Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO₄ backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.     

Mitogen activated protein kinases SakAHOG1 and MpkC collaborate for Aspergillus fumigatus virulence

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen‐activated protein kinases of the high‐osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild‐type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.     

Spermatogenesis in goats with and without scrotum bipartition

The objective of the present research was to quantify the seminiferous epithelium cells, spermatogenesis efficiency and characterize the ultrastrucure of Sertoli cells in goats. Eighteen goats were used and divided into three groups: Group I - goats without bipartition of the scrotum; Group II - animals with bipartition of the scrotum in up to 50% of the testicular length; Group III - goats with bipartition of the scrotum in more than 50% of the testicular length. The goat testes in Group III had a greater number of primary spermatocytes (25.37 ± 4.55 cells per cross sections), spermatids (112 ± 15.12 cells per cross sections), and Sertoli cells (9.46 ± 1.74 cells per cross sections) than the animals in Groups I and II (P<0.05). The spermatogenic mitotic, meiotic, and general efficiency were greater in animals in Group III (1.25 ± 0.28; 5.12 ± 1.63; 6.44 ± 1.96) when compared to those in Groups I and II. Sheet-like processes originated from the Sertoli cell body as simple and smooth structures which involved almost all the surface of germ cells. Slender cord-like processes originated from Sertoli cells and also from the sheet-like processes. The relative frequency of the cycle stages showed differences among the groups of goats studied, and the highest frequency was in Stage 3 (20.68% for goats in Group I, 21.15% for those in Group II, and 16.89% for the animals in Group III). In conclusion, goats with bipartition of the scrotum have a greater number of germ and Sertoli cells per cross section of seminiferous tubule, that indicated a greater sperm production when compared to the other groups, and the ultrastructure of the Sertoli cell process did not present any relationship with bipartition of the scrotum.     

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

Pollen morphology of the Acalyphoideae and Euphorbioideae (Euphorbiaceae) of the Caatinga ecoregion in Brazil

This study dealt with six species of Acalyphoideae and 18 species of Euphorbioideae occurring in the Caatinga ecoregion, with emphasis on endemic species. Pollen samples were obtained from herbarium specimens and were acetolysed and analysed via light and scanning electron microscopy. The pollen of three genera of Acalyphoideae was medium to large, 3-colporate or 3-colpate, with an echinate-perforate exine that was reticulate, bireticulate, and microreticulate. The six genera of Euphorbioideae studied exhibited pollen grains that were small, medium and large; 3-colporate with margines; and an exine with microreticulate, microreticulate-rugulate, microreticulate-caveate, and reticulate ornamentation. The pollen characteristics were more variable in the Acalyphoideae compared to the relatively homogeneous Euphorbioideae. This study provides new data and interpretations of the pollen morphology of two subfamilies of the Euphorbiaceae of the Caatinga ecoregion in Brazil.     

Profile of Central and Effector Memory T Cells in the Progression of Chronic Human Chagas Disease

Background

Chronic Chagas disease presents several different clinical manifestations ranging from asymptomatic to severe cardiac and/or digestive clinical forms. Several studies have demonstrated that immunoregulatory mechanisms are important processes for the control of the intense immune activity observed in the chronic phase. T cells play a critical role in parasite specific and non-specific immune response elicited by the host against Trypanosoma cruzi. Specifically, memory T cells, which are basically classified as central and effector memory cells, might have a distinct migratory activity, role and function during the human Chagas disease.

Methodology/Principal Findings

Based on the hypothesis that the disease severity in humans is correlated to the quality of immune responses against T. cruzi, we evaluated the memory profile of peripheral CD4⁺ and CD8⁺ T lymphocytes as well as its cytokine secretion before and after in vitro antigenic stimulation. We evaluated cellular response from non-infected individuals (NI), patients with indeterminate (IND) or cardiac (CARD) clinical forms of Chagas disease. The expression of CD45RA, CD45RO and CCR7 surface molecules was determined on CD4⁺ and CD8⁺ T lymphocytes; the pattern of intracellular cytokines (IFN-γ, IL-10) synthesized by naive and memory cells was determined by flow cytometry. Our results revealed that IND and CARD patients have relatively lower percentages of naive (CD45RA^high) CD4⁺ and CD8⁺ T cells. However, statistical analysis of ex-vivo profiles of CD4⁺ T cells showed that IND have lower percentage of CD45RA^high in relation to non-infected individuals, but not in relation to CARD. Elevated percentages of memory (CD45RO^high) CD4⁺ T cells were also demonstrated in infected individuals, although statistically significant differences were only observed between IND and NI groups. Furthermore, when we analyzed the profile of secreted cytokines, we observed that CARD patients presented a significantly higher percentage of CD8⁺CD45RA^high IFN-γ-producing cells in control cultures and after antigen pulsing with soluble epimastigote antigens.

Conclusions

Based on a correlation between the frequency of IFN-γ producing CD8+ T cells in the T cell memory compartment and the chronic chagasic myocarditis, we propose that memory T cells can be involved in the induction of the development of the severe clinical forms of the Chagas disease by mechanisms modulated by IFN-γ. Furthermore, we showed that individuals from IND group presented more T_CM CD4⁺ T cells, which may induce a regulatory mechanism to protect the host against the exacerbated inflammatory response elicited by the infection.     

Environmental and Animal Characteristics as Factors Associated with American Cutaneous Leishmaniasis in Rural Locations with Presence of Dogs,Brazil

The aim of the study was to investigate the importance of dogs, other domesticated animals and environmental characteristics as risk factors in the epidemiology of American cutaneous leishmaniasis (ACL). A retrospective survey of cases of human ACL in the last ten years and visits to homes in rural locations were carried out in the municipality of Arapongas (southern Brazil) from 2008 to 2010. ACL in humans was significantly associated with a distance of up to 25 meters from the residence to a forest area (OR 5.08; 95% CI: 1.35–21.04), undergrowth area (OR 6.80; 95% CI: 1.69–45.33) and stream (OR 5.87; 95% CI: 1.15–24.59); banana plants near the residence (OR 5.98; 95% CI: 1.49–39.84), absence of ceiling below the roof in the residence (OR 7.30; 95% CI: 1.26–158.1), the dumping of trash in the forest area (OR 26.33; 95% CI: 7.32–93.46) and presence of ACL in dogs in the surrounding area (OR 4.39; 95% CI: 1.37–13.45). In dogs, ACL was associated with a distance of 25 to 50 meters and 51 to 100 meters, respectively, from the residence to a forest area (OR 2.59; 95% CI: 1.08–5.98; OR 3.29; 95% CI: 1.64–6.62), the presence of a stream up to 25 m from the residence (OR 6.23; 95% CI: 2.34–16.54) and banana plants near the residence (OR 0.45; 95% CI: 0.25–0.80). In the locations studied in the municipality of Arapongas (Brazil), the results reveal that canine infection increases the risk of human infection by ACL and the characteristics surrounding the residence increase the risk of infection in both humans and dogs. Thus, integrated environmental management could be a useful measure to avoid contact between humans and phlebotomines.     

Experimental benznidazole treatment of Trypanosoma cruzi II strains isolated from children of the Jequitinhonha Valley,Minas Gerais,Brazil, with Chagas disease

Trypanosoma cruzi strains from distinct geographic areas show differences in drug resistance and association between parasites genetic and treatment response has been observed. Considering that benznidazole (BZ) can reduce the parasite burden and tissues damage, even in not cured animals and individuals, the goal is to assess the drug response to BZ of T. cruzi II strains isolated from children of the Jequitinhonha Valley, state of Minas Gerais, Brazil, before treatment. Mice infected and treated with BZ in both phases of infection were compared with the untreated and evaluated by fresh blood examination, haemoculture, polymerase chain reaction, conventional (ELISA) and non-conventional (FC-ALTA) serologies. In mice treated in the acute phase, a significant decrease in parasitaemia was observed for all strains. Positive parasitological and/or serological tests in animals treated during the acute and chronic (95.1-100%) phases showed that most of the strains were BZ resistant. However, beneficial effect was demonstrated because significant reduction (p < 0.05%) and/or suppression of parasitaemia was observed in mice infected with all strains (acute phase), associated to reduction/elimination of inflammation and fibrosis for two/eight strains. BZ offered some benefit, even in not cured animals, what suggest that BZ use may be recommended at least for recent chronic infection of the studied region.     

Relationships between taxonomic and functional components of diversity: implications for conservation of tropical freshwater fishes

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