pH dependence of lipid peroxidation and albumin oxidative modification: possible implications to the pH paradox

The hydrogen ion concentration may have an important influence in biological free radical reactions. We studied the effect of an acidic pH on two models of free radical-mediated damage: copper-induced lipid peroxidation in plasma and copper/hydrogen peroxide-induced oxidative modification of albumin. A reduction of pH from 7·4 to 6·6 decreased diene conjugation by 32%, thiobarbituric acid-reactive substances formation by 25% and fluorescence generation by 53% in plasma exposed to cupric chloride. At pH values lower than 6·6 an even greater inhibition of lipid peroxidation in plasma was obtained. Visible fluorescence development in albumin by exposure to site-specific generation of free radicals was also increasingly reduced by decreasing pH values. From pH 7·4 to 6·6 there was a 50% fluorescence generation inhibition. The observed partial protection of lipids and proteins against oxidative damage by an acidic pH alerts to the need for rigorously controlling the pH values when assaying compounds for antioxidant properties in vitro. It may also contribute to the explanation for the protective effect of an acidic pH against anoxic cell injury and for cell death that is precipitated by a rapid return to a normal pH following reperfusion (the ‘pH paradox’).     

Analysis of castor bean ribosome-inactivating proteins and their gene expression during seed development

Ribosome-inactivating proteins (RIPs) are enzymes that inhibit protein synthesis after depurination of a specific adenine in rRNA. The RIP family members are classified as type I RIPs that contain an RNA-N-glycosidase domain and type II RIPs that contain a lectin domain (B chain) in addition to the glycosidase domain (A chain). In this work, we identified 30 new plant RIPs and characterized 18 Ricinus communis RIPs. Phylogenetic and functional divergence analyses indicated that the emergence of type I and II RIPs probably occurred before the monocot/eudicot split. We also report the expression profiles of 18 castor bean genes, including those for ricin and agglutinin, in five seed stages as assessed by quantitative PCR. Ricin and agglutinin were the most expressed RIPs in developing seeds although eight other RIPs were also expressed. All of the RIP genes were most highly expressed in the stages in which the endosperm was fully expanded. Although the reason for the large expansion of RIP genes in castor beans remains to be established, the differential expression patterns of the type I and type II members reinforce the existence of biological functions other than defense against predators and herbivory.     

Detection of Oseltamivir-Resistant Pandemic Influenza A(H1N1)pdm2009 in Brazil: Can Community Transmission Be Ruled Out?

Although surveillance efforts that monitor the emergence of drug-resistant strains of influenza are critical, systematic analysis is overlooked in most developing countries. We report on the occurrence of strains of pandemic influenza A(H1N1)pdm09 with resistance and decreased susceptibility to oseltamivir (OST) in Brazil in 2009, 2011 and 2012. We found 7 mutant viruses, 2 with the mutation S247N and other 5 with the mutation H275Y. Most of these viruses were from samples concentrated in the southern region of Brazil. Some of these resistant viruses were detected prior to the initiation of OST treatment, suggesting that community transmission of mutant viruses may exist. Moreover, we show that one of these OST-resistant (H275Y) strains of A(H1N1)pdm09 was discovered in the tri-border region between Brazil, Argentina and Paraguay, highlighting that this strain could also be found in other Latin American countries. Our findings reinforce the importance of enhanced antiviral resistance surveillance in Brazil and in other Latin American countries to confirm or rule out the community transmission of OST-resistant strains of A(H1N1)pdm09.     

Asymptomatic Cattle Naturally Infected with Mycobacterium bovis Present Exacerbated Tissue Pathology and Bacterial Dissemination

Rational discovery of novel immunodiagnostic and vaccine candidate antigens to control bovine tuberculosis (bTB) requires knowledge of disease immunopathogenesis. However, there remains a paucity of information on the Mycobacterium bovis-host immune interactions during the natural infection. Analysis of 247 naturally PPD+ M. bovis-infected cattle revealed that 92% (n = 228) of these animals were found to display no clinical signs, but presented severe as well as disseminated bTB-lesions at post-mortem examination. Moreover, dissemination of bTB-lesions positively correlated with both pathology severity score (Spearman r = 0.48; p<0.0001) and viable tissue bacterial loads (Spearman r = 0.58; p = 0.0001). Additionally, granuloma encapsulation negatively correlated with M. bovis growth as well as pathology severity, suggesting that encapsulation is an effective mechanism to control bacterial proliferation during natural infection. Moreover, multinucleated giant cell numbers were found to negatively correlate with bacterial counts (Spearman r = 0.25; p = 0.03) in lung granulomas. In contrast, neutrophil numbers in the granuloma were associated with increased M. bovis proliferation (Spearman r = 0.27; p = 0.021). Together, our findings suggest that encapsulation and multinucleated giant cells control M. bovis viability, whereas neutrophils may serve as a cellular biomarker of bacterial proliferation during natural infection. These data integrate host granuloma responses with mycobacterial dissemination and could provide useful immunopathological-based biomarkers of disease severity in natural infection with M. bovis, an important cattle pathogen.     

Inhibition of Leishmania (Leishmania) amazonensis and Rat Arginases by Green Tea EGCG, (+)-Catechin and (?)-Epicatechin: A Comparative Structural Analysis of Enzyme-Inhibitor Interactions

Epigallocatechin-3-gallate (EGCG), a dietary polyphenol (flavanol) from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+)-catechin and (−)-epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L) and rat liver arginase (ARG-1). The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+)-catechin and (−)-epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+)-catechin (IC₅₀ = 0.8 µM) followed by (−)-epicatechin (IC₅₀ = 1.8 µM), gallic acid (IC₅₀ = 2.2 µM) and EGCG (IC₅₀ = 3.8 µM). Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC₅₀ values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.     

Phylogenetic Analysis Reveals a High Prevalence of Sporothrix brasiliensis in Feline Sporotrichosis Outbreaks

Sporothrix schenckii, previously assumed to be the sole agent of human and animal sporotrichosis, is in fact a species complex. Recently recognized taxa include S. brasiliensis, S. globosa, S. mexicana, and S. luriei, in addition to S. schenckii sensu stricto. Over the last decades, large epidemics of sporotrichosis occurred in Brazil due to zoonotic transmission, and cats were pointed out as key susceptible hosts. In order to understand the eco-epidemiology of feline sporotrichosis and its role in human sporotrichosis a survey was conducted among symptomatic cats. Prevalence and phylogenetic relationships among feline Sporothrix species were investigated by reconstructing their phylogenetic origin using the calmodulin (CAL) and the translation elongation factor-1 alpha (EF1α) loci in strains originated from Rio de Janeiro (RJ, n = 15), Rio Grande do Sul (RS, n = 10), Paraná (PR, n = 4), São Paulo (SP, n = 3) and Minas Gerais (MG, n = 1). Our results showed that S. brasiliensis is highly prevalent among cats (96.9%) with sporotrichosis, while S. schenckii was identified only once. The genotype of Sporothrix from cats was found identical to S. brasiliensis from human sources confirming that the disease is transmitted by cats. Sporothrix brasiliensis presented low genetic diversity compared to its sister taxon S. schenckii. No evidence of recombination in S. brasiliensis was found by split decomposition or PHI-test analysis, suggesting that S. brasiliensis is a clonal species. Strains recovered in states SP, MG and PR share the genotype of the RJ outbreak, different from the RS clone. The occurrence of separate genotypes among strains indicated that the Brazilian S. brasiliensis epidemic has at least two distinct sources. We suggest that cats represent a major host and the main source of cat and human S. brasiliensis infections in Brazil.     

Development of a New Approach to Aid in Visual Identification of Murine iPS Colonies Using a Fuzzy Logic Decision Support System

The a priori identification of induced pluripotent stem cells remains a challenge. Being able to quickly identify the most embryonic stem cell-similar induced pluripotent stem cells when validating results could help to reduce costs and save time. In this context, tools based on non-classic logic can be useful in creating aid-systems based on visual criteria. True colonies when viewed at 100x magnification have been found to have the following 3 characteristics: a high degree of border delineation, a more uniform texture, and the absence of a cracked texture. These visual criteria were used for fuzzy logic modeling. We investigated the possibility of predicting the presence of alkaline phosphatase activity, typical of true induced pluripotent stem cell colonies, after 25 individuals, with varying degrees of experience in working with murine iPS cells, categorized the images of 136 colonies based on visual criteria. Intriguingly, the performance evaluation by area under the ROC curve (16 individuals with satisfactory performance), Spearman correlation (all statistically significant), and Cohen''s Kappa agreement analysis (all statistically significant) demonstrates that the discriminatory capacity of different evaluators are similar, even those who have never cultivated cells. Thus, we report on a new system to facilitate visual identification of murine- induced pluripotent stem cell colonies that can be useful for staff training and opens the possibility of exploring visual characteristics of induced pluripotent stem cell colonies with their functional peculiarities. The fuzzy model has been integrated as a web-based tool named “2see-iPS” which is freely accessed at http://genetica.incor.usp.br/2seeips/.     

ANOVA-Like Differential Expression (ALDEx) Analysis for Mixed Population RNA-Seq

Experimental variance is a major challenge when dealing with high-throughput sequencing data. This variance has several sources: sampling replication, technical replication, variability within biological conditions, and variability between biological conditions. The high per-sample cost of RNA-Seq often precludes the large number of experiments needed to partition observed variance into these categories as per standard ANOVA models. We show that the partitioning of within-condition to between-condition variation cannot reasonably be ignored, whether in single-organism RNA-Seq or in Meta-RNA-Seq experiments, and further find that commonly-used RNA-Seq analysis tools, as described in the literature, do not enforce the constraint that the sum of relative expression levels must be one, and thus report expression levels that are systematically distorted. These two factors lead to misleading inferences if not properly accommodated. As it is usually only the biological between-condition and within-condition differences that are of interest, we developed ALDEx, an ANOVA-like differential expression procedure, to identify genes with greater between- to within-condition differences. We show that the presence of differential expression and the magnitude of these comparative differences can be reasonably estimated with even very small sample sizes.     

Increased Frequency of CD4 and CD8 Regulatory T Cells in Individuals under 15 Years with Multibacillary Leprosy

Background

Leprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4⁺/CD8⁺CD25^highFOXP3⁺ and CD4⁺/CD8⁺CD25^highFOXP3^high cells in leprosy patients and household contacts, in both cases under 15 years old.

Methodology/Principal Findings

PBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4⁺/CD8⁺ Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4⁺FOXP3⁺ (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4⁺FOXP3^high (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8⁺FOXP3⁺ (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8⁺FOXP3^high (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients.

Conclusions/Significance

We have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4⁺ and CD8⁺ Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.