Potent Anti-Candida Fraction Isolated from Capsicum chinense Fruits Contains an Antimicrobial Peptide That is Similar to Plant Defensin and is Able to Inhibit the Activity of Different α-Amylase Enzymes

Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 μg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.

     

Increased hip internal abduction moment and reduced speed are the gait strategies used by women with knee osteoarthritis

The purpose of this study was to identify the gait strategies in women with mild and moderate knee osteoarthritis (OA). Forty women diagnosed with OA of the knee and 40 healthy women participated in the study. Toe-out progression angle, trunk lateral lean, hip internal abduction moment and gait speed were measured using Qualisys ProReflex System and two force plates. Principal component analysis was applied to extract features from the gait waveforms data that characterized the waveforms main modes of temporal variation. Discriminant analysis with a stepwise model was conducted to determine which strategies could best discriminate groups. According to the discriminant model, the PC2 of the internal abduction moment of the hip and the gait speed were the most discriminatory variables between the groups. The OA group showed decreased gait speed, decreased hip internal abduction moment during the loading response phase, and increased hip internal abduction moment during the mid and terminal stance phases. Interventions that may increase hip internal abduction moment, such as the strengthening of the hip abductors muscles, may benefit women with knee OA. Training slower than normal gait speeds must be considered in light of potential adverse implications on overall physical function, daily tasks, and safety.     

Synthesis of imidazole-containing analogues of farnesyl pyrophosphate and evaluation of their biological activity on protein farnesyltransferase

With the aim of creating new bisubstrate inhibitors of protein farnesyltransferase (FTase), new carboxylic farnesyl pyrophosphate analogues have been designed and synthesized. The original structures are built around three elements: a prenyl moiety, a 1,4-diacid motif and an imidazole ring. All the compounds were evaluated for their ability to inhibit FTase and compared with the corresponding derivatives lacking the imidazole ring, synthesized for that purpose. These new compounds are not bisubstrate inhibitors probably because the imidazole ring is not in the right position to interact with the zinc atom. However these derivatives display FPP competitive inhibition with a good activity in the carboxylic farnesyl pyrophosphate analogues series.     

Tubulin-interactive stilbene derivatives as anticancer agents

Microtubules are dynamic polymers that occur in eukaryotic cells and play important roles in cell division, motility, transport and signaling. They form during the process of polymerization of α- and β-tubulin dimers. Tubulin is a significant and heavily researched molecular target for anticancer drugs. Combretastatins are natural cis-stilbenes that exhibit cytotoxic properties in cultured cancer cells in vitro. Combretastatin A-4 (3′-hydroxy-3,4,4′, 5-tetramethoxy-cis-stilbene; CA-4) is a potent cytotoxic cis-stilbene that binds to β-tubulin at the colchicine-binding site and inhibits tubulin polymerization. The prodrug CA-4 phosphate is currently in clinical trials as a chemotherapeutic agent for cancer treatment. Numerous series of stilbene analogs have been studied in search of potent cytotoxic agents with the requisite tubulin-interactive properties. Microtubule-interfering agents include numerous CA-4 and transresveratrol analogs and other synthetic stilbene derivatives. Importantly, these agents are active in both tumor cells and immature endothelial cells of tumor blood vessels, where they inhibit the process of angiogenesis. Recently, computer-aided virtual screening was used to select potent tubulin-interactive compounds. This review covers the role of stilbene derivatives as a class of antitumor agents that act by targeting microtubule assembly dynamics. Additionally, we present the results of molecular modeling of their binding to specific sites on the α- and β-tubulin heterodimer. This has enabled the elucidation of the mechanism of stilbene cytotoxicity and is useful in the design of novel agents with improved anti-mitotic activity. Tubulin-interactive agents are believed to have the potential to play a significant role in the fight against cancer.     

Mmi1, the Yeast Homologue of Mammalian TCTP,Associates with Stress Granules in Heat-Shocked Cells and Modulates Proteasome Activity

As we have shown previously, yeast Mmi1 protein translocates from the cytoplasm to the outer surface of mitochondria when vegetatively growing yeast cells are exposed to oxidative stress. Here we analyzed the effect of heat stress on Mmi1 distribution. We performed domain analyses and found that binding of Mmi1 to mitochondria is mediated by its central alpha-helical domain (V-domain) under all conditions tested. In contrast, the isolated N-terminal flexible loop domain of the protein always displays nuclear localization. Using immunoelectron microscopy we confirmed re-location of Mmi1 to the nucleus and showed association of Mmi1 with intact and heat shock-altered mitochondria. We also show here that mmi1Δ mutant strains are resistant to robust heat shock with respect to clonogenicity of the cells. To elucidate this phenotype we found that the cytosolic Mmi1 holoprotein re-localized to the nucleus even in cells heat-shocked at 40°C. Upon robust heat shock at 46°C, Mmi1 partly co-localized with the proteasome marker Rpn1 in the nuclear region as well as with the cytoplasmic stress granules defined by Rpg1 (eIF3a). We co-localized Mmi1 also with Bre5, Ubp3 and Cdc48 which are involved in the protein de-ubiquitination machinery, protecting protein substrates from proteasomal degradation. A comparison of proteolytic activities of wild type and mmi1Δ cells revealed that Mmi1 appears to be an inhibitor of the proteasome. We conclude that one of the physiological functions of the multifunctional protein module, Mmi1, is likely in regulating degradation and/or protection of proteins thereby indirectly regulating the pathways leading to cell death in stressed cells.     

Asymptomatic Cattle Naturally Infected with Mycobacterium bovis Present Exacerbated Tissue Pathology and Bacterial Dissemination

Rational discovery of novel immunodiagnostic and vaccine candidate antigens to control bovine tuberculosis (bTB) requires knowledge of disease immunopathogenesis. However, there remains a paucity of information on the Mycobacterium bovis-host immune interactions during the natural infection. Analysis of 247 naturally PPD+ M. bovis-infected cattle revealed that 92% (n = 228) of these animals were found to display no clinical signs, but presented severe as well as disseminated bTB-lesions at post-mortem examination. Moreover, dissemination of bTB-lesions positively correlated with both pathology severity score (Spearman r = 0.48; p<0.0001) and viable tissue bacterial loads (Spearman r = 0.58; p = 0.0001). Additionally, granuloma encapsulation negatively correlated with M. bovis growth as well as pathology severity, suggesting that encapsulation is an effective mechanism to control bacterial proliferation during natural infection. Moreover, multinucleated giant cell numbers were found to negatively correlate with bacterial counts (Spearman r = 0.25; p = 0.03) in lung granulomas. In contrast, neutrophil numbers in the granuloma were associated with increased M. bovis proliferation (Spearman r = 0.27; p = 0.021). Together, our findings suggest that encapsulation and multinucleated giant cells control M. bovis viability, whereas neutrophils may serve as a cellular biomarker of bacterial proliferation during natural infection. These data integrate host granuloma responses with mycobacterial dissemination and could provide useful immunopathological-based biomarkers of disease severity in natural infection with M. bovis, an important cattle pathogen.     

Bayesian Phylogeography of Crimean-Congo Hemorrhagic Fever Virus in Europe

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis mainly transmitted by ticks that causes severe hemorrhagic fever and has a mortality rate of 5-60%. The first outbreak of CCHF occurred in the Crimean peninsula in 1944-45 and it has recently emerged in the Balkans and eastern Mediterranean. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach, which estimated a mean evolutionary rate of 2.96 x 10^-4 (95%HPD=1.6 and 4.7 x 10^-4) substitutions/site/year for the analysed fragment. All of the isolates segregated into seven highly significant clades that correspond to the known geographical clades: in particular the two new isolates from northern Albania clustered significantly within the Europe 1 clade. Our phylogeographical reconstruction suggests that the global CCHFV clades originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s, when a still circulating but less or non-pathogenic virus emerged in Greece and Turkey, and the second in the early 1900s, when a pathogenic CCHFV strain began to spread in eastern Europe. The most probable location for the origin of this European clade 1 was Russia, but Turkey played a central role in spreading the virus throughout Europe. Given the close proximity of the infected areas, our data suggest that the movement of wild and domestic ungulates from endemic areas was probably the main cause of the dissemination of the virus in eastern Europe.     

Forest Loss and the Biodiversity Threshold: An Evaluation Considering Species Habitat Requirements and the Use of Matrix Habitats

Habitat loss is the main driver of the current biodiversity crisis, a landscape-scale process that affects the survival of spatially-structured populations. Although it is well-established that species responses to habitat loss can be abrupt, the existence of a biodiversity threshold is still the cause of much controversy in the literature and would require that most species respond similarly to the loss of native vegetation. Here we test the existence of a biodiversity threshold, i.e. an abrupt decline in species richness, with habitat loss. We draw on a spatially-replicated dataset on Atlantic forest small mammals, consisting of 16 sampling sites divided between forests and matrix habitats in each of five 3600-ha landscapes (varying from 5% to 45% forest cover), and on an a priori classification of species into habitat requirement categories (forest specialists, habitat generalists and open-area specialists). Forest specialists declined abruptly below 30% of forest cover, and spillover to the matrix occurred only in more forested landscapes. Generalists responded positively to landscape heterogeneity, peaking at intermediary levels of forest cover. Open area specialists dominated the matrix and did not spillover to forests. As a result of these distinct responses, we observed a biodiversity threshold for the small mammal community below 30% forest cover, and a peak in species richness just above this threshold. Our results highlight that cross habitat spillover may be asymmetrical and contingent on landscape context, occurring mainly from forests to the matrix and only in more forested landscapes. Moreover, they indicate the potential for biodiversity thresholds in human-modified landscapes, and the importance of landscape heterogeneity to biodiversity. Since forest loss affected not only the conservation value of forest patches, but also the potential for biodiversity-mediated services in anthropogenic habitats, our work indicates the importance of proactive measures to avoid human-modified landscapes to cross this threshold.