H+-dependent inorganic phosphate transporter in breast cancer cells: Possible functions in the tumor microenvironment

Tumor microenvironment has a high concentration of inorganic phosphate (Pi), which is actually a marker for tumor progression. Regarding Pi another class of transporter has been recently studied, an H⁺-dependent Pi transporter, that is stimulated at acidic pH in Caco2BBE human intestinal cells. In this study, we characterized the H⁺-dependent Pi transport in breast cancer cell (MDA-MB-231) and around the cancer tissue. MDA-MB-231 cell line presented higher levels of H⁺-dependent Pi transport as compared to other breast cell lines, such as MCF-10A, MCF-7 and T47-D. The Pi transport was linear as a function of time and exhibited a Michaelis-Menten kinetic of K_m = 1.387 ± 0.1674 mM Pi and V_max = 198.6 ± 10.23 Pi × h^?1 × mg protein^?1 hence reflecting a low affinity Pi transport. H⁺-dependent Pi uptake was higher at acidic pH. FCCP, Bafilomycin A1 and SCH28080, which deregulate the intracellular levels of protons, inhibited the H⁺-dependent Pi transport. No effect on pHi was observed in the absence of inorganic phosphate. PAA, an H⁺-dependent Pi transport inhibitor, reduced the Pi transport activity, cell proliferation, adhesion, and migration. Arsenate, a structural analog of Pi, inhibited the Pi transport. At high Pi conditions, the H⁺-dependent Pi transport was five-fold higher than the Na⁺-dependent Pi transport, thus reflecting a low affinity Pi transport. The occurrence of an H⁺-dependent Pi transporter in tumor cells may endow them with an alternative path for Pi uptake in situations in which Na⁺-dependent Pi transport is saturated within the tumor microenvironment, thus regulating the energetically expensive tumor processes.     

Quick and accurate detection of Sclerotinia sclerotiorum and Phomopsis spp. in soybean seeds using qPCR and seed‐soaking method

Seed‐borne pathogenic fungi can cause serious damage to soybean crops by reducing the germination, vigour and emergence of the seeds. Special attention should be paid to pathogen detection in seeds to prevent its introduction in disease‐free areas. Considering the importance of rapid and successful diagnosis of seed‐borne pathogenic fungi in soybeans, this study evaluated a method to detect Sclerotinia sclerotiorum and Phomopsis spp. in seeds using quantitative polymerase chain reaction (qPCR). Naturally infested samples were subjected to detection using qPCR and blotter test, and the findings were compared. Using soybean seeds soaked in water, both pathogens were detected at an infestation level up a 0.0625% (one infected seed out of 1,599 healthy seeds) by qPCR. This technique allowed the detection of 300 fg of S. sclerotiorum and 30 fg of Phomopsis spp. DNA in the seed samples. Phomopsis spp. was detected in 40.7% of the evaluated seed batches (81 batches) and S. sclerotiorum was detected in 32.1% of the evaluated batches, although most of the seeds had low infestation levels. It was up to 28.5 times more efficient to use qPCR rather than blotter test to detect pathogens with a low incidence of occurrence in soybean seeds. If routinely used to test healthy seeds, qPCR would contribute to reducing soybean losses due to diseases as well as decreasing the costs required to control those diseases.     

Biogeography of the Lizard Genus Tropidurus Wied-Neuwied, 1825 (Squamata: Tropiduridae): Distribution,Endemism, and Area Relationships in South America

Based on comprehensive distributional records of the 23 species currently assigned to the lizard genus Tropidurus, we investigated patterns of endemism and area relationships in South America. Two biogeographic methods were applied, Parsimony Analysis of Endemicity (PAE) and Brooks Parsimony Analysis (BPA). Two areas of endemism were detected by PAE: the first within the domains of the semiarid Brazilian Caatinga, which includes seven endemic species, and the second in the region of the Serranía de Huanchaca, eastern Bolivia, in which three endemic species are present. The area cladograms recovered a close relationship between the Atlantic Forest and areas of the South American open corridor. The results revealed a close relationship among the provinces Caatinga (Cerrado, Parana Forest (Pantanal+Chaco)). The uplift of the Brazilian Central Plateau in the Late Pliocene-Early Pleistocene (4-2 Myr BP) has been interpreted as a major event responsible for isolation and differentiation of biotas along these areas. However, we emphasize that without the establishment of a temporal framework concerning the diversification history of Tropidurus it is premature to correlate cladogenetic events with specific time periods or putative vicariant scenarios. The limiting factors hampering the understanding of the biogeographic history of this genus include (1) the absence of temporal references in relation to the diversification of distinct clades within Tropidurus; (2) the lack of an appropriate taxonomic resolution of the species complexes currently represented by widely distributed forms; and (3) the need for a comprehensive phylogenetic hypothesis. We suggest that these three important aspects should be prioritized in future investigations.     

Risk Factors for Seroconversion by Leishmania infantum in a Cohort of Dogs from an Endemic Area of Brazil

Visceral leishmaniasis (VL) has recently emerged in various urban and peri-urban areas of Brazil and other countries. Understanding the urbanization of VL requires identification of risk factors associated with human and canine infection. To determine the predictors of risk for canine VL, a survey was conducted of 1,443 dogs, from which a cohort was selected (n = 455) and evaluated for approximately 26 months. Serology was conducted with two enzyme-linked immunosorbent assays (ELISA): one conducted in the Laboratory of Zoonosis of the Belo Horizonte Health Department (LZOON) and the other in the Laboratory of Immunopathology of the Federal University of Ouro Preto (LIMP). A molecular diagnostic method (PCR–restriction fragment length polymorphism) and a structured questionnaire were also used. To identify the factors associated with seroconversion, two time-dependent Cox regression models were performed with different sensitivities (model 1, seroconversion by ELISA/LZOON; model 2, seroconversion by ELISA/LIMP). The overall incidences of seroconversion were 6.5/1000 dogs-months and 11.2/1000 dogs-months for ELISA/LZOON and ELISA/LIMP, respectively. Increased risk of seroconversion was associated with short fur (model 1: hazard ratio [HR] 1.9), the presence of dry leaves (model 1: HR 2.8) or manure (model 1: HR 3.5) in the backyard, dogs sleeping predominantly in the backyard (model 2: HR 2.1), the presence of symptoms (model 2: HR 2.0), and positive molecular results during follow-up (model 2: HR 1.5). Decreased risk was associated with insecticide spraying in the house (model 2: HR 0.5). These results indicate that more-vulnerable domiciles, certain dog behaviors, lack of vector control measures, and positive molecular results were associated with the occurrence of canine VL. Furthermore, it is important to emphasize that PCR-positive dogs should be monitored, owing to the possibility of seroconversion. Identifying risk factors for seroconversion in dogs is crucial for developing adequate strategies for VL prevention and control.     

Validation of Suitable Reference Genes for Expression Studies in Different Pilocarpine-Induced Models of Mesial Temporal Lobe Epilepsy

It is well recognized that the reference gene in a RT-qPCR should be properly validated to ensure that gene expression is unaffected by the experimental condition. We investigated eight potential reference genes in two different pilocarpine PILO-models of mesial temporal lobe epilepsy (MTLE) performing a stability expression analysis using geNorm, NormFinder and BestKepeer softwares. Then, as a validation strategy, we conducted a relative expression analysis of the Gfap gene. Our results indicate that in the systemic PILO-model Actb, Gapdh, Rplp1, Tubb2a and Polr1a mRNAs were highly stable in hippocampus of rats from all experimental and control groups, whereas Gusb revealed to be the most variable one. In fact, we observed that using Gusb for normalization, the relative mRNA levels of the Gfap gene differed from those obtained with stable genes. On the contrary, in the intrahippocampal PILO-model, all softwares included Gusb as a stable gene, whereas B2m was indicated as the worst candidate gene. The results obtained for the other reference genes were comparable to those observed for the systemic Pilo-model. The validation of these data by the analysis of the relative expression of Gfap showed that the upregulation of the Gfap gene in the hippocampus of rats sacrificed 24 hours after status epilepticus (SE) was undetected only when B2m was used as the normalizer. These findings emphasize that a gene that is stable in one pathology model may not be stable in a different experimental condition related to the same pathology and therefore, the choice of reference genes depends on study design.     

Nbn and Atm Cooperate in a Tissue and Developmental Stage-Specific Manner to Prevent Double Strand Breaks and Apoptosis in Developing Brain and Eye

Nibrin (NBN or NBS1) and ATM are key factors for DNA Double Strand Break (DSB) signaling and repair. Mutations in NBN or ATM result in Nijmegen Breakage Syndrome and Ataxia telangiectasia. These syndromes share common features such as radiosensitivity, neurological developmental defects and cancer predisposition. However, the functional synergy of Nbn and Atm in different tissues and developmental stages is not yet understood. Here, we show in vivo consequences of conditional inactivation of both genes in neural stem/progenitor cells using Nestin-Cre mice. Genetic inactivation of Atm in the central nervous system of Nbn-deficient mice led to reduced life span and increased DSBs, resulting in increased apoptosis during neural development. Surprisingly, the increase of DSBs and apoptosis was found only in few tissues including cerebellum, ganglionic eminences and lens. In sharp contrast, we showed that apoptosis associated with Nbn deletion was prevented by simultaneous inactivation of Atm in developing retina. Therefore, we propose that Nbn and Atm collaborate to prevent DSB accumulation and apoptosis during development in a tissue- and developmental stage-specific manner.     

Impact of an Educational Intervention Implanted in a Neurological Intensive Care Unit on Rates of Infection Related to External Ventricular Drains

Background

Studies on the implantation of care routines showed reduction on EVD catheter-related infections rates; however zero tolerance is difficult to be achieved. The objective of this study was to assess the impact of an educational intervention on the maximal reduction on rates of EVD-related infections.

Methodology/Principal Findings

The quasi-experimental (before-after intervention) study occurred in two phases: pre-intervention, from April 2007 to July 2008, and intervention, from August 2008 to July 2010. Patients were followed for 30 days after the removal of the EVD, and EVD-related infections were considered as only those with laboratorial confirmation in the CSF. Observations were made of the care of the EVD and compliance with Hygiene of the Hands (HH), a routine of care was drawn up, training was given, and intervention was made to reduce the time the EVD catheter remained in place.

Results

during the study, 178 patients were submitted to 194 procedures, corresponding to 1217 EVD catheters-day. Gram-negative agents were identified in 71.4% of the infections during the pre-intervention period and in 60% during the intervention period. During the study, EVD-related infection rates were reduced from 9.5% to 4.8% per patient, from 8.8% to 4.4% per procedure, and the incidence density dropped from 14.0 to 6.9 infections per 1000 catheters-day (p = 0.027). The mortality reduced 12% (from 42% to 30%).

Conclusions/Significance

During one year after the fourth intervention, no microbiologically identified infection was documented. In light of these results, educational intervention proved to be a useful tool in reducing these rates and showed also impact on mortality.     

Inequality of Experience of Dental Caries between Different Ethnic Groups of Brazilians Aged 15 to 19 Years

Introduction

The aim of this study was to assess inequality of experience of dental caries, based on race/ethnicity, among Brazilian adolescents aged 15 to 19 years in 2010 and test whether socioeconomic indicators fully explain ethnic differences in dental caries.

Methods

Data from a National Oral Health Survey conducted in Brazil in 2010 was analysed. Race/ethnicity was self-assigned and modified to White, African descents, East Asian descents, Mixed Race and Indigenous descents. The prevalence of caries experience by race/ethnic group in 2010(n = 5,367) was calculated. Further analysis included conceptual hierarchical modelling and mediation analysis.

Results

Caries experience was 76.9% in 15 to 19 year old Brazilians in 2010. While African descents were 32% more likely to have caries experience than Whites, Mixed Race were 69% more likely to have caries experience than Whites. Hierarchical conceptual modelling analysis confirmed the highly significant association between caries and race/ethnicity. Mixed Race and East Asian descents were 1.44 (95% CI 1.24–1.67) and 1.81 (95% CI 1.02–3.20) times more likely to experience caries than Whites after adjusting for age, sex, education and income. The difference in the likelihood of experiencing caries between Whites and African descents was not statistically significant after adjusting for years of education and family income. The results of mediation analysis confirmed that inequality of caries experience between Whites and Mixed Race and East Asian descents was mediated through education and income. The likelihood that Mixed Race and East Asian descents would experience caries compared to Whites was attenuated, by 14.8% and by 9.5% respectively, after adjusting for years of education and income.

Conclusions

Data analysis demonstrated that Whites have benefited more from the significant reduction in dental caries experience in 15 to 19 year old Brazilians, as compared to African descents and Mixed Race. Education and income fully explained ethnic inequalities in experience of dental caries between Whites and African descents, and largely explained inequalities between Whites and Mixed Race.