Biocontrol mechanisms of the Antarctic yeast Debaryomyces hansenii UFT8244 against post-harvest phytopathogenic fungi of strawberries.

The use of yeasts has been explored as an efficient alternative to fungicide application in the treatment and prevention of post-harvest fruit deterioration. Here, we evaluated the biocontrol abilities of the Antarctic yeast strain Debaryomyces hansenii UFT8244 against the post-harvest phytopathogenic fungi Botrytis cinerea and Rhizopus stolonifer for the protection and preservation of strawberry fruit. The strongest inhibition of germination of B. cinerea (57%) was observed at 0 °C, followed by 40% at 25 °C. In addition, germ tubes and hyphae of B. cinerea were strongly surrounded and colonized by D. hansenii. Production of the enzymes ß-1,3-glucanase, chitinase and protease by D. hansenii was detected in the presence of phytopathogenic fungus cell walls. The activity of ß-1,3-glucanase was highest on day 12 of incubation and remained high until day 15. Chitinase and protease activities reached their highest levels on the day 15 of incubation. D. hansenii additionally demonstrated the ability to resist oxidative stress. Our data demonstrated that the main biocontrol mechanisms displayed by D. hansenii were the control of phytopathogenic fungal spore germination, production of antifungal enzymes and resistance to oxidative stress. We conclude that isolate D. hansenii UFT8422 should be further investigated for use at commercial scales at low temperatures.

Postnatal growth in small vulnerable newborns: a longitudinal study of 2 million Brazilians using routine register-based linked data.

BACKGROUND: Preterm, low-birth weight (LBW) and small-for-gestational age (SGA) newborns have a higher frequency of adverse health outcomes, including linear and ponderal growth impairment. OBJECTIVE: To describe the growth trajectories and to estimate catch-up growth during the first 5 y of life of small newborns according to 3 vulnerability phenotypes (preterm, LBW, SGA). METHODS: Longitudinal study using linked data from the 100 Million Brazilian Cohort baseline, the Brazilian National Live Birth System (SINASC), and the Food and Nutrition Surveillance System (SISVAN) from 2011 to 2017. We estimated the length/height-for-age (L/HAZ) and weight-for-age z-score (WAZ) trajectories from children of 6-59 mo using the linear mixed model for each vulnerable newborn phenotype. Growth velocity for both L/HAZ and WAZ was calculated considering the change (Δ) in the mean z-score between 2 time points. Catch-up growth was defined as a change in z-score > 0.67 at any time during follow-up. RESULTS: We analyzed 2,021,998 live born children and 8,726,599 observations. The prevalence of at least one of the vulnerable phenotypes was 16.7% and 0.6% were simultaneously preterm, LBW, and SGA. For those born at term, all phenotypes had a period of growth recovery from 12 mo. For preterm infants, the onset of L/HAZ growth recovery started later at 24 mo and the growth trajectories appear to be lower than those born at term, a condition aggravated among children with the 3 phenotypes. Preterm and female infants seem to experience slower growth recovery than those born at term and males. The catch-up growth occurs at 24-59 mo for males preterm: preterm + AGA + NBW (Δ = 0.80), preterm + AGA + LBW (Δ = 0.88), and preterm + SGA + LBW (Δ = 1.08); and among females: term + SGA + NBW (Δ = 0.69), term + AGA + LBW (Δ = 0.72), term + SGA + LBW (Δ = 0.77), preterm + AGA + LBW (Δ = 0.68), and preterm + SGA + LBW (Δ = 0.83). CONCLUSIONS: Children born preterm seem to reach L/HAZ and WAZ growth trajectories lower than those attained by children born at term, a condition aggravated among the most vulnerable.

UV-C light promotes the reductive cleavage of disulfide bonds in ß-Lactoglobulin and improves in vitro gastric digestion.

ß-Lactoglobulin (ß-Lg) is the main protein in whey and is known for its allergenicity and resistance to the digestion of pepsin and trypsin. The UV-C photoinduced cleavage of disulfide bonds in ß-Lactoglobulin, as promoted by excitation of tryptophan residues (Trp), is shown to induce changes in the protein's secondary structure, significantly reducing the protein's resistance to pepsin digestion. The UV-C light-induced changes in the protein secondary structure are marked by an increase in the contribution of ß-sheet and α-helix structures with a concomitantly smaller contribution of the ß-turn structural motif. The photoinduced cleavage of disulfide bonds in ß-Lg has an apparent quantum yield of ф = 0.0015 ± 0.0003 and was shown by transient absorption laser flash photolysis to arise by two different pathways: a) the reduction of the disulfide bond Cys66Cys160 occurs by direct electron transfer from the triplet-excited 3Trp to the disulfide bond due to the existence of a CysCys/Trp triad (Cys66Cys160/Trp61) and b) the reduction of the buried Cys106Cys119 disulfide bond involves a reaction with a solvated electron originated by the photoejection of electrons from the triplet-excited 3Trp decay. The in vitro gastric digestion index for UV-C-treated ß-Lg is revealed to have increased significantly by 36 ± 4 % and 9 ± 2 % under simulated elderly and young adult digestive conditions, respectively. When compared to the native protein, the peptide mass fingerprint profile of digested UV-C-treated ß-Lg shows a higher content and variety of peptides, including the production of some exclusive bioactive peptides such as PMHIRL and EKFDKALKALPMH.

Assessing the Antitumor Potential of Variants of the Extracellular Carbohydrate Polymer from Synechocystis ΔsigF Mutant.

Cancer is a leading cause of death worldwide with a huge societal and economic impact. Clinically effective and less expensive anticancer agents derived from natural sources can help to overcome limitations and negative side effects of chemotherapy and radiotherapy. Previously, we showed that the extracellular carbohydrate polymer of a Synechocystis ΔsigF overproducing mutant displayed a strong antitumor activity towards several human tumor cell lines, by inducing high levels of apoptosis through p53 and caspase-3 activation. Here, the ΔsigF polymer was manipulated to obtain variants that were tested in a human melanoma (Mewo) cell line. Our results demonstrated that high molecular mass fractions were important for the polymer bioactivity, and that the reduction of the peptide content generated a variant with enhanced in vitro antitumor activity. This variant, and the original ΔsigF polymer, were further tested in vivo using the chick chorioallantoic membrane (CAM) assay. Both polymers significantly decreased xenografted CAM tumor growth and affected tumor morphology, by promoting less compact tumors, validating their antitumor potential in vivo. This work contributes with strategies for the design and testing tailored cyanobacterial extracellular polymers and further strengths the relevance of evaluating this type of polymers for biotechnological/biomedical applications.

Active surveillance and early detection of community transmission of SARS-CoV-2 Mu variant (B.1.621) in the Brazilian Amazon.

Through active surveillance and contact tracing from outpatients, we aimed to identify and characterize SARS-CoV-2 variants circulating in Porto Velho-Rondônia, a city in the Brazilian Amazon. As part of a prospective cohort, we gathered information from 2,506 individuals among COVID-19 patients and household contacts. Epidemiological data, nasopharyngeal swabs, and blood samples were collected from all participants. Nasopharyngeal swabs were tested for antigen rapid diagnostic test and reverse transcription-polymerase chain reaction (RT-PCR) followed by genomic sequencing. Blood samples underwent ELISA testing for IgA, IgG, and IgM antibody levels. From 757 specimens sequenced, three were identified as Mu variant, none of the individuals carrying this variant had a travel history in the previous 15 days before diagnosis. One case was asymptomatic and two presented mild symptoms. Two infected individuals from different households caring viruses with additional amino acid substitutions ORF7a P45L and ORF1a T1055A compared to the Mu virus reference sequence. One patient presented IgG levels. Our results highlight that genomic surveillance for SARS-CoV-2 variants can assist in detecting the emergency of SARS-CoV-2 variants in the community, before its identification in other parts of the country.

Cluster of SARS-CoV-2 Gamma Variant Infections, Parintins, Brazil, March 2021.

High case counts after the Gamma (P. 1) variant of severe acute respiratory syndrome coronavirus 2 emerged in Brazil raised concerns that previously infected persons might become reinfected. Investigation of a cluster of coronavirus disease cases in Parintins, in the Brazilian Amazon, suggested household transmission but did not identify high rates of reinfection.

Analysis of mouse faecal dysbiosis, during the development of cachexia, induced by transplantation with Lewis lung carcinoma cells.

Cachexia (CC) is a complex wasting syndrome that significantly affects life quality and life expectancy among cancer patients. Original studies, in which CC was induced in mouse models through inoculation with BaF and C26 tumour cells, demonstrated that CC development correlates with bacterial gut dysbiosis in these animals. In both cases, a common microbial signature was observed, based on the expansion of Enterobacteriaceae in the gut of CC animals. However, these two types of tumours induce unique microbial profiles, suggesting that different CC induction mechanisms significantly impact the outcome of gut dysbiosis. The present study sought to expand the scope of such analyses by characterizing the CC-associated dysbiosis that develops when mice are inoculated with Lewis lung carcinoma (LLC) cells, which constitutes one of the most widely employed mechanisms for CC induction. Interestingly, Enterobacteriaceae expansion is also observed in LLC-induced CC. However, the dysbiosis identified herein displays a more complex pattern, involving representatives from seven different bacterial phyla, which were consistently identified across successive levels of taxonomic hierarchy. These results are supported by a predictive analysis of gene content, which identified a series of functional/structural changes that potentially occur in the gut bacterial population of these animals, providing a complementary and alternative approach to microbiome analyses based solely on taxonomic classification.

Epidemiologic Findings from Case Investigations and Contact Tracing for First 200 Cases of Coronavirus Disease, Santa Clara County, California, USA.

In January 2020, Santa Clara County, California, USA, began identifying laboratory-confirmed coronavirus disease among residents. County staff conducted case and contact investigations focused on households and collected detailed case demographic, occupation, exposure, and outcome information. We describe the first 200 test-positive cases during January 31-March 20, 2020, to inform future case and contact investigations. Probable infection sources included community transmission (104 cases), known close contact with a confirmed case-patient (66 cases), and travel (30 cases). Disease patterns across race and ethnicity, occupational, and household factors suggested multiple infection risk factors. Disproportionately high percentages of case-patients from racial and ethnic subgroups worked outside the home (Hispanic [86%] and Filipino [100%]); household transmission was more common among persons from Vietnam (53%). Even with the few initial cases, detailed case and contact investigations of household contacts capturing occupational and disaggregated race and ethnicity data helped identify at-risk groups and focused solutions for disease control.

COVID-19 Clinical Phenotypes: Presentation and Temporal Progression of Disease in a Cohort of Hospitalized Adults in Georgia, United States.

BACKGROUND: The epidemiological features and outcomes of hospitalized adults with coronavirus disease 2019 (COVID-19) have been described; however, the temporal progression and medical complications of disease among hospitalized patients require further study. Detailed descriptions of the natural history of COVID-19 among hospitalized patients are paramount to optimize health care resource utilization, and the detection of different clinical phenotypes may allow tailored clinical management strategies. METHODS: This was a retrospective cohort study of 305 adult patients hospitalized with COVID-19 in 8 academic and community hospitals. Patient characteristics included demographics, comorbidities, medication use, medical complications, intensive care utilization, and longitudinal vital sign and laboratory test values. We examined laboratory and vital sign trends by mortality status and length of stay. To identify clinical phenotypes, we calculated Gower's dissimilarity matrix between each patient's clinical characteristics and clustered similar patients using the partitioning around medoids algorithm. RESULTS: One phenotype of 6 identified was characterized by high mortality (49%), older age, male sex, elevated inflammatory markers, high prevalence of cardiovascular disease, and shock. Patients with this severe phenotype had significantly elevated peak C-reactive protein creatinine, D-dimer, and white blood cell count and lower minimum lymphocyte count compared with other phenotypes (P < .01, all comparisons). CONCLUSIONS: Among a cohort of hospitalized adults, we identified a severe phenotype of COVID-19 based on the characteristics of its clinical course and poor prognosis. These findings need to be validated in other cohorts, as improved understanding of clinical phenotypes and risk factors for their development could help inform prognosis and tailored clinical management for COVID-19.

Is Technical-Grade Chlordane an Obesogen?

The prevalence of obesity has tripled in recent decades and is now considered an alarming public health problem. In recent years, a group of endocrine disruptors, known as obesogens, have been directly linked to the obesity epidemic. Its etiology is generally associated with a sedentary lifestyle, a high-fat diet and genetic predisposition, but environmental factors, such as obesogens, have also been reported as contributors for this pathology. In brief, obesogens are exogenous chemical compounds that alter metabolic processes and/or energy balance and appetite, thus predisposing to weight gain. Although this theory is still recent, the number of compounds with suspected obesogenic activity has steadily increased over the years, though many of them remain a matter of debate. Technical-grade chlordane is an organochlorine pesticide widely present in the environment, albeit at low concentrations. Highly lipophilic compounds can be metabolized by humans and animals into more toxic and stable compounds that are stored in fat tissue and consequently pose a danger to the human body, including the physiology of adipose tissue, which plays an important role in weight regulation. In addition, technical-grade chlordane is classified as a persistent organic pollutant, a group of chemicals whose epidemiological studies are associated with metabolic disorders, including obesity. Herein, we discuss the emerging roles of obesogens as threats to public health. We particularly discuss the relevance of chlordane persistence in the environment and how its effects on human and animal health provide evidence for its role as an endocrine disruptor with possible obesogenic activity.

Technical-grade chlordane compromises rat Sertoli cells proliferation, viability and metabolic activity.

Environmental contaminants are a daily presence in human routine. Multiple studies highlight the obesogenic activity of some chemicals. Moreover, these compounds have been suggested as a cause of male subfertility and/or infertility. Technical-grade chlordane (TGC) is classified as an endocrine-disruptor chemical, while its classification as obesogen is controversial. Herein, we studied the influence of TGC on Sertoli cells (SCs) metabolism. Rat Sertoli cells (rSCs) were cultured without and in the presence of increasing concentrations (1, 10 and 1000 nM) of TGC. The viability, proliferation, metabolic activity and the metabolic profile of rSCs was assessed. Expression of key glycolysis-related enzymes, transporters and biomarkers of oxidative damage were also evaluated. Our results show that exposure to higher concentrations of TGC decreases SCs proliferation and viability, which was accompanied by increased glucose consumption associated with an upregulation of Glut3 levels. As a result, pyruvate/lactate production were enhanced thus increasing the glycolytic flux in cells exposed to 1000 nM TGC, although lactate dehydrogenase expression and activity did not increase. Notably, biomarkers associated with oxidative damage remained unchanged after exposure to TGC. This is the first report showing that TGC alters glucose rSCs metabolism and the nutritional support of spermatogenesis with consequences for male fertility.

Conotoxin MVIIA improves cell viability and antioxidant system after spinal cord injury in rats.

This study evaluates whether intrathecal MVIIA injection after spinal cord injury (SCI) elicits neuroprotective effects. The test rats were randomly distributed into six groups- sham, placebo, MVIIA 2.5 µM, MVIIA 5 µM, MVIIA 10 µM, and MVIIA 20 µM-and were administered the treatment four hours after SCI. After the optimal MVIIA dose (MVIIA 10 µM) was defined, the best time for application, one or four hours, was analyzed. Locomotor hind limb function and side effects were assessed. Forty-eight hours after the injury and immediately after euthanasia, spinal cord segments were removed from the test rats. Cell viability, reactive oxygen species, lipid peroxidation, and glutamate release were investigated. To examine the MVIIA mechanism of action, the gene expressions of pro-apoptotic (Bax, nNOS, and caspase-3, -8, -9, -12) and anti-apoptotic (Bcl-xl) factors in the spinal cord tissue samples were determined by real-time PCR, and the activities of antioxidant enzymes were also investigated. Application of intrathecal MVIIA 10 µM four hours after SCI prompted a neuroprotective effect: neuronal death decreased (22.46%), oxidative stress diminished, pro-apoptotic factors (Bax, nNOS, and caspase-3, -8) were expressed to a lesser extent, and mitochondrial viability as well as anti-apoptotic factor (Bcl-xl) expression increased. These results suggested that MVIIA provided neuroprotection through antioxidant effects. Indeed, superoxide dismutase (188.41%), and glutathione peroxidase (199.96%), reductase (193.86%), and transferase (175.93%) expressions increased. Therefore, intrathecal MVIIA (MVIIA 10 µM, 4 h) application has neuroprotective potential, and the possible mechanisms are related to antioxidant agent modulation and to intrinsic and extrinsic apoptotic pathways.

Zika Virus Promotes Neuronal Cell Death in a Non-Cell Autonomous Manner by Triggering the Release of Neurotoxic Factors.

Zika virus (ZIKV) has recently caused a worldwide outbreak of infections associated with severe neurological complications, including microcephaly in infants born from infected mothers. ZIKV exhibits high neurotropism and promotes neuroinflammation and neuronal cell death. We have recently demonstrated that N-methyl-d-aspartate receptor (NMDAR) blockade by memantine prevents ZIKV-induced neuronal cell death. Here, we show that ZIKV induces apoptosis in a non-cell autonomous manner, triggering cell death of uninfected neurons by releasing cytotoxic factors. Neuronal cultures infected with ZIKV exhibit increased levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and glutamate. Moreover, infected neurons exhibit increased expression of GluN2B and augmented intracellular Ca2+ concentration. Blockade of GluN2B-containing NMDAR by ifenprodil normalizes Ca2+ levels and rescues neuronal cell death. Notably, TNF-α and IL-1ß blockade decreases ZIKV-induced Ca2+ flux through GluN2B-containing NMDARs and reduces neuronal cell death, indicating that these cytokines might contribute to NMDAR sensitization and neurotoxicity. In addition, ZIKV-infected cultures treated with ifenprodil exhibits increased activation of the neuroprotective pathway including extracellular signal-regulated kinase and cAMP response element-binding protein, which may underlie ifenprodil-mediated neuroprotection. Together, our data shed some light on the neurotoxic mechanisms triggered by ZIKV and begin to elucidate how GluN2B-containing NMDAR blockade can prevent neurotoxicity.

Data and calculus on isobolographic analysis to determine the antinociceptive interaction between calcium channel blocker and a TRPV1 blocker in acute pain model in mice.

Determining antinociceptive interaction between Phα1ß toxin (a voltage gated calcium channel blocker) and SB366791 (selective TRPV1 antagonist) may have both clinical and mechanistic implications for the pain management. This data in brief article is associated to the research paper "Synergistic antinociceptive effect of a calcium channel blocker and a TRPV1 blocker in an acute pain model in mice". This material supports the isobolographic analysis performed with the above drugs and shows: data of the dose response curves of the agents given as single drug or combination regimens. Mathematics and statistical processing of dose response curves, proportion of drugs dosage to be used in the combination, calculus of theoretical additive DE20 dose as well as experimentally obtained DE20 are provided. It is also presented details of statistical comparison between theoretical and experimentally obtained DE20.

Synergistic antinociceptive effect of a calcium channel blocker and a TRPV1 blocker in an acute pain model in mice.

AIMS: Extensive evidence supports a role for voltage-gated calcium channels (VGCC) and TRPV1 receptors in pain transmission and modulation. We investigated the profile of analgesic interaction between Phα1ß toxin (a VGCC blocker) and SB366791 (selective TRPV1 antagonist) in a model of acute pain induced by capsaicin. Changes in body temperature induced by combination regimens were also evaluated. MAIN METHODS: Isobolographic approach with a fixed dose-ratio of combined drugs was used to determine whether antinociceptive interaction of Phα1ß and SB366791 are subadditive, additive or synergic. Body temperature was obtained by thermal infrared imaging. KEY FINDINGS: Phα1ß and SB366791 interact in a synergistic manner to cause antinociception. We found an interaction index (α) of 0.07 for Phα1ß and SB366791 when these drugs were injected together intraplantarly, which indicates that in vivo interaction between these drugs is greater than additive interaction. Synergism also occurred when intraplantar SB366791 was administered simultaneously with intrathecal Phα1ß (interaction index α=0.06) suggesting a 15 fold rise in potency on the analgesic effect of these drugs when they are added together. It was observed no significant alterations in body temperature of animals treated with this combination regimen. SIGNIFICANCE: Our data reveal that Phα1ß toxin potentiates in 15 fold the antinociceptive action of the TRPV1 blocker SB366791. Therefore, lower doses of these drugs are required to achieve antinociceptive effects when these agents are given in combination.

Análise do extrato aquoso de Arachis hipoagea L. no combate à dislipidemia e ao ganho ponderal de ratos Wistar submetidos à dieta hiperlipídica / Analysis of the aqueous extract of Arachis hipoagea L. to reduce dyslipidemia and weight gain in Wistar rats with high fat diet

O objetivo desse estudo foi avaliar os efeitos do Extrato Aquoso de Amendoim (EAA) no peso, bioquímica sérica e na histologia hepática de ratos Wistar submetidos a dietas normo e hiperlipídicas. A pesquisa foi realizada utilizando 40 ratos Wistar machos, divididos em quatro grupos (n=10): GA (dieta hiperlipídica), GB (dieta hiperlipídica +EAA), GC (dieta normolipídica) e GD (dieta normolipídica +EAA). Após 8 semanas, os animais foram eutanasiados e foram coletadas amostras sanguíneas para a avaliação de dados bioquímicos (Colesterol total e suas frações, triglicerídeos, uréia, creatinina, AST, ALT e glicemia) e fragmentos do fígado para análise histológica. Os animais do grupo GB tiveram um ganho de peso inferior quando comparados ao GA (XGB= versus XGA= p<0,05), já os grupos GC e GD não obtiveram diferenças estatísticas. Os animais que receberam o EAA tiveram uma redução nos níveis de colesterol (XGB= versus XGA= p<0,05 e XGD= versus XGA= p<0,01), dos triglicerídeos (XGB= versus XGA e XGD= versus XGA= p<0,001) e mais discretamente dos níveis de ALT. A glicemia, uréia e creatina permaneceram dentro dos valores de referência. As amostras hepáticas analisadas, dos ratos dos diferentes grupos, não apresentaram alterações histopatológicas. Conclui-se que O EAA apresentou efeitos preventivos sobre o ganho ponderal e dislipidemia.(AU)

The aim of this study was to evaluate the effect of Aqueous Extract from Peanut (EAA) in weight, serum biochemistry and liver histology of Wistar rats with normal hearing and a high fat diet. The survey was conducted using 40 male Wistar rats divided into four groups (n=0): GA (high fat diet), GB (fat diet+EAA), GC (normolipídica diet), and GD (normolipídica diet+EAA). After 8 weeks, the rats were euthanized and blood samples were collected to evaluate biochemical data (total cholesterol and its fractions, triglycerides, urea, creatinine, AST, ALT and glucose) and liver fragments for histological analysis. The animals of the GB group had a lower weight gain when compared with GA (XGB versus XGA= p<0.05), but CG and GD did not obtain statistical differences. The rats that received EAA had a reduction in cholesterol levels (XGB= versus XGA= p<0.05 vs. XGA and XGD= p<0.01), triglycerides (=XGB versus XGA and =XGD versus XGA= p<0,001) and more discreetly ALT levels. Blood glucose, urea and creatine remained within the reference values. Liver samples analyzed, the rats of different groups showed no histopathological changes. In conclusion, the EAA had preventive effects on weight gain and dyslipidemia.(AU)

The Phoneutria nigriventer spider toxin, PnTx4-5-5, promotes neuronal survival by blocking NMDA receptors.

Spider toxins are recognized as useful sources of bioactive substances, showing a wide range of pharmacological effects on neurotransmission. Several spider toxins have been identified biochemically and some of them are specific glutamate receptors antagonists. Previous data indicate that PnTx4-5-5, a toxin isolated from the spider Phoneutria nigriventer, inhibits the N-methyl-d-aspartate receptor (NMDAR), with little or no effect on AMPA, kainate or GABA receptors. In agreement with these results, our findings in this study show that PnTx4-5-5 reduces the amplitude of NMDAR-mediated EPSCs in hippocampal slices. It is well established that glutamate-mediated excitotoxic neuronal cell death occurs mainly via NMDAR activation. Thus, we decided to investigate whether PnTx4-5-5 would protect against various cell death insults. For that, we used primary-cultured corticostriatal neurons from wild type (WT) mice, as well as from a mouse model of Huntington's disease, BACHD. Our results showed that PnTx4-5-5 promotes neuroprotection of WT and BACHD neurons under the insult of high levels of glutamate. Moreover, the toxin is also able to protect WT neurons against amyloid ß (Aß) peptide toxicity. These results indicate that the toxin PnTx4-5-5 is a potential neuroprotective drug.

Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen.

Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.

Phα1ß toxin prevents capsaicin-induced nociceptive behavior and mechanical hypersensitivity without acting on TRPV1 channels.

Phα1ß toxin is a peptide purified from the venom of the armed spider Phoneutria nigriventer, with markedly antinociceptive action in models of acute and persistent pain in rats. Similarly to ziconotide, its analgesic action is related to inhibition of high voltage activated calcium channels with more selectivity for N-type. In this study we evaluated the effect of Phα1ß when injected peripherally or intrathecally in a rat model of spontaneous pain induced by capsaicin. We also investigated the effect of Phα1ß on Ca²âº transients in cultured dorsal root ganglia (DRG) neurons and HEK293 cells expressing the TRPV1 receptor. Intraplantar or intrathecal administered Phα1ß reduced both nocifensive behavior and mechanical hypersensitivity induced by capsaicin similarly to that observed with SB366791, a specific TRPV1 antagonist. Peripheral nifedipine and mibefradil did also decrease nociceptive behavior induced by intraplantar capsaicin. In contrast, ω-conotoxin MVIIA (a selective N-type Ca²âº channel blocker) was effective only when administered intrathecally. Phα1ß, MVIIA and SB366791 inhibited, with similar potency, the capsaicin-induced Ca²âº transients in DRG neurons. The simultaneous administration of Phα1ß and SB366791 inhibited the capsaicin-induced Ca²âº transients that were additive suggesting that they act through different targets. Moreover, Phα1ß did not inhibit capsaicin-activated currents in patch-clamp recordings of HEK293 cells that expressed TRPV1 receptors. Our results show that Phα1ß may be effective as a therapeutic strategy for pain and this effect is not related to the inhibition of TRPV1 receptors.

Endophytic fungi from plums (Prunus domestica) and their antifungal activity against Monilinia fructicola.

Enophytic fungi were isolated from plum (Prunus domestica) leaves, identified with ITS1 and ITS4 primers, and their antagonistic activity was tested against Monilinia fructicola, which causes brown rot, blossom blight, and twig blight of stone fruits, and Colletotrichum gloeosporioides, which causes anthracnose on a variety of fruit crops. The production of antifungal compounds was determined in agar-diffusion and volatile inverted-plate tests. A total of 163 fungi were recovered from 30 plum trees, representing 22 cultivars. Twenty-nine morphotypes were detected, but only 14 species were identified genetically. The most frequently isolated species was Phaeosphaeria nodorum, constituting 86.5% of the total isolates. Four isolates produced inhibitory volatiles to M. fructicola; however, no isolate produced volatiles inhibitory to C. gloeosporioides. The volatiles produced by these fungi were identified as ethyl acetate, 3-methyl-1-butanol, acetic acid, 2-propyn-1-ol, and 2-propenenitrile. The fungal volatiles inhibited growth and reduced width of the hyphae, and caused disintegration of the hyphal content. This is the first study describing fungal endophytes in stone fruits. The P. nodorum strains producing inhibitory volatiles could play a significant role in reduction of M. fructicola expansion in plum tissues. Potential of these strains for biological control of this pathogen on stone fruits warrants further investigation.