Alcohol Use Disorder (AUD) is a major contributor to global mortality and morbidity. Postmortem human brain tissue enables the investigation of molecular mechanisms of AUD in the neurocircuitry of addiction. We aimed to identify differentially expressed (DE) genes in the ventral and dorsal striatum between individuals with AUD and controls, and to integrate the results with findings from genome- and epigenome-wide association studies (GWAS/EWAS) to identify functionally relevant molecular mechanisms of AUD. DNA-methylation and gene expression (RNA-seq) data was generated from postmortem brain samples of 48 individuals with AUD and 51 controls from the ventral striatum (VS) and the dorsal striatal regions caudate nucleus (CN) and putamen (PUT). We identified DE genes using DESeq2, performed gene-set enrichment analysis (GSEA), and tested enrichment of DE genes in results of GWASs using MAGMA. Weighted correlation network analysis (WGCNA) was performed for DNA-methylation and gene expression data and gene overlap was tested. Differential gene expression was observed in the dorsal (FDR < 0.05), but not the ventral striatum of AUD cases. In the VS, DE genes at FDR < 0.25 were overrepresented in a recent GWAS of problematic alcohol use. TheARHGEF15gene was upregulated in all three brain regions. GSEA in CN and VS pointed towards cell-structure associated GO-terms and in PUT towards immune pathways. The WGCNA modules most strongly associated with AUD showed strong enrichment for immune response and inflammation pathways. Our integrated analysis of multi-omics data sets provides further evidence for the importance of immune- and inflammation-related processes in AUD.

(1) Background: Epigenome-wide association studies (EWAS) in peripheral blood have repeatedly found associations between tobacco smoking and aberrant DNA methylation (DNAm), but little is known about DNAm signatures of smoking in the human brain, which may contribute to the pathophysiology of addictive behavior observed in chronic smokers. (2) Methods: We investigated the similarity of DNAm signatures in matched blood and postmortem brain samples (n = 10). In addition, we performed EWASs in five brain regions belonging to the neurocircuitry of addiction: anterior cingulate cortex (ACC), Brodmann Area 9, caudate nucleus, putamen, and ventral striatum (n = 38–72). (3) Results: cg15925993 within the LOC339975 gene was epigenome-wide significant in the ACC. Of 16 identified differentially methylated regions, two (PRSS50 and LINC00612/A2M-AS1) overlapped between multiple brain regions. Functional enrichment was detected for biological processes related to neuronal development, inflammatory signaling and immune cell migration. Additionally, our results indicate the association of the well-known AHRR CpG site cg05575921 with smoking in the brain. (4) Conclusion: The present study provides further evidence of the strong relationship between aberrant DNAm and smoking.

Background & Aims: 3D-Bioprinting of an endocrine pancreas is a promising future curative treatment for selected patients with insulin secretion deficiency. In this study we present an end-to-end integrative, scalable concept extending from the molecular to the macroscopic level. Methods: A hybrid scaffold device was manufactured by 3D-(bio)printing. INS-1 cells with/without endothelial cells were bioprinted in gelatin methacrylate blend hydrogel. Polycaprolactone was 3D-printed and heparin-functionalized as structural scaffold component. In vitro evaluation was performed by viability and growth assays, total mRNA sequencing, and glucose-stimulated insulin secretion. In vivo, xenotransplantation to fertilized chicken eggs was used to investigate vascularization and function, and finite element analysis modeling served to detect boundary conditions and applicability for human islets of Langerhans. Results: Insulin-secreting pseudoislets were formed and resulted in a viable and proliferative experimental model. Transcriptomics revealed upregulation of proliferative and β-cell-specific signaling cascades, downregulation of apoptotic pathways, and overexpression of extracellular matrix proteins and VEGF induced by pseudoislet formation and 3D culture. Co-culture with human endothelial cells created a natural cellular niche resulting in enhanced insulin response after glucose stimulation. Survival and function of the pseudoislets after explantation and extensive scaffold vascularization of both the hydrogel and heparinized polycaprolactone components were demonstrated in ovo. Computer simulations of oxygen, glucose, and insulin flows were used to evaluate scaffold architectures and Langerhans islets at a future transplantation site along neurovascular structures. Conclusion: A defined end-to-end process for multidisciplinary bioconvergence research on a bioartificial endocrine pancreas was developed. A modular, patient-specific device architecture is proposed for future research studies.

Cocaine use disorder (CUD) is characterized by a loss of control over drug intake and is associated with structural, functional, and molecular alterations in the brain. At the molecular level, epigenetic alterations are hypothesized to contribute to the higher-level functional and structural brain changes as observed in CUD. Most evidence of cocaine-associated epigenetic changes comes from animal studies while only a few studies have been performed using human tissue. We investigated epigenome-wide DNA methylation signatures of CUD in human postmortem brain tissue of Brodmann Area 9 (BA9). A total of N = 42 BA9 brain samples were obtained from N = 21 individuals with CUD and N = 21 individuals without a CUD diagnosis. We performed an epigenome-wide association study and analyzed CUD-associated differentially methylated regions (DMRs). To assess the functional role of CUD-associated differential methylation, we performed Gene Ontology enrichment analyses and characterized co-methylation networks using a weighted correlation network analysis. We further investigated epigenetic age in CUD using epigenetic clocks for the assessment of biological age. While no CpG site was associated with CUD at epigenome-wide significance in BA9, we detected a total of 20 CUD-associated DMRs. After annotation of DMRs to genes, we identified NPFFR2 and KALRN for which a previous role in the behavioral response to cocaine in rodents is known. Three of the four identified CUD-associated co-methylation modules were functionally related to neurotransmission and neuroplasticity. Protein-protein interaction networks derived from module hub genes revealed several addiction-related genes as highly connected nodes such as CACNA1C, NR3C1 , and JUN . In BA9, we observed a trend toward epigenetic age acceleration in individuals with CUD remaining stable even after adjustment for covariates. Results from our study highlight that CUD is associated with epigenome-wide differences in DNA methylation levels in BA9 particularly related to synaptic signaling and neuroplasticity. This supports findings from previous studies that report on the strong impact of cocaine on neurocircuits in the human prefrontal cortex. Further studies are needed to follow up on the role of epigenetic alterations in CUD focusing on the integration of epigenetic signatures with transcriptomic and proteomic data.