Ca2+-H+ antiport activity in synaptic vesicles isolated from sheep brain cortex

Synaptic vesicles isolated from sheep brain cortex exhibit an ATP-dependent Ca2+ accumulation that is inhibited by the protonophore uncoupler carbonyl cyanide m-chorophenylhydrazone (CCCP) and completely released by the Ca2+ ionophore ionomycin. This transport activity was sensitive to the V-type ATPase inhibitor, bafilomycin, but not to the P-type ATPase inhibitor, vanadate. We also observed that the proton gradient, established across the synaptic vesicle membranes in the presence of ATP, is partially dissipated by the addition of Ca2+ (100-860 [mu]M) in correlation to an increase of ATP hydrolysis by the H+-pumping ATPase. In contrast, the activity of the H+-ATPase, measured under uncoupling conditions (presence of CCCP), appears to be unaltered by the calcium ion. The Ca2+-induced H+ release visualized by fluorescence quenching of acridine orange correlates well with the Ca2+ uptake determined isotopically. These results indicate that synaptic vesicles accumulate Ca2+, via a low affinity Ca2+-H+ antiport system energized by the protonmotive force originated from the H+-pumping ATPase activity.http://www.sciencedirect.com/science/article/B6T0G-3STG33F-M/1/23212cde5694bae6fc9101190baa18e

Life-history and biochemical responses of Chironomus riparius exposed to different sized microplastics

Freshwater basins are an integral part of microplastics life-cycle, being a repository of plastic micro-debris. In fact, the levels found so far are similar to those found in marine environment and shoreline regions. The deposition and persistence of plastic micro-debris in sediments (lakeshores and riverbanks) makes them long-time available for benthic species. The objective of this study was to investigate developmental, and physiological effects induced by the presence of polyethylene microparticles (PE) in Chironomus riparius, due to its key-role in the freshwater ecosystem. For that partial life cycle tests using different sized polyethylene particles (PE 40-48 μm; PE 125 μm and PE 350 μm) allowed evaluation of effects on C. riparius larval growth and emergence patterns while acute exposures were used to assess effects in parameters related to neurotransmission (AChE); antioxidant defences and biotransformation (CAT, GST total glutathione levels); oxidative damage (LPO); cellular energy allocation (CEA) and immune response (phenoloxidase). Exposure to PE 40-48 μm caused deleterious effects at lower concentrations in comparison with larger particles in several parameters: larval growth and development time of both male and female imagoes and on emergence rate. PE 40-48 μm were then selected to assess effects on physiological homeostasis. Acute exposures to PE 40-48 μm generated alterations in C. riparius larvae antioxidant and biotransformation enzymes activities (CAT, GST and total glutathione) and activation of immune response (induction of phenoloxidase). Larvae exposed to microplastics showed also a depletion in energy reserves. Our study highlights the potential deleterious effect of microplastics for aquatic invertebrate populations. Results will be discussed in terms of effects of different sized plastic particles on different levels of biological organization within freshwater invertebrates and on the needed and ongoing research aiming to address the long term and indirect effects of these particles for natural populations and ecosystem functioning.publishe

Kavezno izlaganje lubina (Dicentrarchus labrax) u procjeni genotoksičnog utjecaja onečišćenja

Genotoxic effects are often the earliest signs of pollution-related environmental disturbance. In this study, we used the comet assay and micronucleus test to assess DNA damage in the erythrocytes of the European sea bass (Dicentrarchus labrax) exposed to environmental pollution in situ. Fish were collected from a fi sh farm in the Trogir Bay and their cages placed at an unpolluted reference site Šolta (Nečujam Bay) and a polluted site Vranjic (Kaštela Bay) for four weeks. A group of fi sh which remained at the fi sh farm Trogir Bay were used as the second control group. Fish exposed at the Vranjic site showed a signifi cantly higher erythrocyte DNA damage, measured by the comet assay, than either control group. Micronucleus induction showed a similar gradient of DNA damage, but did not reach statistical signifi cance. Our results show that cage exposure of a marine fi sh D. labrax can be useful in environmental biomonitoring and confi rm the comet assay as a suitable tool for detecting pollution-related genotoxicity.Genotoksični učinak često je jedan od najranijih pokazatelja štetnog djelovanja onečišćenja okoliša. U ovom radu procijenjeno je oštećenje DNA u eritrocitima lubina (Dicentrarchus labrax) izloženima okolišnom onečišćenju s pomoću komet-testa i mikronukleus-testa. Lubini su prikupljeni na ribogojilištu i kavezno izloženi u periodu od četiri tjedna na dvije postaje različitog stupnja onečišćenja na jadranskoj obali: na kontrolnoj postaji Šolta (zaljev Nečujam) i na onečišćenoj postaji Vranjic (Kaštelanski zaljev). Zasebna skupina lubina skupljena na ribogojilištu poslužila je kao druga kontrola. Rezultati komet-testa pokazali su statistički značajan porast oštećenja DNA na postaji Vranjic u usporedbi s obje kontrolne postaje. Rezultati mikronukleus-testa pokazali su sličan gradijent onečišćenja, iako nisu dosegli statističku značajnost. Ovi rezultati upućuju na primjenjivost kaveznog izlaganja lubina D. labrax u biomonitoringu vodenog okoliša te potvrđuju korisnost komet-testa kao prikladne metode za detekciju genotoksičnog utjecaja onečišćenja

The use of muscle biomarkers for assessing physiological effects of heavy metal pollution in the greater white-toothed shrew (Crocidura russula)

The greater white-toothed shrew Crocidura russula has been used as a sentinel species for estimating environmental risks to human populations. Previous studies in mining areas have focused on the liver of shrews as the primary target of physiological and metabolic changes due to heavy metal pollution. However, populations persist even when detoxification by the liver seems to be compromised and damage is observed. These pollutant-adapted individuals inhabiting contaminated sites may exhibit altered biochemical parameters that confer increased tolerance in various tissues other than the liver. The skeletal muscle tissue of C. russula might be an alternative tissue that allows the survival of organisms inhabiting historically polluted sites due to the detoxification of redistributed metals. Organisms from two heavy metal mine populations and one population derived from an unpolluted site were used to determine the detoxification activities, antioxidant capacity, and oxidative damage, as well as cellular energy allocation parameters and acetylcholinesterase activity (a biomarker of neurotoxicity). Muscle biomarkers differ between shrews from polluted sites and shrews from the unpolluted location, with the mine animals showing: (1) a decreased energy consumption concomitant with increased energy reserves and total available energy; (2) reduced cholinergic activity, suggesting an impairment of neurotransmission at the neuromuscular junction; (3) an overall decrease in detoxification capacity and enzymatic antioxidant response and a higher level of lipid damage. Also, some of these markers differed between females and males. These changes may have resulted from a decreased detoxifying capacity of the liver and could potentially bring about significant ecological effects for this highly active species. Heavy metal pollution induced physiological changes in Crocidura russula showing that skeletal muscle may serve as a backup sink organ allowing rapid species adaptation and evolution.publishe

The sexuality of HIV-positive adolescents: rights and challenges for healthcare

Sexualidade e saúde reprodutiva configuram questões relevantes para o cuidado integral à saúde de pessoas vivendo com HIV. Políticas públicas e serviços de saúde, entretanto, têm dedicado insuficiente atenção ao assunto. O objetivo deste trabalho é compreender como adolescentes e jovens soropositivos lidam com suas experiências sexuais e projetos de namoro, desejo de constituir família e de ter filhos. O estudo qualitativo entrevistou em profundidade 21 adolescentes vivendo com HIV (por transmissão vertical, sexual ou sanguínea) e 13 cuidadores de crianças e jovens, vivendo em São Paulo e em Santos, Brasil. As narrativas descrevem como aprenderam a lidar com a sexualidade e a ansiedade da revelação do diagnóstico nesse contexto. Destacam-se nas narrativas o despreparo, a desinformação sobre prevenção e a falta de apoio para lidar com a situação, assim como o estigma e a discriminação que atravessa grande parte das dificuldades relatadas. O artigo discute criticamente alguns dos desafios postos para uma adequada atenção à questão no Brasil, especialmente a consideração de jovens soropositivos como sujeitos de direitos sexuais, sugerindo diretrizes para a incorporação desta temática a um cuidado integral e humanizado de crianças e jovens vivendo com HIV.Sexuality and reproductive healthcare represent relevant issues for comprehensive care of HIV-positive adolescents. However, public policies and health services give this issue insufficient attention. The scope of this article is to assess how HIV-positive young people and teenagers cope with their sexuality, dating and the urge to have children and start a family. In a qualitative study, in-depth interviews were staged with 21 HIV-positive (contracted by vertical, sexual or intravenous transmission) teenagers and 13 caregivers of children and youths living in Sao Paulo and Santos. The interviews revealed the different ways teenagers cope with their sexuality and with the anxiety of HIV disclosure in this context. Lack of information about HIV prevention, lack of support and skills to cope with their sexuality were revealed in the reports. Furthermore, stigma and discrimination were the most frequently reported difficulties. The main challenges to be faced in Brazil in regard to this issue are discussed, especially the need to consider HIV-positive youth as entitled to sexual rights. Recommendations are also made for incorporating the issue into a humanized and comprehensive care approach for HIV-positive children and young people

The invertebrate lysozyme effector ILYS-3 is systemically activated in response to danger signals and confers antimicrobial protection in C. elegans

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material

High-Throughput Isolation and Mapping of C. elegans Mutants Susceptible to Pathogen Infection

We present a novel strategy that uses high-throughput methods of isolating and mapping C. elegans mutants susceptible to pathogen infection. We show that C. elegans mutants that exhibit an enhanced pathogen accumulation (epa) phenotype can be rapidly identified and isolated using a sorting system that allows automation of the analysis, sorting, and dispensing of C. elegans by measuring fluorescent bacteria inside the animals. Furthermore, we validate the use of Amplifluor® as a new single nucleotide polymorphism (SNP) mapping technique in C. elegans. We show that a set of 9 SNPs allows the linkage of C. elegans mutants to a 5–8 megabase sub-chromosomal region

A transcriptomic snapshot of early molecular communication between Pasteuria penetrans and Meloidogyne incognita

© The Author(s). 2018Background: Southern root-knot nematode Meloidogyne incognita (Kofoid and White, 1919), Chitwood, 1949 is a key pest of agricultural crops. Pasteuria penetrans is a hyperparasitic bacterium capable of suppressing the nematode reproduction, and represents a typical coevolved pathogen-hyperparasite system. Attachment of Pasteuria endospores to the cuticle of second-stage nematode juveniles is the first and pivotal step in the bacterial infection. RNA-Seq was used to understand the early transcriptional response of the root-knot nematode at 8 h post Pasteuria endospore attachment. Results: A total of 52,485 transcripts were assembled from the high quality (HQ) reads, out of which 582 transcripts were found differentially expressed in the Pasteuria endospore encumbered J2 s, of which 229 were up-regulated and 353 were down-regulated. Pasteuria infection caused a suppression of the protein synthesis machinery of the nematode. Several of the differentially expressed transcripts were putatively involved in nematode innate immunity, signaling, stress responses, endospore attachment process and post-attachment behavioral modification of the juveniles. The expression profiles of fifteen selected transcripts were validated to be true by the qRT PCR. RNAi based silencing of transcripts coding for fructose bisphosphate aldolase and glucosyl transferase caused a reduction in endospore attachment as compared to the controls, whereas, silencing of aspartic protease and ubiquitin coding transcripts resulted in higher incidence of endospore attachment on the nematode cuticle. Conclusions: Here we provide evidence of an early transcriptional response by the nematode upon infection by Pasteuria prior to root invasion. We found that adhesion of Pasteuria endospores to the cuticle induced a down-regulated protein response in the nematode. In addition, we show that fructose bisphosphate aldolase, glucosyl transferase, aspartic protease and ubiquitin coding transcripts are involved in modulating the endospore attachment on the nematode cuticle. Our results add new and significant information to the existing knowledge on early molecular interaction between M. incognita and P. penetrans.Peer reviewedFinal Published versio

Caenorhabditis elegans BAH-1 Is a DUF23 Protein Expressed in Seam Cells and Required for Microbial Biofilm Binding to the Cuticle

The cuticle of Caenorhabditis elegans, a complex, multi-layered extracellular matrix, is a major interface between the animal and its environment. Biofilms produced by the bacterial genus Yersinia attach to the cuticle of the worm, providing an assay for surface characteristics. A C. elegans gene required for biofilm attachment, bah-1, encodes a protein containing the domain of unknown function DUF23. The DUF23 domain is found in 61 predicted proteins in C. elegans, which can be divided into three distinct phylogenetic clades. bah-1 is expressed in seam cells, which are among the hypodermal cells that synthesize the cuticle, and is regulated by a TGF-β signaling pathway

Hypoxia and the Hypoxic Response Pathway Protect against Pore-Forming Toxins in C. elegans

Pore-forming toxins (PFTs) are by far the most abundant bacterial protein toxins and are important for the virulence of many important pathogens. As such, cellular responses to PFTs critically modulate host-pathogen interactions. Although many cellular responses to PFTs have been recorded, little is understood about their relevance to pathological or defensive outcomes. To shed light on this important question, we have turned to the only genetic system for studying PFT-host interactions—Caenorhabditis elegans intoxication by Crystal (Cry) protein PFTs. We mutagenized and screened for C. elegans mutants resistant to a Cry PFT and recovered one mutant. Complementation, sequencing, transgenic rescue, and RNA interference data demonstrate that this mutant eliminates a gene normally involved in repression of the hypoxia (low oxygen response) pathway. We find that up-regulation of the C. elegans hypoxia pathway via the inactivation of three different genes that normally repress the pathway results in animals resistant to Cry PFTs. Conversely, mutation in the central activator of the hypoxia response, HIF-1, suppresses this resistance and can result in animals defective in PFT defenses. These results extend to a PFT that attacks mammals since up-regulation of the hypoxia pathway confers resistance to Vibrio cholerae cytolysin (VCC), whereas down-regulation confers hypersusceptibility. The hypoxia PFT defense pathway acts cell autonomously to protect the cells directly under attack and is different from other hypoxia pathway stress responses. Two of the downstream effectors of this pathway include the nuclear receptor nhr-57 and the unfolded protein response. In addition, the hypoxia pathway itself is induced by PFT, and low oxygen is protective against PFT intoxication. These results demonstrate that hypoxia and induction of the hypoxia response protect cells against PFTs, and that the cellular environment can be modulated via the hypoxia pathway to protect against the most prevalent class of weapons used by pathogenic bacteria