Plasma carbonyls do not correlate with lung function or computed tomography measures of lung density in older smokers

Abstract

Oxidative stress and inflammation are hallmarks of chronic obstructive pulmonary disease (COPD). A critical byproduct of oxidative damage is the introduction of carbonyl groups into amino acid residues. We hypothesize that plasma carbonyl content is inversely correlated with lung function and computed tomography (CT) measures of lung density among smokers and is elevated in COPD. Carbonyl was measured in plasma of participants aged 60 years and older by ELISA. Generalized linear and additive models were used to adjust for potential confounders. Among 541 participants (52% male, mean age 67 years, 41% current smokers), mean plasma carbonyl content was 17.9±2.9 nmol ml^?1 and mean forced expiratory volume in one second (FEV₁) was 80.7±20.9% of predicted. Plasma carbonyl content was inversely associated with FEV₁, but this relationship was largely explained by age. Multivariate analyses ruled out clinically meaningful associations of plasma carbonyl content with FEV₁, FEV₁/FVC (forced vital capacity) ratio, severity of airflow obstruction, and CT lung density. Plasma carbonyl content is a poor biomarker of oxidative stress in COPD and emphysema.     

Sex-specific responses in placental fatty acid oxidation,esterification and transfer capacity to maternal obesity

Fatty acid metabolism and oxidation capacity in the placenta, which likely affects the rate and composition of lipid delivered to the fetus remains poorly understood. Long chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), are critical for fetal growth and brain development. We determined the impact of maternal obesity on placental fatty acid oxidation, esterification and transport capacity by measuring PhosphatidylCholine (PC) and LysoPhosphatidylCholine (LPC) containing DHA by mass spectrometry in mother-placenta-baby triads as well as placental free carnitine and acylcarnitine metabolites in women with normal and obese pre-pregnancy BMI. Placental protein expression of enzymes involved in beta-oxidation and esterification pathways, MFSD2a (lysophosphatidylcholine transporter) and OCTN2 (carnitine transporter) expression in syncytiotrophoblast microvillous (MVM) and basal (BM) membranes were determined by Western Blot. Maternal obesity was associated with decreased umbilical cord plasma DHA in LPC and PC fractions in male, but not female, fetuses. Basal membrane MFSD2a protein expression was increased in placenta of males of obese mothers. In female placentas, despite an increased MVM OCTN2 expression, maternal obesity was associated with a reduced MUFA-carnitine levels and increased esterification enzymes. We speculate that lower DHA-PL in fetal circulation of male offspring of obese mothers, despite a significant increase in transporter expression for LPC-DHA, may lead to low DHA needed for brain development contributing to neurological consequences that are more prevalent in male children. Female placentas likely have reduced beta-oxidation capacity and appear to store FA through greater placental esterification, suggesting impaired placenta function and lipid transfer in female placentas of obese mothers.     

Effects of body size,diet, and transience on the demography of the arboreal boid snake Corallus grenadensis on Carriacou (Grenada Grenadines,West Indies)

Remarkably little is known about the demography of snakes in the family Boidae. This lack of information may be attributed, in part, to low population densities on the Neotropical mainland, rendering capture-recapture methods impractical for many species. Conversely, islands support fewer species but snake densities can be much higher. Corallus grenadensis is an arboreal boid endemic to the Grenada Bank and, relative to mainland boids, can be amazingly abundant. As young, its diet is comprised largely of native Anolis lizards, a ubiquitous and abundant food source; it then undergoes an ontogenetic shift in diet to a less abundant resource, rodents. From 2015 to 2019, we marked 254 C. grenadensis and used capture–recapture models to estimate abundance, capture probabilities, survival, and the proportion of transients. We hypothesized that the transient effect would increase with body size (snout–vent length [SVL]), prompted by their ontogenetic shift in diet. Capture probabilities increased with sampling effort and decreased with increasing SVL. Abundance ranged from 96 to 141 individuals and annual resident survival was 0.71, 95% confidence interval (CI) = 0.54–0.83. The proportion of transients increased with increasing SVL, with the estimate being distinguishable from zero starting at ~810 mm SVL, coinciding with the size at which their dietary shift from ectothermic to endothermic prey begins. Ontogenetic dietary shifts are widespread in snakes and occur in at least 11 of 17 species of West Indian boids. Thus, the prominence of transients in our study may be indicative of its demographic and ecological importance among other snake species.     

Assessing the components of the eIF3 complex and their phosphorylation status

The eukaryotic initiation factor 3 (eIF3) is an essential, highly conserved multiprotein complex that is a key component in the recruitment and assembly of the translation initiation machinery. To better understand the molecular function of eIF3, we examined its composition and phosphorylation status in Saccharomyces cerevisiae. The yeast eIF3 complex contains five core components: Rpg1, Nip1, Prt1, Tif34, and Tif35. 2-D LC-MS/MS analysis of affinity purified eIF3 complexes showed that several other initiation factors (Fun12, Tif5, Sui3, Pab1, Hcr1, and Sui1) and the casein kinase 2 complex (CK2) copurify. In Vivo metabolic labeling of proteins with (32)P revealed that Nip1 is phosphorylated. Using 2-D LC-MS/MS analysis of eIF3 complexes, we identified Prt1 phosphopeptides indicating phosphorylation at S22 and T707 and a Tif5 phosphopeptide with phosphorylation at T191. Additionally, we used immobilized metal affinity chromatography (IMAC) to enrich for eIF3 phosphopeptides and tandem mass spectrometry to identify phosphorylated residues. We found that three CK2 consensus sequences in Nip1 are phosphorylated: S98, S99, and S103. Using in vitro kinase assays, we showed that CK2 phophorylates Nip1 and that a synthetic Nip1 peptide containing S98, S99, and S103 competitively inhibits the reaction. Replacement of these three Nip1 serines with alanines causes a slow growth phenotype.     

Mannose receptor (MR) engagement by mesothelin GPI anchor polarizes tumor-associated macrophages and is blocked by anti-MR human recombinant antibody

Tumor-infiltrating macrophages respond to microenvironmental signals by developing a tumor-associated phenotype characterized by high expression of mannose receptor (MR, CD206). Antibody cross-linking of CD206 triggers anergy in dendritic cells and CD206 engagement by tumoral mucins activates an immune suppressive phenotype in tumor-associated macrophages (TAMs). Many tumor antigens are heavily glycosylated, such as tumoral mucins, and/or attached to tumor cells by mannose residue-containing glycolipids (GPI anchors), as for example mesothelin and the family of carcinoembryonic antigen (CEA). However, the binding to mannose receptor of soluble tumor antigen GPI anchors via mannose residues has not been systematically studied. To address this question, we analyzed the binding of tumor-released mesothelin to ascites-infiltrating macrophages from ovarian cancer patients. We also modeled functional interactions between macrophages and soluble mesothelin using an in vitro system of co-culture in transwells of healthy donor macrophages with human ovarian cancer cell lines. We found that soluble mesothelin bound to human macrophages and that the binding depended on the presence of GPI anchor and of mannose receptor. We next challenged the system with antibodies directed against the mannose receptor domain 4 (CDR4-MR). We isolated three novel anti-CDR4-MR human recombinant antibodies (scFv) using a yeast-display library of human scFv. Anti-CDR4-MR scFv #G11 could block mesothelin binding to macrophages and prevent tumor-induced phenotype polarization of CD206(low) macrophages towards TAMs. Our findings indicate that tumor-released mesothelin is linked to GPI anchor, engages macrophage mannose receptor, and contributes to macrophage polarization towards TAMs. We propose that compounds able to block tumor antigen GPI anchor/CD206 interactions, such as our novel anti-CRD4-MR scFv, could prevent tumor-induced TAM polarization and have therapeutic potential against ovarian cancer, through polarization control of tumor-infiltrating innate immune cells.     

Enhanced cellular immunity in shrimp (Litopenaeus vannamei) after 'vaccination'

It has long been viewed that invertebrates rely exclusively upon a wide variety of innate mechanisms for protection from disease and parasite invasion and lack any specific acquired immune mechanisms comparable to those of vertebrates. Recent findings, however, suggest certain invertebrates may be able to mount some form of specific immunity, termed 'specific immune priming', although the mechanism of this is not fully understood (see Textbox S1). In our initial experiments, either formalin-inactivated Vibrio harveyi or sterile saline were injected into the main body cavity (haemocoel) of juvenile shrimp (Litopenaeus vannamei). Haemocytes (blood cells) from V. harveyi-injected shrimp were collected 7 days later and incubated with a 1:1 mix of V. harveyi and an unrelated gram positive bacterium, Bacillus subtilis. Haemocytes from 'vaccinated' shrimp showed elevated levels of phagocytosis of V. harveyi, but not B. subtilis, compared with those from saline-injected (non-immunised) animals. The increased phagocytic activity was characterised by a significant increase in the percentage of phagocytic cells. When shrimp were injected with B. subtilis rather than vibrio, there was no significant increase in the phagocytic activity of haemocytes from these animals in comparison to the non-immunised (saline injected) controls. Whole haemolymph (blood) from either 'immunised' or non-immunised' shrimp was shown to display innate humoral antibacterial activity against V. harveyi that was absent against B. subtilis. However, there was no difference in the potency of antibacterial activity between V. harveyi-injected shrimp and control (saline injected) animals showing that 'vaccination' has no effect on this component of the shrimp's immune system. These results imply that the cellular immune system of shrimp, particularly phagocytosis, is capable of a degree of specificity and shows the phenomenon of 'immune priming' reported by other workers. However, in agreement with other studies, this phenomenon is not universal to all potential pathogens.     

Inference of the Genetic Architecture Underlying BMI and Height with the Use of 20,240 Sibling Pairs

Evidence that complex traits are highly polygenic has been presented by population-based genome-wide association studies (GWASs) through the identification of many significant variants, as well as by family-based de novo sequencing studies indicating that several traits have a large mutational target size. Here, using a third study design, we show results consistent with extreme polygenicity for body mass index (BMI) and height. On a sample of 20,240 siblings (from 9,570 nuclear families), we used a within-family method to obtain narrow-sense heritability estimates of 0.42 (SE = 0.17, p = 0.01) and 0.69 (SE = 0.14, p = 6 × 10⁻⁷) for BMI and height, respectively, after adjusting for covariates. The genomic inflation factors from locus-specific linkage analysis were 1.69 (SE = 0.21, p = 0.04) for BMI and 2.18 (SE = 0.21, p = 2 × 10⁻¹⁰) for height. This inflation is free of confounding and congruent with polygenicity, consistent with observations of ever-increasing genomic-inflation factors from GWASs with large sample sizes, implying that those signals are due to true genetic signals across the genome rather than population stratification. We also demonstrate that the distribution of the observed test statistics is consistent with both rare and common variants underlying a polygenic architecture and that previous reports of linkage signals in complex traits are probably a consequence of polygenic architecture rather than the segregation of variants with large effects. The convergent empirical evidence from GWASs, de novo studies, and within-family segregation implies that family-based sequencing studies for complex traits require very large sample sizes because the effects of causal variants are small on average.     

Stripe rust resistance genes in the UK winter wheat cultivar Claire

Stripe rust resistance in the winter wheat cultivar Claire had remained effective in the UK and Europe since its release in 1999 and consequently has been used extensively in wheat breeding programs. However, in 2012, reports indicated that this valuable resistance may now have been compromised. To characterise stripe rust resistance in Claire and determine which genes may still confer effective resistance a cross was made between Claire and the stripe rust susceptible cultivar Lemhi. A genetic linkage map, constructed using SSR, AFLP, DArT and NBS-AFLP markers had a total map length of 1,730 cM. To improve the definition of two quantitative trait loci (QTL) identified on the long arm of chromosome 2D further markers were developed from wheat EST. Stripe rust resistance was evaluated on adult plants under field and glasshouse conditions by measuring the extent of fungal growth and sporulation, percentage infection (Pi) and the necrotic/chlorotic responses of the plant to infection, infection type (IT). Four QTL contributing to stripe rust adult plant resistance (APR) were identified in Claire, QYr.niab-2D.1, QYr.niab-2D.2, QYr.niab-2B and QYr.niab-7B. For Pi QYr.niab-2D.1 explained up to 25.4 % of the phenotypic variation, QYr.niab-2D.2 up to 28.7 %, QYr.niab-2B up to 21.7 % and QYr.niab-7B up to 13.0 %. For IT the percentages of phenotypic variation explained were 23.4, 31.8, 17.2 and 12.6 %, respectively. In addition to the four QTL conferring APR in Claire, a race-specific, seedling expressed resistance gene was identified on chromosome 3B.     

The eosinophil chemoattractant 5-oxo-ETE and the OXE receptor

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is formed from the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). The cofactor NADP⁺ is a limiting factor in the synthesis of 5-oxo-ETE because of its low concentrations in unperturbed cells. Activation of the respiratory burst in phagocytic cells, oxidative stress, and cell death all dramatically elevate both intracellular NADP⁺ levels and 5-oxo-ETE synthesis. 5-HEDH is widely expressed in inflammatory, structural, and tumor cells. Cells devoid of 5-lipoxygenase can synthesize 5-oxo-ETE by transcellular biosynthesis using inflammatory cell-derived 5-HETE. 5-Oxo-ETE is a chemoattractant for neutrophils, monocytes, and basophils and promotes the proliferation of tumor cells. However, its primary target appears to be the eosinophil, for which it is a highly potent chemoattractant. The actions of 5-oxo-ETE are mediated by the highly selective OXE receptor, which signals by activating various second messenger pathways through the release of the βγ-dimer from G_i/o proteins to which it is coupled. Because of its potent effects on eosinophils, 5-oxo-ETE may be an important mediator in asthma, and, because of its proliferative effects, may also contribute to tumor progression. Selective OXE receptor antagonists, which are currently under development, could be useful therapeutic agents in asthma and other allergic diseases.     

Development of an assay to measure mutagenic non-homologous end-joining repair activity in mammalian cells

Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.