Molecular insights into the determinants of substrate specificity and efflux inhibition of the RND efflux pumps AcrB and AdeB.

Gram-negative bacterial members of the Resistance Nodulation and cell Division (RND) superfamily form tripartite efflux pump systems that span the cell envelope. One of the intriguing features of the multiple drug efflux members of this superfamily is their ability to recognize different classes of antibiotics, dyes, solvents, bile salts, and detergents. This review provides an overview of the molecular mechanisms of multiple drug efflux catalysed by the tripartite RND efflux system AcrAB-TolC from Eschericha coli. The determinants for sequential or simultaneous multiple substrate binding and efflux pump inhibitor binding are discussed. A comparison is made with the determinants for substrate binding of AdeB from Acinetobacter baumannii, which acts within the AdeABC multidrug efflux system. There is an apparent general similarity between the structures of AcrB and AdeB and their substrate specificity. However, the presence of distinct conformational states and different drug efflux capacities as revealed by single-particle cryo-EM and mutational analysis suggest that the drug binding and transport features exhibited by AcrB may not be directly extrapolated to the homolog AdeB efflux pump.

[Mental health surveillance at the Robert Koch Institute - strategies for monitoring the mental health of the population]. / Mental Health Surveillance am Robert Koch-Institut ­ Strategien zur Beobachtung der psychischen Gesundheit der Bevölkerung.

The continuous and systematic surveillance of the health of populations is fundamental for effective public health practice. In light of the growing importance of mental health within population health, a Mental Health Surveillance for Germany is being established at the Robert Koch Institute. Its aim is to continually provide reliable information on the current state and development of the mental health of the population.Three surveillance strategies are currently being pursued: 1) Regular comprehensive assessments aim to describe the mental health status of the population using a wide range of indicators and data sources and to observe long-term developments. They build on existing work in epidemiology and health services research. 2) High-frequency monitoring of a selection of indicators is used for the early detection of trends. 3) A continuous literature review collates current findings on mental health developments in the COVID-19 pandemic on a monthly basis. The latter two strategies were implemented in response to new information needs in the pandemic.This paper describes and discusses these three strategies and their functions, limitations, and potential for development. Their results are communicated through different forms of reporting and serve to identify needs for action and research in public mental health. The further development and long-term operation of the Mental Health Surveillance as a whole has the potential to facilitate the achievement of public mental health objectives and to contribute on different levels to the improvement of population health.

[A Continuous Literature Review for Mental Health Surveillance during the Covid-19 Pandemic in Germany - Method, Selected Results and Lessons Learned]. / Kontinuierliches Literaturreview zur Surveillance der psychischen Gesundheit in Deutschland während der COVID-19-Pandemie ­ Methode, ausgewählte Ergebnisse und Lessons Learned.

In order to provide an up-to-date overview on changes in population's mental health during the COVID-19 pandemic, a continuous literature review was conducted. Building on a rapid review, systematic and hand searches were conducted monthly until December 31, 2022. Studies were assessed for observation periods, risk of bias and outcomes. Trends in depressive symptoms in adults were summarized by vote counting. 102 publications were included from 62 studies in the adult population. Studies declined over the course of the pandemic. Overall, 37% of the studies and 56% of the publications can assess trends in the population reliably. Among evidence for changes in depressive symptoms deteriorations predominated at last. The heterogeneity of results published by the end of 2022 limits evidence syntheses. Evidence of deterioration requires further surveillance. A continuous review can indicate evidence gaps at an early stage.

Epigenetic down regulation of nerve growth factor during alcohol withdrawal.

We investigated the Cytosin-phosphatidyl-Guanin (CpG) island promoter methylation (mean and methylation of individual CpG-sites) of the nerve growth factor (NGF) gene in the blood of alcohol-dependent patients (57 male patients) during withdrawal (days 1, 7 and 14). Methylation and NGF serum levels did not change significantly from days 1-7. From days 7-14, mean methylation increased (F = 30.55, P < 0.001), whereas the NGF serum levels decreased significantly (days 7-14: F = 17.95, P < 0.001). The NGF serum levels were significantly associated with the mean methylation of the investigated CpG-sites (F = 1.55, P < 0.001). These results imply an epigenetic regulation of the NGF gene during alcohol withdrawal.

Indicators for elevated risk factors for alcohol-withdrawal seizures: an analysis using a random forest algorithm.

Alcohol-withdrawal seizures (AWS) are an important and relevant complication during detoxification in alcohol-dependent patients. Therefore, it is important to evaluate the individual risk for AWS. We apply a random forest algorithm to assess possible predictive markers in a large sample of 200 alcohol-dependent patients undergoing alcohol withdrawal. This analysis showed that the combination of homocysteine, prolactin, blood alcohol concentration on admission, number of preceding withdrawals, age and the number of cigarettes smoked may successfully predict AWS. In conclusion, the results of this analysis allow for origination of further research, which should include additional biological and psychosocial parameters as well as consumption behaviour.

Biological markers for alcohol withdrawal seizures: a retrospective analysis.

AIMS: Alcohol withdrawal seizures (AWS) are among the most important possible complications during the detoxification treatment of alcohol-dependent patients. Pharmacological therapy is often used during detoxification, but can cause dangerous side effects [Eur Addict Res 2010;16:179-184]. In separate studies several biological markers have been described as being associated with AWS risk. We investigated the role of homocysteine (HCT), carbohydrate-deficient transferrin (CDT) and prolactin (PRL) as biological markers for the risk of developing AWS. METHODS: The present study included 189 alcohol-dependent patients of whom 51 had a history of AWS. We investigated the HCT, CDT and PRL levels of all patients and calculated sensitivity and specificity. Bayes' theorem was used to calculate positive (PPV) and negative (NPV) predictive values. RESULTS: The highest combined sensitivity and specificity for %CDT was reached at a plasma cutoff value of 3.75%. The combination of HCT at a cutoff value of 23.9 µmol/l and %CDT at a cutoff value of 3.75% showed the best predictive values (sensitivity 47.1%, specificity 88.4%, PPV 0.504, NPV 0.870). CONCLUSION: A combined assessment of HCT and CDT levels can be a useful method to identify patients at a higher risk of AWS, which may lead to a more individualized therapy.

Prolactin serum levels during alcohol withdrawal are associated with the severity of alcohol dependence and withdrawal symptoms.

BACKGROUND: Prolactin serum levels have been described to be elevated during alcohol withdrawal in alcohol-dependent patients and normalize during abstinence. Alterations in prolactin levels may reflect disturbances of dopaminergic neurotransmission which is of crucial importance for alcohol-seeking behavior. METHODS: In this longitudinal observational study, we investigated prolactin serum levels in 99 male patients during the first 14 days of alcohol withdrawal and early abstinence and in 43 healthy controls. To assess the severity of alcohol dependence, the extent of withdrawal symptoms, craving, depressive symptoms, and anxiety, we employed a structured interview including psychologic measurements. RESULTS: Prolactin serum levels were elevated during the whole study period in alcohol-dependent patients compared to the healthy control group. Prolactin levels at admission (first day of alcohol withdrawal) were associated with the severity of alcohol withdrawal (CIWA-Ar) and of alcohol dependence (SESA) but not with the other assessed psychologic parameters. CONCLUSIONS: The presented findings confirm that prolactin is significantly elevated in alcohol-dependent patients during alcohol withdrawal and early abstinence, not showing a rapid decline after cessation of drinking. The association with the severity of withdrawal and dependence may reflect at least partially the individual alterations in the dopaminergic and glutamatergic pathways.

Promoter Methylation of LEP and LEPR before and after Bariatric Surgery: A Cross-Sectional Study.

INTRODUCTION: DNA methylation constitutes one important epigenetic mechanism that regulates gene expression in human cells. With regard to obesity, bariatric surgery-induced weight loss has been associated with promoter methylation changes in several genes. Hyperleptinemia is a characteristic feature of obesity. The underlying regulating mechanisms have not yet been completely elucidated. METHODS: We investigated the methylation of the promoters of the leptin gene (LEP) and the leptin receptor gene (LEPR) as well as leptin expression in pre- and postbariatric surgery patients using a comparative cross-sectional design. RESULTS: Our results revealed significantly higher LEP promoter methylation patterns in prebariatric surgery patients compared to postoperatively. DNA methylation of the LEPR promoter was significantly higher in the postoperative group. Moreover, we found significantly higher leptin serum levels in patients before the bariatric surgery than afterwards. DISCUSSION: These findings strengthen the suggestion that there is an association between LEP expression and LEP methylation in obesity. We suggest that the epigenetic profile of LEP might be influenced by leptin serum levels in the form of a regulating feedback mechanism.

Structural and functional analysis of the promiscuous AcrB and AdeB efflux pumps suggests different drug binding mechanisms.

Upon antibiotic stress Gram-negative pathogens deploy resistance-nodulation-cell division-type tripartite efflux pumps. These include a H+/drug antiporter module that recognizes structurally diverse substances, including antibiotics. Here, we show the 3.5 Å structure of subunit AdeB from the Acinetobacter baumannii AdeABC efflux pump solved by single-particle cryo-electron microscopy. The AdeB trimer adopts mainly a resting state with all protomers in a conformation devoid of transport channels or antibiotic binding sites. However, 10% of the protomers adopt a state where three transport channels lead to the closed substrate (deep) binding pocket. A comparison between drug binding of AdeB and Escherichia coli AcrB is made via activity analysis of 20 AdeB variants, selected on basis of side chain interactions with antibiotics observed in the AcrB periplasmic domain X-ray co-structures with fusidic acid (2.3 Å), doxycycline (2.1 Å) and levofloxacin (2.7 Å). AdeABC, compared to AcrAB-TolC, confers higher resistance to E. coli towards polyaromatic compounds and lower resistance towards antibiotic compounds.

Elevation of homocysteine levels is only partially reversed after therapy in females with eating disorders.

Recent studies have shown elevated homocysteine levels in patients with eating disorders. In a prospective, longitudinal study, we investigated differences of homocysteine plasma levels in patients with anorexia nervosa (N = 12) and bulimia nervosa (N = 17) compared to healthy controls (N = 20) and alteration of homocysteine levels in patients during specific in-patient treatment. We found significantly elevated homocysteine levels in both patient groups (anorexia nervosa and bulimia nervosa) and a non-significant decrease of homocysteine during the 12-week treatment period. Furthermore, we found a significant association between low homocysteine levels and cognitive deficits, pointing toward a beneficial effect of elevated homocysteine levels on cognition in this patient group. We suppose that during effective treatment with significant increase of the body mass index, the observed hyperhomocysteinemia in patients with eating disorders is partially reversible. These findings add further evidence to the hypothesis that homocysteine might be involved in the pathophysiology of anorexia and bulimia nervosa.

Epigenetic dysregulation of dopaminergic genes in eating disorders.

OBJECTIVE: The pathophysiology of eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN) has been linked to an impaired dopaminergic neurotransmission, still the origin of this disturbance remains unknown. The aim of the present study was, therefore, to evaluate whether the expression of dopaminergic genes is altered in the blood of patients suffering from eating disorders and if these alterations can be explained by changes in the promoter specific DNA methylation of the genes. METHOD: We used quantitative real-time PCR to measure both the expression and the promoter specific DNA methylation of the dopamine transporter (DAT), and the D2 (DRD2) and D4 receptor (DRD4) gene in the blood of 46 patients (22 AN, 24 BN) and 30 healthy controls. RESULTS: Patients showed an elevated expression of DAT mRNA when compared with the controls and a downregulation of the DRD2 expression. The upregulation of the DAT gene was accompanied by a hypermethylation of the gene's promoter in the AN and BN group while a significant hypermethylation of the DRD2 promoter was only present in the AN group. No differences in expression or methylation were found for the other dopamine receptors investigated. DISCUSSION: Our study shows a disturbed expression of dopaminergic genes that is accompanied by a dysregulation of the epigenetic DNA methylation. Further studies are necessary to provide more insight into the epigenetic dysregulation of the dopaminergic neurotransmission in the pathophysiology of eating disorders.

Prevalence of Temporomandibular Disorders and Anxiety State Levels in Ballet Dancers A Cross-Sectional Study.

This study evaluated the prevalence of temporomandibular disorders (TMDs) in ballet dancers and explored their association with levels of anxiety. Participants were 51 male and female ballet dancers with a mean age of 31.5 ± 12.6 years. The diagnosis of TMDs was made in accordance with Research Diagnostic Criteria for Temporomandibular Joint Disorders (RDC/TMD). All subjects completed the state part of an anxiety questionnaire (STAI). Data were gathered and analyzed using the R statistical software (version: 3.4.0.) with level of significance set at 5%. The prevalence of all TMDs in the sample was 78%; that is, of the 51 participants, 39 had at least one positive diagnosis. The two most prevalence diagnoses were disk displacement with reduction and arthralgia. Individuals with TMD had a mean anxiety score of 42.55 ± 9.92 on the STAI, whereas for those without TMD it was 44.27 ± 11.89, with no statistically significant difference (p = 0.53). It is concluded that the general prevalence of TMDs in ballet dancer is higher than in the population at large. Disk displacement with reduction and arthralgia are the most common TMDs, and the presence of TMDs does not seem to affect anxiety state levels in this population.

Mining the red deer genome (CerEla1.0) to develop X-and Y-chromosome-linked STR markers.

Microsatellites are widely applied in population and forensic genetics, wildlife studies and parentage testing in animal breeding, among others, and recently, high-throughput sequencing technologies have greatly facilitated the identification of microsatellite markers. In this study the genomic data of Cervus elaphus (CerEla1.0) was exploited, in order to identify microsatellite loci along the red deer genome and for designing the cognate primers. The bioinformatics pipeline identified 982,433 microsatellite motifs genome-wide, assorted along the chromosomes, from which 45,711 loci mapped to the X- and 1096 to the Y-chromosome. Primers were successfully designed for 170,873 loci, and validated with an independently developed autosomal tetranucleotide STR set. Ten X- and five Y-chromosome-linked microsatellites were selected and tested by two multiplex PCR setups on genomic DNA samples of 123 red deer stags. The average number of alleles per locus was 3.3, and the average gene diversity value of the markers was 0.270. The overall observed and expected heterozygosities were 0.755 and 0.832, respectively. Polymorphic Information Content (PIC) ranged between 0.469 and 0.909 per locus with a mean value of 0.813. Using the X- and Y-chromosome linked markers 19 different Y-chromosome and 72 X-chromosome lines were identified. Both the X- and the Y-haplotypes split to two distinct clades each. The Y-chromosome clades correlated strongly with the geographic origin of the haplotypes of the samples. Segregation and admixture of subpopulations were demonstrated by the use of the combination of nine autosomal and 16 sex chromosomal STRs concerning southwestern and northeastern Hungary. In conclusion, the approach demonstrated here is a very efficient method for developing microsatellite markers for species with available genomic sequence data, as well as for their use in individual identifications and in population genetics studies.

Epigenetic regulation and gene expression of vasopressin and atrial natriuretic peptide in alcohol withdrawal.

Disturbances of volume regulating peptides like vasopressin and atrial natriuretic peptide (ANP) have been described in early abstinent patients. Aim of the present study was to evaluate possible alterations of the promoter-related DNA methylation of the ANP and vasopressin precursor genes and the related mRNA-expression of these genes in early alcohol withdrawal. We analyzed blood samples of 57 healthy controls and of 111 patients suffering from alcohol dependence that were admitted for detoxification treatment. Promoter-related DNA methylation and mRNA-expression of vasopressin and ANP genes were assessed using real-time PCR. Vasopressin mRNA-expression was not statistically different between patients and controls. However, we found a significantly elevated promoter-related DNA methylation of the vasopressin gene in patients with alcohol dependence (Mann-Whitney U-test: Z=-2.178, p=0.029). ANP mRNA-expression was significantly elevated in alcoholic patients (Z=-6.240, p<0.001) while promoter-related DNA methylation of ANP was significantly decreased (Z=-2.282, p=0.023). Furthermore, promoter-related DNA methylation of ANP was significantly correlated to the extent of craving measured with the OCDS (r=-0.197, p=0.040). The findings of the present study show significant alterations of the mRNA-expression and promoter-related DNA methylation of vasopressin and especially ANP precursor genes in patients with alcohol dependence. Further studies focusing on longitudinal changes of epigenetic regulation and gene expression of both peptides are needed to clarify the pathophysiological role of these findings.

Elevated cannabinoid 1 receptor mRNA is linked to eating disorder related behavior and attitudes in females with eating disorders.

OBJECTIVE: The endocannabinoid system is involved in the regulation of appetite, food intake and energy balance. METHODS: To study possible differences in CB(1) and CB(2) mRNA expression in eating disorders, 20 patients with anorexia nervosa (AN), 23 with bulimia nervosa (BN) and 26 healthy women were enrolled into the trial (Homocysteine and Eating Disorders, HEaD). RESULTS: We found significantly higher levels of CB(1) receptor mRNA in the blood of patients with AN (DeltaCT: -3.9 (1.0); KW: 11.31; P=0.003) and BN (DeltaCT: -3.7 (1.7)) when compared to controls (DeltaCT: -4.6 (0.6); Dunn's test AN vs. CONTROLS: P<0.05; BN vs. CONTROLS: P<0.001) measured by quantitative real-time PCR. No differences were found regarding the expression of CB(2) receptor mRNA. Higher CB(1) receptor expression was associated with lower scores in several eating disorder inventory-2 (EDI-2) subscales including perfectionism, impulse regulation and drive for thinness. CONCLUSION: Our finding of elevated CB(1)-receptor expression in AN and BN adds further evidence to the hypothesis of impaired endocannabinoid signaling in eating disorders.

Promoter specific methylation of the dopamine transporter gene is altered in alcohol dependence and associated with craving.

Dopaminergic neurotransmission plays a crucial role in the genesis and maintenance of alcohol dependence. Epigenetic regulation via promoter specific DNA methylation of the dopamine transporter gene (DAT) may influence altered dopaminergic neurotransmission in alcoholism. Aim of the present study was to investigate DNA promoter methylation of DAT in early alcohol withdrawal and in relation to alcohol craving. We analyzed blood samples of 76 patients admitted for detoxification treatment and compared them to 35 healthy controls. Methylation specific quantitative real-time PCR was used to measure the promoter specific DNA methylation of the dopamine transporter. We assessed the extent of alcohol craving using the obsessive compulsive drinking scale (OCDS). Compared to healthy controls we found a significant hypermethylation of the DAT-promoter (Mann-Whitney U-test: p=0.001). Ln-transformed methylation of the DAT-promoter was negatively associated with the OCDS (linear regression: Beta=-0.275, p=0.016), particularly with the obsessive subscale (Beta=-0.300, p=0.008). Findings of the present study show that the epigenetic regulation of the DAT-promoter is altered in patients undergoing alcohol withdrawal. Furthermore, hypermethylation of the DAT-promoter may play an important role in dopaminergic neurotransmission and is associated with decreased alcohol craving.

DNA-methylation of the dopamin receptor 2 gene is altered during alcohol withdrawal.

The dopaminergic neurotransmission is known to be of crucial importance in addictive behavior. Epigenetic regulation like methylation of DNA influences the function of dopaminergic transmission. The present study investigated alterations of DNA methylation in the dopamine D2 receptor (DRD2)-gene in patients suffering from alcohol dependence. The study sample consists of 99 alcohol dependent males admitted for alcohol withdrawal treatment and a control group of 33 healthy participants. Blood samples underwent bisulfite sequencing to determine levels of DNA-methylation of the promoter region of the DRD2 gene. Mixed linear modeling was used to test differences between patients and controls, course of methylation during detoxification. While DRD2-gene methylation did not differ significantly between patients and controls, we found a significant increase of DRD2-gene methylation during alcohol withdrawal/early abstinence. Craving, measured with the Obsessive Compulsive Drinking Scale (OCDS), was significantly associated with DRD2-gene methylation. Furthermore, smoking significantly influenced DRD2-gene methylation in both, patients and controls. As in other types of addictive disorders, DRD2-gene methylation is altered during alcohol withdrawal/early abstinence. The findings regarding an association with alcohol craving and tobacco consumption point towards a crucial role of DRD2-gene methylation in the neurobiology of addictive behavior.

Depressive symptoms may explain elevated plasma levels of homocysteine in females with eating disorders.

Elevated plasma homocysteine levels have been found in different psychiatric disorders, including major depression and eating disorders. The aim of the present study was to evaluate whether presence of depression or depressive symptoms is associated with elevated homocysteine levels in patients with eating disorders. Total plasma homocysteine levels were assessed in 44 females with anorexia nervosa (n = 21) or bulimia nervosa (n = 23). Comorbid major depressive disorder (MDD) was diagnosed according to DSM-IV criteria using a semi-structured interview (SCID-I). Furthermore, depressive symptoms were assessed using Beck's depression inventory (BDI). Presence of MDD was not associated with elevated homocysteine levels (t-test: T = 0.42; df = 42; P = 0.68). However, self-rated presence of clinically relevant depressive symptoms (BDI score18) was associated with elevated homocysteine (T = -2.8; df = 42; P = 0.008). Presence of depressive symptoms may explain elevated homocysteine levels previously reported in patients with eating disorders or vice versa. Longitudinal studies are needed to unravel this hen or egg problem.

Alpha-synuclein mRNA levels correspond to beck depression inventory scores in females with eating disorders.

Alpha-synuclein (alpha-Syn) is a neuronal protein involved in the regulation of brain serotonin and dopamine levels. We analyzed the peripheral expression of alpha-Syn mRNA and Beck Depression Inventory scores in female patients suffering from anorexia nervosa (n = 18) or bulimia nervosa (n = 24). We found a significant positive association between alpha-Syn mRNA expression and the total scores of the Beck Depression Inventory (linear regression; R(2) = 0.20; p = 0.003). alpha-Syn may play a pathophysiological role in depressive symptoms associated with eating disorders. Further investigations in patients with depression as a sole diagnosis are needed to support its role in the pathogenesis of major depression.

Hippocampal volume loss in patients with alcoholism is influenced by the consumed type of alcoholic beverage.

AIMS: The individual extent of structural brain tissue changes in patients with alcohol dependence is influenced by genetic factors, gender, age and possibly a dose/duration-effect. Aim of the present study was to investigate different types of alcoholic beverages with regard to hippocampal volume loss in patients suffering from alcoholism. METHODS: We included 52 patients with alcohol dependence and divided them according to their preferred type of beverage consumption (beer, wine, and spirits). Hippocampal volumes were determined using volumetric high-resolution MR imaging. RESULTS: There was a significant difference in hippocampal volumes between patients consuming different beverages (ANOVA: F = 7.454; df = 2; P = 0.0015) with the smallest volumes in the wine group, followed by the spirits group. Furthermore, patients with a preferred spirits consumption showed significantly higher plasma homocysteine levels (ANOVA: F = 3.39; df = 2; P = 0.042). Linear regression analyses revealed an association of homocysteine and hippocampal volume only in the group of patients preferring spirits (R(2) = 0.364; P = 0.008). CONCLUSIONS: Homocysteine-mediated excitotoxicity may be an important pathophysiological mechanism in ethanol-related brain damage, particularly in patients consuming wine and spirits. The extent of brain atrophy in beer consuming patients seems to be more moderate.