions were not performed in 20 CPs proceeded without complications. Were obtained significant differences on all scales of the questionnaire with the control group all CP patients. Assessment of coping with pain in long-terms after various operations was revealed significantly better results and got rid of persistent pain in patients with a complicated course, who underwent surgery. 23 CP patients with a complicated course, as enzyme replacement therapy received in ermital dose of 20,000 IU lipase 3-4 times a day for 3 weeks. The assessment of quality of life before and after therapy with ermital. The intensity of pain significant changes in the groups received. On the other hand the improvement in general health, physical and social functioning.

[Expression of the genes CelA and XylA isolated from a fragment of metagenomic DNA in Escherichia coli].

The glycosyl hydrolase genes cel5A and xyl3A previously isolated by ourselves within a fragment of DNA from the methagenomic library of cow rumen microflora DNA were sub-cloned and expressed in E. coli. The recombinant proteins Cel5A and Xyl3A were purified and characterized. Cellulase Cel5A belongs to the Family 5 glycosyl hydrolases and is a one-module 38.2 kDa enzyme that hydrolyses the 1,4-glycoside bonds of soluble cellulose substrates and amorphous cellulose, showing its maximal activity (31200 u/mg) on lichenan, a soluble substrate with mixed (beta-1,3-1,4) bonds. The end product of the amorphous cellulose hydrolysis is cellobiose. Cel5A is inactive toward the crystal forms of cellulose. Cel5A is an endoglucanase capable of exohydrolysis. The molecular mass of beta-xylosidase Xyl3A belonging to the Family 3 glycosyl hydrolases is 83.7 kDa. The enzyme is active only on xylooligosaccharides, with the maximal activity shown on xylobiose, the end product of the reaction being xylose. No activity on xylane was hitherto observed. Recombinant Cel5A and Xyl3A are stable over a wide range of pH and temperatures, their maximal activity being observed at pH 6.5 and at 55 degrees C.

[Autoimmune reactions in diseases of the digestive system organs].

AIM: To examine content, diagnostic and prognostic role of autologous antibodies in gastrointestinal diseases (GID). MATERIAL AND METHODS: Enzyme immunoassay was used to measure content of autologous antibodies to N+/K+ ATPase (Aab) of gastric parietal cells, mitochondria, microsomes, tissue transglutaminase in blood serum of 196 patients with gastric, gallbladder, small and large intestinal diseases. Aab relations with heterologous antibodies were studied with kits provided by Bektor-Best (Novosibirsk), DRG-Diagnostics, Orgentec (Germany) and others. RESULTS: In GID high circulation of Aab to parietal cells (Ab-PC) was detected in 42% cases, mean content being 217 +/- 32.4 U/ml, 10 U/ml in the control. Maximal concentration (180 = 340 U/ml) occurred in hepatic cirrhosis, celiac disease, atrophic gastritis. In exacerbations of pancreatitis, colelithiasis and duodenal ulcer ab-PC concentration was 190-210 U/ml, in remission--6-12 U/ml. Minimal concentration (8-38 U/ml) was seen in polyps, gastric cancer, nonspecific ulcerative colitis. For primary biliary cirrhosis more typical was high A/ab concentration to mitochondria (in 83%; 200 U.ml), for autoimmune hepatitis--Aab to microsomes (in 81%; 170 U/ml), in celial disease--Aab to tissue transglutaminase (93%, 75 U/ml). High autoantibodies concentration in GID is accompanied with overcirculation of heteroantibodies to infectious-toxic agents confirming their role in development of autoimmune processes. CONCLUSION: GID are associated with high circulation of autologous antibodies--markers of systemic humoral autoimmune reactions differing in duration, severity, site of lesion, form, stage, disease duration. Maximal detection rate and concentration of serum autologous antibodies were observed in hepatic cirrhosis, active hepatitis, celiac disease, atrophic gastritis, exacerbations of cholelithiasis, ulcer, pancreatitis. Estimation of Aab concentration is essential for diagnosis, prognosis of autoimmune diseases, it reflects intensity and duration of autoimmune reactions in GID.

[Cloning and characterization of a large metagenomic DNA fragment containing glycosyl-hydrolase genes].

The problem of search for and characterization of enzymes synthesized by non-cultivated microorganisms is presently being settled by creating metagenomic libraries. A 6000-clone library with the average size of its inserts amounting to 15 bp has been constructed on the basis of total DNA isolated from cow rumen microorganisms. As the result of the screening of the library on plates with different substrates, a clone was selected that efficiently hydrolyzed lichenan and carboxymethylcellulose. The clone contained the recombinant plasmid pBlue-13 bearing a 12071 bp.-long metagenomic fragment carrying ten open reading frames, two of them being identified as glycosyl hydrolase genes. No homology of the metagenomic DNA with any known microorganism genomes was revealed. The amino acid sequence, deduced on the basis of frame 4 and denoted by Xyl3A, bears resemblance with beta-xylosidases of glycosyl hydrolase Family 3. Frame 6 encodes polypeptide Cel5A homologous to cellulases of glycosyl hydrolase Family 5. The amino acid sequences deduced on the basis of seven out of ten open reading frames were homologous to proteins of microorganisms belonging to the Bacteroides sp. family, the bacteria inhabiting mammalian intestines.

[Extracellular glycosyl hydrolase activity of the clostridia producing acetone, butanol, and ethanol].

Production of acetone, butanol, ethanol, acetic acid, and butyric acid by three strains of anaerobic bacteria, which we identified as Clostridium acetobutylicum, was studied. The yield of acetone and alcohols in 6% flour medium amounted to 12.7-15 g/l with butanol constituting 51.0-55.6%. Activities of these strains towards xylan, beta-glucan, carboxymethylcellulose, and crystalline and amorphous celluloses were studied. C. acertobutylicum 6, C. acetoburylicum 7, and C. acertobutylicum VKPM B-4786 produced larger amounts of acetone and alcohols and displayed higher cellulase and hemicellulase activities than the type strain C. acetobutylicum ATCC 824. It was demonstrated that starch in the medium could be partially substituted with plant biomass.

[Change of nitric oxide metabolites in patients with chronic pancreatitis].

72 patients with chronic pancreatitis were investigated. The levels of nitrogen oxide in the blood and urine were examined in the patients with chronic pancreatitis depend on the disease's stage and its complications. The nitrogen oxide metabolites were determined to all patients (by method of V. A. Metelskaya, 2005) The trustworthy differences of NO levels were revealed depend on the chronic pancreatitis complications presence. The patients with the pancreatic hypertension due to pancreatic calcinosis, that was accompanied persistent painful syndrome and relativelly high blood amylase level (amylase increase 3-4 times in comparison with its normal level) were demonstrated the high NO levels. The tendency to the lowering of level NO and painful syndrome with blood amylase activity reduce were revealed in the patients with surgical treatment of the complicated chronic pancreatitis. The lowering of the painful syndrome and the blood amylase activity follow to normalisaton of pancreatic juice outflow and reduce of the inflammation in parenchyma pancreas. So, the revealed connection increase of NO level and pancreatic inflammation may be the additional criterions for the surgical treatment of such patients.

Bacterial acetone and butanol production by industrial fermentation in the Soviet Union: use of hydrolyzed agricultural waste for biorefinery.

Clostridial acetone-butanol fermentation from renewable carbohydrates used to be the largest biotechnological process second only to yeast ethanol fermentation and the largest process ever run under sterile conditions. With the rising prices for mineral oil, it has now the economical and technological potential to replace petrochemistry for the production of fuels from renewable resources. Various methods for using non-food biomass such as cellulose and hemicellulose in agricultural products and wastes have been developed at laboratory scale. To our knowledge, the AB plants in Russia were the only full-scale industrial plants which used hydrolyzates of lignocellosic waste for butanol fermentation. These plants were further developed into the 1980s, and the process was finally run in a continual mode different from plants in Western countries. A biorefinery concept for the use of all by-products has been elaborated and was partially put into practice. The experience gained in the Soviet Union forms a promising basis for the development of modern large-scale processes to replace a considerable fraction of the current chemical production of fuel for our future needs on a sustainable basis.

[Transformation of soil organic matter in microarthropod community from the Northern Taiga of West Siberia].

Recolonization of defaunated soil by springtails as well as by gamasid and oribatid mites and the changes in organic matter content of soil were studied in the northern taiga. After a one-year exposure in gauze bags (1.7 mm mesh), the abundance of microarthropods was higher but the number of species was lower compared to the surrounding soil. Large surface and litter forms did not colonize the samples, while the number of small and/or soil forms was higher. Soil samples inaccessible for microarthropods (0.15 mm mesh) were depleted of organic carbon compared to both surrounding soil and recolonized samples. The content of humic and fulvic acids was higher in the samples inaccessible to microarthropods. Humification processes prevailed in soils in the absence of microarthropods.

Glucose-1-phosphatase (AgpE) from Enterobacter cloacae displays enhanced phytase activity.

Using a screening procedure developed for detection of phytate hydrolysing enzymes, the gene agpE encoding glucose-1-phosphatase was cloned from an Enterobacter cloacae VKPM B2254 plasmid library. Sequence analysis revealed 78% identity on nucleotide and 79% identity on peptide level to Escherichia coli glucose-1-phosphatase characterising the respective gene product as a representative of acid histidine phosphatases harbouring the RH(G/N)RXRP motif. The purified recombinant protein displayed maximum specific activity of 196 U mg(-1) protein against glucose-1-phosphate but was also active against other sugar phosphates and p-nitrophenyl phosphate. High-performance ion chromatography of hydrolysis products revealed that AgpE can act as a 3-phytase but is only able to cleave off the third phosphate group from the myo-inositol sugar ring. Based on sequence comparison and catalytic behaviour against phytate, we propose to classify bacterial acid histidine phosphatases/phytases in the three following subclasses: (1) AppA-related phytases, (2) PhyK-related phytases and (3) Agp-related phytases. A distinguished activity of 32 U mg(-1) of protein towards myo-inositol-hexa-phosphate, which is two times higher than that of E. coli Agp, suggests that possibly functional differences in terms of phytase activity between Agp- and AppA-like acid histidine phosphatases are fluent.

[Thermoanaerobacter ethanolicus gene cluster containing the alpha- and beta-galactosidases genes melA and lacA, and properties of recombinant lacA].

The nucleotide sequence of a 4936 bp Thermoanaerobacter ethanolicus genomic DNA fragment containing the thermostable beta-galactosidase gene lacA and two incomplete open reading frames has been determined. The product of the first frame is highly homologous to alpha-galactosidases (melibiases), the product of the third frame is homologous to the alpha-D-mannosidases. The terminal area of the lacA, immediately following the stop-codon, harbors presumably a transcription termination site. Based on the location of the putative alpha-galactosidase gene melA and of the beta-galactosidase gene lacA on the T. ethanolicus chromosome, their combined transcription could be presumed. The calculated molecular mass of LacA is 86 kDa. LacA belongs to GH family 2 (GH2). Maximal activity of the purified recombinant enzyme was observed between pH values of 5.7 and 6.0 and temperatures of 75-80 degrees C. The highest activity, 480 units mg(-1), was found on lactose (Km 30 mM), the activities on pNPhGal and oNPhGal amounting to 330 and 420 units mg(-1), respectively. Immobilization on aldehyde silochrome increases the thermostability of the enzyme and keeps its high activity.