Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi

The oxidative lesion 8-oxoguanine (8-oxoG) is removed during base excision repair by the 8-oxoguanine DNA glycosylase 1 (Ogg1). This lesion can erroneously pair with adenine, and the excision of this damaged base by Ogg1 enables the insertion of a guanine and prevents DNA mutation. In this report, we identified and characterized Ogg1 from the protozoan parasite Trypanosoma cruzi (TcOgg1), the causative agent of Chagas disease. Like most living organisms, T. cruzi is susceptible to oxidative stress, hence DNA repair is essential for its survival and improvement of infection. We verified that the TcOGG1 gene encodes an 8-oxoG DNA glycosylase by complementing an Ogg1-defective Saccharomyces cerevisiae strain. Heterologous expression of TcOGG1 reestablished the mutation frequency of the yeast mutant ogg1-/- (CD138) to wild type levels. We also demonstrate that the overexpression of TcOGG1 increases T. cruzi sensitivity to hydrogen peroxide (H2O2). Analysis of DNA lesions using quantitative PCR suggests that the increased susceptibility to H2O2 of TcOGG1-overexpressor could be a consequence of uncoupled BER in abasic sites and/or strand breaks generated after TcOgg1 removes 8-oxoG, which are not rapidly repaired by the subsequent BER enzymes. This hypothesis is supported by the observation that TcOGG1-overexpressors have reduced levels of 8-oxoG both in the nucleus and in the parasite mitochondrion. The localization of TcOgg1 was examined in parasite transfected with a TcOgg1-GFP fusion, which confirmed that this enzyme is in both organelles. Taken together, our data indicate that T. cruzi has a functional Ogg1 ortholog that participates in nuclear and mitochondrial BER. © 2012 Furtado et al

Granulocyte-colony stimulating factor (G-CSF) for stroke: an individual patient data meta-analysis

Granulocyte colony stimulating factor (G-CSF) may enhance recovery from stroke through neuroprotective mechanisms if administered early, or neurorepair if given later. Several small trials suggest administration is safe but effects on efficacy are unclear. We searched for randomised controlled trials (RCT) assessing G-CSF in patients with hyperacute, acute, subacute or chronic stroke, and asked Investigators to share individual patient data on baseline characteristics, stroke severity and type, end-of trial modified Rankin Scale (mRS), Barthel Index, haematological parameters, serious adverse events and death. Multiple variable analyses were adjusted for age, sex, baseline severity and time-to-treatment. Individual patient data were obtained for 6 of 10 RCTs comprising 196 stroke patients (116 G-CSF, 80 placebo), mean age 67.1 (SD 12.9), 92% ischaemic, median NIHSS 10 (IQR 5-15), randomised 11 days (interquartile range IQR 4-238) post ictus; data from three commercial trials were not shared. G-CSF did not improve mRS (ordinal regression), odds ratio OR 1.12 (95% confidence interval 0.64 to 1.96, p=0.62). There were more patients with a serious adverse event in the G-CSF group (29.6% versus 7.5%, p=0.07) with no significant difference in all-cause mortality (G-CSF 11.2%, placebo 7.6%, p=0.4). Overall, G-CSF did not improve stroke outcome in this individual patient data meta-analysis

Decolorization of synthetic melanoidins-containing wastewater by a bacterial consortium

The presence of melanoidins in molasses wastewater leads to water pollution both due to its dark brown color and its COD contents. In this study, a bacterial consortium isolated from waterfall sediment was tested for its decolorization. The identification of culturable bacteria by 16S rDNA based approach showed that the consortium composed of Klebsiella oxytoca, Serratia mercescens, Citrobacter sp. and unknown bacterium. In the context of academic study, prevention on the difficulties of providing effluent as well as its variations in compositions, several synthetic media prepared with respect to color and COD contents based on analysis of molasses wastewater, i.e., Viandox sauce (13.5% v/v), caramel (30% w/v), beet molasses wastewater (41.5% v/v) and sugarcane molasses wastewater (20% v/v) were used for decolorization using consortium with color removal 9.5, 1.13, 8.02 and 17.5%, respectively, within 2 days. However, Viandox sauce was retained for further study. The effect of initial pH and Viandox concentration on decolorization and growth of bacterial consortium were further determined. The highest decolorization of 18.3% was achieved at pH 4 after 2 day of incubation. Experiments on fresh or used medium and used or fresh bacterial cells, led to conclusion that the limitation of decolorization was due to nutritional deficiency. The effect of aeration on decolorization was also carried out in 2 L laboratory-scale suspended cell bioreactor. The maximum decolorization was 19.3% with aeration at KLa = 2.5836 h-1 (0.1 vvm)

Acute febrile illness is associated with Rickettsia spp infection in dogs

BACKGROUND: Rickettsia conorii is transmitted by Rhipicephalus sanguineus ticks and causes Mediterranean Spotted Fever (MSF) in humans. Although dogs are considered the natural host of the vector, the clinical and epidemiological significance of R. conorii infection in dogs remains unclear. The aim of this prospective study was to investigate whether Rickettsia infection causes febrile illness in dogs living in areas endemic for human MSF. METHODS: Dogs from southern Italy with acute fever (n = 99) were compared with case–control dogs with normal body temperatures (n = 72). Serology and real-time PCR were performed for Rickettsia spp., Ehrlichia canis, Anaplasma phagocytophilum/A. platys and Leishmania infantum. Conventional PCR was performed for Babesia spp. and Hepatozoon spp. Acute and convalescent antibodies to R. conorii, E. canis and A. phagocytophilum were determined. RESULTS: The seroprevalence rates at first visit for R. conorii, E. canis, A. phagocytophilum and L. infantum were 44.8%, 48.5%, 37.8% and 17.6%, respectively. The seroconversion rates for R. conorii, E. canis and A. phagocytophilum were 20.7%, 14.3% and 8.8%, respectively. The molecular positive rates at first visit for Rickettsia spp., E. canis, A. phagocytophilum, A. platys, L. infantum, Babesia spp. and Hepatozoon spp. were 1.8%, 4.1%, 0%, 2.3%, 11.1%, 2.3% and 0.6%, respectively. Positive PCR for E. canis (7%), Rickettsia spp. (3%), Babesia spp. (4.0%) and Hepatozoon spp. (1.0%) were found only in febrile dogs. The DNA sequences obtained from Rickettsia and Babesia PCRs positive samples were 100% identical to the R. conorii and Babesia vogeli sequences in GenBank®, respectively. Febrile illness was statistically associated with acute and convalescent positive R. conorii antibodies, seroconversion to R. conorii, E. canis positive PCR, and positivity to any tick pathogen PCRs. Fourteen febrile dogs (31.8%) were diagnosed with Rickettsia spp. infection based on seroconversion and/or PCR while only six afebrile dogs (12.5%) seroconverted (P = 0.0248). The most common clinical findings of dogs with Rickettsia infection diagnosed by seroconversion and/or PCR were fever, myalgia, lameness, elevation of C-reactive protein, thrombocytopenia and hypoalbuminemia. CONCLUSIONS: This study demonstrates acute febrile illness associated with Rickettsia infection in dogs living in endemic areas of human MSF based on seroconversion alone or in combination with PCR

miRNA in situ hybridization in circulating tumor cells - MishCTC

irculating tumor cells (CTCs) must be phenotypically and genetically characterized before they can be utilized in clinical applications. Here, we present the first protocol for the detection of miRNAs in CTCs using in situ hybridization (ISH) combined with immunomagnetic selection based on cytokeratin (CK) expression and immunocytochemistry. Locked-Nucleic Acid (LNA) probes associated with an enzyme-labeled fluorescence (ELF) signal amplification approach were used to detect miRNA-21 in CTCs. This protocol was optimized using both epithelial tumor (MDA-MB468) and epithelial non-tumor (MCF-10A) cell lines, and miRNA-21 was selected as the target miRNA because of its known role as an onco-miRNA. Hematopoietic cells do not express miRNA-21; thus, miRNA-21 is an ideal marker for detecting CTCs. Peripheral blood samples were taken from 25 cancer patients and these samples were analyzed using our developed protocol. Of the 25 samples, 11 contained CTCs. For all 11 CTC-positive samples, the isolated CTCs expressed both CK and miRNA-21. Finally, the protocol was applied to monitor miRNA-21 expression in epithelial to mesenchymal transition (EMT)-induced MCF-7 cells, an epithelial tumor cell line. CK expression was lost in these cells, whereas miRNA-21 was still expressed, suggesting that miRNA-21 might be a good marker for detecting CTCs with an EMT phenotype.JJDM thanks the Spanish Ministerio de Economía y Competitividad for a Ramón y Cajal Fellowship (Grant CTQ2012-34778). This research was partially supported by Marie Curie Career Integration Grants within the 7th European Community Framework Program (FP7-PEOPLE-2011-CIG-Project Number 294142 to RMSM and FP7-PEOPLE-2012-CIG-Project Number 322276 toJJDM). Thisresearch wasalso partially supported by Consejeria de Salud de la Junta de Andalucı ́ a (PI0294-2012)

A Worldwide Phylogeography for the Human X Chromosome

BACKGROUND: We reasoned that by identifying genetic markers on human X chromosome regions where recombination is rare or absent, we should be able to construct X chromosome genealogies analogous to those based on Y chromosome and mitochondrial DNA polymorphisms, with the advantage of providing information about both male and female components of the population. METHODOLOGY/PRINCIPAL FINDINGS: We identified a 47 Kb interval containing an Alu insertion polymorphism (DXS225) and four microsatellites in complete linkage disequilibrium in a low recombination rate region of the long arm of the human X chromosome. This haplotype block was studied in 667 males from the HGDP-CEPH Human Genome Diversity Panel. The haplotypic diversity was highest in Africa (0.992+/-0.0025) and lowest in the Americas (0.839+/-0.0378), where no insertion alleles of DXS225 were observed. Africa shared few haplotypes with other geographical areas, while those exhibited significant sharing among themselves. Median joining networks revealed that the African haplotypes were numerous, occupied the periphery of the graph and had low frequency, whereas those from the other continents were few, central and had high frequency. Altogether, our data support a single origin of modern man in Africa and migration to occupy the other continents by serial founder effects. Coalescent analysis permitted estimation of the time of the most recent common ancestor as 182,000 years (56,700-479,000) and the estimated time of the DXS225 Alu insertion of 94,400 years (24,300-310,000). These dates are fully compatible with the current widely accepted scenario of the origin of modern mankind in Africa within the last 195,000 years and migration out-of-Africa circa 55,000-65,000 years ago. CONCLUSIONS/SIGNIFICANCE: A haplotypic block combining an Alu insertion polymorphism and four microsatellite markers on the human X chromosome is a useful marker to evaluate genetic diversity of human populations and provides a highly informative tool for evolutionary studies

The MHC Gene Region of Murine Hosts Influences the Differential Tissue Tropism of Infecting Trypanosoma cruzi Strains

We have previously demonstrated that both parasite genetic variability and host genetic background were important in determining the differential tissue distribution of the Col1.7G2 and JG T. cruzi monoclonal strains after artificial infections in mice. We observed that the JG strain was most prevalent in hearts of mouse lineages with the MHC haplotype H-2d (BALB/c and DBA2), while Col1.7G2 was predominant in hearts from C57BL/6 mice, which have the H-2b haplotype. To assess whether the MHC gene region indeed influenced tissue tropism of T. cruzi, we used the same two parasite strains to infect C57BL/6 (H-2b) and C57BLKS/J (H-2d) mice; the latter strain results from the introgression of DBA2 MHC region into the C57BL/6 background. We also performed ex vivo infections of cardiac explants from four congenic mice lineages with the H-2b and H-2d haplotypes arranged in two different genetic backgrounds: C57BLKS/J (H-2d) versus C57BL/6 (H-2b) and BALB/c (H-2d) versus BALB/B10-H2b (H-2b). In agreement with our former observations, Col1.7G2 was predominant in hearts from C57BL/6 mice (H-2b), but we observed a clear predominance of the JG strain in hearts from C57BLKS/J animals (H-2d). In the ex vivo experiments Col1.7G2 also prevailed in explants from H-2b animals while no predominance of any of the strains was observed in H-2d mice explants, regardless of the genetic background. These observations clearly demonstrate that the MHC region influences the differential tissue distribution pattern of infecting T. cruzi strains, which by its turn may be in a human infection the determinant for the clinical forms of the Chagas disease