Downregulation of GABA<Subscript>A</Subscript> Receptor Recycling Mediated by HAP1 Contributes to Neuronal Death in In Vitro Brain Ischemia

Downregulation of GABAergic synaptic transmission contributes to the increase in overall excitatory activity in the ischemic brain. A reduction of GABA_A receptor (GABA_AR) surface expression partly accounts for this decrease in inhibitory activity, but the mechanisms involved are not fully elucidated. In this work, we investigated the alterations in GABA_AR trafficking in cultured rat hippocampal neurons subjected to oxygen/glucose deprivation (OGD), an in vitro model of global brain ischemia, and their impact in neuronal death. The traffic of GABA_AR was evaluated after transfection of hippocampal neurons with myc-tagged GABA_AR β3 subunits. OGD decreased the rate of GABA_AR β3 subunit recycling and reduced the interaction of the receptors with HAP1, a protein involved in the recycling of the receptors. Furthermore, OGD induced a calpain-mediated cleavage of HAP1. Transfection of hippocampal neurons with HAP1A or HAP1B isoforms reduced the OGD-induced decrease in surface expression of GABA_AR β3 subunits, and HAP1A maintained the rate of receptor recycling. Furthermore, transfection of hippocampal neurons with HAP1 significantly decreased OGD-induced cell death. These results show a key role for HAP1 protein in the downmodulation of GABAergic neurotransmission during cerebral ischemia, which contributes to neuronal demise.     

Re‐evaluation of insect melanogenesis research: Views from the dark side

Melanins (eumelanin and pheomelanin) are synthesized in insects for several purposes including cuticle sclerotization and color patterning, clot formation, organogenesis, and innate immunity. Traditional views of insect immunity detail the storage of pro‐phenoloxidases inside specialized blood cells (hemocytes) and their release upon recognition of foreign bodies. Activated phenoloxidases convert monophenols into reactive quinones in a two‐step enzymatic reaction, and until recently, the mechanism of tyrosine hydroxylation remained a mystery. Herein, we present our interpretations of these enzyme–substrate complexes. The resultant melanins are deposited onto the surface of microbes to immobilize, agglutinate, and suffocate them. Phenoloxidase activity and melanin production are not limited to the blood (hemolymph) or cuticle, as recent evidence points to more diverse, sophisticated interactions in the gut and with the resident symbionts. This review offers insight into the somewhat neglected areas of insect melanogenesis research, particularly in innate immunity, its role in beneficial insects such as pollinators, the functional versatility of phenoloxidases, and the limitations of common experimental approaches that may impede progress inadvertently.     

Simultaneous purification and immobilization of soybean hull peroxidase with a dye attached to chitosan mini-spheres

Soybean hull peroxidase (EC 1.11.1.7, SBP) was simultaneously purified and immobilized by dye affinity chromatography with Reactive Blue 4 attached to chitosan mini-spheres. Under optimized conditions, 96% of SBP was adsorbed to the matrix. Under the most stringent condition, only 49% was desorbed, whereas 2 M NaCl failed to desorb a significant amount of SBP. This behaviour allowed proposing the dye matrix as a support to immobilize SBP from a crude extract. The pH of maximum activity shifted from 7 to 3–5. SBP gained thermostability after immobilization: after 5?h at 85?°C, the remaining activity was 54%, whereas that of the free enzyme was 31%. The optimum temperature for the immobilized SBP was 75?°C, whereas that of the free enzyme was 55?°C. After two months at 4?°C, the activity loss of the immobilized SBP was only 3%. Immobilized SBP removed 80% of 2-bromophenol from wastewater in 180?min and, after five cycles of use, the activity loss was only 12.8%.     

Isolation and chemical characterization of agelaiatoxin8 (AvTx8) from Agelaia vicina wasp venom and its biological effects on GABA neurotransmission

Arthropod venoms are sources of molecules that may be useful tools to investigate molecular mechanisms of putative new medicines and laboratory drugs. Here we show the effects of the compound agelaiatoxin‐8 (AVTx8), isolated from Agelaia vicina venom, on γ‐aminobutyric acid (GABA) neurotransmission in rat brain synaptosomes. Analysis reveals that AvTx8 is composed by 14 amino acid residues with a molecular weight (MW) of 1567 Da. AvTx8 increased GABA release and inhibited GABA uptake in synaptosomes from rat cerebral cortex. AvTx8 inhibited GABA uptake and increased GABA release in the presence of Ca⁺, Na⁺, and K⁺ channel blockers, suggesting that it acts directly on GABA transporters. In addition, AvTx8 significantly decreases GABA binding in synaptic membranes from rat brain cortex, suggesting that it also modulates the activity of GABA receptors. Moreover, AvTx8 decreased GAT‐1– and GAT‐3–mediated GABA uptake in transfected COS‐7 cells. Accordingly, we suggest that AvTx8 modulates GABA neurotransmission and might provide a novel entry point for identifying a new class of GABA‐modulating neuroprotective drugs.     

Exploring the symbiont diversity of ancient western redcedars: arbuscular mycorrhizal fungi of long‐lived hosts

Arbuscular mycorrhizal fungi (AMF) are globally distributed, monophyletic root symbionts with ancient origins. Their contribution to carbon cycling and nutrient dynamics is ecologically important, given their obligate association with over 70% of vascular plant species. Current understanding of AMF species richness and community structure is based primarily on studies of grasses, herbs and agricultural crops, typically in disturbed environments. Few studies have considered AMF interactions with long‐lived woody perennial species in undisturbed ecosystems. Here we examined AMF communities associated with roots and soils of young, mature and old western redcedar (Thuja plicata) at two sites in the old‐growth temperate rainforests of British Columbia. Due to the unique biology of AMF, community richness and structure were assessed using a conservative, clade‐based approach. We found 91 AMF OTUs across all samples, with significantly greater AMF richness in the southern site, but no differences in richness along the host chronosequence at either site. All host age classes harboured AMF communities that were overdispersed (more different to each other than expected by chance), with young tree communities most resembling old tree communities. A comparison with similar clade richness data obtained from the literature indicates that western redcedar AMF communities are as rich as those of grasses, tropical trees and palms. Our examination of undisturbed temperate old‐growth rainforests suggests that priority effects, rather than succession, are an important aspect of AMF community assembly in this ecosystem.     

Screening assay for inhibitors of a recombinant Streptococcus pneumoniae UDP-glucose pyrophosphorylase

The UDP-glucose pyrophosphorylase of Streptococcus pneumoniae (GalU_Spn) is absolutely required for the biosynthesis of capsular polysaccharide, the sine qua non virulence factor of pneumococcus. Since the eukaryotic enzymes are completely unrelated to their prokaryotic counterparts, we propose that the GalU enzyme is a critical target to fight the pneumococcal disease. A recombinant GalU_Spn was overexpressed and purified. An enzymatic assay that is rapid, sensitive and easy to perform was developed. This assay was appropriate for screening chemical libraries for searching GalU inhibitors. This work represents a fundamental step in the exploration of novel antipneumococcal drugs.     

Cell lines authentication and <Emphasis Type="Italic">mycoplasma</Emphasis> detection as minimun quality control of cell lines in biobanking

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.     

Proteomic analysis of serum samples of paracoccidioidomycosis patients with severe pulmonary sequel

BackgroundPulmonary sequelae (PS) in patients with chronic paracoccidioidomycosis (PCM) typically include pulmonary fibrosis and emphysema. Knowledge of the molecular pathways involved in PS of PCM is required for treatment and biomarker identification.Methodology/Principal findingsThis non-concurrent cohort study included 29 patients with pulmonary PCM that were followed before and after treatment. From this group, 17 patients evolved to mild/ moderate PS and 12 evolved severe PS. Sera from patients were evaluated before treatment and at clinical cure, serological cure, and apparent cure. A nanoACQUITY UPLC-Xevo QT MS system and PLGS software were used to identify serum differentially expressed proteins, data are available via ProteomeXchange with identifier PXD026906. Serum differentially expressed proteins were then categorized using Cytoscape software and the Reactome pathway database. Seventy-two differentially expressed serum proteins were identified in patients with severe PS compared with patients with mild/moderate PS. Most proteins altered in severe PS were involved in wound healing, inflammatory response, and oxygen transport pathways. Before treatment and at clinical cure, signaling proteins participating in wound healing, complement cascade, cholesterol transport and retinoid metabolism pathways were downregulated in patients with severe PS, whereas signaling proteins in gluconeogenesis and gas exchange pathways were upregulated. At serological cure, the pattern of protein expression reversed. At apparent cure pathways related with tissue repair (fibrosis) became downregulated, and pathway related oxygen transport became upregulated. Additionally, we identified 15 proteins as candidate biomarkers for severe PS.Conclusions/SignificanceDevelopment of severe PS is related to increased expression of proteins involved in glycolytic pathway and oxygen exchange), indicative of the greater cellular activity and replication associated with early dysregulation of wound healing and aberrant tissue repair. Our findings provide new targets to study mechanisms of PS in PCM, as well as potential biomarkers.     

Analyzing digital color descriptors in wheat

Color is one of the factors used in quality estimation in many agricultural and food products. Currently, the evaluation of color depends on judgments made by human experts. These are subjective and inevitably affected by physical, physiological and environmental conditions. Suitable instrumental is required to provide objectivity and coherence to color measurements and quantitative expressions. It would be very useful to have tools that allow both practical and precise approaches to chromatic evaluation of products for human consumption. This work suggests a methodology which might contribute to solve that constraint and the analysis of environmental influences on this character in two consecutive wheat growing seasons. This research used 18 cultivars, which were sampled in two crop growth stages. On the first part of this study, a spectrophotometer and a digital camera were used for the analysis of color at the phenological state of tillering. Data analysis showed a correlation between both study methodologies, which would make more practical the work of breeders during data collection. The second part of the study of the genotype-environment interaction (GEI) continued using only a digital camera. The phenological state of tillering did not evidence a marked GEI. On the other hand, an appreciable GEI was found at physiological ripeness.