Preventing microalbuminuria with benazepril,valsartan, and benazepril–valsartan combination therapy in diabetic patients with high-normal albuminuria: A prospective,randomized, open-label,blinded endpoint (PROBE) study

BackgroundAngiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) prevent microalbuminuria in normoalbuminuric type 2 diabetic patients. We assessed whether combined therapy with the 2 medications may prevent microalbuminuria better than ACE inhibitor or ARB monotherapy.Methods and findingsVARIETY was a prospective, randomized, open-label, blinded endpoint (PROBE) trial evaluating whether, at similar blood pressure (BP) control, combined therapy with benazepril (10 mg/day) and valsartan (160 mg/day) would prevent microalbuminuria more effectively than benazepril (20 mg/day) or valsartan (320 mg/day) monotherapy in 612 type 2 diabetic patients with high-normal albuminuria included between July 2007 and April 2013 by the Istituto di Ricerche Farmacologiche Mario Negri IRCCS and 8 diabetology or nephrology units in Italy. Time to progression to microalbuminuria was the primary outcome. Analyses were intention to treat. Baseline characteristics were similar among groups. During a median [interquartile range, IQR] follow-up of 66 [42 to 83] months, 53 patients (27.0%) on combination therapy, 57 (28.1%) on benazepril, and 64 (31.8%) on valsartan reached microalbuminuria. Using an accelerated failure time model, the estimated acceleration factors were 1.410 (95% CI: 0.806 to 2.467, P = 0.229) for benazepril compared to combination therapy, 0.799 (95% CI: 0.422 to 1.514, P = 0.492) for benazepril compared to valsartan, and 1.665 (95% CI: 1.007 to 2.746, P = 0.047) for valsartan compared to combination therapy. Between-group differences in estimated acceleration factors were nonsignificant after adjustment for predefined confounders. BP control was similar across groups. All treatments were safe and tolerated well, with a slight excess of hyperkalemia and hypotension in the combination therapy group. The main study limitation was the lower than expected albuminuria at inclusion.ConclusionsRisk/benefit profile of study treatments was similar. Dual renin–angiotensin system (RAS) blockade is not recommended as compared to benazepril or valsartan monotherapy for prevention of microalbuminuria in normoalbuminuric type 2 diabetic patients.Trial registrationEudraCT 2006-005954-62; ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00503152","term_id":"NCT00503152"}}NCT00503152.

Piero Ruggenenti and co-workers study prevention of microalbuminuria in patients with type 2 diabetes.     

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients’ fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.     

In vivo effects of abolishing the single canonical sumoylation site in the C-terminal region of Drosophila p53

Using yeast two-hybrid screens we determined that Drosophila (Dm)p53 interacts with proteins involved in sumoylation (UBA2, UBC9 and PIAS) through different regions of its C-terminal domain. A K302R point mutation within a single canonical sumoylation site of Dmp53 did not abolish the observed interactions. These observations prompted us to analyze whether Dmp53 sumoylation at this site has any functional role in vivo. Genetic assays showed that deleting one copy of genes involved in sumoylation (lwr, Su(var)2-10 or smt3 heterozygosity) enhanced slightly the mutator phenotype of Dmp53. We compared the in vivo effects of wild type and K302R Dmp53 overproduced from transgenes and determined that similar levels of expression of the mutant and wild type proteins resulted in similar phenotype, and the two proteins showed similar cellular localization. The half life and the trans-activator activity of K302R mutant and wild type Dmp53 were also comparable. Lastly, by analyzing wild type and K302R Dmp53 expressed at different levels in animals and in S2 cells we detected no differences between the mobility of the mutant and wild-type protein. From these data we conclude that under normal developmental conditions the loss of SUMO modification at K302 does not affect Dmp53 function significantly.     

Carabid patterns in olive orchards and woody semi-natural habitats: first implications for conservation biological control against <Emphasis Type="Italic">Bactrocera oleae</Emphasis>

The role of carabids (Coleoptera: Carabidae) for pest control service in perennial crop systems has been scarcely investigated. We addressed this knowledge gap exploring activity patterns and traits of adult carabids dwelling olive orchard agroecosystems as potential natural enemies of third instar larvae and pupae of Bactrocera oleae (Diptera: Tephritidae). Olive orchard supported a well-structured carabid assemblage, whose species phenologies revealed a temporal overlapping within the pest cycle. The assemblage of adjacent woody semi-natural habitats is more of conservation interest, but may play a weaker role in B. oleae control provisioning. We suggest the identification of carabids main traits for B. oleae conservation biological control as a cost-effective strategy for addressing future attention and resources only to those predators that satisfy basic requirements. This research may open new scenarios on management interventions to both conserve predators and encourage alternative approaches against olive orchards pest.     

Diagnostic and prognostic potential of the proteomic profiling of serum-derived extracellular vesicles in prostate cancer

Extracellular vesicles (EVs) and their cargo represent an intriguing source of cancer biomarkers for developing robust and sensitive molecular tests by liquid biopsy. Prostate cancer (PCa) is still one of the most frequent and deadly tumor in men and analysis of EVs from biological fluids of PCa patients has proven the feasibility and the unprecedented potential of such an approach. Here, we exploited an antibody-based proteomic technology, i.e. the Reverse-Phase Protein microArrays (RPPA), to measure key antigens and activated signaling in EVs isolated from sera of PCa patients. Notably, we found tumor-specific protein profiles associated with clinical settings as well as candidate markers for EV-based tumor diagnosis. Among others, PD-L1, ERG, Integrin-β5, Survivin, TGF-β, phosphorylated-TSC2 as well as partners of the MAP-kinase and mTOR pathways emerged as differentially expressed endpoints in tumor-derived EVs. In addition, the retrospective analysis of EVs from a 15-year follow-up cohort generated a protein signature with prognostic significance. Our results confirm that serum-derived EV cargo may be exploited to improve the current diagnostic procedures while providing potential prognostic and predictive information. The approach proposed here has been already applied to tumor entities other than PCa, thus proving its value in translational medicine and paving the way to innovative, clinically meaningful tools.Subject terms: Tumour biomarkers, Protein-protein interaction networks     

Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field

For base-paired nucleic acids, variations in ¹ J _NH and the imino ¹H chemical shift are both dominated by hydrogen bond length. In the absence of molecular alignment, the ¹ J _NH coupling for the imino proton then can be approximated by ¹ J _NH = (1.21Hz/ppm)δ_H − 103.5 ± 0.6 Hz, where δ_H represents the chemical shift of the imino proton in ppm. This relation permits imino residual dipolar couplings (RDCs) resulting from magnetic susceptibility anisotropy (MSA) to be extracted from measurement of (¹ J _NH + RDC) splittings at a single magnetic field strength. Magnetic field-induced RDCs were measured for tRNA^Val and the alignment tensor determined from magnetic-field alignment of tRNA^Val agrees well with the tensor calculated by summation of the MSA tensors of the individual nucleobases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jinfa Ying, Alexander Grishaev and Michael P. Latham contributed equally to this work.     

Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix alphaA of the extracellular calcium-binding domain of SPARC and is partially masked by helix alphaC. Previously, we found that the removal of helix alphaC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site approximately 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven alpha2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among alpha chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.     

A Legionella pneumophila Kinase Phosphorylates the Hsp70 Chaperone Family to Inhibit Eukaryotic Protein Synthesis

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