Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

Abstract Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1'. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.     

Galectin‐3 binds Neisseria meningitidis and increases interaction with phagocytic cells

Galectin‐3 is expressed and secreted by immune cells and has been implicated in multiple aspects of the inflammatory response. It is a glycan binding protein which can exert its functions within cells or exogenously by binding cell surface ligands, acting as a molecular bridge or activating signalling pathways. In addition, this lectin has been shown to bind to microorganisms. In this study we investigated the interaction between galectin‐3 and Neisseria meningitidis, an important extracellular human pathogen, which is a leading cause of septicaemia and meningitis. Immunohistochemical analysis indicated that galectin‐3 is expressed during meningococcal disease and colocalizes with bacterial colonies in infected tissues from patients. We show that galectin‐3 binds to N. meningitidis and we demonstrate that this interaction requiresfull‐length, intact lipopolysaccharide molecules. We found that neither exogenous nor endogenous galectin‐3 contributes to phagocytosis of N. meningitidis; instead exogenous galectin‐3 increases adhesion to monocytes and macrophages but not epithelial cells. Finally we used galectin‐3 deficient (Gal‐3^?/?) mice to evaluate the contribution of galectin‐3 to meningococcal bacteraemia. We found that Gal‐3^?/? mice had significantly lower levels of bacteraemia compared with wild‐type mice after challenge with live bacteria, indicating that galectin‐3 confers an advantage to N. meningitidis during systemic infection.     

Access rate to the emergency department for venous thromboembolism in relationship with coarse and fine particulate matter air pollution

Particulate matter (PM) air pollution has been associated with cardiovascular and respiratory disease. Recent studies have proposed also a link with venous thromboembolism (VTE) risk. This study was aimed to evaluate the possible influence of air pollution-related changes on the daily flux of patients referring to the Emergency Department (ED) for VTE, dissecting the different effects of coarse and fine PM. From July 1(st), 2007, to June 30(th), 2009, data about ED accesses for VTE and about daily concentrations of PM air pollution in Verona district (Italy) were collected. Coarse PM (PM(10-2.5)) was calculated by subtracting the finest PM(2.5) from the whole PM(10). During the index period a total of 302 accesses for VTE were observed (135 males and 167 females; mean age 68.3 ± 16.7 years). In multiple regression models adjusted for other atmospheric parameters PM(10-2.5), but not PM(2.5), concentrations were positively correlated with VTE (beta-coefficient = 0.237; P = 0.020). During the days with high levels of PM(10-2.5) (≥ 75(th) percentile) there was an increased risk of ED accesses for VTE (OR 1.69 with 95%CI 1.13-2.53). By analysing days of exposure using distributed lag non-linear models, the increase of VTE risk was limited to PM(10-2.5) peaks in the short-term period. Consistently with these results, in another cohort of subjects without active thrombosis (n = 102) an inverse correlation between PM(10-2.5) and prothrombin time was found (R = -0.247; P = 0.012). Our results suggest that short-time exposure to high concentrations of PM(10-2.5) may favour an increased rate of ED accesses for VTE through the induction of a prothrombotic state.     

A catalytic mechanism for cysteine N-terminal nucleophile hydrolases, as revealed by free energy simulations

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors.     

Complete genome sequence of a novel picobirnavirus, otarine picobirnavirus, discovered in California sea lions

We discovered a novel otarine picobirnavirus in fecal samples of California sea lions. Its genome contains a large segment with two open reading frames (ORFs), ORF1 encoding a putative protein of 163 amino acids with unknown function and ORF2 encoding capsid protein, and a small segment with one ORF encoding RNA-dependent RNA polymerase.     

MUC1 gene polymorphism in three Nelore lines selected for growth and its association with growth and carcass traits

The objective of this study was to describe the VNTR polymorphism of the mucin 1 gene (MUC1) in three Nelore lines selected for yearling weight to determine whether allele and genotype frequencies of this polymorphism were affected by selection for growth. In addition, the effects of the polymorphism on growth and carcass traits were evaluated. Birth, weaning and yearling weights, rump height, Longissimus muscle area, backfat thickness, and rump fat thickness, were analyzed. A total of 295 Nelore heifers from the Beef Cattle Research Center, Instituto de Zootecnia de Sert?ozinho, were used, including 41 of the control line, 102 of the selection line and 152 of the traditional. The selection and traditional lines comprise animals selected for higher yearling weight, whereas control line animals are selected for yearling weight close to the average. Five alleles were identified, with allele 1 being the most frequent in the three lines, especially in the lines selected for higher means for yearling weight. Heterozygosity was significantly higher in the control line. Association analyses showed significant effects of allele 1 on birth weight and weaning weight while the allele 3 exert significant effects on yearling weight and back fat thickness. Despite these findings, application of this marker to marker-assisted selection requires more consistent results based on the genotyping of a larger number of animals in order to increase the accuracy of the statistical analyses.     

Divergences in the Response to Ultraviolet Radiation Between Polar and Non-Polar Ciliated Protozoa

Ultraviolet (UV) radiation has detrimental effects on marine ecosystems, in particular in the polar regions where stratospheric ozone reduction causes higher levels of solar radiation. We analyzed two polar species of Euplotes, Euplotes focardii and Euplotes nobilii, for the sensitivity to UV radiation in comparison with two akin species from mid-latitude and tropical waters. Results showed that they face UV radiation much more efficiently than the non-polar species by adopting alternative strategies that most likely reflect different times of colonization of the polar waters. While E. focardii, which is endemic to the Antarctic, survives for longer exposed to UV radiation, E. nobilii, which inhabits both the Antarctic and Arctic, recovers faster from UV-induced damage.