Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10^-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10^-18 moles of the p24/assay corresponds to ca. 10³ copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10² copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.     

Is the Association between Vitamin D and Cardiovascular Disease Risk Confounded by Obesity? Evidence from the Andhra Pradesh Children and Parents Study (APCAPS)

Background

Evidence of an association between serum vitamin D and cardiovascular disease risk is inconsistent and comes predominantly from studies in high-income settings. We assessed the association between serum levels of 25-hydroxyvitamin D₃ (25(OH)D) and cardiovascular disease risk factors in a population of young Indian adults.

Methods

Cross-sectional analyses of data from APCAPS (Andhra Pradesh Children and Parents Study); a prospective birth cohort study in rural south India. Participants were 1038 (40.3% females) adults aged 18-24 years. Main outcome measures were blood pressures, fasting serum lipids (cholesterols and triglycerides), fasting glucose, insulin, measures of arterial stiffness (aortic augmentation index and aortic pulse wave velocity (aPWV)), carotid intima-media thickness, body mass index (BMI) and body fat (dual X-ray absorptiometry).

Results

Vitamin D deficiency (≤20ng/ml) was observed in 41.1% of this lean (mean BMI: 19.5) and active (mean minutes of moderate or vigorous physical activity per day: 186) population. Vitamin D deficiency was associated with higher median body fat in both males (15.9% body fat in vitamin D deficient males vs. 14.6% in non-deficient males, p<0.05) and females (29.1% body fat in vitamin D deficient females vs. 27.8% in non-deficient females, p<0.05) but no associations were observed between vitamin D deficiency and mean BMI or median fat mass index (FMI). Except a weak inverse association with fasting insulin in males, there was no clear association between serum vitamin D levels and cardiovascular disease risk factors in fully adjusted models.

Conclusions

We did not find clear evidence for an association between serum vitamin D levels and cardiovascular disease risk factors. Our results, consistent with the limited evidence from randomised trials of vitamin D supplementation and Mendelian randomisation experiments, suggest that the postulated link between serum vitamin D and cardiovascular disease may be non-causal. Instead, it may be attributable to confounding by lifestyle factors such as obesity and physical inactivity which may provide more fruitful targets for cardiovascular disease prevention.     

Mobile ER-to-Golgi but not post-Golgi membrane transport carriers disappear during the terminal myogenic differentiation

The organelles of the exocytic pathway undergo a profound reorganization during the myogenic differentiation. Here, we have investigated the dynamics of the membrane trafficking at various stages of the differentiation process by using the green fluorescent protein-tagged, temperature-sensitive vesicular stomatitis virus G protein (tsG-GFP) as a marker. At the restrictive temperature of 39°C, the tsG-GFP located to the endoplasmic reticulum (ER) at each stage of differentiation. Mobile membrane containers moving from the ER to the Golgi elements were seen in myoblasts and myotubes upon shifting the temperature to 20°C. In adult myofibers, in contrast, such containers were not seen although the tsG-GFP rapidly shifted from the ER to the Golgi elements. The mobility of tsG-GFP in the myofiber ER was restricted, suggesting localization in an ER sub-compartment. Contrasting with the ER-to-Golgi trafficking, transport from the Golgi elements to the plasma membrane involved mobile transport containers in all differentiation stages. These findings indicate that ER-to-Golgi trafficking in adult skeletal myofibers does not involve long-distance moving membrane carriers as occurs in other mammalian cell types.     

Serum interleukin-6 and C-reactive protein are markedly elevated in acute decompensated heart failure patients with left ventricular systolic dysfunction

Cytokines play important roles in heart failure (HF). We examined whether cytokine levels are different in acute decompensated heart failure (ADHF) patients between with left ventricular systolic dysfunction (LVSDF) and with preserved LV ejection function (PLVEF). We studied 81 HF patients who were admitted to our hospital with acute decompensation. They were divided into two groups: LVSDF (LVEF) < 45% and PLVEF (LVEF ? 45%). Serum interleukin-6 (IL-6), highly sensitive C-reactive protein (hsCRP), tumor necrosis factor alpha (TNF-α), and IL-18 and plasma brain natriuretic peptide (BNP) were measured on admission and at discharge. On admission, IL-6 and hsCRP were higher in LVSDF than in PLVEF. IL-6 and hsCRP decreased after treatment in LVSDF, but not in PLVEF, while plasma BNP levels decreased in both HF with treatment. There was no difference in TNF-α or in IL-18 level between LVSDF and PLVEF, and they did not change after treatment in either group. In conclusion, cytokine profiles were different in ADHF between those with LVSDF and PLVEF. Activation of IL-6–hsCRP pathway may play a specific role in ADHF with LVSDF.     

Role of hydrogen generation by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in the oral cavity

Some gastrointestinal bacteria synthesize hydrogen (H₂) by fermentation. Despite the presence of bactericidal factors in human saliva, a large number of bacteria also live in the oral cavity. It has never been shown that oral bacteria also produce H₂ or what role H₂ might play in the oral cavity. It was found that a significant amount of H₂ is synthesized in the oral cavity of healthy human subjects, and that its generation is enhanced by the presence of glucose but inhibited by either teeth brushing or sterilization with povidone iodine. These observations suggest the presence of H₂-generating bacteria in the oral cavity. The screening of commensal bacteria in the oral cavity revealed that a variety of anaerobic bacteria generate H₂. Among them, Klebsiella pneumoniae (K. pneumoniae) generated significantly large amounts of H₂ in the presence of glucose. Biochemical analysis revealed that various proteins in K. pneumoniae are carbonylated under standard culture conditions, and that oxidative stress induced by the presence of Fe⁺⁺ and H₂O₂ increases the number of carbonylated proteins, particularly when their hydrogenase activity is inhibited by KCN. Inhibition of H₂ generation markedly suppresses the growth of K. pneumoniae. These observations suggest that H₂ generation and/or the reduction of oxidative stress is important for the survival and growth of K. pneumoniae in the oral cavity.     

Longitudinal Genome-Wide Association of Cardiovascular Disease Risk Factors in the Bogalusa Heart Study

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recent genome-wide association (GWA) studies have pinpointed many loci associated with CVD risk factors in adults. It is unclear, however, if these loci predict trait levels at all ages, if they are associated with how a trait develops over time, or if they could be used to screen individuals who are pre-symptomatic to provide the opportunity for preventive measures before disease onset. We completed a genome-wide association study on participants in the longitudinal Bogalusa Heart Study (BHS) and have characterized the association between genetic factors and the development of CVD risk factors from childhood to adulthood. We report 7 genome-wide significant associations involving CVD risk factors, two of which have been previously reported. Top regions were tested for replication in the Young Finns Study (YF) and two associations strongly replicated: rs247616 in CETP with HDL levels (combined P = 9.7×10⁻²⁴), and rs445925 at APOE with LDL levels (combined P = 8.7×10⁻¹⁹). We show that SNPs previously identified in adult cross-sectional studies tend to show age-independent effects in the BHS with effect sizes consistent with previous reports. Previously identified variants were associated with adult trait levels above and beyond those seen in childhood; however, variants with time-dependent effects were also promising predictors. This is the first GWA study to evaluate the role of common genetic variants in the development of CVD risk factors in children as they advance through adulthood and highlights the utility of using longitudinal studies to identify genetic predictors of adult traits in children.     

Volatile emissions and phenolic compound concentrations along a vertical profile of <Emphasis Type="Italic">Populus nigra</Emphasis> leaves exposed to realistic ozone concentrations

Plants are exposed to increasing levels of tropospheric ozone concentrations. This pollutant penetrates in leaves through stomata and quickly reacts inside leaves, thus making plants valuable ozone sinks, but at the same time triggers oxidation processes which lead to leaf injuries. To counteract these negative effects, plants produce an array of antioxidants which react with ozone and reactive molecules which ozone generates in the leaf tissues. In this study, we measured the effect of an ozone concentration which is likely to be attained in many areas of the world in the near future (80 ppb) on leaves of the vertical profile of the widespread agroforestry species Populus nigra. Changes in (1) physiological parameters (photosynthesis and stomatal conductance), (2) ozone uptake, (3) emission of volatile organic compounds (VOCs, i.e. isoprene, methanol and other oxygenated compounds), (4) concentration of antioxidant surface compounds, and (5) concentration of phenolic compounds were assessed. The aim was to assess whether the defensive pathways leading to isoprenoids and phenolics formation were induced when a moderate and chronic increment of ozone is not able to damage photosynthesis. No visual injuries and minor changes in physiology and ozone uptake were observed. The emission of isoprene and oxygenated six-carbon (C6) volatiles were inhibited by ozone, whereas methanol emission was increased, especially in developing leaves. We interpret these results as suggesting an ontogenetic shift in ozone-treated leaves, leading to a slower development and a faster senescence. Most surface and phenolic compounds showed a declining trend in concentration from the youngest to the fully expanded leaves. Ozone reduced the concentrations of chlorogenic acid derivatives at the leaf surface, whereas in total leaf extracts a metabolic shift towards few phenolics with higher antioxidant capacity was observed.     

Systemic Induction of Salicylates in Salix myrsinifolia(Salisb.)

We studied the induction of salicylates in mature leaves ofSalix myrsinifolia Salisb. (Salicaceae) following severe woundingby a specialist leaf-beetle Phratora vitellinae L. (Chrysomelidae).Levels of individual salicylates and aromatic amino acids andtheir total levels were determined in leaves at different developmentalstages. Induction of salicylates depended on: (1) the individualcompound; (2) the developmental stage of the plant organ; and(3) the genotype of the plant. Induction of salicylates wassystemic: levels of salicylates rose in unwounded young immatureand mature leaves, but no local response was detected in woundedleaves. In addition, there were clear clonal variations in boththe constant and induced levels of salicylates: clones withthe highest levels of salicylates were also most capable ofincreasing this level in response to herbivore attack i.e. notrade-off between constant and induced levels was detected.Furthermore, the levels of three aromatic amino acids, Phe,Tyr and Trp, increased in immature leaves of herbivore-affectedplants, which may indicate induction of enzymes of the shikimatepathway by wounding. The increase in salicylates was suggestedto be a consequence of an increased rate of synthesis ratherthan that of translocation. The induced levels of salicylatesdid not affect the subsequent feeding of highly specializedP. vitellinae. However, the ability to increase levels of salicylatesmay reduce grazing by generalist herbivores which are not ableto tolerate high levels of salicylates. Copyright 2001 Annalsof Botany Company Salix myrsinifolia, dark-leaved willow, Phratora vitellinae, salicylates, phenolic glycosides, herbivory, induced defence