16S rRNA gene primer choice impacts off-target amplification in human gastrointestinal tract biopsies and microbiome profiling.

16S rRNA amplicon sequencing or, more recently, metatranscriptomic analysis are currently the only preferred methods for microbial profiling of samples containing a predominant ratio of human to bacterial DNA. However, due to the off-target amplification of human DNA, current protocols are inadequate for bioptic samples. Here we present an efficient, reliable, and affordable method for the bacteriome analysis of clinical samples human DNA content predominates. We determined the microbiota profile in a total of 40 human biopsies of the esophagus, stomach, and duodenum using 16S rRNA amplicon sequencing with the widely used 515F-806R (V4) primers targeting the V4 region, 68F-338R primers and a modified set of 68F-338R (V1-V2M) primers targeting the V1-V2 region. With the V4 primers, on average 70% of amplicon sequence variants (ASV) mapped to the human genome. On the other hand, this off-target amplification was absent when using the V1-V2M primers. Moreover, the V1-V2M primers provided significantly higher taxonomic richness and reproducibility of analysis compared to the V4 primers. We conclude that the V1-V2M 16S rRNA sequencing method is reliable, cost-effective, and applicable for low-bacterial abundant human samples in medical research.

Gene expression and metabolic activity of Streptococcus mutans during exposure to dietary carbohydrates glucose, sucrose, lactose, and xylitol.

Recent RNA sequencing studies have given us a deeper insight into the cariogenic impact of carbohydrate sources in the bacterium Streptococcus mutans, the principal microbial agent in dental caries etiopathogenesis. The process of dental caries development is facilitated by the ability of this bacterium to ferment some carbohydrates into organic acids contributing to a pH decrease in the oral cavity and the demineralization of the hard tissues of the tooth. Furthermore, in dental caries progression, biofilm formation, which starts and ends with free planktonic cells, plays an important role and has several unique properties called virulence factors. The most cariogenic carbohydrate is sucrose, an easily metabolizable source of energy that induces the acidification and synthesis of glucans, forming typical bacterial cell clumps. We used multifaceted methodological approaches to compare the transcriptomic and metabolomic profiles of S. mutans growing in planktonic culture on preferred and nonpreferred carbohydrates and in fasting conditions. Streptococcus mutans in a planktonic culture with lactose produced the same pH drop as glucose and sucrose. By contrast, xylitol and lactose showed high effectiveness in regulating intracellular polysaccharide metabolism, cell wall structure, and overall virulence involved in the initial phase of biofilm formation and structure but with an opposite pattern compared with sucrose and glucose. Our results confirmed the recent findings that xylitol and lactose play a vital role in biofilm structure. However, they do not reduce its formation, which is related to the creation of a cariogenic environment.

BABA-induced pathogen resistance: a multi-omics analysis of the tomato response reveals a hyper-receptive status involving ethylene.

Prior exposure to microbial-associated molecular patterns or specific chemical compounds can promote plants into a primed state with stronger defence responses. ß-aminobutyric acid (BABA) is an endogenous stress metabolite that induces resistance protecting various plants towards diverse stresses. In this study, by integrating BABA-induced changes in selected metabolites with transcriptome and proteome data, we generated a global map of the molecular processes operating in BABA-induced resistance (BABA-IR) in tomato. BABA significantly restricts the growth of the pathogens Oidium neolycopersici and Phytophthora parasitica but not Botrytis cinerea. A cluster analysis of the upregulated processes showed that BABA acts mainly as a stress factor in tomato. The main factor distinguishing BABA-IR from other stress conditions was the extensive induction of signaling and perception machinery playing a key role in effective resistance against pathogens. Interestingly, the signalling processes and immune response activated during BABA-IR in tomato differed from those in Arabidopsis with substantial enrichment of genes associated with jasmonic acid (JA) and ethylene (ET) signalling and no change in Asp levels. Our results revealed key differences between the effect of BABA on tomato and other model plants studied until now. Surprisingly, salicylic acid (SA) is not involved in BABA downstream signalization whereas ET and JA play a crucial role.

DiaPASEF proteotype analysis indicates changes in cell growth and metabolic switch induced by caspase-9 inhibition in chondrogenic cells.

Caspase-9 is the major apical caspase responsible for triggering the intrinsic apoptotic pathway. Our previous study indicated that specific inhibition of caspase-9 caused microscopically evident alterations in appearance of the primary chondrogenic cultures which cannot be explained by decrease in apoptosis. To describe a complex molecular background of this effect, proteomics analysis of control and caspase-9 inhibitor-treated chondrogenic cultures were performed. Proteins were extracted, identified and quantified using LC-MS in both data dependent and data independent acquisition (DIA) mode. While directDIA analysis of diaPASEF data obtained using timsTOF Pro LC-MS system revealed 7849 protein groups (Q-value <0.01), a parallel analysis of iTRAQ-2DLC-MS3 and conventional DIA-MS data identified only 5146 and 4098 protein groups, respectively, showing diaPASEF a superior method for the study. The detailed analysis of diaPASEF data disclosed 236/551 significantly down-/up-regulated protein groups after caspase-9 inhibition, respectively (|log2FC|>0.58, Q value <0.05). Classification of downregulated proteins revealed changes in extracellular matrix organization, collagen metabolism, and muscle system processes. Moreover, deregulations suggest a switch from glycolytic to lipid based metabolism in the inhibited cells. No essential changes were found in the proteins involved in apoptosis. The data indicate new non-apoptotic participation of caspases in chondrocyte homeostasis with potential applications in cartilage pathophysiology.

L-Aspartate and L-Glutamine Inhibit Beta-Aminobutyric Acid-Induced Resistance in Tomatoes.

Plant diseases caused by pathogens lead to economic and agricultural losses, while plant resistance is defined by robustness and timing of defence response. Exposure to microbial-associated molecular patterns or specific chemical compounds can promote plants into a primed state with more robust defence responses. ß-aminobutyric acid (BABA) is an endogenous stress metabolite that induces resistance, thereby protecting various plants' diverse stresses by induction of non-canonical activity after binding into aspartyl-tRNA synthetase (AspRS). In this study, by integrating BABA-induced changes in selected metabolites and transcript data, we describe the molecular processes involved in BABA-induced resistance (BABA-IR) in tomatoes. BABA significantly restricted the growth of the pathogens P. syringae pv. tomato DC3000 and was related to the accumulation of transcripts for pathogenesis-related proteins and jasmonic acid signalling but not salicylic acid signalling in Arabidopsis. The resistance was considerably reduced by applying amino acids L-Asp and L-Gln when L-Gln prevents general amino acid inhibition in plants. Analysis of amino acid changes suggests that BABA-IR inhibition by L-Asp is due to its rapid metabolisation to L-Gln and not its competition with BABA for the aspartyl-tRNA synthetase (AspRS) binding site. Our results showed differences between the effect of BABA on tomatoes and other model plants. They highlighted the importance of comparative studies between plants of agronomic interest subjected to treatment with BABA.

Achalasia and acromegaly: Co-incidence of these diseases or a new syndrome?

BACKGROUND: Acromegaly is a disorder associated with hypersecretion of growth hormone, most usually caused by a pituitary adenoma. Dysmotility of the gastrointestinal tract has been reported in acromegalic patients. Achalasia is a disorder characterized by aperistalsis of the oesophagus with incomplete lower oesophageal sphincter relaxation and whose aetiology remains unknown. Mutations in some genes have previously been associated with the development of acromegaly or achalasia. The study aims were to analyse mutations in selected genes in a woman having both of these diseases, to identify their aetiological factors, and to suggest explanations for the co-incidence of acromegaly and achalasia. METHODS AND RESULTS: A female patient with acromegaly, achalasia, and a multinodular thyroid gland with hyperplastic colloid nodules underwent successful treatment of achalasia via laparoscopic Heller myotomy, a thyroidectomy was performed, and the pituitary macroadenoma was surgically excised via transnasal endoscopic extirpation. Germline DNA from the leukocytes was analysed by sequencing methods for a panel of genes. No pathogenic mutation in AAAS, AIP, MEN1, CDKN1B, PRKAR1A, SDHB, GPR101, and GNAS genes was found in germline DNA. The somatic mutation c.601C>T/p.R201C in the GNAS gene was identified in DNA extracted from a tissue sample of the pituitary macroadenoma. CONCLUSIONS: We here describe the first case report to our knowledge of a patient with both acromegaly and achalasia. Association of acromegaly and soft muscle tissue hypertrophy may contribute to achalasia's development. If one of these diagnoses is determined, the other also should be considered along with increased risk of oesophageal and colorectal malignancy.

Archaeal community dynamics in biogas fermentation at various temperatures assessed by mcrA amplicon sequencing using different primer pairs.

In this study, the taxonomic and functional diversity of methanogenic archaea in two parallel 120 l fermenters operated at different temperatures and fed with maize silage was estimated by mcrA metabarcoding analysis using two typical primer pairs (ML and MLA) amplifying part of the functional methyl coenzyme M reductase (mcrA) gene. The alpha diversity indices showed that the ML primer pair detected a higher Operational Taxonomic Unit (OTU) abundance compared to the MLA primer pair and methanogen diversity was significantly lower in the 60 °C fermenters. The beta diversity analysis showed the methanogenic community clustered together at 50 °C and 40° and was statistically different from the 60 °C community. Similar, to alpha diversity, beta diversity was also significantly different between primer pairs. At all temperatures analysed, the primer pairs showed a different abundance of the different methanogenic OTUs, e.g. more OTUs relative to Methanoculleus sp. with the ML primer pair, and more OTUs corresponding to Methanobacterium sp. with the MLA primer pair. Moreover, OTUs corresponding to Methanosphaera sp. and Methanobrevibacter sp. were found only by using ML primer pair, while the MLA primer pair detected sequences corresponding to Methanothrix sp.

General Caspase Inhibition in Primary Chondrogenic Cultures Impacts Their Transcription Profile Including Osteoarthritis-Related Factors.

OBJECTIVE: The knowledge about functions of caspases, usually associated with cell death and inflammation, keeps expanding also regarding cartilage. Active caspases are present in the growth plate, and caspase inhibition in limb-derived chondroblasts altered the expression of osteogenesis-related genes. Caspase inhibitors were reported to reduce the severity of cartilage lesions in osteoarthritis (OA), and caspase-3 might represent a promising biomarker for OA prognosis. The objective of this investigation was to decipher the transcriptomic regulation of caspase inhibition in chondrogenic cells. DESIGN: Limb-derived chondroblasts were cultured in the presence of 2 different inhibitors: Z-VAD-FMK (FMK) and Q-VD-OPH (OPH). A whole transcriptome RNA sequencing was performed as the key analysis. RESULTS: The analysis revealed a statistically significant increase in the expression of 252 genes in the FMK samples and 163 genes in the OPH samples compared with controls. Conversely, there was a significant decrease in the expression of 290 genes in the FMK group and 188 in the OPH group. Among the top up- and downregulated genes (more than 10 times changed), almost half of them were associated with OA. Both inhibitors displayed the highest upregulation of the inflammatory chemokine Ccl5, the most downregulated gene was the one for mannose receptors Mrc1. CONCLUSIONS: The obtained datasets pointed to a significant impact of caspase inhibition on the expression of several chondro-/osteogenesis-related markers in an in vitro model of endochondral ossification. Notably, the list of these genes included some encoding for factors associated with cartilage/bone pathologies such as OA.

The oligomeric states of elicitins affect the hypersensitive response and resistance in tobacco.

Successful plant defence against microbial pathogens is based on early recognition and fast activation of inducible responses. Key mechanisms include detection of microbe-associated molecular patterns by membrane-localized pattern recognition receptors that induce a basal resistance response. A well-described model of such responses to pathogens involves the interactions between Solanaceae plants and proteinaceous elicitors secreted by oomycetes, called elicitins. It has been hypothesized that the formation of oligomeric structures by elicitins could be involved in their recognition and activation of defensive transduction cascades. In this study, we tested this hypothesis using several approaches, and we observed differences in tobacco plant responses induced by the elicitin ß-cryptogein (ß-CRY) and its homodimer, ß-CRYDIM. We also found that the C-terminal domain of elicitins of other ELI (true-elicitin) clades plays a significant role in stabilization of their oligomeric structure and restraint in the cell wall. In addition, covalently cross-linking ß-CRYDIM impaired the formation of signalling complexes, thereby reducing its capacity to elicit the hypersensitive response and resistance in the host plant, with no significant changes in pathogenesis-related protein expression. By revealing the details of the effects of ß-CRY dimerization on recognition and defence responses in tobacco, our results shed light on the poorly understood role of elicitins' oligomeric structures in the interactions between oomycetes and plants.

Rapid Multiplex Real-Time PCR Method for the Detection and Quantification of Selected Cariogenic and Periodontal Bacteria.

Dental caries and periodontal diseases are associated with a shift from symbiotic microbiota to dysbiosis. The aim of our study was to develop a rapid, sensitive, and economical method for the identification and quantification of selected cariogenic and periodontal oral bacteria. Original protocols were designed for three real-time multiplex PCR assays to detect and quantify the ratio of 10 bacterial species associated with dental caries ("cariogenic" complex) or periodontal diseases (red complex, orange complex, and Aggregatibacter actinomycetemcomitans). A total number of 60 samples from 30 children aged 2-6 years with severe early childhood caries and gingivitis were tested. In multiplex assays, the quantification of total bacterial (TB) content for cariogenic bacteria and red complex to eliminate differences in quantities caused by specimen collection was included. The mean counts for the TB load and that of ten evaluated specimens corresponded to previously published results. We found a significant difference between the microbial compositions obtained from the area of control and the affected teeth (p < 0.05). Based on this comprehensive microbiological examination, the risk of dental caries or periodontal inflammation may be determined. The test could also be used as a tool for behavioral intervention and thus prevention of the above-mentioned diseases.

Correction to: Intervertebral disc penetration by antibiotics used prophylactically in spinal surgery: implications for the current standards and treatment of disc infections.

Unfortunately, the complete conflict of interest statement was missed out in the original publication. The same is given below.

Intervertebral disc penetration by antibiotics used prophylactically in spinal surgery: implications for the current standards and treatment of disc infections.

PURPOSE: The presence of Propionibacterium acnes in a substantial component of resected disc specimens obtained from patients undergoing discectomy or microdiscectomy has led to the suggestion that this prominent human skin and oral commensal may exacerbate the pathology of degenerative disc disease. This hypothesis, therefore, raises the exciting possibility that antibiotics could play an important role in treating this debilitating condition. To date, however, little information about antibiotic penetration into the intervertebral disc is available. METHODS: Intervertebral disc tissue obtained from 54 microdiscectomy patients given prophylactic cefazolin (n = 25), clindamycin (n = 17) or vancomycin (n = 12) was assayed by high-performance liquid chromatography, with cefaclor as an internal standard, to determine the concentration of antibiotic penetrating into the disc tissue. RESULTS: Intervertebral disc tissues from patients receiving the positively charged antibiotic clindamycin contained a significantly greater percentage of the antibacterial dose than the tissue from patients receiving negatively charged cefazolin (P < 0.0001) and vancomycin, which has a slight positive charge (P < 0.0001). CONCLUSION: Positively charged antibiotics appear more appropriate for future studies investigating potential options for the treatment of low-virulence disc infections. These slides can be retrieved under Electronic Supplementary Material.

Determination of in vitro isoflavone degradation in rumen fluid.

The aim of this study was to determine the degradation of dietary isoflavones in rumen fluid under 2 feeding regimens. The experiments were performed in vitro using a rumen fluid buffer system. The rumen fluid was taken from cows fed either a hay diet or a concentrate-rich diet (the diet consisted of 34.6% maize silage, 17.6% haylage, 12.8% alfalfa hay, and 35.0% supplemental mixture on a dry matter basis). As a source of isoflavones, 40% soybean extract (Biomedica, Prague, Czech Republic) at levels of 5, 25, 50, and 75 mg per 40 mL of rumen fluid was used. Samples of soybean extract were incubated in triplicate at 39°C for 0, 3.0, 6.0, 12.0, and 24.0 h in incubation solution. The metabolism of daidzein and genistein was faster under concentrate-rich diet conditions. In general, production of equol started after 3 to 6 h of incubation and reached the highest rate after approximately 12 h of incubation regardless of the type of diet or concentration of extract. In most of the experiments, production of equol continued after 24 h of incubation. Generally, equol production was greater under the hay diet conditions. Furthermore, experiments with higher amounts of added soybean extract revealed possible inhibitory effects of high levels of isoflavones on the rumen microflora.

Diverse responses of wild and cultivated tomato to BABA, oligandrin and Oidium neolycopersici infection.

Background and Aims: Current strategies for increased crop protection of susceptible tomato plants against pathogen infections include treatment with synthetic chemicals, application of natural pathogen-derived compounds or transfer of resistance genes from wild tomato species within breeding programmes. In this study, a series of 45 genes potentially involved in defence mechanisms was retrieved from the genome sequence of inbred reference tomato cultivar Solanum lycopersicum 'Heinz 1706'. The aim of the study was to analyse expression of these selected genes in wild and cultivated tomato plants contrasting in resistance to the biotrophic pathogen Oidium neolycopersici , the causative agent of powdery mildew. Plants were treated either solely with potential resistance inducers or by inducers together with the pathogen. Methods: The resistance against O. neolycopersici infection as well as RT-PCR-based analysis of gene expression in response to the oomycete elicitor oligandrin and chemical agent ß-aminobutyric acid (BABA) were investigated in the highly susceptible domesticated inbred genotype Solanum lycopersicum 'Amateur' and resistant wild genotype Solanum habrochaites . Key Results: Differences in basal expression levels of defensins, germins, ß-1,3-glucanases, heveins, chitinases, osmotins and PR1 proteins in non-infected and non-elicited plants were observed between the highly resistant and susceptible genotypes. Moreover, these defence genes showed an extensive up-regulation following O. neolycopersici infection in both genotypes. Application of BABA and elicitin induced expression of multiple defence-related transcripts and, through different mechanisms, enhanced resistance against powdery mildew in the susceptible tomato genotype. Conclusions: The results indicate that non-specific resistance in the resistant genotype S. habrochaites resulted from high basal levels of transcripts with proven roles in defence processes. In the susceptible genotype S. lycopersicum 'Amateur', oligandrin- and BABA-induced resistance involved different signalling pathways, with BABA-treated leaves displaying direct activation of the ethylene-dependent signalling pathway, in contrast to previously reported jasmonic acid-mediated signalling for elicitins.

Bacterial community dynamics in a rumen fluid bioreactor during in-vitro cultivation.

To study the various processes in the rumen the in vitro techniques are widely used to realize more controlled and reproducible conditions compared to in vivo experiments. Mostly, only the parameters like pH changes, volatile fatty acids content or metabolite production are monitored. In this study we examine the bacterial community dynamics of rumen fluid in course of ten day cultivation realize under standard conditions described in the literature. Whereas the pH values, total VFA content and A/P ratio in bioreactor were consistent with natural conditions in the rumen, the mean redox-potential values of -251 and -243mV were much more negative. For culture-independent assessment of bacterial community composition, the Illumina MiSeq results indicated that the community contained 292 bacterial genera. In course of ten days cultivation a significant changes in the microbial community were measured when Bacteroidetes to Firmicutes ratio changed from 3.2 to 1.2 and phyla Proteobacteria and Actinobacteria represented by genus Bifidobacterium and Olsenella significantly increased. The main responsible factor of these changes seems to be very low redox potential in bioreactor together with accumulation of simple carbohydrates in milieu as a result of limited excretion of fermented feed and absence of nutrient absorbing mechanisms.