Differences in Cell Morphometry,Cell Wall Topography and Gp70 Expression Correlate with the Virulence of Sporothrix brasiliensis Clinical Isolates

Sporotrichosis is a chronic infectious disease affecting both humans and animals. For many years, this subcutaneous mycosis had been attributed to a single etiological agent; however, it is now known that this taxon consists of a complex of at least four pathogenic species, including Sporothrix schenckii and Sporothrix brasiliensis. Gp70 was previously shown to be an important antigen and adhesin expressed on the fungal cell surface and may have a key role in immunomodulation and host response. The aim of this work was to study the virulence, morphometry, cell surface topology and gp70 expression of clinical isolates of S. brasiliensis compared with two reference strains of S. schenckii. Several clinical isolates related to severe human cases or associated with the Brazilian zoonotic outbreak of sporotrichosis were genotyped and clustered as S. brasiliensis. Interestingly, in a murine subcutaneous model of sporotrichosis, these isolates showed a higher virulence profile compared with S. schenckii. A single S. brasiliensis isolate from an HIV-positive patient not only showed lower virulence but also presented differences in cell morphometry, cell wall topography and abundant gp70 expression compared with the virulent isolates. In contrast, the highly virulent S. brasiliensis isolates showed reduced levels of cell wall gp70. These observations were confirmed by the topographical location of the gp70 antigen using immunoelectromicroscopy in both species. In addition, the gp70 molecule was sequenced and identified using mass spectrometry, and the sequenced peptides were aligned into predicted proteins using Blastp with the S. schenckii and S. brasiliensis genomes.     

Using body geometric morphometrics to identify bluemouth, <Emphasis Type="Italic">Helicolenus dactylopterus</Emphasis> (Delaroche, 1809) populations in the Northeastern Atlantic

In this study, landmark-based geometric morphometric methods were applied to evaluate the possible existence of different bluemouth, Helicolenus dactylopterus (Delaroche, 1809), populations in the NE Atlantic. Fish were obtained from commercial landings in Azores, Madeira, and Peniche (mainland Portugal). Samples from a research survey along the Galician coast (Spain) were also included in this study. A generalized procrustes analysis was done to remove non-shape variation. Then, the obtained shape variables were statistically adjusted to eliminate allometry and permit a comparative analysis of the samples. The discriminant analysis performed to test the homogeneity of the data set regarding sex revealed no shape differences between males and females within the four areas under study. The body shapes of bluemouth differed significantly among the four areas using Procrustes distances (P < 0.01). In this study, a considerable morphological heterogeneity within the Azores group was observed, which could reflect a substructure of the bluemouth population within this area. According to our results, bluemouth from Galicia and Peniche seem to be relatively well separated from each other, despite both areas are relatively close geographically. Bluemouth from Madeira was well differentiated from the rest of locations and presented the highest correct classification rate in the discriminant analysis. In conclusion, morphological differences suggest that different bluemouth populations exist in the NE Atlantic, but an integrated approach, using different methods, would be needed to confirm the population structure of this species.     

Omega-3 fatty acid-derived mediators 17(R)-hydroxy docosahexaenoic acid, aspirin-triggered resolvin D1 and resolvin D2 prevent experimental colitis in mice

Resolvins of the D series are generated from docosahexaenoic acid, which are enriched in fish oils and are believed to exert beneficial roles on diverse inflammatory disorders, including inflammatory bowel disease (IBD). In this study, we investigated the anti-inflammatory effects of the aspirin-triggered resolvin D1 (AT-RvD1), its precursor (17(R)-hydroxy docosahexaenoic acid [17R-HDHA]) and resolvin D2 (RvD2) in dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Our results showed that the systemic treatment with AT-RvD1, RvD2, or 17R-HDHA in a nanogram range greatly improved disease activity index, body weight loss, colonic damage, and polymorphonuclear infiltration in both colitis experimental models. Moreover, these treatments reduced colonic cytokine levels for TNF-α, IL-1β, MIP-2, and CXCL1/KC, as well as mRNA expression of NF-κB and the adhesion molecules VCAM-1, ICAM-1, and LFA-1. Furthermore, AT-RvD1, but not RvD2 or 17R-HDHA, depended on lipoxin A4 receptor (ALX) activation to inhibit IL-6, MCP-1, IFN-γ, and TNF-α levels in bone marrow-derived macrophages stimulated with LPS. Similarly, ALX blockade reversed the beneficial effects of AT-RvD1 in DSS-induced colitis. To our knowledge, our findings showed for the first time the anti-inflammatory effects of resolvins of the D series and precursor 17R-HDHA in preventing experimental colitis. We also demonstrated the relevant role exerted by ALX activation on proresolving action of AT-RvD1. Moreover, AT-RvD1 showed a higher potency than 17R-HDHA and RvD2 in preventing DSS-induced colitis. The results suggest that these lipid mediators possess a greater efficacy when compared with other currently used IBD therapies, such as monoclonal anti-TNF, and have the potential to be used for treating IBD.     

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC₅₀ values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.     

Bradykinin promotes migration and invasion of human immortalized trophoblasts

Maternal undernutrition (MUN) during pregnancy may lead to fetal intrauterine growth restriction (IUGR), which itself predisposes to adult risk of obesity, hypertension, and diabetes. IUGR may stem from insufficient maternal nutrient supply or reduced placental nutrient transfer. In addition, a critical role for maternal stress-induced glucocorticoids (GCs) has been suggested to contribute to both IUGR and the ensuing risk of adult metabolic syndrome. While GC-induced fetal organ defects have been examined, there have been few studies on placental responses to MUN-induced maternal stress. Therefore, we hypothesize that 50% MUN associates with increased maternal GC levels and decreased placental HSD11B. This in turn leads to decreased placental and fetal growth, hence the need to investigate nutrient transporters. We measured maternal serum levels of corticosterone, and the placental basal and labyrinth zone expression of glucocorticoid receptor (NR3C1), 11-hydroxysteroid dehydrogenase B 1 (HSD11B-1) predominantly activates cortisone to cortisol and 11-dehydrocorticosterone (11-DHC) to corticosterone, although can sometimes drive the opposing (inactivating reaction), and HSD11B-2 (only inactivates and converts corticosterone to 11-DHC in rodents) in control and MUN rats at embryonic day 20 (E20). Moreover, we evaluated the expression of nutrient transporters for glucose (SLC2A1, SLC2A3) and amino acids (SLC38A1, 2, and 4). Our results show that MUN dams displayed significantly increased plasma corticosterone levels compared to control dams. Further, a reduction in fetal and placental weights was observed in both the mid-horn and proximal-horn positions. Notably, the placental labyrinth zone, the site of feto-maternal exchange, showed decreased expression of HSD11B1-2 in both horns, and increased HSD11B-1 in proximal-horn placentas, but no change in NR3C1. The reduced placental GCs catabolic capacity was accompanied by downregulation of SLC2A3, SLC38A1, and SLC38A2 expression, and by increased SLC38A4 expression, in labyrinth zones from the mid- and proximal-horns. In marked contrast to the labyrinth zone, the basal zone, which is the site of hormone production, did not show significant changes in any of these enzymes or transporters. These results suggest that dysregulation of the labyrinth zone GC "barrier", and more importantly decreased nutrient supply resulting from downregulation of some of the amino acid system A transporters, may contribute to suboptimal fetal growth under MUN.     

Antibodies to selected pathogens in wild boar (Sus scrofa) from Catalonia (NE Spain)

From 2004 to 2007, blood samples from 273 healthy wild boars (Sus scrofa), culled during the hunting season, were obtained in three areas of Catalonia (NE Spain): Pyrenees, Sant Llorenç del Munt i l’Obac Natural Park (SLM), and Ports de Tortosa i Beseit National Hunting Reserve (PTB). We investigated the presence of antibodies against classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine vesicular disease virus (PVDV), porcine respiratory and reproductive syndrome virus (PRRSV), Aujeszky’s disease virus (ADV), porcine influenza A virus (PIV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Salmonella spp., and Toxoplasma gondii. Four wild boars were suspicious for CSFV, but the infection was discarded with a virus neutralization test, and infection with a border disease virus was confirmed. Negative results were obtained against ASFV and PVDV. Antibodies were detected against PRRSV (3%), ADV (0.8%), PIV (6.4%), PCV2 (64.6%), PPV (54.7%), M. hyopneumoniae (26.6%), E. rhusiopathiae (5.3%), Salmonella spp. (11.3%), and T. gondii (43.5%). In SLM, we detected a higher seroprevalence for PIV and M. hyopneumoniae and a lower seroprevalence for E. rhusiopathiae than in the other two areas. In PTB, seroprevalence was higher for PPV, Salmonella spp., and PCV2. Adult wild boar displayed higher seroprevalence for PPV, PIV, and M. hyopneumoniae, whereas presence of antibodies for Salmonella spp. was higher in juveniles compared with adults and piglets.

     

Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.