Investigating volatile compounds in the Bacteroides secretome

Microorganisms and their hosts communicate with each other by secreting numerous components. This cross-kingdom cell-to-cell signaling involves proteins and small molecules, such as metabolites. These compounds can be secreted across the membrane via numerous transporters and may also be packaged in outer membrane vesicles (OMVs). Among the secreted components, volatile compounds (VOCs) are of particular interest, including butyrate and propionate, which have proven effects on intestinal, immune, and stem cells. Besides short fatty acids, other groups of volatile compounds can be either freely secreted or contained in OMVs. As vesicles might extend their activity far beyond the gastrointestinal tract, study of their cargo, including VOCs, is even more pertinent. This paper is devoted to the VOCs secretome of the Bacteroides genus. Although these bacteria are highly presented in the intestinal microbiota and are known to influence human physiology, their volatile secretome has been studied relatively poorly. The 16 most well-represented Bacteroides species were cultivated; their OMVs were isolated and characterized by NTA and TEM to determine particle morphology and their concentration. In order to analyze the VOCs secretome, we propose a headspace extraction with GC–MS analysis as a new tool for sample preparation and analysis of volatile compounds in culture media and isolated bacterial OMVs. A wide range of released VOCs, both previously characterized and newly described, have been revealed in media after cultivation. We identified more than 60 components of the volatile metabolome in bacterial media, including fatty acids, amino acids, and phenol derivatives, aldehydes and other components. We found active butyrate and indol producers among the analyzed Bacteroides species. For a number of Bacteroides species, OMVs have been isolated and characterized here for the first time as well as volatile compounds analysis in OMVs. We observed a completely different distribution of VOC in vesicles compared to the bacterial media for all analyzed Bacteroides species, including almost complete absence of fatty acids in vesicles. This article provides a comprehensive analysis of the VOCs secreted by Bacteroides species and explores new perspectives in the study of bacterial secretomes in relation the intercellular communication

Underreplicated regions in Drosophila melanogaster are enriched with fast-evolving genes and highly conserved noncoding sequences

Many late replicating regions are underreplicated in polytene chromosomes of Drosophila melanogaster. These regions contain silenced chromatin and overlap long syntenic blocks of conserved gene order in drosophilids. In this report we show that in D. melanogaster the underreplicated regions are enriched with fast-evolving genes lacking homologs in distant species such as mosquito or human, indicating that the phylogenetic conservation of genes correlates with replication timing and chromatin status. Drosophila genes without human homologs located in the underreplicated regions have higher nonsynonymous substitution rate and tend to encode shorter proteinswhen compared with those in the adjacent regions. At the same time, the underreplicated regions are enriched with ultraconserved elements and highly conserved noncoding sequences, especially in introns of very long genes indicating the presence of an extensive regulatory network that may be responsible for the conservation of gene order in these regions. The regions have amodest preference for long noncoding RNAs but are depleted for small nucleolar RNAs, microRNAs, and transfer RNAs. Our results demonstrate that the underreplicated regions have a specific genic composition and distinct pattern of evolution

Microbial Degradation of Poly-3-Hydroxybutyrate in Samples of Agrogenically Changed Soils

Исследована деградация поли-3-гидроксибутирата (П3ГБ) в лабораторных условиях в полевой и огородной почвах с различной структурой микробиоценозов. В огородной почве с высоким содержанием элементов питания общая численность органотрофных бактерий и микромицетов существенно превосходила аналогичные показатели в полевой почве. Выявлены различия в микробных сообществах образцов почв, показано, что в полевой почве доминировали актинобактерии, Arthrobacter и Corynebacterium. В огородной почве доминировали представители родов Bacillus, Micrococcus, Corynebacterium и Pseudomonas. В ходе экспозиции плёнок П3ГБ в почвах микробный состав менялся, в обоих образцах почв увеличивалось количество грамотрицательных палочек. Процесс биоразрушения плёночных образцов П3ГБ в огородной почве протекал в 1,5-1,7 раза активнее по сравнению с полевой почвой (по различию времени достижения одного уровня деградации)Degradation of poly-3-hydroxybutyrate (P3HB) under laboratory conditions was investigated in field and garden soils with different structure of microbial communities. In the vegetable garden soil with a high content of nutrients the total number of organotrophic bacteria and micromycetes was significantly higher than in field soil. The differences in microbial communities of soil samples were estimated. Domination of actinobacteria of Arthrobacter and Corynebacterium genera was found in field soil. In the vegetable garden soil representatives of the genera Bacillus, Micrococcus, Corynebacterium and Pseudomonas dominated. Microbial composition of both soils was changing during the exhibition of P3HB films, with increasing the number of Gram-negative rods. The rate of biodegradation of P3HB film samples in garden soil was 1.5-1.7 times higher then in field soi

Dual Effect of Low-Molecular-Weight Bioregulators of Bacterial Origin in Experimental Model of Asthma

Asthma is one of the most common noncommunicable diseases, affecting over 200 million people. A large number of drugs control asthma attacks, but there is no effective therapy. Identification of reasons for asthma and preventing this disease is a relevant task. The influence of bacterial components is necessary for the normal development of the immune system and the formation of an adequate immune response to antigens. In the absence of microorganisms or their insufficient exposure, the prerequisites are formed for excessive reactivity to harmless antigens. In the present study, we analyzed cellular and humoral factors in a standard mouse model of OVA-induced asthma modified by 5-fold intraperitoneal injection of bacterial cell wall fragments of glucosaminylmuramyl dipeptide (GMDP) 5 μg/animal or 1 μg lipopolysaccharide (LPS) per animal for 5 days before sensitization by ovalbumin (OVA). Preliminary administration of LPS or GMDP to animals significantly reduced goblet cells as well as the number of neutrophils, lymphocytes, and eosinophils in bronchoalveolar lavage, wherein GMDP corrected neutrophilia to a 2-fold degree, and LPS reduced the severity of eosinophilia by 1.9 times. With OVA administration of GMDP or LPS at the sensitization stage, an increase in the total number of bronchoalveolar lavage cells due to neutrophils, macrophages, lymphocytes, and eosinophils in relation to the group with asthma without GMDP or LPS was observed. The administration of GMDP or LPS to normal mice without asthma for 5 days had no statistically significant effect on the change in the number and population composition of cells in bronchoalveolar lavage in comparison with the control group receiving PBS. As a result of a study in a mouse model of asthma, a dual effect of LPS and GMDP was established: the introduction of LPS or GMDP before sensitization reduces neutrophilia and eosinophilia, while the introduction of LPS or GMDP together with an allergen significantly increases neutrophilia and eosinophilia. The study of the immunoglobulin status shows that in normal-asthma mice, GMDP and LPS slightly increase IgA in bronchoalveolar lavage; at the same time, in the asthma model, injections of GMDP or LPS before sensitization contribute to a significant decrease in IgA (2.6 times and 2.1 times, respectively) in BALF and IgE (2.2 times and 2.0 times, respectively) in blood serum. In an experimental model of asthma, the effect of GMDP and LPS was multidirectional: when they are repeatedly administered before sensitization, the bacterial components significantly reduce the severity of the allergic process, while in the case of a joint injection with an allergen, they increase the influx of macrophages, lymphocytes, and neutrophils into the lungs, which can aggravate the course of pathological process. Thus, the insufficient effect of antigens of a bacterial nature, in particular, with prolonged use of antibiotics can be compensated for by substances based on low-molecular-weight bioregulators of bacterial origin to establish the missing signals for innate immunity receptors, whose constant activation at a certain level is necessary to maintain homeostasis

Association of nocturnal intermittent hypoxia with heat shock protein 70 in patients with obstructive sleep apnea: a pilot study

Introduction — Nocturnal intermittent hypoxia in obstructive sleep apnea (OSA) causes cellular stress and consequent change in inducible heat shock protein 70 (HSP70) level. Thus, the objective of this study was to determine the relationship among nocturnal hypoxia and the serum HSP70 level in patients with severe OSA. Material and Methods — The study involved 34 patients with a clinical diagnosis of moderate to severe OSA (24 men and 10 women). Patients without OSA (10 men and 5 women) were included as a control group. The groups were similar in age. The polysomnographic monitoring was carried by standart methodology. Blood sampling for determining the HSP70 level was carried out between 8:00 and 9:00 am after polysomnographic monitoring. Results — The results of this study demonstrated a high apnea/hypopnea index (AHI), which determined the OSA severity and decreased the blood oxygen saturation (SaO2) (p<0.05). Sleep fragmentation in OSA patients confirmed an increase in respiratory arousal index (ArI). The HSP70 level significantly increased in OSA patients compared with the control group. Correlation analysis showed a positive relationship between HSP70 and AHI (R=0.5) in patients with OSAS, as well as a negative relationship between HSP70 and SaO2 (R=-0.3). Conclusion — Our results demonstrated a high level of HSP70 in patients with severe OSA syndrome vs. those without it. In OSA patients, a direct correlation was found between the HSP70 level and AHI, as well as an inverse correlation between the AHI level and SaO2. These findings suggested an association between the level of inducible HSP70 and nocturnal hypoxia in OSA patients

Robertsonian translocation 13/14 associated with rRNA genes overexpression and intellectual disability

Background: The Robertsonian translocations inherited from parents with a normal phenotype are often discovered through children with pathogenesis. The exact causes of pathologies in children with clinical manifestations are often unknown and vary greatly in the reported cases: uniparental disomy, de novo rearrangements, changes in methylation patterns and gene expression, including ribosomal genes. Aim of the study: Molecular-cytogenetic investigation of a clinical case of intellectual disability. Material and methods: GTG-banding, Ag-NOR staining, fluorescent in situ hybridization, PCR, real-time PCR. Results: We describe a family case of a translocation rob (13; 14) and elevated rRNA expression in the proband with developmental delay and in his phenotypically normal mother. We show the loss of the p-arms of original chromosomes and the absence of NORs on the derived chromosome. The whole-chromosome uniparental disomy is excluded. Conclusion: The translocated chromosome in the proband was most likely inherited from the mother and did not come about de novo with normal chromosomes 13 and 14 being obtained from the father. The cause of the pathogenesis in the proband still remains unknown. We hypothesize that it could be caused by impaired imprinting manifesting in altered methylation levels of loci on the derivative chromosome. Keywords: Robertsonian translocation, rRNA, Chromosome 13, Chromosome 14, Intellectual disabilit

Dual Effect of Low-Molecular-Weight Bioregulators of Bacterial Origin in Experimental Model of Asthma

Asthma is one of the most common noncommunicable diseases, affecting over 200 million people. A large number of drugs control asthma attacks, but there is no effective therapy. Identification of reasons for asthma and preventing this disease is a relevant task. The influence of bacterial components is necessary for the normal development of the immune system and the formation of an adequate immune response to antigens. In the absence of microorganisms or their insufficient exposure, the prerequisites are formed for excessive reactivity to harmless antigens. In the present study, we analyzed cellular and humoral factors in a standard mouse model of OVA-induced asthma modified by 5-fold intraperitoneal injection of bacterial cell wall fragments of glucosaminylmuramyl dipeptide (GMDP) 5 μg/animal or 1 μg lipopolysaccharide (LPS) per animal for 5 days before sensitization by ovalbumin (OVA). Preliminary administration of LPS or GMDP to animals significantly reduced goblet cells as well as the number of neutrophils, lymphocytes, and eosinophils in bronchoalveolar lavage, wherein GMDP corrected neutrophilia to a 2-fold degree, and LPS reduced the severity of eosinophilia by 1.9 times. With OVA administration of GMDP or LPS at the sensitization stage, an increase in the total number of bronchoalveolar lavage cells due to neutrophils, macrophages, lymphocytes, and eosinophils in relation to the group with asthma without GMDP or LPS was observed. The administration of GMDP or LPS to normal mice without asthma for 5 days had no statistically significant effect on the change in the number and population composition of cells in bronchoalveolar lavage in comparison with the control group receiving PBS. As a result of a study in a mouse model of asthma, a dual effect of LPS and GMDP was established: the introduction of LPS or GMDP before sensitization reduces neutrophilia and eosinophilia, while the introduction of LPS or GMDP together with an allergen significantly increases neutrophilia and eosinophilia. The study of the immunoglobulin status shows that in normal-asthma mice, GMDP and LPS slightly increase IgA in bronchoalveolar lavage; at the same time, in the asthma model, injections of GMDP or LPS before sensitization contribute to a significant decrease in IgA (2.6 times and 2.1 times, respectively) in BALF and IgE (2.2 times and 2.0 times, respectively) in blood serum. In an experimental model of asthma, the effect of GMDP and LPS was multidirectional: when they are repeatedly administered before sensitization, the bacterial components significantly reduce the severity of the allergic process, while in the case of a joint injection with an allergen, they increase the influx of macrophages, lymphocytes, and neutrophils into the lungs, which can aggravate the course of pathological process. Thus, the insufficient effect of antigens of a bacterial nature, in particular, with prolonged use of antibiotics can be compensated for by substances based on low-molecular-weight bioregulators of bacterial origin to establish the missing signals for innate immunity receptors, whose constant activation at a certain level is necessary to maintain homeostasis

Circadian Rhythms of Melatonin Secretion in Peri- and Postmenopausal Women with Insomnia

The aim of this study was to assess complaints about sleep quality and to investigate circadian rhythms of melatonin secretion in peri- and postmenopausal women. Material and Methods: A total of 146 climacteric women were examined. All patients were divided into 2 groups: Group1 included 72 perimenopausal women and Group 2 included 74 postmenopausal women. Women were surveyed with special questionnaires: PSQI, FFS, ESS, Daytime Feeling and Functioning Scale. Insomnia Severity Index was calculated. Salivary melatonin content was determined (4 times a day) by immunoassay using Microplate Reader EL×808 (USA). Results: Perimenopausal women often complained about difficulties falling asleep (more than 20 minutes from the moment the light was turned off) and difficulties awakening in the morning, while postmenopausal women often complained about snoring and frequent awakenings during sleep(≥2 times). ISI was 21.3±0.54 in Group 1 and 24.8±0.31 in Group 2, which corresponded to insomnia. Daily melatonin secretion in perimenopausal patients with insomnia was altered – the maximal level was registered in the morning hours. The circadian rhythms of melatonin secretion in the group of postmenopausal women did not correlate to the occurrence of insomnia. Conclusion: We can recommend administration of melatonin in the evening time and light therapy in the early morning hours in the complex treatment of SDs in perimenopausal women for normalizing and shifting the chronobiological rhythms of melatonin secretion, and specific therapy is aimed to eliminate snoring for postmenopausal women

Microbial Degradation of Poly-3-Hydroxybutyrate in Samples of Agrogenically Changed Soils

Исследована деградация поли-3-гидроксибутирата (П3ГБ) в лабораторных условиях в полевой и огородной почвах с различной структурой микробиоценозов. В огородной почве с высоким содержанием элементов питания общая численность органотрофных бактерий и микромицетов существенно превосходила аналогичные показатели в полевой почве. Выявлены различия в микробных сообществах образцов почв, показано, что в полевой почве доминировали актинобактерии, Arthrobacter и Corynebacterium. В огородной почве доминировали представители родов Bacillus, Micrococcus, Corynebacterium и Pseudomonas. В ходе экспозиции плёнок П3ГБ в почвах микробный состав менялся, в обоих образцах почв увеличивалось количество грамотрицательных палочек. Процесс биоразрушения плёночных образцов П3ГБ в огородной почве протекал в 1,5-1,7 раза активнее по сравнению с полевой почвой (по различию времени достижения одного уровня деградации)Degradation of poly-3-hydroxybutyrate (P3HB) under laboratory conditions was investigated in field and garden soils with different structure of microbial communities. In the vegetable garden soil with a high content of nutrients the total number of organotrophic bacteria and micromycetes was significantly higher than in field soil. The differences in microbial communities of soil samples were estimated. Domination of actinobacteria of Arthrobacter and Corynebacterium genera was found in field soil. In the vegetable garden soil representatives of the genera Bacillus, Micrococcus, Corynebacterium and Pseudomonas dominated. Microbial composition of both soils was changing during the exhibition of P3HB films, with increasing the number of Gram-negative rods. The rate of biodegradation of P3HB film samples in garden soil was 1.5-1.7 times higher then in field soi

Alteration of rRNA gene copy number and expression in patients with intellectual disability and heteromorphic acrocentric chromosomes

Background: Intellectual disability (ID) is an important medical and social problem that can be caused by different genetic and environmental factors. One such factor could be rDNA amplification and changes in rRNA expression and maturation. Aim of the study: The aim of the present study was to investigate rRNA levels in patients with heteromorphism of the p-arms of acrocentric chromosomes bearing nucleolus organizer regions compared to a healthy control group. Material and methods: Frequencies of p-arms enlargements in patients with ID and in healthy people were analyzed by G-banding screening. rRNA gene copy numbers on affected acrocentric chromosomes in peripheral blood lymphocytes were evaluated in ID patients and healthy bearers using FISH, and in immortalized lymphocytes of one patient – using FISH and real time PCR. Simultaneously, levels of 18S, 28S and 5,8S rRNA in both groups by means of qRT-PCR were investigated. Results: No difference in acrocentric chromosome heteromorphism frequency in patients versus the healthy group were found. However, we found an amplification of rDNA, a significant elevation in 28S and 5.8S rRNA expression and changes in the 28S/18S rRNA ratio in ID patients compared to healthy controls. At the same time, FISH appeared to be not reliable enough for copy number evaluation, but RT-PCR showed rDNA copy changes in heteromorphic cells compared to normal. Conclusion: Our findings indicate a loss of the correct regulation of rDNA activity and processing after amplification. This could disturb the ribosomal apparatus and thus lead to intellectual disability via at least two mechanisms. Keywords: Intellectual disability, Acrocentric chromosomes, Nucleolus organizer region – NOR, Ribosomal DNA – rDNA, Ribosomal RNA – rRN