An improved and extended dual-index multiplexed 16S rRNA sequencing for the Illumina HiSeq and MiSeq platform.

BACKGROUND: Recent advancements in next-generation sequencing (NGS) technology have ushered in significant improvements in sequencing speed and data throughput, thereby enabling the simultaneous analysis of a greater number of samples within a single sequencing run. This technology has proven particularly valuable in the context of microbial community profiling, offering a powerful tool for characterizing the microbial composition at the species level within a given sample. This profiling process typically involves the sequencing of 16S ribosomal RNA (rRNA) gene fragments. By scaling up the analysis to accommodate a substantial number of samples, sometimes as many as 2,000, it becomes possible to achieve cost-efficiency and minimize the introduction of potential batch effects. Our study was designed with the primary objective of devising an approach capable of facilitating the comprehensive analysis of 1,711 samples sourced from diverse origins, including oropharyngeal swabs, mouth cavity swabs, dental swabs, and human fecal samples. This analysis was based on data obtained from 16S rRNA metagenomic sequencing conducted on the Illumina MiSeq and HiSeq sequencing platforms. RESULTS: We have designed a custom set of 10-base pair indices specifically tailored for the preparation of libraries from amplicons derived from the V3-V4 region of the 16S rRNA gene. These indices are instrumental in the analysis of the microbial composition in clinical samples through sequencing on the Illumina MiSeq and HiSeq platforms. The utilization of our custom index set enables the consolidation of a significant number of libraries, enabling the efficient sequencing of these libraries in a single run. CONCLUSIONS: The unique array of 10-base pair indices that we have developed, in conjunction with our sequencing methodology, will prove highly valuable to laboratories engaged in sequencing on Illumina platforms or utilizing Illumina-compatible kits.

[Composition of oropharyngeal microbiota in patients with COVID-19 of different pneumonia severity].

AIM: To identify features of the taxonomic composition of the oropharyngeal microbiota of COVID-19 patients with different disease severity. MATERIALS AND METHODS: The study group included 156 patients hospitalized with confirmed diagnosis of COVID-19 in the clinical medical center of Yevdokimov Moscow State University of Medicine and Dentistry between April and June 2021. There were 77 patients with mild pneumonia according to CT (CT1) and 79 patients with moderate to severe pneumonia (CT2 and CT3). Oropharyngeal swabs were taken when the patient was admitted to the hospital. Total DNA was isolated from the samples, then V3V4 regions of the 16s rRNA gene were amplified, followed by sequencing using Illumina HiSeq 2500 platform. DADA2 algorithm was used to obtain amplicon sequence variants (ASV). RESULTS: When comparing the microbial composition of the oropharynx of the patients with different forms of pneumonia, we have identified ASVs associated with the development of both mild and severe pneumonia outside hospital treatment. Based on the results obtained, ASVs associated with a lower degree of lung damage belong predominantly to the class of Gram-negative Firmicutes (Negativicutes), to various classes of Proteobacteria, as well as to the order Fusobacteria. In turn, ASVs associated with a greater degree of lung damage belong predominantly to Gram-positive classes of Firmicutes Bacilli and Clostridia. While being hospitalized, patients with severe pneumonia demonstrated negative disease dynamics during treatment significantly more often. CONCLUSION: We have observed differences in the taxonomic composition of the oropharyngeal microbiota in patients with different forms of pneumonia developed outside hospital treatment against COVID-19. Such differences might be due to the presumed barrier function of the oropharyngeal microbiota, which reduces the risk of virus titer increase.

A hybrid interferometric system for velocity measurements in shock-wave experiments.

This paper presents a hybrid interferometric system designed to measure the surface velocity of tested specimens in shock-wave experiments. The system integrates the All-Fiber Velocity Interferometer System for Any Reflector (AFVISAR) and the Photonic Doppler Velocimeter (PDV) interferometric channels using a single probing system to measure the velocity of one surface point of specimens under study. This design allows the same optical signal containing the Doppler frequency shift to be processed by the AFVISAR and PDV independent interferometric devices. The interferometric system has been tested in dynamic experiments and provides the velocity measurement accuracy of at least 1.5 m/s with a nanosecond time resolution.

[Intestinal microbiome as a predictor of the anti-PD-1 therapy success: metagenomic data analysis]. / Mikrobiom kishechnika kak prediktor uspekha anti-PD1 terapii: analiz metagenomnykh dannykh.

Anti-PD-1 immunotherapy has a large impact on cancer treatment but the rate of positive treatment outcomes is 40-45% and depends on many factors. One of the factors affecting the outcome of immunotherapy is the gut microbiota composition. This effect has been demonstrated both in model objects and in clinical patients groups. However, in order to identify clear causal relationships between microbiota and anti-PD1 immunotherapy response, it is necessary to expand the number of patients and experimental samples. This work presents an analysis of metagenomic data obtained using whole-genome sequencing of stool samples from melanoma patients (n=45) with different responses to anti-PD1 therapy. The analysis of the differential representation of microbial species has shown a difference in the composition of the microbiota between the experimental groups. Results of this study indicate existence of a strong link between the composition of the gut microbiota and the outcome of anti-PD1 therapy. Expansion of similar research may help develop additional predictive tools for the outcome of anti-PD1 cancer immunotherapy, as well as increase its effectiveness.

Phase states, microstructure and dielectric characteristics of solid solutions (1 - x)NaNbO3 - xCa2Nb2O7 and (1 - x)NaNbO3 - xSr2Nb2O7.

Ceramics of binary systems solid solutions (1 - x)NaNbO3 - xCa2Nb2O7 and (1 - x)NaNbO3 - xSr2Nb2O7 with non-isostructural extreme components were prepared by the solid-phase reactions technique with the following sintering using conventional ceramic technology. It was found that ceramics with x ≤ 0.2 have a perovskite structure. Layered type of structure predominates in the concentration range 0.2 < x ≤ 1. Phase diagrams of both systems at room temperature have been determined in the perovskite area. It was shown that this area contains two concentration regions with the different crystal structures and the morphotropic phase boundary between them. Microstructure and dielectric characteristics of selected solid solutions were investigated. The influence of technological regulations, such as mechanical activation and variation of sintering temperatures, on the formation of the microstructure and dielectric characteristics was studied for the individually selected concentrations (x = 0.1 and x = 0.25). Dielectric characteristics of ceramics revealed the presence of the Maxwell-Wagner polarization and its corresponding relaxation in the solid solutions (1 - x)NaNbO3 - xCa2Nb2O7 at x > 0.20.

[Evaluation of metabolites levels in feces of patients with inflammatory bowel diseases]. / Otsenka urovnia metabolitov v fekaliiakh patsientov s vospalitel'nymi zabolevaniiami kishechnika.

Inflammatory bowel diseases (IBD), which include ulcerative colitis (UC) and Crohn's disease (CD), are chronic intestinal inflammatory disorders with an unknown etiology. They are characterized by chronic recurrent inflammation of the intestinal mucosa and lead to a significant decrease in the quality of life and death of patients. IBD are associated with suppression of normal intestinal microflora, including a decrease in bacteria, producers of short chain fatty acids (SCFAs), exhibiting anti-inflammatory and protective properties. Among the various methods of intestinal microflora correction, fecal microbiota transplantation (FMT), which engrafts the fecal microbiota from a healthy donor into a patient recipient, is of a particular interest. As a result, a positive therapeutic effect is observed, accompanied by the restoration of the normal intestinal microflora of the patient. A significant drawback of the method is the lack of standardization. Metabolites produced by intestinal microflora, namely SCFAs, allow objective assessment of the functional state of the intestinal microbiota and, consequently, the success of the FMT procedure. Using gas chromatography and nuclear magnetic resonance spectroscopy techniques, we have analyzed concentrations and molar ratios of SCFAs in fecal samples of 60 healthy donors. Results were in good accord when comparing two methods as well as with published data. Analysis of SCFAs in feces of patients with UC (19 patients) and CD (17 patients) revealed a general decrease in the concentration of fatty acids in the experimental groups with significant fluctuations in the values in experimental groups compared to control group of healthy donors. On the limited group of IBD patients (6 patients with UC and 5 patients with CD) concentration of SCFAs before and within 30 days of observation after FMT was determined. It was shown that FMT had a significant impact on the SCFAs levels within 1 month term; tendency to reach characteristics of healthy donors is unambiguously traced for both diseases.

[Intestinal microbiom modulates the response to antitumor immunotherapy]. / Vliianie mikrobioma kishechnika na éffektivnost' protivoopukholevoi immunoterapii.

Numerous studies confirm the high degree of involvement of the intestinal microbiota in most processes in the human body. There is evidence for the effect of intestinal microbiota on the success of chemo and immunotherapy of oncological diseases. It is assumed that the intestinal microbiota exhibits an indirect effect on the antitumor therapy through such mechanisms as general immunomodulation, an increase in cells that specifically respond to antigens of both microbial and tumor origin, metabolism, degradation (utilization) of drug compounds. The intestinal microbiota is currently considered as an additional, but important target for studying the effective use of antitumor therapy and reducing its toxicity, as well as a predictor of the success of immunotherapy. In this review, we highlight the results of studies published to date that confirm the relationship between gut microbiome and antitumor efficacy of immune checkpoint inhibitors. Despite the promising and theoretically substantiated conclusions, there are still some discrepancies among the existing data that will have to be addressed in order to facilitate the further development of this direction.

Markers of dysbiosis in patients with ulcerative colitis and Crohn's disease.

AIM: The aim of the study was to study the taxonomic and functional composition of the gut microbiota in ulcerative colitis (UC) and Crohn's disease (CD) patients to identify key markers of dysbiosis in IBD. MATERIALS AND METHODS: Fecal samples obtained from 95 IBD patients (78 UC and 17 CD) as well as 96 healthy volunteers were used for whole-genome sequencing carried out on the SOLiD 5500 W platform. Taxonomic profiling was performed by aligning the reeds, not maped on hg19, on MetaPhlAn2 reference database. Reeds were mapped using the HUNAnN2 algorithm to the ChocoPhlAn database to assess the representation of microbial metabolic pathways. Short-chain fatty acids (SCFA) level were measured in fecal samples by gas-liquid chromatographic analysis. RESULTS: Changes in IBD patients gut microbiota were characterized by an increase in the representation of Proteobacteria and Bacteroidetes phyla bacteria and decrease in the number of Firmicutes phylum bacteria and Euryarchaeota phylum archaea; a decrease in the alpha-diversity index, relative representation of butyrate-producing, hydrogen-utilizing bacteria, and Methanobrevibacter smithii; increase in the relative representation of Ruminococcus gnavus in UC and CD patients and Akkermansia muciniphila in CD patients. Reduction of Butyryl-CoA: acetate CoA transferase gene relative representation in CD patients, decrease of absolute content of SCFA total number as well as particular SCFAs and main SCFAs ratio in IBD patients may indicate inhibition of functional activity and number of anaerobic microflora and/or an change in SCFA utilization by colonocytes. CONCLUSION: the revealed changes can be considered as typical signs of dysbiosis in IBD patients and can be used as potential targets for IBD patients personalized treatment development.

Phenomenological theory of uniaxial relaxor ferroelectrics.

A phenomenological thermodynamic theory of uniaxial relaxor strontium barium niobate [Formula: see text] is developed using the Landau-Devonshire approach with two order parameters. The fourth-order thermodynamic potential allowed to explain the shape of the polarization hysteresis loops experimentally observed at different temperatures. We show that the broad maximum of the dielectric permittivity is not related to the phase transition and arise due to the coupling between polarization and true order parameter which has antiferroelectric nature. We found that the phase transition temperature is much higher than the maximum of the dielectric permittivity and very likely corresponds to so-called Burn's temperature. True order parameter has no simple relation with polar modes.

[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.

[OUTCOME OF PELVIC EXENTERATION FOR LOCALLY ADVANCED CANCER OF THE PELVIC ORGANS].

We analyzed the outcomes of pelvic exenteration in patients with locally advanced cancer of the pelvic organs. During the period from 2006 to 2013 at the Leningrad Regional Oncology Dispensary there were carried out 218 exenterations of the pelvis. Postoperative complications occurred in 68 patients (31.2%), 17 patients died, mortality was 7.8%. The average surgery time was 186 minutes. The average blood loss was 860 ml. In assessing the oncological effectiveness of surgical interventions it was revealed that a 5-year survival rate ranged from 32% in bladder cancer, up to 50% in cervical cancer. Careful selection of patients, multidisciplinary approach to the problem has paramount importance to achieve satisfactory outcomes.

[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.

[Immediate and long-term results of treatment after pelvic exenteration for locally advanced cervical cancer].

Results of treatment after pelvic exenterations were analyzed in 73 patients with locally advanced cervical cancer: 4 patients--Stage IIIB, 24--Stage IVA, 45--with local recurrences after combined (15) or radiation therapy (30) for the period from 2007 to 2012. The median age was 51 years (22 to 77). Variants of pelvic exenterations were as follows: 30 front, 6 rear supralevatory, 37 total (full monoblock removal of the pelvic organs above the pelvic diaphragm): 6 infralevatory and 31 supralevatory. Postoperative complications were recorded in 21 patients (28.8%), postoperative lethality--6.8%. Long-term results of treatment were observed among 34 patients operated in 2007-2009. One-year survival was 50.0%, two-year--47.1%, three-year--41.2%, four-year--38.2%, five-year--38.2%. Best results were observed in the group of primary patients (Stages IIIB, IVA) with only interorgan fistulas. Low survival rate was registered in the group of patients with recurrences after radiotherapy as intraoperatively, despite both data of CT and MRI and the results of express histological examination, it was impossible to assess accurately the lateral edge of the tumor growth.

[Completion technology of pelvic surgery accompanying with cystectomy in elderly and senile patients].

Results of treatment of 204 elderly and senile patients who underwent cystprostatectomy or anterior pelvic exenteration are analyzed. A comparative analysis of two groups of patients whose operation ended with the traditional drainage through the anterior abdominal wall (n = 100), and bilateral perineal drainage (n = 104) is presented. Bilateral perineal drainage after operations on the pelvic organs, accompanied by cystectomy and extended lymphadenectomy in conjunction with the restoration of the peritoneum lateral pelvic walls, improves postoperative recovery of intestinal peristalsis, promotes an earlier reduction in the intensity of pain and morbidity in the early postoperative period. Perineal installation of drains is a simple in design and safe procedure. We recommend bilateral perineal drainage after operations on the pelvic organs, accompanied by cystectomy and extended lymphadenectomy.

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.

[The role of oxidative damage of proteins and lipids in pathogenesis of traumatic disease in severe combined thoracic trauma].

The investigation objective was to study the dynamics of indices of the lipids and proteins oxidative damage, as well as search for possible prognostic criteria in the injured persons with severe combined thoracic trauma. Concentration of carbonyl groups of proteins and malonic dialdehyde was determined on 1-2d, 3-4th and 5-6th day after trauma in the blood plasm of 73 patients, ageing 20 -68 yrs old. While in conditions of massive infusion therapy concentration of the indices investigated do not reflect the oxidative processes intensity. Relative concentration in recalculation on concentration of common protein content constitutes a more demonstratable index. On the 5-6th day after trauma a tendency for normalization of the oxidative damage of lipids and proteins indices was observed in the patients, who have recovered, and while lethal outcome--their further enhancement was noted. There was established a one-direction dynamics of a relative indices in both groups up to 3-4-th day after trauma with a step-by-step its enhancement. Concentration of carbonyl groups of proteins more than 15.86 mcmol/g of protein on the 5-6-th day after trauma ought to be considered a trustworthy criterion of unfavorable prognosis.

[Correction of energy homeostasis in the acute period of concomitant brain injury].

A randomized prospective study of 191 patients with concomitant brain injury (CBI) of different severity has been carried out. All patients underwent surgery and received treatment in reanimation and intensive care departments. The main group consisted of 100 (52.4%) patients treated with cytoflavin in dosage 20-40 ml daily intravenously in drops during 10 days in addition to standard treatment. The comparison group included 91 (47.6%) patients who received standard treatment only. A positive effect of cytoflavin on clinical symptoms and laboratory characteristics of patients with CBI was identified. The decrease in severity measured with the APACHE II, higher activation of consciousness and improvement in the dynamics of neurological symptoms in these patients have resulted in the reduction in the duration of artificial lung ventilation and total hospital stay.

[Pelvic exenteration in surgery for colorectal cancer].

A total of 1436 patients with colorectal cancer underwent resective surgery: 244 (15.6%) received combined interventions, 94 (41.9%) pelvic exenteration (PE), 38 (40.4%) complete PE, 9 (9.6%) of which were infralevator and 29 (30.8%) supralevator. In 56 (59.6%) patients posterior PE was performed, supralevator was performed in 17 (18.1%) cases and infralevator in 39 (41.5%) cases. In 47 (69.1%) of 68 supralevator PE recipients colonic anastomosis was formed. In 21 (38.9%) patients a terminal colostoma was formed, in 29 (76.3%) of 38 patients incontinent urinary diversion was formed. Continent urinary diversion was performed in 9 (23.7%) patients. Twenty six (27.6%) patients had 43 post-operative complications which were lethal in 7 (26.9%) cases.

[Ways to improve the quality of life of elderly and senile patients after the operation by Brikker].

The present study is devoted to improving quality of life of patients in elderly and senile age after operation of Bricker by finding the optimal method of forming ureterointestinal anastomosis. From 2007 to 2009 103 patients of elderly and senile age with diseases requiring removal of the bladder were treated in the Lenigrad Regional Oncology Centre. All the patients were made cystectomy. Patients were divided into two groups: In 1st group, the ureterointestinal anastomosis was formed a classical way "end to side" described Bricker, in the 2nd group ureterointestinal anastomosis was performed by the method of Wallace - "common area". Pathological conditions developed in patients in late postoperative period were as follows: hydronephrosis in early and later stages, obstructive pyelonephritis, frequent attacks of chronic pyelonephritis, chronic renal failure, urinary fistula. Formation of ureterointestinal anastomosis by Wallace during surgery reduces the amount of later postoperative complications. Quality of life was better after the formation of ureterointestinal anastomosis by Wallace.

Influence of digital dermatitis and sole ulcer on dairy cow behaviour and milk production.

The aim of the study was to investigate whether the presence of digital dermatitis (DD) and sole ulcer (SU) in dairy cows was associated with changes in behaviour and milk production. Swedish Red and Swedish Holstein cows (mostly in the first to second lactation) were housed in a cubicle system with automatically scraped passageways. After maintenance claw trimming of all the cows in the herd, 10 cows with DD and 10 cows with SU were selected. For each DD- or SU-affected cow, a healthy control cow, matched according to breed, age, parity and lactation stage, was selected. The behaviour of each of the 20 focal cows was observed for 1 h during 2 to 3 weeks after claw trimming (WACT; period 1) and for 1 h during 5 to 6 WACT (period 2). Milk production parameters: energy-corrected milk (ECM), fat and protein percentages and somatic cell counts (SCCs) were recorded once monthly. Lameness was scored once at the start of the study and cows with SU and DD showed more score 2 lameness (42% v. 31%) than the healthy cows (12%). Most differences in behaviour were found during 2 to 3 WACT when DD- and SU-affected cows were lying less (P = 0.001 and P = 0.012, respectively) than healthy cows. Ruminating while standing was performed more in DD-affected cows (P < 0.001) and tended to be performed more in SU-affected cows (P = 0.079) than their controls. Vigilance was performed more in DD- and SU-affected cows than in healthy cows (P < 0.001 and P = 0.047). Cows with DD produced approximately 5.5 kg less ECM per day both at 2 to 3 WACT (P = 0.022) and at 5 to 6 WACT (P = 0.017) than healthy cows, whereas SU-affected cows tended to produce less ECM at 5 to 6 WACT (P = 0.059). No differences in milk fat and protein or SCC were found. It may be concluded that DD-affected cows showed a stronger behavioural response to the claw disease than the SU-affected cows. This shows the importance of regular claw checking and claw trimming of the cows in order to avoid the negative effects on behaviour and milk production.