Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

IFN-alpha is not sufficient to drive Th1 development due to lack of stable T-bet expression

During inflammatory immune responses, the innate cytokine IL-12 promotes CD4+ Th-1 development through the activation of the second messenger STAT4 and the subsequent expression of T-bet. In addition, type I IFN (IFN-alphabeta), secreted primarily during viral and intracellular bacterial infections, can promote STAT4 activation in human CD4+ T cells. However, the role of IFN-alphabeta in regulating Th1 development is controversial, and previous studies have suggested a species-specific pathway leading to Th1 development in human but not mouse CD4+ T cells. In this study, we found that although both IFN-alpha and IL-12 can promote STAT4 activation, IFN-alpha failed to promote Th1 commitment in human CD4+ T cells. The difference between these innate signaling pathways lies with the ability of IL-12 to promote sustained STAT4 tyrosine phosphorylation, which correlated with stable T-bet expression in committed Th1 cells. IFN-alpha did not promote Th1 development in human CD4+ T cells because of attenuated STAT4 phosphorylation, which was insufficient to induce stable expression of T-bet. Further, the defect in IFN-alpha-driven Th1 development was corrected by ectopic expression of T-bet within primary naive human CD4+ T cells. These results indicate that IL-12 remains unique in its ability to drive Th1 development in human CD4+ T cells and that IFN-alpha lacks this activity due to its inability to promote sustained T-bet expression.     

Endosymbionts of Ctenocephalides felis felis (Siphonaptera: Pulicidae) obtained from dogs captured in Belo Horizonte, Minas Gerais, Brazil

Specimens of fleas Ctenocephalides felis felis (1052 female symbol/448 male symbol), obtained from 150 dogs in Centro de Controle de Zoonoses de Belo Horizonte, Minas Gerais, Brazil, were dissected and examined for endosymbionts. Three protozoan, Nolleria pulicis, a gregarine (Actinocephalidae) and Leptomonas sp., together with one cestode, Dipylidium caninum were identified. Infections by N. pulicis and Leptomonas sp. occurred mainly in the warm-rainy period. The prevalence and distribution of these endosymbionts in fleas derived from Brazil and South America, and the their variation according to sex and season, are reported for the first time.     

Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2.     

Mitochondrial introgressions into the nuclear genome of the domestic cat

Translocation of mtDNA into the nuclear genome, also referred to as numt, was first reported in the domestic cat (Felis catus) by Lopez et al. (1994). The Lopez-numt consisted of a translocation of 7.9 kbp of mtDNA that inserted into the domestic cat chromosome D2 around 1.8 million years ago. More than a decade later, the release of the domestic cat whole-genome shotgun sequences (1.9x coverage) provides the resource to obtain more comprehensive insight into the extent of mtDNA transfer over time in the domestic cat genome. MegaBLAST searches revealed that the cat genome harbors a wide variety of numts (298 320 bp), one-third of which likely correspond to the Lopez-numt tandem repeat, whereas the remaining numts are probably derived from multiple independent insertions, which in some cases were followed by segmental duplication after insertion in the nucleus. Numts were detected across most cat chromosomes, but the number of numts assigned to chromosomes is underestimated due to the relatively high number of numt sequences with insufficient flanking sequence to map. The catalog of cat numts provides a valuable resource for future studies in Felidae species, including its use as a tool to avoid numt contaminations that may confound population genetics and phylogenetic studies.     

Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.     

Effect of germicidal UVC light on fungi isolated from grapes and raisins

AIMS: To examine how UVC affects the different genera of fungi commonly isolated from grapes, with the aim of understanding changes in mycobiota during grape ripening and possible applications for preventing grape decay during storage. METHODS AND RESULTS: Spores of Aspergillus carbonarius, Aspergillus niger, Cladosporium herbarum, Penicillium janthinellum and Alternaria alternata (between 100-250 spores/plate agar) were UVC irradiated for 0 (control), 10, 20, 30, 60, 300 and 600 s. Plates were incubated at 25 degrees C and colonies were counted daily up to 7 days. Alternaria alternata and Aspergillus carbonarius were the most resistant fungi. Conidial germination in these species was reduced by approx. 25% after 10 s of exposure, compared with greater than 70% reduction for the remaining species tested. Penicillium janthinellum spores were the most susceptible at this wavelength. UVC exposures of 300 s prevented growth of all isolates studied, except for Alternaria alternata. CONCLUSIONS: UVC irradiation plays a major role in selecting for particular fungi that dominate the mycobiota of drying grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The UVC irradiation of harvested grapes could prevent germination of contaminant fungi during storage or further dehydration.     

Effect of preharvest fungicides and interacting fungi on Aspergillus carbonarius growth and ochratoxin A synthesis in dehydrating grapes

AIMS: To evaluate the effect of preharvest grape pesticides in Aspergillus section Nigri infection in dehydrating grapes and the final ochratoxin A (OTA) content. Additionally, the effect of coinoculation of moulds frequently isolated from grapes and raisins on Aspergillus section Nigri infection was studied. METHODS AND RESULTS: Fungicide-treated grapes were inoculated with Aspergillus carbonarius, Aspergillus niger aggregate, Eurotium amstelodami and Penicillium janthinellum in different combinations, then dehydrated by reducing a(w) for 20 days. The percentages of colonized grapes treated with fungicides were, in general, lower, but no differences were observed among fungicides. The untreated grapes always showed higher concentrations of OTA, regardless of the inoculum applied. In general, Chorus was the most effective antifungal treatment in reducing OTA accumulation in grapes during dehydration. Penicillium janthinellum reduced Aspergillus section Nigri colonization and OTA accumulation in grapes during dehydration. CONCLUSIONS: The four preharvest fungicides studied reduced the Aspergillus section Nigri growth and OTA production by A. carbonarius during dehydration of grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The success of these chemical treatments might depend on the mycobiota composition of grapes.     

Peripheral disruption of the Grb10 gene enhances insulin signaling and sensitivity in vivo

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.     

Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch

Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.