Chronic Alcohol Consumption Inhibits Autophagy and Promotes Apoptosis in the Liver.

Background: Chronic alcohol consumption is a major cause of liver injury. However, the molecular mechanisms by which alcohol impairs hepatocellular function and induces cell death remain unclear. Macroautophagy (hereafter called 'autophagy') is a degradation pathway involved in the survival or death of cells during conditions of cellular stress. This study examines the effect of chronic alcohol consumption on hepatocellular autophagy in an animal model. Methods: During a 12-week period male Wistar rats were fed a Lieber-DeCarli diet containing 5% alcohol (EtOH group; n=10), or an isocaloric diet (control group; n=10). Hepatic expression of key regulatory autophagy proteins (e.g. Beclin-1, ATG-3, ATG-5, p62/SQSTM1 and LC3) were detected by real-time polymerase chain reaction and Western blot analysis. Markers of cellular stress and apoptotic cell death (e.g. HO-1, caspase-3, PARP-1 and Bcl-2) were determined, and levels of reduced and oxidized glutathione were measured. Results: Chronic alcohol consumption caused cellular and oxidative stress in the liver. Transcriptional and translational expression of Beclin-1 and ATG-5 was significantly impaired. The protein expression of LC3-I and LC3-II was significantly increased, while the ratio of LC3I/II remained unchanged in the EtOH group compared with controls. Hepatocellular expression of p62/SQSTM1 and markers of apoptotic cell death (such as cleaved caspase-3 and cleaved PARP-1) were significantly increased in the EtOH group indicating a disrupted autophagic flux and increased rate of apoptosis in the liver. Conclusions: In this model, chronic alcohol consumption impaired hepatocellular autophagy and induced apoptotic cell death. It appears that changes in autophagy might contribute to alcohol-induced structural and functional hepatocellular injury.

Chronic Alcohol Consumption Leads to a Tissue Specific Expression of Uncoupling Protein-2.

Uncoupling proteins (UCPs) are anion channels that can decouple the mitochondrial respiratory chain. "Mild uncoupling" of internal respiration reduces free radical production and oxidative cell stress. Chronic alcohol consumption is a potent inducer of oxidative stress in multiple tissues and regulates UCP-2 and -4 expression in the brain. To analyse the impact of chronic alcohol intake on UCP-2 expression in tissues with high endogenous UCP-2 contents, male Wistar rats (n=34) were treated with a 12-week 5% alcohol diet. In the lungs and the spleen of rats with a chronic alcohol diet cytochrome c release from mitochondria was significantly increased. Both organs did not show any altered gene and protein expression of UCP-2. Different to cerebral tissue chronic alcohol consumption has no regulatory effect on UCP-2 gene and protein expression in organs with a high endogenous UCP-2 content. Therefore, chronic alcohol consumption leads to a tissue specific expression of UCP-2.

Zoonoses research in the German National Cohort : feasibility of parallel sampling of pets and owners.

Cats and dogs live in more than 20 % of German households and the contact between these pets and their owners can be very close. Therefore, a transmission of zoonotic pathogens may occur. To investigate whether zoonotic research questions can be examined in the context of population-based studies like the German National Cohort (GNC), two studies on different study populations were conducted as part of the feasibility tests of the GNC. The aim of the first study was to quantify the actual exposure of participants of the GNC to cats and dogs. In the second study summarised here the feasibility of the sampling of cats and dogs by their owners was tested. To quantify the exposure of participants of the GNC to cats and dogs 744 study participants of the Pretests of the GNC were asked whether they had contact with animals. Currently 10 % have a dog and 14 % have a cat in their household. These figures confirm that a large proportion of the German population has contact with pets and that there is a need for further zoonoses research. To establish the collection of biological samples from cats and dogs in the context of large-scale population-based studies feasible methods are needed. Therefore, a study was conducted to test whether pet owners can take samples from their cats and dogs and whether the quality of these samples is comparable to samples taken by a qualified veterinarian. A total of 82 dog and 18 cat owners were recruited in two veterinary practices in Hannover and the Clinic for Small Animals at the University of Veterinary Medicine in Hannover. Sampling instructions and sample material for nasal and buccal swabs, faecal samples and, in the case of cat owners, a brush for fur samples, were given to the pet owners. The pet owners were asked to take the samples from their pets at home and to send the samples by surface mail. Swab samples were cultured and bacterial growth was quantified independent of bacterial species. The growth of Gram-positive and Gram-negative bacteria from samples taken by the veterinarian and the pet owners were compared. For Gram-positive bacteria the agreement of laboratory results was 71 % for nasal swabs and 78 % for oral swabs while for Gram-negative bacteria the agreement of laboratory results was 55 % for nasal swabs and 87 % for oral swabs. In conclusion it has been shown that participants of the GNC are exposed to cats and dogs and that the sampling of cats and dogs by their owners is a feasible method which can be a useful tool for zoonoses research in population-based studies.

Chronic alcohol consumption increases the expression of uncoupling protein-2 and -4 in the brain.

BACKGROUND: Chronic alcohol consumption leads to oxidative stress in a variety of cells, especially in brain cells because they have a reduced oxidative metabolism of alcohol. Uncoupling proteins (UCPs) are anion channels of the inner mitochondrial membrane, which can decouple internal respiration. "Mild uncoupling" of the mitochondrial respiratory chain leads to a reduced production of free radicals (reactive oxygen species) and a reduction in oxidative cell stress. The extent to which chronic alcohol consumption regulates UCP-2 and -4 in the brain is still unknown. METHODS: We examined the effects of a 12-week 5% alcohol diet in the brain of male Wistar rats (n = 34). Cerebral gene and protein expression of UCP-2, -4, as well as Bcl-2, and the release of cytochrome c out of the mitochondria were detected by real-time polymerase chain reaction and Western blot analysis. The percentage of degenerated cells was determined by Fluoro-Jade B staining of brain slices. RESULTS: Brains of rats with a chronic alcohol diet showed an increased gene and protein expression of UCP-2 and -4. The expression of the antiapoptotic protein Bcl-2 in the brain of the alcohol-treated animals was decreased significantly, whereas cytochrome c release from mitochondria was increased. In addition increased neurodegeneration could be demonstrated in the alcohol-treated animals. CONCLUSIONS: Chronic alcohol consumption leads to a cerebral induction of UCP-2 and -4 with a simultaneous decrease in the antiapoptotic protein Bcl-2, cytochrome c release from mitochondria and increased neurodegeneration. This study reveals a compensatory effect of UCP-2 and -4 in the brain during chronic alcohol consumption.

Global distribution and evolution of a toxinogenic Burkholderia-Rhizopus symbiosis.

Toxinogenic endobacteria were isolated from a collection of Rhizopus spp. representing highly diverse geographic origins and ecological niches. All endosymbionts belonged to the Burkholderia rhizoxinica complex according to matrix-assisted laser desorption ionization-time of flight biotyping and multilocus sequence typing, suggesting a common ancestor. Comparison of host and symbiont phylogenies provides insights into possible cospeciation and horizontal-transmission events.

The zoonotic potential of Clostridium difficile from small companion animals and their owners.

BACKGROUND: Clostridium difficile infections (CDI) in humans range from asymptomatic carriage to life-threatening intestinal disease. Findings on C. difficile in various animal species and an overlap in ribotypes (RTs) suggest potential zoonotic transmission. However, the impact of animals for human CDI remains unclear. METHODS: In a large-scale survey we collected 1,447 fecal samples to determine the occurrence of C. difficile in small companion animals (dogs and cats) and their owners and to assess potential epidemiological links within the community. The Germany-wide survey was conducted from July 2012-August 2013. PCR ribotyping, Multilocus VNTR Analysis (MLVA) and PCR detection of toxin genes were used to characterize isolated C. difficile strains. A database was defined and logistic regression used to identify putative factors associated with fecal shedding of C. difficile. RESULTS: In total, 1,418 samples met the inclusion criteria. The isolation rates for small companion animals and their owners within the community were similarly low with 3.0% (25/840) and 2.9% (17/578), respectively. PCR ribotyping revealed eight and twelve different RTs in animals and humans, respectively, whereas three RTs were isolated in both, humans and animals. RT 014/0, a well-known human hospital-associated lineage, was predominantly detected in animal samples. Moreover, the potentially highly pathogenic RTs 027 and 078 were isolated from dogs. Even though, C. difficile did not occur simultaneously in animals and humans sharing the same household. The results of the epidemiological analysis of factors associated with fecal shedding of C. difficile support the hypothesis of a zoonotic potential. CONCLUSIONS: Molecular characterization and epidemiological analysis revealed that the zoonotic risk for C. difficile associated with dogs and cats within the community is low but cannot be excluded.

Draft Genome Sequences of Symbiotic and Nonsymbiotic Rhizopus microsporus Strains CBS 344.29 and ATCC 62417.

Specific Rhizopus microsporus pathovars harbor bacterial endosymbionts (Burkholderia rhizoxinica) for the production of a phytotoxin. Here, we present the draft genome sequences of two R. microsporus strains, one symbiotic (ATCC 62417), and one endosymbiont-free (CBS 344.29). The gene predictions were supported by RNA sequencing (RNA-seq) data. The functional annotation sets the basis for comparative analyses.

Protocol for the rapid detection of the urogenital tract mollicutes and Chlamydia with concomitant LGV-(sub)typing.

Urogenital tract infections can be caused by a number of pathogens, some of which, like the obligate intracellular Chlamydia trachomatis, are difficult to culture, or the cell wall-less mollicutes, like M. hominis or Ureaplasma spp. Real-time PCR (qPCR) has become an important diagnostic tool as it enables not only the species-specific detection of the organism but also the quantification essential to define the etiological relevance of a facultative pathogenic bacterium. We developed a set of TaqMan qPCRs for the detection of the species M. genitalium and M. hominis (Mh/Mg-duplex qPCR), U. parvum and U. urealyticum (Uu/Up duplex-PCR), and C. trachomatis (CT-qPCR), and for typing of lymphogranuloma venereum-associated L-serovars of C. trachomatis (LGV-qPCR) as well as a sub-typing of L1, L2, and L3. In addition, the human gap-gene was amplified as quality control of the specimen, and a cryptic plasmid co-amplified in CT-qPCR as an inhibition control. The present protocol focuses on the step-by-step description for the establishment of these TaqMan multiplex qPCRs.

Toxin production by bacterial endosymbionts of a Rhizopus microsporus strain used for tempe/sufu processing.

Mould fungi are not only well known for food spoilage through toxin formation but also for the production of fermented foods. In Asian countries, the fermentation of soy beans and tofu for tempe and sufu production with various Rhizopus strains is widespread. Here we report the finding of toxinogenic bacteria in a starter culture used for sufu production. By means of metabolic profiling of the fungus under standard conditions for tempe and sufu production, we found that toxins of the rhizoxin complex are produced in critical amounts. Considering that rhizoxins are severe toxins with strong antimitotic activity it is important to notice that our findings uncover a health-threatening symbiosis in food processing. A simple PCR method for detecting toxinogenic endofungal bacteria in starter cultures is proposed.

Fungal phytotoxins as mediators of virulence.

Many phytopathogenic fungi exert their destructive effects by producing and secreting toxic low molecular weight compounds. In the past years a large number of novel fungal virulence factors and their modes of action have been identified. This review highlights effective phytotoxin-mediated strategies to distress, weaken or kill the plant host.