Global Gene Expression Profiles Suggest an Important Role for Nutrient Acquisition in Early Pathogenesis in a Plant Model of Pseudomonas aeruginosa Infection

Although Pseudomonas aeruginosa is an opportunistic pathogen that does not often naturally infect alternate hosts, such as plants, the plant-P. aeruginosa model has become a widely recognized system for identifying new virulence determinants and studying the pathogenesis of the organism. Here, we examine how both host factors and P. aeruginosa PAO1 gene expression are affected in planta after infiltration into incompatible and compatible cultivars of tobacco (Nicotiana tabacum L.). N. tabacum has a resistance gene (N) against tobacco mosaic virus, and although resistance to PAO1 infection is correlated with the presence of a dominant N gene, our data suggest that it is not a factor in resistance against PAO1. We did observe that the resistant tobacco cultivar had higher basal levels of salicylic acid and a stronger salicylic acid response upon infiltration of PAO1. Salicylic acid acts as a signal to activate defense responses in plants, limiting the spread of the pathogen and preventing access to nutrients. It has also been shown to have direct virulence-modulating effects on P. aeruginosa. We also examined host effects on the pathogen by analyzing global gene expression profiles of bacteria removed from the intracellular fluid of the two plant hosts. We discovered that the availability of micronutrients, particularly sulfate and phosphates, is important for in planta pathogenesis and that the amounts of these nutrients made available to the bacteria may in turn have an effect on virulence gene expression. Indeed, there are several reports suggesting that P. aeruginosa virulence is influenced in mammalian hosts by the availability of micronutrients, such as iron and nitrogen, and by levels of O₂.     

Inhibition of innate immune response ameliorates Zika virus-induced neurogenesis deficit in human neural stem cells

Global Zika virus (ZIKV) outbreaks and their strong link to microcephaly have raised major public health concerns. ZIKV has been reported to affect the innate immune responses in neural stem/progenitor cells (NS/PCs). However, it is unclear how these immune factors affect neurogenesis. In this study, we used Asian-American lineage ZIKV strain PRVABC59 to infect primary human NS/PCs originally derived from fetal brains. We found that ZIKV overactivated key molecules in the innate immune pathways to impair neurogenesis in a cell stage-dependent manner. Inhibiting the overactivated innate immune responses ameliorated ZIKV-induced neurogenesis reduction. This study thus suggests that orchestrating the host innate immune responses in NS/PCs after ZIKV infection could be promising therapeutic approach to attenuate ZIKV-associated neuropathology.     

Inherent bias toward the null hypothesis in conventional multipoint nonparametric linkage analysis

Traditional nonparametric "multipoint" statistical procedures have been developed for assigning allele-sharing values at a locus of interest to pairs of relatives for linkage studies. These procedures attempt to accommodate a lack of informativity, nongenotyped loci, missing data, and related issues concerning the genetic markers used in a linkage study. However, such procedures often cannot overcome these phenomena in compelling ways and, as a result, assign relevant relative pairs allele-sharing values that are "expected" for those pairs. The practice of assigning expected allele-sharing values to relative pairs in the face of a lack of explicit allele-transmission information can bias traditional nonparametric linkage test statistics toward the null hypothesis of no locus effect. This bias is due to the use of expected values, rather than to a lack of information about actual allele sharing at relevant marker loci. The bias will vary from study to study on the basis of the DNA markers, sample size, relative-pair types, and pedigree structures used, but it can be extremely pronounced and could contribute to a lack of consistent success in the application of traditional nonparametric linkage analyses to complex human traits and diseases. There are several potential ways to overcome this problem, but their foundations deserve greater research. We expose many of the issues concerning allele sharing with data from a large affected-sibling-pair study investigating the genetic basis of autism.     

Dnmt1 deficiency leads to enhanced microsatellite instability in mouse embryonic stem cells

DNA hypomethylation is frequently seen in cancer and imparts genomic instability in mouse models and some tissue culture systems. However, the effects of genomic DNA hypomethylation on mutation rates are still elusive. We have developed a model system to analyze the effects of DNA methyltransferase 1 (Dnmt1) deficiency on DNA mismatch repair (MMR) in mouse embryonic stem (ES) cells. We generated sibling ES cell clones with and without functional Dnmt1 expression, containing a stable reporter gene that allowed us to measure the slippage rate at a mononucleotide repeat. We found that Dnmt1 deficiency led to a 7-fold increase in the microsatellite slippage rate. Interestingly, the region flanking the mononucleotide repeat was unmethylated regardless of Dnmt1 status, suggesting that it is not the local levels of DNA methylation that direct the increase in microsatellite instability (MSI). The enhanced MSI was associated with higher levels of histone H3 acetylation and lower MeCP2 binding at regions near the assayed microsatellite, suggesting that Dnmt1 loss may decrease MMR efficiency by modifying chromatin structure.     

Prevalence and anatomic site of Crassicauda sp. infection,and its use in species identification,in kogiid whales from the mid‐Atlantic United States

The parasitic nematode Crassicauda sp. was initially described in kogiid whales from specimens collected within cervical tissues, uncommon sites of infection for this parasite. Crassicauda sp. has only been reported in Kogia breviceps to date, but no study has yet investigated a large sample of both kogiid species. A 15 yr record of 104 kogiid strandings (K. sima, n = 40; K. breviceps, n = 64) in North Carolina and Virginia, U.S.A. was used to determine the prevalence of Crassicauda sp. across species, within species across sex, and within sex across length and life history categories. Crassicauda sp. was confirmed to be a species‐specific parasite among kogiids infecting only K. breviceps (prevalence = 45%). Within K. breviceps, prevalence was similar (45%) in both immature and mature males, but increased from 10% in immature to 76% in mature females. This study confirmed the cervico‐thoracic distribution of the parasite, and identified a novel site of infection in a previously undescribed exocrine gland associated with the pigmented “false gill slit.” The species‐specific nature of Crassicauda sp. infection, the exocrine gland, and the distinct features of the false gill slit pigmentation associated with the gland, are all useful characters to identify kogiid species in the field.     

Physical activity patterns during weight maintenance following a low-energy density dietary intervention

Objective: The objective was to determine the role of physical activity (PA) and energy intake on weight maintenance among former University of Alabama at Birmingham EatRight Weight Management Program participants. Research Methods and Procedures: Eighty‐nine former participants completed follow‐up visits ≥1 year after completing EatRight. BMI was calculated using measured height and weight. Diet intake was estimated from 4‐day food records. PA was assessed using a module from the Behavioral Risk Factor Surveillance System Survey. Cut‐off points were chosen based on the distribution of minutes of activity. For moderate and total activity, cut‐off points were 0, 1 to 30, 31 to 60, and >60 minutes. For vigorous activity, the categories were 0, 1 to 20, 21 to 30, and >30 minutes. General linear models determined the association of PA with change in weight at follow‐up controlling for gender and total energy intake (kcal/d). Results: At follow‐up, 80% (n = 71) of participants maintained (regained <5% of program end weight) their body weight and 20% had gained weight. Mean weight change was 1.0 ± 6.5 kg. Maintainers consumed 384 fewer kcal/d on average. Maintainers had a lower energy density dietary pattern (1.58 vs. 2.01 kcal/g, p < 0.01). There was no significant difference in PA min/d reported by maintainers and gainers. Discussion: Our results suggest that, despite being minimally active, weight‐reduced individuals can successfully maintain their newly achieved body weight with appropriate caloric intake. Adopting a lower calorie, low energy density dietary pattern may reduce the amount of PA that is truly necessary for weight maintenance.     

A Role for cGMP in Inducible Nitric-oxide Synthase (iNOS)-induced Tumor Necrosis Factor (TNF) α-converting Enzyme (TACE/ADAM17) Activation,Translocation, and TNF Receptor 1 (TNFR1) Shedding in Hepatocytes

We and others have previously shown that the inducible nitric-oxide synthase (iNOS) and nitric oxide (NO) are hepatoprotective in a number of circumstances, including endotoxemia. In vitro, hepatocytes are protected from tumor necrosis factor (TNF) α-induced apoptosis via cGMP-dependent and cGMP-independent mechanisms. We have shown that the cGMP-dependent protective mechanisms involve the inhibition of death-inducing signaling complex formation. We show here that LPS-induced iNOS expression leads to rapid TNF receptor shedding from the surface of hepatocytes via NO/cGMP/protein kinase G-dependent activation and surface translocation of TNFα-converting enzyme (TACE/ADAM17). The activation of TACE is associated with the up-regulation of iRhom2 as well as the interaction and phosphorylation of TACE and iRhom2, which are also NO/cGMP/protein kinase G-dependent. These findings suggest that one mechanism of iNOS/NO-mediated protection of hepatocytes involves the rapid shedding of TNF receptor 1 to limit TNFα signaling.     

Application of Gene Network Analysis Techniques Identifies AXIN1/PDIA2 and Endoglin Haplotypes Associated with Bicuspid Aortic Valve

Bicuspid Aortic Valve (BAV) is a highly heritable congenital heart defect. The low frequency of BAV (1% of general population) limits our ability to perform genome-wide association studies. We present the application of four a priori SNP selection techniques, reducing the multiple-testing penalty by restricting analysis to SNPs relevant to BAV in a genome-wide SNP dataset from a cohort of 68 BAV probands and 830 control subjects. Two knowledge-based approaches, CANDID and STRING, were used to systematically identify BAV genes, and their SNPs, from the published literature, microarray expression studies and a genome scan. We additionally tested Functionally Interpolating SNPs (fitSNPs) present on the array; the fourth consisted of SNPs selected by Random Forests, a machine learning approach. These approaches reduced the multiple testing penalty by lowering the fraction of the genome probed to 0.19% of the total, while increasing the likelihood of studying SNPs within relevant BAV genes and pathways. Three loci were identified by CANDID, STRING, and fitSNPS. A haplotype within the AXIN1-PDIA2 locus (p-value of 2.926×10⁻⁰⁶) and a haplotype within the Endoglin gene (p-value of 5.881×10⁻⁰⁴) were found to be strongly associated with BAV. The Random Forests approach identified a SNP on chromosome 3 in association with BAV (p-value 5.061×10⁻⁰⁶). The results presented here support an important role for genetic variants in BAV and provide support for additional studies in well-powered cohorts. Further, these studies demonstrate that leveraging existing expression and genomic data in the context of GWAS studies can identify biologically relevant genes and pathways associated with a congenital heart defect.