Genome-wide analysis of mitochondrial DNA copy number reveals loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation.

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10-15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10-8) and mtDNA replication (p = 1.2 × 10-7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10-4).

Large trans-ethnic meta-analysis identifies AKR1C4 as a novel gene associated with age at menarche.

STUDY QUESTION: Does the expansion of genome-wide association studies (GWAS) to a broader range of ancestries improve the ability to identify and generalise variants associated with age at menarche (AAM) in European populations to a wider range of world populations? SUMMARY ANSWER: By including women with diverse and predominantly non-European ancestry in a large-scale meta-analysis of AAM with half of the women being of African ancestry, we identified a new locus associated with AAM in African-ancestry participants, and generalised loci from GWAS of European ancestry individuals. WHAT IS KNOWN ALREADY: AAM is a highly polygenic puberty trait associated with various diseases later in life. Both AAM and diseases associated with puberty timing vary by race or ethnicity. The majority of GWAS of AAM have been performed in European ancestry women. STUDY DESIGN, SIZE, DURATION: We analysed a total of 38 546 women who did not have predominantly European ancestry backgrounds: 25 149 women from seven studies from the ReproGen Consortium and 13 397 women from the UK Biobank. In addition, we used an independent sample of 5148 African-ancestry women from the Southern Community Cohort Study (SCCS) for replication. PARTICIPANTS/MATERIALS, SETTING, METHODS: Each AAM GWAS was performed by study and ancestry or ethnic group using linear regression models adjusted for birth year and study-specific covariates. ReproGen and UK Biobank results were meta-analysed using an inverse variance-weighted average method. A trans-ethnic meta-analysis was also carried out to assess heterogeneity due to different ancestry. MAIN RESULTS AND THE ROLE OF CHANCE: We observed consistent direction and effect sizes between our meta-analysis and the largest GWAS conducted in European or Asian ancestry women. We validated four AAM loci (1p31, 6q16, 6q22 and 9q31) with common genetic variants at P < 5 × 10-7. We detected one new association (10p15) at P < 5 × 10-8 with a low-frequency genetic variant lying in AKR1C4, which was replicated in an independent sample. This gene belongs to a family of enzymes that regulate the metabolism of steroid hormones and have been implicated in the pathophysiology of uterine diseases. The genetic variant in the new locus is more frequent in African-ancestry participants, and has a very low frequency in Asian or European-ancestry individuals. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Extreme AAM (<9 years or >18 years) were excluded from analysis. Women may not fully recall their AAM as most of the studies were conducted many years later. Further studies in women with diverse and predominantly non-European ancestry are needed to confirm and extend these findings, but the availability of such replication samples is limited. WIDER IMPLICATIONS OF THE FINDINGS: Expanding association studies to a broader range of ancestries or ethnicities may improve the identification of new genetic variants associated with complex diseases or traits and the generalisation of variants from European-ancestry studies to a wider range of world populations. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by CHARGE Consortium grant R01HL105756-07: Gene Discovery For CVD and Aging Phenotypes and by the NIH grant U24AG051129 awarded by the National Institute on Aging (NIA). The authors have no conflict of interest to declare.

The Role of MicroRNA Expression for Proliferation and Apoptosis of Tumor Cells: Impact of Hypoxia-Related Acidosis.

The metabolic microenvironment in tumors is characterized by hypoxia and acidosis. Extracellular pH sometimes decreases to even below 6.0. Previous experiments showed that tissue pH has an impact on tumor cell proliferation and apoptosis. However, the mechanism of how cell cycle progression is affected by decreased pH is not fully understood yet. One possible mechanism includes changes in the expression of miRNAs. The aim of this study was to analyze the impact of pH-regulated miRNAs (miR-183 and miR-215) on proliferation, apoptosis, and necrosis of tumor cells. Therefore, AT1 prostate and Walker-256 mammary carcinoma cells were transfected with the miRNAs or with the respective antagomirs and incubated at pH 7.4 and 6.6 for 24 h. AT1 cells underwent a G0/G1 cell cycle arrest under acidic conditions and showed a marked reduction of the number of actively DNA-synthesizing cells. In Walker-256 cells, acidosis induced a reduction of apoptosis and additionally a significant increase in necrotic cell death. Transfection of tumor cells with miR-183 or miR-215, which were significantly downregulated under acidic conditions, had no impact on cell death of AT1 or Walker-256 cells. Overexpression of miR-183, which is also downregulated by acidosis, intensified G0/G1 cell cycle arrest in AT1 cells. Previous studies revealed that hypoxia-related tumor acidosis affects the expression of different small noncoding RNAs. However, not all of these acidosis-regulated miRNAs seem to have an impact on proliferation, apoptosis, and necrosis of tumor cells. While miR-215 had no influence, miR-183 seems to be an interesting candidate that could amplify the impact of extracellular acidosis on malignant behavior of tumor cells.

Functional Impact of Acidosis-Regulated MicroRNAs on the Migration and Adhesion of Tumor Cells.

Tumor tissue shows special features in metabolism in contrast to healthy tissue. Besides a distinctive oxygen deficiency, tumors often show a reduced extracellular pH (acidosis) resulting from an intensified glycolysis not only under hypoxic but also under normoxic conditions (Warburg effect). As shown in previous studies, cell migration is increased in AT1 prostate carcinoma cells after incubation at pH 6.6, and this leads to an increased number of lung metastases in vivo. However, the signaling pathway causing these functional changes is still unknown. Possible mediators could be acidosis-regulated microRNAs (miR-7, miR-183, miR-203, miR-215). The aim of the study was therefore to analyze whether a change in the expression of these microRNAs has an impact on the tumor cell migration and adhesion. Studies were performed with AT1 rat prostate cancer cells which were incubated for 24 h at pH 7.4 or 6.6. Keeping AT1 tumor cells at low pH increased the migratory capacity by about 100%. But also the decrease of miR-203 and miR-215 expression (at normal pH) led to an increase in migration velocity by 50%. In contrast, cell adhesion was increased by about 75% at low pH. However, an increase in miR-215 expression at pH 6.6 reduced the adhesion by trend. These results clearly indicated that the extracellular pH has an impact on migration and adhesion of tumor cells. In this mechanism, pH-regulated microRNAs could play a role since changes in the expression of these microRNAs (especially miR-203) are also able to modulate the migratory behavior.

Genome-wide meta-analysis of variant-by-diuretic interactions as modulators of lipid traits in persons of European and African ancestry.

Hypertension (HTN) is a significant risk factor for cardiovascular morbidity and mortality. Metabolic abnormalities, including adverse cholesterol and triglycerides (TG) profiles, are frequent comorbid findings with HTN and contribute to cardiovascular disease. Diuretics, which are used to treat HTN and heart failure, have been associated with worsening of fasting lipid concentrations. Genome-wide meta-analyses with 39,710 European-ancestry (EA) individuals and 9925 African-ancestry (AA) individuals were performed to identify genetic variants that modify the effect of loop or thiazide diuretic use on blood lipid concentrations. Both longitudinal and cross sectional data were used to compute cohort-specific interaction results, which were then combined through meta-analysis in each ancestry. These ancestry-specific results were further combined through trans-ancestry meta-analysis. Analysis of EA data identified two genome-wide significant (p < 5 × 10-8) loci with single nucleotide variant (SNV)-loop diuretic interaction on TG concentrations (including COL11A1). Analysis of AA data identified one genome-wide significant locus adjacent to BMP2 with SNV-loop diuretic interaction on TG concentrations. Trans-ancestry analysis strengthened evidence of association for SNV-loop diuretic interaction at two loci (KIAA1217 and BAALC). There were few significant SNV-thiazide diuretic interaction associations on TG concentrations and for either diuretic on cholesterol concentrations. Several promising loci were identified that may implicate biologic pathways that contribute to adverse metabolic side effects from diuretic therapy.

Simultaneous biosorption of Cd(II), Ni(II) and Pb(II) onto a brown macroalgae Fucus vesiculosus: Mono- and multi-component isotherms, kinetics and thermodynamics.

Due to the anthropic activities, several heavy metal ions are introduced into the environment, impacting ecosystems and local activities. In this context, the biosorption process using algae represents an alternative form for these compounds remediation due to the advantages derived from the biosorbent and process efficiency. Thus, the present study evaluated Cadmium (Cd(II)), Nickel (Ni(II)) and Lead (Pb(II)) remediation from aqueous media in mono- and multi-component systems. The biosorbent was characterized in terms of its morphology and composition and parameters involving equilibrium, kinetics, and thermodynamics were investigated. Lastly, the sample was considered in a real surface water sample remediation impacted by a mining dam rupture. Except for Freundlich, all isotherm models tested satisfactorily adjusted to the experimental data for a mono-component system. The maximum biosorption capacities (qm) were 143.2 ±â€¯7.5, 70.1 ±â€¯1.9, 516.3 ±â€¯12.5 mg g-1 for Cd(II), Ni(II) and Pb(II) ions, respectively. When binary systems were considered, an antagonism effect was observed. The biosorption of Cd(II) was drastically affected by the presence of Ni(II), while Pb(II) biosorption in general was less affected by other metals presence. As observed for the binary system, the worst effect in the ternary system was observed for Cd(II) biosorption, being significantly affected by Ni(II) and Pb(II) presence. Overall, the biosorption order in mono- and multi-component systems was found to be Pb(II) ≫ Cd(II) > Ni(II). The affinity for the metals ions was also observed by Elovich's desorption constant, in which aPb(II)≪aCd(II)aCd(II), achieving an equilibrium passed 49 min. From the stages involved in biosorption process, film diffusion presented the greatest contribution as control-stage obtaining a lower diffusion coefficient in all cases. The process was spontaneous in all temperature range evaluated, considered exothermic for all metal ions evaluated. Iron, manganese and nickel concentrations in real surface water samples were higher than the allowed by the Brazilian National Environment Council (CONAMA). Comparing the hazard index values before and after the biosorption process, a reduction superior to 8 × was observed (HIbefore: 3.36, HIafter: 0.40), in which there was no non-carcinogenic risk imposed to the surrounding population after the treatment applied.

Large-scale pharmacogenomic study of sulfonylureas and the QT, JT and QRS intervals: CHARGE Pharmacogenomics Working Group.

Sulfonylureas, a commonly used class of medication used to treat type 2 diabetes, have been associated with an increased risk of cardiovascular disease. Their effects on QT interval duration and related electrocardiographic phenotypes are potential mechanisms for this adverse effect. In 11 ethnically diverse cohorts that included 71 857 European, African-American and Hispanic/Latino ancestry individuals with repeated measures of medication use and electrocardiogram (ECG) measurements, we conducted a pharmacogenomic genome-wide association study of sulfonylurea use and three ECG phenotypes: QT, JT and QRS intervals. In ancestry-specific meta-analyses, eight novel pharmacogenomic loci met the threshold for genome-wide significance (P<5 × 10-8), and a pharmacokinetic variant in CYP2C9 (rs1057910) that has been associated with sulfonylurea-related treatment effects and other adverse drug reactions in previous studies was replicated. Additional research is needed to replicate the novel findings and to understand their biological basis.

Factors increasing the risk for a severe reaction in anaphylaxis: An analysis of data from The European Anaphylaxis Registry.

BACKGROUND: Preventive measures to decrease the frequency and intensity of anaphylactic events are essential to provide optimal care for allergic patients. Aggravating factors may trigger or increase the severity of anaphylaxis and therefore need to be recognized and avoided. OBJECTIVE: To identify and prioritize factors associated with an increased risk of developing severe anaphylaxis. METHODS: Data from the Anaphylaxis Registry (122 centers in 11 European countries) were used in logistic regression models considering existing severity grading systems, elicitors, and symptoms to identify the relative risk of factors on the severity of anaphylaxis. RESULTS: We identified higher age and concomitant mastocytosis (OR: 3.1, CI: 2.6-3.7) as the most important predictors for an increased risk of severe anaphylaxis. Vigorous physical exercise (OR: 1.5, CI: 1.3-1.7), male sex (OR: 1.2, CI: 1.1-1.3), and psychological burden (OR: 1.4, CI: 1.2-1.6) were more often associated with severe reactions. Additionally, intake of beta-blockers (OR: 1.9, CI: 1.5-2.2) and ACE-I (OR: 1.28, CI: 1.05, 1.51) in temporal proximity to allergen exposition was identified as an important factor in logistic regression analysis. CONCLUSION: Our data suggest it may be possible to identify patients who require intensified preventive measures due to their relatively higher risk for severe anaphylaxis by considering endogenous and exogenous factors.

Application of forward osmosis technology in crude glycerol fermentation biorefinery-potential and challenges.

Forward osmosis (FO) is a low energy-intensive process since the driving force for water transport is the osmotic pressure difference, Δπ, between the feed and draw solutions, separated by the FO membrane, where πdraw > πfeed. The potential of FO in wastewater treatment and desalination have been extensively studied; however, regeneration of the draw solution (thereby generating clean water) requires application of an energy-intensive process step like reverse osmosis (RO). In this study, the potential of applying FO for direct water recirculation from diluted fermentation effluent to concentrated feedstock, without the need for an energy-intensive regeneration step (e.g. RO), has been investigated. Butanol production during crude glycerol fermentation by Clostridium pasteurianum, has been selected as a model process and the effect of cross-flow velocity and the dilution of draw solution on the water flux during short-term experiments (200 min), were investigated. Statistical analysis revealed that the dilution of the draw solution is the most influential factor for the water flux. Subsequent modelling of an integrated FO-fermentation process, showed that water recoveries could lead to substantial financial benefits, although the integrated FO-fermentation process demonstrated lower water flux than expected. FTIR analyses of the membrane surface implied that the decrease in water flux was due to the presence of proteins, polysaccharides and other extracellular polymeric substances on the membrane active layer, indicating the presence of a fouling layer. Based on these findings, possible fouling alleviation strategies and future research directions are discussed and proposed.

Homology to peptide pattern for annotation of carbohydrate-active enzymes and prediction of function.

BACKGROUND: Carbohydrate-active enzymes are found in all organisms and participate in key biological processes. These enzymes are classified in 274 families in the CAZy database but the sequence diversity within each family makes it a major task to identify new family members and to provide basis for prediction of enzyme function. A fast and reliable method for de novo annotation of genes encoding carbohydrate-active enzymes is to identify conserved peptides in the curated enzyme families followed by matching of the conserved peptides to the sequence of interest as demonstrated for the glycosyl hydrolase and the lytic polysaccharide monooxygenase families. This approach not only assigns the enzymes to families but also provides functional prediction of the enzymes with high accuracy. RESULTS: We identified conserved peptides for all enzyme families in the CAZy database with Peptide Pattern Recognition. The conserved peptides were matched to protein sequence for de novo annotation and functional prediction of carbohydrate-active enzymes with the Hotpep method. Annotation of protein sequences from 12 bacterial and 16 fungal genomes to families with Hotpep had an accuracy of 0.84 (measured as F1-score) compared to semiautomatic annotation by the CAZy database whereas the dbCAN HMM-based method had an accuracy of 0.77 with optimized parameters. Furthermore, Hotpep provided a functional prediction with 86% accuracy for the annotated genes. Hotpep is available as a stand-alone application for MS Windows. CONCLUSIONS: Hotpep is a state-of-the-art method for automatic annotation and functional prediction of carbohydrate-active enzymes.

Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells.

Correction of patient-specific induced pluripotent stem cells (iPSC) upon gene delivery through retroviral vectors offers new treatment perspectives for monogenetic diseases. Gene-modified iPSC clones can be screened for safe integration sites and differentiated into transplantable cells of interest. However, the current bottleneck is epigenetic vector silencing. In order to identify the most suitable retroviral expression system in iPSC, we systematically compared vectors from different retroviral genera, different promoters and their combination with ubiquitous chromatin opening elements (UCOE), and several envelope pseudotypes. Lentiviral vectors (LV) pseudotyped with vesicular stomatitis virus glycoprotein were superior to gammaretroviral and alpharetroviral vectors and other envelopes tested. The elongation factor 1α short (EFS) promoter mediated the most robust expression, whereas expression levels were lower from the potent but more silencing-prone spleen focus forming virus (SFFV) promoter. Both full-length (A2UCOE) and minimal (CBX3) UCOE juxtaposed to two physiological and one viral promoter reduced transgene silencing with equal efficiency. However, a promoter-specific decline in expression levels was not entirely prevented. Upon differentiation of transgene-positive iPSC into endothelial cells, A2UCOE.EFS and CBX3.EFS vectors maintained highest transgene expression in a larger fraction of cells as compared with all other constructs tested here. The function of UCOE diminished, but did not fully counteract, vector silencing and possibilities for improvements remain. Nevertheless, the CBX3.EFS in a LV background exhibited the most promising promoter and vector configuration for both high titer production and long-term genetic modification of human iPSC and their progeny.

BMI loci and longitudinal BMI from adolescence to young adulthood in an ethnically diverse cohort.

OBJECTIVE: The association of obesity susceptibility variants with change in body mass index (BMI) across the life course is not well understood. SUBJECTS: In ancestry-stratified models of 5962 European American (EA), 2080 African American (AA) and 1582 Hispanic American (HA) individuals from the National Longitudinal Study of Adolescent to Adult Health (Add Health), we examined associations between 34 obesity single-nucleotide polymorphisms (SNPs) with per year change in BMI, measured by the slope from a growth-curve analysis of two or more BMI measurements between adolescence and young adulthood. For SNPs nominally associated with BMI change (P<0.05), we interrogated age differences within data collection Wave and time differences between age categories that overlapped between Waves. RESULTS: We found SNPs in/near FTO, MC4R, MTCH2, TFAP2B, SEC16B and TMEM18 were significantly associated (P<0.0015≈0.05/34) with BMI change in EA and the ancestry-combined meta-analysis. rs9939609 in FTO met genome-wide significance at P<5e-08 in the EA and ancestry-combined analysis, respectively [Beta(se)=0.025(0.004);Beta(se)=0.021(0.003)]. No SNPs were significant after Bonferroni correction in AA or HA, although five SNPs in AA and four SNPs in HA were nominally significant (P<0.05). In EA and the ancestry-combined meta-analysis, rs3817334 near MTCH2 showed larger effects in younger respondents, whereas rs987237 near TFAP2B, showed larger effects in older respondents across all Waves. Differences in effect estimates across time for MTCH2 and TFAP2B are suggestive of either era or cohort effects. CONCLUSION: The observed association between variants in/near FTO, MC4R, MTCH2, TFAP2B, SEC16B and TMEM18 with change in BMI from adolescence to young adulthood suggest that the genetic effect of BMI loci varies over time in a complex manner, highlighting the importance of investigating loci influencing obesity risk across the life course.

Ana o 3-specific IgE is a good predictor for clinically relevant cashew allergy in children.

INTRODUCTION: Component-resolved diagnostics using specific IgE to 2 S albumins has shown to be a valuable new option in diagnostic procedure. Ana o 3 is a 2 S albumin from cashew. The aim of this study was to investigate the role of Ana o 3-specific serum IgE in the diagnosis of cashew allergy and to identify cut-off levels to replace oral food challenges. Moreover, the value of additional determination of total IgE has been investigated. METHODS: In a multicentre study, we analysed specific IgE to cashew extract and Ana o 3 as well as total IgE in children with suspected cashew allergy using the ImmunoCAP-FEIA and a standardized diagnostic procedure including oral challenges where indicated. RESULTS: A total of 61 patients were included in the study. Forty-two were allergic to cashew, and 19 were tolerant. In receiver operating curves, Ana o 3 discriminates between allergic and tolerant children better than cashew-specific IgE with an area under the curve of 0.94 vs 0.78. The ratio of Ana o 3-specific IgE to total IgE did not further improve the diagnostic procedure. Probability curves for Ana o 3-specific IgE have been calculated, and a 95% probability could be estimated at 2.0 kU/l. CONCLUSION: Specific IgE to Ana o 3 is a valuable tool for the diagnosis of cashew allergy. Considering its positive predictive value, it might allow to make a considerable number of oral challenges superfluous.

Association between gestational diabetes mellitus exposure and childhood adiposity is not substantially explained by offspring genetic risk of obesity.

AIM: To examine the extent to which offspring obesity-associated genetic risk explains the association between gestational diabetes mellitus and childhood adiposity. METHODS: We studied 282 children aged 7-12 years who were enrolled in the Exploring Perinatal Outcomes in Children Study. A genetic risk score for BMI was calculated as the count of 91 established BMI-raising risk alleles. Multivariable linear and logistic regression models were used to estimate associations between the offspring genetic risk score and exposure to gestational diabetes and childhood adiposity (BMI and waist circumference), adjusting for clinical and demographic covariates. The contribution of offspring genetic risk to associations between maternal gestational diabetes and childhood outcomes was estimated by comparing the regression coefficients for the gestational diabetes variable in models with and without the genetic risk score. RESULTS: The offspring BMI genetic risk score was associated with childhood BMI (P = 0.006) and waist circumference (P = 0.02), and marginally with gestational diabetes (P = 0.05). Offspring BMI genetic risk did not contribute significantly to associations between gestational diabetes and childhood BMI [7.7% (95% CI -3.3, 18.8)] or waist circumference [5.8% (95% CI -3.1, 14.8); P = 0.2 for both]. CONCLUSIONS: Offspring obesity genetic risk does not explain a significant proportion of the association between gestational diabetes exposure and childhood adiposity. The association between gestational diabetes and childhood adiposity is probably explained through alternative pathways, including direct intrauterine effects or a shared postnatal environment.

A de novo frameshift in HNRNPK causing a Kabuki-like syndrome with nodular heterotopia.

Kabuki syndrome is a heterogeneous condition characterized by distinctive facial features, intellectual disability, growth retardation, skeletal abnormalities and a range of organ malformations. Although at least two major causative genes have been identified, these do not explain all cases. Here we describe a patient with a complex Kabuki-like syndrome that included nodular heterotopia, in whom testing for several single-gene disorders had proved negative. Exome sequencing uncovered a de novo c.931_932insTT variant in HNRNPK (heterogeneous nuclear ribonucleoprotein K). Although this variant was identified in March 2012, its clinical relevance could only be confirmed following the August 2015 publication of two cases with HNRNPK mutations and an overlapping phenotype that included intellectual disability, distinctive facial dysmorphism and skeletal/connective tissue abnormalities. Whilst we had attempted (unsuccessfully) to identify additional cases through existing collaborators, the two published cases were 'matched' using GeneMatcher, a web-based tool for connecting researchers and clinicians working on identical genes. Our report therefore exemplifies the importance of such online tools in clinical genetics research and the benefits of periodically reviewing cases with variants of unproven significance. Our study also suggests that loss of function variants in HNRNPK should be considered as a molecular basis for patients with Kabuki-like syndrome.

A novel high-temperature combustion interface for compound-specific stable isotope analysis of carbon and nitrogen via high-performance liquid chromatography/isotope ratio mass spectrometry.

RATIONALE: In aqueous samples compound-specific stable isotope analysis (CSIA) plays an important role. No direct method (without sample preparation) for stable nitrogen isotope analysis (δ(15) N SIA) of non-volatile compounds is known yet. The development of a novel HPLC/IRMS interface based on high-temperature combustion (HTC) for both δ(13) C and δ(15) N CSIA and its proof of principle are described in this study. METHODS: To hyphenate high-performance liquid chromatography (HPLC) with isotope ratio mass spectrometry (IRMS) a modified high-temperature combustion total organic carbon analyzer (HTC TOC) was used. A system to handle a continuously large amount of water (three-step drying system), favorable carrier and reaction gas mix and flow, an efficient high-temperature-based oxidation and subsequent reduction system and a collimated beam transfer system were the main requirements to achieve the necessary performance. RESULTS: The proof of principle with caffeine solutions of the system succeeded. In this initial testing, both δ(13) C and δ(15) N values of tested compounds were determined with precision and trueness of ≤0.5 ‰. Further tests resulted in lower working limit values of 3.5 µgC for δ(13) C SIA and 20 µgN for δ(15) N SIA, considering an accuracy of ±0.5 ‰ as acceptable. CONCLUSIONS: The development of a novel HPLC/IRMS interface resulted in the first system reported to be suitable for both δ(13) C and δ(15) N direct CSIA of non-volatile compounds. This highly efficient system will probably open up new possibilities in SIA-based research fields. Copyright © 2016 John Wiley & Sons, Ltd.

A novel tool for stable nitrogen isotope analysis in aqueous samples.

RATIONALE: Bulk stable isotope analysis (BSIA) of dissolved matter (e.g. dissolved organic carbon, total nitrogen bound (TNb ), etc.) is of particular importance since this pool is a prime conduit in the cycling of N and C. Studying the two elemental pools is of importance, as transformation and transport processes of N and C are inextricably linked in all biologically mediated systems. No system able to analyze natural abundance stable carbon and nitrogen isotope composition in aqueous samples (without offline sample preparation) and simultaneously has been reported so far. Extension of the high-temperature combustion (HTC) system, to be capable of measuring TNb stable nitrogen isotope composition, is described in this study. METHODS: To extend the TOC analyzer to be capable of measuring TNb , modifications from the HTC high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS) interface were implemented and expanded. A reduction reactor for conversion of NOx into N2 was implemented into the new developed system. The extension addresses mainly the development of the focusing unit for nitrogen and a degassing device for online separation of TNb from molecular nitrogen (N2 ) prior to injection. RESULTS: The proof of principle of the system was demonstrated with different compound solutions. In this initial testing, the δ15 NAIR-N2 values of the tested compounds were determined with precision and trueness of typically ≤0.5‰. Good results (u ≤ 0.5‰) could be achieved down to a TNb concentration of 40 mgN/L and acceptable results (u ≤ 1.0‰) down to 5 mgN/L. In addition, the development resulted in the first system reported to be suitable for simultaneous and direct δ13 C and δ15 N BSIA of aqueous samples. CONCLUSIONS: The development resulted in the first system shown to be suitable for both δ13 C and δ15 N direct BSIA in aqueous samples. This system could open up new possibilities in SIA-based research fields. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota.

In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function.