Post-translational modifications in MeHg-induced neurotoxicity

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.     

Integration requires a specific interaction of the donor DNA terminal 5'-cytosine with glutamine 148 of the HIV-1 integrase flexible loop

Integration is essential for retroviral replication and gene therapy using retroviral vectors. Human immunodeficiency virus, type 1 (HIV-1), integrase specifically recognizes the terminal sequences of each long terminal repeat (LTR) and cleaves the 3'-end terminal dinucleotide 5'-GT. The exposed 3'-hydroxyl is then positioned for nucleophilic attack and subsequent strand transfer into another DNA duplex (target or chromosomal DNA). We report that both the terminal cytosine at the protruding 5'-end of the long terminal repeats (5'-C) and the integrase residue Gln-148 are critical for strand transfer. Proximity of the 5'-C and Gln-148 was demonstrated by disulfide cross-linking. Cross-linking is inhibited by the inhibitor 5CITEP 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone. We propose that strand transfer requires a conformational change of the integrase-viral (donor) DNA complex with formation of an H-bond between the N-3 of the 5'-C and the amine group of Gln-148. These findings have implications for the molecular mechanisms coupling 3'-processing and strand transfer as well as for the molecular pharmacology of integrase inhibitors.     

Pulsed electromagnetic fields promote repair of focal articular cartilage defects with engineered osteochondral constructs

Articular cartilage injuries are a common source of joint pain and dysfunction. We hypothesized that pulsed electromagnetic fields (PEMFs) would improve growth and healing of tissue-engineered cartilage grafts in a direction-dependent manner. PEMF stimulation of engineered cartilage constructs was first evaluated in vitro using passaged adult canine chondrocytes embedded in an agarose hydrogel scaffold. PEMF coils oriented parallel to the articular surface induced superior repair stiffness compared to both perpendicular PEMF (p = .026) and control (p = .012). This was correlated with increased glycosaminoglycan deposition in both parallel and perpendicular PEMF orientations compared to control (p = .010 and .028, respectively). Following in vitro optimization, the potential clinical translation of PEMF was evaluated in a preliminary in vivo preclinical adult canine model. Engineered osteochondral constructs (∅ 6 mm × 6 mm thick, devitalized bone base) were cultured to maturity and implanted into focal defects created in the stifle (knee) joint. To assess expedited early repair, animals were assessed after a 3-month recovery period, with microfracture repairs serving as an additional clinical control. In vivo, PEMF led to a greater likelihood of normal chondrocyte (odds ratio [OR]: 2.5, p = .051) and proteoglycan (OR: 5.0, p = .013) histological scores in engineered constructs. Interestingly, engineered constructs outperformed microfracture in clinical scoring, regardless of PEMF treatment (p < .05). Overall, the studies provided evidence that PEMF stimulation enhanced engineered cartilage growth and repair, demonstrating a potential low-cost, low-risk, noninvasive treatment modality for expediting early cartilage repair.     

The surface free energy of Leishmania mexicana amazonensis.

Surface charge of Leishmania mexicana amazonensis was investigated by direct zeta-potential determination and ultrastructural cytochemistry, and its surface tension was studied by measurements of the advancing contact angle formed by the parasite monolayers with drops of liquids of different polarities. Both virulent and avirulent promastigotes exhibited negatively charged surfaces with a zeta-potential of about -15 mV. Treatment of these cells with trypsin, alkaline phosphatase, or phospholipase C rendered their surfaces less negatively charged, whereas neuraminidase did not alter the parasite negativeness. Cytochemically, we could observe a reduction in the cationized ferritin binding after the parasite treatment with each of the former enzymes, but not with neuraminidase. The surface free energy of parasites was calculated by taken to account the London dispersion, the Keeson dipole-dipole, and the Debye dipole-induced forces, as well as the surface polarity of the parasites and their zeta-potentials, by considering their adhesion to polystyrene surfaces. The delta G values of -6.4 and -18.1 mJ.m-2 were obtained for avirulent and virulent promstigotes, respectively.     

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.     

In vitro and in vivo characterization of an interleukin‐15 antagonist peptide by metabolic stability, 99mTc‐labeling,and biological activity assays

Interleukin (IL)–15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL‐15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with ^99mTc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL‐2 cells, using 3 different additives to improve the solubility of these peptides. The half‐life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with ^99mTc showed a yield of approximately 99.8%. The ^99mTc‐labeled peptide was stable in a 30‐fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL‐15 overexpression.     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.