Genome Wide Association Study (GWAS) of Chagas Cardiomyopathy in Trypanosoma cruzi Seropositive Subjects

Background

Familial aggregation of Chagas cardiac disease in T. cruzi–infected persons suggests that human genetic variation may be an important determinant of disease progression.

Objective

To perform a GWAS using a well-characterized cohort to detect single nucleotide polymorphisms (SNPs) and genes associated with cardiac outcomes.

Methods

A retrospective cohort study was developed by the NHLBI REDS-II program in Brazil. Samples were collected from 499 T. cruzi seropositive blood donors who had donated between1996 and 2002, and 101 patients with clinically diagnosed Chagas cardiomyopathy. In 2008–2010, all subjects underwent a complete medical examination. After genotype calling, quality control filtering with exclusion of 20 cases, and imputation of 1,000 genomes variants; association analysis was performed for 7 cardiac and parasite related traits, adjusting for population stratification.

Results

The cohort showed a wide range of African, European, and modest Native American admixture proportions, consistent with the recent history of Brazil. No SNPs were found to be highly (P<10⁻⁸) associated with cardiomyopathy. The two mostly highly associated SNPs for cardiomyopathy (rs4149018 and rs12582717; P-values <10⁻⁶) are located on Chromosome 12p12.2 in the SLCO1B1 gene, a solute carrier family member. We identified 44 additional genic SNPs associated with six traits at P-value <10^-6: Ejection Fraction, PR, QRS, QT intervals, antibody levels by EIA, and parasitemia by PCR.

Conclusion

This GWAS identified suggestive SNPs that may impact the risk of progression to cardiomyopathy. Although this Chagas cohort is the largest examined by GWAS to date, (580 subjects), moderate sample size may explain in part the limited number of significant SNP variants. Enlarging the current sample through expanded cohorts and meta-analyses, and targeted studies of candidate genes, will be required to confirm and extend the results reported here. Future studies should also include exposed seronegative controls to investigate genetic associations with susceptibility or resitance to T. cruzi infection and non-Chagas cardiomathy.     

Divergent cytokine response following maximum progressive swimming in hot water

Exercise promotes transitory alterations in cytokine secretion, and these changes are affected by exercise duration and intensity. Considering that exercise responses also are affected by environmental factors, the goal of the present study was to investigate the effect of water temperature on the cytokine response to maximum swimming. Swiss mice performed a maximum progressive swimming exercise at 31 or 38 °C, and plasma cytokine levels were evaluated immediately or 1, 6 or 24 h after exercise. The cytokine profile after swimming at 31 °C was characterized by increased interleukin (IL)‐6 and monocyte chemotactic protein‐1 (MCP‐1) levels, which peaked 1 h after exercise, suggesting an adequate inflammatory milieu to induce muscle regeneration. Transitory reductions in IL‐10 and IL‐12 levels also were observed after swimming at 31 °C. The cytokine response to swimming was modified when the water temperature was increased to 38 °C. Although exercise at 38 °C also led to IL‐6 secretion, the peak in IL‐6 production occurred 6 h after exercise, and IL‐6 levels were significantly lower than those observed after maximum swimming at 31 °C (p = 0·030). Furthermore, MCP‐1 levels were lower and tumour necrosis factor‐α levels were higher immediately after swimming at 38 °C, suggesting a dysregulated pro‐inflammatory milieu. These alterations in the cytokine profile can be attributed in part to reduced exercise total work because exhaustion occurred sooner in mice swimming at 38 °C than in those swimming at 31 °C. Copyright © 2011 John Wiley & Sons, Ltd.     

The reproductive biology of Cybistax antisyphilitica (Bignoniaceae), a characteristic tree of the South American savannah-like “Cerrado” vegetation

Bignoniaceous woody species are very important ecological components of neotropical forests, but the reproductive biology of many species, such as Cybistax antisyphilitica, remains virtually unknown. Most species of Bignoniaceae are characteristically self-sterile, despite typically exhibiting normal pollen tube growth throughout the style, combined with slow rates of ovule penetration, fertilisation and endosperm initiation in selfed pistils. Uniform abortion occurs within a few days of anthesis, indicating the occurrence of late-acting self-incompatibility (LSI). However, breeding system studies have been performed in fewer than 7% of species, and other types of breeding systems (e.g., self-compatibility and apomixis) have been reported in this family. In the present study, the reproductive biology of C. antisyphilitica was investigated by field observation of flower visitors and floral events. Moreover, reproductive biology of this species was examined through experimental pollinations, analyses of pollen tube growth and ovule penetration using fluorescence microscopy, verification of pistil longevity, and a histological analysis of unpollinated vs. self-pollinated pistils. Finally, morphological aspects, quantities and germination were investigated in seeds that resulted from different pollination treatments. Natural pollination was effected by large- and medium-sized bees, and their visiting behaviour favours a high proportion of geitonogamy and no pollen limitation. Self-pollinated flowers produced no fruits, and all of the characteristic post-pollination events cited above were verified, witnessing the occurrence of LSI with post-zygotic rejection of selfed pistils in C. antisyphilitica. Although some indications of extended pistil longevity were found in selfed pistils, this feature seemed to be affected by unidentified environmental factors. The seeds were always monoembryonic and with high viability. A larger variation in the number of viable seeds was found in fruits derived from natural pollination. A low fruit set was observed after both natural and cross-pollination, and most crossed fruits underwent abortion at several points during the juvenile phase, even when protected against herbivory. The formation of surplus flowers/juvenile fruits and the apparently wasteful selfing mechanism control implied in LSI are discussed in the context of the perennial life style of tropical woody plant species.     

Antibacterial activity and lantibiotic post-translational modification genes in Streptococcus spp. isolated from ruminal fluid

The production of bacteriocins is frequently described in high microbial diversity environments. The aims of this study were to screen Streptococcus spp. isolated from rumen for their antibacterial potential and to determine the presence of post-translational modification genes for lantibiotic class of bacteriocins. The isolates were tested for production of antibacterial compounds by the spot-on-lawn assay. Presence of interfering factors and the sensitivity to proteinase K were evaluated. The ruminal bacteria were identified by 16S rRNA gene sequencing and the subspecific discrimination of the isolates belonging to the same specie was performed by PFGE. The presence of lantibiotic post-translational modification genes (lanB, lanC, and lanM) into bacterial genomes was performed by PCR. The bacteriocin-like inhibitory substances showed broad inhibitory activity and the producer cells were identified as S. equinus, S. lutetiensis, and S. gallolyticus. According to PFGE, the isolates identified as S. equinus belong to different strains. Three ruminal isolates showed at least one of the lantibiotic post-translational modification genes, and lanC was more frequently detected (75%). The production of broad-spectrum bacteriocin-like inhibitory substances by rumen strains suggests that antimicrobial peptides may play an important role in competition in the complex ruminal ecosystem.     

Irradiation-induced angiosarcoma and anti-angiogenic therapy: A therapeutic hope?

Angiosarcomas are rare soft-tissue sarcomas of endothelial cell origin. They can be sporadic or caused by therapeutic radiation, hence secondary breast angiosarcomas are an important subgroup of patients. Assessing the molecular biology of angiosarcomas and identify specific targets for treatment is challenging. There is currently great interest in the role of angiogenesis and of angiogenic factors associated with tumor pathogenesis and as targets for treatment of angiosarcomas. A primary cell line derived from a skin fragment of a irradiation-induced angiosarcoma patient was obtained and utilized to evaluate cell biomarkers CD31, CD34, HIF-1alpha and VEGFRs expression by immunocytochemistry and immunofluorescence, drugs cytotoxicity by cell counting and VEGF release by ELISA immunoassay. In addition to previous biomarkers, FVIII and VEGF were also evaluated on tumor specimens by immunohistochemistry to further confirm the diagnosis. We targeted the VEGF–VEGFR-2 axis of tumor angiogenesis with two different class of vascular targeted drugs; caprelsa, the VEGFR-2/EGFR/RET inhibitor and bevacizumab the anti-VEGF monoclonal antibody. We found the same biomarkers expression either in tumor specimens and in the cell line derived from tumor. In vitro experiments demonstrated that angiogenesis plays a pivotal role in the progression of this tumor as cells displayed high level of VEGFR-2, HIF-1 alpha strongly accumulated into the nucleus and the pro-angiogenic factor VEGF was released by cells in culture medium. The evaluation of caprelsa and bevacizumab cytotoxicity demonstrated that both drugs were effective in inhibiting tumor proliferation. Due to these results, we started to treat the patient with pazopanib, which was the unique tyrosine kinase inhibitor available in Italy through a compassionate supply program, obtaining a long lasting partial response. Our data suggest that the study of the primary cell line could help physicians in choosing a therapeutic approach for patient that almost in vitro shows chances of success and that the anti-angiogenetic agents are a reliable therapeutic opportunity for angiosarcomas patients.     

Functional display of Bacillus thuringiensis Cry1Ac toxin on T7 phage

The Cry1Ac toxin from Bacillus thuringiensis was displayed on the surface of T7 phage. The cry1Ac gene was fused to the C-terminal end of T7-10B capsid protein and displayed on the surface of T7 phage as revealed by Western blot analysis of the purified phage particles. The T7-Cry1Ac phages retained toxicity against Manduca sexta larvae. We demonstrated that the T7-Cry1Ac phage interacts with Cry1Ac receptors present in M. sexta BBMV either in solution or in overlay binding assays.     

Mating System and Sleeping Behaviour of the Male and Female <Emphasis Type="Italic">Centris</Emphasis> (<Emphasis Type="Italic">Paracentris</Emphasis>) <Emphasis Type="Italic">burgdorfi</Emphasis> Friese (Apidae,Centridini)

We describe the mating behaviour of males of Centris (Paracentris) burgdorfi, a solitary, univoltine bee, with a wide distribution in Brazil. We also describe the unusual sleeping sites of males and females. The study was performed during two breeding seasons, in northeastern Brazil, where the species nests in aggregations in petrified dunes. Data on mating behaviour were collected through direct observations of the bees at the nesting-emergence site. Males compete intensely for virgin females in the morning, sometimes killing rivals. The high competition for females near the nesting site makes that the male has to take the female to another place to get genital contact. In the evening, males do not use plants to spend the night, instead they aggregate in sleeping clusters inside old burrows in the nesting-emergence area while females sleep in groups on plants that provide the floral oil used in nest construction.     

Low Level Laser Therapy Reduces the Development of Lung Inflammation Induced by Formaldehyde Exposure

Lung diseases constitute an important public health problem and its growing level of concern has led to efforts for the development of new therapies, particularly for the control of lung inflammation. Low Level Laser Therapy (LLLT) has been highlighted as a non-invasive therapy with few side effects, but its mechanisms need to be better understood and explored. Considering that pollution causes several harmful effects on human health, including lung inflammation, in this study, we have used formaldehyde (FA), an environmental and occupational pollutant, for the induction of neutrophilic lung inflammation. Our objective was to investigate the local and systemic effects of LLLT after FA exposure. Male Wistar rats were exposed to FA (1%) or vehicle (distillated water) during 3 consecutive days and treated or not with LLLT (1 and 5 hours after each FA exposure). Non-manipulated rats were used as control. 24 h after the last FA exposure, we analyzed the local and systemic effects of LLLT. The treatment with LLLT reduced the development of neutrophilic lung inflammation induced by FA, as observed by the reduced number of leukocytes, mast cells degranulated, and a decreased myeloperoxidase activity in the lung. Moreover, LLLT also reduced the microvascular lung permeability in the parenchyma and the intrapulmonary bronchi. Alterations on the profile of inflammatory cytokines were evidenced by the reduced levels of IL-6 and TNF-α and the elevated levels of IL-10 in the lung. Together, our results showed that LLLT abolishes FA-induced neutrophilic lung inflammation by a reduction of the inflammatory cytokines and mast cell degranulation. This study may provide important information about the mechanisms of LLLT in lung inflammation induced by a pollutant.     

SIMD parallelization of the WORDUP algorithm for detecting statistically significant patterns in DNA sequences

The development of new techniques in sequencing nucleic acidshas produced a great amount of sequence data and has led tothe discovery of new relationships. In this paper, we studya method for parallelizing the algorithm WORDUP, which detectsthe presence of statistically significant patterns in DNA sequences.WORDUP implements an efficient method to identify the presenceof statistically significant oligomers in a nonhomologous groupof sequences. It is based on a modified version of the Boyer-Moorealgorithm, which is one of the fastest algorithms for stringmatching available in the literature. The aim of the parallelversion of WORDUP presented here is to speed up the computationaltime and allow the analysis of a greater set of longer nucleotidesequences, which is usually impractical with sequential algorithms.