A red wine intervention does not modify plasma trimethylamine N-oxide but is associated with broad shifts in the plasma metabolome and gut microbiota composition.

BACKGROUND: Gut microbiota profiles are closely related to cardiovascular diseases through mechanisms that include the reported deleterious effects of metabolites, such as trimethylamine N-oxide (TMAO), which have been studied as diagnostic and therapeutic targets. Moderate red wine (RW) consumption is reportedly cardioprotective, possibly by affecting the gut microbiota. OBJECTIVES: To investigate the effects of RW consumption on the gut microbiota, plasma TMAO, and the plasma metabolome in men with documented coronary artery disease (CAD) using a multiomics assessment in a crossover trial. METHODS: We conducted a randomized, crossover, controlled trial involving 42 men (average age, 60 y) with documented CAD comparing 3-wk RW consumption (250 mL/d, 5 d/wk) with an equal period of alcohol abstention, both preceded by a 2-wk washout period. The gut microbiota was analyzed via 16S rRNA high-throughput sequencing. Plasma TMAO was evaluated by LC-MS/MS. The plasma metabolome of 20 randomly selected participants was evaluated by ultra-high-performance LC-MS/MS. The effect of RW consumption was assessed by individual comparisons using paired tests during the abstention and RW periods. RESULTS: Plasma TMAO did not differ between RW intervention and alcohol abstention, and TMAO concentrations showed low intraindividual concordance over time, with an intraclass correlation coefficient of 0.049 during the control period. After RW consumption, there was significant remodeling of the gut microbiota, with a difference in ß diversity and predominance of Parasutterella, Ruminococcaceae, several Bacteroides species, and Prevotella. Plasma metabolomic analysis revealed significant changes in metabolites after RW consumption, consistent with improved redox homeostasis. CONCLUSIONS: Modulation of the gut microbiota may contribute to the putative cardiovascular benefits of moderate RW consumption. The low intraindividual concordance of TMAO presents challenges regarding its role as a cardiovascular risk biomarker at the individual level. This study was registered at clinical trials.gov as NCT03232099.

Role prediction of Gram-negative species in the resistome of raw cow's milk.

Extended use of antibiotics in dairy farming for therapeutic and prophylactic reasons, but also the higher prevalence of antibiotic resistant bacteria (ARB) in the farm environment raised the concern of consuming raw cow's milk and its derived products. The aim of this study was to predict by shotgun metagenomic analyses the presence of antibiotic resistance genes (ARGs) mainly correlated with Gram-negative bacteria in antibiotic residue free raw cow's milk derived exclusively from healthy animal from South Tyrol (Northern Italy), chosen as a model system. Assessment of shotgun metagenomic data of reconstructed scaffolds, revealed the existence of Pseudomonas spp. as the most abundant Gram-negative species in the raw cow's milk samples bearing ARGs. Besides, ARGs also linked to lactic acid bacteria such as Lactococcus sp. and Lactobacillus sp. ARGs correlated to microbiome found in milk samples conferred resistance towards aminoglycoside-streptothricin, beta-lactamase, macrolide, tetracycline, carbapenem, cephalosporin, penam, peptide, penem, fluoroquinolone, chloramphenicol and elfamycin antibiotics. Further bioinformatic processing included de-novo reassembly of all metagenomic sequences from all milk samples in one, to reconstruct metagenome assembled genomes (MAGs), which were further used to investigate mobile genetic elements (MGE). Analyses of the reconstructed MAGs showed that, MAG 9 (Pseudomonas sp1.) contained the oriT gene (origin of transfer gene) needed for transferring virulent factors. Although the presence of Pseudomonas is common in raw cow's milk, pasteurization treatment reduces their survivability. Nevertheless, attention should be paid on Pseudomonas spp. due to their intrinsic resistance to antibiotics and their capability of transferring virulent factors to other bacteria.

How multiple farming conditions correlate with the composition of the raw cow's milk lactic microbiome.

Questionnaires on farming conditions were retrieved from 2129 dairy farms and clustered, resulting in 106 representative raw cow's milk samples analysed in winter and summer. Substantiating the efficiency of our survey, some farming conditions affected the milk physicochemical composition. Culturing identified several species of lactic acid bacteria (LAB) per milk, whose number increased through 16S ribosomal RNA (rRNA) gene sequencing and shotgun metagenome analyses. Season, indoor versus outdoor housing, cow numbers, milk substitutes, ratio cattle/rest area, house care system during lactation, and urea and medium-chain fatty acids correlated with the overall microbiome composition and the LAB diversity within it. Shotgun metagenome detected variations in gene numbers and uniqueness per milk. LAB functional pathways differed among milk samples. Focusing on amino acid metabolisms and matching the retrieved annotated genes versus non-starter lactic acid bacteria (NSLAB) references from KEGG and corresponding to those identified, all samples had the same gene spectrum for each pathway. Conversely, gene redundancy varied among samples and agreed with NSLAB diversity. Milk samples with higher numbers of NSLAB species harboured higher number of copies per pathway, which would enable steady-state towards perturbations. Some farming conditions, which affected the microbiome richness, also correlated with the NSLAB composition and functionality.

Potentially probiotic or postbiotic pre-converted nitrite from celery produced by an axenic culture system with probiotic lacticaseibacilli strain.

The present study evaluated the use of the probiotic Lacticaseibacillus paracasei DTA-83 as a nitrite-reducing agent to produce potentially probiotic or postbiotic pre-converted nitrite from celery. The results obtained were compared to those achieved by direct addition of sodium nitrite for the typical reddish color formation in cooked pork sausages and the inhibitory potential against the growth of target microorganisms, including the clostridia group. Regarding the sausages color, similar findings were observed when comparing the use of pre-converted nitrite from celery produced by L. paracasei DTA-83 and the direct addition of sodium nitrite. Additionally, it presented an inhibitory effect against Salmonella spp., which was not observed with the direct addition of nitrite, revealing a potential strategy to control salmonellosis in the matrix. However, a non-equivalent preservative effect against Clostridium perfringens (INCQS 215) was determined. The results highlight a promising alternative to produce probiotic or postbiotic meat ingredients; however, further studies should be conducted to investigate doses that achieve microbial control.

New insights into the variability of lactic acid production in Lachancea thermotolerans at the phenotypic and genomic level.

Non-conventional yeasts are increasingly applied in fermented beverage industry to obtain distinctive products with improved quality. Among these yeasts, Lachancea thermotolerans has multiple features of industrial relevance, especially the production of l(+)-lactic acid (LA), useful for the biological acidification of wine and beer. Since few information is available on this peculiar activity, the current study aimed to explore the physiological and genetic variability among L. thermotolerans strains. From a strain collection, mostly isolated from wine, a huge phenotypic diversity was acknowledged and allowed the selection of a high (SOL13) and a low (COLC27) LA producer. Comparative whole-genome sequencing of these two selected strains and the type strain CBS 6340T showed a high similarity in terms of gene content and functional annotation. Notwithstanding, target gene-based analysis revealed variations between high and low producers in the key gene sequences related to LA accumulation. More in-depth investigation of the core promoters and expression analysis of the genes ldh, encoding lactate dehydrogenase, indicated the transcriptional regulation may be the principal cause behind phenotypic differences. These findings highlighted the usefulness of whole-genome sequencing coupled with expression analysis. They provided crucial genetic insights for a deeper investigation of the intraspecific variability in LA production pathway.

Volatile organic compounds from Starmerella bacillaris to control gray mold on apples and modulate cider aroma profile.

Gray mold caused by Botrytis cinerea is a fungal disease that can determine significant economic losses of apple during the storage phase. An alternative to reduce the use of traditional synthetic fungicides is to employ the yeast Starmerella bacillaris as biological control agent (BCA), also with positive effect on apple juice fermentation for the production of cider. Thus, we aimed to evaluate the safety of 16 S. bacillaris strains and their ability to control B. cinerea. In addition, the fermentation performances in apple juice and the volatile organic compounds (VOCs) profile were assessed, both in single-strain and in sequential fermentations with Saccharomyces cerevisiae. The in vitro assays showed that all S. bacillaris strains can be considered safe from the analyzed virulence factors, and were able to significantly constrain the growth of B. cinerea, reducing mycelial growth of 50% in dual-culture and of 90% through VOCs. Moreover, in vivo antagonistic assays revealed a visible decrease of gray mold rot symptoms on apples confirming the potential of S. bacillaris as BCA. GC-MS analysis of the ciders obtained showed increased concentrations in the sequential fermentation of some higher alcohols and terpenes, positively correlated with the cider aromatic quality, and suggested the involvement of benzyl alcohol, known for its antimicrobial action, in the biocontrol efficacy.

Contribution of non-Saccharomyces yeasts to wine volatile and sensory diversity: A study on Lachancea thermotolerans, Metschnikowia spp. and Starmerella bacillaris strains isolated in Italy.

Saccharomyces cerevisiae starter cultures are largely used in winemaking to repress the wild microorganisms and achieve more predictable and desired outcomes. Notwithstanding, alternative microbial resources received increasing attention for their potential to produce wines with more distinctive and typical features. Our previous survey revealed a great inter- and intra-species diversity in an extensive collection of non-Saccharomyces wine yeasts from multiple regions of Italy. This study aimed to explore the detected biodiversity evaluating the quality of wines obtained by sequential inoculation of specific selected strains of the collection (Lachancea thermotolerans or Metschnikowia spp. or Starmerella bacillaris), and S. cerevisiae EC 1118. Fermentations of natural grape must at laboratory scale were followed by microbiological, chemical and sensorial analysis of the wines. The results indicated that each yeast species and strain exerted a distinctive impact on the wine, giving final products clearly separated with Principal Component Analysis. In particular, L. thermotolerans contributed producing relevant amounts of lactic acid and had the highest potential to reduce ethanol content; the presence of S. bacillaris increased the level of glycerol, and, remarkably, reduced acetaldehyde and total SO2; Metschnikowia spp. promoted the formation of higher alcohols and esters, and reduced volatile phenols. The sensory analysis based on the orthonasal aroma confirmed the separation between the wines obtained with the sequential fermentations and the control with single inoculation of EC 1118, although the three non-Saccharomyces species used could not be clearly distinguishable by the panelists. This study indicates that the use of selected native non-Saccharomyces strains in conjunction with S. cerevisiae positively modulates some relevant chemical parameters, and improves the aromatic intensity of wine, therefore justifying investments in non-conventional yeasts as co-starter cultures.

Untangling Neolithic and Bronze Age mitochondrial lineages in South Asia.

Two key moments shaped the extant South Asian gene pool within the last 10 thousand years (ka): the Neolithic period, with the advent of agriculture and the rise of the Harappan/Indus Valley Civilisation; and Late Bronze Age events that witnessed the abrupt fall of the Harappan Civilisation and the arrival of Indo-European speakers. This study focuses on the phylogeographic patterns of mitochondrial haplogroups H2 and H13 in the Indian Subcontinent and incorporates evidence from recently released ancient genomes from Central and South Asia. It found signals of Neolithic arrivals from Iran and later movements in the Bronze Age from Central Asia that derived ultimately from the Steppe. This study shows how a detailed mtDNA phylogeographic approach, combining both modern and ancient variation, can provide evidence of population movements, even in a scenario of strong male bias such as in the case of the Bronze Age Steppe dispersals.

Application of high-dimensional feature selection: evaluation for genomic prediction in man.

In this study, we investigated the effect of five feature selection approaches on the performance of a mixed model (G-BLUP) and a Bayesian (Bayes C) prediction method. We predicted height, high density lipoprotein cholesterol (HDL) and body mass index (BMI) within 2,186 Croatian and into 810 UK individuals using genome-wide SNP data. Using all SNP information Bayes C and G-BLUP had similar predictive performance across all traits within the Croatian data, and for the highly polygenic traits height and BMI when predicting into the UK data. Bayes C outperformed G-BLUP in the prediction of HDL, which is influenced by loci of moderate size, in the UK data. Supervised feature selection of a SNP subset in the G-BLUP framework provided a flexible, generalisable and computationally efficient alternative to Bayes C; but careful evaluation of predictive performance is required when supervised feature selection has been used.

A K(ATP) channel gene effect on sleep duration: from genome-wide association studies to function in Drosophila.

Humans sleep approximately a third of their lifetime. The observation that individuals with either long or short sleep duration show associations with metabolic syndrome and psychiatric disorders suggests that the length of sleep is adaptive. Although sleep duration can be influenced by photoperiod (season) and phase of entrainment (chronotype), human familial sleep disorders indicate that there is a strong genetic modulation of sleep. Therefore, we conducted high-density genome-wide association studies for sleep duration in seven European populations (N=4251). We identified an intronic variant (rs11046205; P=3.99 × 10(-8)) in the ABCC9 gene that explains ≈5% of the variation in sleep duration. An influence of season and chronotype on sleep duration was solely observed in the replication sample (N=5949). Meta-analysis of the associations found in a subgroup of the replication sample, chosen for season of entry and chronotype, together with the discovery results showed genome-wide significance. RNA interference knockdown experiments of the conserved ABCC9 homologue in Drosophila neurons renders flies sleepless during the first 3 h of the night. ABCC9 encodes an ATP-sensitive potassium channel subunit (SUR2), serving as a sensor of intracellular energy metabolism.

Variability of antiepileptic medication taking behaviour in sudden unexplained death in epilepsy: hair analysis at autopsy.

BACKGROUND: Variable compliance with antiepileptic drugs (AEDs) is a potentially preventable cause of sudden unexplained death in epilepsy (SUDEP). Hair AED concentrations provide a retrospective insight into AED intake variability. METHODS: We compared hair AED concentration variability in patients with SUDEP (n = 16), non-SUDEP epilepsy related deaths (n = 9), epilepsy outpatients (n = 31), and epilepsy inpatients (n = 38). AED concentrations were measured in 1 cm hair segments using high performance liquid chromatography. Individual patient hair AED concentration profiles were corrected for "washout" using linear regression analysis. The coefficient of variation (CV) of the corrected mean hair AED concentration provided an index of variability of an individual's AED taking behaviour. Hair sample numbers varied between subjects, and so weighted regression estimates of the CV were derived for each group. RESULTS: The CV regression estimates for each group were: SUDEP 20.5% (standard error 1.9), non-SUDEP 15.0% (3.9), outpatients 9.6% (1.4), and inpatients 6.2% (2.7). The SUDEP group therefore showed greater hair AED concentration variability than either the outpatient or the inpatient groups (p<0.0001). CONCLUSION: Observed variability of hair AED concentrations, reflecting variable AED ingestion over time, is greater in patients dying from SUDEP than in either epilepsy outpatients or inpatients. SUDEP, at least in a proportion of cases, appears preventable.

Vancomycin therapeutic drug monitoring: is there a consensus view? The results of a UK National External Quality Assessment Scheme (UK NEQAS) for Antibiotic Assays questionnaire.

This study investigated vancomycin therapeutic drug monitoring (TDM) and issues related to patient management. Questionnaires were distributed to 310 participants in the UK National External Quality Assessment Scheme (NEQAS) for Antibiotic Assays. The response rate was 57.4%. The majority (76%) had an 'in-house' assay service based, almost exclusively, in the microbiology department, and a fluorescence polarization immunoassay (FPIA) was used by 97%. Almost half (48.7%) had an assay service available for 24 h/day, 7 days/week and 92.7% expected same-day results. The majority (80%) had issued guidelines for vancomycin use. A 12 hourly initial dosing regimen was used by 89%. Trough assay samples were taken <10 min before the dose by 91.5%. For post-dose assay samples, 44% took a sample at 1 h, 28% at 2 h and the remainder at 'other' times. For trough target ranges, 93% quoted <10 mg/L or 5-10 mg/L. There was no consensus with regard to post-dose assay sample times and 23 ranges were quoted. The majority (74.4%) regarded a trough level of >or=10 mg/L as 'toxic' but 13 concentrations were quoted as toxic post-dose measurements. In conclusion, there was a wide variability and poor consensus with regard to post-dose vancomycin assay sampling times, target ranges and what constituted a toxic level.

Vitamin C therapy ameliorates vascular endothelial dysfunction in treated patients with homocystinuria.

OBJECTIVES: We sought to investigate the effects of short- and long-term vitamin C therapy on endothelial dysfunction in patients with homocystinuria. BACKGROUND: Untreated homocystinuria due to cystathionine beta-synthase deficiency is associated with premature atherothrombotic disease; 25% of untreated patients suffer a vascular event by the age of 16 years and 50% by 29 years. Treatment directed at reducing homocysteine accumulation significantly reduces this risk. However, despite 'optimal' treatment and compliance, hyperhomocysteinaemia usually persists and individuals exhibit endothelial dysfunction indicative of an adverse cardiovascular prognosis. Additional intervention is therefore required to further reduce cardiovascular risk. METHODS: We investigated the endothelial effects of acute (2 g single dose) and chronic (1 g/day for 6 months) administration of oral vitamin C in 5 patients with homocystinuria (mean age 26 years, 1 male) and 5 age- and sex-matched controls. Brachial artery endothelium-dependent flow-mediated dilatation (FMD) and endothelium-independent responses to nitroglycerin (NTG) were measured using high-resolution ultrasonic vessel wall-tracking. RESULTS: Baseline: Plasma total homocysteine was 100.8 +/- 61.6 and 9.2 +/- 1.9 micromol/L in the patient and control groups, respectively (p < 0.001). FMD responses were impaired in the patient group (20 +/- 40 microm) compared with the controls (116 +/- 30 microm) (p < 0.001). Vitamin C administration: FMD responses in the patient group improved both acutely, 160 +/- 65 microm at 4 h (p < 0.001), and chronically, 170 +/- 70 microm at 2 weeks (p < 0.001) and 170 +/- 40 microm at 6 months (p < 0.001). FMD responses in the control group were unaltered (p = 0.526). Within both groups, neither the vascular response to NTG nor plasma homocysteine was altered (p > 0.4). CONCLUSIONS: Vitamin C ameliorates endothelial dysfunction in patients with homocystinuria, independent of changes in homocysteine concentration and should therefore be considered as an additional adjunct to therapy to reduce the potential long-term risk of atherothrombotic disease.

Limitation of the Vitros dry-slide technique in measuring high concentrations of salicylate in plasma.

The linearity of the Vitros dry-slide method for plasma salicylate was assessed in two ways: serial concentrations of sodium salicylate were added to fresh lithium heparin plasma, and the salicylate was determined both neat and in dilution. Vitros salicylate results submitted to the Heathcontrol External Quality Assessment Scheme were compared to the spike value. Similar loss of linearity was observed in both cases. Serious salicylate overdosage requiring specific clinical treatment may have been underestimated.

A clinical performance exercise for medicine--pediatrics residents emphasizing complex psychosocial skills.

PURPOSE: To assess the skills of internal medicine-pediatrics (med-peds) residents in evaluating and counseling patients with complex psychosocial problems using a clinical performance exercise (CPE). METHOD: The authors designed a 13-station CPE [nine standardized-patient (SP) stations and four non-SP stations]. Eight of the SP stations focused on counseling or assessing complex psychosocial needs, and three were videotaped and analyzed for specific verbal and nonverbal communication skills. Residents completed a written task for each station and SPs completed a checklist on interviewing and communication skills and a 52-item patient's-satisfaction survey. All first- and third-year residents (n = 25) from two academic years participated. RESULTS: The range of the average scores on the nine SP stations was 43-75%. The residents performed better with common problems (newborn hospital discharge instructions and cardiac risk-factor counseling) than with more complex problems that are less often encountered in the institution (HIV counseling), or problems less often recognized (adult survivor of childhood sexual abuse). As expected, third-year residents scored better than did first-year residents on the written "plan" part of the SP stations and on the non-SP stations. Third-year and first-year residents had similar scores, however, on measures of verbal and nonverbal communication and patient's satisfaction, and for gathering data and providing information. CONCLUSION: This is the first performance-based evaluation of residents in a combined med-peds residency program. The stations addressed more complex clinical skills than those reported for objective structured clinical evaluations of residents.

Population genetic structure of variable drug response.

Geographic patterns of genetic variation, including variation at drug metabolizing enzyme (DME) loci and drug targets, indicate that geographic structuring of inter-individual variation in drug response may occur frequently. This raises two questions: how to represent human population genetic structure in the evaluation of drug safety and efficacy, and how to relate this structure to drug response. We address these by (i) inferring the genetic structure present in a heterogeneous sample and (ii) comparing the distribution of DME variants across the inferred genetic clusters of individuals. We find that commonly used ethnic labels are both insufficient and inaccurate representations of the inferred genetic clusters, and that drug-metabolizing profiles, defined by the distribution of DME variants, differ significantly among the clusters. We note, however, that the complexity of human demographic history means that there is no obvious natural clustering scheme, nor an obvious appropriate degree of resolution. Our comparison of drug-metabolizing profiles across the inferred clusters establishes a framework for assessing the appropriate level of resolution in relating genetic structure to drug response.

External quality assessment of laboratory performance in analysis of toxicological cases.

A mean of 44 members of the United Kingdom national external quality assessment scheme (UKNEQAS) for toxicology reported analytical findings on 10 toxicological cases circulated between December 1995 and February 2000. Material distributed usually consisted of a 5ml blood and a 20ml urine sample simulated by quantitative addition of drugs and their metabolites to material donated by volunteers and patients. The samples were accompanied by a brief outline of the circumstances surrounding the case. Laboratories were requested to report their analytical findings, list methods of analysis, and provide interpretation of their findings. The mean overall success rate for identification of drugs or their pharmacological group was 76%, failure being largely by laboratories providing an immunoassay-based screening service for a fixed range of drug groups. The latter laboratories indicated that cases would be referred to regional toxicology centres for further investigation or confirmation. The coefficient of variation of measurements was <7% for routine analytes, such as ethanol and paracetamol, but 26-44% for tricyclics and opiates. There were 3% false positive reports. The quantity and content of interpretative comment provided by the laboratories was very variable. A number provide nothing in addition to the analytical result.

A pilot study assessing the influences of charge data and group process on diagnostic test ordering by residents.

PURPOSE: Providing charge data to resident physicians has been shown to reduce the amounts spent on diagnostic testing. This pilot study sought to determine the influences of charge data and group decision making on diagnostic test ordering by internal medicine residents. METHOD: In an interactive workshop, 23 internal medicine residents received a hypothetical case. They completed an 18-item questionnaire estimating charges for diagnostic tests and then "ordered" tests. The residents were then randomly divided into groups that either received charge data, received charge data after ordering tests, or received no charge data. The groups ordered tests by consensus. Tests were weighted for appropriateness (+1 to +6) and inappropriateness (-1 to -6). Analyses compared individual and group decisions and effect of availability of charge data. RESULTS: Residents with access to charge data spent less on tests, but also had lower appropriateness scores. The appropriateness of the diagnostic workup was better by groups than by individuals, but cost more. CONCLUSION: Cost-containment interventions targeted towards doctors in training need to address the effect on quality of care and the influence of the group process in clinical decision making. Group diagnostic decisions may be more costly, but more appropriate.

A survey of extraction techniques for drugs of abuse in urine.

Sixty nine participants in the United Kingdom national external quality assessment scheme for drugs of abuse in urine reported details of their sample extraction technique by questionnaire. Laboratories were categorised by differences in technique and their analytical test results compared for samples containing D-amfetamine 0.4 (4) and 0.8 (3) mg/l, morphine 0.4 (4) and 0.8 (4)mg/l, and benzoylecgonine 0.15/0.2 (2) and 0.45/0.5 (4) mg/l. Values in parentheses are numbers of samples. For amfetamine, there was no significant difference in the frequency of true positive results between liquid-liquid or solid phase extraction and the Toxi-Lab A system at 0.8 mg/l. Toxi-Lab A gave significantly fewer positives when operating below its specified threshold at 0.4 mg/l. Paradoxically, laboratories using >5 ml urine volume performed less well. Acidification of the extract before volume reduction gave significantly more true positives. For extraction of morphine, solid phase systems significantly outperformed both liquid-liquid and the Toxi-Lab A system at both 0.8 and 0.4 mg/l. No significant differences between extraction techniques were demonstrated for analysis of benzoylecgonine.