A rapid and convenient sample preparation method for the analysis of cannabinoids in oral fluid samples.

Cannabis is the world's most used illegal drug. The main psychoactive component of cannabis is Δ9-tetrahydrocannabinol (THC). To aid the identification of cannabis-impaired individuals, a simple but effective workflow for reliable quantification of THC and its metabolites in oral fluid samples collected with the Greiner Bio-One Saliva Collection System is presented. Sampling involves rinsing the oral cavity with an extraction solution containing a citrate buffer stimulating salivary flow. Sample processing targeted the cannabinoid fraction interacting with proteins and other insoluble constituents that can be separated by centrifugation. Approximately 50% of the total amount of cannabinoids included in the oral fluid was recovered from the obtained pellet by extraction with acetonitrile. Liquid chromatography-tandem mass spectrometry was used for cannabinoid quantification. Fitness of the developed workflow for application in forensic and clinical cannabis testing was demonstrated by evaluating multiple performance parameters, including selectivity, linearity, limits of quantification (LOQs), accuracy, precision, matrix effects, extraction recoveries, process efficiencies and stability. Furthermore, sensitivity and specificity of the developed oral fluid-based cannabis test was demonstrated by analysing 195 samples collected either from opioid addicts or persons suspected of driving under the influence of drugs. The accuracy of identifying a person with the presence of THC in blood was found to be 97.9%.

Ethnic Identity and Genome Wide Runs of Homozygosity.

It is long known that inbreeding increases the detrimental effects of recessive sequence variants in "Runs of Homozygosity" (ROHs). However, although the phenotypic association of ROH has been investigated for a variety of traits, the statistical power of the results often remains limited as a sufficiently high number of cases are available for only a restricted number of traits. In the present study, we aim to analyze the association of runs of homozygosity with the trait "in-group ethnic favoritism". This analysis assumes that if ethnic identity is important for an individual, that individual may tend to marry more frequently within their own group and therefore ROH are expected to increase. We hypothesize that an attitude preferring one's own ethnic group may be associated with a stronger tendency of inbreeding and, as a result, with more and longer ROHs. Accordingly, we investigated the association between the attitude to someone's own ethnicity and ROH, using the Wisconsin Longitudinal data (WLS, total N ~ 9000) as discovery data set and the Brisbane Twin data as replication data set (N ~ 8000). We find that both the number as well as the total length of homozygous segments are significantly positively associated with "in-group ethnic favoritism", independent of the method used for ROH calculation.

Factors affecting attitudes toward migrants-An evolutionary approach.

OBJECTIVE: To understand migration from an evolutionary perspective, this phenomenon has so far been mainly investigated in animal species. We therefore aim to investigate the potential evolutionary roots of attitudes toward migrants in humans. METHODS: We used data from the European Social Survey (n = 83 734), analyzing attitudes toward migrants by performing ordinal mixed models. RESULTS: We found that men have a more restrictive attitude toward migration than women, which increases with age and is stronger with a child in the household. Attitude toward migrants is also more skeptical if migrants have a different ethnicity and are from poorer countries. Increasing education and religiousness are associated with a more positive attitude toward migrants, particularly toward migrants of different ethnicity and from poorer countries. DISCUSSION: Although migration flows are a hallmark of the human species, previous findings suggest that (pre-)historic migration flows were at times accompanied by conflict and violence, while at the same time, they insured survival by allowing cultural exchange and the avoidance of inbreeding. Accordingly, we assume that contemporary attitudes toward migration are rooted in our evolutionary past. We discuss the respective behavioral patterns from an evolutionary perspective, arguing that both-a negative attitude as well as openness-make sense.

Fertility Outcomes, Heritability and Genomic Associations of In-Group Preference and In-Group Marriage.

Using data from the Midlife Development in the USA (MIDUS) sample (3070 men and 3182 women) and the Wisconsin Longitudinal Study (WLS; 2240 men and 2346 women), we aimed to investigate whether religious, ethnic and racial in-group preferences as well as religious homogamy are associated with reproductive outcome in terms of number of children. Using data from the MIDUS twin sample, we further estimated the inherited genetic component of in-group attitudes. Additionally, we analyzed the association of ∼50 polygenic scores (PGSs) recently published for the WLS study and in-group attitudes as an indicator of potential pleiotropic effects. We found in both samples that, with one exception, religious though not other in-group attitudes are associated with a higher reproductive outcome. Also, religious homogamy is associated with higher average number of children. The inherited component of all in-group attitudes ranges from ∼21% to 45% (MIDUS twin sample). PGSs associated with religious behavior are significantly positively associated with religious in-group attitudes as well as family attitudes. Further associations are found with PGS on life satisfaction (work) and, negatively, with PGS for any sort of addiction (smoking, alcohol and cannabis use), indicating pleiotropy. We conclude that the positive association between religious in-group attitudes as well as religious homogamy and reproductive outcome may indicate selective forces on religious in-group behavior. As all investigated in-group attitudes, however, have a substantial inherited component, we further speculate that potential previous reproductive benefits of racial and ethnic in-group preferences, if they ever existed, might have been substituted by religious in-group preferences.

Do birds of a feather flock together? Factors for religious heterogamy.

Social cohesion - particularly with regard to the integration of migrants - is primarily measured in terms of education, labour market participation, unemployment, income levels and poverty. When seen from a historical long-term perspective (considering the migrations of Homo sapiens in the past 300,000 years) admixture merged members of diverse groups and forged - in addition to social ties - 'strong biological ties' of kinship, proposing that religious heterogamy is a long-term layer of social cohesion. Accordingly, this study investigated, on the basis of more than 600,000 men and women aged 26-35 years from Austria 2001, Germany (West) 1987, Ireland 2011, Portugal 2011, Romania 2011 and Switzerland 2000, which demographic characteristics foster religious heterogamy, controlling for various confounding factors using linear mixed modelling. By far the most important factor explaining religious heterogamy was the share of adherents to an individual's religious group in their area of residence. It can be concluded that the rate of intermarriage declines with the increasing size of an individual's religious group in their area of residence. From a long-term perspective the lack of familial ties (and conjoint offspring) between religious groups could lead to a lack of social cohesion.

Whole Transcriptome Screening Reveals Myelination Deficits in Dysplastic Human Temporal Neocortex.

Focal cortical dysplasias (FCDs) are local malformations of the human neocortex with strong epileptogenic potential. To investigate the underlying pathomechanisms, we performed a whole human transcriptome screening to compare the gene expression pattern of dysplastic versus nondysplastic temporal neocortex. Tissue obtained from FCD IIIa cases (mean age 20.5 years) who had undergone surgical treatment, due to intractable epilepsy, was compared with nondysplastic specimens (mean age 19.9 years) by means of Affymetrix arrays covering 28 869 genes. We found 211 differentially expressed genes (DEX) among which mainly genes important for oligodendrocyte differentiation and myelination were downregulated in FCD IIIa. These findings were confirmed as functionally important by Database for Annotation, Visualization, and Integrated Discovery (DAVID) analysis. The reduced expression of myelin-associated transcripts was confirmed for FCD Ia, IIa, and IIIa by real-time RT-qPCR. In addition, we found that the density of myelin basic protein mRNA-expressing oligodendrocytes and of 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive myelin fibers was significantly reduced in dysplastic cortex. Moreover, high-resolution confocal imaging and 3D reconstruction revealed that the myelin fiber network was severely disorganized in dysplastic neocortex, indicating a disturbance of myelin sheath formation and maintenance in FCD.

Mutual compensation of the effects of religious and ethnic homogamy on reproduction.

OBJECTIVES: Homogamy, mating based on similarity, has been demonstrated for a great variety of traits such as age, education, religion, and physical and psychological traits. Recently, pro-fertile effects of religious as well as educational homogamy have been reported. We investigate whether ethnic homogamy also has a pro-fertile effect and whether ethnic and religious homogamy interact in their putative effects on reproduction (in terms of average number of offspring). METHODS: We analyzed the association between ethnic as well as religious homogamy and woman's average number of offspring based on census data from ten countries provided by IPUMS international, encompassing a total of 1,485,433 married women aged 46-60 years (who have thus completed or almost completed reproduction) and their spouses. RESULTS: We find a clear pro-fertile but nonadditive effect of both ethnic and religious homogamy, which is most pronounced in the case of double homogamy. Our results further indicate that homogamy for one trait may compensate for heterogamy of the other, albeit countries differ regarding which trait compensates for the other. CONCLUSIONS: We suggest that the interaction between ethnic homogamy, religious homogamy, and reproduction provides an interesting example for gene-culture co-evolution.

MARRIAGE GAP IN CHRISTIANS AND MUSLIMS.

For modern Western societies with a regime of monogamy, it has recently been demonstrated that the socioeconomic status of men is positively associated with being or having been married. This study aims to compare marriage patterns (if a person has been married at least once) for cultures with a tradition of monogamy and polygyny. As no worldwide data on polygyny exist, religion was used as a proxy for monogamy (Christians) vs polygyny (Muslims). The analyses were based on 2000-2011 census data from 39 countries worldwide for 52,339,594 men and women, controlling for sex, sex ratio, age, education, migration within the last 5 years and employment. Overall, a higher proportion of Muslims were married compared with Christians, but the difference in the fraction of married men compared with married women at a certain age (the 'marriage gap') was much more pronounced in Muslims than in Christians, i.e. compared with Christians, a substantially higher proportion of Muslim women than men were married up to the age of approximately 31 years. As expected for a tradition of polygyny, the results indicate that the socioeconomic threshold for entering marriage is higher for Muslim than Christian men, and Muslim women in particular face a negative effect of socioeconomic status on the probability of ever being married. The large 'marriage gap' at a certain age in Muslim societies leads to high numbers of married women and unmarried young men, and may put such polygenic societies under pressure.

The association between religious homogamy and reproduction.

Individuals more strongly affiliated to religion have on average more children than less religious ones. Here, based on census data of 3 658 650 women aged 46-60 years from 32 countries provided by IPUMS International and data from the Wisconsin Longitudinal Study (n = 2400 women, aged 53-57 years), we show that religious homogamy is also associated with higher reproduction in terms of a higher number of children and a lower chance of remaining childless. We argue that, together with the relationship between general religious intensity and number of children, religious homogamy has reproductive consequences. These may impact future demographic developments and could have also played a role in the biological evolution of humans.

Dark chocolate attenuates intracellular pro-inflammatory reactivity to acute psychosocial stress in men: A randomized controlled trial.

Flavanol-rich dark chocolate consumption relates to lower risk of cardiovascular mortality, but underlying mechanisms are elusive. We investigated the effect of acute dark chocolate consumption on inflammatory measures before and after stress. Healthy men, aged 20-50years, were randomly assigned to a single intake of either 50g of flavanol-rich dark chocolate (n=31) or 50g of optically identical flavanol-free placebo-chocolate (n=34). Two hours after chocolate intake, both groups underwent the 15-min Trier Social Stress Test. We measured DNA-binding-activity of the pro-inflammatory transcription factor NF-κB (NF-κB-BA) in peripheral blood mononuclear cells, as well as plasma and whole blood mRNA levels of the pro-inflammatory cytokines IL-1ß and IL-6, and the anti-inflammatory cytokine IL-10, prior to chocolate intake as well as before and several times after stress. We also repeatedly measured the flavanol epicatechin and the stress hormones epinephrine and cortisol in plasma and saliva, respectively. Compared to the placebo-chocolate-group, the dark-chocolate-group revealed a marginal increase in IL-10 mRNA prior to stress (p=0.065), and a significantly blunted stress reactivity of NF-κB-BA, IL-1ß mRNA, and IL-6 mRNA (p's⩽0.036) with higher epicatechin levels relating to lower pro-inflammatory stress reactivity (p's⩽0.033). Stress hormone changes to stress were controlled. None of the other measures showed a significant chocolate effect (p's⩾0.19). Our findings indicate that acute flavanol-rich dark chocolate exerts anti-inflammatory effects both by increasing mRNA expression of the anti-inflammatory cytokine IL-10 and by attenuating the intracellular pro-inflammatory stress response. This mechanism may add to beneficial effects of dark chocolate on cardiovascular health.

Signatures of positive selection in the cis-regulatory sequences of the human oxytocin receptor (OXTR) and arginine vasopressin receptor 1a (AVPR1A) genes.

BACKGROUND: The evolutionary highly conserved neurohypophyseal hormones oxytocin and arginine vasopressin play key roles in regulating social cognition and behaviours. The effects of these two peptides are meditated by their specific receptors, which are encoded by the oxytocin receptor (OXTR) and arginine vasopressin receptor 1a genes (AVPR1A), respectively. In several species, polymorphisms in these genes have been linked to various behavioural traits. Little, however, is known about whether positive selection acts on sequence variants in genes influencing variation in human behaviours. RESULTS: We identified, in both neuroreceptor genes, signatures of balancing selection in the cis-regulative acting sequences such as transcription factor binding and enhancer sequences, as well as in a transcriptional repressor sequence motif. Additionally, in the intron 3 of the OXTR gene, the SNP rs59190448 appears to be under positive directional selection. For rs59190448, only one phenotypical association is known so far, but it is in high LD' (>0.8) with loci of known association; i.e., variants associated with key pro-social behaviours and mental disorders in humans. CONCLUSIONS: Only for one SNP on the OXTR gene (rs59190448) was a sign of positive directional selection detected with all three methods of selection detection. For rs59190448, however, only one phenotypical association is known, but rs59190448 is in high LD' (>0.8), with variants associated with important pro-social behaviours and mental disorders in humans. We also detected various signatures of balancing selection on both neuroreceptor genes.

Paternal age predicts offspring chances of marriage and reproduction.

OBJECTIVES: Mutation-selection balance theory proposes that a balance of forces between constantly arising mildly harmful mutations and selection causes variation in genetic configuration and phenotypic condition. As mutations are predominantly deleterious, the entry of variation due to mutations is kept at low frequencies by selection. It has recently been demonstrated that nearly all de novo mutation are caused by paternal age. METHODS: We examined on basis of the Wisconsin Longitudinal Study (n = 6,182) whether a subject's probability of having ever married as well as having ever reproduced is associated with that subject's father's age at subject's birth. RESULTS: We find that advanced paternal but not maternal age at subject's birth predicts a lower chance of ever being married and a higher chance of childlessness, even controlling for various confounders. CONCLUSIONS: As marriage is a prerequisite of reproduction in this sample, we discuss that mate choice may provide a mechanism to prevent too high mutation load in the progeny.

Functional ADA polymorphism increases sleep depth and reduces vigilant attention in humans.

Homeostatically regulated slow-wave oscillations in non-rapid eye movement (REM) sleep may reflect synaptic changes across the sleep-wake continuum and the restorative function of sleep. The nonsynonymous c.22G>A polymorphism (rs73598374) of adenosine deaminase (ADA) reduces the conversion of adenosine to inosine and predicts baseline differences in sleep slow-wave oscillations. We hypothesized that this polymorphism affects cognitive functions, and investigated whether it modulates electroencephalogram (EEG), behavioral, subjective, and biochemical responses to sleep deprivation. Attention, learning, memory, and executive functioning were quantified in healthy adults. Right-handed carriers of the variant allele (G/A genotype, n = 29) performed worse on the d2 attention task than G/G homozygotes (n = 191). To test whether this difference reflects elevated homeostatic sleep pressure, sleep and sleep EEG before and after sleep deprivation were studied in 2 prospectively matched groups of G/A and G/G genotype subjects. Deep sleep and EEG 0.75- to 1.5-Hz oscillations in non-REM sleep were significantly higher in G/A than in G/G genotype. Moreover, attention and vigor were reduced, whereas waking EEG alpha activity (8.5-12 Hz), sleepiness, fatigue, and α-amylase in saliva were enhanced. These convergent data demonstrate that genetic reduction of ADA activity elevates sleep pressure and plays a key role in sleep and waking quality in humans.

Facial attractiveness is only weakly linked to genome-wide heterozygosity.

Introduction: It has been frequently suggested that overall genomic heterozygosity and, particularly, heterozygosity of loci on the so-called major histocompatibility complex (MHC), which is responsible for the recognition of foreign substances/ pathogens and the recognition of self and non-self, is associated with better health and better resistance to infections and parasites. It has further been speculated that such a potentially beneficial heterozygosity can be detected through body odor and facial attractiveness. Methods: On the basis of genome wide SNP data (713,014 SNPs) of participants from the Wisconsin Longitudinal Study, we therefore investigated whether homozygosity either on the MHC (measured as inbreeding coefficient) or genome-wide (measured as runs of homozygosity and inbreeding coefficient) is associated with rated facial attractiveness. Results: Although we found that the genome-wide average length of homozygous segments and the genome-wide inbreeding coefficient are significantly negatively associated with some measures of facial attractiveness, if corrected for multiple testing, any significant association was no longer formally significant after correction. In addition, the variance in facial attractiveness explained by the genome wide homozygosity is very low (<0.15%). We did not find any significant association between the inbreeding coefficient on the MHC and facial attractiveness. Discussion: We only find a weak association of genome- wide heterozygosity and facial attractiveness.

Increasing pressure on US men for income in order to find a spouse.

In contemporary societies, social status - especially income - is one of the most important determinants of ever marrying among men. Using U.S. census data, we estimated the importance of income for ever marrying among men and women, analyzing birth cohorts from 1890 to 1973. We examined individuals between the ages of 45 and 55, a total of 3.5 million men and 3.6 million women. We find that for men, the importance of income in predicting ever being married increased steadily over time. Income predicted only 2.5% of the variance in ever marrying for those born in 1890-1910, but about 20% for the 1973 cohort. For women, the opposite is true: the higher a woman's income among those born between 1890 and 1910, the lower her odds of ever being married, explaining 6% of the variance, whereas today a woman's income no longer plays a role in ever being married. Thus, our results provide evidence that income may represent a very recent selection pressure on men in the US, a pressure that has become increasingly stronger over time in the 20th and early 21st centuries.

Rapid quality control of black truffles using Direct Analysis in Real Time Mass Spectrometry and Hydrophilic Interaction Liquid Chromatography Mass Spectrometry.

This work is the first to describe the application of Direct Analysis in Real Time Mass Spectrometry for rapid and simple quality control of black truffles. Multivariate analyses (PCA and LDA) of mass spectra provided highly efficient models for the discrimination of four truffle species, namely Tuber melanosporumVittad., Tuber aestivumVittad., Tuber uncinatumChatin and Tuber indicum Cooke etMassee. Full-cross validations showed prediction accuracies up to 99%. Hydrophilic Interaction Liquid Chromatography Mass Spectrometry was used as a reference method and the results of both methods were compared to each other. Multivariate models of the chromatograms also showed excellent results with prediction accuracies of 100%. In a direct comparison of methods, Direct Analysis in Real Time Mass Spectrometry showed minimally lower prediction accuracies, but was significantly faster, more robust and easier to use, making it an excellent choice for rapid quality control of black truffles.

Effects of prenatal stress on hypothalamic-pituitary-adrenal (HPA) axis function over two generations of guinea pigs (Cavia aperea f. porcellus).

Prenatal stress can alter hypothalamic-pituitary-adrenal axis function with potential consequences for later life. The aim of our study was to examine in guinea pigs (Cavia aperea f. porcellus) the effects of stress experienced during F0 pregnancy on glucocorticoid levels in plasma and feces, as well as challenge performance, in F1 offspring (n=44) and fecal glucocorticoid levels in F2 offspring (n=67). F1 animals were either born to F0 dams that had been stressed with strobe light during early to mid pregnancy, resulting in a short term increase but long-term down-regulation of maternal glucocorticoid levels, or to undisturbed F0 dams. The same stressor was used as a challenge for F1 offspring at age 26 days and around 100 days. Basal plasma cortisol concentrations during early F1 development, as well as overall glucocorticoid levels at challenge tests, were lower in F1 animals that were prenatally stressed than in control animals. Fecal cortisol metabolites were initially at lower levels in prenatally stressed F1 animals, relative to control animals, but shifted to higher levels around day 68, with an additional sex difference. Effects were also seen in the F2 generation, as male but not female offspring of prenatally stressed F1 animals had significantly higher levels of cortisol metabolites in feces after weaning. We conclude that stress exposure of F0 dams resulted in lower basal glucocorticoid levels in F1 offspring during the pre-pubertal phase and during stress exposure, but higher glucocorticoid levels in post-adolescent F1 animals. Also in males of F2 generation effects of stress exposure of F0 dams were detected.

Marriage in the Melting Pot: An Evolutionary Approach to European Ancestry, Homogamy, and Fertility in the United States.

To understand marriage patterns, homogamy, and fertility of women of European ancestry in the United States from an evolutionary perspective, we investigated if a prevalence of ancestral homogamy exists, the factors influencing a female preference for an ancestral homogamous vs. heterogamous marriage, and if ancestral homogamous vs. heterogamous marriages have an impact on fertility. Furthermore, we aim to determine the heritability of homogamous vs. heterogamous marriage behavior. We used the census data of 369,121 women in the United States married only once and aged between 46 and 60 years, provided by IPUMS USA (https://usa.ipums.org/usa/). We used linear mixed models to determine the association between the probability of a homogamous vs. heterogamous marriage and the individual fertility of women. We aimed to estimate the heritability (genetics and parental environment) of marriage behavior using a linear mixed model. We found that ancestral heterogamous marriages are more frequent compared to homogamous marriages, but only if all ancestry groups are included. If ancestry is aggregated, homogamous marriages are more frequent compared to heterogamous marriages. Most of the variance (up to 27%) in inter-ancestry marriage and fertility (up to 12%) is explained by ancestry per se, followed by the ratio of individuals of a certain ancestral background in a county (∼6%), indicating a frequency depending selection into marriage: the more individuals of a certain ancestry live in a county, the lower is the tendency to marry someone of a different ancestral background. Furthermore, we found that about 12% (depending to some extent on the clustering) of the marriage behavior is heritable. Being in a homogamous marriage and the income of the spouse are both significantly positively associated with the number of children women have and the probability that women have at least one child, albeit explaining only a very low proportion of the overall variance. The most important factor (in terms of variance explained) for being in an ancestral homogamous vs. heterogamous marriage, for the number of children, and for childlessness is the ancestry of the women. Most children are born to women of Irish, French, and Norwegian ancestry (Irish X̄: 3.24, French X̄: 3.21, and Norwegian X̄: 3.18), the lowest number of children is to women of Latvian, Rumanian, and Russian ancestry (Latvian X̄: 2.26, Rumanian X̄: 2.19, and Russian X̄: 2.35). Albeit, we are not able to distinguish the genetic and social heritability on the basis of our data, only a small heritability for in-group vs. out-group marriage behavior is indicated (∼12% of variance explained).

Rapid quantification of cannabidiol from oils by direct analysis in real time mass spectrometry.

This work is the first to describe the use of Direct Analysis in Real Time Mass Spectrometry (DART-MS) for the rapid quantification of cannabidiol (CBD) in CBD oils. For this study, self-prepared samples spiked with CBD in hemp seed oil as well as commercial CBD oils from the Austrian market with different CBD contents were analyzed. CBD concentrations were between 5 and 30% (m/m) for the spiked samples as well as between 5 and 15% (m/m) for the real samples. The performance of quantification by means of DART-MS was assessed against a validated liquid chromatography-mass spectrometry (LC-MS) method. The correlation of the quantification results of both methods was high with a correlation factor greater than 0.98 and a maximum bias of 9.8%. Furthermore, the relative standard deviation values of the DART-MS measurments were below the tolerable limit of 12%. These results demonstrate that quantification of CBD by DART-MS is reliable and hence suitable as a rapid and cost-effective alternative method for quality control of CBD content in CBD oils.

Rapid differentiation and quality control of tobacco products using Direct Analysis in Real Time Mass Spectrometry and Liquid Chromatography Mass Spectrometry.

This work is the first to describe the use of Direct Analysis in Real Time mass spectrometry (DART-MS) for the rapid and cost-effective quality control of cigars. Multivariate analyses (PCA and LDA) on mass spectra led to highly efficient models for the discrimination of tobacco products. In addition to the geographic origin of the cigars, different pretreatments of the tobacco leaves could also be determined. Cross-validations of the models yielded prediction accuracies up to 92.18%. All results were compared using LC-MS as reference method. Multivariate analyses of chromatograms also yielded powerful models with correctness scores up to 88.1%. However, the models generated with LC-MS were neither able to determine the geographic origin of the tobacco leaves nor different pretreatments. In addition to qualitative analysis, the nicotine content of tobacco leaves was also determined via both methods. While precise quantification was not possible with DART-MS, a correlation coefficient of >0.96 was achieved in direct comparison with LC-MS, which allows semi-quantitative statements about the nicotine content without any problems.