Correction: Lipoprotein signatures of cholesteryl ester transfer protein and HMG-CoA reductase inhibition.

[This corrects the article DOI: 10.1371/journal.pbio.3000572.].

Apolipoprotein A-I concentrations and risk of coronary artery disease: A Mendelian randomization study.

BACKGROUND AND AIMS: Apolipoprotein A-I (apoA-I) infusions represent a potential novel therapeutic approach for the prevention of coronary artery disease (CAD). Although circulating apoA-I concentrations inversely associate with risk of CAD, the evidence base of this representing a causal relationship is lacking. The aim was to assess the causal role of apoA-I using human genetics. METHODS: We identified a variant (rs12225230) in APOA1 locus that associated with circulating apoA-I concentrations (p < 5 × 10-8) in 20,370 Finnish participants, and meta-analyzed our data with a previous GWAS of apoA-I. We obtained genetic estimates of CAD from UK Biobank and CARDIoGRAMplusC4D (totaling 122,733 CAD cases) and conducted a two-sample Mendelian randomization analysis. We compared our genetic findings to observational associations of apoA-I with risk of CAD in 918 incident CAD cases among 11,535 individuals from population-based prospective cohorts. RESULTS: ApoA-I was associated with a lower risk of CAD in observational analyses (HR 0.81; 95%CI: 0.75, 0.88; per 1-SD higher apoA-I), with the association showing a dose-response relationship. Rs12225230 associated with apoA-I concentrations (per-C allele beta 0.076 SD; SE: 0.013; p = 1.5 × 10-9) but not with confounders. In Mendelian randomization analyses, apoA-I was not related to risk of CAD (OR 1.13; 95%CI: 0.98,1.30 per 1-SD higher apoA-I), which was different from the observational association. Similar findings were observed using an independent ABCA1 variant in sensitivity analysis. CONCLUSIONS: Genetic evidence fails to support a cardioprotective role for apoA-I. This is in line with the cumulative evidence showing that HDL-related phenotypes are unlikely to have a protective role in CAD.

Lipoprotein signatures of cholesteryl ester transfer protein and HMG-CoA reductase inhibition.

Cholesteryl ester transfer protein (CETP) inhibition reduces vascular event risk, but confusion surrounds its effects on low-density lipoprotein (LDL) cholesterol. Here, we clarify associations of genetic inhibition of CETP on detailed lipoprotein measures and compare those to genetic inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). We used an allele associated with lower CETP expression (rs247617) to mimic CETP inhibition and an allele associated with lower HMGCR expression (rs12916) to mimic the well-known effects of statins for comparison. The study consists of 65,427 participants of European ancestries with detailed lipoprotein subclass profiling from nuclear magnetic resonance spectroscopy. Genetic associations were scaled to 10% reduction in relative risk of coronary heart disease (CHD). We also examined observational associations of the lipoprotein subclass measures with risk of incident CHD in 3 population-based cohorts totalling 616 incident cases and 13,564 controls during 8-year follow-up. Genetic inhibition of CETP and HMGCR resulted in near-identical associations with LDL cholesterol concentration estimated by the Friedewald equation. Inhibition of HMGCR had relatively consistent associations on lower cholesterol concentrations across all apolipoprotein B-containing lipoproteins. In contrast, the associations of the inhibition of CETP were stronger on lower remnant and very-low-density lipoprotein (VLDL) cholesterol, but there were no associations on cholesterol concentrations in LDL defined by particle size (diameter 18-26 nm) (-0.02 SD LDL defined by particle size; 95% CI: -0.10 to 0.05 for CETP versus -0.24 SD, 95% CI -0.30 to -0.18 for HMGCR). Inhibition of CETP was strongly associated with lower proportion of triglycerides in all high-density lipoprotein (HDL) particles. In observational analyses, a higher triglyceride composition within HDL subclasses was associated with higher risk of CHD, independently of total cholesterol and triglycerides (strongest hazard ratio per 1 SD higher triglyceride composition in very large HDL 1.35; 95% CI: 1.18-1.54). In conclusion, CETP inhibition does not appear to affect size-specific LDL cholesterol but is likely to lower CHD risk by lowering concentrations of other atherogenic, apolipoprotein B-containing lipoproteins (such as remnant and VLDLs). Inhibition of CETP also lowers triglyceride composition in HDL particles, a phenomenon reflecting combined effects of circulating HDL, triglycerides, and apolipoprotein B-containing particles and is associated with a lower CHD risk in observational analyses. Our results reveal that conventional composite lipid assays may mask heterogeneous effects of emerging lipid-altering therapies.

Genetic Determinants of Circulating Glycine Levels and Risk of Coronary Artery Disease.

Background Recent studies have revealed sexually dimorphic associations between the carbamoyl-phosphate synthase 1 locus, intermediates of the metabolic pathway leading from choline to urea, and risk of coronary artery disease ( CAD ) in women. Based on evidence from the literature, the atheroprotective association with carbamoyl-phosphate synthase 1 could be mediated by the strong genetic effect of this locus on increased circulating glycine levels. Methods and Results We sought to identify additional genetic determinants of circulating glycine levels by carrying out a meta-analysis of genome-wide association study data in up to 30 118 subjects of European ancestry. Mendelian randomization and other analytical approaches were used to determine whether glycine-associated variants were associated with CAD and traditional risk factors. Twelve loci were significantly associated with circulating glycine levels, 7 of which were not previously known to be involved in glycine metabolism ( ACADM , PHGDH , COX 18- ADAMTS 3, PSPH , TRIB 1, PTPRD , and ABO ). Glycine-raising alleles at several loci individually exhibited directionally consistent associations with decreased risk of CAD . However, these effects could not be attributed directly to glycine because of associations with other CAD -related traits. By comparison, genetic models that only included the 2 variants directly involved in glycine degradation and for which there were no other pleiotropic associations were not associated with risk of CAD or blood pressure, lipid levels, and obesity-related traits. Conclusions These results provide additional insight into the genetic architecture of glycine metabolism, but do not yield conclusive evidence for a causal relationship between circulating levels of this amino acid and risk of CAD in humans.

Proof of concept for quantitative urine NMR metabolomics pipeline for large-scale epidemiology and genetics.

BACKGROUND: Quantitative molecular data from urine are rare in epidemiology and genetics. NMR spectroscopy could provide these data in high throughput, and it has already been applied in epidemiological settings to analyse urine samples. However, quantitative protocols for large-scale applications are not available. METHODS: We describe in detail how to prepare urine samples and perform NMR experiments to obtain quantitative metabolic information. Semi-automated quantitative line shape fitting analyses were set up for 43 metabolites and applied to data from various analytical test samples and from 1004 individuals from a population-based epidemiological cohort. Novel analyses on how urine metabolites associate with quantitative serum NMR metabolomics data (61 metabolic measures; n = 995) were performed. In addition, confirmatory genome-wide analyses of urine metabolites were conducted (n = 578). The fully automated quantitative regression-based spectral analysis is demonstrated for creatinine and glucose (n = 4548). RESULTS: Intra-assay metabolite variations were mostly <5%, indicating high robustness and accuracy of urine NMR spectroscopy methodology per se. Intra-individual metabolite variations were large, ranging from 6% to 194%. However, population-based inter-individual metabolite variations were even larger (from 14% to 1655%), providing a sound base for epidemiological applications. Metabolic associations between urine and serum were found to be clearly weaker than those within serum and within urine, indicating that urinary metabolomics data provide independent metabolic information. Two previous genome-wide hits for formate and 2-hydroxyisobutyrate were replicated at genome-wide significance. CONCLUSION: Quantitative urine metabolomics data suggest broad novelty for systems epidemiology. A roadmap for an open access methodology is provided.

Biomarker Glycoprotein Acetyls Is Associated With the Risk of a Wide Spectrum of Incident Diseases and Stratifies Mortality Risk in Angiography Patients.

BACKGROUND: Integration of systems-level biomolecular information with electronic health records has led to recent interest in the glycoprotein acetyls (GlycA) biomarker-a serum- or plasma-derived nuclear magnetic resonance spectroscopy signal that represents the abundance of circulating glycated proteins. GlycA predicts risk of diverse outcomes, including cardiovascular disease, type 2 diabetes mellitus, and all-cause mortality; however, the underlying detailed associations of GlycA's morbidity and mortality risk are currently unknown. METHODS: We used 2 population-based cohorts totaling 11 861 adults from the Finnish general population to test for an association with 468 common incident hospitalization and mortality outcomes during an 8-year follow-up. Further, we utilized 900 angiography patients to test for GlycA association with mortality risk and potential utility for mortality risk discrimination during 12-year follow-up. RESULTS: New associations with GlycA and incident alcoholic liver disease, chronic renal failure, glomerular diseases, chronic obstructive pulmonary disease, inflammatory polyarthropathies, and hypertension were uncovered, and known incident disease associations were replicated. GlycA associations for incident disease outcomes were in general not attenuated when adjusting for hsCRP (high-sensitivity C-reactive protein). Among 900 patients referred to angiography, GlycA had hazard ratios of 4.87 (95% CI, 2.45-9.65) and 5.00 (95% CI, 2.38-10.48) for 12-year risk of mortality in the fourth and fifth quintiles by GlycA levels, demonstrating its prognostic potential for identification of high-risk individuals. When modeled together, both hsCRP and GlycA were attenuated but remained significant. CONCLUSIONS: GlycA was predictive of myriad incident diseases across many major internal organs and stratified mortality risk in angiography patients. Both GlycA and hsCRP had shared and independent contributions to mortality risk, suggesting chronic inflammation as an etiological factor. GlycA may be useful in improving risk prediction in specific disease settings.

In vitro characterization of alkylaminophenols-induced cell death.

Alkylaminophenols are synthetic derivatives well known for their anticancer activity. In the previous studies, we described the activity of the series of Alkylaminophenols derivatives and their ability to induce cell death for many cancer cell lines. However, temporal heterogeneity in cell death induced by lead compounds, N-(2-hydroxy-5-nitrophenyl (4'-methylphenyl) methyl) indoline (Compound I) and 2-((3,4-dihydroquinolin-1(2H)-yl) (4-methoxyphenyl) methyl) phenol (Compound II), has never been tested on osteosarcoma cells (U2OS). Here, we address the level of cell-to-cell heterogeneity by examine whether differences in the type of compounds could influence its effects on cell death of U2OS. Here, we applied imaging, computational methods and biochemical methods to study heterogeneity, apoptosis, reactive oxygen species and caspase. Our results demonstrate that the Hill coefficient of dose-response curve of Compound II is greater than compound I in treated U2OS cells. Both Compounds trigger not only apoptotic cell death but also necro-apoptotic and necrotic cell death. The percentage of these sub-populations varies depending on compounds in which greater variance is induced by compound II than Compound I. We also identified the accumulation of compounds-induced reactive oxygen species during the treatment. This resulted in caspase 3/7 activation in turn induced apoptosis. In summary, the screening of Compound I and II molecules for heterogeneity, apoptosis, reactive oxygen species and caspase has identified compound II as promising anti-osteosarcoma cancer agent. Compound II could be a promising lead compound for future antitumor agent development.

Phase-dependent dynamics of the lac promoter under nutrient stress.

To survive, a bacterial population must sense nutrient availability and adjust its growth phase accordingly. Few studies have quantitatively analyzed the single-cell behavior of stress and growth phase-related transcriptional changes in Escherichia coli. To investigate the dynamic changes in transcription during different growth phases and starvation, we analyzed the single-cell transcriptional dynamics of the E. coli lac promoter. Cells were grown under different starvation conditions, including glucose, magnesium, phosphate and thiamine limitations, and transcription dynamics was quantified using a single RNA detection method at different phases. Differences in gene expression over conditions and phases indicate that stochasticity in transcription dynamics is directly connected to cell phase and availability of nutrients. Except for glucose, the pattern of transcription dynamics under all starvation conditions appears to be similar. Transcriptional bursts were more prominent in lag and stationary phase cells starved for energy sources. Identical behavior was observed in exponential phase cells starved for phosphate and thiamine. Noise measurements under all nutrient exhaustion conditions indicate that intrinsic noise is higher than extrinsic noise. Our results, obtained in a relA1 mutational background, which led to suboptimal production of ppGpp, suggest that the single-cell transcriptional changes we observed were largely ppGpp-independent. Taken together, we propose that, under different starvation conditions, cells are able to decrease the trend in cell-to-cell variability in transcription as a common means of adaptation.

Transcription closed and open complex dynamics studies reveal balance between genetic determinants and co-factors.

In E. coli, promoter closed and open complexes are key steps in transcription initiation, where magnesium-dependent RNA polymerase catalyzes RNA synthesis. However, the exact mechanism of initiation remains to be fully elucidated. Here, using single mRNA detection and dual reporter studies, we show that increased intracellular magnesium concentration affects Plac initiation complex formation resulting in a highly dynamic process over the cell growth phases. Mg2+ regulates transcription transition, which modulates bimodality of mRNA distribution in the exponential phase. We reveal that Mg2+ regulates the size and frequency of the mRNA burst by changing the open complex duration. Moreover, increasing magnesium concentration leads to higher intrinsic and extrinsic noise in the exponential phase. RNAP-Mg2+ interaction simulation reveals critical movements creating a shorter contact distance between aspartic acid residues and Nucleotide Triphosphate residues and increasing electrostatic charges in the active site. Our findings provide unique biophysical insights into the balanced mechanism of genetic determinants and magnesium ion in transcription initiation regulation during cell growth.

Origins of transcriptional transition: balance between upstream and downstream regulatory gene sequences.

UNLABELLED: By measuring individual mRNA production at the single-cell level, we investigated the lac promoter's transcriptional transition during cell growth phases. In exponential phase, variation in transition rates generates two mixed phenotypes, low and high numbers of mRNAs, by modulating their burst frequency and sizes. Independent activation of the regulatory-gene sequence does not produce bimodal populations at the mRNA level, but bimodal populations are produced when the regulatory gene is activated coordinately with the upstream and downstream region promoter sequence (URS and DRS, respectively). Time-lapse microscopy of mRNAs for lac and a variant lac promoter confirm this observation. Activation of the URS/DRS elements of the promoter reveals a counterplay behavior during cell phases. The promoter transition rate coupled with cell phases determines the mRNA and transcriptional noise. We further show that bias in partitioning of RNA does not lead to phenotypic switching. Our results demonstrate that the balance between the URS and the DRS in transcriptional regulation determines population diversity. IMPORTANCE: By measuring individual mRNA production at the single-cell level, we investigated the lac promoter transcriptional transition during cell growth phases. In exponential phase, variation in transition rate generates two mixed phenotypes producing low and high numbers of mRNAs by modulating the burst frequency and size. Independent activation of the regulatory gene sequence does not produce bimodal populations at the mRNA level, while it does when activated together through the coordination of upstream/downstream promoter sequences (URS/DRS). Time-lapse microscopy of mRNAs for lac and a lac variant promoter confirm this observation. Activation of the URS/DRS elements of the promoter reveals a counterplay behavior during cell phases. The promoter transition rate coupled with cell phases determines the mRNA and transcriptional noise. We further show that bias in partitioning of RNA does not lead to phenotypic switching. Our results demonstrate that the balance between URS and DRS in transcription regulation is determining the population diversity.