Alcohol-induced autophagy contributes to loss in skeletal muscle mass

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.     

Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Background

The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates.

Methodology and Principal Findings

SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes.

Significance

This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.     

Adipocyte hypertrophy is associated with lysosomal permeability both in vivo and in vitro: role in adipose tissue inflammation

Obesity in both humans and rodents is characterized by adipocyte hypertrophy and the presence of death adipocytes surrounded by macrophages forming "crown-like structures." However, the biochemical pathways involved in triggering adipocyte death as well as the role of death adipocytes in adipose tissue remodeling and macrophage infiltration remain poorly understood. We now show that induction of adipocyte hypertrophy by incubation of mature adipocytes with saturated fatty acids results in lysosomal destabilization and cathepsin B (ctsb), a key lysosomal cysteine protease, activation and redistribution into the cytosol. ctsb activation was required for the lysosomal permeabilization, and its inhibition protected cells against mitochondrial dysfunction. With the use of a dietary murine model of obesity, ctsb activation was detected in adipose tissue of these mice. This is an early event during weight gain that correlates with the presence of death adipocytes, and precedes macrophage infiltration of adipose tissue. Moreover, ctsb-deficient mice showed decreased lysosomal permeabilization in adipocytes and were protected against adipocyte cell death and macrophage infiltration to adipose tissue independent of body weight. These data strongly suggest that ctsb activation and lysosomal permeabilization in adipocytes are key initial events that contribute to the adipocyte cell death and macrophage infiltration into adipose tissue associated with obesity. Inhibition of ctsb activation may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.     

Prevalence of HIV,Hepatitis B and C Infections and an Assessment of HCV-Genotypes and Two IL28B SNPs among People Who Inject Drugs in Three Regions of Nepal

As part of a comprehensive health care programme for people who use drugs in Nepal, HIV and viral hepatitis B and C status—including risk factors, HCV-genotypes and co-infections—as well as two IL28B Single-nucleotide polymorphisms (SNPs) were assessed for a random sample of 401 people who inject drugs in three regions of Nepal: mid-western Terrai (Nepalgunj), the eastern region (Dharan, Biratnagar) and the central region (Kathmandu, Lalitpur and Chitwan). Individuals were included who showed at least a minimum of health care seeking behaviour. This latter criterion was defined by being registered with any organisation offering health services. The average age of the participants was 30.5 yrs, and the average length of intravenous drug use was 8.5 yrs. The prevalence rates of HBsAg, anti-HIV antibodies and HCV-RNA were 3.5%, 13.8% and 41.9%, respectively. Spontaneous HCV clearance was evident in 16% of all of those who tested positive for anti-HCV antibodies. Independent risk factors for HCV-RNA positivity were age, gender, geographical region, duration of injecting drug use, history of imprisonment and HIV co-infection. In the age group ≤24 yrs, the rate of spontaneous HCV clearance was 43.5%. Overall, 59.8% of HCV infections were caused by HCV genotype 3 and 40.2% by HCV genotype 1. No other HCV genotypes were identified in this study. The IL28B SNP rs12979860 and rs8099917 were identified in 122 patients, and 75.4% of all participants had both favourable genotypes rs12979860 C/C and rs8099917 T/T.     

Characterization of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Goose Feces from State Parks in Northeast Ohio

Staphylococcus aureus can colonize a range of species. Although numerous studies have isolated pathogenic bacteria from wild birds, very little is known regarding S. aureus and their potential to spread methicillin-resistant (MRSA) strains. The objective of this study was to determine the presence and molecular characteristics of S. aureus in geese fecal samples collected from ten state parks across Northeast Ohio (NEO). A total of 182 fecal samples from Canada geese (Branta canadensis) were collected in April 2015. Isolates were characterized using multi-locus sequence (MLST) and spa typing, as well as PCR to detect the presence of Panton–Valentine leukocidin (PVL), mecA, and scn genes. Antibiotic susceptibility testing was done via Vitek-2 system. The overall contamination by S. aureus in fecal samples was 7.1% (13/182); 7/182 (3.8%) were MRSA and 6/182 (3.3%) were methicillin-susceptible S. aureus (MSSA). One isolate was positive for PVL. A total of eight different spa types were observed. MLST included ST5, ST8, ST291, ST298, and ST2111. One (7.7%) MSSA isolate was multi-drug resistant. The S. aureus contamination in NEO state parks ranged from 0% (park 1, 4, 8, 9) to 35% (7/20) (park 5). Parks 2, 3, 6, and 7 had 5% (1/20) positive. The results of this study indicate that the feces of geese collected at various state parks in NEO may harbor S. aureus.     

Geographic distribution of livestock-associated Staphylococcus aureus in the United States

Sampling for livestock-associated Staphylococcus aureus (LA-SA) in the United States is haphazard. The diversity of LA-S. aureus in the U.S. appears to be greater than in other countries. We review the epidemiology of LA-S. aureus in U.S. pigs, occupationally-exposed individuals, and environmental samples to assess the diversity and abundance of U.S. LA-S. aureus.