Structural Studies on Ferric Uptake Regulator Proteins from Mycobacterium tuberculosis

Iron is the most abundant trace element in the human body and zinc is the second one. Control of ion homeostasis is of vital importance for mammals and bacteria. Regulation of the ion flux into or out of the cell is a complex and articulated mechanism that still needs to be elucidated. The highly specialized pathogen Mycobacterium tuberculosis has to contend with iron sequestration in order to survive in the human body. Iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. Paucity of this metal triggers an extensive response to increase iron acquisition whereas an excess of it can be toxic for the cell. The control of intracellular iron concentration is also linked to other important processes including oxidative stress response and the regulation of virulence factors. Recent studies have shown that, in patients affected by TB/HIV exposed to high level of dietary iron, the risk of active pulmonary tuberculosis increases. In M. tuberculosis the ferric uptake regulator A (FurA) is activated by Fe2+ to bind specifically to its target DNA sequence thereby repressing the downstream genes. Zinc is also an important element for all living organisms and serves as a cofactor in all six classes of enzymes and also in several regulatory proteins. The intracellular concentration of this metal must be carefully regulated due its toxicity. Compared with eukaryotes, little is known about zinc homeostasis in bacteria. In the tuberculosis genome several genes coding for zinc proteins have been classified but curiously no zinc regulator has been yet defined. Surprisingly, instead, two Fur genes were identified: Mtb furA and Mtb furB, but no clear structural or functional distinction has been reported. In this thesis a careful and detailed structural and biological description of FurA and FurB proteins is presented. Using a variety of biochemical and biophysical methods - including electrophoretic mobility shift assay (EMSA), site directed mutations, isothermal calorimetry (ITC), microPIXE analysis, extended X-ray absorption fine spectroscopy (EXAFS) and X-ray crystallography - we investigated the metal binding sites together with the nature and the structure of these proteins. The combination of these results enable us to distinguish between structurally and functionally distinct metal binding sites, provide a meticulous description and qualitative and quantitative characterization of them, propose biological roles and present for the first time a 3D picture of a zinc uptake regulator

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk

Evaluation of (−)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake

Abstract: Data from dietary intervention studies suggest that intake of (−)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (−)-epicatechin metabolites (SREM), particularly (−)-epicatechin-3′-glucuronide, (−)-epicatechin-3′-sulfate and 3′-O-methyl-(−)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (−)-epicatechin intake. SREMB in urine proved to be a specific indicator of (−)-epicatechin intake, showing also a strong correlation with the amount of (−)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (−)-epicatechin as SREMB in 24 h urine was 10% (IQR 7–13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (−)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (−)-epicatechin

Biomarker-estimated flavan-3-ol intake is associated with lower blood pressure in cross-sectional analysis in EPIC Norfolk

Funder: Mars; doi: http://dx.doi.org/10.13039/100007246Abstract: Flavan-3-ols are a group of bioactive compounds that have been shown to improve vascular function in intervention studies. They are therefore of great interest for the development of dietary recommendation for the prevention of cardio-vascular diseases. However, there are currently no reliable data from observational studies, as the high variability in the flavan-3-ol content of food makes it difficult to estimate actual intake without nutritional biomarkers. In this study, we investigated cross-sectional associations between biomarker-estimated flavan-3-ol intake and blood pressure and other CVD risk markers, as well as longitudinal associations with CVD risk in 25,618 participants of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort. High flavan-3-ol intake, achievable as part of an habitual diet, was associated with a significantly lower systolic blood pressure (− 1.9 (− 2.7; − 1.1) mmHg in men and − 2.5 (− 3.3; − 1.8) mmHg in women; lowest vs highest decile of biomarker), comparable to adherence to a Mediterranean Diet or moderate salt reduction. Subgroup analyses showed that hypertensive participants had stronger inverse association between flavan-3-ol biomarker and systolic blood pressure when compared to normotensive participants. Flavanol intake could therefore have a role in the maintenance of cardiovascular health on a population scale

Biomarker-estimated flavan-3-ol intake is associated with lower blood pressure in cross-sectional analysis in EPIC Norfolk.

Flavan-3-ols are a group of bioactive compounds that have been shown to improve vascular function in intervention studies. They are therefore of great interest for the development of dietary recommendation for the prevention of cardio-vascular diseases. However, there are currently no reliable data from observational studies, as the high variability in the flavan-3-ol content of food makes it difficult to estimate actual intake without nutritional biomarkers. In this study, we investigated cross-sectional associations between biomarker-estimated flavan-3-ol intake and blood pressure and other CVD risk markers, as well as longitudinal associations with CVD risk in 25,618 participants of the European Prospective Investigation into Cancer (EPIC) Norfolk cohort. High flavan-3-ol intake, achievable as part of an habitual diet, was associated with a significantly lower systolic blood pressure (- 1.9 (- 2.7; - 1.1) mmHg in men and - 2.5 (- 3.3; - 1.8) mmHg in women; lowest vs highest decile of biomarker), comparable to adherence to a Mediterranean Diet or moderate salt reduction. Subgroup analyses showed that hypertensive participants had stronger inverse association between flavan-3-ol biomarker and systolic blood pressure when compared to normotensive participants. Flavanol intake could therefore have a role in the maintenance of cardiovascular health on a population scale

Unusual Heme Iron-Lipid Acyl Chain Coordination in Escherichia coli Flavohemoglobin

Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule. Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra. Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron. EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron. EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 Å in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 Å (nonbonding interaction) can be singled out in the lipid-free protein. This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 Å resolution obtained in the present work. Thus, the contributions at 2.7 Å distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid

Identification of Rare Loss-of-Function Genetic Variation Regulating Body Fat Distribution.

CONTEXT: Biological and translational insights from large-scale, array-based genetic studies of fat distribution, a key determinant of metabolic health, have been limited by the difficulty in linking predominantly noncoding variants to specific gene targets. Rare coding variant analyses provide greater confidence that a specific gene is involved, but do not necessarily indicate whether gain or loss of function (LoF) would be of most therapeutic benefit. OBJECTIVE: This work aimed to identify genes/proteins involved in determining fat distribution. METHODS: We combined the power of genome-wide analysis of array-based rare, nonsynonymous variants in 450 562 individuals in the UK Biobank with exome-sequence-based rare LoF gene burden testing in 184 246 individuals. RESULTS: The data indicate that the LoF of 4 genes (PLIN1 [LoF variants, P = 5.86 × 10-7], INSR [LoF variants, P = 6.21 × 10-7], ACVR1C [LoF + moderate impact variants, P = 1.68 × 10-7; moderate impact variants, P = 4.57 × 10-7], and PDE3B [LoF variants, P = 1.41 × 10-6]) is associated with a beneficial effect on body mass index-adjusted waist-to-hip ratio and increased gluteofemoral fat mass, whereas LoF of PLIN4 (LoF variants, P = 5.86 × 10-7 adversely affects these parameters. Phenotypic follow-up suggests that LoF of PLIN1, PDE3B, and ACVR1C favorably affects metabolic phenotypes (eg, triglycerides [TGs] and high-density lipoprotein [HDL] cholesterol concentrations) and reduces the risk of cardiovascular disease, whereas PLIN4 LoF has adverse health consequences. INSR LoF is associated with lower TG and HDL levels but may increase the risk of type 2 diabetes. CONCLUSION: This study robustly implicates these genes in the regulation of fat distribution, providing new and in some cases somewhat counterintuitive insight into the potential consequences of targeting these molecules therapeutically.This study was funded by the United Kingdom’s Medical Research Council through grants MC_UU_12015/1, MC_PC_13046, MC_PC_13048 and MR/L00002/1. This work was supported by the MRC Metabolic Diseases Unit (MC_UU_12012/5) and the Cambridge NIHR Biomedical Research Centre and EU/EFPIA Innovative Medicines Initiative Joint Undertaking (EMIF grant: 115372). R.K.S, D.B.S. and S.O’R. are supported by the Wellcome Trust (WT 210752, WT 219417 and WT 214274 respectively) the MRC Metabolic Disease Unit, the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre and the NIHR Rare Disease Translational Research Collaboration. L.B.L.W. is supported by Sir Henry Wellcome grant 221651/Z/20/Z. I.B. acknowledges funding from an “Expanding excellence in England” award from Research England. K.S.S. is supported by MRC Project Grant L01999X/1. Some computation was enabled through access granted to K.S.S. to the MRC eMedLab Medical Bioinformatics infrastructure, supported by the Medical Research Council (grant number MR/L016311/1). M.McC. is a Wellcome Senior Investigator supported by Wellcome grants 098381, 090532, 106130, 203141. M.McC. declares that the views expressed in this article are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. M.K. is supported by the Gates Cambridge Trust

Hydroxymethylation profile of cell-free DNA is a biomarker for early colorectal cancer.

Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI [87, 93]). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI [83, 94]) and late stage 4 CRC (AUC 94%; 95% CI [89, 98]). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI [89, 94]). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer