👤 William G Hoffmann

The rat pancreatic progenitor cell line B-13 is of interest for research on drug metabolism and toxicity since the cells trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids. In this study we investigated the trans-differentiation and liver-specific functions of B-13/H cells in a three-dimensional (3D) multi-compartment bioreactor, which has already been successfully used for primary liver cell culture. Undifferentiated B-13 cells were inoculated into the bioreactor system and exposed to dexamethasone to promote hepatic trans-differentiation (B-13/HT). In a second approach, pre-differentiated B-13 cells were cultured in bioreactors for 15 days to evaluate the maintenance of liver-typical functions (B-13/HP). During trans-differentiation of B-13 cells into hepatocyte-like cells in the 3D bioreactor system (approach B-13/HT), an increase in glucose metabolism and in liver-specific functions (urea and albumin synthesis; cytochrome P450 [CYP] enzyme activity) was observed, whereas amylase - characteristic for exocrine pancreas and undifferentiated B-13 cells - decreased over time. In bioreactors with pre-differentiated cells (approach B-13/HP), the above liver-specific functions were maintained over the whole culture period. Results were confirmed by gene expression and protein analysis showing increased expression of carbamoyl-phosphate synthase 1 (CPS-1), albumin, CYP2E1, CYP2C11 and CYP3A1 with simultaneous loss of amylase. Immunohistochemical studies showed the formation of 3D structures with expression of liver-specific markers, including albumin, cytokeratin (CK) 18, CCAAT/enhancer-binding protein beta (CEBP-β), CYP2E1 and multidrug resistance protein 2 (MRP2). In conclusion, successful culture and trans-differentiation of B-13 cells in the 3D bioreactor was demonstrated. The requirement for only one hormone and simple culture conditions to generate liver-like cells makes this cell type useful for Show less

Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice. GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing. GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17β-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism. Show less

The opportunistic pathogen Legionella pneumophila employs the Icm/Dot type IV secretion system and ∼300 different effector proteins to replicate in macrophages and amoebae in a distinct 'Legionella-containing vacuole' (LCV). LCVs from infected RAW 264.7 macrophages were enriched by immuno-affinity separation and density gradient centrifugation, using an antibody against the L. pneumophila effector SidC, which specifically binds to the phosphoinositide PtdIns(4)P on the pathogen vacuole membrane. The proteome of purified LCVs was determined by mass spectro-metry (data are available via ProteomeXchange with identifier PXD000647). The proteomics analysis revealed more than 1150 host proteins, including 13 small GTPases of the Rab family. Using fluorescence microscopy, 6 novel Rab proteins were confirmed to localize on pathogen vacuoles harbouring wild-type but not ΔicmT mutant L. pneumophila. Individual depletion of 20 GTPases by RNA interference indicated that endocytic GTPases (Rab5a, Rab14 and Rab21) restrict intracellular growth of L. pneumophila, whereas secretory GTPases (Rab8a, Rab10 and Rab32) implicated in Golgi-endosome trafficking promote bacterial replication. Upon silencing of Rab21 or Rab32, fewer LCVs stained positive for Rab4 or Rab9, implicated in secretory or retrograde trafficking respectively. Moreover, depletion of Rab8a, Rab14 or Rab21 significantly decreased the number of SidC-positive LCVs, suggesting that PtdIns(4)P is reduced under these conditions. L. pneumophila proteins identified in purified LCVs included proteins putatively implicated in phosphorus metabolism and as many as 60 Icm/Dot-translocated effectors, which are likely required early during infection. Taken together, the phagocyte and Legionella proteomes of purified LCVs lay the foundation for further hypothesis-driven investigations of the complex process of pathogen vacuole formation. Show less

Children with attention-deficit/hyperactivity disorder (ADHD) have a higher rate of obesity than children without ADHD. Obesity risk alleles may overlap with those relevant for ADHD. We examined whether risk alleles for an increased body mass index (BMI) are associated with ADHD and related quantitative traits (inattention and hyperactivity/impulsivity). We screened 32 obesity risk alleles of single nucleotide polymorphisms (SNPs) in a genome-wide association study (GWAS) for ADHD based on 495 patients and 1,300 population-based controls and performed in silico analyses of the SNPs in an ADHD meta-analysis comprising 2,064 trios, 896 independent cases, and 2,455 controls. In the German sample rs206936 in the NUDT3 gene (nudix; nucleoside diphosphate linked moiety X-type motif 3) was associated with ADHD risk (OR: 1.39; P = 3.4 × 10(-4) ; Pcorr  = 0.01). In the meta-analysis data we found rs6497416 in the intronic region of the GPRC5B gene (G protein-coupled receptor, family C, group 5, member B; P = 7.2 × 10(-4) ; Pcorr  = 0.02) as a risk allele for ADHD. GPRC5B belongs to the metabotropic glutamate receptor family, which has been implicated in the etiology of ADHD. In the German sample rs206936 (NUDT3) and rs10938397 in the glucosamine-6-phosphate deaminase 2 gene (GNPDA2) were associated with inattention, whereas markers in the mitogen-activated protein kinase 5 gene (MAP2K5) and in the cell adhesion molecule 2 gene (CADM2) were associated with hyperactivity. In the meta-analysis data, MAP2K5 was associated with inattention, GPRC5B with hyperactivity/impulsivity and inattention and CADM2 with hyperactivity/impulsivity. Our results justify further research on the elucidation of the common genetic background of ADHD and obesity. Show less

To identify susceptibility loci for meningioma, we conducted a genome-wide association study of 859 affected individuals (cases) and 704 controls with validation in two independent sample sets totaling 774 cases and 1,764 controls. We identified a new susceptibility locus for meningioma at 10p12.31 (MLLT10, rs11012732, odds ratio = 1.46, P(combined) = 1.88 × 10(-14)). This finding advances our understanding of the genetic basis of meningioma development. Show less

Evidence is sparse about the genetic determinants of major lipids in Pakistanis. Variants (n=45 000) across 2000 genes were assessed in 3200 Pakistanis and compared with 2450 Germans using the same gene array and similar lipid assays. We also did a meta-analysis of selected lipid-related variants in Europeans. Pakistani genetic architecture was distinct from that of several ethnic groups represented in international reference samples. Forty-one variants at 14 loci were significantly associated with levels of HDL-C, triglyceride, or LDL-C. The most significant lipid-related variants identified among Pakistanis corresponded to genes previously shown to be relevant to Europeans, such as CETP associated with HDL-C levels (rs711752; P<10(-13)), APOA5/ZNF259 (rs651821; P<10(-13)) and GCKR (rs1260326; P<10(-13)) with triglyceride levels; and CELSR2 variants with LDL-C levels (rs646776; P<10(-9)). For Pakistanis, these 41 variants explained 6.2%, 7.1%, and 0.9% of the variation in HDL-C, triglyceride, and LDL-C, respectively. Compared with Europeans, the allele frequency of rs662799 in APOA5 among Pakistanis was higher and its impact on triglyceride concentration was greater (P-value for difference <10(-4)). Several lipid-related genetic variants are common to Pakistanis and Europeans, though they explain only a modest proportion of population variation in lipid concentration. Allelic frequencies and effect sizes of lipid-related variants can differ between Pakistanis and Europeans. Show less

Osteopoikilosis is a rare autosomal dominant genetic disorder, characterised by the occurrence of the hyperostotic spots preferentially localized in the epiphyses and metaphyses of the long bones, and in the carpal and tarsal bones 1. Heterozygous LEMD3 gene mutations were shown to be the primary cause of the disease 2. Association of the primarily asymptomatic osteopokilosis with connective tissue nevi of the skin is categorized as Buschke-Ollendorff syndrome (BOS) 3. Additionally, osteopoikilosis can coincide with melorheostosis (MRO), a more severe bone disease characterised by the ectopic bone formation on the periosteal and endosteal surface of the long bones 456. However, not all MRO affected individuals carry germ-line LEMD3 mutations 7. Thus, the genetic cause of MRO remains unknown. Here we describe a familial case of osteopoikilosis in which a novel heterozygous LEMD3 mutation coincides with a novel mutation in EXT1, a gene involved in aetiology of multiple exostosis syndrome. The patients affected with both LEMD3 and EXT1 gene mutations displayed typical features of the osteopoikilosis. There were no additional skeletal manifestations detected however, various non-skeletal pathologies coincided in this group. We investigated LEMD3 and EXT1 in the three-generation family from Poland, with 5 patients affected with osteopoikilosis and one child affected with multiple exostoses. We found a novel c.2203C > T (p.R735X) mutation in exon 9 of LEMD3, resulting in a premature stop codon at amino acid position 735. The mutation co-segregates with the osteopoikilosis phenotype and was not found in 200 ethnically matched controls. Another new substitution G > A was found in EXT1 gene at position 1732 (cDNA) in Exon 9 (p.A578T) in three out of five osteopoikilosis affected family members. Evolutionary conservation of the affected amino acid suggested possible functional relevance, however no additional skeletal manifestations were observed other then those specific for osteopoikilosis. Finally in one member of the family we found a splice site mutation in the EXT1 gene intron 5 (IVS5-2 A > G) resulting in the deletion of 9 bp of cDNA encoding three evolutionarily conserved amino acid residues. This child patient suffered from a severe form of exostoses, thus a causal relationship can be postulated. We identified a new mutation in LEMD3 gene, accounting for the familial case of osteopoikilosis. In the same family we identified two novel EXT1 gene mutations. One of them A598T co-incided with the LEMD3 mutation. Co-incidence of LEMD3 and EXT1 gene mutations was not associated with a more severe skeletal phenotype in those patients. Show less

Deletions within the neurexin 1 gene (NRXN1; 2p16.3) are associated with autism and have also been reported in two families with schizophrenia. We examined NRXN1, and the closely related NRXN2 and NRXN3 genes, for copy number variants (CNVs) in 2977 schizophrenia patients and 33 746 controls from seven European populations (Iceland, Finland, Norway, Germany, The Netherlands, Italy and UK) using microarray data. We found 66 deletions and 5 duplications in NRXN1, including a de novo deletion: 12 deletions and 2 duplications occurred in schizophrenia cases (0.47%) compared to 49 and 3 (0.15%) in controls. There was no common breakpoint and the CNVs varied from 18 to 420 kb. No CNVs were found in NRXN2 or NRXN3. We performed a Cochran-Mantel-Haenszel exact test to estimate association between all CNVs and schizophrenia (P = 0.13; OR = 1.73; 95% CI 0.81-3.50). Because the penetrance of NRXN1 CNVs may vary according to the level of functional impact on the gene, we next restricted the association analysis to CNVs that disrupt exons (0.24% of cases and 0.015% of controls). These were significantly associated with a high odds ratio (P = 0.0027; OR 8.97, 95% CI 1.8-51.9). We conclude that NRXN1 deletions affecting exons confer risk of schizophrenia. Show less

Impaired fetal movement causes malformations, summarized as fetal akinesia deformation sequence (FADS), and is triggered by environmental and genetic factors. Acetylcholine receptor (AChR) components are suspects because mutations in the fetally expressed gamma subunit (CHRNG) of AChR were found in two FADS disorders, lethal multiple pterygium syndrome (LMPS) and Escobar syndrome. Other AChR subunits alpha1, beta1, and delta (CHRNA1, CHRNB1, CHRND) as well as receptor-associated protein of the synapse (RAPSN) previously revealed missense or compound nonsense-missense mutations in viable congenital myasthenic syndrome; lethality of homozygous null mutations was predicted but never shown. We provide the first report to our knowledge of homozygous nonsense mutations in CHRNA1 and CHRND and show that they were lethal, whereas novel recessive missense mutations in RAPSN caused a severe but not necessarily lethal phenotype. To elucidate disease-associated malformations such as frequent abortions, fetal edema, cystic hygroma, or cardiac defects, we studied Chrna1, Chrnb1, Chrnd, Chrng, and Rapsn in mouse embryos and found expression in skeletal muscles but also in early somite development. This indicates that early developmental defects might be due to somite expression in addition to solely muscle-specific effects. We conclude that complete or severe functional disruption of fetal AChR causes lethal multiple pterygium syndrome whereas milder alterations result in fetal hypokinesia with inborn contractures or a myasthenic syndrome later in life. Show less

Purpose of this work was to judge progress of diagnosed bronchial asthma among children and youth that are exposed on tobacco smoke in aspect of number of exacerbation of disease, therapy (doses of corticosteroids) and number of hospitalizations. There were two groups of patients with bronchial asthma under examination, first one were children and youth that are exposed on tobacco smoke, and the other group of children and youth that are not exposed on tobacco smoke. We discovered statistically significant difference in number of exacerbation of disease among those two groups, also bigger therapeutic and prophylactic doses of inhaled steroids during treatment, and much more often hospitalizations among children and youth that are exposed on tobacco smoke. Show less