Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

Paracoccidioidomycosis in people living with HIV/AIDS: A historical retrospective cohort study in a national reference center for infectious diseases,Rio de Janeiro,Brazil

Paracoccidioidomycosis (PCM) is one of the main endemic systemic mycoses in Latin America, usually occurring in rural areas. When PCM occurs simultaneously with underlying immunosuppressive conditions, it can present as an opportunistic disease. Between 2000 and 2017, literature reported around 200 PCM cases in people living with HIV/AIDS (PLWHA). To address research gaps on this co-infection and to study its possible temporal changes in the last decade, we performed an active co-infection case search on the HIV/AIDS and PCM cohorts from a Brazilian reference center database from 1989 to 2019. We found 20 PLWHA among 684 PCM patients (2.92%), predominantly male (70.0%) and urban workers (80.0%). The median age of patients was higher in the 2010–2019 decade (p = 0.006). The occurrence of PCM in PLWHA was lower when compared with other fungal diseases. Although 50.0% of the patients had already been diagnosed with HIV infection and presented CD4+ T cell counts greater than 200/mm³ at the time of PCM diagnosis, the suspicion of immunosuppression in the context of atypical and more severe clinical forms of PCM revealed the diagnosis of HIV infection in 35.0% of the patients. Two (10.0%) patients had an evolution compatible with immune reconstitution inflammatory syndrome (IRIS) after starting antiretroviral therapy (ART).We highlight the importance of considering a PCM diagnosis in PLWHA to prevent a late-onset treatment and progression to severe manifestations and unfavorable outcomes. In addition, HIV investigation is recommended in PCM patients, especially those with atypical and more severe clinical presentations.     

The role of melanin- and carotenoid-based plumage coloration in nest defence in the Great Tit

Although plumage coloration is recognized to convey valuable information about the bearer's parental abilities, few studies have explored the relationship between coloration and nest defence. In this study in Great Tit Parus major, we analysed the relationship between nest defence and melanin‐ as well as carotenoid‐based plumage coloration, after controlling for ecological variables known to influence nest defence. A principal components analysis was applied to classify birds according to how vigorously they defended the nest, and the intensity of nest defence was tested against plumage coloration. Males with a large black tie defended their nests more vigorously, but no such effect was found for yellow breast coloration. This suggests that melanin‐based coloration in the Great Tit is associated with aggression, including both dominance‐aggression and nest defence, whereas carotenoid‐based coloration is not. The challenge in future studies will be to demonstrate whether females use this trait as an ornament to assess male quality and whether they trade off between the different ornaments a male may exhibit.     

Vascular redox imbalance in rats submitted to chronic exercise

Exercise training has been used for treatment/prevention of many cardiovascular diseases, but the mechanisms need to be clarified. Thus, our aim was to compare oxidative stress parameters between rats submitted to a swimming training and sedentary rats (control). Twelve male rats were divided into two groups: control and exercise training. The exercise training had daily 1 h swimming sessions for 8 weeks and a load (5% of its body mass) was placed in rat's tail. Thereafter the animals were killed, aorta and heart were surgically removed and blood was collected. Body mass gain, thiobarbituric acid reactive species (TBARS), carbonyl content, total reactive antioxidant potential (TRAP), total antioxidant reactivity (TAR), superoxide dismutase (SOD) activity and catalase (CAT) activity were evaluted. The trained rats showed a lower body mass gain and no modifications on heart. An increased SOD activity was observed on aorta after the training, but no changes were seen for CAT activity, which led to an increased SOD/CAT ratio. The arterial TBARS was also increased for trained rats. The decrease in TRAP in exercise training was the single modification on plasma. Our findings suggest that the increased SOD activity could play a role in vascular adaptations to exercise training. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein

Alpha-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant alpha-synuclein (alpha-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of alpha-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of alpha-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized alpha-synuclein-C4 molecules. Alpha-synuclein-C4 offers the means for directly probing amyloid formation and interactions of alpha-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.     

Biochemical characterization and molecular cloning of a plasminogen activator proteinase (LV-PA) from bushmaster snake venom

The protein (LV-PA) from bushmaster (Lachesis muta muta) venom is a serine proteinase which specifically activates the inactive proenzyme plasminogen. LV-PA is a single chain glycoprotein with an apparent molecular mass of 33 kDa that fell to 28 kDa after treatment with N-Glycosidase F (PNGase F). Approximately 93% of its protein sequence was determined by automated Edman degradation of various fragments derived from a digestion with trypsin. A cDNA library of L. m. muta was constructed to generate expressed sequence tags (ESTs) and the plasminogen activator precursor cDNA was sequenced. The complete amino acid sequence of the enzyme was deduced from the cDNA sequence. LV-PA is composed of 234 residues and contains a single asparagine-linked glycosylation site, Asn-X-Ser, bearing sugars that account for approximately 10% of the enzyme's total molecular mass of 33 kDa. The sequence of LV-PA is highly similar to the plasminogen activators (PAs) TSV-PA from Trimeresurus stejnegeri venom and Haly-PA from Agkistrodon halys. Furthermore, the mature protein sequence of LV-PA exhibits significant similarity with other viperidae venom serine proteinases which affect many steps of hemostasis, ranging from the blood coagulation cascade to platelet function. The Michaelis constant (Km) and the catalytic rate constant (kcat) of LV-PA on four chromogenic substrates were obtained from Lineweaver-Burk plots. In addition, we used an indirect enzyme-linked immunoabsorbent assay (ELISA) to explore the phylogenetic range of immunological cross-reactivity (using antibodies raised against LV-PA) with analogous serine proteinases from two viperidae venoms and mammals.