Molecular Characterisation and Phylogeny of Tula Virus in Kazakhstan.

Orthohantaviruses are zoonotic pathogens that play a significant role in public health. These viruses can cause haemorrhagic fever with renal syndrome in Eurasia. In the Republic of Kazakhstan, the first human cases were registered in the year 2000 in the West Kazakhstan region. Small mammals can be reservoirs of orthohantaviruses. Previous studies showed orthohantavirus antigens in wild-living small mammals in four districts of West Kazakhstan. Clinical studies suggested that there might be further regions with human orthohantavirus infections in Kazakhstan, but genetic data of orthohantaviruses in natural foci are limited. The aim of this study was to investigate small mammals for the presence of orthohantaviruses by molecular biological methods and to provide a phylogenetic characterization of the circulating strains in Kazakhstan. Small mammals were trapped at 19 sites in West Kazakhstan, four in Almaty region and at seven sites around Almaty city during all seasons of 2018 and 2019. Lung tissues of small mammals were homogenized and RNA was extracted. Orthohantavirus RT-PCR assays were applied for detection of partial S and L segment sequences. Results were compared to published fragments. In total, 621 small mammals from 11 species were analysed. Among the collected small mammals, 2.4% tested positive for orthohantavirus RNA, one sample from West Kazakhstan and 14 samples from Almaty region. None of the rodents caught in Almaty city were infected. Sequencing parts of the small (S) and large (L) segments specified Tula virus (TULV) in these two regions. Our data show that geographical distribution of TULV is more extended as previously thought. The detected sequences were found to be split in two distinct genetic clusters of TULV in West Kazakhstan and Almaty region. TULV was detected in the common vole (Microtus arvalis) and for the first time in two individuals of the forest dormouse (Dryomys nitedula), interpreted as a spill-over infection in Kazakhstan.

Building Scientific Capability and Reducing Biological Threats: The Effect of Three Cooperative Bio-Research Programs in Kazakhstan.

Cooperative research programs aimed at reducing biological threats have increased scientific capabilities and capacities in Kazakhstan. The German Federal Foreign Office's German Biosecurity Programme, the United Kingdom's International Biological Security Programme and the United States Defense Threat Reduction Agency's Biological Threat Reduction Program provide funding for partner countries, like Kazakhstan. The mutual goals of the programs are to reduce biological threats and enhance global health security. Our investigation examined these cooperative research programs, summarizing major impacts they have made, as well as common successes and challenges. By mapping various projects across the three programs, research networks are highlighted which demonstrate best communication practices to share results and reinforce conclusions. Our team performed a survey to collect results from Kazakhstani partner scientists on their experiences that help gain insights into enhancing day-to-day approaches to conducting cooperative scientific research. This analysis will serve as a basis for a capability maturity model as used in industry, and in addition builds synergy for future collaborations that will be essential for quality and sustainment.

Zika virus replication in glioblastoma cells: electron microscopic tomography shows 3D arrangement of endoplasmic reticulum, replication organelles, and viral ribonucleoproteins.

Structural changes of two patient-derived glioblastoma cell lines after Zika virus infection were investigated using scanning transmission electron tomography on high-pressure-frozen, freeze-substituted samples. In Zika-virus-infected cells, Golgi structures were barely visible under an electron microscope, and viral factories appeared. The cytosol outside of the viral factories resembled the cytosol of uninfected cells. The viral factories contained largely deranged endoplasmic reticulum (ER), filled with many so-called replication organelles consisting of a luminal vesicle surrounded by the ER membrane. Viral capsids were observed in the vicinity of the replication organelles (cell line #12537 GB) or in ER cisternae at large distance from the replication organelles (cell line #15747 GB). Near the replication organelles, we observed many about 100-nm-long filaments that may represent viral ribonucleoprotein complexes (RNPs), which consist of the RNA genome and N protein oligomers. In addition, we compared Zika-virus-infected cells with cells infected with a phlebovirus (sandfly fever Turkey virus). Zika virions are formed in the ER, whereas phlebovirus virions are assembled in the Golgi apparatus. Our findings will help to understand the replication cycle in the virus factories and the building of the replication organelles in glioblastoma cells.

Eight Years of Collaboration on Biosafety and Biosecurity Issues Between Kazakhstan and Germany as Part of the German Biosecurity Programme and the G7 Global Partnership Against the Spread of Weapons and Materials of Mass Destruction.

In 2013, the German Federal Foreign Office launched the German Biosecurity Programme with the aim to minimise risks associated with biological substances and pathogens. In this context, the German-Kazakh Network for Biosafety and Biosecurity was established in 2013 and constitutes a successful collaboration between Kazakh and German biomedical organisations, under the co-management of the Bundeswehr Institute of Microbiology (IMB), and the Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH. Ever since then, a network of scientists, stake holders and policymakers has been established, aiming to work on highly pathogenic, potential biological warfare agents with the focus on biosafety and biosecurity, surveillance, detection and diagnostics, networking and awareness raising of these agents in Kazakhstan. Over the past 8 years, the project members trained four PhD candidates, organised over 30 workshops and trainings with more than 250 participants and conducted more than 5,000 PCR assays and 5,000 serological analyses for surveillance. A great success was the description of new endemic areas for Orthohantaviruses, the mixture of two Crimean-Congo haemorrhagic fever virus genetic clusters, new foci and genetic information on tick-borne encephalitis virus and rickettsiae in Kazakh oblasts. The latter even led to the description of two new genogroups. Furthermore, joint contributions to international conferences were made. In this report, we summarise the evolution of the German-Kazakh Network for Biosafety and Biosecurity and critically reflect on the strengths and possible weaknesses. We were able to establish a viable network of biosafety and biosecurity shareholders and to accomplish the aims of the German Biosecurity Programme to lower biosecurity risks by increased awareness, improved detection and diagnostic methods and surveillance. Further, we reflect on forthcoming aspects to lead this interstate endeavour into a sustainable future.

Occurrence of Anti-Rickettsia spp. Antibodies in Hospitalized Patients with Undifferentiated Febrile Illness in the Southern Region of Kazakhstan.

Undifferentiated febrile illness still represents a demanding medical problem all over the world, but primarily in low- and middle-income countries. Scientific and clinical investigations related to undifferentiated febrile illness and rickettsial diseases in Kazakhstan are lacking. This study reflects the investigation of antibodies against spotted fever group (SFG) and typhus group (TG) rickettsiae in patients with undifferentiated febrile illness in the southern region of Kazakhstan (Almaty and Kyzylorda oblasts). Paired serum samples were gathered from 13 hospitals in these two oblasts and explored for the presence of IgM and IgG antibodies against typhus group and IgG antibodies against spotted fever group rickettsiae using ELISA. Patient's questionnaires were statistically analyzed. In total, 802 inpatients from Almaty (N = 9) and Kyzylorda (N = 4) hospitals were included in this research. Based on ELISA results, 250 patients out of 802 (31.2%) from both oblasts had IgG antibodies against SFG rickettsiae. Results from 11 (1.4%) patients indicated acute infection with tick-borne rickettsiosis. Regarding TG rickettsiae (R. typhi), a past infection was detected in 248 (30.9%) febrile patients and acute infection in 22 (2.7%) patients in the two selected oblasts. The data indicated that SFG and TG rickettsioses are present in Kazakhstan. Kazakh physicians should be aware of these emerging diseases in both investigated oblasts because the occurrence of these diseases is not suspected during day-to-day clinical practice. The identification of rickettsial pathogens and implementation of modern laboratory methods for the diagnostics of rickettsioses are in need throughout Kazakhstan.

The prevalence of Borrelia in Ixodes persulcatus in southeastern Kazakhstan.

Borreliosis is one of the most common vector-borne zoonotic diseases in the world. Limited data are available regarding Borrelia spp. and their genotypes in Kazakhstan. The goal of this study was to investigate the prevalence of Borrelia spp. in ixodid ticks collected in the southeastern region of Kazakhstan. A total of 1907 ixodid ticks were collected by flagging vegetation at three collection areas in the Almaty oblast between 2015 and 2018. They were grouped into 407 pools and examined by qPCR for Borrelia burgdorferi sensu lato (s.l.). A conventional PCR with specific primers targeting 16S rRNA gene was used to differentiate B. burgdorferi s.l. genospecies. Sequence analysis of the PCR products was performed for sixteen samples. Lyme borreliosis agents were only detected in adult questing Ixodes persulcatus. The overall B. burgdorferi s.l. prevalence in I. persulcatus estimated as the minimum infection rate reached 10.7 %. Borrelia burgdorferi sensu stricto was not detected in any of the tick pools. Partial 16S rRNA gene sequencing revealed the presence of B. miyamotoi, B. afzelii, and B. garinii. Borrelia afzelii was the dominant genospecies in Almaty oblast. A significantly lower proportion of B. garinii positive tick pools was detected in the Zailiyskiy Alatau as compared to the Dzungarian Alatau (χ2 = 16.243; p = 0.0001) and Yenbekshikazakh district (χ2 = 7.4156; p = 0.0065). The obtained results indicate the epidemiological significance of B. afzelii and B. garinii in southeastern Kazakhstan. These new data aim to improve the diagnostics of Lyme borreliosis and monitoring of tick-borne infections in Kazakhstan.

Prenatally traumatized mice reveal hippocampal methylation and expression changes of the stress-related genes Crhr1 and Fkbp5.

In our previous study, we found that prenatal trauma exposure leads to an anxiety phenotype in mouse pups, characterized by increased corticosterone levels and increased anxiety-like behavior. In order to understand the mechanisms by which aversive in utero experience leads to these long-lasting behavioral and neuroendocrine changes, we investigated stress reactivity of prenatally traumatized (PT) mice, as well as the expression and methylation levels of several key regulatory genes of the stress axis in the dorsal hippocampus (dHPC) of the PT embryo and adult mice. We detected increased corticotropin-releasing hormone receptor 1 (Crhr1) and decreased FK506 binding protein 5 (Fkbp5) mRNA levels in the left dHPC of adult PT mice. These alterations were accompanied by a decreased methylation status of the Crhr1 promoter and an increased methylation status of the Fkbp5 promoter, respectively. Interestingly, the changes in Fkbp5 and Crhr1 mRNA levels were not detected in the embryonic dHPC of PT mice. Together, our findings provide evidence that prenatal trauma has a long-term impact on stress axis function and anxiety phenotype associated with altered Crhr1 and Fkbp5 transcripts and promoter methylation.

The complex interplay of climate, TBEV vector dynamics and TBEV infection rates in ticks-Monitoring a natural TBEV focus in Germany, 2009-2018.

BACKGROUND: Tick-borne encephalitis (TBE) is the most important tick-borne viral disease in Eurasia and causes disease in humans and in a number of animals, among them dogs and horses. There is still no good correlation between tick numbers, weather conditions and human cases. There is the hypothesis that co-feeding due to simultaneous occurrence of larvae and nymphs may be a factor for the increased transmission of the virus in nature and for human disease. Based on long-term data from a natural TBEV focus, phylogenetic results and meteorological data we sought to challenge this hypothesis. METHODS: Ticks from an identified TBE natural focus were sampled monthly from 04/2009 to 12/2018. Ticks were identified and pooled. Pools were tested by RT-qPCR. Positive pools were confirmed by virus isolation and/or sequencing of additional genes (E gene, NS2 gene). Temperature data such as the decadal (10-day) mean daily maximum air temperature (DMDMAT) were obtained from a nearby weather station and statistical correlations between tick occurrence and minimal infection rates (MIR) were calculated. RESULTS: In the study period from 04/2009 to 12/2018 a total of 15,530 ticks (2,226 females, 2,268 males, 11,036 nymphs) were collected. The overall MIR in nymphs over the whole period was 77/15,530 (0.49%), ranging from 0.09% (2009) to 1.36% (2015). The overall MIR of female ticks was 0.76% (17/2,226 ticks), range 0.14% (2013) to 3.59% (2016). The overall MIR of males was 0.57% (13/2,268 ticks), range from 0.26% (2009) to 0.97% (2015). The number of nymphs was statistically associated with a later start of spring/vegetation period, indicated by the onset of forsythia flowering. CONCLUSION: There was no particular correlation between DMDMAT dynamics in spring and/or autumn and the MIR of nymphs or adult ticks detected. However, there was a positive correlation between the number of nymphs and the number of reported human TBE cases in the following months, but not in the following year. The hypothesis of the importance of co-feeding of larvae and nymphs for the maintenance of transmission cycle of TBEV in nature is not supported by our findings.

SARS-CoV-2: International Investigation Under the WHO or BWC.

In late 2019, the novel and highly infectious coronavirus SARS-CoV-2 caused a worldwide outbreak of a severe respiratory infectious disease, known as COVID-19. The disease has started in China and turned into one of the worst pandemics in human history. Due to the very fast global spread of the pathogen, COVID-19 is a great challenge for the Public Health Systems. It had led to a variety of severe limitations in private and public life worldwide. There is a lively public debate about possible sources of SARS-CoV-2. This article aims at providing a better understanding of controversial biological and political issues regarding COVID-19. Recommendations are made for possible actions under the umbrella of the World Health Organization and in respect to the Biological Weapons Convention.

Vectors, molecular epidemiology and phylogeny of TBEV in Kazakhstan and central Asia.

BACKGROUND: In the South of Kazakhstan, Almaty Oblast' (region) is endemic for tick-borne encephalitis, with 0.16-0.32 cases/100,000 population between 2016-2018. The purpose of this study was to determine the prevalence and circulating subtypes of tick-borne encephalitis virus (TBEV) in Almaty Oblast' and Kyzylorda Oblast'. METHODS: In 2015 we investigated 2341 ticks from 7 sampling sites for the presence of TBEV. Ticks were pooled in 501 pools and isolated RNA was tested for the presence of TBEV by RT-qPCR. For the positive samples, the E gene was amplified, sequenced and a phylogenetic analysis was carried out. RESULTS: A total of 48 pools were TBEV-positive by the RT-qPCR. TBEV-positive ticks were only detected in three districts of Almaty Oblast' and not in Kyzylorda Oblast'. The positive TBEV pools were found within Ixodes persulcatus, Haemaphysalis punctata and Dermacentor marginatus. These tick species prevailed only in Almaty Oblast' whereas in Kyzylorda Oblast' Hyalomma asiaticum and D. marginatus are endemic. The minimum infection rates (MIR) in the sampling sites were 4.4% in Talgar, 2.8% in Tekeli and 1.1% in Yenbekshikazakh, respectively. The phylogenetic analysis of the generated sequences indicates that TBEV strains found in Almaty Oblast' clusters in the Siberian subtype within two different clades. CONCLUSIONS: We provided new data about the TBEV MIR in ticks in Almaty Oblast' and showed that TBEV clusters in the Siberian Subtype in two different clusters at the nucleotide level. These results indicate that there are different influences on the circulating TBEV strains in south-eastern Kazakhstan. These influences might be caused by different routes of the virus spread in ticks which might bring different genetic TBEV lineages to Kazakhstan. The new data about the virus distribution and vectors provided here will contribute to an improvement of monitoring of tick-borne infections and timely anti-epidemic measures in Kazakhstan.

Genome-Wide DNA Methylation Patterns in Children Exposed to Nonpharmacologically Treated Prenatal Depressive Symptoms: Results From 2 Independent Cohorts.

BACKGROUND: Maternal depressive symptoms are a common phenomenon during pregnancy and are related to negative outcomes for child development and health. Modifications in child DNA methylation are discussed as an underlying mechanism for the association between prenatal depressive symptoms and alterations in child outcomes. However, formerly reported genome-wide associations have yet to be replicated. METHODS: In an epigenome-wide association study (EWAS), alterations of DNA methylation related to maternal prenatal depressive symptoms were investigated in buccal cell samples from 174 children (n = 52 exposed to prenatal depressive symptoms; 6-9 years old) of the German longitudinal study FRAMES-FRANCES. Whole blood samples from the independent, age-comparable ARIES subsample of the ARIES/ALSPAC study (n = 641; n = 159 exposed to prenatal depressive symptoms; 7-8 years old) were examined as a confirmation sample. Depressive symptoms were assessed with the Edinburgh Postnatal Depression Scale. DNA methylation was analyzed with the Infinium Human Methylation 450k BeadChip. Modifications in single CpGs, regions, and biological pathways were investigated. Results were adjusted for age and birth outcomes as well as postnatal and current maternal depressive symptoms. Analyses were performed for the whole sample as well as separated for sex. RESULTS: The EWAS yielded no differentially methylated CpG or region as well as no accordance between samples withstanding correction for multiple testing. In pathway analyses, no overlapping functional domain was found to be enriched for either sample. A comparison of current and former findings suggests some overlapping methylation modifications from infancy to childhood. Results suggest that there might be sex-specific differential methylation, which should be further investigated in additional studies. CONCLUSIONS: The current, mainly nonsignificant, results challenge the assumption of consistent modifications of DNA methylation in children exposed to prenatal depressive symptoms. Despite the relatively small sample size used in this study, this lack of significant results may reflect diverse issues of environmental epigenetic studies, which need to be addressed in future research.

Serological investigation of orthohantaviruses in patients with fever of unknown origin in Kazakhstan.

OBJECTIVE: Orthohantaviruses are geographically widely distributed and present various clinical manifestations from mild symptoms to the severe form of haemorrhagic fever with renal syndrome (HFRS) in Eurasia. Official registration of HFRS in Kazakhstan started in the year 2000. However, the true prevalence of human infections by orthohantaviruses within Kazakhstan is unknown. The aim of this study was to investigate of the seroprevalence of orthohantavirus infections in patients with fever of unknown origin (FUO) in two regions, Almaty and Kyzylorda region. METHODS: Paired serum samples from 802 patients with FUO were screened for the presence of orthohantavirus IgG and IgM antibodies by ELISA. Positive samples were further tested by immunoblotting and indirect immunofluorescence tests (IIFT) to determine the respective orthohantavirus serotypes. Suspected acute serum samples were additionally checked by a RT-PCR to identify viral RNA. RESULTS: In total 178/802 (22.2%) serum samples reacted with orthohantavirus IgG antibodies and 4/802 (0.5%) with IgM antibodies. All positive samples were tested by immunoblotting which resulted in 2.9% positive samples with IgG antibodies against Puumala (PUUV), Hantaan (HTNV) and Dobrava (DOBV) virus serotypes in Almaty region and 5.4% to PUUV and DOBV serotypes in Kyzylorda region, respectively. In the IFFT, 1.9% positive samples from Almaty and 3.1% from Kyzylorda were confirmed for PUUV and DOBV serotypes. Out of four IgM ELISA positive samples only three were positive against PUUV in the immunoblot and showed weak positive reactivity for the Saaremaa (SAAV), PUUV and HTNV serotypes in the IFFT. CONCLUSIONS: This study demonstrates the presence of orthohantavirus infections among patients with FUO in Kazakh regions that were so far considered as non-endemic. The healthcare system needs to be prepared accordingly in order to be capable of detecting cases and providing adequate management of patients.

Salivary and hair cortisol as biomarkers of emotional and behavioral symptoms in 6-9 year old children.

The aim of the present work is to investigate the association of salivary and cumulative cortisol levels with emotional and behavioral symptoms in a Franconian Cognition and Emotion Studies (FRANCES) general population cohort of 158 6- to 9 year old children. Salivary cortisol values were measured by one-day diurnal cortisol profile, whereas cumulative cortisol was estimated via one-month hair cortisol concentrations (rHCC). Nearly all significant associations of clinical symptoms with child cortisol indices were age dependent: We report emotional symptoms being associated with lower rHCC in younger children (6.06-7.54 years). In older children (7.55-9.41 years) behavioral problems were further associated with higher rHCC and lower salivary cortisol awakening responses. In summary, child clinical symptoms were stronger associated with markers of hair cortisol compared to salivary cortisol. To picture developmental mechanisms, we suggest longitudinal designs for cortisol measures of stress systems in children and adolescents.

Prevalence of Rickettsia species in ticks including identification of unknown species in two regions in Kazakhstan.

BACKGROUND: Over 60 years ago clinical patterns resembling tick-borne rickettsioses have been described for the first time in Kazakhstan. Since 1995 the incidence of clinical cases of tick-borne rickettsioses in humans seems to be rising but studies on epidemiological data regarding the occurring etiological agents, tick vector species, prevalence and distribution throughout Kazakhstan are still scarce to date. The aim of the study was molecular investigation of ticks for spotted-fever group rickettsiae in the endemic Kyzylorda region and the so far considered as non-endemic Almaty region. A total of 2341 ticks was collected in the two regions in Kazakhstan and sorted in 501 pools: Ixodes persulcatus (243); Dermacentor marginatus (129); Haemaphysalis punctata (104); Hyalomma asiaticum (17); Dermacentor reticulatus (3); and Rhipicephalus turanicus (5). Pools were tested for Rickettsia spp. using real-time PCR. For positive samples multilocus sequence typing (MLST) was performed. RESULTS: The calculated minimum infection rate (MIR) for rickettsiae in the investigated ticks in Almaty region varied between 0.4-15.1% and 12.6-22.7% in the Kyzylorda region. At least four different Rickettsia species were identified in the two selected regions of Kazakhstan. Two of these are already known to science: Rickettsia raoultii and R. slovaca, the latter being reported for the first time in Almaty region One new form, "Candidatus R. yenbekshikazakhensis", was described by MLST of six gene fragments in Almaty region and one new genotype, "genotype R. talgarensis" was detected using three gene fragments. CONCLUSIONS: Kazakh physicians should be aware of rickettsioses after tick bites in both regions studied. Both, R. raoultii and R. slovaca should be included in the diagnostics. The role for human diseases has further to be investigated for the newly described rickettsiae, "Candidatus R. yenbekshikazakhensis" and "Genotype R. talgarensis".

Associations of prenatal depressive symptoms with DNA methylation of HPA axis-related genes and diurnal cortisol profiles in primary school-aged children.

Epigenetic DNA modifications in genes related to the hypothalamic-pituitary-adrenal (HPA) axis are discussed as a mechanism underlying the association between prenatal depression and altered child HPA activity. In a longitudinal study, DNA methylation changes related to prenatal depressive symptoms were investigated in 167 children aged 6 to 9 years. At six candidate genes, 126 cytosine-guanine dinucleotides were considered without correcting for multiple testing due to the exploratory nature of the study. Further associations with the basal child HPA activity were examined. Children exposed to prenatal depressive symptoms exhibited lower bedtime cortisol (p = .003, ηp2 = 0.07) and a steeper diurnal slope (p = .023, ηp2 = 0.06). For total cortisol release, prenatal exposure was related to lower cortisol release in boys, and higher release in girls. Furthermore, prenatal depressive symptoms were associated with altered methylation in the glucocorticoid receptor gene (NR3C1), the mineralocorticoid receptor gene (NR3C2), and the serotonin receptor gene (SLC6A4), with some sex-specific effects (p = .012-.040, ηp2 = 0.03-0.04). In boys, prenatal depressive symptoms predicted bedtime cortisol mediated by NR3C2 methylation, indirect effect = -0.07, 95% confidence interval [-0.16, -0.02]. Results indicate relations of prenatal depressive symptoms to both child basal HPA activity and DNA methylation, partially fitting a mediation model, with exposed boys and girls being affected differently.

Seroepidemiological and molecular investigations of infections with Crimean-Congo haemorrhagic fever virus in Kazakhstan.

OBJECTIVES: The aim of this study was to detect the seroprevalence of Crimean-Congo haemorrhagic fever virus (CCHFV) in patients with fever of unknown origin (FUO) in endemic (Kyzylorda) and non-endemic (Almaty) oblasts of Kazakhstan. METHODS: Paired serum samples from 802 patients with FUO were collected. Serum samples were investigated by ELISA to detect IgG and IgM antibodies against CCHFV. Sera with suspected acute infection were further investigated by RT-PCR to detect the viral RNA. RESULTS: IgG antibodies were detected in 12.7% of the sera from both oblasts. Acute infection was shown by IgM ELISA in four patients from Kyzylorda, with only one developing severe CCHF. Viral RNA was found by RT-PCR in the other three patients' sera. Phylogenetic analysis of partial L and S segments revealed CCHFV genotype Asia 2 and a possible reassortment between the genotypes Asia 1/Asia 2. Animal husbandry, such as working with cattle and horses, was significantly associated with CCHFV seropositivity. CONCLUSIONS: The antibodies and viral RNA detected in sera indicate that mild or even asymptomatic CCHFV infections are presented in Kazakhstan. This study describes the circulation of CCHFV in the so far non-endemic Almaty oblast for the first time. In conclusion, physicians treating patients with FUO in Kazakhstan should be aware of mild CCHF.

Prenatal Alcohol Exposure Is Associated With Adverse Cognitive Effects and Distinct Whole-Genome DNA Methylation Patterns in Primary School Children.

Prenatal alcohol exposure (PAE) is known to elicit a broad range of systemic effects, including neurophysiological alterations that result in adverse behavioral and cognitive outcomes. However, molecular pathways underlying these long-term intrauterine effects remain to be investigated. Here, we tested a hypothesis that PAE may lead to epigenetic alterations to the DNA resulting in attentional and cognitive alterations of the children. We report the results of the study that included 156 primary school children of the Franconian Cognition and Emotion Studies (FRANCES) cohort which were tested for an objective marker of PAE, ethyl glucuronide (EtG) in meconium at birth. Thirty-two newborns were found to be exposed to alcohol with EtG values above 30 ng/g (EtG+). Previously we described PAE being associated with lower IQ and smaller amplitude of the event-related potential component P3 in go trials (Go-P3), which indicates a reduced capacity of attentional resources. Whole-genome methylation analysis of the buccal cell DNA revealed 193 differentially methylated genes in children with positive meconium EtG, that were clustered into groups involved in epigenetic modifications, neurodegeneration, neurodevelopment, axon guidance and neuronal excitability. Furthermore, we detected mediation effects of the methylation changes in DPP10 and SLC16A9 genes on the EtG related cognitive and attention-related deficits. Our results suggest that system-wide epigenetic changes are involved in long-term effects of PAE. In particular, we show an epigenetic mediation of PAE effects on cognition and attention-related processes.

[Epigenetic modifications in children associated with maternal emotional stress during pregnancy]. / Affektive Belastungen der Mutter in der Schwangerschaft und assoziierte epigenetische Veränderungen beim Kind: Eine Übersicht.

Besides typical physical and hormonal changes during pregnancy, this life period is often associated with an increased emotional and mental stress for women. For the child, the time in utero is regarded as a critical developmental period since adverse stimuli during pregnancy can have lasting consequences for the fetal and postnatal health and development. Thus, prenatal depression, anxiety and stress are considered as risk factors for developmental delay, emotional and behavioral problems. Epigenetic modifications, especially modifications in DNA methylation, are discussed as a possible biological mechanism that could explain the association between prenatal emotional stress and altered developmental and health outcomes of the child. This review summarizes evidence for DNA methylation changes related to prenatal emotional stress from studies with a candidate-gene approach as well as epigenome-wide association studies. Problematic issues are discussed and recommendations for future research are presented.

Attention, cognitive control and motivation in ADHD: Linking event-related brain potentials and DNA methylation patterns in boys at early school age.

In order to better understand the underpinnings of attention-deficit/hyperactivity disorder (ADHD), we targeted the relationship of attentional, cognitive control and motivational processes with DNA methylation patterns of 60 candidate genes in boys at early school age. Participants (6 to 8 years; N = 82) were selected from a German longitudinal cohort (FRANCES). ADHD-related behaviour was assessed via maternal ratings. Performance and event-related potential measures (inter alia Cue-P3 and Nogo-P3), which were recorded in a motivational go/nogo task, indicated diminished attentional orienting, reduced inhibitory response control and a larger motivational effect on performance in ADHD already at this relatively young age. Methylation patterns were analysed in buccal cell DNA with the Illumina HumanMethylation 450K array. For CpG sites at genes of the dopaminergic (COMT, ANKK1) and the neurotrophic (BDNF, NGFR) system, associations with the Nogo-P3 as well as ADHD symptom severity were found suggesting that these systems are involved in response control deficits in ADHD. Methylation effects related to both functional aspects and ADHD behaviour were also observed for DPP10 and TPH2. Epigenetic mechanisms may play a role in ADHD-associated deficits but findings need to be replicated in larger samples and are limited by the fact that only peripheral methylation could be considered.

Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.