Evaluation of YO-PRO-1 as an early marker of apoptosis following radiofrequency ablation of colon cancer liver metastases

Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal. Conclusion: This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.     

Antioxidant and Cytotoxic Activity of Hydroethanolic Extract from Jacaranda decurrens Leaves

Background and Purpose

Leaves of Jacaranda decurrens are used in traditional Brazilian medicine to treat metabolic diseases related to increased reactive oxygen species. The present study evaluated the antioxidant and cytotoxic potential of hydroethanolic extract from the leaves of Jacaranda decurrens subsp. symmetrifoliolata.

Experimental Approach

Phenolic compounds, flavonoids and saponins were evaluated in an ethanol∶water (80∶20, v/v) extract from the leaves of Jacaranda decurrens subsp. symmetrifoliolata (E-Jds). The antioxidant activity of E-Jds was investigated by assessing the following: 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity; protection against 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced hemolysis of erythrocytes; in vitro and in vivo malondialdehyde dosage; and the ability to activate antioxidant enzymes. K562 leukemia cells were used for the cytotoxic evaluation of E-Jds and for the assessment of the cell death profile through flow cytometry.

Key Results

Phenolic and flavonoid compounds were quantified as 14.38% and 2.15%, respectively, of E-Jds. These phenolic and flavonoid compounds proved to be able to scavenge DPPH free radicals with an IC₅₀ of 9.3±3.3 µg/mL, to protect up to 50% of erythrocytes against AAPH-induced hemolysis and to reduce in vitro and in vivo malondialdehyde levels up to 84% and 22%, respectively. E-Jds also increased glutathione peroxidase enzyme activity, with a concomitant decrease in superoxide dismutase and catalase activity, and exhibited dose-dependent cytotoxic activity on K562 erythroleukemia cells with cell death occurring via both late apoptosis and necrosis.

Conclusions

E-Jds exhibits in vitro and in vivo antioxidant potential, which may be the mechanism mediating the metabolic activities reported in folk medicine. Furthermore, the cytotoxic activity identified in this study contributes with the knowledge of antiproliferative activities that have been described in the literature for the genus Jacaranda.     

Prevalence and Risk Factors for Echinococcal Infection in a Rural Area of Northern Chile: A Household-Based Cross-Sectional Study

Background

Hydatidosis is a zoonotic disease of worldwide distribution caused by Echinococcus granulosus. Our study aimed to determine the prevalence of human and canine echinococcosis as well as the associated risk factors in a rural area of the Limarí province in northern Chile.

Methodology/Principal Findings

A cross-sectional study was conducted between August and November 2009 using a stratified sampling design in each of the five districts of the province. In the selected villages, up to 10 households were sampled. Serum and fecal samples from an adult family member and a dog were collected from each participating household. Risk factors were assessed by standardized questionnaires. Seroprevalence was assessed using a multi-step approach: an ELISA for screening, IFA, IHA and western blot for confirmation of results, respectively. The prevalence of echinococcal infection in dogs was determined by coproantigen genus specific ELISA. Chi-square, Fisher tests and logistic regressions were used to assess risk factors for human seropositivity and dog copropositivity. A seroprevalence of 2.6% (10/403) and coproprevalence of 28% (26/93) was recorded for humans and dogs respectively. Contact with dogs and dog feces were risk factors for human seropositivity while dog copropositivity was associated with home slaughter of livestock (OR = 3.35; CI 90%: 1.16–6.85) and households de-worming dogs (OR = 2.82; CI 90%: 1.33–8.43).

Conclusions/Significance

Echinococcal infection of humans and their dogs is common in Limarí province. Risk factors for human seropositivity were related to contact with domestic dogs and their feces, whereas those for dogs were home slaughter of livestock and the practice of de-worming dogs.     

Biocompatible Anionic Polymeric Microspheres as Priming Delivery System for Effetive HIV/AIDS Tat-Based Vaccines

Here we describe a prime-boost regimen of vaccination in Macaca fascicularis that combines priming with novel anionic microspheres designed to deliver the biologically active HIV-1 Tat protein and boosting with Tat in Alum. This regimen of immunization modulated the IgG subclass profile and elicited a balanced Th1-Th2 type of humoral and cellular responses. Remarkably, following intravenous challenge with SHIV89.6P_cy243, vaccinees significantly blunted acute viremia, as compared to control monkeys, and this control was associated with significantly lower CD4⁺ T cell depletion rate during the acute phase of infection and higher ability to resume the CD4⁺ T cell counts in the post-acute and chronic phases of infection. The long lasting control of viremia was associated with the persistence of high titers anti-Tat antibodies whose profile clearly distinguished vaccinees in controllers and viremics. Controllers, as opposed to vaccinated and viremic cynos, exhibited significantly higher pre-challenge antibody responses to peptides spanning the glutamine-rich and the RGD-integrin-binding regions of Tat. Finally, among vaccinees, titers of anti-Tat IgG1, IgG3 and IgG4 subclasses had a significant association with control of viremia in the acute and post-acute phases of infection. Altogether these findings indicate that the Tat/H1D/Alum regimen of immunization holds promise for next generation vaccines with Tat protein or other proteins for which maintenance of the native conformation and activity are critical for optimal immunogenicity. Our results also provide novel information on the role of anti-Tat responses in the prevention of HIV pathogenesis and for the design of new vaccine candidates.     

Gain- and Loss-of-Function Mutations in the Breast Cancer Gene GATA3 Result in Differential Drug Sensitivity

Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.     

Stable Isotope Evidence of Puma concolor (Felidae) Feeding Patterns in Agricultural Landscapes in Southeastern Brazil

We evaluated puma (Puma concolor) feeding patterns in southeastern Brazilian agricultural landscapes using carbon and nitrogen stable isotope analyses of hair collected from fecal samples (N = 64). We classified the samples into three groups: feeding patterns based on forest remnants, on the agricultural matrix or both. We observed a predominance of consumption of C₃ prey (~47% of individuals) in the area with the highest proportion of forest coverage. Conversely, C₄ prey were highly consumed (~40% of individuals) where the agricultural matrix was predominant. The δ¹³C values for pumas in both areas indicated that their food resources come from both forest remnants and the agricultural matrix and that some individuals preferentially consumed C₄ prey, indicating that food resources from the agricultural matrix make up most of their diet (~46% of prey individuals). The wide range of puma's δ¹⁵N values in both areas indicated a diet based on different types of prey. However, the C₄ group had higher values, indicating that both pumas and their prey feed on enriched resources from the agricultural matrix. The results confirm the high behavioral plasticity of pumas in using highly anthropogenic habitats. The stable isotope analyses conducted in this study yielded new information on large carnivore trophic ecology that might be useful in the development of new conservation strategies in disturbed areas.     

Predicting domain-domain interactions using a parsimony approach

We propose a novel approach to predict domain-domain interactions from a protein-protein interaction network. In our method we apply a parsimony-driven explanation of the network, where the domain interactions are inferred using linear programming optimization, and false positives in the protein network are handled by a probabilistic construction. This method outperforms previous approaches by a considerable margin. The results indicate that the parsimony principle provides a correct approach for detecting domain-domain contacts.