Effects of anthocyanin and carotenoid combinations on foliage and immature fruit color of Capsicum annuum L

Shades ranging from violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. High-performance liquid chromatography and mass spectrometry analysis of violet and black fruit tissue identified a single anthocyanin that was determined to be delphinidin-3-p-coumaroyl-rutinoside-5-glucoside. Leaf tissue of a black-pigmented foliage genotype contained the same anthocyanin found in fruit but at a considerably higher concentration in comparison to violet and black fruit tissue. Fruit chlorophyll concentration was approximately 14-fold higher in black fruit in comparison to violet fruit that contained relatively little chlorophyll. Beta-carotene, lutein, violaxanthin, and neoxanthin carotenoid concentrations in black fruit were also significantly greater in comparison to violet fruit. High concentrations of delphinidin in combination with chlorophyll and accessory carotenoid pigments produced the characteristic black pigmentation observed in fruits and leaves of selected genotypes. Anthocyanins were accumulated in the outer mesocarp of violet and black fruit and in the palisade and mesophyll cells of black leaves. Consistent with chlorophyll content of respective genotypes, chloroplast density was greater in cells of black fruits. Utilizing Capsicum pigment variants, we determine the biochemical factors responsible for violet versus black-pigmented pepper tissue in the context of described pepper color genes.     

Mechanisms of peroxisome proliferator-induced DNA hypomethylation in rat liver

Genomic hypomethylation is a consistent finding in both human and animal tumors and mounting experimental evidence suggests a key role for epigenetic events in tumorigenesis. Furthermore, it has been suggested that early changes in DNA methylation and histone modifications may serve as sensitive predictive markers in animal testing for carcinogenic potency of environmental agents. Alterations in metabolism of methyl donors, disturbances in activity and/or expression of DNA methyltransferases, and presence of DNA single-strand breaks could contribute to the loss of cytosine methylation during carcinogenesis; however, the precise mechanisms of genomic hypomethylation induced by chemical carcinogens remain largely unknown. This study examined the mechanism of DNA hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP (di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5 months), the level of genomic hypomethylation increased by approximately 2-fold, as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5 months) DNA methylation did not change. Global DNA hypomethylation in livers from WY-14,643 group was accompanied by the accumulation of DNA single-strand breaks, increased cell proliferation, and diminished expression of DNA methyltransferase 1, while the metabolism of methyl donors was not affected. In contrast, none of these parameters changed significantly in rats fed DEHP. Since WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent of loss of DNA methylation may be related to the carcinogenic potential of the chemical agent, and that accumulation of DNA single-strand breaks coupled to the increase in cell proliferation and altered DNA methyltransferase expression may explain genomic hypomethylation during peroxisome proliferator-induced carcinogenesis.     

Sampling-Based Approaches to Improve Estimation of Mortality among Patient Dropouts: Experience from a Large PEPFAR-Funded Program in Western Kenya

Background

Monitoring and evaluation (M&E) of HIV care and treatment programs is impacted by losses to follow-up (LTFU) in the patient population. The severity of this effect is undeniable but its extent unknown. Tracing all lost patients addresses this but census methods are not feasible in programs involving rapid scale-up of HIV treatment in the developing world. Sampling-based approaches and statistical adjustment are the only scaleable methods permitting accurate estimation of M&E indices.

Methodology/Principal Findings

In a large antiretroviral therapy (ART) program in western Kenya, we assessed the impact of LTFU on estimating patient mortality among 8,977 adult clients of whom, 3,624 were LTFU. Overall, dropouts were more likely male (36.8% versus 33.7%; p = 0.003), and younger than non-dropouts (35.3 versus 35.7 years old; p = 0.020), with lower median CD4 count at enrollment (160 versus 189 cells/ml; p<0.001) and WHO stage 3–4 disease (47.5% versus 41.1%; p<0.001). Urban clinic clients were 75.0% of non-dropouts but 70.3% of dropouts (p<0.001). Of the 3,624 dropouts, 1,143 were sought and 621 had their vital status ascertained. Statistical techniques were used to adjust mortality estimates based on information obtained from located LTFU patients. Observed mortality estimates one year after enrollment were 1.7% (95% CI 1.3%–2.0%), revised to 2.8% (2.3%–3.1%) when deaths discovered through outreach were added and adjusted to 9.2% (7.8%–10.6%) and 9.9% (8.4%–11.5%) through statistical modeling depending on the method used. The estimates 12 months after ART initiation were 1.7% (1.3%–2.2%), 3.4% (2.9%–4.0%), 10.5% (8.7%–12.3%) and 10.7% (8.9%–12.6%) respectively.

Conclusions/Significance Abstract

Assessment of the impact of LTFU is critical in program M&E as estimated mortality based on passive monitoring may underestimate true mortality by up to 80%. This bias can be ameliorated by tracing a sample of dropouts and statistically adjust the mortality estimates to properly evaluate and guide large HIV care and treatment programs.     

Variation in the bitter-taste receptor gene TAS2R38, and adiposity in a genetically isolated population in Southern Italy

Objective: Variation in the bitter‐taste receptor gene, TAS2R38 confers the ability to taste 6‐n‐propylthiouracil (PROP). The objective of this study was to relate TAS2R38 haplotypes and PROP‐tasting phenotypes to adiposity in a genetically isolated population. We hypothesized that the nontaster phenotype would be associated with higher BMI and waist circumference (WC) in females, and that dietary restraint would mediate this relationship. Methods and Procedures: Participants were 540 healthy inhabitants of the genetically isolated village of Carlantino in southern Italy who were 15–89 years of age at the time of the study. Haplotype analyses were performed and PROP tasting was assessed using a filter paper method. Height, weight, and WC were measured and restrained eating was assessed using a brief questionnaire. Results: Nontaster females had higher BMI and WC than females who were phenotypic tasters, and this relationship was specific to females with low dietary restraint. Regression analysis showed that BMI declined by 1.7 units across taster groups in females when the model included the PROP by restraint interaction. PROP phenotype was not significantly associated with WC in the regression models. Polymorphisms in TAS2R38 were not associated with BMI or WC in females. Neither TAS2R38 haplotype nor PROP phenotype was strongly related to BMI or WC in males. Discussion: These data support previous findings of a relation between the nontaster phenotype and higher BMI in females that is modified by dietary restraint. Assessment of PROP phenotypes might provide unique information about adiposity that is not captured by haplotype analysis alone.     

Regulation of the murine TR2/HVEM gene expression by IRF

TR2 (TNFR-related 2, HVEM, or TNFRSF-14), a member of the TNFR family, is involved in a number of immune responses. While TR2 is expressed on the surface of T cells during the resting state, little is known regarding how expression of the TR2 gene is regulated. To understand the mechanisms regulating the expression of TR2 in T cells, we analyzed the 5' flanking region of TR2. We identified an important region for the activity of the TR2 promoter using site directed mutagenesis. Using EMSA analysis, we found that IRF-2 was bound to the promoter region of the TR2 gene during the resting state of EL-4 T cells. Transfection of IRF-2 expression plasmid and of dominant negative IRF-2 mutant further confirmed our results. Together, these data demonstrate that IRF-2 is involved in the regulation of TR2 expression in EL-4 T cells.     

Antioxidant protein 2 prevents methemoglobin formation in erythrocyte hemolysates.

Antioxidant protein 2 (AOP2) is a member of a family of thiol-specific antioxidants, recently renamed peroxiredoxins, that evolved as part of an elaborate system to counteract and control detrimental effects of oxygen radicals. AOP2 is found in endothelial cells, erythrocytes, monocytes, T and B cells, but not in granulocytes. AOP2 was found solely in the cytoplasm and was not associated with the nuclear or membrane fractions; neither was it detectable in plasma. Further experiments focused on the function of AOP2 in erythrocytes where it is closely associated with the hemoglobin complex, particularly with the heme. An investigation of the mechanism of this interaction demonstrated that the conserved cysteine-47 in AOP2 seems to play a role in AOP2-heme interactions. Recombinant AOP2 prevented induced as well as noninduced methemoglobin formation in erythrocyte hemolysates, indicating its antioxidant properties. We conclude that AOP2 is part of a sophisticated system developed to protect and support erythrocytes in their many physiological functions.     

Molecular Characterization of Novel Red Green Nonsulfur Bacteria from Five Distinct Hot Spring Communities in Yellowstone National Park

We characterized and compared five geographically isolated hot springs with distinct red-layer communities in Yellowstone National Park. Individual red-layer communities were observed to thrive in temperatures ranging from 35 to 60°C and at pH 7 to 9. All communities were dominated by red filamentous bacteria and contained bacteriochlorophyll a (Bchl a), suggesting that they represented novel green nonsulfur (GNS) bacteria. The in vivo absorption spectra of individual sites were different, with two sites showing unusual Bchl a protein absorption bands beyond 900 nm. We prepared and analyzed 16S rRNA libraries from all of these sites by using a combination of general bacterial primers and new GNS-specific primers described here. These studies confirmed the presence of novel GNS-like bacteria in all five communities. All GNS-like clones were most similar to Roseiflexus castenholzii, a red filamentous bacterium from Japan that also contains only Bchl a. Phylogenies constructed by using GNS-like clones from Yellowstone red-layer communities suggest the presence of a moderately diverse new “red” cluster within the GNS lineage. Within this cluster, at least two well-supported subclusters emerged: YRL-A was most similar to Roseiflexus and YRL-B appeared to be novel, containing no known isolates. While these patterns showed some site specificity, they did not correlate with observed Bchl a spectrum differences or obvious features of the habitat.     

13C content of ecosystem respiration is linked to precipitation and vapor pressure deficit

Variation in the carbon isotopic composition of ecosystem respiration ('¹³C_R) was studied for 3 years along a precipitation gradient in western Oregon, USA, using the Keeling plot approach. Study sites included six coniferous forests, dominated by Picea sitchensis, Tsuga heterophylla, Pseudotsuga menziesii, Pinus ponderosa, and Juniperus occidentalis, and ranged in location from the Pacific coast to the eastern side of the Cascade Mountains (a 250-km transect). Mean annual precipitation across these sites ranged from 227 to 2,760 mm. Overall '¹³C_R varied from -23.1 to -33.1‰, and within a single forest, it varied in magnitude by 3.5-8.5‰. Mean annual '¹³C_R differed significantly in the forests and was strongly correlated with mean annual precipitation. The carbon isotope ratio of carbon stocks (leaves, fine roots, litter, and soil organic matter) varied similarly with mean precipitation (more positive at the drier sites). There was a strong link between '¹³C_R and the vapor saturation deficit of air (vpd) 5-10 days earlier, both across and within sites. This relationship is consistent with stomatal regulation of gas exchange and associated changes in photosynthetic carbon isotope discrimination. Recent freeze events caused significant deviation from the '¹³C_R versus vpd relationship, resulting in higher than expected '¹³C_R values.