The C-terminal extension of the beta 7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation

The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. beta 7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the alpha ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in beta subunit maturation. The effect of blm10 Delta on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4Cterminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.info:eu-repo/semantics/publishedVersio

Common variation in FAM155A is associated with diverticulitis but not diverticulosis

Colonic diverticulosis is a very common condition. Many patients develop diverticulitis or other complications of diverticular disease. Recent genome-wide association studies (GWAS) consistently identified three major genetic susceptibility factors for both conditions, but did not discriminate diverticulititis and diverticulosis in particular due the limitations of registry-based approaches. Here, we aimed to confirm the role of the identified variants for diverticulosis and diverticulitis, respectively, within a well-phenotyped cohort of patients who underwent colonoscopy. Risk variants rs4662344 in Rho GTPase-activating protein 15 (ARHGAP15), rs7609897 in collagen-like tail subunit of asymmetric acetylcholinesterase (COLQ) and rs67153654 in family with sequence similarity 155 A (FAM155A) were genotyped in 1,332 patients. Diverticulosis was assessed by colonoscopy, and diverticulitis by imaging, clinical symptoms and inflammatory markers. Risk of diverticulosis and diverticulitis was analyzed in regression models adjusted for cofactors. Overall, the variant in FAM155A was associated with diverticulitis, but not diverticulosis, when controlling for age, BMI, alcohol consumption, and smoking status (ORadjusted 0.49 [95% CI 0.27-0.89], p = 0.002). Our results contribute to the assessment specific genetic variants identified in GWAS in the predisposition to the development of diverticulitis in patients with diverticulosis

A Variant of COL3A1 (rs3134646) Is Associated With Risk of Developing Diverticulosis in White Men

BACKGROUND: Colonic diverticulosis is one of the most common gastroenterological disorders. Although diverticulosis is typically benign, many individuals develop diverticulitis or other aspects of diverticular disease. Diverticulosis is thought to stem from a complex interaction of environmental, dietary, and genetic factors; however, the contributing genetic factors remain unknown. OBJECTIVE: The aim of our present study was to determine the role of genetic variants within genes encoding for collagens of the connective tissue in diverticulosis. DESIGN: This was a transsectional genetic association study. SETTINGS: This study was conducted at three tertiary referral centers in Germany and Lithuania. PATIENTS: Single-nucleotide polymorphisms in COL3A1 (rs3134646, rs1800255) and COL1A1 (rs1800012) were genotyped in 422 patients with diverticulosis and 285 controls of white descent by using TaqMan assays. MAIN OUTCOME MEASURES: The association of colonoscopy-proven diverticulosis with genetic polymorphisms with herniations was assessed in multivariate models. RESULTS: The rs3134646, rs1800255, and rs1800012 variants were significantly associated with the risk of developing diverticulosis in the univariate model; however, these associations were not significant in the multivariate logistic regression analysis including additional nongenetic variables. When selectively analyzing sexes, the genotype AA (AA) in rs3134646 remained significantly associated with diverticulosis in men (OR, 1.82; 95% CI, 1.04-3.20; p = 0.04). LIMITATIONS: Because a candidate approach was used, additional relevant variants could be missed. Within our cohort of patients with diverticulosis, only a small proportion had diverticular disease and thus, we could not examine the variants in these subgroups. Functional studies, including the analysis of the involved collagens, are also warranted. CONCLUSIONS: Our study shows that a variant of COL3A1 (rs3134646) is associated with the risk of developing colonic diverticulosis in white men, whereas rs1800255 (COL3A1) and rs1800012 (COL1A1) were not associated with this condition after adjusting for confounding factors. Our data provide novel valuable insights in the genetic susceptibility to diverticulosis. See Video Abstract at http://links.lww.com/DCR/A504

Differential miRNA and Protein Expression Reveals miR-1285, Its Targets TGM2 and CDH-1, as Well as CD166 and S100A13 as Potential New Biomarkers in Patients with Diabetes Mellitus and Pancreatic Adenocarcinoma

Early detection of PDAC remains challenging due to the lack of early symptoms and the absence of reliable biomarkers. The aim of the present project was to identify miRNA and proteomics signatures discriminating PDAC patients with DM from nondiabetic PDAC patients. Proteomics analysis and miRNA array were used for protein and miRNA screening. We used Western blotting and Real-Time Quantitative Reverse Transcription polymerase chain reaction (qRT-PCR) for protein and miRNA validation. Comparisons between experimental groups with normal distributions were performed using one-way ANOVA followed by Tukey’s post hoc test, and pairwise tests were performed using t-tests. p ≤ 0.05 was considered statistically significant. Protein clusters of differentiation 166 (CD166), glycoprotein CD63 (CD63), S100 calcium-binding protein A13 (S100A13), and tumor necrosis factor-β (TNF-β) were detected in the proteomics screening. The miRNA assay revealed a differential miRNA 1285 regulation. Previously described target proteins of miR-1285 cadherin-1 (CDH-1), cellular Jun (c-Jun), p53, mothers against decapentaplegic homolog 4 (Smad4), human transglutaminase 2 (TGM2) and yes-associated protein (YAP), were validated via Western blotting. miR-1285-3p was successfully validated as differentially regulated in PDAC + DM via qRT-PCR. Overall, our data suggest miRNA1285-3p, TGM2, CDH-1, CD166, and S100A13 as potential meaningful biomarker candidates to characterize patients with PDAC + DM. Data are available via ProteomeXchange with the identifier PXD053169

Development and Validation of Corynebacterium DNA Microarrays

We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains

Efficacy of stapler versus hand-sewn closure after distal pancreatectomy (DISPACT): a randomised, controlled multicentre trial

Background The ideal closure technique of the pancreas after distal pancreatectomy is unknown. We postulated that standardised closure with a stapler device would prevent pancreatic fistula more effectively than would a hand-sewn closure of the remnant. Methods This multicentre, randomised, controlled, parallel group-sequential superiority trial was done in 21 European hospitals. Patients with diseases of the pancreatic body and tail undergoing distal pancreatectomy were eligible and were randomly assigned by central randomisation before operation to either stapler or hand-sewn closure of the pancreatic remnant. Surgical performance was assessed with intraoperative photo documentation. The primary endpoint was the combination of pancreatic fistula and death until postoperative day 7. Patients and outcome assessors were masked to group assignment. Interim and final analysis were by intention to treat in all patients in whom a left resection was done. This trial is registered, ISRCTN18452029. Findings Between Nov 16, 2006, and July 3, 2009, 450 patients were randomly assigned to treatment groups (221 stapler; 229 hand-sewn closure), of whom 352 patients (177 stapler, 175 hand-sewn closure) were analysed. Pancreatic fistula rate or mortality did not differ between stapler (56 [32%] of 177) and hand-sewn closure (49 [28%] of 175; OR 0.84, 95% CI 0.53-1.33; p=0.56). One patient died within the first 7 days after surgery in the hand-sewn group; no deaths occurred in the stapler group. Serious adverse events did not differ between groups. Interpretation Stapler closure did not reduce the rate of pancreatic fistula compared with hand-sewn closure for distal pancreatectomy. New strategies, including innovative surgical techniques, need to be identified to reduce this adverse outcome