Exploring the interplay between the core microbiota, physicochemical factors, agrobiochemical cycles in the soil of the historic tokaj mád wine region.

A Hungarian survey of Tokaj-Mád vineyards was conducted. Shotgun metabarcoding was applied to decipher the microbial-terroir. The results of 60 soil samples showed that there were three dominant fungal phyla, Ascomycota 66.36% ± 15.26%, Basidiomycota 18.78% ± 14.90%, Mucoromycota 11.89% ± 8.99%, representing 97% of operational taxonomic units (OTUs). Mutual interactions between microbiota diversity and soil physicochemical parameters were revealed. Principal component analysis showed descriptive clustering patterns of microbial taxonomy and resistance gene profiles in the case of the four historic vineyards (Szent Tamás, Király, Betsek, Nyúlászó). Linear discriminant analysis effect size was performed, revealing pronounced shifts in community taxonomy based on soil physicochemical properties. Twelve clades exhibited the most significant shifts (LDA > 4.0), including the phyla Verrucomicrobia, Bacteroidetes, Chloroflexi, and Rokubacteria, the classes Acidobacteria, Deltaproteobacteria, Gemmatimonadetes, and Betaproteobacteria, the order Sphingomonadales, Hypomicrobiales, as well as the family Sphingomonadaceae and the genus Sphingomonas. Three out of the four historic vineyards exhibited the highest occurrences of the bacterial genus Bradyrhizobium, known for its positive influence on plant development and physiology through the secretion of steroid phytohormones. During ripening, the taxonomical composition of the soil fungal microbiota clustered into distinct groups depending on altitude, differences that were not reflected in bacteriomes. Network analyses were performed to unravel changes in fungal interactiomes when comparing postveraison and preharvest samples. In addition to the arbuscular mycorrhiza Glomeraceae, the families Mycosphaerellacae and Rhyzopodaceae and the class Agaricomycetes were found to have important roles in maintaining soil microbial community resilience. Functional metagenomics showed that the soil Na content stimulated several of the microbiota-related agrobiogeochemical cycles, such as nitrogen and sulphur metabolism; steroid, bisphenol, toluene, dioxin and atrazine degradation and the synthesis of folate.

Monitoring and recovery of hyperglycaemia-induced endothelial dysfunction with rheopheresis in diabetic lower extremity ulceration with hyperviscosity.

AIMS: Rheopheresis is an extracorporeal haematotherapy that improves haemorheological status by filtering proteins that enhance blood viscosity. It also has anti-inflammatory effects by removing inflammatory cytokines. Our study aims to examine the effects of rheopheresis on the endothelial status in diabetic lower extremity ulceration. METHODS: In vitro experiments were performed in a HUVEC model to mimic hyperglycaemic stress. We determined the changes in gene expression levels of IL-6, IL-8, TNF-alpha, endothelin convertase enzyme, ET-1, and NO synthase, as well as the ROS and intracellular GSH levels upon hyperglycaemia. In in vivo studies, two rheopheresis procedures were performed on seven patients with diabetic lower extremity ulceration with hyperviscosity, and we measured the changes in plasma concentrations of ET-1, TXB2, SOD enzyme activity, and extracellular components of the glutathione pool depending on treatments. RESULTS: Our results showed that hyperglycaemia increases endothelial expression of inflammatory cytokines, ET-1, and endothelin convertase enzyme, while NO synthase was decreased. As a result of rheopheresis, we observed decreased ET-1 and TXB2 concentrations in the plasma and beneficial changes in the parameters of the glutathione pool. CONCLUSION: To summarize our results, hyperglycaemia-induced oxidative stress and endothelial inflammation can be moderated by rheopheresis in diabetic lower extremity ulceration with hyperviscosity.

Circulating microRNA sequencing revealed miRNome patterns in hematology and oncology patients aiding the prognosis of invasive aspergillosis.

Invasive aspergillosis (IA) may occur as a serious complication of hematological malignancy. Delays in antifungal therapy can lead to an invasive disease resulting in high mortality. Currently, there are no well-established blood circulating microRNA biomarkers or laboratory tests which can be used to diagnose IA. Therefore, we aimed to define dysregulated miRNAs in hematology and oncology (HO) patients to identify biomarkers predisposing disease. We performed an in-depth analysis of high-throughput small transcriptome sequencing data obtained from the whole blood samples of our study cohort of 50 participants including 26 high-risk HO patients and 24 controls. By integrating in silico bioinformatic analyses of small noncoding RNA data, 57 miRNAs exhibiting significant expression differences (P < 0.05) were identified between IA-infected patients and non-IA HO patients. Among these, we found 36 differentially expressed miRNAs (DEMs) irrespective of HO malignancy. Of the top ranked DEMs, we found 14 significantly deregulated miRNAs, whose expression levels were successfully quantified by qRT-PCR. MiRNA target prediction revealed the involvement of IA related miRNAs in the biological pathways of tumorigenesis, the cell cycle, the immune response, cell differentiation and apoptosis.

Effects of phytonutrient-supplemented diets on the intestinal microbiota of Cyprinus carpio.

In the aquaculture sector, a strategy for the more efficient use of resources and proper disease control is needed to overcome the challenges of meat production worldwide. Modulation of the gastrointestinal tract microbiota is a promising approach for promoting animal health and preventing infection. This feeding experiment was conducted to discover the phytonutrient-induced changes in the gastrointestinal tract microbiota of common carp (Cyprinus carpio). Acclimatized animals aged 7 months (30 weeks) were divided randomly into five experimental groups to investigate the effects of the applied feed additives. The dietary supplements were manufactured from anthocyanin-containing processing wastes from the food industry, specifically the production of Hungarian sour cherry extract, synbiotics from fermented corn, and fermentable oligosaccharides from Hungarian sweet red pepper seeds and carotenoids from Hungarian sweet red pepper pulps, applied at a dose of 1%. The gut contents of the animals were collected at four time points throughout the 6-week study period. To track the compositional and diversity changes in the microbiota of the carp intestinal tract, V3-V4 16S rRNA gene-based metagenomic sequencing was performed. The growth performance of common carp juveniles was not significantly affected by supplementation of the basal diet with plant extracts. Phytonutrients improve the community diversity, increase the Clostridium and Lactobacillus abundances and decrease the abundances of potentially pathogenic and spoilage bacteria, such as Shewanella, Pseudomonas, Acinetobacter and Aeromonas. The phyla Proteobacteria, Tenericutes and Chlamydiae were positively correlated with the body weight, whereas Spirochaetes and Firmicutes exhibited negatively correlations with the body weight. We hypothesize that the application of phytonutrients in aquaculture settings might be a reasonable green approach for easing the usage of antibiotics.

Nutraceuticals Induced Changes in the Broiler Gastrointestinal Tract Microbiota.

Effects of nutraceuticals on the intestinal microbiota are receiving increased attention; however, there are few studies investigating their effects on broiler meat production. The aim of this study was to implement feeding strategies and carry out a comprehensive trial examining the interplay between natural biologically active compounds such as carotenoids, anthocyanins, fermentable oligosaccharides, and synbiotics and the gastrointestinal tract microbiota. Our feeding program was applied to an intensive production system with a flock of 1,080 Ross 308 broilers. Aging induced significant changes through the feeding experiment. Nutraceuticals were shown to modulate broiler intestinal diversity and differentially enriched Lactobacillus, Enterococcus, Campylobacter, and Streptococcus in the core microbiome during the different stages of broiler rearing. Additionally, they did not remarkably affect animal growth performance; nevertheless, a positive correlation was found between body weight and Corynebacteriales and Pseudomonadales Furthermore, a diet high in carotenoid, fermentable oligosaccharide, and anthocyanin contents affected the number of beneficial genera such as Faecalibacterium, Lactobacillus, Blautia, and Ruminococcus With this comprehensive trial, we revealed that nutraceuticals induced modulations in broiler gastrointestinal tract microbiota. We believe that plant-derived immunostimulants, recycled from plant food waste products, can supplement antibiotic-free broiler meat production.IMPORTANCE In this trial, nutraceuticals were manufactured from waste products of food industry processing of Hungarian red sweet pepper and sour cherry and incorporated into the diet of poultry to investigate their effects on broilers' growth and the broiler gastrointestinal tract microbiota. To avoid the generation of food waste products, we believe that this approach can be developed into a sustainable, green approach that can be implemented in commercial antibiotic-free poultry to provide safe and high-quality meat.

Free circulating mircoRNAs support the diagnosis of invasive aspergillosis in patients with hematologic malignancies and neutropenia.

Fungal infections represent a worrisome complication in hematologic cancer patients and in the absence of disease specific symptoms, it is important to establish new biological indicators, which can be used during mould-active prophylaxis. Recently, miRNAs have appeared as candidate diagnostic and prognostic markers of several diseases. A pilot clinical study was performed to evaluate the diagnostic utility of 14 microRNAs which can be related to invasive fungal infections. Based on our data miR-142-3p, miR-142-5p, miR-26b-5p and miR-21-5p showed significant overexpression (p < 0.005) due to invasive aspergillosis in hemato-oncology patients with profound neutropenia. A tetramiR assay was designed to monitor peripheral blood specimens. Optimal cut-off was estimated by using the median value (fold change 1.1) of the log10 transformed gene expressions. The biomarker panel was evaluated on two independent sample cohorts implementing different antimicrobial prophylactic strategies. The receiver operating characteristic analysis with area under the curve proved to be 0.97. Three miRNAs (miR-142-5p, miR-142-3p, miR-16-5p) showed significant expression alterations in episodes with sepsis. In summary, the tetramiR assay proved to be a promising diagnostic adjunct with sufficient accuracy and sensitivity to trace invasive aspergillosis in hemato-oncology patients.

Allithiamine Alleviates Hyperglycaemia-Induced Endothelial Dysfunction.

Diabetes mellitus-related morbidity and mortality is a rapidly growing healthcare problem, globally. Several nutraceuticals exhibit potency to target the pathogenesis of diabetes mellitus. The antidiabetic effects of compounds of garlic have been extensively studied, however, limited data are available on the biological effects of a certain garlic component, allithiamine. In this study, allithiamine was tested using human umbilical cord vein endothelial cells (HUVECs) as a hyperglycaemic model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometric analysis using antibodies against specific marker proteins including CD31, CD45, CD54, and CD106. The non-cytotoxic concentration of allithiamine was determined based on MTT, apoptosis, and necrosis assays. Subsequently, cells were divided into three groups: incubating with M199 medium as the control; or with 30 mMol/L glucose; or with 30 mMol/L glucose plus allithiamine. The effect of allithiamine on the levels of advanced glycation end-products (AGEs), activation of NF-κB, release of pro-inflammatory cytokines including IL-6, IL-8, and TNF-α, and H2O2-induced oxidative stress was investigated. We found that in the hyperglycaemia-induced increase in the level of AGEs, pro-inflammatory changes were significantly suppressed by allithiamine. However, allithiamine could not enhance the activity of transketolase, but it exerts a potent antioxidant effect. Collectively, our data suggest that allithiamine could alleviate the hyperglycaemia-induced endothelial dysfunction due to its potent antioxidant and anti-inflammatory effect by a mechanism unrelated to the transketolase activity.

Tendentious effects of automated and manual metagenomic DNA purification protocols on broiler gut microbiome taxonomic profiling.

Here, we developed protocols to improve sensitivity, rigor and comparability of 16S rRNA gene amplification-based next-generation sequencing (NGS) results. A thorough study was performed by evaluating extraction efficiency with respect to the yield, purity, fragmentation of the purified DNA, and sequencing metrics considering the number of quality reads, amplicon sequence variants (ASVs), community structure and biodiversity. We identified batch-effects that significantly bias broiler gastrointestinal tract (GIT) community compositions and made recommendations to improve sensitivity, consistency, and cross-study comparability. We found that the purity of the extracted nucleic acid had a strong effect on the success rate of downstream library preparations. The preparation of stool bacterial suspensions from feces showed a significant positive influence on community biodiversity by enriching Gram-negative bacteria and cataloguing low abundant taxa with greater success than direct processing of fecal material. Applications relying on the automated Roche MagNa Pure 24 magnetic-bead based method provided results with high consistency therefore it seems to be the optimal choice in large-scale studies for investigating broiler GIT microbiota.

Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species.

BACKGROUND: Fungal bloodstream infections (BSI) may be serious and are associated with drastic rise in mortality and health care costs. Candida spp. are the predominant etiological agent of fungal sepsis. The prompt and species-level identification of Candida may influence patient outcome and survival. The aim of this study was to develop and evaluate the CanTub-simplex PCR assay coupled with Tm calling and subsequent high resolution melting (HRM) analysis to barcode seven clinically relevant Candida species. METHODS: Efficiency, coefficient of correlation and the limit of reliable detection were estimated on purified Candida EDTA-whole blood (WB) reference panels seeded with Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, Candida guilliermondii, Candida dubliniensis cells in a 6-log range. Discriminatory power was measured on EDTA-WB clinical panels on three different PCR platforms; LightCycler®96, LightCycler® Nano, LightCycler® 2.0. Inter- and intra assay consistencies were also calculated. RESULTS: The limit of reliable detection proved to be 0.2-2 genomic equivalent and the method was reliable on broad concentration ranges (106-10 CFU) providing distinctive melting peaks and characteristic HRM curves. The diagnostic accuracy of the discrimination proved to be the best on Roche LightCycler®2.0 platform. Repeatability was tested and proved to be % C.V.: 0.14 ± 0.06 on reference- and % C.V.: 0.14 ± 0.02 on clinical-plates accounting for a very high accuracy. Reproducibility was % C.V.: 0.11 between reference- and % C.V.: 0.12between clinical-panels which is highly acceptable. CONCLUSION: Our assay demonstrates recent advances on Tm calling and HRM analysis for the molecular identification of relevant Candida species. This unique, simplex PCR assay may be capable to outperform conventional phenotypic methods by reducing time and providing accurate and reliable results directly from blood (2 h) or from whole blood culture bottles (12-24 h).

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions.

Combining standard clinical methods with PCR showed improved diagnosis of invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia.

BACKGROUND: We assessed the diagnostic value of standard clinical methods and combined biomarker testing (galactomannan assay and polymerase chain reaction screening) in a prospective case-control study to detect invasive pulmonary aspergillosis in patients with hematological malignancies and prolonged neutropenia. METHODS: In this observational study 162 biomarker analyses were performed on samples from 27 febrile neutropenic episodes. Sera were successively screened for galactomannan antigen and for Aspergillus fumigatus specific nucleic acid targets. Furthermore thoracic computed tomography scanning was performed along with bronchoscopy with lavage when clinically indicated. Patients were retrospectively stratified to define a case-group with "proven" or "probable" invasive pulmonary aspergillosis (25.93 %) and a control-group of patients with no evidence for of invasive pulmonary aspergillosis (74.07 %). In 44.44 % of episodes fever ceased in response to antibiotic treatment (group II). Empirical antifungal therapy was administered for episodes with persistent or relapsing fever (group I). 48.15 % of patients died during the study period. Postmortem histology was pursued in 53.85 % of fatalities. RESULTS: Concordant negative galactomannan and computed tomography supported by a polymerase chain reaction assay were shown to have the highest discriminatory power to exclude invasive pulmonary aspergillosis. Bronchoalveolar lavage was performed in 6 cases of invasive pulmonary aspergillosis and in 15 controls. Although bronchoalveolar lavage proved negative in 93 % of controls it did not detect IPA in 86 % of the cases. Remarkably post mortem histology convincingly supported the presence of Aspergillus hyphae in lung tissue from a single case which had consecutive positive polymerase chain reaction assay results but was misdiagnosed by both computed tomography and consistently negative galactomannan assay results. For the galactomannan enzyme-immunoassay the diagnostic odds ratio was 15.33 and for the polymerase chain reaction assay it was 28.67. According to Cohen's kappa our in-house polymerase chain reaction method showed a fair agreement with the galactomannan immunoassay. Combined analysis of the results from the Aspergillus galactomannan enzyme immunoassay together with those generated by our polymerase chain reaction assay led to no misdiagnoses in the control group. CONCLUSION: The data from this pilot-study demonstrate that the consideration of standard clinical methods combined with biomarker testing improves the capacity to make early and more accurate diagnostic decisions.