Phenotypic antimicrobial resistance (AMR) of avian pathogenic Escherichia coli (APEC) from broiler breeder flocks between 2009 and 2018.

Colibacillosis is one of the most important diseases in poultry production. The use of antimicrobials remains a therapeutic cornerstone for avian pathogenic E. coli (APEC), thereby contributing to the development of antimicrobial resistance (AMR). The aim of this study was to characterize AMR in broiler breeder flocks reared under commercial conditions. Data covering 10 years, from 2009 to 2018, were used to evaluate the phenotypic AMR of 264 APEC isolates obtained from 158 broiler breeder flocks of a large company in Portugal. The APEC isolates were tested against eleven antimicrobials by the Kirby-Bauer disc diffusion test. The annual proportion of AMR was calculated by dividing the number of APEC isolates with phenotypic resistance by the total number of APEC isolated that year. Similarly, the overall AMR of the whole period was calculated. The relationship of antimicrobial resistance with time (years) was investigated with a generalized linear model using logistic regression. The overall AMR of the 10-year period was: amoxicillin 78%, ampicillin 73.5%, tetracycline 63.3%, doxycycline 56.4%, apramycin 34.5%, neomycin 68.2%, enrofloxacin 32.6%, flumequine 39.4%, co-trimoxazole 47.7%, florfenicol 46.6% and lincospectin 66.3%. Over time, a significant decrease in AMR was observed for amoxicillin and ampicillin, neomycin, flumequine, co-trimoxazole, florfenicol and lincospectin. Multidrug resistance (MDR) decreased from 100% in 2009 to 48% in 2018. Only seven (2.7%) APEC strains were fully susceptible to all tested antimicrobials. The decrease over time of AMR in APEC likely reflects the efficacy of manifold improvements in broiler breeder production systems. A further reduction in AMR is still desirable. RESEARCH HIGHLIGHTSDecreasing trend of antimicrobial resistance in avian pathogenic E. coli over time.Over 50% of isolates still resistant to amoxicillin, tetracycline and doxycycline.Multidrug resistance decreased from 100% in 2009 to 48% in 2018.Further reduction of antimicrobial resistance in broiler breeders desirable.

Particle size analysis of pristine food-grade titanium dioxide and E 171 in confectionery products: Interlaboratory testing of a single-particle inductively coupled plasma mass spectrometry screening method and confirmation with transmission electron microscopy.

Titanium dioxide is a white colourant authorised as food additive E 171 in the EU, where it is used in a range of alimentary products. As these materials may contain a fraction of particulates with sizes below 100 nm and current EU regulation requires specific labelling of food ingredient to indicate the presence of engineered nanomaterials there is now a need for standardised and validated methods to appropriately size and quantify (nano)particles in food matrices. A single-particle inductively coupled plasma mass spectrometry (spICP-MS) screening method for the determination of the size distribution and concentration of titanium dioxide particles in sugar-coated confectionery and pristine food-grade titanium dioxide was developed. Special emphasis was placed on the sample preparation procedure, crucial to reproducibly disperse the particles before analysis. The transferability of this method was tested in an interlaboratory comparison study among seven experienced European food control and food research laboratories equipped with various ICP-MS instruments and using different software packages. The assessed measurands included the particle mean diameter, the most frequent diameter, the percentage of particles (in number) with a diameter below 100 nm, the particles' number concentration and a number of cumulative particle size distribution parameters (D0, D10, D50, D99.5, D99.8 and D100). The evaluated method's performance characteristics were, the within-laboratory precision, expressed as the relative repeatability standard deviation (RSDr), and the between-laboratory precision, expressed as the relative reproducibility standard deviation (RSDR). Transmission electron microscopy (TEM) was used as a confirmatory technique and served as the basis for bias estimation. The optimisation of the sample preparation step showed that when this protocol was applied to the relatively simple sample food matrices used in this study, bath sonication turned out to be sufficient to reach the highest, achievable degree of dispersed constituent particles. For the pristine material, probe sonication was required. Repeatability and reproducibility were below 10% and 25% respectively for most measurands except for the lower (D0) and the upper (D100) bound of the particle size distribution and the particle number concentration. The broader distribution of the lower and the upper bounds could be attributed to instrument-specific settings/setups (e.g. the timing parameters, the transport efficiency, type of mass-spectrometer) and software-specific data treatment algorithms. Differences in the upper bound were identified as being due to the non-harmonised application of the upper counting limit. Reporting D99.5 or D99.8 instead of the effectively largest particle diameter (D100) excluded isolated large particles and considerably improved the reproducibility. The particle number-concentration was found to be influenced by small differences in the sample preparation procedure. The comparison of these results with those obtained using electron microscopy showed that the mean and median particle diameter was, in all cases, higher when using spICP-MS. The main reason for this was the higher size detection limit for spICP-MS plus the fact that some of the analysed particles remained agglomerated/aggregated after sonication. Single particle ICP-MS is a powerful screening technique, which in many cases provides sufficient evidence to confirm the need to label a food product as containing (engineered) titanium dioxide nanomaterial according to the current EU regulatory requirements. The overall positive outcome of the method performance evaluation and the current lack of alternative standardised procedures, would indicate this method as being a promising candidate for a full validation study.

Comparison of three explorative methods for identifying building surface materials contributing pollutants to stormwater.

Runoff from building and structure surfaces may contribute to the pollution of urban stormwater and, thereby, to the degradation of the receiving water quality. Various micropollutants have been found in surface runoff from buildings in the urban environment, including metals and organic micropollutants. Effective methods for identification of such pollutants and their sources are the prerequisites for the development of control measures. In this paper, three different methods for the identification of building surface materials acting as sources of metals (Cd, Cr, Cu, Ni, Pb and Zn), nonylphenols and phthalates are presented: (i) screening of the material composition, (ii) laboratory leaching experiments with synthetic rainwater, and (iii) open-air pilot testing of material panels exposed to actual rainfall and runoff. These three methods cover a wide span of experimental aspects, including, e.g., size of material samples, resource demands, and control of influential factors. Nine materials commonly used on building and structure surfaces in the urban environment were tested: metal sheets of zinc, copper, galvanised steel, coated corrugated steel and stainless steel; and, four different roofing membranes of bitumen as well as polyvinyl chloride (PVC). The experimental results indicated that all three methods were meritorious in providing some information contributing to the identification of pollutant sources. The screening of material composition for targeted pollutants is relatively quick and inexpensive, but may fail to identify minor sources of pollutants, or may identify the substances present in the material, but not released in contact with water. Laboratory leaching was generally effective in identifying sources of substances present in surface runoff, but was unsuitable for estimating the magnitude of actual concentrations in building runoff. Open-air pilot studies of material samples (exposed area = 2 m2) were thought to provide the results corresponding well to concentrations in runoff from actual building surfaces, but required relatively large financial and labour resources. Thus, the choice of the method for pollutant identification should be based on study objectives, and some benefits may be achieved using more than one method in an integrated manner; e.g., composition screening and lab or open-air leaching of targeted materials.

Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants.

The multi-C2 domain protein otoferlin is required for hearing and mutated in human deafness. Some OTOF mutations cause a mild elevation of auditory thresholds but strong impairment of speech perception. At elevated body temperature, hearing is lost. Mice homozygous for one of these mutations, OtofI515T/I515T, exhibit a moderate hearing impairment involving enhanced adaptation to continuous or repetitive sound stimulation. In OtofI515T/I515T inner hair cells (IHCs), otoferlin levels are diminished by 65%, and synaptic vesicles are enlarged. Exocytosis during prolonged stimulation is strongly reduced. This indicates that otoferlin is critical for the reformation of properly sized and fusion-competent synaptic vesicles. Moreover, we found sustained exocytosis and sound encoding to scale with the amount of otoferlin at the plasma membrane. We identified a 20 amino acid motif including an RXR motif, presumably present in human but not in mouse otoferlin, which reduces the plasma membrane abundance of Ile515Thr-otoferlin. Together, this likely explains the auditory synaptopathy at normal temperature and the temperature-sensitive deafness in humans carrying the Ile515Thr mutation.

Tailored access to the hepatobiliary system in post-bariatric patients: a tertiary care bariatric center experience.

BACKGROUND: In bariatric surgery patients, pancreaticobiliary access via endoscopic retrograde cholangiopancreatography (ERCP) is technically challenging and the optimal approach for the evaluation and treatment of biliary tree-related pathologies has been debated. Besides laparoscopy-assisted ERCP (LA-ERCP) as standard of care, EUS-directed transgastric ERCP (EDGE) and hepaticogastrostomy (HGS) with placement of a fully covered metal stent have emerged as novel techniques. The objective of this study was to evaluate safety and efficacy of three different endoscopic approaches (LA-ERCP, EDGE, and HGS) in bariatric patients. METHODS: In this retrospective review, consecutive patients with Roux-en-Y gastric bypass (RYGB) and Sleeve Gastrectomy (SG) who underwent from 2013 to 2019 a LA-ERCP, an EDGE, or a HGS at a tertiary care reference center for bariatric surgery were analyzed. Patient demographics, type of procedure and indication, data regarding cannulation and therapeutic intervention of the common bile duct (procedure success), and clinical outcomes were analyzed. RESULTS: A total of 19 patients were included. Indications for LA-ERCP, EDGE, or HGS were mostly choledocholithiasis (78.9%) and in a few cases papillitis stenosans. Eight patients (57.1%) with LA-ERCP underwent concomitant cholecystectomy. Procedure success was achieved in 100%. Adverse events (AEs) were identified in 15.7% of patients (all ERCP related). All AEs were rated as moderate and there were no serious AEs. CONCLUSION: This case series indicates that ERCP via a transgastric approach (LA-ERCP, EDGE, or HGS) is a minimally invasive, effective, and feasible method to access the biliary tree in bariatric patients. These techniques offer an appealing alternative treatment option compared to percutaneous transhepatic cholangiography and drainage- or deep enteroscopy-assisted ERCP. In bariatric patients who earlier had a cholecystectomy, EUS-guided techniques were the preferred treatment options for biliary pathologies.

Influence of salt concentration and iodized table salt on the microbiota of fermented cucumbers.

The fermentation of vegetables is a traditional preservation method, that experiences a renaissance even in domestic households. Table salt is added to the fermentation batches to favor the growth of lactic acid bacteria usually. On an industrial scale, the fermentation brine is typically prepared with non-iodized table salt. In our study, we investigated the microbiota of cucumber fermentations using culture-dependent and -independent methods. We could show that the fermentation process of cucumbers and the involved microbiota is influenced by the concentration of table salt and not by the use of iodized table salt. Therefore, we conclude that the use of iodized table salt does not negatively affect the fermentation process. We could verify that iodine permeates the cucumbers by diffusion, leading to satisfactory iodine concentrations in the final food product. The industrial use of iodized table salt in food fermentations could contribute to maintain a constant iodine supply to the general public.

The valuation of forest ecosystem services as a tool for management planning - A choice experiment.

Forest owners and managers deal with an increasing demand for forest ecosystem services (ES). In addition, a recent change can be observed from a governmental top-down approach to bottom-up initiatives, including efforts of the local population to have a say in forest management decisions. Matching supply and demand is seen as a basic condition for the sustainable utilization of forest ES. Against this background, we address the following research questions: (i) How can the preferences on the supply and demand side of forest ES be consistently determined? (ii) In how far do these preferences vary due to regional and societal differences? (iii) How can the supply and demand of forest ES be matched by forest management alternatives? We conducted a survey in Switzerland with foresters and the wider population to compare attitudes and preferences of the supply and demand side of forest ES. The core of the study is a choice experiment (CE) to elicit the population's willingness to pay (WTP) for specific forest management alternatives, and the respective willingness to accept (WTA) on the foresters' side. To address spatial and societal heterogeneity, we compare different geographic forest zones and settlement areas.

Epidemiology of Human Parechovirus Type 3 Upsurge in 2 Hospitals, Freiburg, Germany, 2018.

In 2018, a cluster of pediatric human parechovirus (HPeV) infections in 2 neighboring German hospitals was detected. Viral protein 1 sequence analysis demonstrated co-circulation of different HPeV-3 sublineages and of HPeV-1 and -5 strains, thereby excluding a nosocomial outbreak. Our findings underline the need for HPeV diagnostics and sequence analysis for outbreak investigations.

Influence of iodized table salt on fermentation characteristics and bacterial diversity during sauerkraut fermentation.

The effect of iodine present in 1.0% table salt in combination with the use of starter cultures in sauerkraut fermentations were investigated in order to determine whether iodine interferes with lactic acid bacteria responsible for the fermentation. The effect of iodine was tested in fermentations performed using selected starter cultures or without starters (spontaneous fermentation). Lactobacillus plantarum and Leuconostoc mesenteroides used as starters at levels of ca. 1 × 107 cfu ml-1 led to a quick establishment of lactic acid bacteria (LAB) as predominant microorganisms, reaching 1 × 109 cfu ml-1 after 24 h decreasing the pH to below 4.0. In contrast, LAB counts in control fermentations without starters increased slower from 1 × 105 cfu ml-1 to 1 × 109 cfu ml-1 and a pH reduction below 4.0 was achieved only after 3 days fermentation. A metagenomic investigation showed a more diverse bacterial community in fermentations without starters, consisting of enterobacteria and pseudomonads in the first days of fermentation, and of LAB such as lactococci in the later stages. In fermentations with starters, lactobacilli predominated. Leuconostocs also occurred, but at much lower sequence abundance than lactobacilli, and thus were not able to predominate. Determination of iodine in the fermentation with starter bacteria and with iodized salt showed that the fermentation did not affect iodine concentration. The use of iodized salt did not statistically significantly influence microbial populations in the fermentation. Thus, there is no basis for the popular held belief that the use of iodized salt inhibits the growth of the bacteria important for the sauerkraut fermentation. A statistically near significant effect (p = 0.06), however, was noted for the effect of iodine on yeasts and mould populations in the fermentations performed without starter cultures. As sauerkraut is usually produced without starters, this should be further investigated.

Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines.

Allicin (diallyl thiosulfinate) from garlic is a highly potent natural antimicrobial substance. It inhibits growth of a variety of microorganisms, among them antibiotic-resistant strains. However, the precise mode of action of allicin is unknown. Here, we show that growth inhibition of Escherichia coli during allicin exposure coincides with a depletion of the glutathione pool and S-allylmercapto modification of proteins, resulting in overall decreased total sulfhydryl levels. This is accompanied by the induction of the oxidative and heat stress response. We identified and quantified the allicin-induced modification S-allylmercaptocysteine for a set of cytoplasmic proteins by using a combination of label-free mass spectrometry and differential isotope-coded affinity tag labeling of reduced and oxidized thiol residues. Activity of isocitrate lyase AceA, an S-allylmercapto-modified candidate protein, is largely inhibited by allicin treatment in vivo Allicin-induced protein modifications trigger protein aggregation, which largely stabilizes RpoH and thereby induces the heat stress response. At sublethal concentrations, the heat stress response is crucial to overcome allicin stress. Our results indicate that the mode of action of allicin is a combination of a decrease of glutathione levels, unfolding stress, and inactivation of crucial metabolic enzymes through S-allylmercapto modification of cysteines.

Delta-Like Ligand 4 Modulates Liver Damage by Down-Regulating Chemokine Expression.

Disrupting Notch signaling ameliorates experimental liver fibrosis. However, the role of individual Notch ligands in liver damage is unknown. We investigated the effects of Delta-like ligand 4 (Dll4) in liver disease. DLL4 expression was measured in 31 human liver tissues by immunohistochemistry. Dll4 function was examined in carbon tetrachloride- and bile duct ligation-challenged mouse models in vivo and evaluated in hepatic stellate cells, hepatocytes, and Kupffer cells in vitro. DLL4 was expressed in patients' Kupffer and liver sinusoidal endothelial cells. Recombinant Dll4 protein (rDll4) ameliorated hepatocyte apoptosis, inflammation, and fibrosis in mice after carbon tetrachloride challenge. In vitro, rDll4 significantly decreased lipopolysaccharide-dependent chemokine expression in both Kupffer and hepatic stellate cells. In bile duct ligation mice, rDll4 induced massive hepatic necrosis, resulting in the death of all animals within 1 week. Inflammatory cell infiltration and chemokine ligand 2 (Ccl2) expression were significantly reduced in rDll4-receiving bile duct ligation mice. Recombinant Ccl2 rescued bile duct ligation mice from rDll4-mediated death. In patients with acute-on-chronic liver failure, DLL4 expression was inversely associated with CCL2 abundance. Mechanistically, Dll4 regulated Ccl2 expression via NF-κB. Taken together, Dll4 modulates liver inflammatory response by down-regulating chemokine expression. rDll4 application results in opposing outcomes in two models of liver damage. Loss of DLL4 may be associated with CCL2-mediated cytokine storm in patients with acute-on-chronic liver failure.

Making the invisible visible: a systematic review of sexual minority women's health in Southern Africa.

BACKGROUND: Over the past two decades research on sexual and gender minority (lesbian, gay, bisexual and transgender; LGBT) health has highlighted substantial health disparities based on sexual orientation and gender identity in many parts of the world. We systematically reviewed the literature on sexual minority women's (SMW) health in Southern Africa, with the objective of identifying existing evidence and pointing out knowledge gaps around the health of this vulnerable group in this region. METHODS: A systematic review of publications in English, French, Portuguese or German, indexed in PubMed or MEDLINE between the years 2000 and 2015, following PRISMA guidelines. Additional studies were identified by searching bibliographies of identified studies. Search terms included (Lesbian OR bisexual OR "women who have sex with women"), (HIV OR depression OR "substance use" OR "substance abuse" OR "mental health" OR suicide OR anxiety OR cancer), and geographical specification. All empirical studies that used quantitative or qualitative methods, which contributed to evidence for SMW's health in one, a few or all of the countries, were included. Theoretical and review articles were excluded. Data were extracted independently by 2 researchers using predefined data fields, which included a risk of bias/quality assessment. RESULTS: Of 315 hits, 9 articles were selected for review and a further 6 were identified through bibliography searches. Most studies were conducted with small sample sizes in South Africa and focused on sexual health. SMW included in the studies were racially and socio-economically heterogeneous. Studies focused predominately on young populations, and highlighted substance use and violence as key health issues for SMW in Southern Africa. CONCLUSIONS: Although there are large gaps in the literature, the review highlighted substantial sexual-orientation-related health disparities among women in Southern Africa. The findings have important implications for public health policy and research, highlighting the lack of population-level evidence on the one hand, and the impact of criminalizing laws around homosexuality on the other hand.

Hepatocyte fate upon TGF-ß challenge is determined by the matrix environment.

Primary hepatocytes are a versatile tool to investigate all aspects of liver function, and frequently used in drug development and testing. Upon TGF-ß challenge, hepatocytes either undergo an epithelial to mesenchymal transition (EMT) or apoptosis: culture on stiff collagen (monolayer) results in EMT whereas hepatocytes in a soft collagen matrix (sandwich) undergo programmed cell death. In this study, we analyzed the transcriptional programs triggered by TGF-ß under different culture conditions. Our results indicate that TGF-ß initiates an identical transcription profile in hepatocytes irrespective of the cellular environment. The fact that we nevertheless observe two vastly different phenotypes indicates that the matrix environment rather than the TGF-ß induced expression signature is the major determinant of the hepatocellular response. To confirm the impact of the surrounding matrix environment on the hepatocytes׳ phenotype in response to TGF-ß signaling, we studied the effect of Snail overexpression and knockout in both culture conditions. Neither genetic manipulation showed an impact on hepatocytes' fate upon TGF-ß treatment, confirming the crucial role of the surrounding matrix. Our findings provide novel insights into the hepatocellular basis of the fate decision between EMT and apoptotic cell death, and might explain why liver cells can react in very different manners to identical stimuli when tissue remodeling has changed the matrix environment, as occurs in a fibrotic liver.

Coordinated expression of fdxD and molybdenum nitrogenase genes promotes nitrogen fixation by Rhodobacter capsulatus in the presence of oxygen.

Rhodobacter capsulatus is able to grow with N2 as the sole nitrogen source using either a molybdenum-dependent or a molybdenum-free iron-only nitrogenase whose expression is strictly inhibited by ammonium. Disruption of the fdxD gene, which is located directly upstream of the Mo-nitrogenase genes, nifHDK, abolished diazotrophic growth via Mo-nitrogenase at oxygen concentrations still tolerated by the wild type, thus demonstrating the importance of FdxD under semiaerobic conditions. In contrast, FdxD was not beneficial for diazotrophic growth depending on Fe-nitrogenase. These findings suggest that the 2Fe2S ferredoxin FdxD specifically supports the Mo-nitrogenase system, probably by protecting Mo-nitrogenase against oxygen, as previously shown for its Azotobacter vinelandii counterpart, FeSII. Expression of fdxD occurred under nitrogen-fixing conditions, but not in the presence of ammonium. Expression of fdxD strictly required NifA1 and NifA2, the transcriptional activators of the Mo-nitrogenase genes, but not AnfA, the transcriptional activator of the Fe-nitrogenase genes. Expression of the fdxD and nifH genes, as well as the FdxD and NifH protein levels, increased with increasing molybdate concentrations. Molybdate induction of fdxD was independent of the molybdate-sensing regulators MopA and MopB, which repress anfA transcription at micromolar molybdate concentrations. In this report, we demonstrate the physiological relevance of an fesII-like gene, fdxD, and show that the cellular nitrogen and molybdenum statuses are integrated to control its expression.

Redox proteomics uncovers peroxynitrite-sensitive proteins that help Escherichia coli to overcome nitrosative stress.

Peroxynitrite is a highly reactive chemical species with antibacterial properties that are synthesized in immune cells. In a proteomic approach, we identified specific target proteins of peroxynitrite-induced modifications in Escherichia coli. Although peroxynitrite caused a fairly indiscriminate nitration of tyrosine residues, reversible modifications of protein thiols were highly specific. We used a quantitative redox proteomic method based on isotope-coded affinity tag chemistry and identified four proteins consistently thiol-modified in cells treated with peroxynitrite as follows: AsnB, FrmA, MaeB, and RidA. All four were required for peroxynitrite stress tolerance in vivo. Three of the identified proteins were modified at highly conserved cysteines, and MaeB and FrmA are known to be directly involved in the oxidative and nitrosative stress response in E. coli. In in vitro studies, we could show that the activity of RidA, a recently discovered enamine/imine deaminase, is regulated in a specific manner by the modification of its single conserved cysteine. Mutation of this cysteine 107 to serine generated a constitutively active protein that was not susceptible to peroxynitrite.

GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida.

Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse Pseudomonas putida that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system's activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators. All of them were applied not only to directly control Cas9 and its lethal effects, but also to modulate the expression of two of its inhibitors: the AcrIIA4 anti-CRISPR protein and the transcriptional repressor TetR. Either upon direct induction of the endonuclease or under non-induced conditions of its inhibitors, the presence of Cas9 suppressed cell survival which could be exploited beyond biocontainment in situations where further CRISPR genome editing is undesirable.

Pancreatic beta-cell IL-22 receptor deficiency induces age-dependent dysregulation of insulin biosynthesis and systemic glucose homeostasis.

The IL-22RA1 receptor is highly expressed in the pancreas, and exogenous IL-22 has been shown to reduce endoplasmic reticulum and oxidative stress in human pancreatic islets and promote secretion of high-quality insulin from beta-cells. However, the endogenous role of IL-22RA1 signaling on these cells remains unclear. Here, we show that antibody neutralisation of IL-22RA1 in cultured human islets leads to impaired insulin quality and increased cellular stress. Through the generation of mice lacking IL-22ra1 specifically on pancreatic alpha- or beta-cells, we demonstrate that ablation of murine beta-cell IL-22ra1 leads to similar decreases in insulin secretion, quality and islet regeneration, whilst increasing islet cellular stress, inflammation and MHC II expression. These changes in insulin secretion led to impaired glucose tolerance, a finding more pronounced in female animals compared to males. Our findings attribute a regulatory role for endogenous pancreatic beta-cell IL-22ra1 in insulin secretion, islet regeneration, inflammation/cellular stress and appropriate systemic metabolic regulation.

Liver and pancreatic-targeted interleukin-22 as a therapeutic for metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the most prevalent cause of liver disease worldwide, with a single approved therapeutic. Previous research has shown that interleukin-22 (IL-22) can suppress ß-cell stress, reduce local islet inflammation, restore appropriate insulin production, reverse hyperglycemia, and ameliorate insulin resistance in preclinical models of diabetes. In clinical trials long-acting forms of IL-22 have led to increased proliferation in the skin and intestine, where the IL-22RA1 receptor is highly expressed. To maximise beneficial effects whilst reducing the risk of epithelial proliferation and cancer, we designed short-acting IL-22-bispecific biologic drugs that successfully targeted the liver and pancreas. Here we show 10-fold lower doses of these bispecific biologics exceed the beneficial effects of native IL-22 in multiple preclinical models of MASH, without off-target effects. Treatment restores glycemic control, markedly reduces hepatic steatosis, inflammation, and fibrogenesis. These short-acting IL-22-bispecific targeted biologics are a promising new therapeutic approach for MASH.

Nonnative disulfide bond formation activates the σ32-dependent heat shock response in Escherichia coli.

Formation of nonnative disulfide bonds in the cytoplasm, so-called disulfide stress, is an integral component of oxidative stress. Quantification of the extent of disulfide bond formation in the cytoplasm of Escherichia coli revealed that disulfide stress is associated with oxidative stress caused by hydrogen peroxide, paraquat, and cadmium. To separate the impact of disulfide bond formation from unrelated effects of these oxidative stressors in subsequent experiments, we worked with two complementary approaches. We triggered disulfide stress either chemically by diamide treatment of cells or genetically in a mutant strain lacking the major disulfide-reducing systems TrxB and Gor. Studying the proteomic response of E. coli exposed to disulfide stress, we found that intracellular disulfide bond formation is a particularly strong inducer of the heat shock response. Real-time quantitative PCR experiments showed that disulfide stress induces the heat shock response in E. coli σ(32) dependently. However, unlike heat shock treatment, which induces these genes transiently, transcripts of σ(32)-dependent genes accumulated over time in disulfide stress-treated cells. Analyzing the stability of σ(32), we found that this constant induction can be attributed to an increase of the half-life of σ(32) upon disulfide stress. This is concomitant with aggregation of E. coli proteins treated with diamide. We conclude that oxidative stress triggers the heat shock response in E. coli σ(32) dependently. The component of oxidative stress responsible for the induction of heat shock genes is disulfide stress. Nonnative disulfide bond formation in the cytoplasm causes protein unfolding. This stabilizes σ(32) by preventing its DnaK- and FtsH-dependent degradation.

Selective and programmed cleavage of GPI-anchored proteins from the surface membrane by phospholipase C.

Many surface proteins of eukaryotic cells are tethered to the membrane by a GPI-anchor which is enzymatically cleavable. Here, we investigate cleavage and release of different GPI-proteins by phospholipase C from the outer membrane of the ciliate Paramecium tetraurelia. Our data indicate that different GPI-proteins are not equally cleaved as proteins of the surface antigen family are preferentially released in vitro compared to several smaller GPI-proteins. Likewise, the analysis of culture medium indicates exclusive in vivo release of surface antigens by two phospholipase C isoforms (PLC2 and PLC6). This suggests that phospholipase C shows affinity for select groups of GPI-anchored proteins. Our data also reveal an up-regulation of PLC isoforms in GPI-anchored protein cleavage during antigenic switching. As a consequence, silencing of these PLCs leads to a drastic decrease of antigen concentration in the medium. These results suggest a higher order of GPI-regulation by phospholipase C as cleavage occurs programmed and specific for single GPI-proteins instead of an unspecific shedding of the entire surface membrane GPI-content.