Microbial enrichment and multiplexed microfiltration for accelerated detection of Salmonella in spinach

To attain Salmonella detection thresholds in spinach suspensions using enrichment media requires at least 24 hr. Separation and concentration of selected microorganisms via microfiltration and microfugation reduce time for sample preparation, especially when working with large volumes of vegetable suspensions. This facilitates accelerated detection of Salmonella in spinach suspensions, and may contribute to effectively monitoring this pathogen before it reaches the consumer. We report a microfiltration-based protocol for accelerated sample preparation to concentrate and recover ≤1 colony forming unit (CFU) Salmonella/g pathogen-free spinach. Store-bought samples of spinach and a spinach plant subjected to two environmental conditions (temperature and light exposure) during its production were tested. The overall procedure involves extraction with buffer, a short enrichment step, prefiltration using a nylon filter, crossflow hollow fiber microfiltration, and retentate centrifugation to bring microbial cells to detection levels. Based on 1 CFU Salmonella/g frozen spinach, and a Poisson distribution statistical analyses with 99% probability, we calculated that 3 hr of incubation, when followed by microfiltration, is sufficient to reach the 2 log concentration required for Salmonella detection within 7 hr. Longer enrichment times (5 hr or more) is needed for concentrations lower than 1 CFU Salmonella/g of ready to eat spinach. The recovered microbial cells were identified and confirmed as Salmonella using both polymerase chain reaction (PCR) and plating methods. Different environmental conditions tested during production did not affect Salmonella viability; this demonstrated the broad adaptability of Salmonella and emphasized the need for methods that enable efficient monitoring of production for the presence of this pathogen.     

Detoxification of olefinic epoxides and nucleotide excision repair of epoxide-mediated DNA damage: Insights from animal models examining human sensitivity to 1,3-butadiene

1,3-Butadiene (BD) is a well-documented mutagen and carcinogen in rodents and is currently classified as a probable carcinogen in humans. Studies investigating workers exposed to BD indicate that, in some plants, there may be an increased genetic risk, and that polymorphisms in biotransformation and DNA repair proteins may modulate genetic susceptibility. To investigate the role of genetic polymorphisms in microsomal epoxide hydrolase (mEH) or nucleotide excision repair (NER) in contributing to the mutagenicity of BD, we conducted a series of experiments in which mice lacking mEH or NER activity were exposed to BD by inhalation or to the reactive epoxide metabolites of BD (epoxybutene-EB or diepoxybutane-DEB) by i.p. injection. Genetic susceptibility was measured using the Hprt cloning assay. Both deficient strains of mouse were significantly more sensitive to the mutagenic effects of BD and the injected epoxides. These studies provide support for the critical role that mEH plays in the biotransformation of BD, and the role that NER plays in maintaining genomic integrity following exposure to BD. Additional studies are needed to examine the importance of base excision repair (BER) in maintaining genomic integrity, the differential formation of DNA and protein adducts in deficient strains, and the potential for enhanced sensitivity to BD genotoxicity in mice either lacking or deficient in both biotransformation and DNA repair activity.     

Recurrent locus-specific mutation resulting from a cryptic ectopic insertion in Neurospora

New mutations are found among approximately 20% of progeny when one or both parents carry eas allele UCLA191 (eas(UCLA), easily wettable, hydrophobin-deficient, linkage group II). The mutations inactivate the wild-type allele of cya-8 (cytochrome aa3 deficient, linkage group VII), resulting in thin, "transparent" mycelial growth. Other eas alleles fail to produce cya-8 mutant progeny. The recurrent cya-8 mutations are attributed to repeat-induced point mutation (RIP) resulting from a duplicated copy of cya-8+ that was inserted ectopically at eas when the UCLA191 mutation occurred. As expected for RIP, eas(UCLA)-induced cya-8 mutations occur during nuclear proliferation prior to karyogamy. When only one parent is eas(UCLA), the new mutations arise exclusively in eas(UCLA) nuclei. Mutation of cya-8 is suppressed when a long unlinked duplication is present. Stable cya-8 mutations are effectively eliminated in crosses homozygous for rid, a recessive suppressor of RIP. The eas(UCLA) allele is associated with a long paracentric inversion. A discontinuity is present in eas(UCLA) DNA. The eas promoter is methylated in cya-8 progeny of eas(UCLA), presumably by the spreading of methylation beyond the adjoining RIP-inactivated duplication. These findings support a model in which an ectopic insertion that created a mutation at the target site acts as a locus-specific mutator via RIP.     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.     

Parent overweight predicts daughters' increase in BMI and disinhibited overeating from 5 to 13 years

Objective: To assess whether parental overweight status and disinhibited overeating are predictive of daughters’ accelerated weight gain and disinhibited overeating. Research Methods and Procedures: Participants were part of a longitudinal study of girls (N = 197) and their parents. Measured height and weight were used to calculate BMI [weight (kilograms)/height (meters)²]. Parents’ disinhibited eating behavior was assessed using the Eating Inventory. Girls’ disinhibited eating was assessed using a behavioral protocol to measure eating in the absence of hunger. Girls were classified based on parental overweight at study entry into four groups: neither, mother only, father only, or both parents overweight. Results: Girls with both parents overweight had the most rapid increases in BMI from 5 to 13 years of age; BMI increased most slowly among the neither parent overweight group, with intermediate increases in BMI among mother only and father only overweight groups. Daughters with both parents overweight at study entry were eight times more likely to be overweight at age 13, controlling for daughters’ weight at age 5. Girls with both parents overweight had higher levels of disinhibited eating across all ages than all other groups. Although girls in all parental weight status groups showed increases in disinhibited eating over time, girls with both parents overweight had larger increases in disinhibited eating over time compared with all other groups. Discussion: Girls growing up in families differing in parental overweight had divergent developmental trajectories for BMI and disinhibited overeating. Findings reveal the need to focus prevention efforts on overweight parents of young children.     

Long-Term Carriage of Medicopsis romeroi,an Agent of Black-Grain Mycetoma,Presenting as Phaeohyphomycosis in a Renal Transplant Patient

Mycopathologia - Medicopsis species are rare fungal pathogens that frequently resist common antifungal therapies and are difficult to identify morphologically as conidia are produced in pycnidia, a...     

Mechanisms of HP1-mediated gene silencing in Drosophila

Heterochromatin Protein 1 (HP1) is a structural component of silent chromatin at telomeres and centromeres. Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner. Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes. HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9. The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber. To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E. coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats. Association of lacI-HP1 with the repeats resulted in silencing of both reporter genes and correlated with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin. Chromatin immunoprecipitation experiments demonstrated that HP1 spread bi-directionally from the tethering site and associated with the silenced reporter transgenes. To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated. Silencing was minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner.     

Mitochondrial beta-oxidation in Aspergillus nidulans

Beta-oxidation (beta-ox) occurs exclusively in the peroxisomes of Saccharomyces cerevisiae and other yeasts, leading to the supposition that fungi lack mitochondrial beta-ox. Here we present unequivocal evidence that the filamentous fungus Aspergillus nidulans houses both peroxisomal and mitochondrial beta-ox. While growth of a peroxisomal beta-ox disruption mutant (DeltafoxA) was eliminated on a very long-chain fatty acid (C(22:1)), growth was only partially impeded on a long-chain fatty acid (C(18:1)) and was not affected at all on short chain (C4-C6) fatty acids. In contrast, growth of a putative enoyl-CoA hydratase mutant (DeltaechA) was abolished on short-chain and severely restricted on long- and very long-chain fatty acids. Furthermore fatty acids inhibited growth of the DeltaechA mutant but not the DeltafoxA mutant in the presence of an alternate carbon source (lactose). Disruption of echA led to a 28-fold reduction in 2-butenoyl-CoA hydratase activity in a preparation of organelles. EchA was also required for growth on isoleucine and valine. The subcellular localization of the FoxA and EchA proteins was confirmed through the use of red and green fluorescent protein fusions.     

Differing roles of mitochondrial nitric oxide synthase in cardiomyocytes and urothelial cells

The existence of mitochondrial nitric oxide (NO) synthase (mtNOS) has been controversial since it was first reported in 1995. We have addressed this issue by making direct microsensor measurements of NO production in the mitochondria isolated from mouse hearts. Mitochondrial NO production was stimulated by Ca2+ and inhibited by blocking electrogenic Ca2+ uptake or by using NOS antagonists. Cardiac mtNOS was identified as the neuronal isoform by the absence of NO production in the mitochondria of mice lacking the neuronal but not the endothelial or inducible isoforms. In cardiomyocytes from dystrophin-deficient (mdx) mice, elevated intracellular Ca2+, increased mitochondrial NO production, slower oxidative phosphorylation, and decreased ATP production were detected. Inhibition of mtNOS increased contractility in mdx but not in wild-type cardiomyocytes, indicating that mtNOS may protect the cells from overcontracting. mtNOS was also implicated in radiation-induced cell damage. In irradiated rat/mouse urinary bladders, we have evidence that mitochondrially produced NO damages the urothelial "umbrella" cells that line the bladder lumen. This damage disrupts the permeability barrier thereby creating the potential to develop radiation cystitis. RT-PCR and Southern blot analyses indicate that mtNOS is restricted to the umbrella cells, which scanning electron micrographs show are selectively damaged by radiation. Simultaneous microsensor measurements demonstrate that radiation increases NO and peroxynitrite (ONOO-) production in these cells, which can be prevented by transfection with manganese superoxide dismutase (MnSOD) or instillation of NOS antagonists during irradiation or irradiation of bladders devoid of mtNOS. These studies demonstrate that mtNOS is in the cardiomyocytes and urothelial cells, that it is derived from the neuronal isoform, and that it can be either protective or detrimental.