A Novel Approach for Single Step Analyte Fractionation of Raw Milk Prior to Antbiotica Residue Trace Analysis as an Alternative to QuEchERS Based Extraction.

BACKGROUND: Antibiotic residues in milk are a well-known hazard in the dairy food chain. Detection methods for these residues, such as non-specific microbiological inhibitor tests or group-specific receptor tests, respectively, are relatively inexpensive, easy to use and widely applied to ensure food safety. In contrast, specific detection by liquid chromatography tandem mass spectrometry (LC-MS/MS) - although a critical, complimentary method to confirm the results of non-specific testing-is relatively costly, time consuming and laborious. Furthermore, sample processing before LC-MS/MS analysis requires unique preparation procedures for different groups of antibiotic compounds. OBJECTIVE: To simplify and speed up specific antibiotic residue detection, a low-cost, passive and single-step method to fractionate analytes in raw milk was developed. METHODS: Untreated raw milk was fractionated into its water and fat/protein phases using a FraMiTrACR® AB fractionation unit. The water fraction was then analyzed by LC-MS/MS. The analyte fractionation method was evaluated against a QuEChERS based method for sample preparation. RESULTS: Our method allows qualitative and quantitative detection of substances from the Penicillin, Cephalosporin, Macrolide, Lincosamide, Sulfonamide, Tetracycline and Fluoroquinolone groups of antibiotics. Detection limits are below the legally prescribed maximum residue levels, allowing reliable, specific and rapid validation of a positive result in non-specific microbiological inhibitor tests. CONCLUSION: Analyte fractionation by FraMiTrACR® AB is a faster alternative to QuEChERS based sample preparation for the detection of antibiotic substances in milk. HIGHLIGHT: This method describes a low-cost, environmentally friendly, passive and single-step milk analyte fractionation. As an alternative to QuEChERS based preparation, this fractionation method simplifies and speeds up the process for specific antibiotic residue detection.

Impact on birth weight of maternal smoking throughout pregnancy mediated by DNA methylation.

BACKGROUND: Cigarette smoking has severe adverse health consequences in adults and in the offspring of mothers who smoke during pregnancy. One of the most widely reported effects of smoking during pregnancy is reduced birth weight which is in turn associated with chronic disease in adulthood. Epigenome-wide association studies have revealed that smokers show a characteristic "smoking methylation pattern", and recent authors have proposed that DNA methylation mediates the impact of maternal smoking on birth weight. The aims of the present study were to replicate previous reports that methylation mediates the effect of maternal smoking on birth weight, and for the first time to investigate whether the observed mediation effects are sex-specific in order to account for known sex-specific differences in methylation levels. METHODS: Methylation levels in the cord blood of 313 newborns were determined using the Illumina HumanMethylation450K Beadchip. A total of 5,527 CpG sites selected on the basis of evidence from the literature were tested. To determine whether the observed association between maternal smoking and birth weight was attributable to methylation, mediation analyses were performed for significant CpG sites. Separate analyses were then performed in males and females. RESULTS: Following quality control, 282 newborns eventually remained in the analysis. A total of 25 mothers had smoked consistently throughout the pregnancy. The birthweigt of newborns whose mothers had smoked throughout pregnancy was reduced by >200g. After correction for multiple testing, 30 CpGs showed differential methylation in the maternal smoking subgroup including top "smoking methylation pattern" genes AHRR, MYO1G, GFI1, CYP1A1, and CNTNAP2. The effect of maternal smoking on birth weight was partly mediated by the methylation of cg25325512 (PIM1); cg25949550 (CNTNAP2); and cg08699196 (ITGB7). Sex-specific analyses revealed a mediating effect for cg25949550 (CNTNAP2) in male newborns. CONCLUSION: The present data replicate previous findings that methylation can mediate the effect of maternal smoking on birth weight. The analysis of sex-dependent mediation effects suggests that the sex of the newborn may have an influence. Larger studies are warranted to investigate the role of both the identified differentially methylated loci and the sex of the newborn in mediating the association between maternal smoking during pregnancy and birth weight.

Expert and self-assessment of lifetime symptoms and diagnosis of major depressive disorder in large-scale genetic studies in the general population: comparison of a clinical interview and a self-administered checklist.

Major depression disorder (MDD) is a complex neuropsychiatric disorder and an increasing number of genetic risk variants are being identified. Investigation of their influence in the general population requires accurate and efficient assessment of depressive symptoms. Here, clinical interviews conducted by clinicians are the gold standard. We investigated whether valid and reliable clinical phenotypes can be obtained efficiently using self-administered instruments. Lifetime depressive symptoms and lifetime MDD diagnosis were assessed in 464 population-based individuals using a clinical interview and a structured, self-administered checklist. Analyses were carried out of the following: (i) intraclass correlations (ICC) between checklist and interview; (ii) sensitivity/specificity of the checklist; and (iii) the association of interview and checklist with a positive family history of MDD (FH-MDD+). The correspondence of the self-administered checklist with the clinical interview was good for most depressive symptoms (ICC=0.60-0.80) and moderate for MDD diagnosis (ICC=0.45). With the consecutive inclusion of MDD diagnostic criteria, sensitivity decreased from 0.67 to 0.46, whereas specificity remained high (0.95). For checklist and interview, strong associations were found between FH-MDD+ and most depressive symptoms and MDD diagnosis (all odds ratio≥1.83). The self-administered checklist showed high reliability for both the assessment of lifetime depressive symptoms and screening for individuals with no lifetime diagnosis of MDD. However, attention is warranted when the aim is to identify MDD cases. The positive association between depressive symptomatology and FH-MDD+ indicates the usefulness of both instruments to assess patients in genetic studies. Our data suggest that the more time-efficient and cost-efficient self-administered instruments also allow for the assessment of depressive symptoms accurate enough to investigate the influence of MDD genetic risk variants in the general population.

Investigation of SHANK3 in schizophrenia.

The postsynaptic scaffolding protein SHANK3 is essential for the normal function of glutamatergic synapses in the brain. Emerging evidence suggests that impaired plasticity of glutamatergic synapses contributes to the pathology of schizophrenia (SCZ). To investigate whether variants in the SHANK3 gene contribute to the etiology of SCZ, we sequenced SHANK3 in 500 affected individuals (cohort C1). In total, we identified 48 variants and compared them to European controls from the 1000 Genomes Project and the Exome Variant Server. Five variants showed significant differences in frequencies between patients and controls. We were able to follow three of them up in an independent cohort (C2) comprising 993 SCZ patients and 932 German controls. We could not confirm an association for three of these variants (rs140201628, rs1557620, and rs61729471). Two rare variants with predicted functional relevance were identified in further SCZ individuals of cohort C1: c.3032G>T (p.G1011V) and c.*27C>T. The latter variant was found in one additional SCZ individual and the p.G1011V variant was identified in two additional SCZ individuals from cohort C2. The p.G1011V variant was the most interesting variant in our study; together with previous studies this variant has been identified in 4 out of 1,524 SCZ patients and in 4 out of 2,147 individuals with autism spectrum disorder (ASD), but not in 2468 European Sanger-sequenced controls. Therefore, we consider this variant a promising candidate variant for follow-up studies in larger samples and functional investigations. © 2017 Wiley Periodicals, Inc.

Association between neuropeptide Y receptor Y2 promoter variant rs6857715 and major depressive disorder.

Stress increases the risk for major depressive disorder (MDD), overeating, and alcohol dependence (AD). The neuropeptide Y system is one of the best-known modulators of the stress response, and some of its effects are mediated through the neuropeptide Y receptor Y2 (NPY2R). The functional NPY2R variant rs6857715 (C-599T) has been implicated in both obesity and AD, but with opposing alleles. The present study explored whether rs6857715 is also associated with MDD. Analysis of the overall sample (595 MDD cases; 1295 controls) showed an association with the AD risk allele C [P=0.020, odds ratio (OR) (C-allele)=1.18]. The association remained significant after excluding MDD patients with AD/alcohol abuse [P=0.038, OR (C-allele)=1.18]; increased weight/appetite [P=0.006, OR (C-allele)=1.23]; or both [P=0.008, OR (C-allele)=1.25]. The present findings suggest that the NPY2R rs6857715 C-allele makes a genuine contribution toward MDD.

Genome-wide Association for Major Depression Through Age at Onset Stratification: Major Depressive Disorder Working Group of the Psychiatric Genomics Consortium.

BACKGROUND: Major depressive disorder (MDD) is a disabling mood disorder, and despite a known heritable component, a large meta-analysis of genome-wide association studies revealed no replicable genetic risk variants. Given prior evidence of heterogeneity by age at onset in MDD, we tested whether genome-wide significant risk variants for MDD could be identified in cases subdivided by age at onset. METHODS: Discovery case-control genome-wide association studies were performed where cases were stratified using increasing/decreasing age-at-onset cutoffs; significant single nucleotide polymorphisms were tested in nine independent replication samples, giving a total sample of 22,158 cases and 133,749 control subjects for subsetting. Polygenic score analysis was used to examine whether differences in shared genetic risk exists between earlier and adult-onset MDD with commonly comorbid disorders of schizophrenia, bipolar disorder, Alzheimer's disease, and coronary artery disease. RESULTS: We identified one replicated genome-wide significant locus associated with adult-onset (>27 years) MDD (rs7647854, odds ratio: 1.16, 95% confidence interval: 1.11-1.21, p = 5.2 × 10-11). Using polygenic score analyses, we show that earlier-onset MDD is genetically more similar to schizophrenia and bipolar disorder than adult-onset MDD. CONCLUSIONS: We demonstrate that using additional phenotype data previously collected by genetic studies to tackle phenotypic heterogeneity in MDD can successfully lead to the discovery of genetic risk factor despite reduced sample size. Furthermore, our results suggest that the genetic susceptibility to MDD differs between adult- and earlier-onset MDD, with earlier-onset cases having a greater genetic overlap with schizophrenia and bipolar disorder.

Rapporteur summaries of plenary, symposia, and oral sessions from the XXIIIrd World Congress of Psychiatric Genetics Meeting in Toronto, Canada, 16-20 October 2015.

The XXIIIrd World Congress of Psychiatric Genetics meeting, sponsored by the International Society of Psychiatric Genetics, was held in Toronto, ON, Canada, on 16-20 October 2015. Approximately 700 participants attended to discuss the latest state-of-the-art findings in this rapidly advancing and evolving field. The following report was written by trainee travel awardees. Each was assigned one session as a rapporteur. This manuscript represents the highlights and topics that were covered in the plenary sessions, symposia, and oral sessions during the conference, and contains major notable and new findings.

Analysis of Rare Variants in the Alcohol Dependence Candidate Gene GATA4.

BACKGROUND: Common variants in the gene GATA binding protein 4 (GATA4) show association with alcohol dependence (AD). The aim of this study was to identify rare variants in GATA4 in order to elucidate the role of this gene in AD susceptibility. Identification of rare variants may provide a more complete picture of the allelic architecture at this risk locus. METHODS: Sanger sequencing of all 6 coding exons of GATA4 was performed in 528 patients and 517 controls. Four in silico prediction tools were used to determine the effect of a DNA variant on the amino acid sequence and protein function. Five variants were included in the replication step. Of these, 4 were successfully genotyped in our replication cohort of 655 patients and 1,501 controls. All patients fulfilled DSM-IV criteria for AD, and all individuals were of German descent. RESULTS: In the discovery step, 19 different heterozygous variants were identified. Four patient-specific and potentially functionally relevant variants were followed up. Only the variant S379S (c.1137C>T) remained patient specific (1/1,166 patients vs. 0/1,997 controls). None of the variants showed a statistically significant association with AD. CONCLUSIONS: The present study elucidated the role of GATA4 in AD susceptibility by identifying rare variants via Sanger sequencing and subsequent replication. Although novel patient-specific rare variants of GATA4 were identified, none received support in the independent replication step. However, given previous robust findings of association with common variants, GATA4 remains a promising candidate gene for AD.

Perceived stress and hair cortisol: Differences in bipolar disorder and schizophrenia.

INTRODUCTION: Bipolar disorder (BD) and schizophrenia (SCZ) are psychiatric disorders with shared and distinct clinical and genetic features. In both disorders, stress increases the risk for onset or relapse and dysregulation of the hypothalamus-pituitary-adrenal (HPA) axis has been reported. The latter is frequently investigated by measuring changes in the hormonal end product of the HPA axis, i.e., the glucocorticoid cortisol, whose concentration exhibits diurnal variation. The analysis of hair cortisol concentration (HCC) is a new method, which allows assessment of cumulative cortisol secretion over the preceding three months. AIMS: To explore whether perceived stress and HCC: (i) differ between BD patients, SCZ patients, and controls; (ii) change over disease course; and iii) are associated with an increased genetic risk for BD or SCZ. METHODS: 159 SCZ patients, 61 BD patients and 82 controls were included. Assessment included psychopathology, perceived stress, and HCC. Inpatients with an acute episode (38 BD and 77 SCZ) were assessed shortly after admission to hospital and at 3 and 6 months follow-up. Outpatients in remission and controls were assessed at one time point only. Polygenic risk scores for BD and SCZ were calculated based on results of the Psychiatric Genomic Consortium. RESULTS: (i) Perceived stress was higher in BD and SCZ patients compared to controls (p<0.02), and was lower in outpatients in remission compared to inpatients on admission. HCC was higher in BD patients compared to SCZ patients and controls (p<0.02), and higher in inpatients on admission than in outpatients in remission (p=0.0012). In BD patients (r=0.29; p=0.033) and SCZ patients (r=0.20; p=0.024) manic symptoms were correlated with HCC. (ii) In both BD and SCZ inpatients, perceived stress decreased over the 6 month study period (p=0.048), while HCC did not change significantly over the 6 month study period. (iii) In controls, but not in the patient groups, the genetic risk score for BD was associated with HCC (r=0.28, p=0.023). CONCLUSIONS: While our results are consistent with previous reports of increased perceived stress in BD and SCZ, they suggest differential involvement of the HPA axis in the two disorders. The genetic study supports this latter finding, and suggests that this effect is present below the threshold of manifest disorder.

New-born females show higher stress- and genotype-independent methylation of SLC6A4 than males.

BACKGROUND: Research has demonstrated an association between exposure to early life stress and an increased risk of psychiatric disorders in later life, in particular depression. However, the mechanism through which early life stress contributes to disease development remains unclear. Previous studies have reported an association between early life stress and altered methylation of the serotonin transporter gene (SLC6A4), a key candidate gene for several psychiatric disorders. These differences in methylation are influenced by sex and genetic variation in the SLC6A4-linked polymorphic region (5-HTTLPR). Furthermore, one study indicated that stress during pregnancy may induce methylation changes in SLC6A4 in the newborn. The present study is the first to investigate whether early life stress during pregnancy impacts on SLC6A4 methylation in newborns, taking into account the influence of genetic variation and sex. METHODS: Cord blood was obtained from newborns with high (n = 45) or low (n = 45) early life stress, defined as maternal stress during pregnancy. The effect on methylation of early life stress, 5-HTTLPR genotype, and sex was assessed at four cytosin-phosphate-guanine dinucleotide (CpG) sites in the promoter associated CpG island north shore (CpG 1 to 4). The epigenetic analyses focused on these CpG sites, since research has shown that CpG island shore methylation has functional consequences. RESULTS: Significant sex-specific methylation was observed, with females displaying higher methylation levels than males (p < 0.001). Importantly, this effect was influenced by neither early life stress nor genotype. CONCLUSIONS: The present data suggest that sex-specific methylation of SLC6A4 is present at birth, and is independent of early life stress and 5-HTTLPR genotype. This may contribute to the sex-specific prevalence of depression.

XRCC5 as a risk gene for alcohol dependence: evidence from a genome-wide gene-set-based analysis and follow-up studies in Drosophila and humans.

Genetic factors have as large role as environmental factors in the etiology of alcohol dependence (AD). Although genome-wide association studies (GWAS) enable systematic searches for loci not hitherto implicated in the etiology of AD, many true findings may be missed owing to correction for multiple testing. The aim of the present study was to circumvent this limitation by searching for biological system-level differences, and then following up these findings in humans and animals. Gene-set-based analysis of GWAS data from 1333 cases and 2168 controls identified 19 significantly associated gene-sets, of which 5 could be replicated in an independent sample. Clustered in these gene-sets were novel and previously identified susceptibility genes. The most frequently present gene, ie in 6 out of 19 gene-sets, was X-ray repair complementing defective repair in Chinese hamster cells 5 (XRCC5). Previous human and animal studies have implicated XRCC5 in alcohol sensitivity. This phenotype is inversely correlated with the development of AD, presumably as more alcohol is required to achieve the desired effects. In the present study, the functional role of XRCC5 in AD was further validated in animals and humans. Drosophila mutants with reduced function of Ku80-the homolog of mammalian XRCC5-due to RNAi silencing showed reduced sensitivity to ethanol. In humans with free access to intravenous ethanol self-administration in the laboratory, the maximum achieved blood alcohol concentration was influenced in an allele-dose-dependent manner by genetic variation in XRCC5. In conclusion, our convergent approach identified new candidates and generated independent evidence for the involvement of XRCC5 in alcohol dependence.

BRENDA in 2013: integrated reactions, kinetic data, enzyme function data, improved disease classification: new options and contents in BRENDA.

The BRENDA (BRaunschweig ENzyme DAtabase) enzyme portal (http://www.brenda-enzymes.org) is the main information system of functional biochemical and molecular enzyme data and provides access to seven interconnected databases. BRENDA contains 2.7 million manually annotated data on enzyme occurrence, function, kinetics and molecular properties. Each entry is connected to a reference and the source organism. Enzyme ligands are stored with their structures and can be accessed via their names, synonyms or via a structure search. FRENDA (Full Reference ENzyme DAta) and AMENDA (Automatic Mining of ENzyme DAta) are based on text mining methods and represent a complete survey of PubMed abstracts with information on enzymes in different organisms, tissues or organelles. The supplemental database DRENDA provides more than 910 000 new EC number-disease relations in more than 510 000 references from automatic search and a classification of enzyme-disease-related information. KENDA (Kinetic ENzyme DAta), a new amendment extracts and displays kinetic values from PubMed abstracts. The integration of the EnzymeDetector offers an automatic comparison, evaluation and prediction of enzyme function annotations for prokaryotic genomes. The biochemical reaction database BKM-react contains non-redundant enzyme-catalysed and spontaneous reactions and was developed to facilitate and accelerate the construction of biochemical models.

BKM-react, an integrated biochemical reaction database.

BACKGROUND: The systematic, complete and correct reconstruction of genome-scale metabolic networks or metabolic pathways is one of the most challenging tasks in systems biology research. An essential requirement is the access to the complete biochemical knowledge - especially on the biochemical reactions. This knowledge is extracted from the scientific literature and collected in biological databases. Since the available databases differ in the number of biochemical reactions and the annotation of the reactions, an integrated knowledge resource would be of great value. RESULTS: We developed a comprehensive non-redundant reaction database containing known enzyme-catalyzed and spontaneous reactions. Currently, it comprises 18,172 unique biochemical reactions. As source databases the biochemical databases BRENDA, KEGG, and MetaCyc were used. Reactions of these databases were matched and integrated by aligning substrates and products. For the latter a two-step comparison using their structures (via InChIs) and names was performed. Each biochemical reaction given as a reaction equation occurring in at least one of the databases was included. CONCLUSIONS: An integrated non-redundant reaction database has been developed and is made available to users. The database can significantly facilitate and accelerate the construction of accurate biochemical models.