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.

     

Non-native prey species supporting fish assemblage biomass in a Neotropical reservoir

The present study aimed to investigate the role of four non-native invertebrates in supporting fish biomass as well as their influence on the carbon flow into the Volta Grande reservoir food web. The fish samples were carried out quarterly between October 2015 and July 2016 using gillnets. At the sampled sites, four non-native invertebrates (golden mussel, Asian clam, trumpet snail and Amazonian prawn), which are potential prey for fish in the Volta Grande reservoir, were collected by targeted sampling using a Petersen-type bottom dredger and semi-circular sieves. The gut contents of the fish were collected and analyzed under stereoscope, and samples of muscle tissue of these fish, as well as the four non-native invertebrate species sampled, were submitted for isotopic analysis. Results obtained by the present study, by both gut content and stable isotopic analyses, pointed to a trophic structure where non-native species represent not only a strong component of the fish community, but also their main carbon source. Based on gut contents and isotopic mixing models, we found that together, non-native prey are essential carbon sources for the fish fauna, fuelling more than 40.0% of the biomass in four dominant fish species. The consumption rate of non-native bivalves by the native omnivorous fish Leporinus friderici suggested these filter-feeding organisms potentially constitute an important trophic connection between littoral consumers and pelagic energy sources. In addition, non-native prey were also prominent carbon sources for non-native fish, fuelling more than half of the biomass in peacock bass and silver croaker, suggesting these prey have a fundamental role in maintaining non-native fish populations in this system. Our results may help to understand fundamental ecological issues bringing to light the extent to which these new combinations of species influence the energy flow and ecosystem properties of the Volta Grande reservoir.

     

R-Spondin1 enhances wnt signaling and decreases weight loss in short bowel syndrome zebrafish

BackgroundR-spondins, including R-spondin 1 (RSPO1), are a family of Wnt ligands that help to activate the canonical Wnt/β-catenin pathway, which is critical for intestinal epithelial cell proliferation and maintenance of intestinal stem cells. This proliferation underpins the epithelial expansion, or intestinal adaptation (IA), that occurs following massive bowel resection and short bowel syndrome (SBS). The purpose of this study was to identify if recombinant human RSPO1 (rhRSPO1) could be serially administered to SBS zebrafish to enhance cellular proliferation and IA.MethodsAdult male zebrafish were assigned to four groups: sham + PBS, SBS + PBS, sham + rhRSPO1, and SBS + rhRSPO1. Sham fish had a laparotomy alone. SBS fish had a laparotomy with distal intestinal ligation and creation of a proximal stoma. Fish were weighed at initial surgery and then weekly. rhRSPO1 was administered post-operatively following either a one- or two-week dosing schedule with either 3 or 5 intraperitoneal injections, respectively. Fish were harvested at 7 or 14 days with intestinal segments collected for analysis.ResultsRepeated intraperitoneal injection of rhRSPO1 was feasible and well tolerated. At 7 days, intestinal epithelial proliferation was increased by rhRSPO1. At 14 days, SBS + rhRSPO1 fish lost significantly less weight than SBS + PBS fish. Measurements of intestinal surface area were not increased by rhRSPO1 administration but immunofluorescent staining for β-catenin and gene expression for cyclin D1 was increased.ConclusionsIntraperitoneal injection of rhRSPO1 decreased weight loss in SBS zebrafish with increased β-catenin + cells and cyclin D1 expression at 14 days, indicating improved weight maintenance might result from increased activation of the canonical Wnt pathway.     

The citrus plant pathogen Xanthomonas citri has a dual polyamine-binding protein

ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.     

Examining the reliability of the flexor carpi radialis V-wave at different levels of muscle contraction

This study examined the reliability and scaling of the flexor carpi radialis (FCR) V-wave during submaximal and maximal voluntary muscle contractions (MVC). 23 participants were tested on three separate sessions. For each session, participants performed isometric wrist flexions at five contraction levels (20, 40, 60, 80 and 100 %MVC). When the target contraction level was reached, a supramaximal electrical stimulus was applied to the median nerve in order to elicit an FCR V-wave. Across all participants, the FCR V-wave amplitude, normalized to its superimposed M-wave amplitude, increased from 0.030 ± 0.001 to 0.143 ± 0.015 (P < 0.001) as the muscle contraction increased from 20 to 100 %MVC. Contraction level did not influence the reliability of evoking the FCR V-wave, as the V-wave demonstrated both stability and consistency. With the exception of a single day main effect during the 20 %MVC condition, V:M_sup was not different across days or trials (P > 0.05) indicating measurement stability. High reliability co-efficients (0.827–0.913) at each contraction level signified measurement consistency. This study establishes that FCR V-waves can be reliably evoked during both submaximal and maximal muscle contractions and suggests the possibility for FCR V-wave recordings to be used to document neuromuscular adaptations associated with factors such as training or fatigue.     

Dislodgement effect of natural semiochemicals released by disturbed triatomines: a possible alternative monitoring tool

The quick detection of domestic and peridomestic triatomines in their environments becomes difficult without the use of dislodgement substances that flush them out from their shelters. At present, tetramethrin 0.2% is being widely used in control programs. Although it is an efficient dislodging agent, its toxicity might affect the health of captured triatomines, of other insects and, to a lesser extent, of other animals, including humans. Here, we tested if semiochemicals released by disturbed adults of Triatoma infestans and/or Rhodnius prolixus can make larvae of the same species exit from their refuges. In a walking olfactometer we found that: 1) larvae of T. infestans were repelled by the odors released by disturbed adults of their own species and of R. prolixus, 2) larvae of R. prolixus did not change their behavior in the presence of odors released by adults of both species, and 3) activity levels were not modulated by these odors in any of both species. Besides, in pseudo‐natural conditions we found an increased flushing‐out activity of larvae of T. infestans when their shelters were sprayed with isobutyric acid or 3‐pentanol, and of larvae of R. prolixus when sprayed with 3‐methyl1butanol. We succeeded in this work to dislodge larvae of triatomines from artificial shelters using natural volatile compounds, allowing the capture of live bugs for further investigations (e.g., xenodiagnosis or genetic studies) and favoring ecological aspects (e.g., minimizing environmental insecticide‐contamination and non‐targeted mortality).     

Thiopurine S-Methyltransferase Gene Polymorphisms in a Healthy Slovak Population and Pediatric Patients with Inflammatory Bowel Disease

Thiopurine methyltransferase (TPMT) is a key component in thiopurine metabolism. There is an insufficient evidence about the distribution of the genotype frequencies of TPMT variants and frequencies of TPMT alleles associated with intermediate and deficient activity in a healthy Slovak population and pediatric patients with inflammatory bowel disease (IBD). TPMT variant alleles (*1,*2, *3A, *3B, and *3C) were determined in 114 children treated for IBD and in 281 healthy volunteers. Mutant alleles were present in 9/114 (7.89%) in the IBD patients and in 23/281 (8.19%) of probands. The distribution of the most frequent variants of TPMT gene was similar in a healthy population and patients with IBD.     

Development and Validation of a Discriminative Dissolution Test for Betamethasone Sodium Phosphate and Betamethasone Dipropionate Intramuscular Injectable Suspension

The intramuscular administration of the injectable suspension betamethasone sodium phosphate (BSP) and betamethasone dipropionate (BD) has immediate therapeutic activity due to solubilized BSP and prolonged activity resulting from the slow release of BD micro-crystals. The purpose of this study was to develop and validate a dissolution method for BD in intramuscular injectable suspensions with detection by high-performance liquid chromatography (HPLC) method. Five commercial products presented a distribution of particle sizes, ranging between 7.43 and 40.25 μm as measured by laser diffraction. It was also found that particle sizes differed between batches of the same product. The different products were tested using the paddle apparatus, with stirring speeds of 25 and 50 rpm in 300 mL of phosphate buffer; simulated body fluid, muscle fluid, and synovial fluid were used as biorelevant dissolution media at 37 ± 0.5°C. It was verified that not only does average particle size affect the dissolution rate, but also the mode and the polydispersity index of the particles. Discriminatory power was obtained using the in vitro dissolution method with 0.1 M sodium phosphate buffer pH 7.4 containing 0.1% sodium lauryl sulfate and a stirring speed of 50 rpm. The HPLC-method is linear, precise, selective, and accurate for the quantification of BSP and BD in dissolution profile testing. This dissolution method can be utilized as a method to control the quality of these injectable suspensions.Key words: dipropionate betamethasone, dissolution test, intramuscular injectable suspensions, simulated muscular fluid, sodium phosphate betamethasone     

Farsenyl pyrophosphate synthase is a potential molecular drug target of risedronate in Babesia bovis

A cDNA encoding farnesyl pyrophosphate synthase of Babesia bovis (BbFPPS) has been isolated, cloned and characterized as molecular drug target. Sequence analysis revealed that BbFPPS contains an open reading frame of 1011 bp with predicted 336 amino acids and molecular mass of 38 kDa. Antiserum raised in mice against recombinant BbFPPS expressed in Escherichia coli specifically reacted with native protein of B. bovis parasites by Western blot analysis and indirect immunofluorescent test. Enzymatic assay using recombinant BbFPPS revealed that the K_m value of the enzyme for isopentenyl pyrophosphate and dimethylallyl pyrophosphate was 2.494 ± 1.536 μM. Risedronate inhibited the activity of BbFPPS yielding IC₅₀ value of 8.4 ± 1.2 nM. Furthermore, the in vitro growth of B. bovis was significantly inhibited in the presence of a micromolar concentration of risedronate (IC₅₀ = 4.02 ± 0.91 μM). No regrowth of B. bovis was observed at 10 μM of risedronate in the subsequent viability test. These results demonstrate that BbFPPS is the molecular target of risedronate, which could inhibit the in vitro growth of B. bovis.     

Zooplankton species richness–productivity relationship: Confronting monotonic positive and hump‐shaped models from a local perspective

This study examined two models that are most frequently used to describe the relationship between species richness and productivity (SPR): monotonic positive and hump‐shaped models. We assessed zooplankton community diversity in response to algal productivity. The relationship between net primary productivity (NPP) and rarefied species richness was examined by fitting the data to two models and comparing them using the Akaike information criterion (AICc). Macrophyte banks with the highest net primary productivity had the highest zooplankton abundance. Our results pointed to a hump‐shaped model as the best fit to describe the relationship between zooplankton species richness and primary productivity (ΔAICc > 4). Thus, the diversity was lower at the extremes of productivity and higher at intermediate levels of productivity. We suggest that this relationship might occur because when the resource supply rates are low, environmental conditions are stressful, whereas a high availability of resources enhances competitive exclusion. Two observations supported this statement: (i) the total abundance of the community positively correlated with NPP (P < 0.05), indicating that less productive sites had few consumers and the raised productivity tended to favour the total abundance; (ii) NPP was negatively correlated with evenness (P < 0.05), indicating that productivity increased the dominance of certain species in the communities. Therefore, we challenged two of the models most frequently used to explain SPR, and discuss some mechanisms underlying a hump‐shaped SPR.