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The fungus Paracoccidioides brasiliensis has been isolated from nine-banded armadillos ( Dasypus novemcinctus ) in different regions where paracoccidiodomycosis (PCM) is endemic. The link between PCM and these animals has provided the first valuable clue in the effort to elucidate the ecological niche of P . brasiliensis . The present study was aimed at correlating P . brasiliensis infection in armadillos with local ecological features and, if possible, the presence of the fungus in the soil in the Botucatu hyperendemic area of PCM. In this region the mean temperature ranges from 14.8 to 25.8°C and the annual average precipitation is 1520 mm. The sites where 10 infected animals (positive group) were collected were studied and compared with the sites where five uninfected animals were found. The occurrence of the fungus in soil samples collected from the positive armadillos' burrows and foraging sites was investigated by the indirect method of animal inoculation. Environmental data from the sites of animal capture, such as temperature, rainfall, altitude, vegetation, soil composition, presence of water and proximity of urban areas, were recorded. All 37 soil samples collected from the sites had negative fungal cultures. Positive animals were found much more frequently in sites with disturbed vegetation, such as riparian forests and artificial Eucalyptus or Pinus forests, in altitudes below 800 m, near water sources. The soil type of the sites of positive animals was mainly sandy, with medium to low concentrations of organic matter. The pH was mainly acidic at all the sites, although the concentrations of aluminum cations (H+Al) were lower at the sites where positive animals were found. Positive armadillos were also captured in sites very close to urban areas. Our data and previous studies indicate that P . brasiliensis occurs preferentially in humid and shady disturbed forests in a strong association with armadillos. [ABSTRACT FROM AUTHOR]

Abstract: Although it is long known that the tricyclic antidepressants amitriptyline, nortriptyline and imipramine inhibit the noradrenaline transporter and α1-adrenoceptors with similar affinities, which may lead to self-cancelling actions, the selectivity of these drugs for α1-adrenoceptor subtypes is unknown. The present study investigates the selectivity of amitriptyline, nortriptyline and imipramine for human recombinant and rat native α1-adrenoceptor subtypes. The selectivity of amitriptyline, nortriptyline and imipramine was investigated in HEK-293 cells expressing each of the human α1-subtypes and in rat native receptors from the vas deferens (α1A), spleen (α1B) and aorta (α1D) through [3H]prazosin binding, and noradrenaline-induced intracellular Ca2+ increases and contraction assays. Amitriptyline, nortriptyline and imipramine showed considerably higher affinities for α1A- (∼25- to 80-fold) and α1D-adrenoceptors (∼10- to 25-fold) than for α1B-adrenoceptors in both contraction and [3H]prazosin binding assays with rat native and human receptors, respectively. In addition, amitriptyline, nortriptyline and imipramine were substantially more potent in the inhibition of noradrenaline-induced intracellular Ca2+ increases in HEK-293 cells expressing α1A- or a truncated version of α1D-adrenoceptors which traffics more efficiently towards the cell membrane than in cells expressing α1B-adrenoceptors. Amitriptyline, nortriptyline and imipramine are much weaker antagonists of rat and human α1B-adrenoceptors than of α1A- and α1D-adrenoceptors. The differential affinities for these receptors indicate that the α1-adrenoceptor subtype which activation is most increased by the augmented noradrenaline availability resultant from the blockade of neuronal reuptake is the α1B-adrenoceptor. This may be important for the behavioural effects of these drugs. [Copyright &y& Elsevier]

Soil is the principal habitat and reservoir of fungi that act on ecological processes vital for life on Earth. Understanding soil fungal community structures and the patterns of species distribution is crucial, considering climatic change and the increasing anthropic impacts affecting nature. We evaluated the soil fungal diversity in southeastern Brazil, in a transitional region that harbors patches of distinct biomes and ecoregions. The samples originated from eight habitats, namely: semi-deciduous forest, Brazilian savanna, pasture, coffee and sugarcane plantation, abandoned buildings, owls' and armadillos' burrows. Forty-four soil samples collected in two periods were evaluated by metagenomic approaches, focusing on the high-throughput DNA sequencing of the ITS2 rDNA region in the Illumina platform. Normalized difference vegetation index (NDVI) was used for vegetation cover analysis. NDVI values showed a linear relationship with both diversity and richness, reinforcing the importance of a healthy vegetation for the establishment of a diverse and complex fungal community. The owls' burrows presented a peculiar fungal composition, including high rates of Onygenales, commonly associated with keratinous animal wastes, and Trichosporonales, a group of basidiomycetous yeasts. Levels of organic matter and copper influenced all guild communities analyzed, supporting them as important drivers in shaping the fungal communities' structures.

Hepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A-H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.

The HIV subtype B is the most frequent in Brazil. The HIV subtype B' codes the amino acids glicine-tryptophan-glicine (GWG) instead of glicine-proline-glicine on the tip of gp120 V3 loop. This variant was associated to a slower HIV progression in mono-infected patients; however, there is no information in coinfected patients. This study evaluated the infection progression of HIV variant B' on the hepatitis C virus presence. RNA isolated from plasma of the 601 infected patients were used to human immunodeficiency virus (HIV) subtyping and to classify the virus according their syncytium-inducing ability. The HIV infection progression was evaluated by clinical and laboratorial data. The results showed a significant association between HIV B' variant and CD4 count and time of AIDS in HIV mono-infected patients. Notwithstanding the fact that we did not find a direct association between GWG variant and AIDS and in HIV coinfected patients no mitigating effect due to GWG presence was found. We did observe that the association between GWG variant and CD4 counts is lost in coinfected patients. This is first work showing influence of the HIV GWG variant in coinfected patients. Nevertheless, the presence of the GWG variant can indicate a better prognostic in the mono-infected patients.

The cauda epididymis (CE), the site of sperm storage until the ejaculation, is densely innervated by the sympathetic nervous system. Contraction of CE smooth muscle via α 1 -adrenoceptors ( α 1 -ARs) plays a key role during the seminal emission phase of ejaculation and α 1 -AR antagonism has been suggested as a nonhormonal and reversible male contraceptive target. Since the α 1 -AR subtype mediating contraction of rat CE is not known, this study investigates the expression and role of α 1 -AR subtypes on the proximal and distal rat CE duct contraction to norepinephrine in vitro. Alpha 1a , α 1b , and α 1d transcripts were detected by real-time quantitative polymerase chain reaction in proximal and distal CE segments and α 1a and α 1d were shown to predominate over α 1b The inhibition of [ 3 H]prazosin specific binding to intact CE segments from proximal and distal CE by RS 100329 and 5-methylurapidil ( α 1A -selective) and BMY 7378 ( α 1D -selective) showed that α 1A - and α 1D -ARs are expressed at similar densities. Norepinephrine-induced contractions of CE were competitively antagonized with high affinity by RS 100329 (p K B ≈ 9.50) and 5-methylurapidil (p K B ≈ 9.0) and with low affinity by BMY 7378 (p K B ≈ 7.0) and the α 1B -selective L-765,314 (p A 2 < 7.0), suggesting contractions are mediated by α 1A -ARs. The clinically used α 1A/D -ARs antagonist tamsulosin potently (p A 2 ≈ 10.0) inhibited the norepinephrine-induced CE contractions. Altogether, our results show that α 1A - and α 1D -ARs are expressed in the CE duct and α 1A -AR is the main subtype mediating contraction to norepinephrine. Our results highlight the importance of α 1A -AR in the peripheral control of ejaculation and strengthen the α 1A -AR as a target for a nonhormonal approach to male contraception., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Loss of response on repetitive drug exposure (i.e., tachyphylaxis) is a particular problem for the vasoconstrictor effects of medications containing oxymetazoline (OXY), an α1-adrenoceptor (AR) agonist of the imidazoline class. One cause of tachyphylaxis is receptor desensitization, usually accompanied by phosphorylation and internalization. It is well established that α1A-ARs are less phosphorylated, desensitized, and internalized on exposure to the phenethylamines norepinephrine (NE), epinephrine, or phenylephrine (PE) than are the α1B and α1D subtypes. However, here we show in human embryonic kidney-293 cells that the low-efficacy agonist OXY induces G protein-coupled receptor kinase 2-dependent α1A-AR phosphorylation, followed by rapid desensitization and internalization (∼40% internalization after 5 minutes of stimulation), whereas phosphorylation of α1A-ARs exposed to NE depends to a large extent on protein kinase C activity and is not followed by desensitization, and the receptors undergo delayed internalization (∼35% after 60 minutes of stimulation). Native α1A-ARs from rat tail artery and vas deferens are also desensitized by OXY, but not by NE or PE, indicating that this property of OXY is not limited to recombinant receptors expressed in cell systems. The results of the present study are clearly indicative of agonist-directed α1A-AR regulation. OXY shows functional selectivity relative to NE and PE at α1A-ARs, leading to significant receptor desensitization and internalization, which is important in view of the therapeutic vasoconstrictor effects of this drug and the varied biologic process regulated by α1A-ARs.

Although it is long known that the tricyclic antidepressants amitriptyline, nortriptyline and imipramine inhibit the noradrenaline transporter and alpha(1)-adrenoceptors with similar affinities, which may lead to self-cancelling actions, the selectivity of these drugs for alpha(1)-adrenoceptor subtypes is unknown. The present study investigates the selectivity of amitriptyline, nortriptyline and imipramine for human recombinant and rat native alpha(1)-adrenoceptor subtypes. The selectivity of amitriptyline, nortriptyline and imipramine was investigated in HEK-293 cells expressing each of the human alpha(1)-subtypes and in rat native receptors from the vas deferens (alpha(1A)), spleen (alpha(1B)) and aorta (alpha(1D)) through [(3)H]prazosin binding, and noradrenaline-induced intracellular Ca(2+) increases and contraction assays. Amitriptyline, nortriptyline and imipramine showed considerably higher affinities for alpha(1A)- (approximately 25- to 80-fold) and alpha(1D)-adrenoceptors (approximately 10- to 25-fold) than for alpha(1B)-adrenoceptors in both contraction and [(3)H]prazosin binding assays with rat native and human receptors, respectively. In addition, amitriptyline, nortriptyline and imipramine were substantially more potent in the inhibition of noradrenaline-induced intracellular Ca(2+) increases in HEK-293 cells expressing alpha(1A)- or a truncated version of alpha(1D)-adrenoceptors which traffics more efficiently towards the cell membrane than in cells expressing alpha(1B)-adrenoceptors. Amitriptyline, nortriptyline and imipramine are much weaker antagonists of rat and human alpha(1B)-adrenoceptors than of alpha(1A)- and alpha(1D)-adrenoceptors. The differential affinities for these receptors indicate that the alpha(1)-adrenoceptor subtype which activation is most increased by the augmented noradrenaline availability resultant from the blockade of neuronal reuptake is the alpha(1B)-adrenoceptor. This may be important for the behavioural effects of these drugs., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Interleukin 13 (IL-13) is immunoregulatory in many diseases, including cancer. The protective or suppressive role of CD1-restricted natural killer T cells (NKT cells) in tumor immunosurveillance and immunity is well documented. Interleukin 12 (IL-12) can activate type I NKT cells to produce interferon-gamma (IFN-gamma), whereas type II NKT cells may produce IL-13. The high-affinity chain of IL-13Ralpha2 may act as negative inhibitor, suppressing the action of IL-13 and helping to maintain tumor immunosurveillance. We constructed an mIL-13Ralpha2-Fc chimera in a eukaryotic expression vector and confirmed the identity of the recombinant protein by immunoblot analysis and binding to IL-13 in chemiluminescent ELISA. Such DNA vaccine was tested against syngeneic B16F10-Nex2 murine melanoma. In vivo experiments showed a protective effect mediated by high production of IFN-gamma and down-regulation of anti-inflammatory interleukins mainly by NKT 1.1(+) T cells. Biochemoterapy in vivo with plasmid encoding mIL-13Ralpha2-Fc in association with plasmid encoding IL-12 and the 7A cyclopalladated drug led to a significant reduction in the tumor evolution with 30% tumor-free mice. We conclude that IL-12 gene therapy, followed by continuous administration of IL-13Ralpha2-Fc gene along with 7A-drug has antitumor activity involving the high production of proinflammatory cytokines and low immune suppression, specifically by NK1.1(+)T cells producing IL-13 and IL-10.

From a 0.72-kb fragment universally generated in Paracoccidioides brasiliensis strains, primers were designed and tested on genomic DNA of this and other pathogenic fungi. They were specific and highly sensitive for P. brasiliensis DNA. Positive results were obtained when these were tested in clinical samples.

Paracoccidioides brasiliensis isolates from 10 nine-banded armadillos (Dasypus novemcinctus) were comparable with 19 clinical isolates by sequence analysis of the PbGP43 gene and ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and by random amplified polymorphic DNA. In this original ITS study, eight isolates differed by one or three sites among five total substitution sites.